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Age-related reductions in vaccine-induced B cells in aging indicate that germinal centers (GCs), the anatomical site where the development of humoral responses takes place, may lose efficacy with age. We have investigated the baseline follicular and GC composition in nonhuman primates (NHPs) with respect to their age. There was a marked reduction in follicular area in old animals. We found significantly lower normalized numbers of follicular PD1 hi CD4 T (Tfh) and proliferating (Ki67 hi ) GC B cells with aging, a profile associated with significantly higher numbers of potential follicular suppressor FoxP3 hi Lag3 hi CD4 T cells. Furthermore, a positive correlation was found between Tfh and follicular CD8 T cells (fCD8) only in young animals. Despite the increased levels of circulating preinflammatory factors in aging, young animals had higher numbers of monocytes and granulocytes in the follicles, a profile negatively associated with numbers of Tfh cells. Multiple regression analysis showed an altered association between GC B cells and other GC immune cell populations in old animals suggesting a differential mechanistic regulation of GC activity in aging. Our data demonstrate defective baseline GC composition in old NHPs and provide an immunological base for further understanding the adaptive humoral responses with respect to aging., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Integrin α 4 β 7 mediates the trafficking of leukocytes, including CD4 + T cells, to lymphoid tissues in the gut. Virus mediated damage to the gut is implicated in HIV and SIV mediated chronic immune activation and leads to irreversible damage to the immune system. We employed an immuno-PET/CT imaging technique to evaluate the impact of an anti-integrin α 4 β 7 mAb alone or in combination with ART, on the distribution of both SIV infected cells and CD4 + cells in rhesus macaques infected with SIV. We determined that α 4 β 7 mAb reduced viral antigen in an array of tissues of the lung, spleen, axillary, and inguinal lymph nodes. These sites are not directly linked to α 4 β 7 mediated homing; however, the most pronounced reduction in viral load was observed in the colon. Despite this reduction, α 4 β 7 mAb treatment did not prevent an apparent depletion of CD4 + T cells in gut in the acute phase of infection that is characteristic of HIV/SIV infection. However, α 4 β 7 mAb appeared to facilitate the preservation or restoration of CD4 + T cells in gut tissues at later stages of infection. Since damage to the gut is believed to play a central role in HIV pathogenesis, these results support further evaluation of α 4 β 7 antagonists in the study and treatment of HIV disease.

Glycosylation of Env defines pathogenic properties of simian immunodeficiency virus (SIV). We previously demonstrated that pathogenic SIVmac239 and a live-attenuated, quintuple deglycosylated Env mutant (Δ5G) virus target CD4 + T cells residing in different tissues during acute infection. SIVmac239 and Δ5G preferentially infected distinct CD4 + T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Here, we studied the host responses relevant to SIV targeting of CXCR3 + CCR5 + CD4 + T cells in SLOs. Genome-wide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239-infected animals. Consistent with robust expression of CXCL10, CXCR3 + T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs was secondary to the induction of CD14 + CD16 + monocytes and MAC387 + macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387 + macrophages, T cells, dendritic cells (DCs), and residential macrophages near high endothelial venules, the results highlight critical roles of innate/inflammatory responses in SIVmac239 infection. Restricted infection in SLOs by Δ5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DC lineages. IMPORTANCE We previously demonstrated that a pathogenic SIVmac239 virus and a live-attenuated, deglycosylated mutant Δ5G virus infected distinct CD4 + T cell subsets in SLOs and the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Accordingly, infections with SIVmac239, but not with Δ5G, deplete CXCR3 + CCR5 + CD4 + T (Th1) cells during the primary infection, thereby compromising the cellular immune response. Thus, we hypothesized that distinct host responses are elicited by the infections with two different viruses. We found that SIVmac239 induced distinctly higher levels of inflammatory Th1 responses than Δ5G. In particular, SIVmac239 infection elicited robust expression of CXCL10, a chemokine for CXCR3 + cells, in CD14 + CD16 + monocytes and MAC387 + macrophages recently infiltrated in SLOs. In contrast, Δ5G infection elicited only modest inflammatory responses. These results suggest that the glycosylation of Env modulates the inflammatory/Th1 responses through the monocyte/macrophage subsets and elicits marked differences in SIV infection and clinical outcomes., (Copyright © 2017 American Society for Microbiology.)

α4β7 integrin-expressing CD4(+) T cells preferentially traffic to gut-associated lymphoid tissue (GALT) and have a key role in HIV and simian immunodeficiency virus (SIV) pathogenesis. We show here that the administration of an anti-α4β7 monoclonal antibody just prior to and during acute infection protects rhesus macaques from transmission following repeated low-dose intravaginal challenges with SIVmac251. In treated animals that became infected, the GALT was significantly protected from infection and CD4(+) T cell numbers were maintained in both the blood and the GALT. Thus, targeting α4β7 reduces mucosal transmission of SIV in macaques.

The inhibitory receptor programmed death-1 (PD-1) has been shown to regulate CD8 T cell function during chronic SIV infection; however, its role on CD4 T cells, specifically in the gut-associated lymphoid tissue, is less well understood. In this study, we show that a subset of CD4 T cells expresses high levels of PD-1 (PD-1(hi)) in the rectal mucosa, a preferential site of virus replication. The majority of these PD-1(hi) CD4 T cells expressed Bcl-6 and CXCR5, markers characteristic of T follicular helper cells in the lymph nodes. Following a pathogenic SIV infection, the frequency of PD-1(hi) cells (as a percentage of CD4 T cells) dramatically increased in the rectal mucosa; however, a significant fraction of them did not express CXCR5. Furthermore, only a small fraction of PD-1(hi) cells expressed CCR5, and despite this low level of viral coreceptor expression, a significant fraction of these cells were productively infected. Interestingly, vaccinated SIV controllers did not present with this aberrant PD-1(hi) CD4 T cell enrichment, and this lack of enrichment was associated with the presence of higher frequencies of SIV-specific granzyme B(+) CD8 T cells within the lymphoid tissue, suggesting a role for antiviral CD8 T cells in limiting aberrant expansion of PD-1(hi) CD4 T cells. These results highlight the importance of developing vaccines that enhance antiviral CD8 T cells at sites of preferential viral replication and support the need for developing therapeutic interventions that limit expansion of SIV(+)PD-1(hi) CD4 T cells at mucosal sites as a means to enhance viral control., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Prevention of HIV acquisition and replication requires long lasting and effective immunity. Given the state of HIV vaccine development, innovative vectors and immunization strategies are urgently needed to generate safe and efficacious HIV vaccines. Here, we developed a novel lentivirus-based DNA vector that does not integrate in the host genome and undergoes a single-cycle of replication. Viral proteins are constitutively expressed under the control of Tat-independent LTR promoter from goat lentivirus. We immunized six macaques once only with CAL-SHIV-IN- DNA using combined intramuscular and intradermal injections plus electroporation. Antigen-specific T cell responses were monitored for 47 weeks post-immunization (PI). PBMCs were assessed directly ex vivo or after 6 and 12 days of in vitro culture using antigenic and/or homeostatic proliferation. IFN-γ ELISPOT was used to measure immediate cytokine secretion from antigen specific effector cells and from memory precursors with high proliferative capacity (PHPC). The memory phenotype and functions (proliferation, cytokine expression, lytic content) of specific T cells were tested using multiparametric FACS-based assays. All immunized macaques developed lasting peripheral CD8+ and CD4+ T cell responses mainly against Gag and Nef antigens. During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes. These responses contracted but then reemerged later in absence of antigen boost. Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI. Altogether, our study demonstrated that a single immunization with a replication-limited DNA vaccine elicited persistent vaccine-specific CM and EM CD8+ and CD4+ T cells with immediate and readily inducible effector functions, in the absence of ongoing antigen expression.

