Here we describe a broadly applicable nongenetic approach that simultaneously addresses the multiple challenges of CAR T as a therapy for solid tumors. The approach massively reprograms CAR T cells by exposing them to stressed target cancer cells which have been exposed to the cell stress inducer disulfiram (DSF) and copper (Cu)(DSF/Cu) plus ionizing irradiation (IR). The reprogrammed CAR T cells acquired early memory-like characteristics, potent cytotoxicity, enhanced in vivo expansion, persistence, and decreased exhaustion. Tumors stressed by DSF/Cu and IR also reprogrammed and reversed immunosuppressive TME in humanized mice. The reprogrammed CAR T cells, derived from peripheral blood mononuclear cells (PBMC) of healthy or metastatic breast cancer patients, induced robust, sustained memory and curative anti-solid tumor responses in multiple xenograft mouse models, establishing proof of concept for empowering CAR T by stressing tumor as a novel therapy for solid tumor.The poor efficacy of chimeric antigen receptor T-cell therapy (CAR T) for solid tumor is due to insufficient CAR T cell tumor infiltration,  In contrast, results from clinical trials of CAR T for solid tumors have been disappointing3. Many factors contribute to the poor efficacy of CAR T for solid tumors. These include insufficient infiltration, expansion, persistence, and effector function, resulting in the ultimate exhaustion of adoptively transferred CAR T cells and an immunosuppressive tumor microenvironment (TME)4. In addition, intrinsic target antigen heterogeneity and/or antigen loss due to selective pressure by targeted therapies6 also contribute to the resistance of solid tumors to CAR T8. Significant efforts have been made to genetically engineer modified CAR T cells to promote more effective treatments for solid tumors. These include, but are not limited to, altering an array of tumor-specific CAR T cells to express (i) the p40 IL23 subunit to promote proliferation and survival9, (ii) the dominant-negative TGF-β receptor to promote proliferation10, (iii) the IL-8 receptor, CXCR1 or CXCR2, to enhance migration and persistence in the TME11, (iv) the anti-PD-L1 antibody, PD-1 dominant negative receptor, or PD-1–knockout alteration to block PD-1/PD-L1 signaling in CAR T cells13, and (v) immunostimulatory RNA RN7SL1 to activate RIG-I/MDA5 signaling and promote expansion and effector-memory differentiation of CAR-T cells. The cumulative effects of these alterations enhance myeloid cell and dendritic cell (DC) activity, reverse immunosuppressive TME conditions, and prime endogenous T cells to reject tumor cells with CAR-directed antigen loss14.Chimeric antigen receptor T-cell therapy (CAR T) has achieved unprecedented success as a novel immunotherapy with curative potential for certain hematologic cancersCM) is highly concordant with longer-term in vivo persistence and favorable clinical outcomes in neuroblastoma15. Moreover, stem-like memory T cells (TSCM) have been shown to play a critical role in mediating early anti-leukemic responses and long-term immune surveillance against relapse of leukemia in patients for up to 3 years16. Importantly, sustained remission of chronic lymphocytic leukemia (CLL) has been associated with elevated frequency of CD19 CAR T cells with memory-like characteristics. More striking, the presence of an early memory T cell population in the pre-manufacturing leukapheresis product predicted, with 100% accuracy, responders vs. non-responders to CD19 CAR T therapy among CLL patients17. These results underscore an essential requirement for successful CAR T, namely, a high frequency of early memory CAR T cells. However, genetically engineering one or even several genes produces a limited effect, and it is difficult to engineer a genetic solution that simultaneously addresses all or most of the existing barriers to treating solid tumors. Moreover, even though the efficacy of these genetically modified CAR T cells is improved compared to the parental CAR T cells, they still yield low curative outcomes in preclinical models as well as in clinical trials for solid tumors2. Thus, we sought to develop a more broadly applicable non-genetic approach that could overcome many if not all the obstacles that prevent CAR T from achieving long-lasting complete solid tumor rejection.The percentage of CAR T cells with central memory phenotype 19. It is well established that DSF/Cu targets the p97 segregase adaptor nuclear protein localization protein 4 (NPL4), which is essential for protein turnover and involved in multiple regulatory and stress-response signaling pathways20. By blocking NPL4/p97, DSF/Cu activates endoplasmic reticulum (ER) stress via upregulating the inositol-requiring enzyme 1 alpha (IRE1α)–X-box-binding protein 1 (XBP1) axis, leading to autophagic apoptosis21. Recently, we found that by combining ionizing radiation (IR) with DSF/Cu, we induced more robust immunogenic cell death (ICD) of differentiated cancer cells and cancer stem cells than could be achieved with either method alone22. The molecular characteristics of ICD include the release or cell-surface expression of highly immune-stimulatory damage-associated molecular pattern molecules (DAMPs). These molecules stimulate antigen-presenting cells (APCs), which boost CAR T expansion and activity23 and activate dendritic cells (DCs), leading to subsequent development and activation of endogenous effector T cells and memory T cells24 (unpublished data). In addition, DAMPs function as ligands for pattern recognition receptors (PRRs) and PRR agonists, which promote expansion and effector-memory differentiation of CAR-T cells14. Therefore, we hypothesized that we could use DSF/Cu and IR–stressed target cancer cells to reprogram the CAR T cells into early-memory T cells with more robust expansion, activation, persistence, and effector function, while simultaneously converting the immunosuppressive TME to an immunostimulatory TME by inducing the “hot death” (ICD) of cancer cells and release of pro-inflammatory cytokines and chemokines.Disulfiram (DSF) is an irreversible pan-aldehyde dehydrogenase (ALDH) inhibitor approved by the FDA in 1951 for treating alcoholismin vitro and in vivo could reprogram not only healthy donor or metastatic breast cancer patient donor–derived CAR T cells, but also the TME, thereby improving the efficacy of these effectors against solid tumors. This “one-punch” massive impact approach, which simultaneously addresses the multiple barriers to CAR T cell therapy, was assessed in vitro and in vivo by monitoring reprogrammed and non-reprogrammed CAR T cells for evidence of early memory T-phenotype switch, expansion, infiltration, effector function, and persistence; and by analyzing reprogrammed TME to identify the number of infiltrated immune cells, immune cell subtypes, cytokine/chemokine types, and levels. More importantly, CAR T cells targeting different antigens and a panel of stressed solid tumors were evaluated for curative therapeutic potential using various established tumor cell lines and patient-derived xenograft (PDX)–derived solid tumor mouse models.This study investigated whether target cancer cells stressed by DSF/Cu and IR (DSF/Cu+IR) 25, we investigated a broad spectrum of stresses that can be induced by DSF/Cu+IR treatment of cancer cells. RNA-sequencing (RNA-seq) analysis revealed that genes related to ER, oxidative, chemical, and heat shock stress were upregulated in DSF/Cu+IR–treated vs. untreated target human breast cancer cells SUM159 . Specifically, DSF/Cu+IR–induced ER stress in target cells was detected by increased p-eIF2a and p-IRE1α 26 , defined as CD45RA−CD62L+ cells, and found the percentage to be Significantly higher in normal donor PBMC-derived B7-H3 CAR T cells following repetitive co-culture in separate experiments with 3 types of stressed cancer cells (stressed repetitive co-culture) compared with non-stressed cancer cells (regular repetitive co-culture) , which had been co-cultured with the stressed tumor cells. The percentage of preexisting stem-like memory T cells (TSCM), defined as CD45RA+CD62L+ cells, ranged from 0.61% to9.04% in PBMCs from these 20 patients. To facilitate this analysis, we categorized the findings into two groups, i.e., low TSCM in PBMC (low TSCM/PBMC<2%) and high TSCM PBMC (high TSCM/ PBMC>2%). The rate of PBMC-derived CAR T cells with TSCM phenotype was Significantly higher in both low and high TSCM groups following 48h of co-culture with DSF/Cu+IR–stressed target breast cancer cells (SUM159) compared with B7-H3 CAR T cells co-cultured with untreated non-stressed SUM159 cells. Notably, the stressed target cells drove the percentage of stem-like memory T cells (TSCM%) in CAR T cells derived from the low TSCM/PBMC group to levels as high as the non-reprogrammed CAR T cells derived from the high TSCM/PBMC group  , c, d. Aor cells . Next, wMC group . These d T cells .DSF/Cu+IR-stressed-target cells  cell lines compared to repetitive co-culture with non-stressed tumor cells , as well as in normal donor PBMC-derived CSPG4 CAR T cells following repetitive co-culture with stressed vs. non-stressed triple negative breast cancer (TNBC) and head and neck squamous cell carcinoma (HNSCC) cell lines . Then, strong and sustained cytotoxicity on a panel of different target cancer cells and increased release levels of IFN-γ and TNF-α were found in normal donor PBMC-derived B7-H3 CAR T cells or CSPG4 CAR T cells, respectively, following repetitive co-culture with stressed tumor cells . Moreover, following co-culture with DSF/Cu+IR–stressed target cells (SUM149), the profoundly enhanced proliferation and cytotoxic activity of CAR T cells was shown to be time-dependent, i.e., detected as early as 6h, and gradually became more apparent over 48h as evidenced by increased numbers of CAR T cells and fewer target cells DSF/Cu+IR–stressed TNBC and a similarly stressed PDAC cell line may have contributed to enhanced target cell cytolysis  and IR delivery of DSF/Cu to determine if we could enhance the antitumor efficacy of normal donor derived-CAR T cells in mice bearing human cancer cell line-derived xenografts (Based on promising nografts . In the nografts . Strikinnografts . MoreoveSCM (CD45RA+CD62L+), T (CD45RA−CD62L+CM) and tumor-infiltrated CAR T cells (CD3+CD45+), in addition to having fewer exhausted CAR T cells (CD3+CD45+CTLA-4+/PD-1+) and higher pro-inflammatory cytokines/chemokines in tumor tissues compared to mice treated with B7-H3 CAR T cells only  was Significantly higher in mice treated with RP–CSPG4 CAR T cells compared to NRP–CSPG4 CAR T cells  of the mice rejected the challenge, maintaining long-lasting, tumor-free survival . To undells only , e, f, g, CSPG4.  