Adoptive immunotherapy with virus-specific cytotoxic T cells (VSTs) has evolved over the last three decades as a strategy to rapidly restore virus-specific immunity to prevent or treat viral diseases after solid organ or allogeneic hematopoietic cell-transplantation . Since the early proof-of-principle studies demonstrating that seropositive donor-derived T cells, specific for the commonest pathogens post transplantation, namely cytomegalovirus or Epstein-Barr virus (EBV) and generated by time- and labor-intensive protocols, could effectively control viral infections, major breakthroughs have then streamlined the manufacturing process of pathogen-specific T cells (pSTs), broadened the breadth of target recognition to even include novel emerging pathogens and enabled off-the-shelf administration or pathogen-naive donor pST production. We herein review the journey of evolution of adoptive immunotherapy with nonengineered, natural pSTs against infections and virus-associated malignancies in the transplant setting and briefly touch upon recent achievements using pSTs outside this context. Prolonged immune deficiency and especially delayed T-cell reconstitution posttransplant plays an important role in the outcome of antiviral prophylaxis and treatment, affecting the transplant-related mortality, all-cause mortality, and even relapse.4 To address this major limitation, sophisticated antiviral approaches have been explored, including adoptive transfer of T cells. In the current review, we describe the preclinical and clinical achievements of adoptive immunotherapy with pathogen-specific T cells (pSTs) in the allo-HCT setting and discuss the challenges and striking developments toward broader application. We also summarize recent achievements using nonengineered and genetically engineered pSTs against infections and virus-associated malignancies, beyond the transplant setting.Opportunistic infections/reactivations remain the Achilles heel of allogeneic hematopoietic cell transplantation  significantly increasing morbidity and mortality among allograft recipients.5 Nevertheless, broader application of DLIs in this context has been limited mainly due to their low content in virus-specific T cells compared with the higher frequency of alloreactive T cells that can cause severe graft-versus-host-disease (GvHD).6 To circumvent such an unwanted event, the use of conditionally eliminated DLIs being genetically modified to express suicide genes has been explored providing promising results.7In 1994, 5 patients with monoclonal EBV-post-transplant lymphoproliferative disease (EBV-PTLD) achieved complete remission after the infusion of peripheral blood mononuclear cells (PBMCs) from their EBV-seropositive transplant donors, demonstrating that donor lymphocyte infusions (DLIs) are effective in restoring virus-specific immunity and controlling viral infection.8 In a multicenter study, Heslop et al. demonstrated high efficacy of EBV-CTLs as prophylaxis (0/101 patients developed PTLD) or treatment of PTLD (11/13 patients controlled PTLD) in allograft recipients and also persistence of functional CTLs for up to 9 years.9Later on, Rooney et al prepared and administered the first donor-derived, EBV-specific cytotoxic T-lymphocyte lines (EBV-CTLs), successfully preventing or controlling EBV reactivation in ten allograft recipients, without evidence of GvHD.10 major obstacles for timely delivery to a patient developing PTLD. Since then, several groups have explored the development of faster and less sophisticated approaches and the manufacturing process has been significantly shortened and simplified. Such approaches involve (i) direct selection of donor VSTs with multimers that select T cells directed against specific viral peptides in the context of a specific HLA class I molecule11or IFN-γ capture, where specific T cells are selected on the basis of IFN-γ secretion upon viral stimulation12 and (ii) ex vivo expansion of the endogenous antigen-specific T cells by specific T-cell stimulation with mixtures of overlapping peptides13  significantly increased cytotoxic activity against CMV in vitro. GvHD developed in 7 patients before the first infusion, in 3 patients during the T-cell therapy course or after the last dose, while 4 patients did not develop GvHD. Neither CMV viremia nor disease developed in any treated patient.15CMV-specific T-cell generation went also through various stages of protocol refinements. In the first proof-of-principle study by Riddell et al, virus-specific T-cell clones from a healthy donor generated ex vivo from autologous CMV-infected fibroblasts and adoptively transferred into an allo-HCT recipient, prevented CMV infection without causing GvHD.+CMV-specific T-cell clones using donor PBMCs stimulated with CMV lysate, which were infused in 25 haploidentical HCT recipients. Cellular therapy proved to be safe and efficient in accelerating the recovery of endogenous CD8+CMV-specific responses, clinically translating into fewer CMV reactivation episodes and lower antigenemia levels.16Also, Perruccio et al generated CD4+- CD8+-CMV-specific CTLs using donor PBMCs pulsed with CMV lysate. The cellular products were safely infused to 8 HCT recipients who presented ganciclovir-resistant CMV viremia. Significant viral load reduction was observed in 7 patients, whereas in the 2 patients with the highest viral loads, the antiviral effect was only transient. A second T-cell infusion in one of the 2 patients resulted in complete response.17 Similar results with excellent response rates were reported by other groups as well.18Alternatively, Einsele et al generated both CD4+ and CD8+ T cells were infused into 9 children with systemic AdV infection post-HCT. Five of 6 evaluable patients managed to successfully clear viremia. T-cell infusion was well tolerated in all patients, except one case with skin GvHD grade II.19 In another phase I/II multicenter study, Ip et al demonstrated safety and feasibility of the preemptive administration of AdV-specific T cells in high-risk pediatric patients after allo-HCT to control viremia. Eight children received the cellular products and all successfully controlled the infection, while 1 patient developed stage II GvHD 2 months postinfusion.20 Feucht et al showed that adoptive transfer of TH1 cells is a safe and effective alternative therapeutic approach for refractory AdV disease or viremia after HCT, resulting, in patients with antigen-specific T-cell responses (14/23), in 86% (12/14) complete remissions and 100% survival (versus 9.5% in nonresponders).21 Clinical trials and preclinical studies with pSTs are summarized in Tables Many studies targeted AdV, another common pathogen, especially in the pediatric HCT field. Feuchtinger et al isolated AdV-specific donor T cells based on IFN-γ secretion after in vitro antigenic stimulation. The cellular products which combined both CD4The high response rates, along with the low toxicity observed in numerous early phase clinical trials of adoptively transferring VSTs Table Candida albicans-specific T-cell products.105 Nevertheless, clinical use of pSTs against mold infections was at that time hampered, due to the complex, lengthy, labor-intense, and costly manufacturing procedures.107as prophylaxis or treatment against the main pathogens causing morbidity and mortality in immunocompromised hosts—namely CMV, EBV, and AdV—set the rationale for developing pSTs targeting less common, albeit well-recognized and potentially lethal for this particular population, opportunistic pathogens. In particular, since mid-2000s, manufacturing by ex vivo expansion or direct isolation, were separately employed or combined to enable the generation of AF- or BK polyomavirus (BKV),110 human herpesviruses [varicella zoster virus (VZV),111human herpesvirus 6 (HHV6), respiratory viruses [influenza,113human parainfluenza virus-3 (HPIV3),115human metapneumovirus (hMPV), herpes simplex virus type 1 (HSV-1),117human papillomavirus (HPV),120Zika virus,121norovirus,122 fungi such as AF, Aspergillus flavus, Aspergillus terreus, C. albicans, Candida krusei, Fusarium solani, Fusarium oxysporum, Rhizopus oryzae, Lomentospora prolificans126 and recently the more complex Mycobacteria127  molecules, have since then facilitated the production process and provided a springboard to extend the breadth of pathogens that can be targeted by T cells. Indeed, over the last years, the pST cell therapy platform has been simplified and adapted to an array of pathogens which complicate immune-deficient states, including viruses such as 16 reported that 9 of 10 haploidentical HCT patients treated with AF-specific T-cell clones survived invasive aspergillosis versus 7 of 13 individuals receiving conventional antifungal therapy. Other cases of transplanted patients treated with adoptive immunotherapy with donor-derived, monovalent virus-specific T cells (VSTs) against less common pathogens, included three patients who received in different studies, donor-derived JC-VSTs as treatment for the JC virus-associated progressive multifocal leukoencephalopathy (PML) and managed to clear JC achieving remarkable clinical improvement.65 In an another patient who was treated with IFN-γ capture-enriched BKV-STs from his HLA-haploidentical donor for drug-resistant BKV hemorrhagic cystitis, the treatment led to full resolution of symptoms and viremia.47 Those cases affirmed that the adoptive transfer of either isolated or ex vivo expanded T cells against less common pathogens, is safe and can restore pathogen-specific immune competence and control of opportunistic pathogen-associated diseases.In the transplant setting, Perrucio et alDespite the compelling results of single-targeting VSTs, transplanted patients usually experience reactivations from more than one opportunistic pathogens, thus making the generation of monovalent pSTs through multiple, time- labor-, and cost-intensive manufacturing processes, each time a new pathogen is being reactivated, rather impractical.51 By stimulating PBMCs with either a null adenoviral vector (Ad5f35null)50 or the Ad5f35pp65 vector encoding the CMV-pp65 antigen,51 and subsequently with autologous, EBV-transformed cell lines (EBV-LCLs) modified with the same vector(s) to artificially present adenoviral and/or (in the case of Ad5f35pp65) CMV-pp65 antigens, along with the constitutively expressed EBV antigens, demonstrated that the production of a single-cell product simultaneously targeting 2 or 3 viruses was feasible. Moreover, bi- or tri-VSTs presented clinically measurable antiviral activity after their administration to immunocompromised individuals, however, the long manufacturing procedure of 10 weeks limited wider applicability. By a similar approach, Hanley et al expanded tri-VSTs from virus-naive cord blood units (CBUs) using monocyte-derived dendritic cells (DCs) and Ad5f35pp65-transduced LCLs.139 The administration of tri-VSTs derived from a 20% fraction of the CBU to patients receiving a single CBU transplantation was safe and effective in preventing viral disease in the highest-risk patients.52 To simplify and accelerate the tri-VST production from seropositive donors, DCs were nucleofected with plasmids encoding CMV, EBV, and AdV antigens and used as antigen-presenting cells (APCs) for T-cell stimulation.53 This approach considerably shortened the manufacturing of tri-VSTs to 9–11 days.Leen et al pioneered the generation of bivalent and trivalent VSTs.54; (ii) PBMCs infected with an AdV vector-based antigen presentation platform141; (iii) DCs pulsed with overlapping peptide pools of immunogenic viral proteins143; and (iv) phytohemagglutinin (PHA) blasts and K562 cells genetically modified to express co-stimulatory molecules.142 Among the various multi-VST generation methods, DCs-based approaches have been clinically tested, providing substantial responses as prophylaxis/treatment against VZV,54 EBV, CMV, AdV, BKV, and HHV6.57Following the clinical success of tri-VSTs, expansion protocols for the production of tetravalent, pentavalent, and even heptavalent VSTs have been developed Table , employi107 Preclinical efforts, including ours, have been made towards the development of fungus-specific T-cell products,145 yet not clinically translated. To further unleash the potential of adoptive immunotherapy with antigen-specific T cells and enable the simultaneous treatment of multiple viral and fungal infections by an “all-in-one” single T-cell product, we and others, have developed refined strategies for manufacturing multipathogen-specific T-cell products (multi-pSTs). By either ex vivo expansion or magnetic isolation, T-cell products with specificity against a plethora of viruses and the main fungi affecting