Unlabelled: The impact of Epstein-Barr virus (EBV) on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA-1) is an important target for vaccination because it is the only protein expressed in all EBV-associated malignancies. We have designed and tested two therapeutic EBV vaccines that target the rhesus (rh) lymphocryptovirus (LCV) EBNA-1 to determine if ongoing T cell responses during persistent rhLCV infection in rhesus macaques can be expanded upon vaccination. Vaccines were based on two serotypes of E1-deleted simian adenovirus and were administered in a prime-boost regimen. To further modulate the response, rhEBNA-1 was fused to herpes simplex virus glycoprotein D (HSV-gD), which acts to block an inhibitory signaling pathway during T cell activation. We found that vaccines expressing rhEBNA-1 with or without functional HSV-gD led to expansion of rhEBNA-1-specific CD8(+) and CD4(+) T cells in 33% and 83% of the vaccinated animals, respectively. Additional animals developed significant changes within T cell subsets without changes in total numbers. Vaccination did not increase T cell responses to rhBZLF-1, an immediate early lytic phase antigen of rhLCV, thus indicating that increases of rhEBNA-1-specific responses were a direct result of vaccination. Vaccine-induced rhEBNA-1-specific T cells were highly functional and produced various combinations of cytokines as well as the cytolytic molecule granzyme B. These results serve as an important proof of principle that functional EBNA-1-specific T cells can be expanded by vaccination., Importance: EBV is a common human pathogen that establishes a persistent infection through latency in B cells, where it occasionally reactivates. EBV infection is typically benign and is well controlled by the host adaptive immune system; however, it is considered carcinogenic due to its strong association with lymphoid and epithelial cell malignancies. Latent EBNA-1 is a promising target for a therapeutic vaccine, as it is the only antigen expressed in all EBV-associated malignancies. The goal was to determine if rhEBNA-1-specific T cells could be expanded upon vaccination of infected animals. Results were obtained with vaccines that target EBNA-1 of rhLCV, a virus closely related to EBV. We found that vaccination led to expansion of rhEBNA-1 immune cells that exhibited functions fit for controlling viral infection. This confirms that rhEBNA-1 is a suitable target for therapeutic vaccines. Future work should aim to generate more-robust T cell responses through modified vaccines.

Mother-to-infant transmission (MTIT) of HIV is a serious global health concern, with over 300,000 children newly infected in 2011. SIV infection of rhesus macaques (RMs) results in similar rates of MTIT to that of HIV in humans. In contrast, SIV infection of sooty mangabeys (SMs) rarely results in MTIT. The mechanisms underlying protection from MTIT in SMs are unknown. In this study we tested the hypotheses that breast milk factors and/or target cell availability dictate the rate of MTIT in RMs (transmitters) and SMs (non-transmitters). We measured viral loads (cell-free and cell-associated), levels of immune mediators, and the ability to inhibit SIV infection in vitro in milk obtained from lactating RMs and SMs. In addition, we assessed the levels of target cells (CD4+CCR5+ T cells) in gastrointestinal and lymphoid tissues, including those relevant to breastfeeding transmission, as well as peripheral blood from uninfected RM and SM infants. We found that frequently-transmitting RMs did not have higher levels of cell-free or cell-associated viral loads in milk compared to rarely-transmitting SMs. Milk from both RMs and SMs moderately inhibited in vitro SIV infection, and presence of the examined immune mediators in these two species did not readily explain the differential rates of transmission. Importantly, we found that the percentage of CD4+CCR5+ T cells was significantly lower in all tissues in infant SMs as compared to infant RMs despite robust levels of CD4+ T cell proliferation in both species. The difference between the frequently-transmitting RMs and rarely-transmitting SMs was most pronounced in CD4+ memory T cells in the spleen, jejunum, and colon as well as in central and effector memory CD4+ T cells in the peripheral blood. We propose that limited availability of SIV target cells in infant SMs represents a key evolutionary adaptation to reduce the risk of MTIT in SIV-infected SMs.

The programmed cell death-1 (PD-1)/programmed cell death ligand-1 pathway has been shown to limit cell-mediated effector functions during chronic viral infections impeding clearance of pathogens. As a strategy to reverse this exhaustion and increase T cell polyfunctionality, PD-1 ligands were blocked in vivo using a recombinant macaque PD-1 fused to a macaque Ig-Fc (rPD-1-Fc) in SIVmac239-infected rhesus macaques during the early chronic phase of infection, either alone or in combination with antiretroviral therapy. In vitro blockade showed improvement of Ag-specific CD4(+) and CD8(+) T cells from monkeys chronically infected with SIV. Of note, a prolonged 5-d blockade in culture was beneficial for both gag-specific CD4(+) and CD8(+) T cells based on proliferation and dual cytokine production. Although the in vivo administration of rPD-1-Fc induced enhanced SIV-specific CD4 and CD8 T cell proliferation both in the blood and gut, it failed to alter plasma viremia. However, rPD-1-Fc administration in the context of antiretroviral therapy interruption induced a significant delay of viral load rebound. In addition, rPD-1-Fc administration in MamuA*001(+) monkeys led to both an increase in the frequencies and Ki67 expression of GagCM9(+) CD8(+) T cells in the blood and rectal mucosa and polyfunctionality of GagCM9(+) CD8(+) T cells in blood. In conclusion, however, our data suggest that PD-1/programmed cell death ligand-1 blockade using soluble rPD-1-Fc instead of anti-PD-1 mAb, although effective in rescuing the effector function of SIV-specific CD4(+) and CD8(+) T cells during the early chronic phase of infection, has limited clinical benefit.

Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4(+) and/or CD8(+) T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4(+) T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8(+) T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines.

PD-1 expression is generally associated with exhaustion of T cells during chronic viral infections based on the finding that PD-1 expressing cells respond poorly to antigen activation and blockade of PD-1/PD-ligand interaction restores such antigen specific responses in vitro. We tested this hypothesis by examining PD-1 expression on virus-specific CD8 T cells and total T cells in vivo to determine whether PD-1 expression constitutes a reliable marker of immune exhaustion during SIV infection. The expression of PD-1 and Ki67 was monitored longitudinally on T cell subsets in peripheral blood, bone marrow, lymph node and rectal biopsy specimens from rhesus macaques prior to and post infection with pathogenic SIVmac239. During the course of infection, a progressive negative correlation was noted between PD-1 density and Ki67 expression in p11CM(+) CD8(+) T cells, as seen in other studies. However, for total and memory CD4 and CD8 T cells, a positive correlation was observed between PD-1 and Ki67 expression. Thus, while the levels of non-proliferating PD-1(+) p11CM(+) CD8 T cells were markedly elevated with progressing infection, such an increase was not seen on total T cells. In addition, total memory PD1(+) T cells exhibited higher levels of CCR5 than PD-1(-) T cells. Interestingly, few PD-1(+) CD8(+) T cells expressed CCR7 compared to PD-1(+) CD4 T cells and PD-1(-) T cells. In conclusion, overall PD1(+) T cells likely represent a particular differentiation stage or trafficking ability rather than exhaustion and in the context of chronic SIV infection, the level of PD-1 expression by T cells does not by itself serve as a reliable marker for immune exhaustion.

IL-21 regulates Th17 cell homeostasis, enhances the differentiation of memory B cells and antibody-secreting plasma cells, and promotes the maintenance of CD8(+) T-cell responses. In this study, we investigated the phenotype, function, and frequency of blood and intestinal IL-21-producing cells in nonhuman primates that are hosts of progressive (rhesus macaques [RMs]) and nonprogressive (sooty mangabeys [SMs]) SIV infection. We found that, in both species, memory CD4(+)CD95(+)CCR6(-) T cells are the main IL-21 producers, and that only a small fraction of CD4(+)IL-21(+) T cells produce IL-17. During chronic SIV infection of RMs, CD4(+)IL-21(+) T cells were significantly depleted in both blood and rectal mucosa, with the extent of this depletion correlating with the loss of Th17 cells. Furthermore, treatment with IL-21 increased the in vivo levels of Th17 cells in SIV-infected RMs. In contrast, normal levels of CD4(+)IL-21(+) T cells were found in SIV-infected SMs. Collectively, these data indicate that depletion of IL-21-producing CD4(+) T cells distinguishes progressive from nonprogressive SIV infection of RMs and SMs, and suggest that depletion of CD4(+)IL-21(+) T cells is involved in the preferential loss of Th17 cells that is associated with SIV disease progression. Further preclinical studies of IL-21 as a potential immunotherapeutic agent for HIV infection may be warranted.

We have previously shown that interleukin-21, a pleiotropic C γ-chain signaling cytokine, induces the expression of the cytotoxic molecules granzyme B (GrB) and perforin in vitro in CD8 T cells and NK cells of chronically HIV infected individuals. In this pilot study, four chronically SIV infected rhesus macaques (RM) in late-stage disease were given two doses of recombinant MamuIL-21, 50 μg/kg, intravenously 7 days apart, followed by one subcutaneous dose, 100 μg/kg, 23 days after the second dose. Three animals served as controls. After each dose of IL-21, increases were noted in frequency and mean fluorescence intensity of GrB and perforin expression in memory and effector subsets of CD8 T cells in peripheral blood (PB), in peripheral and mesenteric lymph node (LN) cells, in PB memory and effector CD4 T cells and in NK cells. Frequencies of SIV-gag specific CD107a(+)IFN-γ(+) CD8 T cells increased 3.8-fold in PB and 1.8-fold in LN. In addition, PB CD27(+) memory B cells were 2-fold higher and serum SIV antibodies increased significantly after IL-21 administration. No changes were observed in markers of T cell activation, T cell proliferation or plasma virus load. Thus, administration of IL-21 to chronically SIV infected viremic animals was safe, well tolerated and could augment the cytotoxic potential of T cells and NK cells, promote B cell differentiation with increases in SIV antibody titers without discernable increase in cellular activation. Further studies are warranted to elucidate the effects and potential benefit of IL-21 administration in the context of SIV/HIV infection and in SIV/HIV vaccine design., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Human immunodeficiency virus (HIV) infection is characterized by a progressive loss of memory CD4(+) T cells in multiple tissues, especially at mucosal surfaces where most of these cells reside. Although antiretroviral therapy (ART) suppresses viral replication and promotes the recovery of peripheral CD4(+) T cells, HIV-infected patients fail to fully reconstitute the CD4(+) T-cell pool at mucosal sites. IL-15 has been shown to preferentially expand memory-phenotype T cells and promote their migration to nonlymphoid tissues. Here we examined IL-15 treatment in combination with highly active ART in chronically SIV-infected rhesus macaques and found that IL-15 delayed viral suppression and failed to enhance ART-induced total and antigen-specific CD4(+) T-cell reconstitution at mucosal and lymphoid sites. IL-15 was able to induce the transient proliferation of SIV-specific, CMV-specific, and total memory CD8(+) T cells, but not of SIV-specific or total CD4(+) T cells. Moreover, upon treatment interruption, macaques receiving combined IL-15+ART lost CD4(+) T cells faster than those receiving ART alone. These results suggest that the combination of IL-15 with highly active ART is not more efficient than ART alone in promoting CD4(+) T-cell recovery in HIV-infected individuals and may accelerate CD4+ T-cell loss after treatment interruption.