Likewise, CSPG4. . In vivo T cells .Extended Fig3a). Again, RP–B7-H3 CAR T treatment resulted in complete tumor regression in 100% of mice while NRP–B7-H3 CAR T treatment inhibited tumor growth Significantly but did not cause complete tumor regression . In vivo expansion and persistence of B7-H3 CAR T cells at early (day 8) or late (day 92) time points after CAR T infusion of each mouse group were positively reflected in their antitumor efficacy . RP–B7-H3 CAR T-treated mice had long-lasting tumor-free survival . To further confirm the strength and duration of the immunologic memory response that was previously observed in the SUM159 mouse model . Subsequently, we asked the question, if there is no accessible tumor for IR and /or i.t. injection of DSF/Cu, could this approach be applied ex vivo to enhance CAR T cell antitumor efficacy? To this end, we determined in vivo the anti-solid tumor activity of magnetic bead-sorted B7-H3 CAR T cells following 48h in vitro co-culture with PANC-1 cells pre-stressed by DSF/Cu and IR, designated ex vivo reprogrammed (ex vivo RP) CAR T cells  CAR T cells, and non-cocultured (untreated) B7-H3 CAR T cells served as controls.Last, we assessed whether the same strategy could boost the anti-tumor activity of B7-H3 CAR T cells in a PDAC xenograft model, namely, PDAC PANC-1 cell line-derived xenografts in mice  in only 40–60% of mice, respectively  of mice treated by ex vivo RP–B7-H3 CAR T cells, as measured by tumor rejection and/or remaining tumor-free, while 20% (1/5) developed tumors . To gain knowledge about the effect of 3 in dimension, theour approach in immunocompetent mice, we investigated DSF/Cu+IR–stressed tumor-induced TME changes in humanized mice bearing TNBC SUM159 cell-derived xenografts . When th DSF/Cu+IR+B7-H3 CAR T treatment was initiated. Owing to the rapid response to this therapy, the tumors shrank quickly in mice  and 10 (n=5 mice/group) after CAR T cell infusion. At both time points, we found that DSF/Cu+IR+B7-H3 CAR T cell-treated mice had higher levels of infiltrated T cells (CD3+CD45+)(+CTLA-4+) in the TME , CAR T cD3+CD45+), and DCsD3+CD45+), as well the TME  compared the TME , a disti the TME . These pExtended Fig5a, b), (ii) time-dependent increases of IL-6 release, most noticeably detected in DSF/Cu+IR–stressed SUM159 cells , as well as the predominant increase of IL-6 compared to other cytokines/chemokines in TME of NSG or NSG-humanized mice treated with DSF/Cu+IR  JAK1, p-JAK2, p-STAT3, p-STAT5 and its target anti-apoptotic protein BCL-2 . We then demonstrated that the JAK inhibitors (JAKi), ruxolitinib and/or momelotinib, decreased TCM% in B7-H3 CAR T cells in a dose-dependent manner . More important, the JAK inhibitors completely abolished, in a dose-dependent fashion, the effects of increasing TCM% and expansion of B7-H3 CAR T cells by DSF/Cu+IR–stressed cancer cells .Next, we sought to identify the main responder when CAR T cells encounter DSF/Cu+IR–stressed cancer cells. As indicated earlier, DSF/Cu+IR induces cell stress responses leading to upregulation of an array of pro-inflammatory chemokine and cytokine genes in target cancer cells . Of thesSF/Cu+IR  +Fig5h, SF/Cu+IR , i. It iExtended Fig5f).However, JAKi decreased the target cell killing capacity of B7-H3 CAR T cells, regardless of co-culture with non-stressed or stressed tumor cells (SCM in PBMC (Extended Table 2) was evaluated. The RP–B7-H3 CAR T cells and NRP–B7-H3 CAR T cells derived from each patient (n=20) were compared for therapeutic responses in the orthotopic TNBC SUM159 mouse model  mice with complete tumor rejection than in partial responder (PR) mice with reduced tumor sizes in RP–CAR T cell-treated mice . Furthermore, B7-H3 CAR T cells derived from PBMC with low TSCM% (0.85%), obtained from a 75-year-old metastatic TNBC patient, were tested in a TNBC PDX mouse model . Subsequent RP–B7-H3 CAR T cell treatment resulted in complete tumor rejection in 100% of the treated mice. In sharp contrast, treatment with NRP–B7-H3 CAR T cells showed non-significant tumor growth inhibition compared to untreated mice (In vivo expansion and persistence of B7-H3 CAR T cells was significantly higher in mice treated with RP–B7-H3 CAR T cells than with NRP–B7-H3 CAR T cells derived from this patient (It appears that the superior anti-tumor efficacy of RP–B7-H3 CAR T cells correlates with (i) increased CAR T cell expansion in vivo ; (ii) moCAR TEFF , and  or “hot death” of targeted cancer cells accompanied by the release of highly immune stimulatory DAMPs, pro-inflammatory cytokines, and chemokines, which in turn, yielded reprogrammed (RP) CAR T cells and TME22  CAR T cells showed upregulated expression of memory T cell-related genes  and greater in vivo persistence15.  This finding is consistent with a recent clinical report that early memory T cells play a determinant role in the clinical success of CD19 CAR T cells in CLL17. Second, RP–CAR T cells acquired higher levels of expression of proliferation-related and T cell activation genes , and increased release of IFN-γ and TNF-α . Furthermore, as an indication of long and sustained immunological memory responses against tumors from the persistent residual CAR T cells, first-time rechallenged TNBC tumors were rejected in 100% of mice and PDAC tumors in 60% of mice , while even second-time rechallenged TNBC tumors were rejected in 60% of mice  (+) (+CTLA-4+) in humanized mice  , CAR T c(+CD45+)  and DCs 45+) (+) , and fewzed mice .in vivo reprogramming of B7-H3 CAR T cells via stressed tumor in TNBC-bearing mice by evaluating the efficacy of RP–B7-H3 CAR T cells derived from 20 patients with metastatic breast cancer, most of whom were heavily pretreated (Extended Table 2). RP–B7-H3 CAR T treatment resulted in 8/20 mice exhibiting a complete response (CR), accompanied by an increased number of early CAR T memory cells and fewer CAR T effector memory and exhausted CAR T cells, and in 12/20 mice exhibiting a partial response (PR), while NRP B7-H3 CAR T cell-treated mice exhibited 0/20 CR, accompanied by fewer CAR T early memory cells and increased CAR T effector memory cells and exhaustion. These results establish the correlation between increased CAR T early memory cells in complete vs partial responder mice and concur with the finding, as defined by the transcriptional profiles, that early vs. late memory CAR T cellular products from CLL are associated with a clinical response17. Most notably, when we treated mice bearing PDX with RP–B7-H3 CAR T cells derived from a metastatic TNBC patient, CR was obtained in 100% of mice, accompanied by increased CAR T cell in vivo expansion and long-lasting tumor-free survival. At the same time, compared to untreated mice, PR was observed with Significant but diminished survival in mice treated by NRP–B7-H3 CAR T derived from the same patient. These data support further optimization of the in vivo reprogramming CAR T strategy to increase the ability to induce CR, spiking promise that this type of CAR T cell can offer the clinical benefit of CR for metastatic breast cancer patients.In this study, we investigated the impact of STAT signaling axis was at least partially attributed to the reprogramming of CAR T cells by stressed cancer cells . This observation was further confirmed by activating the JAK/STAT pathway in CAR T cells. Co-culturing CAR T cells with stressed cancer cells and JAKi, ruxolitinib and/or momelotinib, completely abolished the DSF/Cu+IR-induced effects of increasing TCM% and cell expansion . However, while JAKi inhibited the killing capacity of RP-CAR T cells, it also inhibited the killing capacity of NRP-CAR T cells to the same extent (data were not shown). Therefore, it appears that the mechanisms of reprogramming CAR T cells by stressed-cancer cells is not limited solely to activation of the JAK/STAT axis, warranting further investigation.Initially, our data indicated that activation of the JAK/ex vivo reprogrammed CAR T cells presented potent antitumor activity in vivo, like that observed in vivo in tumor-bearing mice with the combination of intratumoral treatment with DSF/Cu+IR and systemic CAR T .Notably, the data obtained from humanized mice demonstrate that DSF/Cu+IR–stressed cancer cells not only reprogram CAR T cells, but also enhance the infiltration of T and DC cells into tumors. These results imply that besides reprogramming CAR T cells, stressed cancer cells are highly likely to activate endogenous immune antitumor responses as the result of increased DAMPs and pro-inflammatory cytokines and chemokines present within the TME. Furthermore, when the reprogrammed CAR T cells target B7-H3, an immune checkpoint protein, even more pronounced anti-tumor immune responses are elicited6.Therefore, this approach may simultaneously empower both passive and active immunity. Passive immunity in the host patient is mediated by the adoptive transfer of CAR T- or T cell receptor (TCR)-engineered T cells or tumor-infiltrating lymphocytes (TILs). Active immunity occurs when endogenous T cells recognize a variety of tumor antigens to prevent immune escape from CAR T- or TCR-engineered T cell therapy aimed at one or a few tumor antigen target(s) which may be downregulated or lostWe have developed a nongenetic approach to reprogram and enhance the efficacy of CAR T cells and reverse the immunosuppressive TME by stimulating CAR T cells with DSF/Cu+IR–stressed target cancer cells. The reprogrammed CAR T cells acquire characteristics that promote therapeutic efficacy against solid tumors, including, but not limited to, robust cell expansion, long-term persistence, greater effector function, and decreased exhaustion. Intratumoral injection of DSF/Cu+ tumoral IR caused the release of elevated pro-inflammatory cytokines/chemokines, increased the infiltration of CAR T, T cells, and DC, and empowered the CAR T cells in the TME.This unique “one-punch” massive impact approach overcomes most of the major obstacles that currently impede CAR T efficacy against solid tumors, yielding effectors that completely reject an array of distinct types of solid tumors, including TNBC, PDAC, and TNBC PDX, as observed in 100% of the mice tested in this study. In addition, the persistent residual RP–CAR T cells demonstrate long-term immunological memory responses as 60–100% of treated mice were protected from first-time tumor rechallenges and 60% were protected from second tumor rechallenges. Thus, stressing target cancer cells by DSF/Cu+IR is a timely approach with substantial translational potential for enhancing the efficacy of CAR T of solid tumors and inducing long-term immunological anti-tumor memory responses.37. In some cases, the cell lines were stably transduced with m-cherry and firefly luciferase. The SUM149 and SUM159 cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS. The PANC-1 and PDAC-6 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS. All cells were cultured at 37°C in 5% CO2 humidified atmosphere.Human triple negative breast cancer (TNBC) cell lines, SUM149 and SUM159, were acquired from the Duke Comprehensive Cancer Center Cell Culture Facility and Asterand Bioscience, Inc., respectively. The human pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and the head and neck squamous cell carcinoma (HNSCC) cell line PCI-13 were purchased from American Type Culture Collection (ATCC). The human PDAC cell line PDAC-6 was established at MGH using ascites fluid from a patient with metastatic PDAC™ 594 (cat#302646), CD276 (B7-H3)-APC (cat#351005), CD11b-Pacific Blue (cat#301315), CD33-PerCP/Cyanine5.5 (cat#366616), CD163-BV785 (cat#333632) and Zombie Red™ Fixable Viability Kit (cat# 423110) were purchased from Biolegend. ATF-6 (cat#65880S), Phospho-eIF2α (Ser51) (cat#3398), Bcl-2 (cat#15071S), β-Actin (#3700), STAT5 (cat#25656S), Phospho-Stat5 (Tyr694) (cat#9356S), STAT3 (cat#9139S), JAK1 (cat#3344S), Phospho-Jak2 (Tyr1007/1008) (cat#3771), Phospho-Stat3 (Tyr705) (cat#9145), Phospho-Jak1(Tyr1034/1035) (cat#74129) and Jak2 (cat#3230) were obtained from Cell Signaling Technology. CD3-FITC (cat#555339) and CD28 (cat# 556620) were purchased from Biosciences, and CD3 (cat#130-093-387) from Miltenyi Biotec. Phospho-IRE1 alpha (Ser724) (cat# PA585647) and FITC-Labeled Human B7-H3 (4Ig) / B7-H3b Protein (cat# B7B-HF2E7-25ug) were obtained from Invitrogen and Acro Biosytems, respectively.R-Phycoerythrin AfiniPure F(ab’) Fragment Goat Anti-Mouse IgG (H+L) (cat#115-116-146), Allophycocyanin (APC) AfiniPure F(ab’) Fragment Goat Anti-Mouse IgG (H+L) (cat#115-136-146), Fluorescein (FITC) AfiniPure F(ab’) Fragment Goat Anti-Mouse IgG (H+L) (cat# 115-096-146) were obtained from Jackson ImmunoResearch. CD3-PE-Cy7 (cat#300420), CD8-APC-Cy7 (cat#344714), CD4-FITC (cat#357406), CD45RA-PE-Cy5 (cat#304110), CD45RO-APC (cat#304210), CD62L-PE (cat#304806), CD27-BV421 (cat#302824), CD45RA-FITC (cat#304148), PD1 (CD279) -APC (cat#329907), CCR7 (CD197) -BV421 (cat#353208), CD261 -PE (cat#307206), CD262 -APC (cat#307408), CD95 (Fas)-BV785 (cat#305646), CD127 (IL-7Rα)-PE (cat#351304), TNFα-PE (cat#502909), IFNγ-APC (cat#502512), Granzyme B-Pacific Blue (cat#515408), CD62L-BV711 (cat#304860), CD253 (TRAIL)-PE (cat#308206), CD45-PE (cat#368510), CD366 (Tim-3)-Pacific Blue (cat#345042), CD279 (PD-1)-PerCP/Cyanine5.5 (cat#367410), CD152 (CTLA-4)-BV605 (cat#369610), CD25-PE/DazzleThe 6-to-8-week-old NSG mice were obtained from the Massachusetts General Hospital (MGH) COX7 animal facility or Jackson laboratory. The MGH Institutional Animal Care and Use Committee approved the animal studies described herein.5 cells/well  or 4×105 cells/well  in 2 mL RPMI 1640 medium containing 10% FBS and permitted to grow overnight. The following day, DSF/Cu (0.2 μM/1 μM) or DMSO was added and cultured for 24h, followed by 12 Gy IR. The cells were then resuspended, washed twice with PBS, and set up immediately in 6-well plates at a density of 5×105 cells/well. After 6–12 h, the tumor cells were co-cultured with CAR T cells at different E:T ratios.Tumor cells were plated in 6-well culture plates at a density of 3×10in vitro treatment, cells were irradiated with a single dose of IR (12 Gray (Gy)). For tumors treated in vivo, one 12 Gy fraction was delivered locally to each mouse over the area of the tumor, while the remaining body was shielded with a lead drape. The X-RAD 320 Biological Irradiator  was used for these experiments.For the For cell surface staining, the cells were harvested, washed twice with PBS, and then incubated with an appropriate amount of fluorochrome-conjugated or unconjugated antibodies for 20 min or 60 min at 4°C in 2% PBB (2% BSA in PBS buffer). Fcγ fragment-specific secondary antibodies were used accordingly if necessary. For intracellular staining, the surface-stained cells were fixed and permeabilized in Fixation/Permeabilization solution (BD Biosciences) for 20 min at 4°C followed by two washings with Perm/Wash buffer (BD Biosciences). After intracellular staining was performed, the cells were incubated for 30 min at 4°C in Perm/Wash buffer followed by two more washings with Perm/Wash buffer. To measure the number of CAR T cells that had infiltrated tumor tissues and spleens, the tumor samples were digested with collagenase IV  and deoxyribonuclease (DNase) I  for 30 min at 37°C. Tumor digests and spleens were filtered through 70 μm cell strainers to obtain a single-cell suspension. ACK lysing buffer (Themo Fisher) was used to lyse the red blood cells according to the manufacturer’s instructions. Viability dye was used in each sample. The cells were analyzed by flow cytometry using the LSR II cytometer (BD Biosciences) or BD Accuri C6 flow cytometer (BD Biosciences). The data were analyzed with FlowJo software.5 cells/well  or 7.5×106 cells/well (PANC-1) in 2mL complete CAR T medium31. After 6–12 h, the tumor cells were co-cultured with CAR T cells at the indicated E:T ratios.The stressed target cells were resuspended, washed with PBS twice, and set up immediately in 6-well culture plates at a density of 5×105 cells/well  or 7.5×106 cells/well (PANC-1) in 2 mL of complete CAR T medium and grown overnight. Then, 2.5×105 CAR T cells (E: T=1:3 or 1:2) were added to the tumor cells. The CAT T cells eliminated 100% of the tumor cells within 2–5 days of co-culture . Next, the CAR T cells were collected and resuspended in 2 mL complete CAR T medium and transferred to wells containing either 5×105  or 7.5×105 (PANC-1) untreated tumor cells that had been seeded 12–14 h previously, and the co-culture was continued for 3 days. . The procedure was repeated once more , and the cells remaining from R3 were then collected for analysis of exhaustion-related markers .The tumor cells  were seeded in 6-well plates at a density of 5×10in vitro, cells from R1 of the repetitive co-culture assay were resuspended and washed twice with PBS. Thereafter, the cells were cultured in 2mL complete CAR T medium supplemented with 5 ng/mL IL-15 and 10 ng/mL IL-7. After 3 days, the cells were collected and stained with CD3, CD4, CD8, CD62L, and CD45RA antibodies and then analyzed using flow cytometry.To evaluate the phenotypic transformation of CAR T cells Cells collected at the end of the co-culture assays were stained to quantify tumor cells with B7-H3-specific mAb376.96 and CAR T cells with CD3-specific antibody. Counting beads (Thermo Fisher Scientific) were used based on the manufacturer’s instructions to obtain the absolute numbers of both residue tumor cells and CAR T cells.Cell culture supernatants were quantified for secreted cytokines  according to the manufacturer’s instructions using ELISA kit (Biolegend).Human cytokines and chemokines found in the cell culture supernatant and tumor tissue homogenates were quantified using a customized 25-plex Luminex panel (Thermo Fisher Scientific) containing the following analytes: CCL11, GM-CSF, Granzyme B, HSP60, IFNα, IFNγ, IL-1, IL-3, IL-4, IL-6, IL7, IL8 (CXCL8), IL-9, IL-10, IL-12p70, IL-15, IL-17A, IL-18, CXCL10, M-CSF, CXCL9, CCL3, CCL4, CCL5, TNFα. Data were acquired on a MAGPIX instrument and analyzed using the ProcartaPlex analysis app.The tumor tissue was dissected and washed briefly with chilled PBS. The samples were placed in vessels containing lysis buffer, RIPA buffer (Thermo Fisher Scientific) and 1/50 (vol./vol.) of protease inhibitor (Thermo Fisher Scientific), and then disrupted for 30s in the homogenizer. The cultured cells were detached and washed briefly with chilled PBS before adding the lysis buffer. All samples were placed on ice for 30 min and then centrifuged for 20 min to collect the supernatant without disturbing the debris. The protein concentration was quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.38.Performed as describedTotal RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions.The total RNA content of both tumor cells and CAR T cells was extracted as described above, and the library was prepared by LC Sciences company. Raw data from both tumor cells and CAR T cells was normalized in three replicates for GSEA analysis. GSEA was performed using the GSEA_4.1.0 software (Broad Institute) on genes that differentially expressed on non-stressed vs. stressed-SUM159 cells; or NRP B7-H3 CAR T cells vs. RP B7-H3 CAR T cells.39.Performed as described31.CAR T cells were generated as described+ T cell Dynabeads  via positive selection. The magnetic bead isolated CAR T cells were used in Western blotting or ex vivo experiments in mice.CAR T cells were co-cultured with DSF/Cu and IR-stressed tumor cells for 48 h to allow for reprogramming. The CAR T cells were subsequently isolated from the co-cultures as CD3TNBC cell line-derived orthotopic mouse model: SUM149 (2×106) or SUM159 (2×106) tumor cells in 100 μL FBS-free RPMI medium were injected into the mammary fat pads of 6-to-8-week-old female NSG mice. When the tumors became palpable, the mice were divided into groups using a stratified randomization strategy (n = 5 mice/group) and treated as indicated.PDAC xenograft mouse model: PANC-1 (2×106) tumor cells in 100 μL FBS-free RPMI medium were injected subcutaneously (s.c.) into the right thigh of 8-to-9-week-old male NSG mice. When the tumors became palpable, the mice were divided into groups using a stratified randomization strategy (n = 5 mice/group) and treated as indicated.PDAC orthotopic xenograft mouse model: mcherry/Luc-PANC-1 (2×106) tumor cells were orthotopically engrafted into the pancreas of 8-to-9-week-old male NSG mice. Tumors were measured by bioluminescence imaging (BLI) and divided into groups using a stratified randomization strategy (n=5 mice/group).Humanized mouse bearing TNBC orthotopic tumor model: SUM159 (2×106) tumor cells in 100 μL FBS-free RPMI medium were injected into the mammary fat pads of 6-to-8-week-old female NSG mice. After 21 days, the mice were divided into groups using a stratified randomization strategy (n = 10 mice/group) and humanized by i.v. injection of 2×107 human PBMCs per mouse from a heathy donor, and the mice were treated with the same donor-derived CAR T as indicated.TNBC-PDX orthotopic mouse model : A 3×3×3 mm piece of in vivo expanded PDX was orthotopically engrafted into the mammary fat pad of 6-to-8-week-old female NSG mice. When tumors developed, the mice were divided into groups using a stratified randomization strategy (n ≥ 6 mice/group) and treated as indicated. Tumor volumes were measured using a digital caliper and calculated as described31.Five distinct xenograft models were used in this study:Treated tumors were intratumorally injected (i.t.) at multiple sites with 200 μL DSF/Cu (1.5 μM/1 μM) or vehicles (DMSO/PBS), followed by 12 Gy local IR on the next day.Freshly thawed and expanded (within 2–3 days) CAR T cells were injected i.v. into the mouse tail vein once at the indicated dose.Fifty μL of peripheral blood was collected once a week from each mouse, after which red blood cells were removed using ACK lysing buffer (Themo Fisher). T cells were assessed with CD3 and CD45 antibodies or CD62L, CD45RA, PD-1, LAG3, and Tim3 antibodies and quantified using CountBright Absolute Counting Beads (Thermo Fisher) on a BD LSRII flow machine.Statistical analysis was performed using GraphPad Prism v.8 software. The one-way ANOVA was used to determine the comparisons among three or more groups at a certain condition. The student’s t-test was applied to the comparison between two groups. Additional two-way ANOVA was adopted as specified. Overall survival was calculated using Kaplan-Meier methods and log-rank tests. All data are expressed as mean ± standard deviation (SD) unless specified otherwise. Results were obtained from two or three independent experiments. Differences between groups were considered Significant when p<0.05.


