Vaccine-Mediated Inhibition of the Transporter Associated with Antigen Processing Is Insufficient To Induce Major Histocompatibility Complex E-Restricted CD8+ T Cells in Nonhuman Primates.

Major histocompatibility complex E (MHC-E) is a highly conserved nonclassical MHC-Ib molecule that tightly binds peptides derived from leader sequences of classical MHC-Ia molecules for presentation to natural killer cells. However, MHC-E also binds diverse foreign and neoplastic self-peptide antigens for presentation to CD8+ T cells. Although the determinants of MHC-E-restricted T cell priming remain unknown, these cells are induced in humans infected with pathogens containing genes that inhibit the transporter associated with antigen processing (TAP). Indeed, mice vaccinated with TAP-inhibited autologous dendritic cells develop T cells restricted by the murine MHC-E homologue, Qa-1b. Here, we tested whether rhesus macaques (RM) vaccinated with viral constructs expressing a TAP inhibitor would develop insert-specific MHC-E-restricted CD8+ T cells. We generated viral constructs coexpressing SIVmac239 Gag in addition to one of three TAP inhibitors: herpes simplex virus 2 ICP47, bovine herpes virus 1 UL49.5, or rhesus cytomegalovirus Rh185. Each TAP inhibitor reduced surface expression of MHC-Ia molecules but did not reduce surface MHC-E expression. In agreement with modulation of surface MHC-Ia levels, TAP inhibition diminished presentation of MHC-Ia-restricted CD8+ T cell epitopes without impacting presentation of peptide antigen bound by MHC-E. Vaccination of macaques with vectors dually expressing SIVmac239 Gag with ICP47, UL49.5, or Rh185 generated Gag-specific CD8+ T cells classically restricted by MHC-Ia but not MHC-E. These data demonstrate that, in contrast to results in mice, TAP inhibition alone is insufficient for priming of MHC-E-restricted T cell responses in primates and suggest that additional unknown mechanisms govern the induction of CD8+ T cells recognizing MHC-E-bound antigen.IMPORTANCE Due to the near monomorphic nature of MHC-E in the human population and inability of many pathogens to inhibit MHC-E-mediated peptide presentation, MHC-E-restricted T cells have become an attractive vaccine target. However, little is known concerning how these cells are induced. Understanding the underlying mechanisms that induce these T cells would provide a powerful new vaccine strategy to an array of neoplasms and viral and bacterial pathogens. Recent studies have indicated a link between TAP inhibition and induction of MHC-E-restricted T cells. The significance of our research is in demonstrating that TAP inhibition alone does not prime MHC-E-restricted T cell generation and suggests that other, currently unknown mechanisms regulate their induction.

The Role of MHC-E in T Cell Immunity Is Conserved among Humans, Rhesus Macaques, and Cynomolgus Macaques.

MHC-E is a highly conserved nonclassical MHC class Ib molecule that predominantly binds and presents MHC class Ia leader sequence-derived peptides for NK cell regulation. However, MHC-E also binds pathogen-derived peptide Ags for presentation to CD8+ T cells. Given this role in adaptive immunity and its highly monomorphic nature in the human population, HLA-E is an attractive target for novel vaccine and immunotherapeutic modalities. Development of HLA-E-targeted therapies will require a physiologically relevant animal model that recapitulates HLA-E-restricted T cell biology. In this study, we investigated MHC-E immunobiology in two common nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM). Compared to humans and MCM, RM expressed a greater number of MHC-E alleles at both the population and individual level. Despite this difference, human, RM, and MCM MHC-E molecules were expressed at similar levels across immune cell subsets, equivalently upregulated by viral pathogens, and bound and presented identical peptides to CD8+ T cells. Indeed, SIV-specific, Mamu-E-restricted CD8+ T cells from RM recognized antigenic peptides presented by all MHC-E molecules tested, including cross-species recognition of human and MCM SIV-infected CD4+ T cells. Thus, MHC-E is functionally conserved among humans, RM, and MCM, and both RM and MCM represent physiologically relevant animal models of HLA-E-restricted T cell immunobiology.

Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques.

Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present. Furthermore, viral vector-mediated expression of hNTCP on hepatocytes in vivo renders rhesus macaques permissive to HBV infection. These in vivo macaque HBV infections are characterized by longitudinal HBV DNA in serum, and detection of HBV DNA, RNA, and HBV core antigen (HBcAg) in hepatocytes. Together, these results show that expressing hNTCP on macaque hepatocytes renders them susceptible to HBV infection, thereby establishing a physiologically relevant model of HBV infection to study immune clearance and test therapeutic and curative approaches.

