The differences in amendment effect on pH SOC, TN, POXC, and nitrate were less distinct in the fine-textured Piedmont soil than the coarse-textured Coastal soil. Overall, the results indicate that, unlike HA, HR and HB will have beneficial effects on soil health and productivity, therefore potentially improving soil’s resilience to changing climate.Application of crop residues and biochar have been demonstrated to improve soil biological and chemical properties in agroecosystems. However, the integrated effect of organic amendments and hydrological cycles on soil health indicators are not well understood. In this study, we quantified the impact of hemp residue (HR), hemp biochar (HB), and hardwood biochar (HA) on five hydrolytic enzymes, soil microbial phospholipid (PLFA) community structure, pH, permanganate oxidizable carbon (POXC) soil organic carbon (SOC), and total nitrogen (TN). We compared two soil types, Piedmont and Coastal Plain soils of North Carolina, under (i) a 30-d moisture cycle maintained at 60% water-filled pore space (WFPS) (D-W1), followed by (ii) a 7-day alternate dry-wet cycle for 42 days (D-W2), or (iii) maintained at 60% WFPS for 42 days (D-W3) during an aerobic laboratory incubation. Results showed that HR and HB significantly increased the geometric mean enzyme activity by 1-2-fold in the Piedmont soil under the three moisture cycles and about 1.5-fold under D-W in the Coastal soil. In the presence of HA, the measured soil enzyme activities were significantly lower than control under the moisture cycles in both soil types. The shift in microbial community structure was distinct in the Coastal soil but not in the Piedmont soil. Under D-W2, HR and HB significantly increased POXC (600–700 mg POXC kg Cannabis sativa (L.)] for cannabidiol (CBD) has become a center of attention among farmers in the United States following the signing of the 2018 Farm Bill into law, which re-established it as an agricultural commodity when tetrahydrocannabinol (THC) concentration is below 0.3%  soil health indicators in response to hydrological cycles in two soil types located in North Carolina, USA.Secale cereale) and crimson clover (Trifolium incarnatum) as winter cover crops, while the soil collected at CEFS, which is from the Coastal Plain and hereinafter mentioned as “Coastal”, was previously cultivated under buckwheat (Fagopyrum esculentum) in summer followed by rye and crimson clover as winter cover crops. Piedmont soil is a sandy clay loam soil classified as fine, mixed, active, thermic Ultic Hapludalfs Alfisol, while Coastal soil is loamy sand soil classified as fine-loamy, siliceous, subactive, thermic Aquic Paleudults Ultisols. The 60 yr average annual precipitation and temperature are 1291 mm and 16°C, respectively for the Coastal soil and 1179 mm and 15°C, respectively for the Piedmont soil.Soil (0–15 cm) for this study was collected in March 2019 from the North Carolina Agricultural and Technical (N.C. A&T) State University Research Farm in Greensboro  and the Small Farm Unit of the Center for Environmental Farming Systems (CEFS) in Goldsboro , North Carolina. The soil from N.C. A&T, which is in the Piedmont region and hereinafter mentioned as “Piedmont”, was previously cultivated under hemp in 2018 summer before rye (3--N) was determined using field moist samples. The soil properties are presented in Soil samples were passed through a 2 mm mesh sieve, visible crop residues were removed, and sieved soils were stored at 4 ˚C for 7 days prior to incubation set up. A portion of the soil was air-dried to determine the pH [, total SOC, TN, and Mehlich (III) P. Nitrate-N (NOQuercus sp.). The hemp and hardwood biochar were produced using slow pyrolysis at a temperature of 800 ˚C for 1 h. Elemental analysis of biochar samples for C, H, and N was determined using a CHN analyzer 2400 series II, , while the chemical analysis was determined after digestion with HNO3/HF using an Optima 8300 Inductively coupled plasma—optical emission spectrometry . The Brunauer-Emmett-Teller (BET) surface area analysis of the biochar was carried out using the ASAP 2020 BET Surface Area and Porosity Analyzer  at the Joint School of Nanoscience and Nanoengineering at N.C. A&T State University. The chemical properties of the amendments are presented in total:Ntotal), i.e., 46.7, 67.6, and 229 for hemp residue (HR), hemp biochar (HB), and hardwood biochar (HA), respectively. The BET surface area of the hemp biochar is 73 m2 g-1 while the hardwood biochar has a value of 656 m² g-1.Hemp residues from the 2018 harvest season were collected from N.C. A&T research farm in Greensboro. Hardwood biomass was obtained from an oak tree . The soils were destructively sampled at the end of each moisture cycle for further analysis as mentioned below.Four treatments were used for the incubation study. This includes soils amended with (i) HR, (ii) HB, (iii) HA, and (iv) control (no amendment). A soil microcosm laboratory incubation experiment was designed under varying moisture cycles, i.e., (i) a 30-d moisture cycle maintained at 60% water-filled pore space (WFPS) (D-W1); followed by (ii) a longer moisture cycle of a 7-d alternate dry-wet (5 and 60% WFPS) cycle for 42-d (D-W2); or (iii) a 42-d moisture cycle maintained at 60% WFPS (D-W3) . The exp2 and 4 mL of 0.5 M NaOH or 0.1 M Tris(hydroxymethyl)aminomethane buffer depending on the enzyme, and the suspension was filtered using a Whatman No. 2v filter. The p-nitrophenol product released was measured colorimetrically at 400 nm using a visible spectrophotometer. Soil microbial community structure, using phospholipid fatty acids (PFLA) analysis, was determined using fresh soil at the Ward Laboratories Inc. . Briefly, soil lipid was extracted with one-phase chloroform:methanol:phosphate (1:2:0.8) buffer, with PLFA eluted from silicic acid columns in methanol, following removal of non-polar lipids by washing with chloroform and acetone [The potential activities of five soil hydrolytic enzymes, β-glucosidase, β-glucosaminidase, acid phosphatase, phosphodiesterase, and arylsulfatase, associated with C, N, P, and S cycling, respectively, were determined –22. Brie acetone . An aliq acetone . Microbi acetone , which a3--N), a chemical soil health indicator, was determined in 2 M KCl extracts  after shaking at 180 rev min-1 for 1 h and filtered using Q2 filter papers . The NO3--N concentration was determined colorimetrically using the Vanadium (III) acid method at 540 nm [-1 KMnO4 solution was added to 2.5 g of air-dried soil and placed on a shaker for 180 rev min-1 for 2 min. Thereafter, the soil was left undisturbed for 10 min and 0.5 mL of the supernatant was quantitively transferred into a second 50 mL centrifuge tube containing 49.5 mL of deionized water. The absorbance values were read at 550 nm using a Synergy HTX multimode microplate reader. The TC and TN, which are soil biochemical indicators, were measured using a CHN analyzer 2400 series II, . The TC is equivalent to the total SOC because these soils did not contain carbonates.Soil pH, a chemical soil health indicator,  was measured with a benchtop Thermo Orion 420A Plus pH meter . Inorganic nitrate-nitrogen (NOt 540 nm  using a t 540 nm . BrieflyF-statistic) was not significant, a simple effect comparison of the interaction (LSMESTIMATE) was done. Pearson correlation test was performed to test the linear relationships (p<0.05) among the response variables for each soil type using the CORR procedure in SAS Studio University Edition. To distinguish the treatment effects on the response variables as a function of the soil type, amendment, and moisture cycle, principal component analysis (PCA) was performed after standardizing the data using the JMP package of SAS 9.3 .Statistical analysis was completed using a three-way analysis of variance (ANOVA) as a completely randomized design with the GLIMMIX procedure in SAS Studio University Edition . The fixed factors were soil type, moisture cycle, and amendment type. The data was assessed to meet the assumptions of normality and constant variance, log-transformed if required and back-transformed. Differences between means were separated using Tukey-Kramer method (p<0.05). Where the three-way interaction (p-nitrophenol g-1 soil) and HR-amended soil (10.7 μg p-nitrophenol g-1 soil) but higher than in HB-amended soil (8.3 μg p-nitrophenol g-1 soil) at D-W1 (p-nitrophenol g-1 soil). Similarly, in the Piedmont soil, HA significantly reduced the β-glucosidase activity (2.32 μg p-nitrophenol g-1 soil), while it was 75–97% higher in HR-amended soil and about 40% higher in HB-amended soil compared to the control at D-W1 and D-W2. At D-W3, the β-glucosidase activity in HR-amended soil (12.1 μg p-nitrophenol g-1 soil) was higher than in control (9.76 μg p-nitrophenol g-1 soil) and HB-amended (10.1 μg p-nitrophenol g-1 soil) Piedmont soil  the change in soil physical conditions, (ii) the amount of readily decomposable C, or (iii) the presence of toxic compounds such as polycyclic aromatic hydrocarbons, polar pyrolysis condensates or residual pyrolysis oil ,38,39. HWhile the effects of HR and HB on overall enzyme activities were less distinct in the Coastal soil except under D-W3, the geometric mean of enzyme activities in the Piedmont soil was consistently highest in HR-amended soils followed by HB-amended soils, except at D-W3 where there was no significant difference between HB-amended and control soils . MicrobiThe PLFA microbial concentrations responded to a different degree than did soil enzymes across the soil types. The two-way interactions of amendment × moisture cycle (p = 0.011) and soil type × moisture cycle (p = 0.004) were significant for the total PLFA microbial biomass. Changes in the concentrations of total microbial, bacterial, and fungal PLFAs biomass were more distinct in the Coastal soil as compared to the Piedmont soil . In the Long-term D-W events and constant moisture reduced the concentration of total and bacterial PLFA biomass by approximately 50–60% in the Coastal soil but not in the Piedmont soil . In D-W1+) concentration, which increases with decreasing pH, have also been linked with the presence or absence of soil microbial communities, suggesting that the microbial communities in the two soils may be potentially different [Soils with lower fertility are likely to experience a higher frequency of fluctuations in the active soil microbial biomass due to lower labile C and/or available nutrients when perturbed. Our results are in line with those of , suggestifferent . This miifferent . On the ifferent . Howeverifferent , found nIn our study, shifts in microbial community composition, F:B, Gram-positive (G+):Gram-negative (G-), and saturated fatty acid to unsaturated fatty acid (sat:unsat) ratios were calculated . In geneThe measured G(+):G(-) PLFA ratios were lower in amended Coastal soil when averaged over hydrological cycles but remained unchanged in the Piedmont soil . LikewisBoth G(+):G(-) and sat:unsat PLFA concentrations have been used to describe microbial shifts in soils in response to nutrient fluctuations or environmental conditions ,51. It iChanges in the soil chemical and biochemical properties are presented in 3--N was obtainable in HA-amended soils at the end of the incubation at D-W2 and D-W3 in the Coastal soil (3--N in the HA-amended soils (1.8–7.1 mg N kg-1) was between 167–175% higher than control soils in the Coastal soil. Contrarily, no changes in NO3--N among the amended soils were observed in the Piedmont soil (34–53 mg N kg-1). The effect of biochar on NO3--N was notable in the Coastal soil with lower inherent NO3--N levels (24.3 mg kg-1) while there were