Caenorhabditis elegans and genetic screens for systemic RNA interference-defective mutants have identified RNA transporters  and a signaling protein (SID-3). Here, we report that SID-4 is nck-1, a C. elegans NCK-like adaptor protein. sid-4 null mutations cause a weak, dose-sensitive, systemic RNA interference defect and can be effectively rescued by SID-4 expression in target tissues only, implying a role in double-stranded RNA import. SID-4 and SID-3 (ACK-1 kinase) homologs interact in mammals and insects, suggesting that they may function in a common signaling pathway; however, a sid-3; sid-4 double mutants showed additive resistance to RNA interference, suggesting that these proteins likely interact with other signaling pathways as well. A bioinformatic screen coupled to RNA interference sensitivity tests identified 23 additional signaling components with weak RNA interference-defective phenotypes. These observations suggest that environmental conditions may modulate systemic RNA interference efficacy, and indeed, sid-3 and sid-4 are required for growth temperature effects on systemic RNA interference silencing efficiency.RNA interference is sequence-specific gene silencing triggered by double-stranded RNA. Systemic RNA interference is where double-stranded RNA, expressed or introduced into 1 cell, is transported to and initiates RNA interference in other cells. Systemic RNA interference is very efficient in  Caenorhabditis elegans, experimentally introduced dsRNA is mobile (systemic RNAi-defective (Sid) mutants that has led to the identification of dsRNA transport and signal transduction proteins (RNA interference (RNAi) is sequence-specific gene silencing triggered by natural or experimentally introduced double-stranded RNA (dsRNA)  (sid-4.Characterization of the to cells . In sid-lencing) . Here, wrde-10, -11, and -12 and rrf-1, which fail to produce abundant secondary siRNAs resulting in dose-dependent RNAi-silencing defects ; HC119 sid-4(qt17); HC158 sid-4(qt33); and HC160 sid-4(qt35). Samples were sheared to 250 bp and prepared according to NEB Ultra DNA library kit E7370S. Sample concentrations were quantified via qPCR (Kapa Kit KK4824) and sequenced (Illumina), recovering a total of 250 million reads with 80× coverage. Reads were aligned using bowtie2 and exon variants on linkage group X were called using Samtools. Coding sequence variants in only a single gene, nck-1, were identified in all 4 strains.Genomic DNA was extracted from each of the 4 sid-4A (ZK470.5A) and sid-4B (ZK470.5B) cDNA constructs (Open Biosystems) were verified and individually injected (4.5 ng/μl) with a Pmyo-2::mCherry (10 ng/μl) coinjection marker (PCFJ90) and 1 kb DNA ladder (10 ng/μl) (New England Biolabs) into sur-5::NLS-GFP; sid-4(ok694) (HC1133). For each isoform plasmid, 3 recovered independent complex extrachromosomal array lines  were analyzed.Full-length 1 adult progeny were scored for sensitivity to RNAi. Bacteria containing the empty-vector L4440 was used as the control for all feeding RNAi experiments. The following phenotypes were scored for each food: gfp, GFP silencing; dpy-11, body length ; bli-1, full-body (hyp7) blisters; unc-45, paralysis; unc-22, twitching; act-5, F1 larval growth; fkh-6, F1 fertility ; pos-1, F2 embryonic viability.L4-young adults were placed on RNAi food  at room sid-4(ok694); qtEX159 sid-4::gfp young adults were cultured on bli-1 RNAi plates for 3 days. RNAi-sensitive blistered animals were counted and RNAi “resistant” nonblistered animals were collected and scored for GFP expression. All resistant worms expressing detectable GFP were then mounted for widefield microscopy and scored for GFP expression in the hypodermis.HC1139 pal-1 dsRNA was made by amplifying a 1.2-kb region from the pal-1 plasmid . A T7-flash Ampliscribe kit was used to make single-stranded sense and anti-sense RNA strands, which were annealed, quantified, diluted as necessary, and injected into either 1 or both gonad arms of sid-4(ok694) and N2 animals. Six hours after injection, each recovered animal was singled to an OP50 plate. To score embryonic lethality, the adult was removed after 24 h and laid eggs counted. Three days later, the number of hatched progeny were counted.C. elegans orthologs of these proteins as well as to identify viable mutations. Fifty-three mutant strains were ordered from the C. elegans Genetic stock Center (cgc.umn.edu). To test each strain for RNAi defects, young adult hermaphrodites were placed on fkh-6 RNAi plates and dpy-11 RNAi plates. Three days later, the fkh-6 RNAi plates were scored for presence of laid eggs and the dpy-11 RNAi plates were scored for Dpy progeny.All known mammalian NCK and ACK physical interactors were obtained from BioGrid (build 3.4.140) and Human protein reference databases (release 9) and all mammalian network information for both was obtained from PathCards (version 4.4) (sid-4 locus (nck-1) in each isolate: sid-4(qt15) [S338L], sid-4(qt17) [G162E], sid-4(qt33) [Y250-stop], and sid-4(qt35) [E132K] (sid-4/nck-1 (ok694)   Fig. 1)sid-4 locnck-1/nckα and nck-2/nckβ, may function redundantly as well as have distinct roles in signal transduction  with isoform-specific cDNA constructs driven by their endogenous promoters.The mammalian NCK homologs, d NCK-1B . NCK-1A d NCK-1B . The scrd NCK-1B . The lacon sites  ; sur-5::gfp hermaphrodites grown on bacteria expressing gfp dsRNA are resistant or partially resistant to silencing ; sur-5::gfp strain; however, all identified transgenic progeny were developmentally arrested. This phenotype is unlikely to be related to dsRNA transport; thus, we did not investigate further.To score RNAi efficacy in multiple tissues, we monitored the expression of a ilencing . We thenNAi food ; the sur strains . This resid-4 mutants partially disable RNAi silencing. To directly compare sid-4 systemic silencing defects to that of other Sid mutants, we crossed the deletion allele sid-4(ok694) into the transgenic HC57 background used for the screen. In this transgenic strain, pharyngeal expressed gfp dsRNA autonomously silences pharyngeal GFP and systemically silences GFP in anterior body wall muscle cells. When cultured on gfp dsRNA expressing bacteria, the ingested dsRNA silences GFP in all body wall muscle cells. A sid-1 mutant in this transgenic background disrupts the silencing of the body wall muscle GFP from both pharyngeal expressed gfp dsRNA and ingested bacterial gfp dsRNA  and sid-4(ok694) show incomplete systemic RNAi defects  to all these RNAi foods . sid-4(qt33) and sid-4(ok694) worms were completely or nearly completely resistant to fkh-6 (gonad), unc-45 (muscle), and bli-1 (hypodermis); completely or mostly sensitive to act-5 (intestine) and unc-22 (muscle); and partially resistant to pos-1 (germline) and dpy-11 (hypodermis). The discrepancy in sensitivity to dpy-11 between qt33 (nonsense) and ok694 (deletion) indicates that the nonsense mutant qt33 may have residual activity, perhaps reflecting tissue-specific read-through translation of the stop codon  on the same panel of RNAi foods  mutants that are suppressed by high levels of pharyngeal expressed dsRNA can produce wild-type silencing in the pharynx  phenotype, embryonic lethality. Because the germline is syncytial, injections into the anterior or posterior gonad do not require dsRNA transport for effective silencing. If sid-4 mutants are weak RNAi defective, then wild-type and sid-4 mutant worms should show distinct embryonic lethality frequencies in response to decreasing injected dsRNA dose. We injected both gonad arms to eliminate the effect of gonad to gonad transfer.le cells  indicatisid-4(ok694) mutants showed proportionate changes in embryonic lethality when injected with changing concentrations of pal-1 dsRNA  mutants for systemic RNAi defects (Sid), we injected single gonad arms with either a high or low pal-1 dsRNA dose. Consistent with effective systemic RNAi, there was no detectable difference in embryonic lethality among the progeny of wild-type worms injected with pal-1 dsRNA into either a single or both gonad arms at either high or low dsRNA dose (sid-4(ok694) animals injected with the high concentration of pal-1 dsRNA into either a single or both gonad arms was nearly identical (sid-4(ok694) animals injected with the low concentration of pal-1 dsRNA into single gonad arm was half that of animals injected into both gonad arms  animals with a sid-4::gfp construct maintained as a mitotically unstable extrachromosomal array. About 82% of the progeny inherited the array and were GFP positive and formed full-body blisters when cultured on bli-1 RNAi food. The remaining 18% of the progeny did not inherit the array and were mostly GFP negative and fully resistant to bli-1 RNAi. bli-1 is expressed in the large syncytial cell hyp7 , which produces the full-body blister in bli-1 RNAi-treated animals ; sid-4(ok694) double mutant on a broad panel of RNAi foods (unc-22 (muscle) and pos-1 (germline), the sensitivity of both single mutants was intermediate, and in both cases, the double mutant was less sensitive  with both an NCK and an ACK ortholog . Among 116 C. elegans orthologs of these proteins, 80 viable mutants have been described and we tested 52 by feeding RNAi for reduced sensitivity to fkh-6 and dpy-11 RNAi food  deletion mutant more thoroughly, we scored the RNAi phenotypes of the progeny of adult animals placed on a panel of RNAi foods targeting a variety of tissues (fkh-6 RNAi food fail to lay any eggs and while ver-1(ok1783) animals lay eggs ; sid-3(tm342) double mutant on the same panel of RNAi foods . These results indicate that sid-3 and sid-4 do not contribute to systemic RNAi at 25°C and are required primarily for the low temperature enhanced penetrance of systemic RNAi.Efficient systemic RNAi requires signaling proteins, indicating that physiological or environmental conditions may modulate systemic RNAi. We previously reported that temperature affects systemic RNAi; we noted more efficient silencing of body wall muscle GFP in response to exported pharyngeal  at 25°C . Thus, t mutants . We founge trial , which cC. elegans and understanding what conditions regulate their activity will lead to further insight into the mechanisms and functions of systemic RNAi. Unlike the dsRNA transporters SID-1 and SID-2, these 2 proteins have been implicated in a variety of presumably systemic RNAi-independent functions in C. elegans, including cell migration , pak-1(p21 activated kinase), and cdc-42. It should be emphasized that only viable alleles of candidate SID-3–SID-4-interacting proteins were tested and for many of these genes null alleles are inviable . Among the tested RNAi foods, sid-4 mutants were most resistant to fkh-6 silencing  vs. the variable and relatively sparse GFP expression in the weak mutants (sid-3, sid-4, and sid-5, as well as a partial loss-of-function sid-1 alleles; sid-3 and sid-4 are dose-dependent SIDs, that is, increasing the dose of dsRNA bypasses the dsRNA import defect.The original large visual screen identified many alleles in sid-3- and sid-4-dependent process is most apparent at lower growth temperatures, confirming that the temperature effect on silencing is due to more efficient systemic RNAi at lower growth temperature.Furthermore, and unexpectedly, we discovered that combining 2 weak mutants resulted in a stronger Sid phenotype, suggesting that multiple independent processes contribute to dsRNA import. Some of these processes may involve putative NCK/ACK1-interacting proteins, several of which have weak RNAi-defective phenotypes. Finally, the jkac252_Supplemental_FiguresClick here for additional data file.jkac252_SupplementalTable_S1Click here for additional data file.


