Divergent Kinetics of Proliferating T Cell Subsets in Simian Immunodeficiency Virus (SIV) Infection: SIV Eliminates the “First Responder” CD4+ T Cells in Primary Infection

Although increased lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been reported in blood, there is little information on cell turnover in tissues, particularly in primary SIV infection. Here we examined the levels of proliferating T cell subsets in mucosal and peripheral lymphoid tissues of adult macaques throughout SIV infection. To specifically label cells in S-phase division, all animals were inoculated with bromodeoxyuridine 24 h prior to sampling. In healthy macaques, the highest levels of proliferating CD4⁺ and CD8⁺ T cells were in blood and, to a lesser extent, in spleen. Substantial percentages of proliferating cells were also found in intestinal tissues, including the jejunum, ileum, and colon, but very few proliferating cells were detected in lymph nodes (axillary and mesenteric). Moreover, essentially all proliferating T cells in uninfected animals coexpressed CD95 and many coexpressed CCR5 in the tissues examined. Confocal microscopy also demonstrated that proliferating cells were substantial viral target cells for SIV infection and viral replication. After acute SIV infection, percentages of proliferating CD4⁺ and CD8⁺ T cells were significantly higher in tissues of chronically infected macaques and macaques with AIDS than in those of the controls. Surprisingly, however, we found that proliferating CD4⁺ T cells were selectively decreased in very early infection (8 to 10 days postinoculation [dpi]). In contrast, levels of proliferating CD8⁺ T cells rapidly increased after SIV infection, peaked by 13 to 21 dpi, and thereafter remained significantly higher than those in the controls. Taken together, these findings suggest that SIV selectively infects and destroys dividing, nonspecific CD4⁺ T cells in acute infection, resulting in homeostatic changes and perhaps continuing loss of replication capacity to respond to nonspecific and, later, SIV-specific antigens.     

Anti-α4 Antibody Treatment Blocks Virus Traffic to the Brain and Gut Early,and Stabilizes CNS Injury Late in Infection

Four SIV-infected monkeys with high plasma virus and CNS injury were treated with an anti-α4 blocking antibody (natalizumab) once a week for three weeks beginning on 28 days post-infection (late). Infection in the brain and gut were quantified, and neuronal injury in the CNS was assessed by MR spectroscopy, and compared to controls with AIDS and SIV encephalitis. Treatment resulted in stabilization of ongoing neuronal injury (NAA/Cr by 1H MRS), and decreased numbers of monocytes/macrophages and productive infection (SIV p28⁺, RNA⁺) in brain and gut. Antibody treatment of six SIV infected monkeys at the time of infection (early) for 3 weeks blocked monocyte/macrophage traffic and infection in the CNS, and significantly decreased leukocyte traffic and infection in the gut. SIV – RNA and p28 was absent in the CNS and the gut. SIV DNA was undetectable in brains of five of six early treated macaques, but proviral DNA in guts of treated and control animals was equivalent. Early treated animals had low-to-no plasma LPS and sCD163. These results support the notion that monocyte/macrophage traffic late in infection drives neuronal injury and maintains CNS viral reservoirs and lesions. Leukocyte traffic early in infection seeds the CNS with virus and contributes to productive infection in the gut. Leukocyte traffic early contributes to gut pathology, bacterial translocation, and activation of innate immunity.     

The role of CD101-expressing CD4 T cells in HIV/SIV pathogenesis and persistence

Despite the advent of effective antiretroviral therapy (ART), human immunodeficiency virus (HIV) continues to pose major challenges, with extensive pathogenesis during acute and chronic infection prior to ART initiation and continued persistence in a reservoir of infected CD4 T cells during long-term ART. CD101 has recently been characterized to play an important role in CD4 Treg potency. Using the simian immunodeficiency virus (SIV) model of HIV infection in rhesus macaques, we characterized the role and kinetics of CD101⁺ CD4 T cells in longitudinal SIV infection. Phenotypic analyses and single-cell RNAseq profiling revealed that CD101 marked CD4 Tregs with high immunosuppressive potential, distinct from CD101^- Tregs, and these cells also were ideal target cells for HIV/SIV infection, with higher expression of CCR5 and α4β7 in the gut mucosa. Notably, during acute SIV infection, CD101⁺ CD4 T cells were preferentially depleted across all CD4 subsets when compared with their CD101^- counterpart, with a pronounced reduction within the Treg compartment, as well as significant depletion in mucosal tissue. Depletion of CD101⁺ CD4 was associated with increased viral burden in plasma and gut and elevated levels of inflammatory cytokines. While restored during long-term ART, the reconstituted CD101⁺ CD4 T cells display a phenotypic profile with high expression of inhibitory receptors (including PD-1 and CTLA-4), immunsuppressive cytokine production, and high levels of Ki-67, consistent with potential for homeostatic proliferation. Both the depletion of CD101⁺ cells and phenotypic profile of these cells found in the SIV model were confirmed in people with HIV on ART. Overall, these data suggest an important role for CD101-expressing CD4 T cells at all stages of HIV/SIV infection and a potential rationale for targeting CD101 to limit HIV pathogenesis and persistence, particularly at mucosal sites.     

