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CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and β-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.

The COVID-19 pandemic is a global health emergency, and the development of a successful vaccine will ultimately be required to prevent the continued spread and seasonal recurrence of this disease within the human population. However, very little is known about either the quality of the adaptive immune response or the viral Ag targets that will be necessary to prevent the spread of the infection. In this study, we generated recombinant Vaccinia virus expressing the full-length spike protein from SARS-CoV-2 (VacV-S) to evaluate the cellular and humoral immune response mounted against this viral Ag in mice. Both CD8+ and CD4+ T cells specific to the SARS-CoV-2 spike protein underwent robust expansion, contraction, and persisted for at least 40 d following a single immunization with VacV-S. Vaccination also caused the rapid emergence of spike-specific IgG-neutralizing Abs. Interestingly, both the cellular and humoral immune responses strongly targeted the S1 domain of spike following VacV-S immunization. Notably, immunization with VacV-expressing spike conjugated to the MHC class II invariant chain, a strategy previously reported by us and others to enhance the immunogenicity of antigenic peptides, did not promote stronger spike-specific T cell or Ab responses in vivo. Overall, these findings demonstrate that an immunization approach using VacV or attenuated versions of VacV expressing the native, full-length SARS-CoV-2 spike protein could be used for further vaccine development to prevent the spread of COVID-19.

CD8(+) T cells are key mediators of antiviral and antitumor immunity. The isolation and study of antigen-specific CD8(+) T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, due to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of antigen-specific CD8(+) T cells, particularly well-suited for samples with limited primary cells. Cells are stimulated ex vivo with antigen of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence from the paired TCR alpha and beta chains from these antigen-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8(+) T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using Cytomegalovirus-specific CD8(+) T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel antigens and characterize antigen-specific CD8(+) T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.

Rhesus cytomegalovirus (RhCMV) strain 68-1 vectored vaccines elicit MHC-II-and MHC-E-, but not classical, MHC-Ia-restricted CD8+ T cell responses. This is due to the presence of a viral MHC-E ligand encoded by Rh67 (UL40 in HCMV) and the combined deletion or inactivation of Rh157.5/.4 (UL128/UL130 in HCMV) and Rh158–Rh161 (members of the UL146 family of CXC chemokines). Importantly, the ability to elicit MHC-E-restricted CD8+ T cell responses correlates with the efficacy of RhCMV-vectored vaccines to control and clear infection by highly virulent simian immunodeficiency virus (SIV). To investigate whether HCMV encodes additional modulators of unconventional CD8+ T-cell priming that are not conserved in non-human primate CMVs, we inserted non-conserved HCMV genes into 68-1 and monitored the induction of the unconventional CD8+ T cells. This screen revealed that the UL18 protein of HCMV prevented the induction of MHC-II and MHC-E restricted CD8+ T cells resulting in a classical, MHC-Ia-targeted T cell response. UL18 is a MHC-Ia homolog and associates with cellular β2 microglobulin and peptides. However, unlike host MHC-Ia, this trimeric complex does not engage with the T cell receptor, but binds with high affinity to the inhibitory receptor LIR-1 widely expressed on leukocytes. Indeed, 68-1 recombinants expressing UL18 mutants lacking LIR-1-binding no longer interfered with the induction of MHC-II and MHC-E restricted CD8+ T cells. Thus, HCMV UL18 appears to prevent unconventional T cell priming by binding to LIR-1. In the event that this mechanism is conserved in the human host, UL18 could interfere with HCMV/HIV vaccine efficacy as the ability to elicit MHC-E-restricted CD8+ T cells is likely required for protection against HIV.