CD4(+) T cell depletion is a fundamental component of HIV infection and AIDS pathogenesis and is not always reversed following antiretroviral therapy (ART). In this study, the SIV-infected rhesus macaque model was used to assess recombinant simian IL-7 in its glycosylated form (rsIL-7gly) to enhance regeneration of CD4(+) T cells, particularly the crucial central memory compartment, after ART. We assessed the impact of rsIL-7gly administration as single injections and as a cluster of three doses. Irrespective of the dosing strategy used, the rsIL-7gly administration transiently increased proliferation of both central memory and naive cells, in both CD4(+) and CD8(+) subsets, without increasing SIV levels in the blood. Administration of rsIL-7gly at intervals of 4-6 wk maximized the proliferative response to therapy but resulted in only transient increases in peripheral blood T cell counts. Although more frequent rsIL-7gly "clustered" dosing (three times weekly with 2 wk of rest and then repeat) induced only an initial proliferative burst by CD4(+) T cells, this dosing strategy resulted in sustained increases in peripheral blood CD4(+) T cell counts. The clustered rsIL-7gly treatment regimen was shown to increase the half-life of a BrdU label among memory T cells in the blood when compared with that of macaques treated with ART alone, which is consistent with enhanced cell survival. These results indicate that dosing intervals have a major impact on the response to rsIL-7gly in SIV-positive ART-treated rhesus macaques and that optimum dosing strategies may be ones that induce CD4(+) T cell proliferation initially and provide increased CD4(+) T cell survival.

The optimization of immune responses (IR) induced by HIV DNA vaccines in humans is one of the great challenges in the development of an effective vaccine against AIDS. Ideally, this vaccine should be delivered in a single dose to immunize humans. We recently demonstrated that the immunization of mice with a single dose of a DNA vaccine derived from pathogenic SHIV(KU2) (Delta4SHIV(KU2)) induced long-lasting, potent, and polyfunctional HIV-specific CD8(+) T-cell responses (G. Arrode, R. Hegde, A. Mani, Y. Jin, Y. Chebloune, and O. Narayan, J. Immunol. 178:2318-2327, 2007). In the present work, we expanded the characterization of the IR induced by this DNA immunization protocol to rhesus macaques. Animals immunized with a single high dose of Delta4SHIV(KU2) DNA vaccine were monitored longitudinally for vaccine-induced IR using multiparametric flow cytometry-based assays. Interestingly, all five immunized macaques developed broad and polyfunctional HIV-specific T-cell IR that persisted for months, with an unusual reemergence in the blood following an initial decline but in the absence of antibody responses. The majority of vaccine-specific CD4(+) and CD8(+) T cells lacked gamma interferon production but showed high antigen-specific proliferation capacities. Proliferative CD8(+) T cells expressed the lytic molecule granzyme B. No integrated viral vector could be detected in mononuclear cells from immunized animals, and this high dose of DNA did not induce any detectable autoimmune responses against DNA. Taken together, our comprehensive analysis demonstrated for the first time the capacity of a single high dose of HIV DNA vaccine alone to induce long-lasting and polyfunctional T-cell responses in the nonhuman primate model, bringing new insights for the design of future HIV vaccines.

Depletion of CD8(+) lymphocytes during acute simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) results in irreversible prolongation of peak-level viral replication and rapid disease progression, consistent with a major role for CD8(+) lymphocytes in determining postacute-phase viral replication set points. However, we report that CD8(+) lymphocyte depletion is also associated with a dramatic induction of proliferation among CD4(+) effector memory T (T(EM)) cells and, to a lesser extent, transitional memory T (T(TrM)) cells, raising the question of whether an increased availability of optimal (activated/proliferating), CD4(+)/CCR5(+) SIV "target" cells contributes to this accelerated pathogenesis. In keeping with this, depletion of CD8(+) lymphocytes in SIV(-) RMs led to a sustained increase in the number of potential CD4(+) SIV targets, whereas such depletion in acute SIV infection led to increased target cell consumption. However, we found that the excess CD4(+) T(EM) cell proliferation of CD8(+) lymphocyte-depleted, acutely SIV-infected RMs was completely inhibited by interleukin (IL)-15 neutralization, and that this inhibition did not abrogate the rapidly progressive infection in these RMs. Moreover, although administration of IL-15 during acute infection induced robust CD4(+) T(EM) and T(TrM) cell proliferation, it did not recapitulate the viral dynamics of CD8(+) lymphocyte depletion. These data suggest that CD8(+) lymphocyte function has a larger impact on the outcome of acute SIV infection than the number and/or activation status of target cells available for infection and viral production.

Acute SIV infection is characterized by explosive infection of memory CD4 T cells in peripheral and mucosal tissues. Interestingly, relatively few memory CD4 T cells are infected until as late as days 7-8 after challenge. However, by day 10 postinfection, most of the memory CD4 T cells are infected and carry viral DNA. The rapidity with which infection expands within 2-3 days to encompass virtually the entire memory CD4 T cell compartment suggests significant alterations in the susceptibility of memory CD4 T cells to infection during this period. The mechanism(s) underlying this increased permissiveness to infection is not known. In this study, we show that IL-15 secretion significantly correlates with the up-regulated expression of CD4 on memory CD4 T cells that is associated with increased permissiveness to SIV infection. Activation and proliferation of memory CD8, but not memory CD4 T cells, preceded the amplification of viral infection. Although memory CD4 T cells did not express normal activation markers, they displayed a significant up-regulation in the density of CD4 but not CCR5 expression between days 7 and 10 postinfection that correlated with increased plasma IL-15 levels and infection in these cells. Culture of purified CD4 T cells with IL-15 and/or SIV was associated with a significant increase in the expression of CD4 and infection of these sorted cells. Our results demonstrate that IL-15 contributes to the increased susceptibility of memory CD4 T cells to SIV during the early phase of acute SIV infection.

Membrane-anchored C-peptides (for example, maC46) derived from human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 effectively inhibit HIV-1 entry in cell lines and primary human CD4+ cells in vitro. Here we evaluated this gene therapy approach in animal models of AIDS. We adapted the HIV gp41-derived maC46 vector construct for use in rhesus monkeys. Simian immunodeficiency virus (SIV and SHIV) sequence-adapted maC46 peptides, and the original HIV-1-derived maC46 expressed on the surface of established cell lines blocked entry of HIV-1, SIVmac251 and SHIV89.6P. Furthermore, primary rhesus monkey CD4+ T cells expressing HIV sequence-based maC46 peptides were also protected from SIV entry. Depletion of CD8+ T cells from PBMCs enhanced the yield of maC46-transduced CD4+ T cells. Supplementation with interleukin-2 (IL-2) increased transduction efficiency, whereas IL-7 and/or IL-15 provided no additional benefit. Phenotypic analysis showed that maC46-transduced and expanded cells were predominantly central memory CD4+ T cells that expressed low levels of CCR5 and slightly elevated levels of CD62L, beta7-integrin and CXCR4. These findings show that maC46-based cell surface-expressed peptides can efficiently inhibit primate immunodeficiency virus infection, and therefore serve as the basis for evaluation of this gene therapy approach in an animal model for AIDS.