Patients with STAT1 gain-of-function (GOF) mutations suffer from an inborn error of immunity hallmarked by chronic mucocutaneous candidiasis (CMC). The pathogenesis behind this complex and heterogeneous disease is still incompletely understood. Beyond the well-recognized Th17 failure, linked to the STAT1/STAT3 dysbalance-driven abrogation of antifungal defense, only little is known about the consequences of augmented STAT1 signaling in other cells, including, interestingly, the innate immune cells. STAT1-mediated signaling was previously shown to be increased in STAT1 GOF CD14+ monocytes. Therefore, we hypothesized that monocytes might represent important co-orchestrators of antifungal defense failure, as well as various immunodysregulatory phenomena seen in patients with STAT1 GOF CMC, including autoimmunity. In this article, we demonstrate that human STAT1 GOF monocytes are characterized by proinflammatory phenotypes and a strong inflammatory skew of their secretory cytokine profile. Moreover, they exhibit diminished CD16 expression, and reduction of classical (CD14++C16-) and expansion of intermediate (CD14++16+) subpopulations. Amongst the functional aberrations, a selectively enhanced responsiveness to TLR7/8 stimulation, but not to other TLR ligands, was noted, which might represent a contributing mechanism in the pathogenesis of STAT1 GOF-associated autoimmunity. Importantly, some of these features extend to STAT1 GOF monocyte-derived dendritic cells and to STAT1 GOF peripheral myeloid dendritic cells, suggesting that the alterations observed in monocytes are, in fact, intrinsic due to STAT1 mutation, and not mere bystanders of chronic inflammatory environment. Lastly, we observe that the proinflammatory bias of STAT1 GOF monocytes may be ameliorated with JAK inhibition. Taken together, we show that monocytes likely play an active role in both the microbial susceptibility and autoimmunity in STAT1 GOF CMC. Hypermorphic mutations in signal transducer and activator of transcription 1 gene  in humans cause an inborn error of immunity which is hallmarked by chronic mucocutaneous candidiasis (CMC). This complex syndrome of immune dysregulation displays a broad phenotype beyond the microbial susceptibility, including autoimmunity (often severe and life threatening), malignancy and vascular abnormalities . While tCandida is impaired (The Janus Activating Kinase (JAK)/STAT pathway is utilized by both adaptive and innate immune cells in response to signals from many various cytokines and hormones. It encompasses seven structurally homologous STAT proteins which are involved in a range of essential cellular processes, such as growth, differentiation, metabolism and immune functions –10. STATimpaired , 14, 15.ex-vivo STAT1 GOF monocytes.Although more than 100 STAT1 GOF mutations in over 400 patients have been identified worldwide to date , only twSTAT1 mutation. All patients were non-consanguineous, of Caucasian ethnicity, 3 males and 5 females, the median age was 45 years (range 8–52 years). All patients suffered from CMC of various severity, three had clinically manifest autoimmunity and six had autoantibodies against organ-nonspecific antigens. All patients received antifungal prophylaxis. Three patients received selective Janus kinases (JAK) 1/2 inhibitor ruxolitinib , however majority of samples for this study were obtained prior to ruxolitinib initiation, unless indicated otherwise. No patients received any other immunomodulatory compounds at the time of sampling or during a relevant period prior to sampling. The mutations within the cohort include previously described heterozygous mutations causing amino acid change in p.E29A, p.Y68C, p.A267V, p.T288N, p.N357D and p.M390T  for 5, 15, 30, 60 and 120 minutes or left untreated at 37°C. Intracellular signaling was prevented by using 4% paraformaldehyde without methanol for 10 minutes at room temperature. Erythrocytes were lysed using 0,1% Triton-X for 20 minutes  at 37°C, leukocytes were permeabilized with ice-cold 80% methanol for 30 minutes and stained with anti-phosphoSTAT1-BV421 (Tyr701) (clone 4a) and anti-phosphoSTAT3-PE (Tyr705) (clone 4/5-STAT3) , anti CD14-APC (63D3), CD66b-PC7 (clone G10F5)  in case of whole blood. The samples were acquired on BD Fortessa (BD Biosciences), and data analysis was performed using FlowJo (TreeStar).Monocytes and dendritic cells from peripheral blood were stained with the following antibodies: Lin-FITC , CD16-A700 (clone 3G8), CD11c-APC (clone BU15), CD14-PE-DyLight594 (clone MEM-15) CD5-APC (L17F12) (Exbio), HLA-DR-PerCP (clone L243) (BD Biosciences), CD123-PE-Cy7 (clone 6H6), CCR2-BV421 (clone K036C2), CD1c-BV510 (clone L161), CD141-BV421 (clone M80) CD163-BV510 (GHI/61) (Biolegend), for 30 minutes in the dark. The cells were then lysed, centrifuged, resuspended and data were acquired on BD Fortessa (BD Biosciences), and data analysis was performed using FlowJo (TreeStar).Monocytes and dendritic cells from peripheral blood were stained with the following antibodies: Lin-FITC , CD16-A700 (clone 3G8), CD11c-APC (clone BU15), CD14-PE-DyLight594 (clone MEM-15), CD86-PE (clone BU63) (Exbio), HLA-DR-PerCP (clone L243) (BD Biosciences), PD-L1-BV510 (clone 29E.2A3), PD-1-BV421 (clone EH12.2H7), CD123-PE-Cy7 (clone 6H6), CD40-BV650 (clone 5C3) (Biolegend) for 30 minutes in the dark. The cells were then lysed, centrifuged, resuspended and data were acquired on BD Fortessa (BD Biosciences), and data analysis was performed using FlowJo (TreeStar).6C. albicans or left untreated for 24 hours. Then CD86 expression was detected by staining the peripheral blood with Lin-FITC , CD16-A700 (clone 3G8), CD11c-APC (clone BU15), CD14-PE-DyLight594 (clone MEM-15), CD86-PE (clone BU63), HLA-DR-PerCP (clone L243) (BD Biosciences), CD123-PE-Cy7 (clone 6H6), CD1c-BV510 (clone L161) and CD141-BV421 (clone M80) (Biolegend) for 30 minutes in the dark. The cells were then lysed, centrifuged, resuspended and data were acquired on BD Fortessa (BD Biosciences), and data analysis was performed using FlowJo (TreeStar).Peripheral blood was stimulated with LPS (1ug/ml), zymosan (100ng/ml), R848 (1ug/ml), polyI:C (50ug/ml), IFNγ (1ug/ml), heat-killed 1x10For intracellular cytokine detection, 100ul of peripheral blood from heparin coated tubes was stained against lineage specific markers  conjugated with FITC, CD16-Alexa Fluor 700, CD11c-APC, CD14-PE-DyLight594 (Exbio), HLA-DR-PerCP (BD Biosciences), CD1c-BV510, CD141-BV421 (BioLegend), and CD123-PE-Cy7 (ThermoFisher Scientific). After RBC were lysed with BD Lysing solution (BD Biosciences), cells were fixed and permeabilized using FixPerm kit (ThermoFisher Scientific). Cytokines were stained using anti IL-6-PE (clone MQ2-13A5) (Biolegend), IL-1β-PE (clone CRM56) – (ThermoFisher Scientific) TNFα-PE (clone MAb11) (Exbio) or CXCL10-PE (clone J034D6), respectively. When indicated, peripheral blood was stimulated with R848 (1ug/ml) for 4 hours and 1 μl/ml Brefeldin A (Biolegend) was added for the last 3 h of the incubation (BD Biosciences). CXCL10 production was detected as described above.For CD16 extracellular detection, whole blood was stained with the following antibodies: Lin-FITC , CD16-A700 (clone 3G8), CD11c-APC (clone BU15), CD14-PE-DyLight594 (clone MEM-15) (Exbio), CCR2-BV421 (clone K036C2) (Biolegend), HLA-DR-PerCP (clone L243) (BD Biosciences). For intracellular CD16 detection the whole blood was lysed, fixed and permeabilized before CD16-A700 staining. Data was acquired on BD Fortessa (BD Biosciences), and data analysis was performed using FlowJo (TreeStar).6/mL concentration and the next day the phenotype and cell-free supernatant was collected for cytokine production assessment. Cells were stained with anti, CD14-PEDy590 (clone MEM15), CD86-PerCP (clone BU63), CD16-A700 (clone 3G8) (Exbio), HLA-DR-A700 (clone L243), CD40-BV650 (clone 5C3), PD-L1-BV510 (clone 29E.2A3) (BioLegend). The samples were collected using BD Fortessa (BD Biosciences) and BD FACSDiva software (BD Biosciences) was used for signal acquisition. Cytokine production was determined byLuminex xMAP technology.Monocyte-derived DCs (cDCs) were generated from adherent monocytes cultured in IL-4 (20ng/mL) and GM-CSF (500IU/mL)  presence for 6 days. The cytokines were replenished on day 3. On day 6, the cells were harvested, seeded in 96-well plates at 1x10Peripheral blood was collected into blood tubes without anticoagulant agent and centrifuged at 3000rpm, 5 minutes. The serum was collected, aliquoted and stored at 80°C. The serum cytokine, chemokine and growth factor quantification were performed by LUMINEX xMAP technology.t-test was also applied for data analysis using GraphPad Prism 8. Values of p<0.05 (*), p<0.01 (**) p<0.001 (***) and p<0.0001 (****) were considered statistically significant.The results obtained from at least three independent experiments are given as the medians ± SDs. Not all patients were involved in all experiments due to the limited amount of blood available per sample. Statistical analysis was performed using non-parametric one-way analysis of variance (ANOVA) with multiple comparisons Dunn’s post-test where applicable. A two-tailed paired Wilcoxon or unpaired Mann-Whitney Firstly, STAT1 phosphorylation assays in monocytes after IFNα and IFNγ stimulation were performed. As expected, most of the patients displayed increased levels of phosphorylated STAT1 (pSTAT1), except for patients carrying p.Y68C mutation , intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes  were indCandida antigens. TLR7/8-stimulated STAT1 GOF monocytes expressed increased surface levels of CD86 and produced higher amounts of CXCL10 (Upon stimulation of STAT1 GOF monocytes with various Toll-like receptors (TLR) ligands, increased response was observed to R848 (imidazoquinoline), a dual TLR8 and TLR7 synthetic agonist, but not to lipopolysaccharide (LPS), polyI:C, fungal zymosan or Bone marrow-derived monocytes are precursors of myeloid DCs, which constitute the majority of blood DCs in the healthy. As such, circulating DCs should also exhibit signs of dysregulation inflicted by STAT1 GOF mutation. Multiple subsets of DCs are recognized, each defined by unique combination of their surface markers. These include DC1 (CD14-CD1c-CD141+CD16-), DC2 (CD14-CD1c+CD5+CD163-), DC3 (CD14-CD1c+CD5-CD163+) and DC4 (CD14-CD1c-CD141-CD16+) as subtypes of mDCs (CD11c+HLA-DR+CD14-), and plasmacytoid DCs  , 25. TheIn a similar experimental setup as above, we examined mDCs’ responsiveness to various TLR ligands (in-vitro monocyte-derived DC model (moDCs) was employed. After six days of cultivation from adherent monocytes, the generated STAT1 GOF moDCs expressed higher levels of CD86, CD40 and reduced amounts of PD-L1 To ascertain that the changes in monocytes and DCs are genotype-driven and not secondary to chronic infections, inflammation or treatment, an Monocytes are major producers of various stimulatory, pro-migratory cytokines and growth factors. Having previously established changes in STAT1 GOF mutant monocytes’ phenotype and secretory activities, we set out to explore the patients’ serum cytokine profiles. Out of 57 cytokines, chemokines and growth factors (GF) six were excluded: four  were undetectable and two (lactoferrin and CXCL4) exceeded the detection maximum. Out of 51 analytes, 17 cytokines/chemokines/GFs were differentially produced in the STAT1 GOF group compared to HDs  compared to HDs  and expansion of intermediate (CD14++16+) subpopulations, monocytes adopt proinflammatory phenotypes and exhibit a strong inflammatory skew within their secretory cytokine profile. We also describe a selective enhancement of TLR7/8 responsiveness. Importantly, these features extended to STAT1 GOF monocyte-derived dendritic cells, and some to blood myeloid dendritic cells, which differentiate from monocytes. Finally, in one patient, from whom sequential samples were available, upstream JAK inhibition ameliorated the proinflammatory bias of STAT1 GOF monocytes.In this article, we demonstrate that human peripheral monocytes harboring heterozygous hypermorphic mutations in CXCL10, as well as CXCL10 serum increase were detected in STAT1 GOF patients’ monocytes and DCs, in concordance with others . In. Invia tvia immune synapses (The hereby observed loss of surface CD16 from monocytes, mDCs and moDCs is unclear as to its origin and significance. CD16 is a transmembrane Fc gamma type III low-affinity receptor for IgG expressed predominantly on natural killer cells, neutrophils, monocytes, macrophages and a subset of T cells. It is associated with phagocytosis, secretion of inflammatory cytokines and clearance of immune complexes . A well-synapses . Moreovesynapses . Downregsynapses . Previousynapses . It is tCXCL10, through STAT1 activation (via a proinflammatory TLR7/TLR8-MyD88-MEK pathway (Monocytes have been associated with autoimmunity  and autotivation . Moreovetivation  and, contivation . Importa pathway . Interes pathway . Thus, wThe interpretation of the hereby observed skew of the subtypes of STAT1 GOF DCs is largely hypothetical, as only limited understanding currently exists regarding functions and biology of individual DCs subsets. For example, the diminished pool of pDCs, characterized by their antiviral sensing and interferon production, may contribute to the viral susceptibility of STAT1 GOF patients. The pDCs’ decrease may be caused by type I IFN negative feedback loop , potentiin vitro generated STAT1 GOF monocyte-derived DCs retain the abnormal features of primary patients’ monocytes, including increased surface expression of CD86, decreased expression of PD-L1 and CD16, and increased production of inflammatory cytokines. Additionally, chronic stimulation in monocytes was shown to result in cellular exhaustion characterized by reduction of CD86 and elevation of PD-L1 (Although we were unable to provide direct evidence that the phenotypical alterations observed in STAT1 GOF monocytes are mechanistically linked to the augmented STAT1 signaling, and not merely secondary to the overall chronic inflammatory milieu or salvage regulatory mechanisms, several indices would suggest so. Most importantly, we show that of PD-L1 –52, whicIn summary, the dataset presented here indicates a proinflammatory skew and abnormal functions of STAT1 GOF monocytes of patients with CMC, which may directly contribute to both their microbial susceptibility and autoimmunity. These observations instate monocytes as prominent co-orchestrators of STAT1 GOF-associated immune dysregulation. As our data suggest, the aberrations seen in monocytes are likely carried onto other monocyte-derived cells, which outlines and substantiates a direction of possible future research in STAT1 GOF towards the DCs or macrophage biology.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethical Committee of the second Faculty of Medicine, Charles University in Prague, and University Hospital in Motol, Czech Republic. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.MB conceived the hypothesis, treated the patients and co-wrote the manuscript.IZ design the study, performed experiments, analyzed and visualized data and reviewed the manuscript. TM and AS treated the patients, provided biological material and reviewed the manuscript. ZP conceived the hypothesis, designed the study, performed phosphoflow experiments, analyzed and visualized data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The study was funded by kind support from Jeffrey Model Foundation. We thank the patients and healthy volunteers for the blood samples used in this study. We confirm that this manuscript has not been published elsewhere and is not under consideration by another journal.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.