no distinct differences in the Piedmont soil with higher NO3--N values (54.5 mg kg-1)  [3--N retention and subsequent availability. In addition, the other biochar characteristics such as pore structure and surface area have also been linked to high retention characteristics of biochar [-1 compared to 73 m2 g-1 for HB, which likely contributed to a higher NO3--N concentration at the end of the incubation. Also, the higher amounts of NO3--N in HA-amended Coastal soils under continuous drying and rewetting cycle were reduced under continuous moisture  . Surpris17–0.27) . The HA  biochar . The surmoisture . The redAlthough the HA biochar used in our study reduced microbial activity and N availability, its high surface area, and low H:C ratio indicates it would be effective for remediating contaminated soils and improving soil health. Several studies ,59 have Coastal soils amended with HR and HB were characterized by high POXC values under D-W1 and D-W2 but not under D-W3 . On the Understanding SOC dynamics under variable moisture conditions in amended soils is important for better management. Drying-rewetting condition (D-W2) seemed to facilitate the loss of active C in clay Piedmont soil compared to sandy Coastal soil  but undeWhile changes in SOC are known to be slow, previous studies indicated that biochar addition mostly increases SOC because of its high OC content per mass  even thoThe linkages between soil function, community structure, and chemical composition were assessed using PCA and correlation analyses Figs –9. Our iFurthermore, the correlation analysis showed that PLFA biomarkers were weakly associated with measures of nutrient cycling and other studied soil chemical and biochemical variables Figs  and 9. IThe negative relationship between N and enzyme activity can be explained by the differences in N sorption and retention by biochar which affects microbial activity. This was evidenced by the higher amount of extractable nitrate from HA amended soils. For example, Rajkovich et al.  found the.g., β-glucosidase, β -glucosaminidase, acid phosphatase, arylsulfatase, and phosphodiesterase), though the extent varied with soil type within the same moisture cycle. On the other hand, the application of hardwood biochar consistently led to a decline in soil nutrient cycling shown by the reduced soil enzymatic activities irrespective of the soil type or moisture cycle because of its high surface area and low H:C ratio. The microbial biomass PLFA revealed unclear effects among amended soils, although changes were more distinct in the sandy soil  than the clay soil (Piedmont), and at D-W2 and D-W3, indicating the strong impact of soil type and moisture cycle. It thus appears that under repeated D-W cycle, the application of hemp biochar would likely enhance biological soil health under fluctuating and dynamic hydrological events, especially in coarse texture soils. Based on our current study, the application of hemp biochar has the potential to enhance soil health, considering the positive effect on soil microbial functional diversity and promote soil C accrual in Piedmont and Coastal Plain soils in North Carolina.Impacts of three different amendment types  on soil microbial community structure and function, and biochemical soil health indices at three different moisture cycles were examined in a laboratory incubation experiment in two soil types from North Carolina. Our study demonstrates that both hemp residue and hemp biochar occasionally improved soil microbial enzyme activities (S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


In the present study, we investigated the influence of sequence variation in the recognition arms of the RNA/PNAzyme complex on the RNA cleavage activity of the artificial enzymes. The base pairs closing the 3-nucleotide bulge region on both sides of the bulge as well as the neighbouring nucleobases were shown to significantly influence the RNA cleavage activity. Elongation of the RNA/PNAzyme complex was shown to be tolerated, although potentially prohibitive for catalytic turnover. The specificity of PNAzyme action was clearly demonstrated by the significantly reduced or absent cleavage activity in complexes containing mismatches. Further investigation into 2- and 4-nucleotide RNA bulges indicated that formation of 3-nucleotide bulges in the target RNA gives the optimal cleavage rates, while some potential off-target cleavage of formed 4-nucleotide bulges of select sequences should be considered.The development of Zn 2+ dimethyl-dppz PNAzymes to cleave RNA target sequences with under 20 minute half-lives is critically dependent on the bulge-closing base pairs as well as their stacking interactions with the neighbouring nucleobases.The ability of Zn A thorough understanding of the intricacies of the sequence–activity relationships in the RNA/PNAzyme complexes can shed light on various aspects of PNAzyme action, while also being critical for the effective identification of novel RNA targets and for understanding possible off-target effects. The present study takes a particular interest in the bulge-closing base pairs on both sides of the bulge, as well as the sequence and length of the shorter recognition arm of the RNA/PNAzyme complex. Moreover, in addition to the cleavage of 3-nucleotide RNA bulges, which have been studied in detail,15 we demonstrate the cleavage of 2-nucleotide RNA bulges and study the sequence-dependence in 4-nucleotide bulges.Artificial ribonucleases of various designs have been reported, including both metal-freei.e. the long recognition arm.15 In such systems, the AAA bulge is cleaved site-specifically with an approximately 1 h half-life  showed slightly lower activity. Rather strikingly, the cleavage activity suffered a major loss when the AAA bulge was closed with a UA (RNA 5/PNAzyme V) or AT base pair (RNA 4/PNAzyme IV), where the RNA cleavage half-lives were estimated to be in the 4 and 11 h range, respectively . MS anal15 the nucleobase preceding the bulge-closing base pair has been either of the two pyrimidine bases in the RNA and the complementary purine in the PNA strand. As such, it should be carefully considered in the selection of novel RNA targets in future investigations whether instead of UC or CC (pyrimidine-C), perhaps AG or GG (purine-G) sequence would be tolerated equally well in the bulge-closing region of the long recognition arm. It should be noted that the activity of more complex triplex-forming Cu2+-neocuproine PNAzymes has indeed been higher with purine–purine (AG) rather than purine-pyrimidine (AC) sequence in the RNA target in the same bulge-closing region.18The overall dependence of RNA cleavage activity on the bulge-closing base pairs (most to least favourable CG > GC > UA > AT) suggests that the strength of hydrogen bonding is key. Moreover, there seems to be an additional benefit if the pyrimidine is in the RNA and the purine in the PNA strand. Herein as well as in prior reports,25 The bulge nucleobases can point out into the solvent, fold back onto adjacent hydrophobic surfaces, or they may take part in the continuous stacking of the surrounding double strands, favouring kinking of the helix.25 As such, the bulge-closing base pair is clearly changing the structural arrangement, which is also evident in the comparison of the circular dichroism (CD) spectra of these four complexes , but the activity remained very low for all five bulge sequences  once again outperformed those with the AT and UA pairs . Interestingly, this change in the position next to the bulge-closing base pair in the short recognition arm led to a slightly lower cleavage rate. As such, the GC sequence in RNA 13 closing the bulge in the short recognition arm was favoured over GG in RNA 20. It remains unclear whether the strength of hydrogen bonding in the position next to the bulge-closing base pair is critical, or perhaps GU (purine–pyrimidine) would be tolerated equally well, hence allowing the possibility of a UA base pair in the RNA/PNAzyme complex next to the bulge-closing GT wobble.Moreover, a further change was then introduced by replacing the GT wobble with an AT bulge-closing base pair, which was then followed by a GC base pair (RNA 6/PNAzyme VII). A further decrease in the RNA cleavage rate was observed. The cleavage still took place at a single site between the second and third nucleotide in the bulge  since the complex stability will be higher. The previously studied complexes (e.g. RNA 1/PNAzyme I) consist of two recognition arms comprising 7 Watson–Crick base pairs on the one side and 1 GT wobble followed by 3 Watson–Crick base pairs on the other side. The elongated complexes studied herein contained elongated short recognition arms. The RNA 24/PNAzyme XI and RNA 25/PNAzyme XII complexes have two and four additional Watson–Crick base pairs at the RNA 3′ terminus. Elongation of the complex in the short recognition arm led to virtually the same extent of cleavage as the shorter system in the presence of equimolar PNAzyme  in the long recognition arm would likely also adversely affect turnover.While the rate of cleavage is unaffected by elongation of the short recognition arm, the length of the complex can be more critical for catalytic turnover, 15 While the latter is evidently a better target, the possibility of off-target cleavage of the former should be considered. Moreover, the rate of cleavage of 3-nucleotide bulges is greatly increased when the AAA bulge is replaced with UUA, AUA or GUA bulges .15 It is likely that the sequence of the 4-nucleotide bulges would also have a significant influence on the cleavage rate, but this sequence-dependence requires further investigation.Prior reports have indicated that the cleavage of the AAAA bulge can be several times slower than the AAA bulge.15 the preservation of the preferred 5′-U/A-3′ or A/A cleavage site was clearly important also in 4-nucleotide bulges, as the presence of uridine in the position next to the GT wobble (AAAU bulge in RNA 8) led to a slight decrease in the cleavage rate and two consecutive uridines next to the GT wobble  were even more detrimental to the cleavage rate  were cleaved more efficiently when the complex contained the CG and GC bulge-closing base pairs instead of UA and AT pairs. Thus, the bulge-closing base pair was suggested to have a critical role in defining the structural arrangement of the bulge nucleotides, while interactions involving the base pair preceding the bulge-closing base pair are also likely to be important.The bulge-closing base pair in the short recognition arm was equally influential, as the highest rates were obtained with a GT wobble, and both AT and GC Watson–Crick base pairs had a negative effect on the cleavage rate. The activity was also affected by the succeeding base pair.2+-dimethyl-dppz-PNAzymes. For rapid site-specific cleavage, the RNA targets are suggested to require the sequence 5′-Py-C-bulge-G-C-3′ , although the feasibility of the RNA sequence Pu-G (Pu = purine) instead of Py-C preceding the bulge and G-U following the bulge could also be evaluated. Variations in the rest of the sequence are likely to be tolerated. Furthermore, the RNA/PNAzyme complex can be longer than the standard 11 base pairs, albeit the catalytic turnover might be affected if either of the cleaved fragments forms more than 7 base pairs with the PNAzyme. Consequently, these structural demands must be taken into consideration in the selection of RNA target sequences for PNAzymes in future studies. Although RNA targets forming 3-nucleotide bulges are clearly more efficiently cleaved, potential off-target sequence cleavage should be considered, as the PNAzymes were shown to also cleave 4-nucleotide RNA bulges of select sequences with down to 1 h half-lives.These sequence–activity relationships shed light on the structural demands for efficient cleavage of RNA targets by the Zn15 Oligoribonucleotides RNA 26–28 were purchased purified from integrated DNA Technologies, Inc . RNA 13, 16, 24 and 25 were synthesised as previously reported.15 Concentrations of RNA sequences were determined by UV absorption at 260 nm on a Varian Cary 300 UV-Vis dual beam spectrophotometer  using extinction coefficients obtained by the nearest neighbour approximation.26All reagents and solvents used were of analytical commercial quality. RNA cleavage experiments were performed in nuclease-free water for molecular biology purchased from Sigma-Aldrich . Oligoribonucleotides RNA 1–23 were purchased from Dharmacon . RNA 1 and 13 were purchased purified. RNAs 2–12 and 14–23 were purified as previously reported.