Phenotypic and functional studies of the programmed death-1 (PD-1) molecule on CD4(+) and CD8(+) T cells were performed on peripheral blood mononuclear cells from uninfected and simian immunodeficiency virus (SIV)-infected rhesus macaques. These data demonstrated a rapid upregulation of PD-1 expression on tetramer-positive CD8(+) T cells from MamuA.01(+) SIV-infected macaques upon infection. Upregulation of PD-1 on total CD8(+) T cells was not detectable. In contrast, CD4(+) T-cell PD-1 expression was markedly higher in total CD4(+) T cells during chronic, but not acute, infection and there was a correlation between the level of PD-1 expression on naive and central memory CD4(+) T cells and the levels of viral loads. Such association was emphasized further by a marked decrease of PD-1 expression on tetramer-positive CD8 T cells as well as on CD4(+) T cells on longitudinal samples collected before and after the initiation of antiretroviral therapy and downregulation of viral replication in vivo. Cloning of PD-1 and its two ligands from several non-human primate species demonstrated > 95% conservation for PD-1 and PD-L2 and only about 91% homology for PD-L1. Functional studies using soluble recombinant PD-1 protein or PD-1-immunoglobulin G fusion proteins induced marked increases in the SIV-specific proliferative responses of both CD4(+) and CD8(+) T cells from rhesus macaques. The results of these studies serve as a foundation for future in vivo trials of the use of rMamu-PD-1 to potentially enhance and/or restore antiviral immune responses in vivo.

Naturally SIV-infected sooty mangabeys (SMs) remain asymptomatic despite high virus replication. Elucidating the mechanisms underlying AIDS resistance of SIV-infected SMs may provide crucial information to better understand AIDS pathogenesis. In this study, we assessed the determinants of set-point viremia in naturally SIV-infected SMs, i.e., immune control of SIV replication versus target cell limitation. We depleted CD4+ T cells in 6 naturally SIV-infected SMs by treating with humanized anti-CD4 mAb (Cdr-OKT4A-huIgG1). CD4+ T cells were depleted almost completely in blood and BM and at variable levels in mucosal tissues and LNs. No marked depletion of CD14+ monocytes was observed. Importantly, CD4+ T cell depletion was associated with a rapid, significant decline in viral load, which returned to baseline level at day 30-45, coincident with an increased fraction of proliferating and activated CD4+ T cells. Throughout the study, virus replication correlated with the level of proliferating CD4+ T cells. CD4+ T cell depletion did not induce any changes in the fraction of Tregs or the level of SIV-specific CD8+ T cells. Our results suggest that the availability of activated CD4+ T cells, rather than immune control of SIV replication, is the main determinant of set-point viral load during natural SIV infection of SMs.

Background: Our lab has previously shown that adoptive transfer of in vitro expanded autologous purified polyclonal CD4(+) T cells using anti-CD3/CD28 coated beads induced antiviral responses capable of controlling simian immunodeficiency virus (SIV) replication in vivo., Results: Expansion on anti-CD3/28 coated beads was found to induce a true polyclonal expansion as CFSE labeled cells uniformly showed dilution of the dye over several days of culture, in contrast to aliquots of the same cells subjected to mitogen stimulation. Of interest was the finding that CD4(+) T cells collected before and during early chronic SIV infection or AIDS stage did not show any or only modest differences in proliferative response or expansion kinetics. The reason for such excellent expansion properties was analyzed by the quantitation of telomerase activity in aliquots of expanding CD4(+) T cells from sample collected at various times post-infection. First, anti-CD3/28 expanded CD4(+) T cells exhibited telomerase levels 2- to 20-fold higher than the starting population of CD4(+) T cells. Moreover, while telomerase activity in ex vivo tested CD4(+) T cells was found to decrease following SIV infection and disease progression, anti-CD3/28 expansion appeared to restore significant levels of telomerase activity as no difference was noted in telomerase expression between CD4(+) T cells expanded from samples collected before or during the chronic SIV infection. When such expanded and CFSE labeled T cells were autologously transferred to monkeys, evidence for extended survival in vivo was provided as CFSE labeled cells were detected to relatively high levels in blood and spleen at 1 week post-infection., Conclusion: In summary, the data suggest that anti-CD3/28 mediated expansion of CD4(+) T cells retains its immunotherapeutic potential not only during the early stages of lentiviral infection but also at more advanced stages of disease.

IL-2 is an important cytokine required for the physiological function of CD4(+) T cells. Immunological unresponsiveness-anergy- of CD4(+) T cells is characterized by the inability of these cells to synthesize IL-2. Both progressive HIV infection leading to AIDS in humans and SIV infection in rhesus macaques (RM) are associated with dysregulation of IL-2 synthesis. In certain nonhuman primate species, such as sooty mangabeys (SM), SIV infection does not lead to AIDS. We have shown that this is associated with the resistance of the CD4(+) T cells from SM to undergo anergy in vitro. In this study, we show that CD4(+) T cells from SM spontaneously synthesize 2- to 3-fold higher levels of IL-2 than corresponding cells from RM. Proximal IL-2 promoter constructs derived from SM show significantly higher activity than the RM-derived constructs in primary CD4(+) T cells, which is associated with an element at approximately nt -200. Activity of both constructs was up-regulated by p300 and down-regulated by CREB to a similar degree. Chromatin immunoprecipitation analysis showed significantly higher binding of p300 and lower binding of CREB to the SM promoter in vivo. Two single nucleotide substitutions present in the SM sequence around position -200 and -180 seem to increase the affinity of these sites for the binding of transcription factors, one of which was identified as Oct-1. These unique characteristics of the proximal IL-2 promoter in SM therefore can represent one of the mechanisms contributing to the resistance of these cells to undergo anergy.