The development of safe and effective vaccines to respond to COVID-19 pandemic/endemic remains a priority. We developed a novel subunit protein-peptide COVID-19 vaccine candidate (UB-612) composed of: (i) receptor binding domain of SARS-CoV-2 spike protein fused to a modified single-chain human IgG1 Fc; (ii) five synthetic peptides incorporating conserved helper and cytotoxic T lymphocyte (Th/CTL) epitopes derived from SARS-CoV-2 structural proteins , and one universal Th peptide; (iii) aluminum phosphate as adjuvant. The immunogenicity and protective immunity induced by UB-612 vaccine were evaluated in four animal models: Sprague–Dawley rats, AAV-hACE2 transduced BALB/c mice, rhesus and cynomolgus macaques. UB-612 vaccine induced high levels of neutralizing antibody and T-cell responses, in all animals. The immune sera from vaccinated animals neutralized the SARS-CoV-2 original wild-type strains and multiple variants of concern, including Delta and Omicron. The vaccination significantly reduced viral loads, lung pathology scores, and disease progression after intranasal and intratracheal challenge with SARS-CoV-2 in mice, rhesus and cynomolgus macaques. UB-612 has been tested in primary regimens in Phase 1 and Phase 2 clinical studies and is currently being evaluated in a global pivotal Phase 3 clinical study as a single dose heterologous booster. The COVID-19 pandemic remains a significant concern driven by the continued emergence of variants of concern (VOCs) and the short duration of protection from natural infection or vaccination. The most recent VOC, Omicron B.1.1.529) and its sub-lineages  have quickly spread globally breaking through the immune protection generated by vaccines and/or natural infections . In resp29 and itMost authorized COVID-19 vaccines were designed to elicit binding and neutralizing antibodies (NAbs) using full-length spike (S) protein or receptor binding domain (RBD) as immunogen. The binding and NAb responses are strongly correlated with protection against SARS-CoV-2 infection, clinical manifestations, and disease progression in COVID-19 . The RBDIn UB-612 vaccine composition, a Chinese Hamster Ovary (CHO)-cell-produced RBD fused to a modified single-chain human IgG1 Fc (sFc) protein  serves as the immunogen to generate humoral responses. UB-612 vaccine also contains five promiscuous synthetic S2/M/N peptides composed of helper and cytotoxic T-cell (Th/CTL) epitopes derived from S2 subunit of S protein (3 peptides), membrane , and nucleocapsid  to induce cross-reactive cell-mediated immune responses against SARS-CoV-2; and one proprietary Th peptide, derived/modified from measles virus fusion protein to catalyze T-cell activation [This report evaluated the immunogenicity and protective efficacy of the UB-612 vaccine candidate against SARS-CoV-2 infection in four animal models, including rats, adeno-associated virus (AAV) transduced mice expressing human angiotensin-converting enzyme 2 (AAV/hACE2), rhesus and cynomolgus macaques.The UB-612 vaccine formulation  is comprised of RBD-sFc protein and five conserved Th/CTL S2/M/N peptides as immunogen. RBD-sFc protein was produced from a stable CHO cell line. To identify the desirable CTL T-cell epitopes in SARS-CoV-2 S, M and N proteins, we employed “Epitope Prediction and Analysis Tools” , 14. CpGTo prepare UB-612 vaccine for animal studies, RBD-sFc protein was first formulated with UBITh®1a Th peptide and adsorbed on Adju-Phos adjuvant (InvivoGen) at 1.6 μg/mL, before mixing with five S2/M/N T-cell peptides (4 μg/mL each) in CpG1 excipient (4 μg/mL). In UB-612 formulation, the weight:weight ratio of immunogens is 88% of RBD-sFc protein and 12% of T-cell peptides in each dose for rat , mouse and macaque immunization studies.nd immunization, mice were transduced by intratracheal (IT) inoculation with 3 × 1011 vector genomes (vg) of AAV6/hACE2. To transduce extrapulmonary organs, 1 × 1012 vg of AAV9/hACE2 was injected intraperitoneally [6 PFU (100 μL) of SARS-CoV-2 hCoV-19/Taiwan/4/2020 strain by intranasal (IN) inoculation. Mice were weighed daily for four days, and lung tissues were collected on Day 5 post-challenge for pathology evaluation. Mouse challenge experiments were performed in ABSL-3 facility. All mouse procedures were approved by institutional animal care and use committee (IACUC) at UBIAsia and Academia Sinica in Taiwan.The immunogenicity and protective immunity of UB-612 vaccine were evaluated in an AAV/hACE2 transduced BALB/c mouse model . Four grtoneally . Two weeWe conducted two non-human primate (NHP) immunogenicity and protection studies in rhesus macaques (3-dose) and cynomolgus macaques (2-dose), respectively.rd immunization) with 106 50% Tissue Culture Infectious Dose (TCID50) of SARS-CoV-2 WH-09/human/2020/CHN (WT) strain by IT at ABSL-3 facility. Virus loads in lungs were evaluated on Day 7 post-challenge using RT–PCR, expressed in viral RNA genome copies/gram of lung tissue. All procedures were approved by JOINN’s IACUC.In rhesus macaques study similar to a previously established COVID-19 NHP model , 19, found immunization (Day 55), animals were challenged by both intratracheal and intranasal routes with a total of 1.0 × 105 TCID50 of SARS-CoV-2 USA/WA1/2020 (WA) strain, which was ∼5-fold higher dose than that used in a similar cynomolgus macaque challenge study at BIOQUAL for the authorized COVID-vaccine NVX-CoV2373 [4 TCID50) and IT (5 × 104 TCID50) administrations. On Days 0, 3, 5, and 8 post-challenge, bronchoalveolar lavage (BAL) and nasal swabs were collected to assess viral shedding by subgenomic messenger RNA (sgmRNA) RT–PCR. All procedures were approved by Biomere and BIOQUAL IACUCs.Three groups (n = 5) of cynomolgus macaques  were immunized with 30 or 100 μg of UB-612 vaccine or saline on Days 0 and 28 at Biomere . The immunized animals were transferred to BIOQUAL’s ABSL-3 facility  for challenge study. Sera and peripheral blood mononuclear cell (PBMCs) were collected on Days 0, 14, 28 and 50 to evaluate RBD-specific IgG, NAb and cell-mediated immune responses. Four weeks after the 2RBD-specific IgG and RBD:hACE-2 binding inhibition antibody responses in mice, rats and macaques were evaluated by ELISA. Three types of SARS-CoV-2 neutralization assays were conducted to evaluate the NAb responses: live virus microneutralization, live virus cytopathic effect (CPE), and pseudovirus neutralization assays. Live virus microneutralization assays were performed using original SARS-CoV-2 WT and WA strains, Delta and Omicron BA.1 variants. The live virus CPE neutralization assays were performed using D614G, Alpha (B.1.1.7), Gamma (P.1), Beta (B.1.351), and Delta (B.1.617.v2) variants. Pseudovirus neutralizations were conducted using Vesicular stomatitis virus (VSV)-based pseudoviruses expressing S protein from WT strain, three VOCs: Alpha (B.1.1.7), Beta (B.1.351) and Gamma (P.1), two Variants of Interest (VOIs) Epsilon (B.1.429) and Iota (B.1.526). All live virus neutralization assays were performed in BSL-3 laboratory. The detailed methods are described in Supplemental Materials.nd immunization by T-cell ELISPOT and intracellular cytokine staining (ICS). PBMCs were cultured and stimulated in vitro with mixed S2/M/N peptides described previously [The S2/M/N peptide specific IFN-γ and IL-4 T-cell responses in cynomolgus macaques were evaluated in PBMCs collected at 3 weeks after the 2eviously  and in S50 in mouse lungs and N gene sgmRNA levels in BAL fluid and nasal swabs of macaques, post-challenge. The detailed virus TCID50 and sgmRNA RT–PCR detection methods are described in Supplemental Materials.The viral loads were detected as virus TCIDAt the end of mouse and macaque challenge studies, animals were euthanized, and lung tissues were collected, trimmed, processed, embedded, sectioned, and stained with Hematoxylin and Eosin. Histopathology was examined under microscope and the histopathology scores were determined by microscopic examination .P-values <0.05 were considered as significant differences.The results and analyses are descriptive. The data presented in the graphs are group geometric mean titers/values (GMT) with standard deviation (SD). An unpaired t-test or one-way ANOVA was performed for statistical analyses using GraphPad Prism. The UB-612 vaccine contains RBD-sFc and conserved T-cell epitopes derived from S2, M and N proteins  , 14. RBDSynthetic T-cell peptides in UB-612 include three S2 peptides , one N peptide , and one M peptide , containing Th and/or CTL epitopes with known MHC-I and/or MHC-II binding and good manufacturability characteristics . Each peThe UB-612 vaccine was first demonstrated that it could elicit high levels of RBD-specific IgG, neutralizing antibody and T-cell responses in rats .After two immunizations with UB-612 vaccine in mice, the RBD-specific IgG, RBD:hACE-2 binding blocking and neutralizing antibodies against a wild-type live virus hCoV-19/Taiwan/4/2020, were induced in all three dose groups  with a dose-dependent manner .Figure nd immunization, mice were transduced with a recombinant AAV/hACE2. Two weeks later, the transduced mice were intranasally challenged with 106 TCID50 of SARS-CoV-2 strain hCoV-19/Taiwan/4/2020  and started to wane six weeks post the 2nd immunization (Day 70) , the rhesus macaques were challenged by IT route with SARS-CoV-2 WT strain (106 TCID50). In UB-612 vaccine groups , viral RNA loads in lungs were significantly reduced compared to saline group , Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.v2) VOCs. However, the titers against Gamma (P.1) and Beta (B.1.351) variants were reduced  and then started to wane on Day 70 before the 3rd booster dose. After the 3rd immunization, NAb titers against all viruses were significantly increased surpassing the peak titers after the 2nd immunization.VSV-based-pseudoviruses were generated to express individual S protein from WT strain, three VOCs: Alpha (B.1.1.7), Beta (B.1.351) and Gamma (P.1), and two VOIs: Epsilon (B.1.429) and Iota (B.1.526), to evaluate the breadth of neutralizing activities. Macaque sera from the 100 µg dose group, collected on Days 42 and 70 (two- and six-weeks