−1) was purchased from Biotage . Peptide nucleic acid monomers, Fmoc-PNA-A(Bhoc)-OH, Fmoc-PNA-G(Bhoc)-OH, Fmoc-PNA-C(Bhoc)-OH and Fmoc-PNA-T-OH, were purchased from Link Technologies Ltd . Fmoc-l-Dap(Mtt)-OH , Fmoc-l-Lys(Boc)-OH, N,N′-diisopropylcarbodiimide (DIC) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolopyridinium 3-oxide hexafluorophosphate (HATU) were purchased from Iris Biotech GmbH . Ethyl cyano(hydroxyimino) acetate (Oxyma) was purchased from Merck-Millipore . Acetic anhydride and 2,6-lutidine were purchased from Alfa Aesar . N-Methylpyrrolidone (NMP), dichloromethane (DCM), triisopropylsilane (TIS), piperidine and N-methylmorpholine (NMM) were purchased from Sigma-Aldrich . Trifluoroacetic acid (TFA) was purchased from Fisher Scientific . Neocuproine and 3,4-diaminobenzoic acid were purchased from Fluorochem .Rink Amide resin  were prepared as previously reported.15 The RP-HPLC purity analysis of purified PNA conjugates is available in ESI-S1.−1 sinapic acid in acetonitrile–water  containing 0.1% TFA).PNA oligomers were prepared on a Biotage Initiator+ Alstra microwave peptide synthesiser as previously reported.m/z: PNAzyme I C151H181N73O38 [M + H]+1 calculated 3625.34, observed 3625.36. PNAzyme II C162H194N80O41 [M + H]+1 calculated 3916.56, observed 3916.92. PNAzyme III C161H194N78O41 [M + H]+1 calculated 3876.56, observed 3877.13. PNAzyme IV C162H195N77O42 [M + H]+1 calculated 3891.56, observed 3891.73. PNAzyme V C162H194N80O40 [M + H]+1 calculated 3900.57, observed 3901.06. PNAzyme VI C150H180N74O37 [M + H]+1 calculated 3610.46, observed 3611.66. PNAzyme VII C150H181N71O38 [M + H]+1 calculated 3585.45, observed 3585.60. PNAzyme VIII C150H181N71O38 [M + H]+1 calculated 3585.45, observed 3585.29. PNAzyme IX C151H181N73O37 [M + H]+1 calculated 3609.46, observed 3609.30. PNAzyme X C151H182N70O39 [M + H]+1 calculated 3600.45, observed 3600.53. PNAzyme XI C173H207N87O42 [M + H]+1 calculated 4175.68, observed 4176.44. PNAzyme XII C194H234N96O49 [M + H]+1 calculated 4692.88, observed 4693.48.MALDI-MS 26 and the reported extinction coefficient for dipyridophenazine.27Concentrations of PNA conjugates were determined by UV absorption at 260 nm on a Varian Cary 300 UV-Vis dual beam spectrophotometer , using extinction coefficients obtained by the nearest neighbour approximation,15 but for clarity we requote them in ESI-S2.2+ ion binding.RNA cleavage experiments were performed and analysed as previously reported,−1 linear gradient from 0 to 80% acetonitrile in 8.6 mM TEA + 100 mM HFIP buffer over 15 min at 40 °C on Waters Xevo G2-XS QTof. Ionization mode: ESI negative. Source capillary voltage: 2 kV. Source desolvation temperature: 320 °C. Full scan with fragmentation, mass range: 400–2000 Da/0.5 s, sensitivity mode. Deconvoluted using 5 ppm tolerance. The MS spectra of the cleaved fragments are shown in ESI-S3.RNA cleavage experiments were performed as detailed above, but with a 22 hour incubation time. The cleaved fragments were then identified by LC-MS analysis of the quenched reaction aliquots as follows. HRMS spectra were collected by elution of the aliquots of RNA cleavage reactions on an Oligonucleotide BEH C18  column using a 0.4 mL min2+ ions in 10 mM phosphate buffer containing 100 mM NaCl and 0.1 mM EDTA at pH 7.0. The spectra were smoothed over 5 points. The spectra are shown in ESI-S4.†CD spectra of RNA/PNAzyme complexes were measured between 230 and 370 nm on a Jasco J-1500 CD Spectrometer using 10 mm path length cuvettes. The spectra were recorded as an average of five scans at 25 °C and normalised by subtracting the background buffer scans. The samples of RNA/PNAzyme complexes (4 μM) were analysed in the absence of ZnConceptualisation – O. L., M. M. and R. S. Formal analysis – O. L. and R. S. Funding acquisition – R. S. Investigation – O. L and K. K. with contributions from P. B., M. B. Methodology – O. L., M. M., R. S. Project administration – O. L., R. S. Resources – U. T., R. S. Supervision – M. B., U. T., R. S. Visualisation – O. L. Writing - original draft – O. L. Writing – review and editing – O. L. and R. S. with contributions from all.Since performing this work, Olivia Luige has become an employee of AstraZeneca.There are no conflicts to declare.RA-012-D1RA08319H-s001


In the last decade, anti-PD-1 and anti-PD-L1 checkpoint inhibitors (CIs) have demonstratedto be clinically active as a single agent in several types of solid tumors and hematologicalmalignancies. However, the clinical benefit is observed only in a fraction of patients and,in the majority of cases, only for a limited period of time .in vivo inimmunocompetent murine models of triple negative breast cancer (TNBC) and B-cell lymphomathe effects of different types and dosages of chemotherapeutics over the immune cellorchestra.Chemotherapeutics are known to have profound effects over several myeloid, innate, helperand effector immune cell subsets . As CI cTwo main findings emerged: first, we observed that vinca alcaloids (V), at low dosages, cangenerate and activate new APCs. Secondly, we found that intermitted, medium dosagecyclophosphamide  can generate new CD3+CD4+ and CD3+CD8+T cellclones. In our models, a “two-hit” approach (V plus C140) significantly improved CIpreclinical activity against local and disseminated neoplastic growth -6.Our models included mice with neoplastic lesions showing an immune infiltrate predominantlyincluding lymphoid cells and others with and an immune infiltrate with high proportions ofmyeloid cells. In both cases, antibody-mediated depletion of CD3+CD4+ or of CD3+CD8+ T cellsabrogated the preclinical efficacy of the V plus C140 plus CI combinatorial therapy. Thesedata suggest that anti-cancer activity was largely related to these immune T cells .Tcf1+stem-like CD8+ T-cells [Single-cell transcriptome analysis of >50,000 intratumoural immune cells, after V plus C140and CI combinatorial therapy, showed a gene signature suggestive of a change resulting fromexposure to a mitogen, ligand, or an antigen for which it is specific, as well asAPC-to-T-cell adhesion . This tr T-cells  and alte T-cells . The proTCF1+ stem-like CD8+ T-cells; c) CXCL13 andCCR5 overexpressing, neo-antigen reactive CD8+ T cells [As V and C140 dosages found to significantly improve CI preclinical activity are suitablefor a combinatorial use in cancer patients, clinical trials in TNBC and B-cell lymphoma arenow planned to confirm the efficacy of this “two-hit” plus CI therapy. It will be ofinterest to investigate in enrolled patients a number of emerging candidate biomarkers ofimmune cell activation, including: a) APC activation patterns; b) the generation of new T cells ; e) pre- T cells ; and f) T cells . The goa


Vibrio cholerae O1 in aquatic reservoirs in Bangladesh apparently increases coinciding with the occurrence of seasonal cholera epidemics. In between epidemics, these bacteria persist in water mostly as dormant cells, known as viable but non-culturable cells (VBNC), or conditionally viable environmental cells (CVEC), that fail to grow in routine culture. CVEC resuscitate to active cells when enriched in culture medium supplemented with quorum sensing autoinducers CAI-1 or AI-2 which are signal molecules that regulate gene expression dependent on cell density. V. cholerae O1 mutant strains with inactivated cqsS gene encoding the CAI-1 receptor has been shown to overproduce AI-2 that enhance CVEC resuscitation in water samples. Since V. cholerae non-O1 non-O139 (non-cholera-vibrios) are abundant in aquatic ecosystems, we identified and characterized naturally occurring variant strains of V. cholerae non-O1 non-O139 which overproduce AI-2, and monitored their co-occurrence with V. cholerae O1 in water samples. The nucleotide sequence and predicted protein products of the cqsS gene carried by AI-2 overproducing variant strains showed divergence from that of typical V. cholerae O1 or non-O1 strains, and their culture supernatants enhanced resuscitation of CVEC in water samples. Furthermore, prevalence of V. cholerae O1 in the aquatic environment was found to coincide with an increase in AI-2 overproducing non-O1 non-O139 strains. These results suggest a possible role of non-cholera vibrios in the environmental biology of the cholera pathogen, in which non-O1 non-O139 variant strains overproducing AI-2 presumably contribute in resuscitation of the latent pathogen, leading to seasonal cholera epidemics. Importance. Toxigenic Vibrio cholerae which causes seasonal epidemics of cholera persists in aquatic reservoirs in endemic areas. The bacteria mostly exist in a dormant state during inter-epidemic periods, but periodically resuscitate to the active form. The resuscitation is enhanced by signal molecules called autoinducers (AIs). Toxigenic V. cholerae can be recovered from water samples that normally test negative for the organism in conventional culture, by supplementing the culture medium with exogenous AIs. V. cholerae belonging to the non-O1 non-O139 serogroups which do not cause cholera are also abundant in natural waters, and they are capable of producing AIs. In this study we characterized V. cholerae non-O1 non-O139 variant strains which overproduce an autoinducer called AI-2, and found that the abundance of the cholera pathogen in aquatic reservoirs correlates with an increase in the AI-2 overproducing strains. Our results suggest a probable role of these variant strains in the environmental biology and epidemiology of toxigenic V. cholerae, and may lead to novel means for surveillance, prevention and control of cholera.Prevalence of toxigenic  Vibrio cholerae is the aquatic ecosystem, and comprises more than 250 O-serogroups. However, only toxigenic strains of V. cholerae O1 and rarely that of O139 cause cholera in humans and may spread as epidemics -deoxycytidine triphosphate  [Presence of defined mutations in the n N16961 , was des strains  Figs. ThCi/mmol) . Colony Ci/mmol) . The colCi/mmol) .V. cholerae genomes were sequenced at the genomics facility of icddr,b using Illumina based technology. For whole genome sequencing, Illumina Nextera® XT DNA library Preparation Kit  was used as per manufacturer’s instructions, to prepare Genomic fragment libraries, and sequencing was conducted with an Illumina MiSeq sequencer. The sequences were assembled, and aligned with reference sequences using software available at Illumina BaseSpace (https://basespace.illumina.com/lab) on-line.V. cholerae O1 in conventional enrichment culture were subjected to this assay as described previously [Environmental water samples which were initially found negative for eviously . In brieeviously . Water sV. cholerae O1 and that of the AI-2 overproducing non-O1 non-O139 strains was tested by Pearson’s