Dysregulation of both the cell cycle within the CD4(+) T cells and T cell responses is characteristic for pathogenic HIV infection in humans and experimental SIV infection in rhesus macaques. However, SIV infection in sooty mangabeys does not lead to either an AIDS-like disease or such CD4(+) T cell dysregulation. A previous study has highlighted a potential role for cell cycle regulatory proteins in these distinct clinical outcomes. This study was performed to characterize the effect of SIV infection on the expression of cell cycle-related molecules in CD4(+) T cells of rhesus macaques and sooty mangabeys in attempts to define activation-induced gene expression patterns associated with disease resistance or susceptibility. First, T cell receptor (TCR)-mediated cell activation induced gene expression profiles that were unique to CD4(+) T cells from SIV-naive sooty mangabeys and rhesus macaques. More importantly, distinct and reproducible gene expression patterns were detected in CD4(+) T cells as a result of in vivo SIV infection in animals from each of the two species. In addition, SIV infection in both species showed significant differential effects on TCR activation-induced expression with a reproducible alteration of 10 genes highlighted by discordant effects on expression of Cyclin D3, Cyclin B, and RAD17. Therefore SIV infection in rhesus macaques and sooty mangabeys exhibits distinct and reproducible effects on cell cycle regulation in CD4(+) T cells during T cell activation that may be the basis for disease susceptibility vs. resistance in these two species, respectively.

Background: Our laboratory has previously shown that adoptive transfer of in vitro-expanded autologous purified polyclonal CD4(+) T cells using anti-CD3/CD28-coated beads induced antiviral responses capable of controlling SIV replication in vivo., Methods: As CD4(+) T cells comprise several phenotypic and functional lineages, studies were carried out to optimize the in vitro culture conditions for maximal CD4(+) T-cell expansion, survival and delineate the phenotype of these expanded CD4(+) T cells to be linked to maximal clinical benefit., Results and Conclusions: The results showed that whereas anti-monkey CD3gamma/epsilon was able to induce T-cell proliferation and expansion in combination with antibodies against multiple co-stimulatory molecules, monkey CD3epsilon cross reacting antibodies failed to induce proliferation of macaque CD4(+) T cells. Among co-stimulatory signals, anti-CD28 stimulation was consistently superior to anti-4-1BB, CD27 or ICOS while the use of anti-CD154 failed to deliver a detectable proliferation signal. Increasing the relative anti-CD28 co-stimulatory signal relative to anti-CD3 provided a modest enhancement of expansion. Additional strategies for optimization included attempts to neutralize free radicals, enhancement of glucose uptake by T cells or addition of T-cell stimulatory cytokines. However, none of these strategies provided any detectable proliferative advantage. Addition of 10 autologous irradiated feeder cells/expanding T cell provided some enhancement of expansion; however, given the high numbers of T cell needed, this approach was deemed impractical and costly, and lower ratios of feeder to expanding T cells failed to provide such benefit. The most critical parameter for efficient expansion of purified CD4(+) T cells from multiple monkeys was the optimization of space and culture conditions at culture inception. Finally, anti-CD3/28-expanded CD4(+) T cells uniformly exhibited a central memory phenotype, absence of CCR5 expression, marked CXCR4 expression in vitro, low levels of caspase 3 but also of Bcl-2 expression.

HIV infection selectively targets CD4+ effector memory T (T EM) cells, resulting in dramatic depletion of CD4+ T cells in mucosal effector sites in early infection. Regeneration of the T EM cell compartment is slow and incomplete, even when viral replication is controlled by antiretroviral therapy (ART). Here, we demonstrate that IL-15 dramatically increases in vivo proliferation of rhesus macaque (RM) CD4+ and CD8+ T EM cells with little effect on the naive or central memory T (T CM) cell subsets, a response pattern that is quite distinct from that of either IL-2 or IL-7. T EM cells produced in response to IL-15 did not accumulate in blood. Rather, 5-bromo-2'-deoxyuridine (BrdU) labeling studies suggest that many of these cells rapidly disperse to extralymphoid effector sites, where they manifest (slow) decay kinetics indistinguishable from that of untreated controls. In RMs with uncontrolled SIV infection and highly activated immune systems, IL-15 did not significantly increase CD4+ T EM cell proliferation, but with virologic control and concomitant reduction in immune activation by ART, IL-15 responsiveness was again observed. These data suggest that therapeutic use of IL-15 in the setting of ART might facilitate specific restoration of the CD4 + T cell compartment that is the primary target of HIV with less risk of exhausting precursor T cell compartments or generating potentially deleterious regulatory subsets.

Objective: HIV infection in humans and experimental SIV infection in rhesus macaques leads to the loss of recall antigen responses, immunological anergy in CD4 T cells and AIDS. In contrast, natural infection in sooty mangabeys with SIV does not lead to disease despite high viral loads accompanied by resistance of their CD4 T cells to undergo immunological anergy. The objective of the study was to further elucidate the mechanisms that contribute to anergy resistance in CD4 T cells from sooty mangabeys and identify whether the anergy resistance is a function of a specific subset or the entire cell population., Materials and Methods: Susceptibility or resistance to anergy was analyzed in antigen specific and defined CD4 T cell subsets from peripheral blood of sooty mangabeys and rhesus macaques by using an in vitro anergy inducing protocol; expression of the anergy associated gene GRAIL was measured., Results: Antigen specific CD4 T cells from SIV disease resistant sooty mangabey are relatively resistant to the development of anergy. This resistance is associated with a lack of an upregulation of GRAIL. Conversely, rhesus macaque CD4 T cells are sensitive to anergy induction associated with upregulation of GRAIL. Furthermore the anergy resistant phenotype of sooty mangabey CD4 T cells predominantly resides in central memory cells defined either as CD4+CD45RA-CD62L+ or CD4+CD28+CD95+CCR7+., Conclusions: The maintenance of recall responses in sooty mangabeys is associated with the resistance of central memory CD4 T cells to the induction of anergy which may represent an important mechanism underlying SIV disease resistance in this species.

Using tetanus toxoid (TT) and influenza (Flu) immunization of rhesus macaques as a model, the effect of IL-2 and IL-15 on the generation and maintenance of antigen specific memory T cells was evaluated following primary and secondary immunization. Daily cytokine administration expanded primarily effector but not memory cells, while spacing cytokine administration to q3-7 days markedly enhanced TT and Flu specific memory responses. Following primary immunization, TT specific CD4 and influenza matrix protein (Flu-MP) specific CD8 effector responses were enhanced by IL-2 administration but CD8 specific memory responses were no different from cytokine non-treated monkeys. In contrast, expansion of Flu specific CD8 cells with IL-15 was only modest but resulted in significantly elevated levels of memory cells at 6 months. IL-15 also significantly enhanced early and late TT specific CD4 responses. The highest levels of primary effector and memory T cells were observed following alternate administration of both IL-2 and IL-15. Following booster immunization, however, only IL-15 appeared able to enhance CD8 T cell responses while IL-2 or IL2/IL-15 administration were less effective.