severely immunocompromised patients (AF and Candida)151 have been generated147 .Single- or multitargeting T-cell therapies against fungi have lagged well behind VSTs, mainly due to the complexity and length of the manufacturing procedures.C. albicans, despite being profoundly different fungal pathogens, may induce CD4+ memory Th1 cells cross-protective against both pathogens, probably through molecular mimicry; the epitope p41 of the AF’s cell wall glucanase Crf1 has high homology to Chr1 epitope of Candida albicans, both being recognized by the same T-cell receptor (TCR).153 We and others have shown that such cross-reactivity may extend also to other Aspergillus, Candida, Fusarium, and Mucorales species,123 an important feature that will further augment the efficacy of multi-pSTs, induce a rapid restoration of a “broad repertoire” immunity, broaden the target patient population and reduce the manufacturing costs.There is accumulating evidence that AF and Multi-pSTs is an individualized product that for the time being can only be manufactured in specialized centers. Refined manufacturing procedures require at least 15 days for release, making these products unavailable for urgent administration, as in cases where an infection rapidly evolves or/and the stem cell donor is not accessible. The establishment of “off-the shelf,” third-party banks can help overcome such issues. With this strategy, pSTs are manufactured in advance from seropositive, transplant-eligible healthy donors, then being readily available for urgent use in partially HLA-matched patients with active infections  had a complete response. The investigators found an association between the number of matches and patient outcome. TCR spectratyping for in vivo tracking of the infused VSTs showed either a gradual increase in the peak signal with each infusion or a maximum signal, 4 or 24 hours after each infusion. The cells were detected until 7 days postinfusion. These results were recapitulated by several other groups.73 In a most recent study by the Memorial Sloan Kettering group, 33 HCT- and 13 SOT-individuals with rituximab-refractory PTLD, received 3 weekly VST infusions, with minimum 2 HLA allele-matching, followed by a 3-week observational period. The infusions were well-tolerated, but additional cycles were required for response (68% and 54% in the HCT and SOT recipients). In contrast to the study by Haque et al, this study showed no correlation between HLA matching and response.68The third-party bank approach was first studied in solid organ transplant (SOT) recipients where donor blood is not generally available for pST manufacture. Haque et al83 The same group later reported the generation of a pentavalent product additionally targeting BKV and HHV6. The cells were administered to 38 HCT recipients with drug refractory CMV (n = 17), EBV (n = 2), AdV (n = 7), BKV (n = 16), and HHV6 (n = 3) infections. While VST products and patients matched at 1–7 HLA alleles, there were only 3 cases of grade-I, de novo GvHD. Ninety-two percent of patients responded to therapy, including patients with viral diseases; EBV-PTLD (n = 1), CMV colitis (n = 2), encephalitis (n = 1), AdV enteritis (n = 11), respiratory tract infection (n = 2), HHV6 encephalitis (n = 1), BKV-hemorrhagic cystitis (n = 14), and nephritis (n = 2). VST responses were confirmed to be of third-party donor origin in 11 cases and shown to persist in vivo for up to 12 weeks postinfusion.87The use of multitargeting, off-the-shelf VSTs was first investigated by the Baylor group to treat patients with persistent CMV (n = 19), EBV (n = 18), and AdV (n = 9) infections, reaching objective response rates 74%, 79%, and 67%, respectively.154To facilitate the establishment of third-party cell banks and make pSTs broadly available, investigators sought to use donors with HLA types found at high frequency in the respective population Figure . Given t66 In addition, using a scale-up manufacturing protocol, by starting with a unit of blood it was feasible to generate >2000 VST doses for infusion.More recently, the Baylor group generated a bank of CMV-STs from just 8 healthy donors. Donors were carefully selected based on an algorithm that compared 666 HCT recipient HLA types with the HLA types of a pool of diverse, healthy donors. This minibank was sufficient to provide VST matching at ≥2 HLA antigens to 28 of 29 patients referred.155 off-the-shelf cell products are attracting increased interest. Currently, ElevateBio and Allovir biotech companies have joined forces to explore the use of off-the-shelf multi-VSTs as prophylaxis or treatment of CMV, AdV, EBV, BKV or HHV6 viral infections post-HCT , as treatment of BKV infection post kidney transplant , as treatment of BKV-associated hemorrhagic cystitis post-HCT , or as treatment of respiratory viral infections from community acquired respiratory viruses  post-HCT (phase I/II NCT04933968).As adoptive therapy with either third-party VSTs or transplant donor VSTs seems to trigger comparable antiviral responses that might be mediated by restoration of endogenous virus-specific immunity,157 Gamma-delta T cells represent also an interesting treatment option for viral reactivations, yet overall understudied as a platform for antiviral immunotherapy.An attractive alternative cell source for adoptive immunotherapy, especially as third-party, off-the-shelf approach, are the unconventional γδ T cells, an early innate arm of the immune system and key players in the fight against viruses. Their unique property of major histocompatibility complex (MHC)-independent recognition of their targets allows effective antitumor and antiviral control without GvHD. Their potential has mainly been explored within the context of cancer treatment, using either unmanipulated or genetically engineered γδ T cells by the introduction of αβ TCRs, chimeric antigen receptors (CARs), and drug-resistance genes.161 First results of the safety and efficacy profile of CBU-derived tri-VSTs look promising.52 A second approach is the donor vaccination. The rationale lies in the establishment of specific immunity by vaccination of a seronegative donor and subsequent generation and adoptive transfer of pSTs to the transplant recipient in cases of refractory and persistent viremia posttransplant.162 CMV-pp65 specific CTLs ex vivo expanded from a vaccinated CMV-sero-negative stem cell donor (Towne strain CMV vaccine) were administered in a haploidentical HCT recipient with a refractory CMV disease; CMV DNA decreased after the CTL infusion while CMV-specific cytotoxicity increased.162 However, this strategy has the limitations of usually not timely identification before transplantation of the most appropriate donor to be vaccinated early enough to develop immunological memory, the lack of effective vaccines against the commonest pathogens posttransplant and the logistical, ethical, and medical concerns on donor vaccination for the benefit of the recipient.163 A third approach to overcome the “pathogen-naive” donor issue is to generate genetically engineered pSTs with a transgenic TCR to redirect patient’s (autologous) or seronegative donor’s T cells against a desirable pathogen175  by priming and repeated stimulations with APCs in the presence of various cytokines.212; and (iv) the absence of normal negative regulation, such as TCR downregulation in proportion to the strength of initial antigen recognition, stemming from the extrinsic promoter-driven expression of transgenic TCR.213 Orthotopic TCR replacement by a transgenic one may resolve some of the above issues and enable TCR-based therapies.214However, this approach entails several challenges, including (i) limited access to patients expressing specific HLAs; (ii) epitope restriction potentially increasing the risk of immune escape; (iii) risk for TCR mispairing between the endogenous and the transgenic TCR, mounting a potential harmful specificity190 EBV,191 hepatitis B virus (HBV),192 HCV,194 and AF188 have been described  have also been proposed against infections and infectious diseases. To date, few anti-infectious CARs against CMV,d Figure . These p222 This creates the paradox of precluding from the potential benefits of adoptive immunotherapy, the patients who are most likely to experience severe infections and are those with an unmet need for effective treatment of opportunistic infections.In patients receiving high-dose and long-term immunosuppression, such as HCT patients with GvHD or SOT patients, who are in fact, the most vulnerable to opportunistic infections, pSTs become functionally impaired in vivo.177 or calcineurin mutants to disrupt its docking to calcineurin inhibitors.179 The latter approach is being currently evaluated in an open-label, nonrandomized, multicenter phase I trial assessing the safety of tacrolimus-resistant autologous EBV-STs in SOT patients with PTLD (NCT03131934).To this end, several groups have been exploring mechanisms to genetically engineer pSTs and render them resistant to immunosuppressants Table . Two tea181 multitargeting VSTs,182 multi-pSTs,183 or tacrolimus-resistant single-targeting VSTs184  and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). We and others have generated either glucocorticoid-resistant single-targeting VSTs,224 Antigenic competition may favor the preferential outgrowth of high frequency VSTs (such as those directed against EBV and CMV antigens) over the low frequency antigen-specific T cells (such as those directed against AF and BKV), resulting in products dominated by responses to a single or a restricted repertoire of pathogens. Although in several reports, antigenic competition noticeably decreased the frequency of antigen-specific T cells in a multi-pST product as compared to single-targeting pSTs,225 other reports have shown that antigenic competition could be mitigated by the direct stimulation of PBMCs with pepmixes and culture conditions.57 Indeed, multivalent cell products simultaneously targeting a broad spectrum of pathogens (up to 7)58 could expand in vivo, in response to viral challenge and produce clinically beneficial effects against multiple pathogens  frequencies of subsets of antigen-specific T cells in the starting PBMC population.ns Table . However226 as well as in the real world.227 Therefore, prophylactic administration of pSTs for CMV reactivation prevention may not be necessary. However, mutations in the genes encoding for the CMV terminase complex may confer resistance of the virus to letermovir, and in this context, VSTs remain important in limiting drug-refractory CMV disease. Moreover, letermovir prophylaxis may be associated with impaired CMV-specific immunity.228 In patients who lack effective CMV-specific T-cell immunity, delayed-onset CMV infection has been shown to occur at higher frequency compared to patients with reactivation following letermovir229; thus, there may be room for pST therapy in those patients. As far as other dsDNA viruses are concerned , there are limited if any, antiviral drugs available. Current medical prophylaxis regimens are ineffective at preventing viral reactivation and often associated with toxicities. Therefore, the prophylactic administration of pSTs against these viruses in high-risk patients early post-HCT, remains rational. However, given as treatment, pSTs may be more cost-effective as some patients may not develop infections or acute GvHD requiring high-dose steroids may occur before infection, thus abrogating the pSTs potential.Recently, letermovir as primary prophylaxis to prevent clinically significant CMV infection in HCT seropositive patients has been associated with significant clinical benefit, leading to decreased clinically significant CMV reactivation both in a phase III trialThe compelling results on the management of viral complications posttransplant with adoptive immunotherapy has stimulated research with the use of pSTs in other immunocompromised patient populations, outside the context of transplantation.232 or genetically modified with a dominant-negative TGF-β receptor to become resistant to otherwise inhibitory concentrations of TGF-β in the tumor microenvironment,233 could be generated from patients with type 2 latency tumors. Upon administration, EBV-STs elicited objective clinical benefit in patients with Hodgkin- and non-Hodgkin lymphomas233 or nasopharyngeal carcinoma (NPC)237 without major toxicities.The impressive results of EBV-STs in type II latency tumors posttransplant, has attracted researchers’ interest to the evaluation of adoptive transfer of EBV-STs as treatment of less-immunogenic EBV-related malignancies, occurring outside the transplantation context. Despite the low immunogenicity of the targeted