Allogeneic stem cell transplantation in fully MHC-matched Mauritian cynomolgus macaques recapitulates diverse human clinical outcomes.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a critically important therapy for hematological malignancies, inborn errors of metabolism, and immunodeficiency disorders, yet complications such as graft-vs.-host disease (GvHD) limit survival. Development of anti-GvHD therapies that do not adversely affect susceptibility to infection or graft-vs.-tumor immunity are hampered by the lack of a physiologically relevant, preclinical model of allogeneic HSCT. Here we show a spectrum of diverse clinical HSCT outcomes including primary and secondary graft failure, lethal GvHD, and stable, disease-free full donor engraftment using reduced intensity conditioning and mobilized peripheral blood HSCT in unrelated, fully MHC-matched Mauritian-origin cynomolgus macaques. Anti-GvHD prophylaxis of tacrolimus, post-transplant cyclophosphamide, and CD28 blockade induces multi-lineage, full donor chimerism and recipient-specific tolerance while maintaining pathogen-specific immunity. These results establish a new preclinical allogeneic HSCT model for evaluation of GvHD prophylaxis and next-generation HSCT-mediated therapies for solid organ tolerance, cure of non-malignant hematological disease, and HIV reservoir clearance.

Contribution of Calpain and Caspases to Cell Death in Cultured Monkey RPE Cells.

Purpose: AMD is the leading cause of human vision loss after 65 years of age. Several mechanisms have been proposed: (1) age-related failure of the choroidal vasculature leads to loss of RPE; (2) RPE dysfunctions due to accumulation of phagocytized, but unreleased A2E (N-retinylidene-N-retinylethanolamine); (3) zinc deficiency activation of calpain and caspase proteases, leading to cell death. The purpose of the present study is to compare activation of calpain and caspase in monkey RPE cells cultured under hypoxia or with A2E. Methods: Monkey primary RPE cells were cultured under hypoxic conditions in a Gaspak pouch or cultured with synthetic A2E. Immunoblotting was used to detect activation of calpain and caspase. Calpain inhibitor, SNJ-1945, and pan-caspase inhibitor, z-VAD-fmk, were used to confirm activation of the proteases. Results: (1) Hypoxia and A2E each decreased viability of RPE cells in a time-dependent manner. (2) Incubation under hypoxia alone induced activation of calpain, but not caspases. SNJ-1945 inhibited calpain activation, but z-VAD-fmk did not. (3) Incubation with A2E alone induced activation of calpain, caspase-9, and caspase-3. SNJ-1945 inhibited calpain activation. z-VAD-fmk inhibited caspase activation, suggesting no interaction between calpain and caspases. Conclusions: Hypoxia activated the calpain pathway, while A2E activated both calpain and caspase pathways in monkey RPE cells. Such knowledge may be utilized in the treatment of AMD if inhibitor drugs against calpain and/or caspase are used to prevent RPE dysfunction caused by hypoxia or A2E.

Cross-Species Rhesus Cytomegalovirus Infection of Cynomolgus Macaques.

Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68-1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells in vitro, RhCMV 68-1 failed to productively infect MCM in vivo, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68-1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68-1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68-1 and 68-1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics.

Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques.

Prevention of mother-to-child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested HIV-1-specific human neutralizing monoclonal antibodies (NmAbs) as a post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with the simian-human immunodeficiency virus SHIVSF162P3. On days 1, 4, 7 and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h after antibody administration. Replicating virus was found in multiple tissues by day 1 in animals that were not treated. All NmAb-treated macaques were free of virus in blood and tissues at 6 months after exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged after CD8(+) T cell depletion. These results suggest that early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs.

TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection.

HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.

Broadly targeted CD8⁺ T cell responses restricted by major histocompatibility complex E.

Major histocompatibility complex E (MHC-E) is a highly conserved, ubiquitously expressed, nonclassical MHC class Ib molecule with limited polymorphism that is primarily involved in the regulation of natural killer (NK) cells. We found that vaccinating rhesus macaques with rhesus cytomegalovirus vectors in which genes Rh157.5 and Rh157.4 are deleted results in MHC-E-restricted presentation of highly varied peptide epitopes to CD8αß(+) T cells, at ~4 distinct epitopes per 100 amino acids in all tested antigens. Computational structural analysis revealed that MHC-E provides heterogeneous chemical environments for diverse side-chain interactions within a stable, open binding groove. Because MHC-E is up-regulated to evade NK cell activity in cells infected with HIV, simian immunodeficiency virus, and other persistent viruses, MHC-E-restricted CD8(+) T cell responses have the potential to exploit pathogen immune-evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.

Technical advance: liposomal alendronate depletes monocytes and macrophages in the nonhuman primate model of human disease.