CD4(+) T-cell dysfunction highlighted by defects within the intracellular signaling cascade and cell cycle has long been characterized as a direct and/or indirect consequence of human immunodeficiency virus (HIV) infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques (RM). Dysregulation of the M phase of the cell cycle is a well-documented effect of HIV or SIV infection both in vivo and in vitro. In this study the effect of SIV infection on the modulation of two important regulators of the M phase-polo-like kinases Plk3 and Plk1-was investigated. We have previously shown that Plk3 is markedly downregulated in CD4(+) T cells from SIV-infected disease-susceptible RM but not SIV-infected disease-resistant sooty mangabeys (SM), denoting an association of downregulation with disease progression. Here we show that, in addition to the downregulation, Plk3 exhibits aberrant activation patterns in the CD4(+) T cells from SIV-infected RM following T-cell receptor stimulation. Interestingly, in vitro SIV infection of CD4(+) T cells leads to the upregulation, rather than downregulation, of Plk3, suggesting that different mechanisms operate in vitro and in vivo. In addition, CD4(+) T cells from RM with high viral loads exhibited consistent and significant upregulation of Plk1, concurrent with an aberrant activation-induced Plk1 response, suggesting complex mechanisms of SIV-induced M-phase abnormalities in vivo. Altogether this study presents a novel mechanism underlying M-phase defects observed in CD4(+) T cells from HIV or SIV-infected disease-susceptible humans and RM which may contribute to aberrant T-cell responses and disease pathogenesis.

Imaging of adoptively transferred cells in vivo by magnetic resonance imaging (MRI) could provide important information on disease-related patterns of lymphocyte homing in nonhuman primate models of AIDS. As a preliminary study to assess the feasibility of visualizing activated rhesus T cells by MRI, anti-CD3/CD28-expanded CD4+ T lymphocytes were labeled in vitro with monocrystalline iron oxide nanoparticles (MION). Intracellular incorporation of MION was determined by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrography (ICP-MS). Pretreatment with colchicine did not affect MION labeling, suggesting that cellular uptake of MION occurred by adsorptive pinocytosis or receptor-mediated endocytosis. TEM analysis revealed that MION were intracellularly compartmentalized exclusively in the cytoplasm and did not cause any measurable physiologic effects on T-cell function, including viability, proliferation, synthesis of select cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma), activation antigens (CD25 and CD69), adhesion molecules (alpha4beta7 and CD49d), and susceptibility to in vitro infection with simian immunodeficiency virus mac239. A sensitivity of 0.05% (1 MION-labeled T cell in 2000 unlabeled cells) could be achieved using T2-weighted gradient echo imaging. Furthermore, under these experimental conditions, the MRI signal did not decrease in proliferating MION-labeled CD4+ T cells over a period of 120 hours. These results indicate that intracellular labeling with MION can be a useful technique for noninvasively monitoring trafficking patterns of adoptively transferred leukocyte subsets in real-time by MRI in nonhuman primate models of AIDS.

Adoptive transfer of autologous preinfection-collected peripheral blood mononuclear cells (PBMCs) or activated CD4(+) T cells was performed in simian immunodeficiency virus (SIVmac239)-infected monkeys following short-term antiviral therapy with PMPA (9-R-[2-phosphonylmethoxypropyl] adenine). Short-term chemotherapy alone led to a transient decrease in plasma and cellular proviral DNA loads and transient rescue of gag/pol and env cytotoxic T-lymphocyte precursors (pCTLs). However, cessation of therapy allowed for SIV infection to resume its clinical course. PMPA chemotherapy coupled with infusions of either autologous pre-SIV infection-collected PBMCs or activated CD4(+) T cells led to extended control of plasma and cellular proviral DNA loads after infusion, in spite of the fact that the transfused cells were not primed against SIV. However, qualitatively different antiviral defenses were induced by infusion of unfractionated and unmanipulated PBMCs versus purified and activated CD4(+) T cells: PBMC infusions significantly favored development of SIVenv-specific pCTLs, neutralizing antibodies, and secretion of soluble noncytotoxic suppressor factors of SIV replication. In contrast, activated CD4(+) T cells predominantly promoted CTL responses to SIVgag/pol and SIVenv. In addition, infusion of influenza-primed activated CD4(+) T cells markedly enhanced influenza-specific pCTL responses, whereas infusion of similarly influenza-primed unfractionated PBMCs enhanced such pCTL responses only modestly, suggesting that the predominant immune defect after SIV infection lies in the T helper cell compartment rather than the effector cell compartment. Thus, adoptive immunotherapy with autologous "SIV naïve" CD4(+) lymphocytes was sufficient to rescue cell-mediated immune responses and induce long-term anti-SIV control and immune responses in the absence of continued antiviral chemotherapy.

Human immunodeficiency virus infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques (RM) leads to a generalized loss of immune responses involving perturbations in T-cell receptor (TCR) signaling. In contrast, naturally SIV-infected sooty mangabeys (SM) remain asymptomatic and retain immune responses despite relatively high viral loads. However, SIV infection in both RM and SM led to similar decreases in TCR-induced Lck phosphorylation. In this study, a protein tyrosine kinase (PTK) differential display method was utilized to characterize the effects of in vivo SIV infection on key signaling molecules of the CD4(+) T-cell signaling pathways. The CD4(+) T cells from SIV-infected RM, but not SIV-infected SM, showed chronic downregulation of baseline expression of MLK3, PRK, and GSK3, and symptomatically SIV-infected RM showed similar downregulation of MKK3. In vitro TCR stimulation with or without CD28 costimulation of CD4(+) T cells did not lead to the enhancement of gene transcription of these PTKs. While the CD4(+) T cells from SIV-infected RM showed a significant increase of the baseline and anti-TCR-mediated ROR2 transcription, SIV infection in SM led to substantially decreased anti-TCR-stimulated ROR2 transcription. TCR stimulation of CD4(+) T cells from SIV-infected RM (but not SIV-infected SM) led to the repression of CaMKKbeta and the induction of gene transcription of MLK2. Studies of the function of these molecules in T-cell signaling may lead to the identification of potential targets for specific intervention, leading to the restoration of T-cell responses.