antigens, autologous EBV-STs, either nonengineered238 Preliminary results from a phase I study have reported sustained clinical improvement in 7 of 10 patients with MS treated with autologous EBV-STs, without experiencing any serious adverse event, including disease exacerbation.240 Therefore, an academic phase-I  and a commercial phase-I/II (Atara Biotherapeutics: product ATA188) clinical trial, assessing the safety and feasibility or/and efficacy of adoptive cell therapy with autologous or allogeneic EBV-STs in patients with a first clinical episode highly suggestive of MS or progressive MS, respectively, are currently running (NCT02912897 and NCT03283826).EBV-STs have also been proposed as an alternative cell-based approach for the management of multiple sclerosis (MS). Indeed, EBV has been very recently proposed as the leading cause of MS.242 Theoretically, in this patient population, pSTs could serve as a bridging therapy before HCT. So far, only 6 of 63 patients with PID treated with pST therapy, received the cell products before HCT.243 Notwithstanding the very small number of patients, this study provided the first evidence that individuals with PID could benefit, albeit at higher doses, from adoptive immunotherapy with pSTs, and proceed to transplant expeditiously.Patients with primary immunodeficiency disorders (PIDs), a group of congenital defects of immunity such as severe combined immunodeficiency (SCID), are susceptible to opportunistic infections frequently caused by common pathogens such as CMV, EBV, AdV, VZV, and respiratory viruses, even before HCT.175, and recently -E7 antigen,173, demonstrating robust tumor regression and objective clinical responses in 6 of 12 patients, including 4 of 8 patients with anti-PD-1 refractory disease in a first-in-human phase I trial in collaboration with Kite-Gilead. E7 TCR-T cells are currently being tested in the ph-II arm of that trial (NCT02858310) in patients with metastatic HPV-associated epithelial cancers. In an academic setting, the Baylor group is clinically investigating the safety and efficacy of patient-derived HPV-STs, which are being ex vivo expanded to target both the E6- and E7- antigens through their native TCR, in patients with relapsed HPV-associated cancers. HPV-STs are also genetically modified to become TGF-beta resistant and combined with the immune-checkpoint inhibitor Nivolumab, to reduce antigen escape-mediated relapses and T-cell exhaustion (NCT02379520).In a similar context, Hinrichs’s group developed autologous genetically engineered TCR-Ts directed against the HPV-E688 They later treated 2 more patients with autologous HLA B*3502- and HLA-A*0201-epitope-restricted MCPyV-STs.89 Notwithstanding the observed tumor lesion regressions, both patients relapsed, thus confirming the risk of tumor escape from single-epitope-targeting T cells. In the gene-engineered T-cell field, Bluebird bio and Fred Hutchinson Cancer Research Center are actively recruiting patients with metastatic or unresectable MCPyV-associated MCC to be treated with autologous A2-MCC1-expressing TCR-Ts, specific for an HLA-A02-restricted viral oncoprotein (NCT03747484).There are limited data on adoptive transfer of VSTs in Merkel cell carcinoma (MCC), an aggressive skin cancer, typically caused by the Merkel cell polyomavirus (MCPyV). Chapuis’s group first observed a complete response in 2 of 3 metastatic lesions of a 67-year-old man with metastatic MCPyV-expressing MCC who was treated with autologous ex vivo expanded, HLA-A*2402-epitope-restricted specific T cells (MCPyV-STs).+ HCC.170 A single infusion of only 104 per kg HBsAg-specific TCR-Ts led to a reduction of serum HBsAg levels by >90% within 30 days, without any detectable on/off-target toxicities. Importantly, the risk of inducing a severe liver injury due to on-target, off-tumor lysis of normal infected hepatocytes was minimized in this patient in whom HBsAg was exclusively produced by HCC extrahepatic metastasis and not by the transplanted liver; yet a possibility that it should always be taken into consideration. Lion TCR biotechnology company reported the first results of this approach on 8 patients who had not met the criteria for liver transplantation, showing a reduction or stabilization of circulating HBsAg and HBV DNA levels in most of the patients after HBV-TCR-T-cell infusion,172 while post-liver transplantation data are still pending (NCT02719782). Importantly, immunosuppressive drug-resistant HBV-TCR-Ts have been recently developed for immune therapy of HCC in liver transplant patients.185The proof-of-concept of using TCR-Ts redirected to target HBV antigen was assessed in a patient who developed hepatocellular carcinoma (HCC) relapse 10 years following liver transplantation for HBV244 As BKV and JC virus are closely related and share several immunogenic proteins, adoptive transfer of allogeneic JC-STs or BKV-STs has also been explored as treatment of PML.86 Preliminary, albeit very promising, results of those trials demonstrated safety of the infusion of partially matched VSTs as well as alleviation of clinical symptoms, progressive decline of viral load in cerebrospinal fluid and an unprecedented survival beyond 1 year in a number of PML patients with poor expectation for survival.86Progressive multifocal leukoencephalopathy (PML) is a devastating and generally fatal neurologic disease occurring in profoundly immunocompromised patients due to brain infection with JC polyomavirus and lacking effective treatment, unless cellular immunity is restored.245 Functional HIV-specific T cells (HIV-STs), both natural246 or genetically redirected,200 have been manufactured from HIV+ or HIV-seronegative donors  is successfully used to suppress HIV-1 replication, viral latency is responsible for the persistence of a reservoir of infected CD4+ T cells which lead to systemic viremia should ART is ceased, thus constituting major barrier to HIV cure.rs Table  and are as treatment against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated life-threatening coronavirus disease (COVID-19)138 (Table 247 Repeated off-the-shelf CoV-2-ST infusions (Allovir’s product ALVR109) also resulted in clinical and virologic improvement when administered to treat refractory severe COVID-19 pneumonia in a heart transplant patient.81 Our group assessed in a randomized 2:1 phase I/II trial, the safety and efficacy of the administration of ex vivo expanded, partially HLA-matched CoV-2-STs in addition to standard of care (SoC) in 57 patients with severe COVID-19 as compared to 30 patients who received SoC-only (control arm) (NCT05447013). The add-on treatment with CoV-2-STs was well tolerated and increased the likelihood of recovery by day 30, shortened the time to recovery and lowered by 51% the risk of mortality than SoC-alone, suggesting that the off-the-shelf immunotherapy with CoV-2-STs can serve as a safe and effective treatment in a real-world environment for severe COVID-19.79The urgency of the pandemic has prompted scientists from different fields  to address the problem. Leveraging the VST platform has been recently proposed 186 or tacrolimus187 have also been proposed , and the exhaustion due to sustained antigenic stimulation in vivo, represents the main reason for antigen-positive relapse and a major limitation.9 The remarkable difference in their lifespan mainly stems from the fact that pSTs are nonengineered cells “per se” and as “natural” pSTs, their transmitting signaling via native TCR involved in cognate antigen recognition and T-cell activation, remains unadulterated. In contrast, artificial CARs result in an abnormal or rewired TCR signaling253 as well as an unconstrained ligand-independent constitutive signaling, known as tonic signaling,254 being incriminated in the exhaustion, poor persistence or the toxicities associated with CAR-Ts255. Indeed, CD19-CAR-T cells with disrupted endogenous TCR, although similarly functional to TCR+/CAR-Ts, presented only limited persistence in vivo.256 Another important factor on the difference in persistence between the engineered and nonengineered, antigen-specific T-cell products, is the starting source of T cells; healthy donor- and quiescent memory pool-derived pSTs have a clear intrinsic survival advantage over CAR-Ts, which mainly derive from lymphopenic, usually heavily pretreated patients and contain higher frequencies of effector memory cells. Numerous studies have shown that T-cell products enriched for early lineage cells expand better and may delay CAR T-cell exhaustion, leading to improved persistence of the CAR-Ts.251Unlike CAR-Ts, pSTs have been shown to persist for up to 9 years.257  or without vaccination boosting (NCT05432635) or allogeneic dual TCR/CAR-specific T cells . In parallel, Atara Bio expects IND for off-the-shelf, allogeneic CD19-CAR EBV-STs (product A3219) in the last quarter of 2022. If proved safe and effective, this platform will improve longevity of remissions after CAR-T-cell therapy in the future.To overcome the significant hindrance of limited CAR-T function and persistence, the Baylor group proposed the introduction of CARs  into EBV- or tri-VSTs (either patient- or HCT donor-derived), as a platform to generate CAR-equipped pSTs259 or BIII as the second-line therapy for EBV-PTLD,260 implicating that such approaches, albeit a major breakthrough, have not yet become a standard treatment. This could be attributed to the fact that the majority of clinical trials conducted so far were early phase clinical trials, under academic grant funding, including in their majority limited number of patients. However, the progress made the last decade on simplifying the manufacturing process and speeding up the production along with the extended applications beyond the transplant setting, have attracted the interest of biotech and pharmaceutical companies and paved the way for late phase, controlled, prospective clinical trials studying the efficacy of adoptive immunotherapy. The results from Trace , the first, multinational, ph-III trial, sponsored by the European Commission, and other nonrandomized (NCT03394365) or randomized ph-III trials sponsored by Atara Biotherapeutics, Allovir  and Tessa Therapeutics (NCT02578641) are expected to generate strong clinical evidence on safety and efficacy of adoptive immunotherapy and enable the industrial investment and ultimately, regulatory approval. To facilitate academic medicine developers targeting an unmet medical need to optimize their development plans, European Medicines Agency (EMA) has recently launched the “PRIME” project, offering early and proactive support at improving clinical trial designs and enabling accelerated assessment of medicines application (https://www.ema.europa.eu/en/human-regulatory/research-development/prime-priority-medicines).The ability of adoptive immunotherapy to restore antiviral immunity and clear viral infections as a “living drug” platform, has been unequivocally shown and over the last three decades, the manufacture and clinical application of antigen-specific T cells has greatly evolved. The high and durable responses of pathogen-specific T cells, the broad coverage of antiviral spectrum, the good safety profile, the fast manufacturing, the increasing applicability due to third-party cell banks, have introduced a new era in the management of opportunistic infections. pSTs, eventually become part of the conventional antiviral treatment, falling under ECIL’s recommendations as regards EBV-PTLD and refractory CMV infection or disease post-HCT. Nevertheless, for cellular therapies, the strength of recommendation in combination with the level of evidence does not exceed level BIIu in refractory CMV disease261 Adoptive immunotherapy with pSTs may ultimately become a standard treatment for opportunistic infections post-HCT while serve as a platform for targeting novel emerging pathogens in the future.By the future incorporation of adoptive transfer of pSTs into clinical practice and acquisition of real world data, the adoptive transfer of pSTs may obtain a higher level of recommendation and evidence in the treatment’s guidelines. Moreover, patient risk stratification by monitoring the pathogen-specific immune reconstitution posttransplant by functional assays can guide the clinical management of patients at high-risk for morbidity and mortality from opportunistic reactivations and select the best candidates for pathogen-specific immunotherapy.Conceptualization, writing, review and editing, AP, MA, GK, IT and EY.The authors have no conflicts of interest to disclose.Funding for this project was provided by the T2EDK-02437 Research, Technology Development and Innovation (RTDI) State Aid Action “RESEARCH - CREATE -INNOVATE.”