A critical question facing the field of transplantation is how to control effector T cell (Teff) activation while preserving regulatory T cell (Treg) function. Standard calcineurin inhibitor-based strategies can partially control Teffs, but breakthrough activation still occurs, and these agents are antagonistic to Treg function. Conversely, mechanistic target of rapamycin (mTOR) inhibition with sirolimus is more Treg-compatible but is inadequate to fully control Teff activation. In contrast, blockade of OX40L signaling has the capacity to partially control Teff activation despite maintaining Treg function. We used the nonhuman primate graft-versus-host disease (GVHD) model to probe the efficacy of combinatorial immunomodulation with sirolimus and the OX40L-blocking antibody KY1005. Our results demonstrate significant biologic activity of KY1005 alone (prolonging median GVHD-free survival from 8 to 19.5 days), as well as marked, synergistic control of GVHD with KY1005 + sirolimus (median survival time, >100 days; P < 0.01 compared to all other regimens), which was associated with potent control of both TH/TC1 (T helper cell 1/cytotoxic T cell 1) and TH/TC17 activation. Combined administration also maintained Treg reconstitution [resulting in an enhanced Treg/Teff ratio (40% over baseline) in the KY1005/sirolimus cohort compared to a 2.9-fold decrease in the unprophylaxed GVHD cohort]. This unique immunologic signature resulted in transplant recipients that were able to control GVHD for the length of analysis and to down-regulate donor/recipient alloreactivity despite maintaining anti-third-party responses. These data indicate that combined OX40L blockade and sirolimus represents a promising strategy to induce immune balance after transplant and is an important candidate regimen for clinical translation.

One of the central challenges of transplantation is the development of alloreactivity despite the use of multiagent immunoprophylaxis. Effective control of this immune suppression–resistant T-cell activation represents one of the key unmet needs in the fields of both solid-organ and hematopoietic stem cell transplant (HCT). To address this unmet need, we have used a highly translational nonhuman primate (NHP) model to interrogate the transcriptional signature of T cells during breakthrough acute graft-versus-host disease (GVHD) that occurs in the setting of clinically relevant immune suppression and compared this to the hyperacute GVHD, which develops in unprophylaxed or suboptimally prophylaxed transplant recipients. Our results demonstrate the complex character of the alloreactivity that develops during ongoing immunoprophylaxis and identify 3 key transcriptional hallmarks of breakthrough acute GVHD that are not observed in hyperacute GVHD: (1) T-cell persistence rather than proliferation, (2) evidence for highly inflammatory transcriptional programming, and (3) skewing toward a T helper (Th)/T cytotoxic (Tc)17 transcriptional program. Importantly, the gene coexpression profiles from human HCT recipients who developed GVHD while on immunosuppressive prophylactic agents recapitulated the patterns observed in NHP, and demonstrated an evolution toward a more inflammatory signature as time posttransplant progressed. These results strongly implicate the evolution of both inflammatory and interleukin 17–based immune pathogenesis in GVHD, and provide the first map of this evolving process in primates in the setting of clinically relevant immunomodulation. This map represents a novel transcriptomic resource for further systems-based efforts to study the breakthrough alloresponse that occurs posttransplant despite immunoprophylaxis and to develop evidence-based strategies for effective treatment of this disease.