correlation coefficient and calculating significance using the online calculator at https://www.socscistatistics.com/pvalues/pearsondistribution.aspx.General statistical analysis of data was done using the data analysis program built-in Microsoft Excel (MS office version 2007) and were expressed as mean ± standard deviation. The differences were analyzed by two-tailed t-test, and values of p<0.05 were considered significant. Correlation between the increase in prevalence of V. cholerae non-O1 non-O139 strains 126V0117, 107V1216, and 86V1216 respectively.The whole genome shotgun projects for various strains have been deposited at DDBJ/ENA/GenBank under the Accession numbers JADNQP000000000, JADNQQ00000000, and JADNQR000000000, for Experimental protocols were reviewed and approved by the Research Review Committee, and Ethics Review Committee of the International Centre for Diarrhoeal Disease Research, Bangladesh (Protocol numbers PR-15029 and PR-07018).V. cholerae produces at least three autoinducers namely CAI-1 and AI-2, and DPO [V. cholerae present in environmental water samples [V. cholerae non-O1 non-O139 variant strains which might overproduce either of the two autoinducers CAI-1 and AI-2 associated with CVEC resuscitation, we initially analyzed 61 different isolates of V. cholerae non-O1 non-O139 from water samples using two bioluminescent reporter strains MM920 and BB170 [V. cholerae O1 in enrichment culture. The two reporter strains MM920 and BB170 expressed bioluminescence specifically in response to autoinducer CAI-1 and AI-2 respectively, and were used to estimate these autoinducers in the culture supernatants of different strains. As control samples, in these assays we included culture supernatant of E. coli DH5α strains harboring recombinant plasmids carrying genes for the synthesis of CAI-1 or AI-2. For comparison, we also included spent culture supernatants of a typical V. cholerae O1 strain C6706 and a V. cholerae non-O1 non-O139 strain 126V0117. As negative controls we also included derivatives of strain C6706 in which the indigenous genes for synthesis of CAI-1 or AI-2 had been deleted [V. cholerae non-O1 designated as 86V-1216 and 107V-1216, both of which overproduced the autoinducer AI-2 in their culture supernatants  and C6706 (4019194 bp), found in the GenBank data base. A total of six genes of the quorum sensing pathway , and five housekeeping genes encoding 6-phosphofructokinase, glucose-6-phosphate dehydrogenase, citrate synthase, malate dehydrogenase, and NAD kinase, carried by the non-O1 non-O139 strains and the V. cholerae O1 strains N16961 and C6706 were compared by multiple sequence alignment. The products of the 5 house keeping genes were found to be conserved with no change at the amino acid level. On the other hand, the predicted cqsS gene products in the two non-O1 non-O139 strains 86V1216, and 107V1216 were significantly altered in several positions compared to the control O1 and non-O1 non-O139 strains. In particular, a cysteine residue at position 324 of the predicted cqsS protein was replaced by an Arginine residue  suddenly after 5h of incubation. Culture supernatants of recombinant E. coli strains expressing either CAI-1 or AI-2, and that of V. cholerae strain C6706 carrying deletion in its cqsS gene were also used in parallel assays, and resuscitation of CVEC also occurred in the presence of these culture supernatants. However, when enriched similarly but using spent medium of V. cholerae strain C6706 deleted of the AI synthase genes, and that of E. coli DH5α with the empty vector pTAC, aliquots of these same water samples were mostly found negative for culturable V. cholerae .These isolates were initially subjected to a PCR assay for the be assay . IsolateqsS gene , and werqsS gene . Remarkain water , and theV. cholerae O1 and its ecological relation to the highly ubiquitous non-O1 non-O139 V. cholerae. These results might help to explain the observed increased abundance of the pathogen in its aquatic reservoirs periodically which presumably enhances transmission leading to seasonal cholera epidemics. Quorum sensing autoinducers (AIs) of V. cholerae such as CAI-1 and AI-2 have been shown to act as resuscitation factors for CVEC, the dormant environmental form of V. cholerae O1 found in water samples, when assayed in the laboratory [V. cholerae O1 mutant strains may overproduce AI-2 which resuscitate dormant toxigenic V. cholerae in such water samples [V. cholerae belong to serogroups other than O1 or O139, collectively known as V. cholerae non-O1 non-O139, presence and periodic blooms of probable AI-overproducing non-O1 non-O139 strains would significantly influence the resuscitation process. Therefore, in this study we sought to identify and genetically characterize V. cholerae non-O1 non-O139 strains which overproduce one or more AIs  was statistically significant (p<0.001). Besides, any water sample that was positive for V. cholerae O1 also yielded a high proportion (>5 out of 10 isolates tested) of V. cholerae non-O1 non-O139 isolates which overproduced AI-2.The environmental prevalence of luctuate . When thV. cholerae O1 that exist in aquatic reservoirs periodically resuscitate prior to occurrence of seasonal epidemics [V. cholerae non-O1 non-O139, which are abundant in natural waters, and have genetic variants that overproduce AI-2.In the Ganges Delta regions of Bangladesh and India, cholera epidemics occur in a seasonal pattern with usually two epidemics each year ,2, and dpidemics –6. The rpidemics . We presV. cholerae [V. cholerae in resuscitation of CVEC can not be ruled out. The resuscitation of CVEC could occur either in the aquatic reservoir due to AI-2 produced by diverse bacterial species or inside the host through possible exposure to AI molecules produced by the normal intestinal microbiota present in humans ingesting V. cholerae. However, the observed correlation of V. cholerae O1 prevalence with that of non-O1 strains overproducing AI-2 in the environment suggest significant contribution of the non-O1 non-O139 V. cholerae. It may be mentioned that in our previous studies [V. cholerae strains were found to produce autoinducers at a relatively lower concentration compared to the AI-2 overproducing mutant strains  PCR results and (b) an autoradiograph derived from colony blot hybridization assays is shown.Primers were designed to exclude strains carrying the defined mutation from amplification of the (DOCX)Click here for additional data file.S2 FigV. cholerae O1 strains. Strains VCN23 and VCN27 are environmental non-O1 non-O139 strains which were positive in the PCR assay for the cqsS gene, whereas strains designated as VCN42, VCN54, VCN56, VCN72, VCN87, and VNC 29 were V. cholerae non-O1 non-O139 strains which were negative in the PCR assay but positive in the DNA probe assay for cqsS gene. C6706 is a laboratory strain of V. cholerae O1.Recombinant plasmid pJZ365 carries the gene for AI-2 synthase required for biosynthesis of AI-2. Strains VCO3 and VCO9 are environmental (DOCX)Click here for additional data file.


This study contributes to the characterization of CD8+ HLA-DR+ T cell mediated-mechanisms involved in the anti-tumor response in breast cancer patients who respond to neoadyuvant chemotherapy (NACT). Cultures with plasma from responder (R), but not from non-responder (NR), patients increased the intracellular production of cytotoxic molecules and pro-inflammatory cytokines in CD8+HLA-DR+ T cells. However, the addition of neutralizing anti-IFN-γ or anti-IL-12 antibodies to cultures with plasma from R patients decreased this effector function on CD8+HLA-DR+ T cells. These results suggest the presence of soluble factors in the plasma of R patients inducing the effector function of CD8+HLA-DR+ T cells.Cytotoxic T lymphocyte (CTLs) activation is an independent predictor of response to neoadjuvant chemotherapy (NACT) in breast cancer (BC) patients. Here, we go deeper into the function of CD8+ HLA-DR+ T cells from NACT treated HER2 negative BC patients. Flow cytometry analysis revealed that CD8+ HLA-DR+ T cell percentage was increased in NACT responder (R) compared to non-responder (NR) patients. R patients with ER-/PR- hormone receptors had the highest CD8+ HLA-DR+ T cell frequencies, while no differences were found when patients were classified according to cancer stage or menopause status. Interestingly, the cytotoxicity and production of anti-tumor cytokines were enhanced when CD8+ HLA-DR+ T cells from healthy donors were cultured with plasma from R, but not from NR patients. The induced anti-tumor profile of CD8+ HLA-DR+ T cells was associated with plasmatic IL-12 and IFN-γ levels, increased cytokines in R patients. IL-12 or IFN-γ neutralization decreased cytotoxic activity and TNF-α production by cultured CD8+ HLA-DR+ T cells in R plasma presence. All these data suggest that an effective response to NACT in BC patients is associated with increased IL-12 or IFN-γ levels involved in the induction of cytotoxic and pro-inflammatory mechanisms in CD8+ HLA-DR+ T cells. Breast cancer (BC) remains the most common malignancy and a major cause of cancer-related death in women worldwide ,2. Due tPrevious studies in triple negative breast cancer (TNBC) have shown an increase of TILs in 85% of patients after NACT and this increase was associated with an improved prognosis . FurtherIt has recently been shown that HLA-DR expression on CD8+ T cells reflects tumor immune status and is associated with a reduction in tumor size and tumor cell migration . SaraivaTo explore the role of CD8+ HLA-DR+ T cells in HER2 negative BC patients after NACT, we firstly compared the percentage of these cells in patients who responded and who did not respond to NACT. Our goal was to decipher the effector molecules of this subpopulation and soluble factors that can activate it. To achieve this objective, we firstly analyzed the influence of plasma content on the induction of effector cytotoxic functions, activating healthy donor CD8+ HLA-DR+ T cells in the presence of plasma from responder (R) and non-responder (NR) patients. Then, after comparing the plasmatic concentration of IFN-γ and IL-12 in R and NR patients’ plasma, we neutralized the effect of these differentially expressed cytokines in activating cultures.Peripheral blood from 18 healthy donors (HD), as controls, and 73 BC patients (post-NACT) were collected in Vacutainer tubes with heparin . All patients recruited were HER2 negative and received NACT with antracyclines and taxanes. Patients were selected according to convenience sampling and to the criteria specified below: female patients, 18 years old, stages II-III who were potentially resectable in addition to having an evaluation of pathological response according to the criteria of residual cancer burden (RCB). The expressions of estrogen (ER) and progesterone (PR) receptor were evaluated and patients were classified as ER+ PR+ (DP), ER- PR- (DN) and ER+ PR- or ER- PR+ (SP). A summary of patient characteristics is given in n = 10), those that who had a minimal residual burden ; and NR for patients who maintained a moderate  or marked  residual disease post-treatment.Tumor response was evaluated in surgical specimen using the RCB as previously reported ,22. PatiPBMCs were isolated from the heparinized peripheral blood of HD and BC patients using a Ficoll-Histopaque gradient  and were cryopreserved. After thawing, PBMCs were stained with the following anti-human antibodies: CD3-PE , CD8-Vioblue  and HLA-DR-APC-Cy7 (Miltenyi Biotech). Data acquisition and analysis was performed on a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotech). For data analysis, live