Targeted radionuclide therapy (TRT) delivers cancer-selective radiopharmaceuticals to eradicate cancer cells while sparing healthy tissue. The recent development of combinatory treatments is a growing research field in nuclear medicine to enhance cancer cytotoxicity of TRT. Among promising combinatorial strategies, this review focuses on the rationale, efficacy, and safety of targeting the mammalian target of rapamycin complex 1 (mTORC1) to improve systemic radiation with radiolabeled ligands in cancer patients.Radioligand therapy (RLT) represents an effective strategy to treat malignancy by cancer-selective delivery of radioactivity following systemic application. Despite recent therapeutic successes, cancer radioresistance and insufficient delivery of the radioactive ligands, as well as cytotoxicity to healthy organs, significantly impairs clinical efficacy. To improve disease management while minimizing toxicity, in recent years, the combination of RLT with molecular targeted therapies against cancer signaling networks showed encouraging outcomes. Characterization of the key deregulated oncogenic signaling pathways revealed their convergence to activate the mammalian target of rapamycin (mTOR), in which signaling plays an essential role in the regulation of cancer growth and survival. Therapeutic interference with hyperactivated mTOR pathways was extensively studied and led to the development of mTOR inhibitors for clinical applications. In this review, we outline the regulation and oncogenic role of mTOR signaling, as well as recapitulate and discuss mTOR complex 1 (mTORC1) inhibition to improve the efficacy of RLT in cancer. External beam radiation therapy (EBRT) represents the standard-of-care treatment of locally confined tumors either alone or in combination with other treatment modalities. However, patients with disseminated metastatic disease are unlikely to benefit from this approach, and systemic therapy is required for effective treatment. Targeted radionuclide therapy (TRT), such as radioligand therapy (RLT), represents another treatment strategy using ionizing radiation for spread cancer lesions by cancer-selective delivery of the radio-pharmaceuticals following systemic application . RLT als177Lu]Lu-PP-F11N demonstrated tumor-specific accumulation with low adverse reactions in MTC patients Lu-RM2 in nude mice bearing prostate cancer PC-3 tumors Lu-PSMA-617 in androgen-dependent prostate cancer LNCaP cells [177Lu]Lu-PP-F11N in human A431/CCKBR cells [177Lu]Lu-PP-F11N in the A431/CCKBR xenograft nude mouse model. Importantly, the radioligand uptake in healthy organs including the stomach, which expresses CCKBR, and the kidney were not influenced by the everolimus treatment. Notably, in the same study, indirect mTORC1 inhibitor metformin significantly increased the uptake of [177Lu]Lu-PP-F11N in A431/CCKBR cells. However, in vivo validation showed only a marginal increase in tumor uptake, which was not statistically significant. These findings suggest that metformin is less potent in vivo, and further research is needed to provide evidence of the effectiveness of metformin treatment in combination with RLT. Nevertheless, mTORC1 inhibition by everolimus has the potential to substantially improve the response of CCKBR-positive cancers to RLT. Indeed, a subsequent study in the same animal model demonstrated significantly improved therapeutic efficacy and extended survival of the everolimus and [177Lu]Lu-PP-F11N-treated mice as compared with the monotherapies without significant adverse effects [177Lu]Lu-DOTATATE treatment did not produce benefit over the monotherapy, and the metastases were found both in the combination and everolimus-treated groups, suggesting that everolimus could promote malignancy [177Lu]Lu-DOTATATE in Levis rats did not indicate increased renal or hematological toxicity as compared with the monotherapies, prompting further characterization of the efficacy of this combination Lu-Octreotate [177Lu]Lu-DOTATATE in 41 patients with NETs [177Lu]Lu-DOTATATE was terminated due to toxicity in three patients and progression in another three patients [As a registered drug, everolimus is often used in clinics for the treatment of certain cancers. Early evaluation of the safety profile of everolimus in 24 patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs) showed that it was not influenced by the previous treatment with [treotate . Partialith NETs . Notablyith NETs . Furtherith NETs . The compatients . One patIn human cancers, hyperactivated mTORC1 signaling pathways support carcinogenesis and survival at multiple levels, as well as drive radiotherapy resistance. Thus, targeting mTORC1 represents a promising strategy for the development of combinatory treatments to improve the therapeutic efficacy of RLT. To efficiently deplete mTORC1 activity during RLT, the genetic background of the cancer patients should be taken into consideration before treatment. This will allow the exclusion of the potential non-responders with not only a low level of the targeted receptor but also with mTORC1 inhibitor-insensitive cancers. The rapalog resistance can be determined by assessing the overexpression or activation of the mTORC1 substrates, including S6K1 and 4EBP1 or regulated proteins, such as cyclin D1 and HIF-1α ,92,93. R


The impact of statins on COVID-19 remains unclear. This study aims to investigate whether statin exposure assessed both in the population and in well-defined cohorts of COVID-19 patients may affect the risk and severity of COVID-19 using nationwide Swedish population-based register data. A population ≥ 40 years was selected by age/sex-stratified random sampling from the Swedish population on 1 Jan 2020. COVID-19 outcomes were identified from the SmiNet database, the National Patient Register and/or Cause-of-Death Register and linked with the National Prescribed Drug Register and sociodemographic registers. Statin exposure was defined as any statin prescriptions in the year before index date. In Cox regressions, confounding was addressed using propensity score ATT (Average Treatment effect in the Treated) weighting. Of 572,695 individuals in the overall cohort, 22.3% had prior statin treatment. After ATT weighting, protective effects were observed among statin user for hospitalization and COVID-19 death in the overall cohort and onset cohort. In the hospitalized cohort, statin use was only associated with lower risk for death , but not ICU admission. Statin-treated individuals appear to have lower COVID-19 mortality than nonusers, whether assessed in the general population, from COVID-19 onset or from hospitalization. Various risk factors for severe forms of COVID-19 infection have been identified, including older age, male sex, hypertension, obesity and comorbidities such as cardiovascular disease, diabetes mellitus, and hypertension4. The 2019 ESC/EAS guidelines for managing dyslipidemia recommend the use of statins for patients with established cardiovascular disease to mitigate overall cardiovascular disease risk5. The main mechanism by which statins reduce cardiovascular disease risk is to lower the blood cholesterol level, primarily low-density lipoprotein (LDL), but there are also several important pleiotropic mechanisms such as anti-inflammatory effects, reduction of reactive oxygen species and platelet activity, and lipid regulatory effects that may be important in patients with cardiovascular disease6. Some of these may represent mechanisms for modulating the host response among COVID-19 patients. Statins could confer protective effects among COVID-19 patients by stabilizing and promoting regress of atherosclerotic plaques along with anti-inflammatory effects, inhibitor effects on leucocyte–endothelial interaction and inflammatory gene transcription7. Additional potential statin mechanisms for a beneficial effect on COVID-19 include plasma membrane effects. Since statins inhibit cholesterol biosynthesis, they could influence the organization of plasma membrane lipid rafts on SARS-CoV-2 target cells in a way that decreases virus adsorption and/or egress of virus particles from infected cells, analogously to the effect statins have on HIV infection8, thus reducing the severity of COVID-19 infection. A potential concern with statins and COVID-19 is that statins may increase the expression of ACE29—the membrane receptor that allows SARS-CoV-2 to gain entry into host cells, particularly in lung, heart, vasculature, brain, gastrointestinal tract and kidney—thus suggesting that statins could instead predispose to severe infection and adverse outcomes among COVID-19 patients10. On the other hand, ACE2 has both lung- and cardio-protective roles indicating that an increase in its expression may actually ameliorate the COVID-19 outcome12.The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has affected nearly 432 million people worldwide and has led to over 5.9 million confirmed COVID-19-related deaths7. Subsequently, more studies have been conducted, and meta-analyses performed18. Although recent meta-analyses have estimated both no pooled effect of statins7 and a protective effect13, based on different selections of studies, two carefully conducted meta-analyses noted that pooling of results from studies with adjustment for confounders showed more protective results , whereas pooling crude estimates did not16. Overall, many of the studies on statins and COVID-19 were small and with limited or no adjustment for confounders. A majority were based on convenience samples of COVID-19 patients, often hospitalized cohorts, with unclear selection mechanisms—especially those in the earlier phases of the pandemic. The timing of statin exposure was often not clearly assessed. Non-severe outcomes have more rarely been studied concerning statins, but alleviation of COVID-19 symptoms has been reported19. The effects of statins on outcomes in patients with COVID-19 thus remain to a large extent unclear14 and deficiencies in many retrospective studies such as differential handling of comorbidities and confounders and selection issues.To date, published studies show heterogeneous findings concerning statin use and COVID-19 disease. A meta-analysis based on a few early studies initially reported significant risk reduction for fatal or severe COVID-19 with statins The current study uses a large linked Swedish population-based register database to investigate whether statin exposure both in the general population and in well-defined cohorts of COVID-19 patients may affect multiple outcomes, including the risk, severity and mortality in the COVID-19 study, taking into account comorbidities and confounding factors.20. For the current study, a cohort study population including individuals aged 40 and above, free of COVID-19, was obtained by age-sex stratified random sampling of approximately 10% of the Swedish national population on 1 Jan 2020.This study used data from the SCIFI-PEARL (Swedish Covid-19 Investigation for Future Insights—a Population Epidemiology Approach using Register Linkage) project, described in detail elsewhereWe defined 3 study cohorts, with different index dates: (1) overall cohort of all study subjects, with index date 1 Jan 2020; (2) onset cohort of subjects with COVID-19 onset, with index date defined as 2 days prior to the earliest of first positive SARS-CoV-2 polymerase chain reaction (PCR) test results from SmiNet  or COVID-19 International Classification of Diseases, version 10, Swedish edition (ICD-10-SE) code (U07.1 or U07.2) in a healthcare encounter  in our data; (3) hospitalized cohort of all subjects hospitalized for COVID-19, with index date at the admission date. We obtained information on pre-index drug use from the National Prescribed Drug Register, sociodemographic data from Statistics Sweden and comorbidities from the National Patient Register. All information was linked to study subjects using unique personal identifiers.The main exposure was statin use (ATC code C10AA) in the year prior to index date with the unexposed group including those without any prescription for statin in this time period. In addition, in a sensitivity analysis we also identified statin user based on at least three filled prescriptions in the year before the index date. Prescriptions for chronic drug use are generally dispensed for 3-month periods  each time prescriptions are filled in Sweden. Individuals were grouped as those who regularly used (exposed) and those who did not regularly use statins (unexposed) according to this definition.COVID-19 test-positivity: Individuals with positive PCR test result for SARS-CoV-2 in SmiNet. Event date was the test sampling date.COVID-19 diagnosis: Individuals with healthcare encounter with COVID-19 ICD-10 code in the Patient Register or the Cause-of-Death register, or positive test result for SARS-CoV-2 in SmiNet. Event date was defined as the earliest of these.COVID-19 hospitalization: Individuals admitted to hospital based on primary or secondary diagnosis with COVID-19 ICD-10 code in the Patient Register. Event date was the date of hospital admission.COVID-19 ICU admission: Individuals who were transferred to or were directly admitted in the Intensive Care Unit (ICU) with COVID-19 diagnosis based on the Swedish Intensive Care Register. Event date was the date of ICU admission.COVID-19 mortality: Individuals identified from Cause-of-death register with COVID-19 ICD-10 code as the underlying or contributing cause of death. Event date was the date of death.Follow-up for each cohort started on the day after index date and extended for each subject to the earliest of outcome, death, emigration or end of follow-up on 20 Jan 2021. The outcomes investigated were:Descriptive statistics are presented as means with standard deviation for continuous variables and counts with percentage for categorical variables. We calculated standardized mean difference (SMD) to evaluate the balance between treated and untreated groups before and after propensity score weighting. The unadjusted number of events and event rates (per 1000 person-years) with 95% exact Poisson Confidence Intervals (CI) for COVID-19 endpoints were computed for statin users and non-users. The cumulative incidence of COVID-19 endpoints was estimated as one minus the Kaplan–Meier estimate of the survival function and are presented with 95% CIs.21. The propensity scores were estimated using gradient boosting (twang package in R) with shrinkage set to 0.01, interaction depth 322. The number of trees was optimized to give optimal weighted balance between the treatment groups as determined by minimizing the average weighted SMD across the variables in the propensity score model. The ATT weight-adjusted Cox proportional hazards regression model used robust standard errors to account for the weights. The models estimated HR with 95% CI. p values below 0.05 were considered statistically significant, with no adjustment for multiple comparisons. We used R software (version 4.0.2) for statistical analyses. We also stratified the analysis by sex and performed a sensitivity analysis using a different exposure definition, as described above.In the primary analyses, Cox regressions with statin exposure as the sole independent variable were used. We computed hazard ratios (HRs) unadjusted and adjusted using propensity scores for weighting to estimate the average treatment effect for the treated (ATT) using ATT weights, also known as standardized mortality ratio (SMR) weights, arrhythmia, heart failure, diabetes mellitus, chronic kidney disease, respiratory disease, cancer, liver disease, obesity, neurological disease, dementia, and autoimmune disease) and prior medications (including angiotension converting enzyme inhibitors (ACEI), angiotension receptor blockers (ARB), calcium channel blockers (CCB), diuretics, betablockers, ezetimib, novel oral anticoagulants (NOACs), platelet P2Y12 receptor inhibitors, metformin, sulfonylurea, insulin, glucosidase inhibitors, pioglitazone, dipeptidyl peptidase 4 (DPP-4) inhibitors, glucagon-like peptide 1 (GLP-1) receptor agonists, sodium-glucose co-transporter-2 (SGLT2) inhibitors, neuroleptics, and respiratory treatments), see 22.Covariates in the propensity score included age, sex, education, marital status, employment, disposable income, country of birth, comorbidities Ethical approval for this research was obtained from the Swedish Ethical Review Authority (EPM), no. 2020-01800, 2020-05829, 2021-00267, 2021-00829, 2021-02106, 2021-04098. All study procedures involving human participants followed the ethical standards of the institutional research committee, as well as the 1964 Helsinki Declaration and its amendments, or equivalent ethical standards. In Sweden, informed consent is generally not required for large-scale, registry-based studies. In this study, the Ethical Committee did not require the research team to collect informed consent from all individuals. Additionally, all linked information has been anonymized and de-identified by the National Board of Health and Welfare and Statistics Sweden, respectively, before reaching University of Gothenberg and the research team.Among 572,695 individuals aged ≥ 40 years in the overall population cohort, 22.3% n = 127,542) were statin users   Table.When we stratified the analyses by sex, after ATT weight adjustment, the hazard ratio for COVID-19 death in the overall population cohort was significantly reduced for both men  and women  with prior statin exposure Table  Table.30 and align with evidence summarized by recent meta-analyses and systematic reviews on the association of statin use and the potential protective effects against progression and severity of COVID-1932. A recent Swedish cohort study using register data with a smaller sample size limited to the Stockholm Region similarly showed statin use to be a protective factor for COVID-19 death29. That study had shorter follow-up than ours, but similar to our main exposure definition, defined statin exposure broadly as any statin treatment initiated before the pandemic, not regular statin use).This study investigated the impact of prior statin treatment on a range of five different COVID-19 outcomes in three different Swedish population-based cohorts—general population, individuals with COVID-19 onset and a COVID-19 hospitalization cohort. Our main findings show that prior statin treatment was significantly associated with a reduced risk of COVID-19 test-positivity, diagnosis, hospitalization and mortality in the 3 studied population cohorts. These findings strengthen and support evidence on the hypothesis of pleiotropic protective effects of statins in COVID-19 from prior cohort studies35. Statins have potential effects to reduce the cytokine release syndrome in COVID-19 by inhibiting Toll-like receptor 4 (TLR4) and down-modulating macrophage activity38. Statins have further been demonstrated to suppress the expression of both TLR2 and TLR4, leading to an immune response shift towards an anti-inflammatory response39.As noted previously, plausible biological and clinical mechanisms, both direct and indirect, by which statins might protect against COVID-19 disease or severity have been proposed40. Statins may well cause a reduction of syncytia by reducing plasma membrane cholesterol levels, thus reducing viral infection. Preliminary results indicate that statins indeed can reduce SARS-CoV-2 infection of lung epitheial cells .Beyond their cholesterol-lowering effect, statins could also affect the plasma membrane composition, where cholesterol is a dominating component. This effect is particularly important for enveloped viruses like SARS-CoV-2 that must pass the plasma membrane twice upon cell infection and again upon egress of newly synthesized virus particles. Recent studies have demonstrated that cholesterol is important in forming syncytia, multinucleated cells, that characterizes SARS-CoV-2 cell entry36 of potential relevance for infection and lung protection. Statins may diminish the complications of COVID-19 by improving endothelial function, reducing serum PAI-1 levels and attenuating TGF-and VEGF in lung tissue34. Furthermore, statins can constrain SARS-CoV-2 reproduction by restraining the main protease (Mpro) and RNA-dependent RNA polymerase (RdRp)34. Moreover, the effectiveness of statin treatment in significantly decreasing hospitalizations and deaths has been shown for influenza and Ebola virus diseases42.Statins also exert pleiotropic effects affecting inflammation and oxidative stress43. Although statin treatment is commonly considered safe and well-tolerated, statins may induce liver injury44. Current European guidance for the diagnoses and management of cardiovascular disease during the COVID-19 pandemic recommends temporarily restraining statin treatment in patients with high liver enzymes45. Future studies should preferably assess both positive and negative statin effects in COVID-19 patients.Alongside their potential benefits in COVID-19, the side effects of statin treatment need to be considered, such as elevated creatinine kinase (CK) and elevated serum glucose levels, which have been reported in severe COVID-19 patients50. Residual confounding may be an issue, and more carefully adjusted studies show more consistent protective results16. We have investigated individuals at three stages in the COVID-19 disease progression using 3 different cohorts: healthy before disease onset, from COVID-19 onset, and from COVID-19 hospitalization—with careful adjustment for confounding using propensity score analysis with ATT weighting. Protective effects of statins were seen for COVID-19 death for all three cohorts and in our overall population cohort and COVID-19 onset population also for the less severe outcomes , supporting a protective effect of statin on COVID-19 infection and severity. Despite some indications of an overall stronger protection in women than in men, we did not really observe consistent sex differences, which would have been consistent with the overall higher risk for severe COVID-19 outcomes seen in men during the pandemic51.Conflicting findings of the beneficial effects of prior statin treatment on COVID-19 have been reported in retrospective studies. Recent cohort studies have reported increased, decreased and unaffected risk for COVID-19 severity and mortality52, which may include considering discontinuation, especially in cases of severe illness and frailty. A hospital-based study in the US showed that the odds of mortality decreased by 35% in statin-users who continued to use statins upon hospital admission for COVID-19 compared with those for which the. statin therapy was discontinued53. The effect of discontinuation of statin treatment on the risk of COVID-19 in a population needs to be investigated in further detail.Despite current guidelines recommending continued statin treatment in patients who were previously taking these drugs because of the known effects of statins on ACE2, statin treatment may be discontinued in patients with COVID-19. In addition, guidelines suggest an individualized approach in deciding prescribing statins to older peopleOur study has several strengths and limitations that merit discussion. Using a population-based database in Sweden, we investigated the impact of statin use both in the population and in well-defined cohorts of COVID-19 patients for several different clinical COVID-19 outcomes. The completeness of data from nationwide administrative registries  and efficient linkages of different register data for COVID-19 patients to identify test-positive disease, clinical diagnoses and severe COVID-19 outcomes  are fundamental strengths of this study and support generalization of the results. In this study, we outlined statin exposure in the prior year into two different ways. Our main exposure definition was any statin exposure in the prior year, consistent with definitions commonly used in many studies. As a sensitivity analysis, we also used a more specific exposure definition, regular statin use, defined as ≥ 3  prescriptions in the prior year, to better capture individuals with likely ongoing regular statin use at index date, recognizing the variable adherence to actual daily statin intake in real-life data. Our sensitivity analysis with this definition gave very similar results, suggesting that the main exposure definition has adequate specificity for the hypothesized true exposure. We assume that short-term mechanisms of current use at the index would most likely be relevant for the observed effects we report. However, the study also has some limitations. Some exposure misclassification is possible, both of statin exposed and unexposed individuals, but this is unlikely to be related to the outcomes , and potential bias would thus tend to be towards the null. Unless the exposure misclassification is substantial, and the effect in the misclassified individuals is highly different, such bias would also have limited impact. Misclassification errors in identifying COVID-19 outcomes are also possible, especially under ascertainment of test-positive milder disease, but it is unlikely this differs between statin-treated and untreated. For the more severe outcomes, this issue is likely of less concern. Although propensity score weighting was used to minimize potential confounding, we cannot exclude some remaining bias from patient selection, treatment indication and residual confounding due to unmeasured confounders such as health risk behaviour  and laboratory parameters (dyslipidemia). Nevertheless, the exposure cohorts were quite well balanced after propensity score weighting, and the results were consistent across the studied cohorts and follow-up periods from different index time points.Findings from this study provide broad evidence of a beneficial effect of prior statin exposure on COVID-19 mortality in all the three cohort populations studied , as well as less strong effects on other COVID-19 outcomes along the COVID-19 progression spectrum of outcomes that we studied. Together with prior available scientific evidence, both mechanistic and epidemiological, our findings support potential investigation into repurposing of statins for COVID-19 protection.Supplementary Information.