Background : The Berlin patient is thus far the only individual considered cured of HIV. Three aspects of the Berlin Patient's treatment are thought to have contributed to his cure following allogeneic hematopoietic stem cell transplantation (HCT): 1) the myeloablative conditioning regimen, 2) transplantation with HIV-resistant cells, and 3) graft-versus-host disease (GVHD). This cure occurred in the context of HCT from an unrelated donor whose cells contained two copies of the CCR5delta32 mutation, which rendered them resistant to CCR5-tropic viruses (the vast majority of the transmitted variants). He also developed GVHD, which is thought to have contributed to his cure by inducing a graft-versus-viral-reservoir (GVVR) effect. While it is still unknown which of these factors was critical to the cure achieved in this patient, the viral rebound observed following allogeneic transplantation with wild-type cells in two Boston patients suggests that HIV-resistance factors may be key to achieving a permanent HIV cure through HCT. Our lab has previously shown that transplantation with autologous unmodified hematopoietic stem cells is not sufficient to eradicate the viral reservoir in a non-human primate (NHP) model of infection. However, the relative contributions of myeloablative conditioning, GVVR, and HIV resistant cells to the clearance of the HIV reservoir have not been rigorously determined. To dissect the impact of each of these factors on the viral reservoir, we have developed the first NHP model of allogeneic bone marrow transplantation (BMT) in Simian/Human Immunodeficiency Virus (SHIV)-infected, combined antiretroviral therapy (cART)-treated rhesus macaques. Methods : We intravenously infected 6 animals with SHIV-1157ipd3N4 and left them untreated for six months, followed by six months of cART (PMPA, FTC, Raltegravir). 3 animals served as untransplanted controls, and 3 animals received haplo-identical BMT following myeloablative total body irradiation (1020 Gy) without cART discontinuation. Donor chimerism was monitored by molecular analysis and by flow cytometry. Plasma viral RNA was measured by RT-PCR, and cell-associated DNA and RNA were quantified by qPCR in 6 tissues longitudinally, and in >20 tissues at necropsy. Results : All animals showed peak SHIV plasma viral loads (1.5e7 copies/ml) 10-11 days post-infection, subsequently reaching viral set points. 1 of 6 animal controlled viremia before cART initiation. In all other animals, plasma viral RNA became undetectable 2-3 weeks post cART initiation. We euthanized the transplant recipients at day 47, 29, and 9 post-transplant, due to infection, graft-versus host disease, and renal complications, respectively. Analysis of whole blood and gated CD4+ T cells showed acquisition of 100% donor blood chimerism at day 29, with lower T cell chimerism in 1 animal. Despite undetectable SHIV plasma viremia post-transplant, the cell-associated SHIV DNA reservoir persisted in multiple tissues, including lymphoid, hematopoietic and major organs, gut and CNS, and was even increased in certain tissues of transplanted animals compared to untransplanted controls. Conclusions : Our results indicate that the DNA reservoir persists and may even increase early after transplantion, despite control of peripheral viremia. Thus, allogeneic HCT is likely associated with an initial loss of anti-HIV immunity leading to an increase in the size of the viral reservoir. This may also explain the rebound observed in the Boston patients. Thus, we propose that reservoir eradication will require additional HIV resistance factors and/or anti-HIV strategies post-transplant to enhance 1) donor cell resistance, and 2) the targeted killing of infected cells. Disclosures Kiem: Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Kean: Regeneron: Research Funding; Bristol Myers Squibb: Consultancy; Juno: Research Funding; Kymab Ltd: Research Funding.

Microglia are the macrophage population residing in the parenchyma of the central nervous system (CNS), and are thought to play critical roles in CNS development, homeostasis and defense against pathogens. Microglia are capable of rapidly responding to microbial pathogens through engagement of their Toll-like receptors (TLRs). We first compared the efficiency of these responses in primary microglia acutely isolated from adult and neonatal mice. While the cytokine and chemokine responses of adult microglia were generally higher than those of neonatal cells stimulated ex vivo through TLRs, the nitric oxide response of neonatal microglia was markedly enhanced relative to the adult cells. We then went on to identify culture conditions such as exposure to M-SCF or GM-CSF that markedly enhanced the nitric oxide response of microglia, particularly those from the adult CNS. Finally, we demonstrate that the differential nitric oxide response of neonatal and adult microglia is not only limited to the mouse, but also extends to rat microglia.

(-)-Epigallocatechin gallate (EGCG), a catechin polyphenol compound, represents the main ingredient of green tea extract. Although EGCG has been shown to be growth inhibitory in a number of tumor cell lines, it is not clear whether the effect is cancer-specific. In this study we compared the effect of EGCG on the growth of SV40 virally transformed WI38 human fibroblasts (WI38VA) with that of normal WI38 cells. The IC50 value of EGCG was estimated to be 120 and 10 microM for WI38 and WI38VA cells, respectively. Thus, EGCG at 40 microM completely inhibited the growth of WI38VA cells, but had little or no inhibitory effect on the growth of WI38 cells. Similar differential growth inhibition was also observed between a human colorectal cancer cell line (Caco-2), a breast cancer cell line (Hs578T) and their respective normal counterparts. EGCG at a concentration range of 40-200 microM induced a significant amount of apoptosis in WI38VA cultures, but not in WI38 cultures, as determined by terminal deoxynucleotidyl transferase assay. After exposure to EGCG at 200 microM for 8 h, more than 50% of WI38VA cells in a confluent culture became apoptotic. In contrast, less than 1% of WI38 cells displayed apoptotic labeling under the same condition. EGCG did not affect the serum-induced expression of c-fos and c-myc genes in normal WI38 cells. However, it significantly enhanced their expression in transformed W138VA cells. It is possible that differential modulation of certain genes, such as c-fos and c-myc, may cause differential effects of EGCG on the growth and death of cancer cells.