The immunopathogenesis of retroviral diseases: No immunophenotypic alterations in T,B, and NK cell subsets in SIVmac239-challenged rhesus macaques protected by SIVΔnef vaccination

Abstract: Immunophenotype analysis was used to characterize circulating lymphocyte subset levels in both rhesus monkeys that were chronically infected with SIV_mac239 and in those that had resisted SIV_mac239 infection as a result of prior vaccination with an attenuated SIV strain. Alterations in T, NK, and B cell subsets were compared with those previously identified in humans chronically infected with HIV [8–11, 14, 22]. The well-known decrease in CD4⁺ cell levels was observed in the SIV_mac239-infected animals. However, these animals had relatively little activation of circulating CD8⁺ T cells as compared with uninfected monkeys. This contrasts with chronically HIV-infected humans who have substantial activation of circulating CD8⁺ cells as evidenced by elevated HLA-DR and CD38 antigen expression on CD8⁺ cells as well as substantially increased percentages and numbers of total CD8⁺ cells. NK cells of the SIV_mac239-infected animals, on the other hand, demonstrated the same changes recently described in HIV-infected humans, i.e., a decrease in circulating percentages and a decreased amount of FcRIII (CD 16). B cell percentages were markedly increased in the SIV_mac239-infected animals, a finding also noted in some children with HIV infection but not in HIV-infected adults. SIVΔnef-vaccinated/SIV_mac239-challenged animals showed none of the immune alterations found in the SIV_mac239-infected monkeys, providing further confirmation of lack of SIV disease in these vaccinated animals.     

Factors contributing to spontaneous Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected macaques

Background A cohort of SIV‐infected macaques had been used to investigate the effect of dietary supplement, immune status, SIV/AIDS disease progression and serum micronutrients levels on spontaneous acquisition of Enterocytozoon bieneusi infection in SIV‐infected macaques. Methods Twenty‐four SIV‐infected macaques were randomized into 2 groups. One group received a vitamin/mineral supplementation and a second group received a placebo. Both groups were examined for E. bieneusi infection. Results SIV‐infected macaques were more prone to acquire E. bieneusi with the progression of SIV/AIDS, and the increased shedding of infectious spores was directly associated with decreased CD4 lymphocyte and increased circulating SIV, in both supplemented and unsupplemented groups of animals. Dietary supplementation, body composition factors and serum micronutrients levels however had no association with the acquisition of E. bieneusi infection in these animals. Conclusions Acquisition of E. bieneusi infection is related to SIV disease progression, CD4 counts and viral load but independent of changes in body composition and serum micronutrient levels.     

CD4 Depletion in SIV-Infected Macaques Results in Macrophage and Microglia Infection with Rapid Turnover of Infected Cells

In rhesus macaques (RMs), experimental depletion of CD4⁺ T-cells prior to SIV infection results in higher viremia and emergence of CD4-independent SIV-envelopes. In this study we used the rhesus recombinant anti-CD4 antibody CD4R1 to deplete RM CD4⁺ T-cells prior to SIVmac₂₅₁ infection and investigate the sources of the increased viral burden and the lifespan of productively infected cells. CD4-depleted animals showed (i) set-point viral load two-logs higher than controls; (ii) macrophages constituting 80% of all SIV vRNA⁺ cells in lymph node and mucosal tissues; (iii) substantial expansion of pro-inflammatory monocytes; (iv) aberrant activation and infection of microglial cells; and (v) lifespan of productively infected cells significantly longer in comparison to controls, but markedly shorter than previously estimated for macrophages. The net effect of CD4⁺ T-cell depletion is an inability to control SIV replication and a shift in the tropism of infected cells to macrophages, microglia, and, potentially, other CD4-low cells which all appear to have a shortened in vivo lifespan. We believe these findings have important implications for HIV eradication studies.     

Conditional CD8+ T cell escape during acute simian immunodeficiency virus infection

CD8⁺ T cell responses rapidly select viral variants during acute human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. We used pyrosequencing to examine variation within three SIV-derived epitopes (Gag_386-394GW9, Nef_103-111RM9, and Rev_59-68SP10) targeted by immunodominant CD8⁺ T cell responses in acutely infected Mauritian cynomolgus macaques. In animals recognizing all three epitopes, variation within Rev_59-68SP10 was associated with delayed accumulation of variants in Gag_386-394GW9 but had no effect on variation within Nef_103-111RM9. This demonstrates that the entire T cell repertoire, rather than a single T cell population, influences the timing of immune escape, thereby providing the first example of conditional CD8⁺ T cell escape in HIV/SIV infection.     