post-dose 2) and Day 77 (one-week post-dose 3), neutralized all six viruses tested. The 3Taken together, UB-612 vaccine induced broadly neutralizing antibodies against multiple SARS-CoV-2 variants in macaques.nd NHP study, cynomolgus macaques received 30 or 100 μg of UB-612 or saline on Days 0 and 28  variant. Two-dose UB-612 also elicited NAb responses against Delta (nd immunization (Day 50) by IFN-γ and IL-4 T-cell ELISpots and intracellular cytokine staining (ICS). IFN-γ secreting spots were detected in both 30 and 100 μg dose groups  and IN (4 × 104 TCID50) infections with SARS-CoV-2 WA strain at three weeks after the 2nd immunization (Day 55). After challenge, viral sgmRNA loads were measured in BAL . Cynomolgus macaques immunized with two doses of UB-612 demonstrated strong immune responses and were protected against SARS-CoV-2 IN/IT challenge. Our results are consistent with previous studies which have shown that RBD-Fc or RBD vaccines induce high levels of NAbs in NHPs. A yeast-expressed RBD-based vaccine formulated with 3M-052-alum significantly reduced viral loads in respiratory tracts and lung inflammation in rhesus macaques in a SARS-CoV-2 challenge study, with NAbs peaking after the 3cination .rd dose booster immunization with the RBD may improve neutralizing antibodies in nonhuman primates [rd dose UB-612 homologous booster generated high titers of RBD-specific IgG and potent NAb responses. Furthermore, UB-612 induced potent NAbs against multiple VOCs in live virus and pseudovirus neutralization assays, including Alpha, Beta, Gamma, Delta, and Omicron BA.1. Similarly, in our Phase 1 clinical study conducted in Taiwan, a booster dose of UB-612 vaccine administered 7–9 months after primary vaccination increased NAb levels by 131-, 61- and 49-fold against the ancestral SARS-CoV-2, Omicron BA.1, and BA.2 variants, respectively [An additional 3primates . The booectively . Based oectively  althoughUB-612 protein/peptide subunit vaccine has several key differentiators distinguishing it from the available COVID-19 vaccine options. (1) Longevity of NAbs: in clinical trials, UB-612 induced long lasting NAbs with a half-life of 187 days (post primary immunization), which was higher than, 40 days for an inactivated vaccine , 68 daysIn summary, UB-612 vaccine elicited high levels of neutralizing antibodies with breadth across multiple major SARS-CoV-2 variants including Alpha, Beta, Gamma, Delta and Omicron  and a ThClick here for additional data file.


These findings indicated that the autologous PBMC-based vaccine could induce functional STLV-1-specific CTLs in vivo.A small proportion of human T-cell leukemia virus type-1 (HTLV-1)-infected individuals develop adult T-cell leukemia/lymphoma, a chemotherapy-resistant lymphoproliferative disease with a poor prognosis. HTLV-1-specific cytotoxic T lymphocytes (CTLs), potential anti-tumor/virus effectors, are impaired in adult T-cell leukemia/lymphoma patients. Here, using Japanese monkeys naturally infected with simian T-cell leukemia/T-lymphotropic virus type-1 (STLV-1) as a model, we demonstrate that short-term-cultured autologous peripheral blood mononuclear cells (PBMCs) can serve as a therapeutic vaccine to activate such CTLs. In a screening test, STLV-1-specific CTL activity was detectable in 8/10 naturally STLV-1-infected monkeys. We conducted a vaccine study in the remaining two monkeys with impaired CTL responses. The short-term-cultured PBMCs of these monkeys spontaneously expressed viral antigens, in a similar way to PBMCs from human HTLV-1 carriers. The first monkey was subcutaneously inoculated with three-day-cultured and mitomycin C (MMC)-treated autologous PBMCs, and then boosted with MMC-treated autologous STLV-1-infected cell line cells. The second monkey was inoculated with autologous PBMC-vaccine alone twice. In addition, a third monkey that originally showed a weak STLV-1-specific CTL response was inoculated with similar autologous PBMC-vaccines. In all three vaccinated monkeys, marked activation of STLV-1-specific CTLs and a mild reduction in the STLV-1 proviral load were observed. Follow-up analyses on the two monkeys vaccinated with PBMCs alone indicated that STLV-1-specific CTL responses peaked at 3–4 months after vaccination, and then diminished but remained detectable for more than one year. The significant reduction in the proviral load and the control of viral expression were associated with CTL activation but also diminished 6 and 12 months after vaccination, respectively, suggesting the requirement for a booster. The vaccine-induced CTLs in these monkeys recognized epitopes in the STLV-1 Tax and/or Envelope proteins, and efficiently killed autologous STLV-1-infected cells  in vivo, where Tax is undetectable by serological means. These findings imply that HTLV-1 expression in vivo may be too scarce to restore the impaired CTLs but could be recognized by CTLs once they are activated. Notably, short-term culture of peripheral blood mononuclear cells (PBMCs) from HTLV-1-infected individuals induces viral antigens and can activate antigen-presenting cells. In the present study, using Japanese monkeys that were naturally infected with simian T-cell leukemia/T-lymphotropic virus type-1 (STLV-1) closely related to HTLV-1, we demonstrated that vaccination with short-term-cultured autologous PBMCs evoked functional STLV-1-specific CTL responses in the subgroup of monkeys that originally exhibited impaired STLV-1-specific CTL activities. These results imply that short-term-cultured autologous PBMCs could act as an anti-ATL vaccine, without being limited by HLA.Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL) in a small proportion of infected individuals. ATL patients exhibit impaired HTLV-1-specific cytotoxic T-lymphocyte (CTL) responses, even in the early stages of the disease. Activation of these CTLs may induce anti-ATL effects, as suggested by the favorable clinical outcomes in a recent clinical study of a Tax peptide-pulsed dendritic cell vaccine in ATL patients with limited HLA alleles. Paradoxically, HTLV-1 expression is extremely low  A small percentage of human T-cell leukemia virus type-1 (HTLV-1)-infected individuals develop adult T-cell leukemia/lymphoma (ATL), an aggressive lymphoproliferative disease with a poor prognosis owing to its rapid progression, frequent relapses, and immune suppression , 3. Howe+ HTLV-1 Tax-specific CTLs have anti-tumor potential . Bec. Becin v+ STLV-1-specific CTL responses in naturally STLV-1-infected non-human primates that share striking similarities with HTLV-1 infection.In the present study, we evaluated STLV-1-specific CTL responses in Japanese monkeys and found a few monkeys with impaired STLV-1-specific CTL responses. The impaired CTL responses in these monkeys were successfully restored by immunization with short-term-cultured autologous PBMCs. This is the first report to show that short-term-cultured autologous PBMCs can act as an effective vaccine to evoke CD8+ CTL activities that are restricted to MHC-I, we prepared IL-2-dependent STLV-1-infected T-cell lines (ILTs) from each monkey as positive control targets for a CTL assay. We also prepared STLV-1-negative herpesvirus papio-infected B-cell lines (LCL) from these monkeys as negative control targets. The cell surface phenotype and STLV-1 antigen expression of the cell lines established from three representative monkeys are shown in ˗CD8˗, whereas ILT-1640 and ILT-2312 were CD4+CD8˗. The LCLs were weakly positive for CD20, but not CD3  was heterogeneous. To evaluate CD8 not CD3 . STLV-1  not CD3 .#2312) clearly produced IFNγ following stimulation with autologous ILT cells compared with barely detectable levels of IFNγ following stimulation with LCL cells, indicating that these monkeys had functional STLV-1-specific CTLs. By contrast, monkey #1686 lacked an STLV-1-specific CTL response, as the PBMCs from this monkey did not respond to autologous ILT or LCL cells, whereas they clearly produced IFNγ when stimulated with phorbol myristate acetate (PMA) and ionomycin.To evaluate STLV-1-specific CTL responses, PBMCs from each monkey were pre-stimulated with formalin-fixed autologous ILT cells in culture for 2 weeks, and then interferon gamma (IFNγ) production in response to secondary stimulation with autologous ILT or LCL cells was evaluated by flow cytometry Figs  and S1. To examine Tax-specificity of the STLV-1-specific CTLs in these monkeys, CTLs were expanded in the PBMC culture from monkey #2312 in the presence of IL-2, and their tumor necrosis factor (TNF)-α-producing responses against autologous LCLs that had been pulsed with a series of synthetic oligopeptides corresponding to sTax were analyzed . The CTLWe further examined STLV-1-specific CTL responses in a total of 10 monkeys using autologous STLV-1-infected cells or LCL cells as targets. The results are summarized in We then analyzed the two monkeys showing impaired STLV-1-specific CTL responses for any differences with the other monkeys that had detectable CTL responses. The age, sex, and STLV-1 antibody titers of the tested monkeys are summarized in + T lymphocytes from the PBMCs samples resulted in vigorous p19 production even in the samples that had not released p19 in the presence of CD8+ cells  were mitomycin C (MMC)-treated and subcutaneously inoculated into the inguinal skin of monkey #1686. Two months after the initial vaccination, PBMCs from monkey #1686 showed mild proliferation during culture, but STLV-1-specific CTL activity was unclear (6), over 80% of which expressed STLV-1 antigen . We confirmed that STLV-1 expression could be induced in the PBMCs of monkey #2330 following short-term culture . MMC-treine dose , and itsine dose . In PBMCine dose . The spoine dose .Thus, the impaired STLV-1-specific CTL response in the naturally STLV-1-infected monkey #2330 could be restored by inoculation with short-term-cultured MMC-treated autologous PBMCs alone, leading to a transient reduction in the PVL and the control of viral expression in STLV-1-infected cells.