Despite high viral loads, T cells from sooty mangabey (SM) monkeys that are naturally infected with SIV but remain clinically asymptomatic, proliferate and demonstrate normal Ag-specific memory recall CD4(+) T cell responses. In contrast, CD4(+) T cells from rhesus macaques (RM) experimentally infected with SIV lose Ag-specific memory recall responses and develop immunological anergy. To elucidate the mechanisms for these distinct outcomes of lentiviral infection, highly enriched alloreactive CD4(+) T cells from humans, RM, and SM were anergized by TCR-only stimulation (signal 1 alone) and subsequently challenged with anti-CD3/anti-CD28 Abs (signals 1 + 2). Whereas alloreactive CD4(+)T cells from humans and RM became anergized, surprisingly, CD4(+) T cells from SM showed marked proliferation and IL-2 synthesis after restimulation. This resistance to undergo anergy was not secondary to a global deficiency in anergy induction of CD4(+) T cells from SM since incubation of CD4(+) T cells with anti-CD3 alone in the presence of rapamycin readily induced anergy in these cells. The resistance to undergo anergy was reasoned to be due to the ability of CD4(+) T cells from SM to synthesize IL-2 when incubated with anti-CD3 alone. Analysis of phosphorylated kinases involved in T cell activation showed that the activation of CD4(+) T cells by signal 1 in SM elicited a pattern of response that required both signals 1 + 2 in humans and RM. This function of CD4(+) T cells from SM may contribute to the resistance of this species to SIV-induced disease.

Both increased lymphocyte renewal with subsequent exhaustion of the immune system and impaired T-cell renewal have been put forth to account for CD4+ T-cell depletion and development of AIDS in HIV-1-infected humans and SIV-infected nonhuman primates. In the present study, telomeric terminal restriction fragment length and telomerase activity were used as measures of proliferative activity of T lymphocytes from three nonhuman primate species before and after being infected with SIV. In peripheral blood T cells, our data show both species and T-cell-subset-specific differences in proliferative activity accompanied by different patterns of disease progression. A significant postinfection increase in telomerase/proliferative activity in CD4+ T cells from seropositive sooty mangabeys and from normal progressor rhesus macaques was associated with asymptomatic infection or delayed disease progression, respectively, whereas a decrease in telomerase/proliferative activity detected in CD4+ T cells postinfection from SIVsmmPBj14-infected pigtailed macaques was associated with rapid CD4+ T-cell depletion and disease progression. The levels of telomerase activity observed in CD4+ T cells from peripheral blood closely parallelled those seen in CD4+ T cells in lymph node samples from selected animals. Our data suggest that an increase in proliferative activity of T lymphocytes in vivo may be associated with a favorable course of SIV infection in nonhuman primates.

Although CD4+ T-cell activation has long been shown to promote infection and replication of simian immunodeficiency virus (SIV) and HIV, recent studies have documented that not all activated CD4+ T cells from human and nonhuman primates are susceptible to infection with HIV/SIV, respectively. Activation of CD4+ T cells with anti-CD3 + anti-CD28 conjugated beads led to induction of a state of anti-viral resistance to infection with strains of viruses that primarily use CCR5 as a coreceptor. The studies reported herein were designed to address the mechanism by which anti-CD3 + anti-CD28-induced stimulation in turn induced antiviral resistance. Results of these studies show that the anti-viral resistance induced by activation of CD4+ T cells with anti-CD3 + anti-CD28 is primarily conferred by the synthesis of tumor necrosis factor-alpha (TNF-alpha), and highlight a unique regulatory role for TNF-alpha in regulating synthesis of MIP-1alpha, MIP-1beta, and regulated-on-activation normal T-expressed and secreted cells, which contributes to this state of antiviral resistance to R5-tropic strains of HIV/SIV. However, while TNF-alpha has a protective role in antiviral resistance of activated CD4+ T cells to R5-tropic viruses, it enhances CXCR4 expression of CD4+ T cells and mediates increased susceptibility to infection with X4-tropic strains of HIV and recombinant SIVs. The results of the studies reported herein also suggest that it is not the Th1 v/s Th2 cytokine profile but the mode of CD4+ T-cell activation that dictates the synthesis of distinct cytokines which regulate the expression of chemokines and chemokine receptors which in turn regulate and confer susceptibility/resistance to R5 v/s X4-tropic HIV and SIV.

Previous studies have shown that in vitro culture of human CD4+ T cells with antibodies to CD3 and CD28 immobilized on beads induced an antiviral effect to HIV-1 infection. Herein, we have used CD4+ T cells from nonhuman primates to address issues critical for use of such cells for therapy and immune reconstitution of humans and nonhuman primates infected with HIV and simian immunovirus (SIV). These studies include definition of the kinetics of the antiviral effect, the relative stability of the acquired phenotype, and whether such activated and expanded CD4+ T cells retain their immune function. Results of our studies show that antiviral effect is induced rapidly following activation with anti-CD3/CD28-coated beads. Additionally, the antiviral effect is not stable in these cells and requires continuous culture with anti-CD3/CD28 beads. Removal of CD4+ T cells from anti-CD3/CD28 stimulation renders these cells susceptible to infection, demonstrating that the resistant phenotype is not stable in these cultures. However, anti-CD3/CD28 expanded CD4+ T cells do retain immune function. Thus, although these findings imply a note of caution for therapeutic strategies aimed at providing patients with virus-resistant CD4+ T cells, the present study suggests that transfusion of such cells with retained immune function may have immune restoration capability.

Isolated intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) from jejunum of SIV infected animals were examined for alterations in basal cytokine expression by RT-PCR. Remarkable changes in IFNgamma and IL-10 RNA levels were observed in IEL and LPL in SIV infection while IL-4 and IL-2 RNA levels remained unaltered. In addition, the CD4+ and CD8+ LPL were examined for intracellular cytokine production following mitogenic activation by flowcytometry. Both CD4+ and CD8+ T lymphocytes in intestinal mucosa retained the potential to produce IFNgamma in response to mitogenic stimulation in vitro, without a remarkable change in IL-4 production. The dominant IFNgamma cytokine response could be one of the major contributing factors in SIV associated enteropathy.

Monocytes from SIV/SMM infected sooty mangabeys and rhesus macaques were incubated in vitro with live SIV/SMM. The reverse transcriptase (RT) activity in the supernatant fluids of the monocyte cultures of the former species was higher than the RT activity in the latter species. No differences were found in the supernatant fluid of similar cultures of CD4+ T cells from both these species. Autologous (but not allogeneic) CD8+ T cells from SIV infected mangabeys and rhesus macaques inhibited SIV replication in vitro. The suppression appeared more marked in monocytes from the mangabey species. These in vitro differences may relate to the clinically asymptomatic state of the sooty mangabeys and the disease-susceptible state of the rhesus macaques.