This review highlights the multifunctional properties of perlecan (HSPG2) and its potential roles in repair biology. Perlecan is ubiquitous, occurring in vascular, cartilaginous, adipose, lymphoreticular, bone and bone marrow stroma and in neural tissues. Perlecan has roles in angiogenesis, tissue development and extracellular matrix stabilization in mature weight bearing and tensional tissues. Perlecan contributes to mechanosensory properties in cartilage through pericellular interactions with fibrillin-1, type IV, V, VI and XI collagen and elastin. Perlecan domain I - FGF, PDGF, VEGF and BMP interactions promote embryonic cellular proliferation, differentiation, and tissue development. Perlecan domain II, an LDLR-like domain interacts with lipids, Wnt and Hedgehog morphogens. Perlecan domain III binds FGF-7 and 18 and has roles in the secretion of perlecan. Perlecan domain IV, an immunoglobulin repeat domain, has cell attachment and matrix stabilizing properties. Perlecan domain V promotes tissue repair through interactions with VEGF, VEGF-R2 and α2β1 integrin. Perlecan domain-V LG1-LG2 and LG3 fragments antagonize these interactions. Perlecan domain V promotes reconstitution of the blood brain barrier damaged by ischemic stroke and is neurogenic and neuroprotective. Perlecan-VEGF-VEGFR2, perlecan-FGF-2 and perlecan-PDGF interactions promote angiogenesis and wound healing. Perlecan domain I, III and V interactions with platelet factor-4 and megakaryocyte and platelet inhibitory receptor promote adhesion of cells to implants and scaffolds in vascular repair. Perlecan localizes acetylcholinesterase in the neuromuscular junction and is of functional significance in neuromuscular control. Perlecan mutation leads to Schwartz-Jampel Syndrome, functional impairment of the biomechanical properties of the intervertebral disc, variable levels of chondroplasia and myotonia. A greater understanding of the functional working of the neuromuscular junction may be insightful in therapeutic approaches in the treatment of neuromuscular disorders. Tissue engineering of salivary glands has been undertaken using bioactive peptides (TWSKV) derived from perlecan domain IV. Perlecan TWSKV peptide induces differentiation of salivary gland cells into self-assembling acini-like structures that express salivary gland biomarkers and secrete α-amylase. Perlecan also promotes chondroprogenitor stem cell maturation and development of pluripotent migratory stem cell lineages, which participate in diarthrodial joint formation, and early cartilage development. Recent studies have also shown that perlecan is prominently expressed during repair of adult human articular cartilage. Perlecan also has roles in endochondral ossification and bone development. Perlecan domain I hydrogels been used in tissue engineering to establish heparin binding growth factor gradients that promote cell migration and cartilage repair. Perlecan domain I collagen I fibril scaffolds have also been used as an FGF-2 delivery system for tissue repair. With the availability of recombinant perlecan domains, the development of other tissue repair strategies should emerge in the near future. Perlecan co-localization with vascular elastin in the intima, acts as a blood shear-flow endothelial sensor that regulates blood volume and pressure and has a similar role to perlecan in canalicular fluid, regulating bone development and remodeling. This complements perlecan’s roles in growth plate cartilage and in endochondral ossification to form the appendicular and axial skeleton. Perlecan is thus a ubiquitous, multifunctional, and pleomorphic molecule of considerable biological importance. A greater understanding of its diverse biological roles and functional repertoires during tissue development, growth and disease will yield valuable insights into how this impressive proteoglycan could be utilized successfully in repair biology. This review highlights the interactive properties of perlecan (HSPG2) and its potential applications in repair biology. Perlecan is often referred to as a large heparan sulfate proteoglycan (HS-PG) but exists as a HS/chondroitin sulfate (CS) hybrid form in most tissues, endothelial cells however synthesize a monosubstituted HS glycoform . KeratinPerlecan colocalizes with elastin in blood vessels and this contributes to their visco-elastic properties in vasculogenesis during establishment of early vascular networks and tissue development  and is aPerlecan is a modular multifunctional PG with five distinct domains. Domain I is unique to perlecan and its GAG chains bind fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) promoting cellular proliferation, differentiation and tissue development  Figure . PerlecaDrosophila (domain V homologue unc-52) DNA sequences in bacterial and mammalian expression systems  and four EGF-like repeats . These L systems . Human d systems , or huma biology .Endogenously produced perlecan domain V released by MMPs from full-length perlecan also promotes tissue repair through angiogenic interactions with VEGF, VEGF-R2, α2β1 integrin, ECM-1 and progranulin . LG1-LG2Perlecan is a large multi-domain ECM HSPG detectable in four cell blastocysts during embryogenesis, its expression changes spatiotemporally during implantation of the embryo and in the placentation stage of embryonic development . Perlecain vitro. The HS side chains of perlecan have critical roles to play in the vessel wall, as an interleukin (IL)-2 receptor for vascular SMCs . These are predominantly avascular tissues devoid of a basement membrane, however the PCM of chondrocytes has been suggested to represent an intrinsic basement membrane around each cell . Atomic Perlecan has critical roles in basement membrane in the blood brain barrier and important roles in NMJ assembly and function , 6. PerlHSPG2 gene resulting in skeletal dysplasia and neuromuscular hyperactivity   and Dyssactivity . In thisactivity . DDSH  mice die just after birth mainly due to respiratory failure  mice were developed that express the perlecan transgene only in cartilage using the Col2a1promoter and enhancer  mice, perlecan is only expressed in cartilage but not the synovium. This results in less development of osteophytic spurs in the tibial and femoral joint margins , including upregulation of FRNK  and down regulation of FAK signalling .HS’s interactions with FGF-2, PDGF, VEGF, HGF, BMP2, GM-CSF, angiopoietin-3, and activin A illustrate the potential of perlecan domain I as a co-receptor for growth factor delivery and receptor activation. The ITIM megakaryocyte-platelet receptor (G6b-B-R) is an additional ligand for the HS chains of perlecan domain I . G6b-B-RPerlecan attached to endothelial cells in the lumen of blood vessels acts as a shear flow sensor that interacts with Ca2+ or Na+ regulating charge density at the endothelial cell surface. This also regulates membrane polarization in endothelial cells and SMCs, ion transport regulates vasoconstriction and relaxation in blood vessels  and is cin situ has been shown to improve wound healing through the re-mobilization of sequestered growth factors locally at sites of tissue repair , laminin-111 , and fibronectin (RGDSP) have been incorporated into HA scaffolds with RGDSP and TWSKV peptide HA scaffolds significantly accelerating cell proliferation . Perlecain vivo . FGF-10 in vivo .Once damaged, articular cartilage has a notoriously poor ability to repair itself . PerlecaT which is anchored to the synaptic basal lamina by non-fibrillar collagen Q (ColQ) (The duration of synaptic transmission of impulses at the NMJ is chiefly controlled by acetylcholinesterase (AChE), an enzyme which hydrolyzes acetylcholine (ACh) to choline and acetate . PerlecaQ (ColQ) . ColQ, aQ (ColQ) .in vitro and in pre-clinical models of the NMJ  and endovascular clot removal (thrombectomy) treatments. Whilst these primary treatments remove the underlying cause of the stroke, they do little to reverse or prevent subsequent brain injury. Perlecan domain V is potentially one such therapy whose efficacy has been experimentally demonstrated in both young and aged rodent models of ischemic stroke, where it has been reported to have neuroprotective, neuroreparative and functionally restorative effects . These bvia the downstream activation of PI3K-Akt, MEK-ERK and HIF-1α  model that induces mild cortical lesion  is essentially dependent on how quickly the first dose of perlecan domain V is administered after ischemic stroke induction. A recent report by Nakamura et al.  found mi3 Similarly, 2 hours of oxygen-glucose deprivation (OGD) in astrocytes and neuron cultures substantially increases LG3 levels. LG3 is anti-apoptotic as it decreases Caspase 3 after OGD-reperfusion  that could be potentially harnessed in repair biology. Emerging improvements in implant design, therapeutic blood brain barrier repair strategies following stroke, modeling of artificial neural stem cell niches and neuromuscular synaptic interface tissues may find application in the treatment of neurodegenerative disorders . Acetylc