single cells were analyzed to select lymphocytes based on their morphology by forward- versus side-scatter (FSC-SSC) dotplot. By combining anti-CD3 and anti-CD8, we identified CD3+ CD8+ (CD8+ T cells).and CD3+ CD8-  . Then, tFresh isolated PBMCs from HD were cultured in 96 well-plates  with 25% of plasma from R and NR patients in RMPI-1640  supplemented with 10% FBS and 1% Penicillin/Streptomycin (Biowest) during 72 h in 5% CO2 at 37 °C as previously described . For bloThe percentage of CD8+ HLA-DR+ T cells producing cytokines was measured by intracellular flow cytometry. After surface staining with anti-CD8-PECy-7  CD3-Viogreen (Miltenyi Biotech) and anti-HLA-DR-FITC (Immunotools) antibodies, PBMCs were intracellularly stained with anti-IL-10-PE , anti-IFN-γ-APC (BD Biosciences), anti-TNF-α-PE (Biolegend), anti-perforin-APC (Biolegend) or anti-Granzyme-B-PE (eBioscience) antibodies after fixation and permeabilization according to the manufacturer’s instructions (BD Biosciences). Data acquisition and analysis were performed on a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotech) using FlowJo version 10 .Concentrations of IL-12 (Immunotools) and IFN-γ  from R and NR patients were determined using specific ELISA kits according to the manufacturers’ instructions and using the specific standard curves of recombinant molecules. The limits of detection were as follows: 15.62 pg/mL for IL-12 and 6.25 pg/mL for IFN-γ.t-test (paired or unpaired) or the Mann-Whitney test according to normal distribution. Comparisons of three or more groups were tested with a one-way analysis of variance (ANOVA) and the Bonferroni post-hoc test. Correlation analyses were determined with Pearson’s or Spearman’s correlation according to normal distribution. ROC curve for CD8+ HLA-DR+ T cells were performed to assign a threshold to differentiate R from NR patients. The determined area under the curve, sensitivity and specificity were obtained. The cut-off point for CD8+ HLA-DR+ T cells was determined by a ROC curve analysis, considering the expression value that corresponded to the maximum sensitivity and specificity. p-values < 0.05 were considered statistically significant.Statistical analyses were performed using GraphPad Prism 7 software. Normal data distribution was assessed by the Kolmogorov-Smirnov test. Variables were presented as mean ± SEM or median (interquartile range-IQR-) according to normal or non-normal distribution respectively. Comparisons between the two groups were tested with the Student’s We compared the percentage of CD8+ T cells expressing HLA-DR in R and NR patients, and HD. Our results showed that R patients had higher levels of CD8+ HLA-DR+ than NR or HD A,B. HoweThe percentage of CD8+ HLA-DR+ T cells was not statistically different when R patients were grouped according to cancer stage or menopause status, while this percentage was increased in DN patients when they were grouped by expression of hormone receptors E–G.To find a statistically valid cut-off point of CD8+ HLA-DR+ T cells to differentiate response to NACT, a ROC curve analysis with all patients was performed, leading to >18.05 as the value for NACT responder patients. The area under the ROC curve was 0.926, sensitivity was 60.38% and specificity was 94.12% H.To determine whether plasma soluble factors in BC patients could regulate the percentage and the cytotoxic/pro-inflammatory activity of CD8+ HLA-DR+ T cells, we cultured HD PBMCs in the presence of plasma from HD, R or NR patients. We found that the percentage of CD8+ HLA-DR+ T cells and the MFI of HLA-DR on CD8+ T cells was comparable in the three groups B–E.Interestingly, we found a positive correlation between the production of intracellular perforin, but not granzyme B, in CD8+ HLA-DR+ T cells after culturing PBMCs in the presence of plasma from HD, R or NR patients and the percentage of CD8+ HLA-DR+ T cells found in peripheral blood in R and NR patients . The proThe percentage of CD8+ HLA-DR+ T cells with intracellular IFN-γ and TNF-α was higher and IL-10 was lower when PBMCs were cultured in the presence of R than in NR plasma A–C.We found a positive correlation between the production of IFN-γ, but not of IL-10 and TNF-α, in CD8+ HLA-DR+ T cells after culturing PBMCs in the presence of plasma from R or NR patients and the percentage of CD8+ HLA-DR+ T cells in peripheral blood of all patients –C. HowevWe also found a positive correlation between the levels of IFN-γ and TNF-α, but a negative correlation between IL-10 levels and the production of perforin and granzyme B by CD8+ HLA-DR+ T cells obtained after culturing PBMCs in the presence of plasma from R or NR patients A–F.To identify plasmatic factors that may contribute to the effector profile of CD8+ HLA-DR+ T cells in the R patients, we compared the plasmatic IL-12 and IFN-γ concentrations of R and NR patients by ELISA. IL-12 and IFN-γ concentrations were significantly higher in R than in NR patients A,B. We aWe next investigated whether plasmatic IL-12 and IFN-γ were responsible for inducing the production of effector cytotoxic and pro-inflammatory molecules by CD8+ HLA-DR+ T cells. For this purpose, we added neutralizing anti-IL-12 or anti-IFN-γ antibodies before culturing PBMCs with plasma from R patients. Anti-IL-12 or anti-IFN-γ antibodies, alone and in combination, decreased similarly the percentage of CD8+ HLA-DR+ T cells producing perforin, granzyme B and TNF-α A–C. InteWe observed that CD8+ HLA-DR+ T cells were increased in BC patients who responded to NACT. Plasma from R but not from NR patients can enhance the percentage of intracellular cytotoxic granules and pro-inflammatory cytokines only in CD8+ HLA-DR+ T cells. We found that plasmatic levels of IFN-γ and IL-12 were higher in R than in NR patients and were correlated with the cytotoxic and pro-inflammatory activity of plasma-cultured CD8+ HLA-DR+ T cells. When we blocked plasmatic IFN-γ and IL-12, a significantly decreased percentage of intracellular cytotoxic granules and pro-inflammatory cytokines were found on CD8+ HLA-DR+ T cells. These results suggest that NACT induces an anti-tumor response, activating the effector cytotoxicity of CD8+ T cells through IFN-γ and IL-12.We observed higher percentages of CD8+ HLA-DR+ T cells in peripheral blood from R patients, suggesting, in line with studies in other neoplasias, an increased activation of these cells ,15. We cCulturing HD PBMCs with plasma from patients, we found that plasma from R patients did not increase the expression of HLA-DR on CD8+ T cells. However, these experimental conditions cannot rule out the possibility that the upregulation of HLA-DR on peripheral CD8+ T cells may have been occurred in the tumor microenvironment or under tumor influence in R patients. It is tempting to speculate that the increase in HLA-DR expression on CD8+ T cells reflects their activation status and may be induced by released tumor peptides during an effective response to NACT. It is also possible that a longer culture is required to increase the expression of HLA-DR under in vitro conditions. For instance, a higher percentage of activated CD8+ HLA-DR+ T cells after 5 days of culture with HIV peptides has been reported . HoweverOur results show that plasma from R patients increased the intracellular content of cytotoxic granules and pro-inflammatory cytokines in CD8+ HLA-DR+ T. A possible explanation for this is that the action of NACT generates a pro-inflammatory context that activates an effective cytotoxic response in CD8+ HLA-DR+ T cells from R patients. To confirm the cytotoxic capacity of CD8+ HLA-DR+ T cells after activation with R plasma from BC patients, a co-culture with autologous tumor cells would be necessary. In line with this, a previous report reported CD8+ HLA-DR+ T cell activation with the production of IFN-γ and granzyme B after co-culturing with glioma cells . MoreoveHere, we have showed that culturing PBMCs with NR plasma induces a higher percentage of CD8+ HLA-DR+ T cells producing intracellular IL-10. It is possible that CD8+ HLA-DR+ T cells from NR patients, without induced cytotoxic activity but with immunosuppressive potential, may be related with tumor progression. These results are in concordance with those of Noble et al., who showed that CD8+ T-cells can secrete anti-inflammatory cytokines, such as IL-10, with suppressor activity .In this study, we have found an increased activity of CD8+ HLA-DR+ T cells in R patients classified as DN. We have also found that plasma IFN-γ and IL-12 levels were comparable between patients were segregated according to the expression of hormone receptors (data not shown). Moreover, no differences in the percentage of CD8+ HLA-DR+ T cells that produce granzyme B or perforin were found (data not shown). We are aware that more samples would be needed to find a definitive conclusion. In addition to this cytotoxic potential, CD8+ HLA-DR+ T cells have been found to have the machinery needed for antigen processing and loading on HLA-DR molecules and, therefore, may express co-stimulatory molecules for an effective T cell activation .Three of our results suggest that plasmatic IFN-γ and IL-12 are partly responsible for induced cytotoxic and pro-inflammatory activity on CD8+ HLA-DR+ T cells. First of all, we determined that plasmatic levels of IFN-γ and IL-12 were higher in R than in NR patients. Second, we found a positive correlation between the percentage of intracellular cytotoxic granules and pro-inflammatory cytokines with the plasmatic levels of IFN-γ and IL-12. Finally, the blocking of these cytokines reduced the percentage of intracellular cytotoxic and pro-inflammatory content on cultured CD8+ HLA-DR+ T cells. It’s difficult to know if circulating plasma levels of IFN-γ and IL-12 are the cause or the consequence of the CD8+ HLA-DR+ T cell activation. It is tempting to speculate that the released tumor peptides during an effective response to NACT can stimulate APCs, which produce IL-12 and activate IFN-γ production on CD8+ HLA-DR+ T cells, as previously reported on CD8+ T cells . CompariOur findings suggest that plasma levels of IFN-γ and IL-12 could be used as a biomarker to identify responder patients after NACT. In this line, increased levels of plasma IFN-γ only in NACT responder patients on BC patients have already been reported . AdditioDespite the contribution of these results to our understanding of the role of this subset of cytotoxic cells, our study has some limitations. One is that samples were obtained at the end of NACT patients were included and the percentage of CD8+ HLA-DR+ T cells in pre-NACT patients is not known. Another is that our findings cannot be extrapolated to primary tumors since the phenotype and the functional mechanisms of CD8+ HLA-DR+ T cells can differ in the tumor microenvironment. Additionally, with our current approach, we cannot predict the function of CD8+ HLA-DR+ T cells with other BC therapies. Finally, the limited number of samples does not allow to analyze subgroups of BC patients.Our findings not only contribute to the characterization of the mechanisms involved in the anti-tumor response of CD8+ HLA-DR+ T cells but also suggest that activating and/or expanding these cells could be used for future therapeutic purposes. Particularly, we speculate that increasing IFN-γ and IL-12 could improve response to NACT. We are currently validating the use of the levels of CD8+ HLA-DR+ T cells in another cohort of BC and other tumor patients as a biomarker of response to NACT. Furthermore, it would be interesting to compare the frequencies of CD8+ HLA-DR+ T cells in blood and the primary tumor tissue collected at the same time point.