+ cell-depleted PBMCs from monkey #2425 expressed slightly lower levels of STLV-1 antigens than monkey #2330 in vitro  and this level gradually decreased after the second vaccine dose, but remained at detectable levels for more than one year  could act as a vaccine to abrogate immune tolerance and/or newly evoke STLV-1-specific CTL responses; our results therefore highlight the strong immunogenic potential of STLV-1-expressing PBMCs.It has been suggested that HTLV-1-infected cells are immunogenic because Tax functions to promote DC maturation in vitro –50. Our  , in accordance with the recommendations of the Weatherall report, “The use of non-human primates in research”: http://www.acmedsci.ac.uk/more/news/the-use-of-non-human-primates-in-research/.Ten STLV-1-seropositive Japanese macaques   were used: CD3-Pacific Blue (SP34-2), CD4-FITC (L200), CD4-APC/H7 (L200), CD8-BV510 (SK1), HLA-DR-PerCP/Cy5.5 (L243), CD86-FITC (FUN-1), CCR4-PE (1G1), and CD20-APC/Cy7 (L27). For IFNγ cytokine flow cytometry, APC-conjugated mouse anti-human IFNγ mAb  and APC-conjugated mouse IgG1 antibody (MOPC-21) were purchased from BioLegend . Unconjugated GIN7 mAb , recogni+ cells using Dynabeads coated with anti-CD8 antibodies  and cultured for more than 3 months in the presence of 30 U/ml rhIL-2 . The ILTs were maintained in RPMI1640  containing 10% fetal bovine serum ) and 30 U/ml rhIL-2. In monkeys #1936 and #2290, ILTs could not be induced, and alternative autologous STLV-1-infected cell lines were established by co-culturing autologous CD8+ cell-depleted PBMCs with MMC-treated ILT-1686 cells.PBMCs were isolated by applying density gradient centrifugation using Ficoll-Paque PLUS  following the sedimentation of red blood cells in 1.5% dextran . To establish ILTs, the PBMCs were depleted of CD8+ cells using Dynabeads coated with anti-mouse antibodies, after incubation with anti-CD3 antibody (BD Biosciences), were forced infected with herpesvirus papio in the supernatant of the S594 cell line, which is a herpesvirus papio-producing baboon B-cell line . The LCLs were maintained in RPMI1640 with 10% FBS and antibiotics.To establish herpesvirus papio-infected LCLs, PBMCs that had been depleted of CD3Phenotypic analyses of ILTs and LCLs were performed by staining cells with CD3-Pacific Blue, CD4-FITC, CD8-BV510, and CD20-APC/Cy7 for 15 min at room temperature. The cells were then washed with phosphate-buffered saline (PBS) containing 1% FBS and fixed in PBS with 1% formaldehyde. To detect intracellular STLV-1 proteins, the PBMCs were permeabilized using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set . The permeabilized cells were incubated with mAbs to anti-HTLV-1 p19 (GIN7) , Tax (1A6 cells/ml) were pre-stimulated with formalin-fixed autologous ILTs (5×105 cells/ml) in the presence of 30 U/ml rhIL-2 for 13–14 days, and then 2×105 cells were re-stimulated with or without 1×105 autologous ILTs or LCLs in the presence of 10 μg/ml brefeldin A (Sigma Aldrich) for 6 h. These cells were first stained with CD86-FITC, CCR4-PE, HLA-DR-PerCP/Cy5.5, CD4-APC/H7, CD3-Pacific Blue, and CD8-BV510 and then permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences), followed by staining with APC-conjugated mouse anti-human IFNγ mAb or control mouse IgG.PBMCs (1×106 cells) using DNeasy Blood & Tissue kits . The following primer sets were used in this study: STLV pX2 (5′- TGG ATA CCC AGT CTA CGT ATT TGG AGA CTG T-3′) and STLV pX3 (5′-GAG CCG ATA ACG CGT CCA TCA ATG GGG TCC-3′) for STLV-1 pX, and Mf RAG1-F (5′-TCA AAG TCA TGG GCA GCT ATT GT-3′) and Mf RAG1-R (5′-AGG GAA TTC AAG ATG CTC AGA A-3′) for the RAG1 gene. The PCR assays were performed in 20-μl reaction mixtures consisting of 50 ng of genomic DNA, 2.5 mM MgCl2, 0.4 μM each pX primer, and 1× LightCycler FastStart DNA Master SYBR Green 1. Amplification for pX was performed with an initial denaturation step at 95°C for 10 min, followed by 45 cycles of denaturation for 1 s at 95°C, annealing for 10 s at 61°C, elongation for 10 s at 72°C, and denaturation of primer dimers for 2 s at 86°C. The thermal cycling conditions for the RAG1 gene consisted of an initial denaturation at 95°C for 10 min and 30 cycles of denaturation at 95°C for 1 s, annealing at 60°C for 10 s, and elongation at 72°C for 10 s. The PVL was calculated as: [(copy number of pX gene)/(copy number of RAG1 gene/2)] × 100. The PVL of a reference sample was measured along with the test samples to standardize the results among assays.The STLV-1 PVL was measured using LightCycler FastStart DNA Master SYBR Green 1  on a LightCycler 2.0 Instrument (Roche Diagnostic). Genomic DNA was extracted from PBMCs , according to the manufacturer’s instructions, and were quantified for STLV-1 tax and STLV-1 bZIP factor (SBZ) RNA expression by real-time PCR as previously described . GAPDH m5 cells/200 μl) were cultured in RPMI1640 containing 10% FBS and antibiotics for 4–7 days in the wells of a 96-well round-bottom tissue culture plate. To examine the effect of CD8+ cells on the PBMCs, PBMCs were divided into two groups before culture, and CD8+ cells were depleted from one group and resuspended in the same volume as before depletion. The supernatant was collected after culture, and the concentration of STLV-1 p19 protein was measured by HTLV p19 Antigen ELISA (ZeptoMetrix).PBMCs  were used as an immunogen for the second vaccination.Naturally STLV-1-infected Japanese monkeys #1686, #2330, and #2425 were used for the vaccine experiments. Approximately 1×10in vitro by stimulating PBMCs (1×106 cells/ml) from monkeys #2330 and #2425 with MMC- or formalin-treated autologous ILT-cells (5×105 cells/ml) in the presence of 30 U/ml rhIL-2 for the first two weeks, then further culturing the cells in the presence of 100 U/ml rhIL-2 with periodical stimulation with MMC-treated or fixed autologous ILT cells. These CTLs were subjected to functional analyses.STLV-1-specific CTLs induced after vaccination were expanded The amino acid sequence of STLV-1 Tax was determined based on the DNA sequence of the pX region of the STLV-1 proviral DNA from one of the ILT lines, and five amino acid-overlapping 15-mer synthetic peptides spanning the entire STLV-1 Tax protein (residues 1–354) were obtained through the custom service of Cosmo Bio Tokyo, Japan . SimilarTo identify the target epitope recognized by CTLs derived from monkeys, autologous LCL cells were incubated with 10 μM pooled or single peptides for 3 h, then further incubated with CTLs overnight. TNF-α concentrations in the supernatants were evaluated using BD Opt EIA Human TNF-α ELISA sets (BD Biosciences).To confirm the direct killing function of the CTLs induced by vaccination, target cells were labeled with 5 μM carboxylflorescein succinimidyl ester  for 15 min at room temperature, washed, and cultured alone or co-cultured with the same number of CTLs overnight, and then subjected to flow cytometry. As a control, target cells and CTLs were separately cultured overnight, and then mixed on ice just before flow cytometry. In some experiments, CFSE-labelled target cells that had been co-cultured with or without CTLs for 6 h were further stained with PerCP-Cyanine5.5-conjugated annexin V (BioLegend) to evaluate apoptosis, followed by flow cytometry.S1 TablePVL, proviral load. U.D., undetectable.(PDF)Click here for additional data file.S2 TableFive amino acid-overlapping 15-mer synthetic peptides spanning the entire STLV-1 Tax protein. Red font indicates amino acids that differ from HTLV-1 Tax (GenBank accession #M10085).(PDF)Click here for additional data file.S3 TableAmino acid sequences of HTLV-1 Env (Env) peptides (A) (GenBank accession #M37747) and STLV-1 Env (sEnv) peptides (B) (GenBank accession #LC490324) used. Red font in B indicates amino acids that differ from HTLV-1 Env.(PDF)Click here for additional data file.S4 TableFive amino acid-overlapping 15-mer synthetic peptides spanning the entire SBZ protein (GenBank accession #LC490324 and #LC490325).(PDF)Click here for additional data file.S1 Fig+ cell proportions were evaluated among the PBMC-#1640 fraction that was gated out from the co-cultured CD3loCD86hi ILT-1640 cells (A) and CD3˗HLA-DR+ LCL-1640 cells (B).The PBMCs isolated from monkey #1640 were cultured with formalin-fixed autologous STLV-1-infected cells (ILT-#1640) in the presence of rhIL-2 for 2 weeks, and then further cultured in the presence of brefeldin A for 6 h as they were  or after secondary stimulation with ILT-1640 cells (PBMC-#1640+ILT-1640), LCL-1640 cells (PBMC-#1640+LCL-1640), or phorbol myristate acetate (PMA) and ionomycin (PBMC-#1640+PMA+Ionomycin) as indicated. ILT-1640 and LCL-1640 were simultaneously cultured alone as controls. The resulting cells were first stained with monoclonal antibodies against CD3, CD4, CD8, CD86, and HLA-DR, and were then fixed and permeabilized for further staining with monoclonal antibodies against IFNγ or control mouse IgG. The intracellular IFNγ(PDF)Click here for additional data file.S2 FigA. PBMCs from the indicated STLV-1-infected monkeys were divided into two aliquots of the same volume, and then CD8+ cells were depleted from one aliquot (open bar) but not the other (closed bar). The cells were cultured for 4–7 days, and the p19 level in the supernatants was measured by ELISA and presented as the mean and SD of duplicate samples. B. The results in A were indicated as relative values (%) against CD8+ cell-depleted samples in each monkey.(PDF)Click here for additional data file.S3 FigA. The levels of stax and SBZ mRNA in the total (top) or CD8+ cell-depleted (bottom) PBMCs from monkeys #2330 (left) and # 2425 (right) were analyzed before and 2 and 4 days after culture by quantitative RT-PCR as described in the Methods. The relative values standardized against Si-2 are indicated as the mean and SD of triplicate samples. U.D., undetectable, N.T., not tested. B. Spontaneous STLV-1 antigen expression in the CD8+ cell-depleted PBMCs from monkeys #2330 (top) and #2425 (bottom) following in vitro culture for 3 days was analyzed by flow cytometry. Mouse IgG (control), or anti-HTLV-1 Tax, Env, or p19 monoclonal antibodies cross-reacting with STLV-1 antigens were used for intracellular staining.(PDF)Click here for additional data file.