To estimate prior severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among skilled nursing facility (SNF) staff in the state of Georgia and to identify risk factors for seropositivity as of fall 2020.Baseline survey and seroprevalence of the ongoing longitudinal Coronavirus 2019 (COVID-19) Prevention in Nursing Homes study.The study included 14 SNFs in the state of Georgia.In total, 792 SNF staff employed or contracted with participating SNFs were included in this study. The analysis included 749 participants with SARS-CoV-2 serostatus results who provided age, sex, and complete survey information.We estimated unadjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) for potential risk factors and SARS-CoV-2 serostatus. We estimated adjusted ORs using a logistic regression model including age, sex, community case rate, SNF resident infection rate, working at other facilities, and job role.Staff working in high-infection SNFs were twice as likely  to be seropositive as those in low-infection SNFs. Certified nursing assistants and nurses were 3 times more likely to be seropositive than administrative, pharmacy, or nonresident care staff: unadjusted OR, 2.93  and unadjusted OR, 3.08 . Logistic regression yielded similar adjusted ORs.Working at high-infection SNFs was a risk factor for SARS-CoV-2 seropositivity. Even after accounting for resident infections, certified nursing assistants and nurses had a 3-fold higher risk of SARS-CoV-2 seropositivity than nonclinical staff. This knowledge can guide prioritized implementation of safer ways for caregivers to provide necessary care to SNF residents. SNF residents are at extremely high risk for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) transmission from close contact with staff during activities of daily living (ADL) assistance, rehabilitative care, and respiratory care. Additionally, residents suffer from high morbidity and mortality related to COVID-19 because they tend to be older and have more chronic medical conditions than the general population.Skilled nursing facilities (SNFs), commonly referred to as nursing homes, are settings at high risk for outbreaks of respiratory diseases like coronavirus 2019 (COVID-19). Nevertheless, outbreaks continued to occur in SNFs, some of which may have resulted from inadvertent SARS-CoV-2 transmission via nursing home staff. Both residents and staff may have transmitted and acquired SARS-CoV-2 from each other. SNFs may have amplified SARS-CoV-2 transmission between May 25 and November 22, 2020; the 572,135 COVID-19 cases reported by SNFs during this period accounted for a large proportion of total infections.To limit the introduction and transmission of SARS-CoV-2 in SNFs and protect vulnerable residents, on March 13, 2020, the Centers for Medicare and Medicaid Services (CMS) recommended restricting resident visitation outside of end-of-life situations. Resident-care SNF staff have different types and intensities of contact with residents  compared to hospital staff contacts with patients. Previous work identifying risk factors for hospital staff  may not apply to SNF staff. Risks to nonresident care SNF staff are also uncertain. With widespread SARS-CoV-2 transmission, reliance on self-reported symptomatic disease is insufficient for evaluation of specific risk factors; two-thirds of asymptomatic SNF staff may be SARS-CoV-2 seropositive after outbreaks in SNFs where they work. Our objectives were to estimate prior SARS-CoV-2 infection among SNF staff at 14 nursing homes in the state of Georgia and to identify risk factors for seropositivity as of fall 2020.Considering that SNF staff may both initiate SNF transmission chains and also acquire infections at SNFs, identifying risk factors for SNF staff infection would support targeted infection control and prevention efforts. Currently, evidence for risk factors is scarce. Job role may be important, with some evidence of increased risk for nursing assistants and social workers.1 online). All facilities provided postacute and long-term care services. No facilities cared for ventilator-dependent residents. All facilities followed CMS-required visitor restrictions for the duration of recruitment and data collection, which were part of the ongoing longitudinal COVID-19 Prevention in Nursing Homes (COPING) study. Participants were recruited from August 25 to November 22, 2020 for the first study visit, which was the focus of analysis. This study was evaluated and approved by the Emory University IRB (#00000900). All participants provided consent.We recruited participants from 14 SNFs selected from 54 facilities affiliated with 4 healthcare systems in Georgia , COVID-19 symptom history, workplace masking and social distancing, and occupational activities . After completing the survey and under study staff supervision, SNF staff used lancets to self-collect dried blood spot samples for SARS-CoV-2 serology testing. Staff provided sex, age, and their residential ZIP code upon sample collection. Race and ethnicity were not available for this analysis. Sample cards air dried for 15 minutes before packaging, storage, and shipment to the testing laboratory  within 2 days of acquisition. A qualitative enzyme-linked immunosorbent assay  was used to test samples for the presence of anti–SARS-CoV-2 spike protein immunoglobulin G. to provide a population denominator. To assess facility-based exposure to SARS-CoV-2, we used data on confirmed SNF resident COVID-19 cases reported to CMS. The documented number of total resident cases from the first week of reporting and new cases from the first week of reporting until 2 weeks before the serology test was used to approximate facility infection rates. The first week of reporting was either May 24 or May 31, 2020, for each participating facility. Facility-specific bed sizes from CMS data provided a denominator to account for differences in facility size and to calculate facility resident COVID-19 cases per 100 beds. For both potential SARS-CoV-2 exposures, the 2-week window before serology testing accounted for the typical minimum lag between infection and seroconversion.To assess community-based exposure to SARS-CoV-2, we used confirmed COVID-19 case data from the Georgia Department of Public Health. The number of documented COVID-19 cases occurring in a participant’s residential ZIP code tabulation area (ZCTA) from March 1, 2020, to 2 weeks before the serology test was used to approximate staff’s cumulative community exposure to SARS-CoV-2 (termed community case rates). ZCTA-specific population estimates were obtained from the 2019 American Community SurveyParticipant seropositivity by facility and frequency of COVID-19 symptoms experienced by seropositive participants were calculated. To examine risk factors for staff seropositivity, we a priori identified age, sex, job role, community exposure (known contact with community cases and cumulative community infection rates), facility case rates [dichotomized into high-burden  and low-burden among SNF residents based on the approximate sample median], known contact with cases in facilities, and working at multiple facilities as risk factors to consider. Age was categorized into 4 groups: <40, 40–49, 50–59, and ≥60 years. Staff responses were mapped to 6 job categories to compare types of contact with residents: (1) healthcare administration, pharmacy, and other nonresident care; (2) resident activities, environmental services, and food services; (3) social work and physical, occupational, respiratory, and speech therapy; (4) certified nursing assistants; (5) nurses ; and (6) physicians and advanced practice providers. We estimated unadjusted odds ratios (ORs) and 95% confidence interval (95% CIs) for these and other potential risk factors and SARS-CoV-2 serostatus.We used logistic regression to estimate adjusted ORs for seropositivity, including all a priori identified variables, shifts worked monthly (≤10 or 10+), proportion of work time directly caring for residents (≤50% or >50%), and a facility-level random intercept (fully adjusted model). We could not examine interactions between job role and facility resident COVID-19 burden in the model due to insufficient sample size. We determined that age, sex, job role, community cases, and facility resident cases were established risk factors to be retained in the model. The other variables were assessed for potential removal and retained if >10% change in odds ratios (ORs) from the fully adjusted ORs occurred.1 online). Cumulative facility resident infection rates ranged from 0.4 to 106.7 infections per 100 beds . Cumulative community case rates ranged from 0.94 to 13.10 cases per 100 ZCTA population .Of 2,053 eligible SNF staff, we enrolled 792 staff (38.6%) and included 749 in this analysis; 23.5% were SARS-CoV-2 seropositive. Seropositivity by facility varied from 5.8% to 48.0% Table . Only 47Unadjusted odds ratios for SARS-CoV-2 seropositivity . Although smaller, our study captured a variety of geographic and temporal diversity in community and facility infections.Our findings of heightened risk for certified nursing and nurses are supported by similar findings from a recent serosurvey of 1,500 SNF staff in Rhode Island.This study had several limitations. We used self-reported data on exposures, which introduces possible recall error into our risk factor assessment, and we do not have data on race or ethnicity. Duties for the same job title may vary by facility, and we cannot ascertain the specific activities of participants who reported working in multiple facilities, although only 11% of SNF staff reported such work. We also cannot determine where SNF staff acquired infection . Asymptomatic and presymtomatic transmission may facilitate unrecognized transmission and contacts in both the community and the workplace; individuals may be less vigilant with infection prevention measures in the absence of respiratory symptoms. Regardless, it seems clear that early in this pandemic, SNFs provide conditions to amplify SARS-CoV-2 transmission beyond what is observed in the community. In this study, we addressed the lack of evidence for risk factors for SNF staff SARS-CoV-2 seropositivity, and our findings provide insight into differences between acute-care and long-term care risk factors.Overall, US-based SNF staff and residents have been severely affected by the COVID-19 pandemic. Our study helps define and quantify the risks to SNF staff. The consistency between our work and the earlier Rhode Island study suggests that the work activities of certified nursing assistants and nurses put these personnel at higher risk for SARS-CoV-2 infection early in the pandemic. We did not find community infections to be a significant risk factor; however, we are limited in capturing heterogeneity in individual-level community-derived risk. Given their elevated risks for infection, certified nursing assistants and nurses may have needed better implementation of recommended infection prevention measures in their existing workflows given the types of interactions required to serve SNF residents and interact with other staff . Further study is urgently needed to determine and implement safer ways to provide necessary care to senior populations in SNFs and avoid transmission of respiratory viruses.