PD-L1 and NKG2A in melanoma tissue with clinicopathologic characteristics and survival in Indonesian primary nodular melanoma patients.Biomarker mRNA levels have been suggested to be predictors of patient survival and therapy response in melanoma cases. This study aimed to investigate the correlations between the mRNA expression levels of PD-L1-patients were 15.800 ± 2.345 and 28.945 ± 4.126 months, respectively. However, this difference was not significant statistically (p = 0.086). Upregulated and normoregulated NKG2A patients differed very little in median survival time . Expression of PD-L1 and NKG2A were strongly correlated . No clinicopathologic associations with PD-L1 and NKG2A mRNA levels were observed. These results suggest that PD-L1 may have potential as a prognostic factor. Although an unlikely prognostic factor, NKG2A may become an adjunct target for therapy. The strong correlation between PD-L1 and NKG2A suggests that anti-PD-1 and anti-NKG2A agents could be effective in patients with PD-L1 upregulation. The mRNA levels of these two genes may help direct choice of immunotherapy and predict patient outcomes.Thirty-one tissue samples were obtained; two were excluded from survival analysis due to Breslow depth of less than 4 mm. The median survival of upregulated and normoregulated The online version contains supplementary material available at 10.1186/s13104-021-05623-7. Among cutaneous malignancies, melanoma results in the highest mortality; statistics revealed that melanomas caused 287,723 new cases and 60,712 deaths worldwide in 2018 [PD-1/PD-L1, help improve the immune response to cancerous cells. Nivolumab, an anti-PD-1 antibody, has been approved by the FDA for the treatment of advanced melanomas [NKG2A antibody that enhances NK cell and CD8+ T cell activity [Agents that block immune checkpoints, such as elanomas . Other iactivity , are alsPD-1 immunotherapy, clinicians commonly test tumor tissues for PD-L1 expression by using immunohistochemistry (IHC). Tumors expressing PD-L1 respond better to anti-PD-1/PD-L1 agents compared with non-expressers [PD-L1 expression on survival are conflicting [Before starting anti-pressers . Howeverflicting . Detectiflicting , 8.. reported that mRNA levels of PD-L1/2 show potential in predicting survival and response toward immunotherapy in metastatic melanoma [NKG2A should also be investigated.Researchers are exploring new methods to predict therapy responses and survival in melanoma patients. Several studies have investigated the use of biomarker mRNA levels as an alternative parameter –11. Guptmelanoma . Given tPD-L1 and NKG2A, as well as the associated clinicopathologic characteristics.Immuno-oncological research is still rare in Indonesia. Most Indonesian patients are treated with surgical resection, dacarbazine chemotherapy, and radiotherapy. Information on the expression of immune-checkpoint molecules is needed to gauge the potential efficacy of using immunotherapy agents in Indonesia. Thus, this study aimed to investigate the prognostic role of mRNA levels of Formalin-fixed paraffin-embedded (FFPE) tissue samples from patients diagnosed in 2012–2019 with primary cutaneous nodular melanoma were collected from the archives of the Department of Anatomical Pathology, Dr. Sardjito Hospital, which is the main cancer referral center in Yogyakarta, Indonesia. Cases with prior chemotherapy or radiotherapy, incomplete clinical data, and degraded specimens were excluded. Thirty-one samples were analyzed in this retrospective cohort study, and all patients were of Javanese ethnicity.PD-L1 and NKG2A expression quantification was conducted using AccuPower® GreenStar™ RT-qPCR PreMix on an Exicycler™ 96  with primer pairs and thermocycler conditions as previously described by Vassilakopoulou et al. and Meckawy et al. [PD-L1 and NKG2A were calculated from the quantification cycle (Cq) values of the gene targets and normalized against GAPDH as an internal control. Subsequent normalization was performed using the ΔΔCq values of RNA derived from healthy skin tissues. Age, sex, tumor location, Breslow thickness, greatest diameter, lymph node involvement, and stage were retrieved from medical records. Pathological data, including the presence of necrosis, lymphovascular invasion, tumor-infiltrating lymphocytes (TILs), and mitotic index, were obtained from hematoxylin–eosin and Ki-67 IHC stained slides. Survival status (living or deceased) was determined through telephone calls at the point of follow-up of the study (until April 2020).RNA was extracted from FFPE tissues using GeneAll® Ribospin™ II . Real-time polymerase chain reaction (RT-PCR) for y et al. , 14. ThePD-L1 and NKG2A levels; conversely, samples were classified as upregulated if the expression was above the mean. Comparison of mRNA level averages based on categorical clinicopathologic characteristics was performed using Mann–Whitney U tests. Spearman correlation was used to analyze associations between the expression of PD-L1 and NKG2A and continuous clinicopathologic features. Kaplan–Meier analysis and log-rank tests with Cox regression were used to determine hazard ratios (HRs) for survival analysis. To minimize the effect from Breslow thickness, two samples was excluded from survival analysis due to depth of less than 4 mm.Samples were classified as normoregulated if the expression was lower than or equal to the mean of the The characteristics of the subjects are presented in Table PD-L1 and NKG2A was not significantly associated with the patients’ clinicopathologic characteristics , 2.429 (p = 0.101), and 1.011 (p = 0.981) , while the other HRs decreased to 1.017 (p = 0.951) for stage and 0.590 (p = 0.391) for NKG2A upregulation. However, the differences were not statistically significant.In the Cox univariate regression analysis, higher stage, upregulated  p = 0.76, 2.429  compared with upregulated expressers     cases with PD-L1 overexpression tended to have lower survival rates and (2) PD-L1 and NKG2A levels were strongly correlated.In this study, we compared the clinicopathologic characteristics and overall survival of Indonesian primary nodular melanoma cases with different mRNA levels of PD-L1 tended to have lower overall survival, with an approximately two to threefold higher HR compared to normal expressers. This trend was not proven statistically significant, possibly due to the small sample size. However, the increase in the HR for PD-L1 upregulation in multivariate regression suggests this trend may exist in the overall population of melanoma patients in Indonesia.Melanoma patients with upregulated PD-L1 molecule interacts with PD-1 receptors on T cells, causing anergy, exhaustion, and apoptosis [PD-L1 expression. Melanoma cell line studies show that cells with upregulated PD-L1 demonstrate highly invasive and aggressive behavior [PD-L1 on IHC staining had lower median survival time compared with the subgroup with negative or indeterminate PD-L1 status (9.7 months vs. 11.6 months) [The poptosis . Melanombehavior . In a st months) .., who observed that higher PD-L1 mRNA levels reflect better prognoses for melanoma patients treated with anti-PD-1 agents [PD-L1 respond well to anti-PD-1 antibodies, hence the increase in survival [PD-L1 expression is a negative prognostic factor in patients with melanoma treated with conventional chemotherapy. However, when treated with anti-PD-1 antibodies, patients with high levels of PD-L1 respond well and have good outcomes. Therefore, the choice of therapy also affects the performance of PD-L1 as a prognostic factor.Our results seem to oppose those of Gupta et al1 agents . This disurvival . The resPD-L1 independently  or in response to TILs (reactive expression). TILs can secrete interferon-gamma, which induces the expression of PD-L1 in tumor cells [PD-L1 expression is accompanied by the presence of TILs, the expression is likely reactive, and vice versa. Due to the strongly positive correlation between PD-L1 and TILs, studies report that most melanomas express PD-L1 reactively [PD-L1 mRNA levels than the group with TILs. This finding suggests that the cases with upregulated PD-L1 in our study are likely constitutive expressers.Tumor cells can express or cells . When PDactively . HoweverPD-L1 expression may have different prognostic implications. When previous studies divided patients based on PD-L1 expression and the presence of TILs, patients with constitutive PD-L1 expression without lymphocyte infiltrates showed the poorest outcomes, followed by reactive PD-L1 expressers, those with PD-L1(−) without TILs, and, finally, those with PD-L1(−) and TILs [PD-L1 showed poorer prognoses than the groups with normoregulated PD-L1. However, survival did not differ significantly when the cases were divided further based on TIL status, likely because none of the patients were treated using immunotherapy, in which the presence of TILs predicts improved response [These two modes of and TILs , 19. In response . AnotherNKG2A mRNA did not appear to affect survival, given the highly insignificant results in univariate and multivariate regression analysis. NKG2A is an inhibitory receptor found on NK cells [NKG2A receptors and impair the function of NK cells. Trials in mouse models indicate that monalizumab is ineffective as a single therapy but highly effective when used together with other immunotherapy agents that promote activated TILs, such as anti-PD-1 or cancer vaccines [NKG2A as an adjunct treatment for other immunotherapies. Our results reinforce the idea that NKG2A may not be an independent therapeutic target and prognostic factor but may play a role in combination therapy.Upregulated NK cells . Cancer vaccines . One escvaccines . This fiNKG2A and PD-L1 were strongly correlated. This finding indicates that tumors with high PD-L1 expression would also likely express NKG2A strongly and, thus, respond well to anti-NKG2A agents. When NKG2A expression was combined with the TIL parameter, the distribution of survival curves obtained resembled the curves for PD-L1 combined with TILs. NKG2A upregulation with and without the presence of TILs may have different pathogeneses and prognostic implications, like PD-L1.The mRNA expressions of NKG2A and PD-L1 and clinicopathologic characteristics in this work resembles the findings of several previous studies [The lack of correlation between the expression of  studies , 18. ThePD-L1 and NKG2A in the prognosis and therapy of Asian patients who have not previously received immune checkpoint inhibitors.The findings of this study must be interpreted with caution especially due to the small sample size. However, our results support the findings of several studies that show that mRNA profiles may serve as a prognostic factor in melanoma cases , 24. FurPD-L1 and NKG2A with clinicopathologic characteristics and survival in primary nodular melanoma patients in Yogyakarta, Indonesia. Patients with upregulated PD-L1 expression had shorter median overall survival, although insignificant statistically. PD-L1 and NKG2A mRNA levels were positively correlated.We investigated the correlations between the mRNA levels of PD-L1 expression. NKG2A was not proven to be an independent predictive factor but may serve as an adjunct target for therapy. The strong correlation between PD-L1 and NKG2A suggests that anti-PD1 and anti-NKG2A agents may be effective in patients with PD-L1 upregulation. Studies with larger subject groups are needed to confirm the patterns of PD-L1 expression in Asian cases.Our findings suggest that the therapy regimen and presence of TILs may affect the prognostic role of Our study was limited by its small sample size and homogenous ethnic population. Results among diverse Indonesian and Asian populations may differ. TILs examination did not discriminate between lymphocyte subtypes.Additional file 1: Table S1. Comparison of PD-L1 and NKG2A expression levels based on clinicopathologic characteristics and overall survival.Additional file 2: Table S2. Spearman correlations between continuous variables.Additional file 3: Table S3. Univariate and multivariate Cox regression results for stage, PD-L1 upregulation, and NKG2A upregulation.Additional file 4: Fig. S1. Kaplan–Meier survival curves comparing the survival of patients with primary nodular melanoma with or without TILs.Additional file 5: Fig. S2. Kaplan–Meier survival curves comparing the survival of patients with primary nodular melanoma based on PD-L1 expression and the presence of TILs.Additional file 6: Fig. S3. Kaplan–Meier survival curves comparing the survival of patients with primary nodular melanoma based on NKG2A expression and the presence of TILs.