Nonhuman primates are critical animal models for the study of human disorders and disease and offer a platform to assess the role of immune cells in pathogenesis via depletion of specific cellular subsets. However, this model is currently hindered by the lack of reagents that safely and specifically ablate myeloid cells of the monocyte/macrophage Lin. Given the central importance of macrophages in homeostasis and host immunity, development of a macrophage-depletion technique in nonhuman primates would open new avenues of research. Here, using LA at i.v. doses as low as 0.1 mg/kg, we show a >50% transient depletion of circulating monocytes and tissue-resident macrophages in RMs by an 11-color flow cytometric analysis. Diminution of monocytes was followed rapidly by emigration of monocytes from the bone marrow, leading to a rebound of monocytes to baseline levels. Importantly, LA was well-tolerated, as no adverse effects or changes in gross organ function were observed during depletion. These results advance the ex vivo study of myeloid cells by flow cytometry and pave the way for in vivo studies of monocyte/macrophage biology in nonhuman primate models of human disease.

Tertiary mutations stabilize CD8+ T lymphocyte escape-associated compensatory mutations following transmission of simian immunodeficiency virus.

Compensatory mutations offset fitness defects resulting from CD8(+) T lymphocyte (CD8(TL))-mediated escape, but their impact on viral evolution following transmission to naive hosts remains unclear. Here, we investigated the reversion kinetics of Gag(181-189)CM9 CD8(TL) escape-associated compensatory mutations in simian immunodeficiency virus (SIV)-infected macaques. Preexisting compensatory mutations did not result in acute-phase escape of the SIVmac239 CD8(TL) epitope Gag(181-189)CM9 and instead required a tertiary mutation for stabilization in the absence of Gag(181-189)CM9 escape mutations. Therefore, transmitted compensatory mutations do not necessarily predict rapid CD8(TL) escape.

Calpain protease causes hypoxia-induced proteolysis in cultured human retina.

PURPOSE/AIM: Calpain proteases are known to be involved in retinal cell death in animal models. The purpose of the present study was to test for calpain activation in human retinas cultured under hypoxic conditions. MATERIALS AND METHODS: Calpain activation was detected by immunoblotting for calpain substrates in human and monkey retinas cultured in gas generating pouches to reduce oxygen. RESULTS: Hypoxia caused activation of calpains as measured by accumulation of the calpain-specific 145 kDa α-spectrin breakdown product. Opsin-1 (photoreceptor marker) and vimentin (Müller cell marker) were degraded. Calpain inhibitor SNJ-1945 ameliorated these changes. Results were similar to comparative data from cultured monkey retinas. CONCLUSIONS: In cultured human retina, hypoxia caused activation of calpain and subsequent proteolysis of critical substrates. The efficacy of SNJ-1945 in ameliorating these changes indicated that it might be useful to test as a drug for protecting against pathologic proteolysis of photoreceptor and Müller cells.

Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms.

CD8(+) T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of antipathogen immunity. We found that simian immunodeficiency virus (SIV) protein-expressing rhesus cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8(+) T cells that recognize unusual, diverse, and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope-specific CD8(+) T cell responses is suppressed by the RhCMV-encoded Rh189 gene (corresponding to human CMV US11), and the promiscuous MHC class I- and class II-restricted CD8(+) T cell responses occur only in the absence of the Rh157.5, Rh157.4, and Rh157.6 (human CMV UL128, UL130, and UL131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8(+) T cell epitope recognition.

Calpain, not caspase, is the causative protease for hypoxic damage in cultured monkey retinal cells.

PURPOSE: Cell death occurring in human retina during AMD, high IOP, and diabetic retinopathy could be caused by activation of calpain or caspase proteolytic enzymes. The purpose of the present study was to determine whether calpains and/or caspase-3 were involved in cell death during retinal hypoxia in a monkey model. METHODS: Dissociated monkey retinal cells were cultured for two weeks and subjected to 24-hour hypoxia/24-hour reoxygenation. TUNEL staining and immunostaining for Müller and photoreceptor markers were used to detect which retinal cell types were damaged. RESULTS: Culturing dissociated monkey retina cells for two weeks resulted in proliferation of Müller cells and maintenance of some rod and cone photoreceptor cells, as identified by vimentin, recoverin, and rhodopsin immunocytochemical staining. Hypoxia/reoxygenation increased the number of cells staining positive for TUNEL. Immunoblotting showed that the calpain-specific 145 kDa α-spectrin breakdown product (SBDP) increased in hypoxic cells, but no caspase-specific 120 kDa α-spectrin breakdown product was detected. TUNEL staining and proteolysis were significantly reduced in the retinal cells treated with 10 and 100 µM calpain inhibitor SNJ-1945. Caspase inhibitor, z-VAD, did not inhibit cell damage from hypoxia/reoxygenation. Intact pro-caspase-3 was in fact cleaved by activated calpain during hypoxia/reoxygenation to pre 29 kDa caspase-3 and 24 kDa inactive fragments. No 17 and 12 kDa fragments, which form the active caspase-3 hetero-dimer, were detected. Calpain-induced cleavage of caspase was inhibited by SNJ-1945. CONCLUSIONS: Calpain, not caspase-3, was involved in hypoxic damage in cultured monkey retinal cells.