Ovarian cancers rank first in both aggressiveness and dismal prognosis among gynecological neoplasms. The poor outcome is explained by the fact that most patients present with late-stage disease and progress through the first line of treatment. Ovarian neoplasms, especially epithelial ovarian cancers, are diagnosed at advanced/metastatic stages, often with a high angiogenesis index, one of the hallmarks of ovarian cancers with rapid progression and poor outcome as resistance to anti-angiogenic therapy develops. Despite therapy, the metastatic progression of aggressive ovarian cancer is a spectacularly selective function of tumor cells aided and abetted by the immune, mesenchymal and angiogenic components of the tumor microenvironment (TME) that enforces several pro-metastatic event(s) via direct and indirect interactions with stromal immune cells, cancer-associated fibroblasts (CAFs), and vascular endothelial cells. Since transdifferentiation of tumor endothelium is one of the major sources of CAFs, we hypothesized that ovarian CAF plays a critical role in resisting anti-angiogenic effects via direct crosstalk with endothelium and hence plays a direct role in the development of resistance to anti-angiogenic drugs. To test the hypothesis, we set up a hybrid ex vivo model for co-culture comprising Patient-Derived ex vivo primary CAFs from ovarian tumor samples and human umbilical vein endothelial cells (HUVEC). Patient-Derived CAFs were characterized by the mRNA and protein expression of positive , negative , functional  and immunological markers  associated with CAFs by qRT-PCR, flow cytometry, Western blot, and ICC. Data from our HUVEC-on-CAF ex vivo Hybrid Co-Culture (HyCC) study demonstrate the pro-angiogenic effect of Patient-Derived ovarian CAFs by virtue of their ability to resist the effect of anti-angiogenic drugs, thereby aiding the development of resistance to anti-angiogenic drugs. Ascertaining direct experimental proof of the role of CAFs in developing resistance to specific anti-angiogenic drugs will provide an opportunity to investigate new drugs for counteracting CAF resistance and "normalizing/re-educating" TME in aggressive ovarian cancers. Our data provide a unique experimental tool for the personalized testing of anti-angiogenic drugs, positively predicting the development of future resistance to anti-angiogenic drugs well before it is clinically encountered in patients. Angiogenesis, the formation of new vessels from pre-existing vasculature, is one of the hallmarks of ovarian neoplasms and a phenotype essential for the progression of the disease ,2. DurinOvarian neoplasms account for a fifth of cancer deaths in women, causing more deaths than any other cancer of the reproductive system (American Cancer Society statistics 2022). According to the estimates of the American Cancer Society for ovarian cancers for 2022, 1) close to 20K women will get newly diagnosed, and (2) over 12K patients will die from ovarian cancer.  With 75% close toCombination vascular targeted therapy is a recognized promising approach for patients with platinum-resistant recurrent ovarian cancers . Three cCAFs comprise the bulk of TME and share the most versatility in origin and function, making CAF clinically and therapeutically relevant ,25,26,27Considering the role of CAFs in tumor angiogenesis and the development of resistance to anti-angiogenic drugs in ovarian neoplasms, we hypothesized that ovarian CAF plays a critical role in resisting anti-angiogenic effects via direct crosstalk with endothelium. Our HyCC model of Patient-Derived ex vivo primary CAFs and HUVEC endothelial cells tested our hypothesis to prove that CAFs impart resistance to anti-angiogenic drugs. For the first time, we proved the ability of Patient-Derived primary CAFs to resist lenvatinib’s effect. Our HyCC model can be used to evaluate the crosstalk of Patient-Derived CAFs with endothelium during the course of developing resistance to anti-angiogenic drugs in ovarian neoplasms well before it is encountered in clinics. All experimental protocols were approved by the institutional and/or licensing committee(s). The informed consent(s) (IRB approved: Protocol Number Study: 2017.053-100399_ExVivo001) was obtained from 27 subjects and/or their legal guardian(s). Tissue samples were collected in designated collection media during the time of surgery in accordance with the guidelines of the pathology department in accordance with relevant guidelines and regulations. All methods were carried out in accordance with relevant guidelines and regulations. We included samples from consecutive consented patients with ovarian tumors at any stage/grade of the disease undergoing surgery with or without pre-treatment/history of any previous carcinoma. Both tumor and tumor-adjacent normal tissue were obtained as provided by the pathologist, depending upon the availability of the tissue on a case-to-case basis.Human uterine fibroblasts  and HUVEC cells were procured from ATCC (cat # PCS-100-013) and were cultured according to the standard cell culture procedures as per ATCC. Other cell lines for qRT-PCR were procured from ATCC. Matrigel was procured from BD biosciences, USA. Fluorescence vital stains, DiI, and DiO were bought from Molecular Probes, USA. Lenvatinib was procured from Selleckchem, PA, USA. Fibroblasts Antibody (TE-7) and recombinant Anti-S100A4 antibodies were procured from NOVUS and Abcam, respectively. PD-L1 [Clone 22C3] was bought from Agilent-Dako. PD-L2 (D7U8C) and CD31 were procured from Cell Signaling, USA. Cytokeratin 8 and 18 (B22.1 and B23.1), Ep-CAM/Epithelial Specific Antigen (Ber-EP4), Actin, Smooth Muscle (1A4), and Vimentin (SP20) were bought from Cell Marque, USA. Primers for qRT-PCR  of primary CAF are presented below:F: CGT TAC TAC TGC TGA GCG TGAR: GCC CAT CAG GCA ACT CGT AAACTA-2/SMAF: ATT GCA GTG GTT ATC ATC GGA GTGR: CTG GTT GTT GGA GTT CAG AAG TGGCD31F: AGC ACT TCA GGA GGT TAC ATC TR: CTT GCC TCT TGG TTG CTG TCTCD44F: CTT CAG TGG TCC ATT TGG TGR: CCC TTT GTT CTC GGC TTC CAGCD45F: GAA GGT CCT CTA CTT ATC CGC C R: TGA TGC CCT CAC ACT TGA CCA G CD90/THY1F: AGC GAG TGA GAA CCT ACT GGA R: CGC GTT GTG ATC TCC TTC TGA EpCAMF: GGA AGT GCC TGT TCC AGC AAT G R: TGT CTG CCA GTC TTC CCT GAA G FAP-AF: TCA AGG CTG AGA ACG GGA AG R: CGC CCC ACT TGA TTT TGG AG GAPDHF: TGG CAG TAC CCC ATG TCT GAA R: CCA AGA CCG TCA CAA AAA GGC PDGFRAF: ACC TAC TGG CAT TTG CTG AAC G R: ATA GAC AAT TAG TGC AGC CAG GT PD-L1F: CAG AAC TAA AGG AGC TGC TGA CC R: CTT GGA AGT CCA CCT CGT TGT C S100A4Tumor samples were collected at the time of surgery/biopsy as a part of a protocol approved by the institutional and/or licensing committee. We included tumor samples from patients with ovarian neoplasms at any stage/grade of the disease undergoing surgery/biopsy with or without pre-treatment/history of any previous carcinoma. We did not include any bone-marrow transplant patients or patients with liquid tumors. We tested the expression of Ki67, cl-caspase3 (cl-c3), and cl-PARP in representative tumor samples from patients with ovarian cancers by dual IHC stain for Ki67  and cl-c3  as well as cl-PARP . The morphological features are presented by H&E stain. The expression of markers for angiogenic blood vessels (CD31) and fibroblast/CAFs  was tested by IHC along with the expression of proliferation (Ki67), apoptotic markers (cl-PARP and cl-c3) in the tumor sample at day zero. The expression of PD-L1, PD-L2, and PD-1 was also tested both in the tumor and TME components. The cord formation assay was performed, as reported earlier . In shorhttps://ccrod.cancer.gov/confluence/display/ROB2/Home) (access on 9 August 2022) ) at 4 h and 18 h ]) ]) B,C. Intehttps://ccrod.cancer.gov/confluence/display/ROB2/Home [(access o on 9 August 2022)]) (https://ccrod.cancer.gov/confluence/display/ROB2/Home [(accessed on 9tAugust 2022)]) ( 2022)]) B,C. No c 2022)]) A, irresp 2022)]) B,C. InteSince the presence of CAF caused the cord formation of HUVEC cells on gel and protected it from the anti-angiogenic effect of lenvatinib both in sequential and simultaneous HyCC, we tested the effect of CAF in the initiation of cord formation independent of matrigel. Sequential HyCC of HUVEC cells on CAF (cultures “on-plate”) was performed in the presence of lenvatinib . To findIn a progressing angiogenic tumor, CAFs are directly involved in the formation of new vessels in various solid tumors. Literature showed that CAFs actively promote tumor angiogenesis by pro-angiogenic signals via secretomic soluble signaling factors, including VEGF, stromal-derived factor1 (SDF-1), YAP, and HIF-1alpha pathways (See ). It hasA tumor is known to be one of the sources of CAFs in many solid tumors ,40. OvarActivated CAFs are known to express various positive markers, including SMA, FAP-A, and platelet-derived growth factor receptor-A . PDGFR aThe induction of CAFs in a progressing tumor is contributed by tumor cells which in turn supports tumorigenesis, progressive disease, and drug resistance in response to treatment . CAFs arTumor vessels are known to be distinct from endothelial cell-lined vessels because tumor cells integrate into the endothelium or even mimic and replace it in vasculogenic mimicry vessels ,27,63. CTargeting “fibroblast addiction” in primary and metastatic tumors has been long recognized as a novel therapeutic target for drug discovery . With thNCT03797326, NCT04781088, NCT04519151, NCT02788708, and NCT05114421) [Lenvatinib, an oral multi-targeted TKI of VEGFR1-3, FGFR, PDGFR-β, RET, and KIT, is the representative TKI clinically targeted in ovarian cancers and has been approved by the FDA in combination with pembrolizumab for microsatellite stable recurrent endometrial cancer in 2019  with a n5114421) . In this5114421) .Using a novel HyCC model, we identified that Patient-Derived ovarian CAFs could initiate an immediate angiogenic response in HUVEC cells and specifically resist the effect of the anti-angiogenic drug lenvatinib. Our HyCC model of Patient-Derived ex vivo primary CAFs and HUVEC endothelial cells proved that ovarian CAFs impart resistance to lenvatinib. Here, our data provide the first direct experimental evidence to explain the mechanistic role of ovarian CAF in the development of resistance to anti-angiogenic drugs. The ex vivo HyCC model can potentially evaluate the crosstalk of Patient-Derived endothelium towards developing resistance to anti-angiogenic drugs in ovarian cancers. Our data present a unique experimental tool for personalized testing of anti-angiogenesis drugs in predicting the development of future resistance to anti-angiogenesis drugs, within 3 months of surgery, well before it is clinically encountered in patients. The data in support of CAF-driven resistance to anti-angiogenic drugs are still coming, and it has only just begun.