Peripheral NKT Cells in Simian Immunodeficiency Virus-Infected Macaques

NKT cells are a specialized population of T lymphocytes that have an increasingly recognized role in immunoregulation, including controlling the response to viral infections. The characteristics of NKT cells in the peripheral blood of macaques during simian immunodeficiency virus (SIV) or chimeric simian/human immunodeficiency virus (HIV) (SHIV) infection were assessed. NKT cells comprised a mean of 0.19% of peripheral blood lymphocytes across the 64 uninfected macaques studied. Although the range in the percentages of NKT cells was large (0 to 2.2%), levels were stable over time within individual macaques without SIV/SHIV infection. The majority of NKT cells in macaques were CD4⁺ (on average 67%) with smaller populations being CD8⁺ (21%) and CD4/CD8 double positive (13%). A precipitous decline in CD4⁺ NKT cells occurred in all six macaques infected with CXCR4-tropic SHIV_mn229 early after infection, with a concomitant rise in CD8⁺ NKT cells in some animals. The depletion of CD4⁺ NKT cells was tightly correlated with the depletion of total CD4⁺ T cells. R5-tropic SIV_mac251 infection of macaques resulted in a slower and more variable decline in CD4⁺ NKT cells, with animals that were able to control SIV virus levels maintaining higher levels of CD4⁺ NKT cells. An inverse correlation between the depletion of total and CD4⁺ NKT cells and SIV viral load during chronic infection was observed. Our results demonstrate the infection-driven depletion of peripheral CD4⁺ NKT cells during both SHIV and SIV infection of macaques. Further studies of the implications of the loss of NKT cell subsets in the pathogenesis of HIV disease are needed.     

Resistance to Infection,Early and Persistent Suppression of Simian Immunodeficiency Virus SIVmac251 Viremia,and Significant Reduction of Tissue Viral Burden after Mucosal Vaccination in Female Rhesus Macaques

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIV_mac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIV_mac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8⁺ T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4⁺ central memory and CD4⁺/α4β7⁺ T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4⁺, and CD8⁺ T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.     

Asymptomatic Simian Immunodeficiency Virus Infection Decreases Blood CD4+ T Cells by Accumulating Recirculating Lymphocytes in the Lymphoid Tissues

Declining blood CD4⁺ T-cell counts mark the progress of simian immunodeficiency virus (SIV) disease in macaques and model the consequences of untreated human immunodeficiency virus infection in humans. However, blood lymphocytes are only a fraction of the recirculating lymphocyte pool, and their numbers are affected by cell synthesis, cell depletion, and distribution among blood and lymphoid tissue compartments. Asymptomatic, SIV-infected macaques maintained constant and nearly normal numbers of recirculating lymphocytes despite the decline in CD4⁺ T-cell counts. Substantial depletion was detected only when blood CD4⁺ T-cell counts fell below 300/μl. In asymptomatic animals, changes in CD4⁺ T-cell distribution were more important than lymphocyte depletion for controlling the blood cell levels.     

Resistance to Intranasal Infection with Balamuthia mandrillaris Amebae is T‐Cell Dependent

T and B cell‐deficient BALB/c SCID mice become severely ill and die of amebic encephalitis after intranasal infection with Balamuthia mandrillaris, while adult immunocompetent BALB/c wild‐type (WT) mice are resistant. To further investigate the role of lymphocytes in protection from Balamuthia amebic encephalitis (BAE), SCID mice were reconstituted with and WT mice selectively depleted of lymphocytes before infection. Reconstitution of SCID mice with whole spleen cells from WT mice rendered the recipients as resistant to BAE as WT mice. SCID mice that had received spleen cells depleted of CD4⁺ T cells remained susceptible. When adult WT mice were depleted of both CD4⁺ and CD8⁺ T cells or of CD4⁺ T cells alone, these mice also became susceptible to BAE. Depletion of CD8⁺ T cells alone increased susceptibility only marginally. All morbidity and mortality data were underpinned by histological analysis of the brain.     