PMM2, the encoding gene for the phosphomannomutase 2 (PMM2). This enzyme catalyzes the conversion of mannose-6-P to mannose-1-P required for the synthesis of GDP-Man and Dol-P-Man, donor substrates for glycosylation reactions. The identified variants were c.422G>A (R141H) and c.178G>T, the former being the most frequent PMM2 pathogenic mutation and the latter a previously uncharacterized variant restricted to the Latino population with conflicting interpretations of pathogenicity and that we here report causes leaky non-functional alternative splicing (p.V60Cfs*3).We report on a Mexican mestizo with a multisystemic syndrome including neurological involvement and a type I serum transferrin isoelectric focusing (Tf IEF) pattern. Diagnosis of PMM2-CDG was obtained by clinical exome sequencing (CES) that revealed compound heterozygous variants in  Screening for N-glycosylation defects mostly occurs by serum transferrin isoelectric focusing (Tf IEF)  (HSF Donor site (matrix GT) 84.54 > 74.03 (−12.43%) and MaxEnt Donor site 6.41 > −0.21 (−103.28%)).PMM2 was PCR amplified to detect alternative splicing isoforms. The human PMM2 coding region spans over 738 bp and codes for a 246 aminoacid protein. Amplification of PMM2 with primers PMM2s and PMM2as encompasses the coding sequence plus short stretches from the 5′- and 3´-UTR for a predicted amplicon of 951 bp. PCR product analysis in agarose gel showed that the healthy control and the mother had one single PMM2 amplicon with the expected wt size (PMM2–1). Nonetheless, the patient and the father presented two additional amplicons with approximately 450 bp increase in size, termed PMM2–2 and PMM2–3, respectively  mutation, but not the c.178G>T variant  mutation, it is known that this type of sequencing may not detect minor isoforms. Because of this, we designed an allele-specific PCR to detect the c.178G>T mutation. The wt PMM2 amplicon from the control and the patient's PMM2–1 were used as template. An allele-specific-PCR product was detected only in the patient , activating two intronic cryptic donor splice sites and causing partial retention of intron 2. The PMM2–2 and PMM2–3 are characterized by isoforms with a 455 bp and 479 bp partial intron 2 retention, respectively. In both cases, abrogation of the wt donor splice site and partial intron retention leads to a frameshift that causes a premature stop codon +6 bp from the mutation  mutation , the mosvariants ,16. The PMM2 variants were found: the well-known c.422G>A (p.R141H) pathogenic variant and the c.178G>T variant that is annotated as likely pathogenic, according to ACMG/AMP guidelines, but with missing data on functional characterization or published reports. Interestingly, according to GnomAD exomes and genomes database [This report presents a patient with a mild clinical phenotype and IEF type I pattern consistent with CDG, noting that no inverted nipples or abnormal fat pads were observed as frequently occurs in PMM2-CDG. Therefore, CES was performed and two database , the c.1PMM2 cDNA PCR amplicons of the patient and the father. Confirmation was obtained by Sanger sequencing, demonstrating that alternatively spliced isoforms PMM2–2 and PMM2–3 were derived from the paternal allele as they exclusively carry the c.178G>T mutation.To further characterize the mechanism of pathogenicity of the c.178G>T variant and in view that it is located −1 from the donor splicing site in exon 2, we considered that there was a high probability that this variant caused alternative splicing. This was first evidenced in gel-electrophoresis of PMM2–2 and PMM2–3 further revealed that the c.178G>T variant disrupts the normal donor splice site and results in partial intron 2 retention due to activation of two cryptic donor splice sites, causing ORF change and the generation of a premature stop codon p.V60Cfs*3 in both cases (c.178+1G>A (ClinVar 929132) and c.178+2T>G (ClinVar 553770), potentially affecting the same canonical donor splicing site, have been reported but without studies evaluating alternative splicing consequences.Sequencing of th cases C. We conPMM2–1 amplicons, the use of an allele-specific PCR allowed the identification of isoforms with constitutive splicing that present this mutation  and that alongside the well-characterized c.422G>A (p.R141H) mutation genetically confirms a PMM2-CDG diagnosis. In most disease-related genes, mutations affecting splicing are not fully characterized because mutation screening is restricted to gDNA. For example, of the 28 pathogenic or likely-pathogenic variants of splicing , only 9 6PMM2 c.178G>T mutation is a Latino pathogenic variant that causes leaky non-functional alternative splicing inducing frameshift mutations (p.V60Cfs*3). Mutations should be studied concerning their potential disruption of splicing, particularly if they affect canonical splicing sites.In conclusion, the CAG-D and I-MD: conceptualization. CAG-D, MR-M, CE-V, SM-H, RS-M, MEC-M FO-R, AG-J, K-B, CM-G, KC-S, LLF-L, MJ-O, A-MR and CA-V: investigation. CAG-D, CA-V and I-MD: data curation. CAG-D and IM-D: writing original draft. CAG-D, HMM-M, CA-V and IM-D: review &editing. IM-D provided project administration and funding acquisition. IM-D, CA-V and HMM-M supervision and validation.


After SARS-CoV-2 infection, antibody and T-cell responses develop rapidly and persist over time in both immunocompetent and transplanted patients.The development and persistence of SARS-CoV-2-specific immune response in immunocompetent (IC) and immunocompromised patients is crucial for long-term protection. Immune response to SARS-CoV-2 infection was analysed in 57 IC and 15 solid organ transplanted (TX) patients. Antibody responses were determined by ELISA and neutralization assay. T-cell response was determined by stimulation with peptide pools of the Spike, Envelope, Membrane, and Nucleocapsid proteins with a 20-h Activation Induced Marker (AIM) and 7-day lymphoproliferative assays. Antibody response was detected at similar levels in IC and TX patients. Anti-Spike IgG, IgA and neutralizing antibodies persisted for at least one year, while anti-Nucleocapsid IgG declined earlier. Patients with pneumonia developed higher antibody levels than patients with mild symptoms. Similarly, both rapid and proliferative T-cell responses were detected within the first two months after infection at comparable levels in IC and TX patients, and were higher in patients with pneumonia. T-cell response persisted for at least one year in both IC and TX patients. Spike, Membrane, and Nucleocapsid proteins elicited the major CD4 A novel coronavirus named SARS-CoV-2 has been identified as the causative agent of a global outbreak of a respiratory tract disease, referred to as COVID-19  in the presence of co-stimulator molecules CD28 and CD49d  for 30 min at 4 °C. After rising with PBS and staining in PBS 5% FCS with CD4 APC Cy7 (BD Biosciences), CD8 V500 (BD Biosciences), CD25 PECy7 (BD Biosciences) and CD137 PECy5 (BD Biosciences) antibodies for 30 min at 4 °C. Finally, cells were washed and resuspended in PBS 1% paraformaldehyde. +CD137+ CD4+ or CD8+ detected in PBMC incubated with actin peptides from the frequency of CD25+CD137+ CD4+ or CD8+ detected in PBMC incubated with SARS-CoV-2 peptides. To determine the cut-off for antigen-specific T-cell frequency, samples from seven SARS-CoV-2-seronegative subjects were tested. A value < 0.05% antigen-specific T-cells was considered negative while a value ≥ 0.05% was considered positive.Antigen-specific T-cell frequency was determined by subtracting the frequency of CD25Flow cytometry analyses were performed with a FACS Canto II flow cytometer and BD DIVA software (BD Biosciences). A representative pseudocolor plot analysis is shown in Supernatant concentrations of cytokines and chemokines were measured in duplicate using BioPlex Pro Human Cytokine Screening Panel  according to the manufacturer’s instructions. All the cells’ supernatants were inactivated for 60 min at 56 °C at the same time before analysis. The supernatants were measured undiluted and culture medium was used as diluent for standards and control. All the results are analysed with BIO-PLEX manager software 6.0.To evaluate antigen-specific proliferative response, PBMCs  were stimulated in triplicate in 96-well round-bottom plates with SARS-CoV-2 and human actin peptide pools at a final concentration of 0.1 µg/mL for 7 days. Culture medium was the same as the AIM assay supplemented with 5% heat inactivated human serum AB (Sigma), 1 mM Sodium Pyruvate , 100 µM non-essential amino acids (Euroclone), and 50 µM 2-Mercaptoethanol (Gibco). After culture, cells were washed, stained with Live/Dead Fixable Violet Dye (Invitrogen) and subsequently with CD3 PerCP 5.5 (BD), CD4 APC Cy7 (BD), CD8 FITC (BD), CD25 PECy7 (BD), CD278 (ICOS) APC (Invitrogen). Finally, cells were washed and resuspended in PBS 1% paraformaldehyde.+ICOS+ CD3+CD4+ or CD3+CD8+ detected in PBMC incubated with actin peptides from the percentage of CD25+ICOS+ T-cell subsets detected in PBMC incubated with SARS-CoV-2 peptides. To determine the cut-off for antigen-specific CPI, samples from five SARS-CoV-2-seronegative subjects were tested. A CPI <1.5% was considered negative while a value ≥1.5% was considered positive.A Cell Proliferation Index (CPI) for antigen-specific expanded T-cells was determined by subtracting the percentage of CD25Flow cytometry analyses were performed with a FACS Canto II flow cytometer and BD DIVA software (BD Biosciences). A representative pseudocolor plot analysis is shown in Statistical analyses were performed with GraphPad Prism 6. The Mann–Whitney U-test or Wilcoxon signed-rank test were applied for unpaired or paired comparison, respectively, while the Friedman test was applied for multiple comparisons.As reported in p < 0.001) and late (p < 0.001) time points, whereas anti-S IgA and anti-N IgG were higher in IC patients with pneumonia only at the early time point. In TX patients, no significant difference was observed for anti-S IgG, anti-S IgA, and anti-N IgG between patients with pneumonia  was observed. The three antibody subclasses declined at late time points in TX patients with pneumonia, but did not change significantly in both IC and TX patients with mild symptoms, (only 2/13 IC patients with mild symptoms did not display anti-S and anti-N antibodies at the late time point).Peak antibody response was detected within the first two months after infection and a significantly higher level was detected in IC than in TX patients with pneumonia for anti-S and anti-N IgG (p = 0.060) and late (p = 0.048) time points  (+ T cells (in IC p = 0.016), although SARS-CoV-2 CD8+ T-cell levels were lower than CD4+  at similar levels. At the early time point, CD4= 0.013) A. A simihan CD4+ B. + and CD8+ T-cell responses (p < 0.001). Nevertheless, as also observed for rapid T-cell activation, SARS-CoV-2-specific T-cell proliferation was detected mainly in the CD4+ than the CD8+ T-cell subset  and Th2 (IL-4 and IL-5) cytokines and IL-10 was evaluated after 20 h stimulation with S protein in IC and TX patients.As observed with the AIM assay, PBMCs from IC patients with pneumonia produced the higher levels of both Th1 and Th2 cytokines at the early time point (+ T-cell response directed more against the M than S protein (p = 0.020), but no difference was observed at the late time point  elicited the major T-cell response, we considered IC and TX patients together and analysed the T-cell response by dividing the subjects into patients with pneumonia (me point A. No sigresponse B and in + and CD8+ T-cell proliferative response (p < 0.001 for CD4+ and CD8+) or M proteins  in the pneumonia group at the early time point. Additionally, in mildly symptomatic patients, CD4+ and CD8+ T-cells proliferated in response to the S protein significantly more than to the M protein (p = 0.020) at the early time point post infection  patients with pneumonia or mild symptoms. However, conversely to antibody levels, antigen specific T-cell responses did not decrease significantly within 12 months after infection both in IC and TX patients. Here, we show that all patients with pneumonia and most patients with mild symptoms (both IC and TX) developed detectable and persistent anti-S IgG and IgA antibodies from two months until at least one year after infection , while anti-N IgG levels disappeared at the late time point in most TX patients. Notably, IC but not TX patients with pneumonia developed higher IgG, IgA, and Nt Ab levels than patients with mild symptoms. Only two TX patients, who had isolated gastrointestinal symptoms, did not develop a detectable antibody response. We could hypothesize that in these two patients the viral load was not sufficient to elicit a systemic antibody response, but triggered an immune reaction at the local mucosal level only. At the early time point, rapid CD4Interestingly, both the Nt Ab and T-cell responses developed at similar levels in IC and TX patients, although, among patients with pneumonia, we observed a higher anti-S and anti-N IgG antibody levels in IC patients. This observation could be explained by the fact that, according to the oxygen supply required, the IC patients analysed in our study had more severe pneumonia than the TX patients. Therefore, we could hypothesize that IC pneumonia patients received a higher inflammatory stimulus, which may have contributed to the induction of a higher but not more functional (since the Nt Ab response was not significantly different) IgG antibody response. Although the levels of antibodies were sustained overtime especially in pneumonia patients, at present the level required for protection against secondary infections or disease is unknown.