Increased loss of CCR5+ CD45RA- CD4+ T cells in CD8+ lymphocyte-depleted Simian immunodeficiency virus-infected rhesus monkeys

Previously we have shown that CD8(+) T cells are critical for containment of simian immunodeficiency virus (SIV) viremia and that rapid and profound depletion of CD4(+) T cells occurs in the intestinal tract of acutely infected macaques. To determine the impact of SIV-specific CD8(+) T-cell responses on the magnitude of the CD4(+) T-cell depletion, we investigated the effect of CD8(+) lymphocyte depletion during primary SIV infection on CD4(+) T-cell subsets and function in peripheral blood, lymph nodes, and intestinal tissues. In peripheral blood, CD8(+) lymphocyte-depletion changed the dynamics of CD4(+) T-cell loss, resulting in a more pronounced loss 2 weeks after infection, followed by a temporal rebound approximately 2 months after infection, when absolute numbers of CD4(+) T cells were restored to baseline levels. These CD4(+) T cells showed a markedly skewed phenotype, however, as there were decreased levels of memory cells in CD8(+) lymphocyte-depleted macaques compared to controls. In intestinal tissues and lymph nodes, we observed a significantly higher loss of CCR5(+) CD45RA(-) CD4(+) T cells in CD8(+) lymphocyte-depleted macaques than in controls, suggesting that these SIV-targeted CD4(+) T cells were eliminated more efficiently in CD8(+) lymphocyte-depleted animals. Also, CD8(+) lymphocyte depletion significantly affected the ability to generate SIV Gag-specific CD4(+) T-cell responses and neutralizing antibodies. These results reemphasize that SIV-specific CD8(+) T-cell responses are absolutely critical to initiate at least partial control of SIV infection.     

Biologically-Directed Modeling Reflects Cytolytic Clearance of SIV-Infected Cells In Vivo in Macaques

The disappointing outcomes of cellular immune-based vaccines against HIV-1 despite strong evidence for the protective role of CD8⁺ T lymphocytes (CTLs) has prompted revisiting the mechanisms of cellular immunity. Prior data from experiments examining the kinetics of Simian Immunodeficiency Virus (SIV) clearance in infected macaques with or without in vivo CD8 depletion were interpreted as refuting the concept that CTLs suppress SIV/HIV by direct killing of infected cells. Here we briefly review the biological evidence for CTL cytolytic activity in viral infections, and utilize biologically-directed modeling to assess the possibility of a killing mechanism for the antiviral effect of CTLs, taking into account the generation, proliferation, and survival of activated CD4⁺ and CD8⁺ T lymphocytes, as well as the life cycle of the virus. Our analyses of the published macaque data using these models support a killing mechanism, when one considers T lymphocyte and HIV-1 lifecycles, and factors such as the eclipse period before release of virions by infected cells, an exponential pattern of virion production by infected cells, and a variable lifespan for acutely infected cells. We conclude that for SIV/HIV pathogenesis, CTLs deserve their reputation as being cytolytic.     

CD4+ T Cells Support Production of Simian Immunodeficiency Virus Env Antibodies That Enforce CD4-Dependent Entry and Shape Tropism In Vivo

CD4⁺ T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4⁺ T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4⁺ T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV⁺) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV⁺ plasma neutralization. However, plasma from SIV-infected CD4⁺ T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4⁺ T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4⁺ target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.     

In Vivo CD8+ T-Cell Suppression of SIV Viremia Is Not Mediated by CTL Clearance of Productively Infected Cells

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.     

NK cells and monocytes modulate primary HTLV-1 infection

We investigated the impact of monocytes, NK cells, and CD8⁺ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1_WT) or to the HTLV-1_p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8⁺ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8⁺ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1_WT. In contrast, HTLV-1_p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1_WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1_p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1_WT and HTLV-1_p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 “don’t-eat-me” signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.     

Un vivo depletion of murine CD8 positive T cells impairs survival during infection with a highly virulent strain ofCryptococcus neoformans

Cell-mediated immunity plays an important but incompletely understood role in host defense againstCryptococcus neoformans. Because of their multiple capacities as cytokine-secreting cells, cytotoxic cells, and antigen-specific suppressor cells, CD8 positive T lymphocytes could potentially either enhance or impair host defense againstC. neoformans. To determine whether CD8 T cells enhance or inhibit host defence during an infection with a highly virulent strain ofC. neoformans, we examined the effect of in vivo CD8 cell depletion on suNival and on the number of organisms in mice infected by either the intratracheal or intravenous routes. Adequacy of depletion was confirmed both phenotypically and functionally. Regardless of the route of infection, we found that survival of mice depleted of CD8 T cells was significantly reduced compared to undepleted mice. Surprisingly, however, CD8 depletion did not alter organism burden measured by quantitative CFU assay in mice infected by either route. These data demonstrate that CD8 positive T cells participate in the immune response to a highly virulent strain ofC. neoformans. By contrast to minimally virulent isolates that do not cause a life threatening infection, the immune response to a highly virulent isolate does not alter the burden of organisms, but does enhance host defense as it is necessary for the optimal survival of infected mice.Abbreviations ³H-TdR ³H-thmidine - CFU colony forming units - FITC Fluorescein isothiocyanate - MLR mixed lymphocyte reaction - PBS phosphate buffered saline