+ T-cell response for the M protein in IC patients with pneumonia at the early time point). Conversely, we observed that the S protein induced the major proliferative CD4+ and CD8+ T-cell responses. We observed no difference in Th1 and Th2 cytokines production in IC and TX patients, except that IC patients with pneumonia produced the higher levels of cytokines. This is in line with the higher rapid T-cell activation observed in this group of patients with the AIM assay. Previous reports reported Th2-skewed cytokine production in SARS-CoV infected patients developing pneumonia, but no difference was found in the quality of cytokines detected in S-specific T-cells in patients developing pneumonia after SARS-CoV-2 infection [Regarding the antigen specificity of the T-cell response, while T-cells elicited by the E protein were negligible, we did not find a significant difference in the rapid response for the S, M, or N proteins  patients, as reported by Weiskopf et al. [+ T-cell response directed to the M protein only in IC pneumonia patients and we can support that the “effector” T-cell response appears the same for all SARS-CoV-2 proteins analysed , while the major reactivity of the proliferative T-cell response is directed towards the S protein. In addition, the proliferative assay appears more sensitive in detecting the CD4+ and, particularly, CD8+ T-cell responses compared to the AIM assay, especially at the late time point.We observed a dominance of rapid CD4f et al. . Concernf et al. . We obseDan et al. reported a duration of the immune response until at least 5–8 months post symptoms ; we exteDifferent studies have reported a significantly higher risk of fatal outcomes among TX patients developing COVID-19 as compared to the healthy population ,25,26,27There are some limitations in this study such as the small sample size and cross-sectional analysis conducted in IC patients with mild symptoms at early and late time points.In conclusion, despite immunosuppressive therapy, TX patients developed a robust immune response after SARS-CoV-2 infection, comparable to that observed in IC patients. The immune response persists until one year after SARS-CoV-2 infection, and it is likely that the immunological memory could persist for a long time and may protect both the IC and TX populations from SARS-CoV-2 secondary infections ,35. Futu


The non-classical human leukocyte antigen G (HLA-G) is a potent regulatory protein involved in the induction of immunological tolerance. This is based on the binding of membrane-bound as well as soluble HLA-G to inhibitory receptors expressed on various immune effector cells, in particular NK cells and T cells, leading to their attenuated functions. Despite its restricted expression on immune-privileged tissues under physiological conditions, HLA-G expression has been frequently detected in solid and hematopoietic malignancies including urological cancers, such as renal cell and urothelial bladder carcinoma and has been associated with progression of urological cancers and poor outcome of patients: HLA-G expression protects tumor cells from anti-tumor immunity upon interaction with its inhibitory receptors by modulating both the phenotype and function of immune cells leading to immune evasion. This review will discuss the expression, regulation, functional and clinical relevance of HLA-G expression in urological tumors as well as its use as a putative biomarker and/or potential therapeutic target for the treatment of renal cell carcinoma as well as urothelial bladder cancer. During the last two decades it has been generally accepted that altered immune responses and immune evasion strategies are characteristic hallmarks of cancer. It has been demonstrated that the immune system within a tumor undergoes changes in its cellular composition, functionality and localization . While eNovel cancer immunotherapies, such as immune checkpoint (ICP) inhibitors and adoptive cell therapy, have been developed during the last decade and demonstrated a therapeutic efficacy in hematologic tumors, but also in some solid cancers. However, the response rates to these therapies, in particular to solid tumors, still need to be improved and only a few patients achieve durable response rates due to intrinsic and/or acquired resistances mediated by immune evasion strategies of the tumor cells. This could be driven by both cellular and molecular suppressive networks within the TME, but also within the tumor, such as e.g. the loss of tumor antigens, downregulated expression of HLA class I molecules and antigen processing machinery (APM) and interferon (IFN) pathway components as well as upregulation of various immune checkpoint (ICP) molecules, like PD-L1, B7-H4, B7-H6, LAG-3, TIM-3, VISTA, HLA-E or HLA-G –10./LILRB2 , (iii) killer cell immunoglobulin-like receptor, two domains long cytoplasmic tail, 4/KIR2DL4 (CD158D), (iv) killer cell lectin-like receptor, subfamily c, member 1/KLRC1  and (v) natural killer cell receptor By55 (CD160). LILRB1 is expressed on monocytes, dendritic cells (DCs), B cells, NK cells and T cells; LILRB2 on cells of myeloid origin, KIR2DL4 on NK cells, KLRC1 on approximately 50% of NK cells and on a subset of CD8+ T cells and CD160 is expressed on NK cells. Furthermore, HLA-G can be expressed and secreted from non-cancer cells, such as human mesenchymal stem cells (hMSCs). These hMSCs can inhibit both NK cell cytotoxicity and T lymphocyte alloproliferation  and is localized like the classical HLA class Ia molecules within the cluster of the major histocompatibility complex (MHC) on the short arm of chromosome 6 at region 6p21.3. However, it has distinct properties to HLA class Ia molecules  such as a highly restricted and tightly regulated expression, which is under physiological conditions limited to immune-privileged tissues/organs with confined local immune and inflammatory responses  preventiferation . In addiferation .The expression of LILRB1/ILT2 and LILRB2/ILT4 on different immune effector cells, endothelial cells and tumor cells and the effects of the interaction with HLA-G have been recently summarized by Carosella and co-authors , while tHLA-G gene leading to at least seven different HLA-G protein encoding splice variants (HLA-G1-HLA-G7) reviewed by Hviid, 2006  the IPD and IMGT/HLA database  enlists id, 2006 . Next toid, 2006 –37 (Tab2-microglobulin (β2-m) and these complexes can even present a limited and cell type-specific peptide repertoire towards CD8+ cytotoxic T lymphocytes (CTL)  , which i2-m lacking one or two α domains, e.g. HLA-G2 (has α1 and α3), HLA-G3 (α1), HLA-G4 (α1 and α2), HLA-G6 (α1 and α3), and HLA-G7 (α1)  . Not onl-G7 (α1) , 41. It -G7 (α1) . Their u-G7 (α1) .3) group to a cytosine residue in CpG dinucleotides enriched CpG islands (+-expressing vs. HLA-G-non-expressing cells. Treatment with histone deacetylation inhibitors (HDACi), like sodium butyrate or trichostatin A (TSA), induced histone hyperacetylation, which was associated with a reversion of HLA-G repression , RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) –61. A nuIt has been recently demonstrated that HLA-G is crucial for tumor immune evasion and is also associated with malignant transformation . A pathoSince HLA-G is frequently associated with an advanced tumor stage and a poor prognosis of patients, a diagnostic and prognostic potential has been suggested for HLA-G in different cancer types.Renal cell carcinoma (RCC) is the most common kidney tumor with an incidence of 2% of tumors in the Western world . Based oDuring the last decades, the therapeutic options of RCC improved starting from unspecific cytokine treatment using high doses of IL-2 for lymphocyte activated killer cell generation , followeIt is noteworthy that HLA-G neoexpression and/or LILRB2/ILT4 expression in ccRCCs has been recently linked to aberrant expression of the vascular endothelial factor (VEGF)A and VEGFC . Thus, rWhile HLA-G expression could not be detected in healthy renal tissue, the pathophysiological HLA-G expression in RCC lesions ranged between 30 and 60% and was found at the cell surface as well as in the cytoplasm. Furthermore, the HLA-G plasma levels were statistically significant increase in RCC patients Table 3.Omitting early reports applying only RCC cell lines, studies analyzing RCC patient cohorts could demonstrate a statistically significant correlation between HLA-G expression and higher tumor grading and staging in RCC patients using IHC and ELISA. These data suggest that HLA-G might be an interesting prognostic marker for RCC.Furthermore, a link between the pathophysiologic HLA-G expression and the frequency and composition of the immune cell infiltration was reported . The HLAAnalysis of mRNA and protein levels revealed a discordant HLA-G mRNA and protein expression pattern frequently occurred suggesting a post-transcriptional regulation of HLA-G in this tumor entity , 98. Furth most common tumor worldwide with a high incidence in the Western world. It is more prevalent in male than female  is the 10n female . The inin female . There en female , 102. A n female , 104. InIn addition, repeated applications of Bacillus Calmette Guerin (BCG) can induce HLA-G neoexpression extrinsically leading to acquired resistance to further BCG-based instillation therapy . Few stuTo best of our knowledge meaningful studies with sufficient BC or RCC patient cohort sizes in regard to elevated HLA-G protein levels in urine samples and its suitability as a potential prognostic marker has not yet been performed.A strong intratumoral heterogeneity exists in RCC for the expression of ICPs, such as PD-L1, B7-H3, PD-L2 and HLA-G, in primary RCC lesions with a highly variable HLA-G expression either between patients’ tumor samples or at different areas within a tumor tissue , 108. TrImmunohistochemical analysis of 75 primary bladder transitional cell carcinoma (TCC) lesions demonstrated a HLA-G expression in 51 of 75 tumors while the normal bladder lacks HLA-G expression. However, the expression varied from negative to 100% positive . A similDue to the frequent pathophysiological HLA-G expression in solid and hematopoietic tumor entities a clinical relevance has been suggested. In HLA-G gene have been described, which are important parameters for cancer and clinical correlations  of the immune cells were CD3ectively .+ and CD8+ cells and a non-statistically significant higher number of CD4+ and CD56+ cells. However, the T cell activation markers CD69 and CD25 did not show a statistically significant difference between HLA-G-negative and HLA-G-positive RCC samples  expressed on T cells, its ligand CD274, the programmed cell death protein ligand 1  expressed on tumor cells and immune cells , the cytotoxic T-lymphocyte-associated antigen (CTLA-4) expressed on T cells  and its immunological counterparts B7-1/B7-2 (CD80/CD86) expressed on antigen presenting cells . Several ICP inhibitors targeting CTLA4 and the PD1/PD-L1 axes have been recently approved by the EMA and/or the FDA. Their application can result in an activation of different immune cells accompanied by tumor cell elimination, leading to the remarkable success of this therapeutic approach in different cancers including RCC and BC –121. Recin silico analyses of RCC TCGA data also identified B7-H3, B7-H5, HVEM, CD40, CD70 and ILT2 (on tumor cells) as putative novel ICP axes (Next to HLA-G and PD-L1 (B7-H1) ICP axes . Indeed,ICP axes .+ CTL, CD4+ T helper cells, B cells and other APC, HLA-G appears to be a potent candidate for further anti-tumor immunotherapy aiming on inhibition of immune tolerance/suppression/evasion exerted by HLA-G expressing tumors.However, the currently available ICPi are mostly restricted to T cells, since CTLA-4 and PD-1 are expressed on T cells . During in vitro studies, the downregulation of HLA-G protein levels, e.g. by overexpression of HLA-G regulatory miRs  . It is k+ T cell receptors and even may cause apoptosis of immune effector cells as well as the functional inhibition of immune effector cells. HLA-G suppresses proliferation of CD4+ T lymphocytes  show a more mature and aggressive CTL phenotype with a higher cytolytic capacity compared to ILT2-negative peripheral blood precursors or CD8+ PD-1+ TIL. Since HLA-G is able to nearly completely block their activity, this cell pool is an interesting target to release their cytolytic capacity by therapeutic HLA-G inhibition in HLA-G expressing tumors , which are characterized by high expression of HLA-G and other tolerogenic molecules, such as ILT2 and ILT4 , the suppression of IFN-ƴ release by NK cells  and Dr. Werner Jackstädt Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.