GagCM9-Specific CD8+ T Cells Expressing Limited Public TCR Clonotypes Do Not Suppress SIV Replication In Vivo

Several lines of evidence suggest that HIV/SIV-specific CD8+ T cells play a critical role in the control of viral replication. Recently we observed high levels of viremia in Indian rhesus macaques vaccinated with a segment of SIVmac239 Gag (Gag45–269) that were subsequently infected with SIVsmE660. These seven Mamu-A*01+ animals developed CD8+ T cell responses against an immunodominant epitope in Gag, GagCM9, yet failed to control virus replication. We carried out a series of immunological and virological assays to understand why these Gag-specific CD8+ T cells could not control virus replication in vivo. GagCM9-specific CD8+ T cells from all of the animals were multifunctional and were found in the colonic mucosa. Additionally, GagCM9-specific CD8+ T cells accessed B cell follicles, the primary residence of SIV-infected cells in lymph nodes, with effector to target ratios between 20–250 GagCM9-specific CD8+ T cells per SIV-producing cell. Interestingly, vaccinated animals had few public TCR clonotypes within the GagCM9-specific CD8+ T cell population pre- and post-infection. The number of public TCR clonotypes expressed by GagCM9-specific CD8+ T cells post-infection significantly inversely correlated with chronic phase viral load. It is possible that these seven animals failed to control viral replication because of the narrow TCR repertoire expressed by the GagCM9-specific CD8+ T cell population elicited by vaccination and infection

Humanized Rag1−/−γc−/− Mice Support Multilineage Hematopoiesis and Are Susceptible to HIV-1 Infection via Systemic and Vaginal Routes

Several new immunodeficient mouse models for human cell engraftment have recently been introduced that include the Rag2−/−γc−/−, NOD/SCID, NOD/SCIDγc−/− and NOD/SCIDβ2m−/− strains. Transplantation of these mice with CD34+ human hematopoietic stem cells leads to prolonged engraftment, multilineage hematopoiesis and the capacity to generate human immune responses against a variety of antigens. However, the various mouse strains used and different methods of engrafting human cells are beginning to illustrate strain specific variations in engraftment levels, duration and longevity of mouse life span. In these proof-of-concept studies we evaluated the Balb/c-Rag1−/−γ−/− strain for engraftment by human fetal liver derived CD34+ hematopoietic cells using the same protocol found to be effective for Balb/c-Rag2−/−γc−/− mice. We demonstrate that these mice can be efficiently engrafted and show multilineage human hematopoiesis with human cells populating different lymphoid organs. Generation of human cells continues beyond a year and production of human immunoglobulins is noted. Infection with HIV-1 leads to chronic viremia with a resultant CD4 T cell loss. To mimic the predominant sexual viral transmission, we challenged humanized Rag1−/−γc−/− mice with HIV-1 via vaginal route which also resulted in chronic viremia and helper T cell loss. Thus these mice can be further exploited for studying human pathogens that infect the human hematopoietic system in an in vivo setting

Follicular Regulatory CD8 T Cells Impair the Germinal Center Response in SIV and Ex Vivo HIV Infection.

During chronic HIV infection, viral replication is concentrated in secondary lymphoid follicles. Cytotoxic CD8 T cells control HIV replication in extrafollicular regions, but not in the follicle. Here, we show CXCR5hiCD44hiCD8 T cells are a regulatory subset differing from conventional CD8 T cells, and constitute the majority of CD8 T cells in the follicle. This subset, CD8 follicular regulatory T cells (CD8 TFR), expand in chronic SIV infection, exhibit enhanced expression of Tim-3 and IL-10, and express less perforin compared to conventional CD8 T cells. CD8 TFR modestly limit HIV replication in follicular helper T cells (TFH), impair TFH IL-21 production via Tim-3, and inhibit IgG production by B cells during ex vivo HIV infection. CD8 TFR induce TFH apoptosis through HLA-E, but induce less apoptosis than conventional CD8 T cells. These data demonstrate that a unique regulatory CD8 population exists in follicles that impairs GC function in HIV infection

Low levels of SIV-specific CD8+ T cells in germinal centers characterizes acute SIV infection.

CD8+ T cells play an important role in controlling of HIV and SIV infections. However, these cells are largely excluded from B cell follicles where HIV and SIV producing cells concentrate during chronic infection. It is not known, however, if antigen-specific CD8+ T cells are excluded gradually as pathogenesis progresses from early to chronic phase, or this phenomenon occurs from the beginning infection. In this study we determined that SIV-specific CD8+ T cells were largely excluded from follicles during early infection, we also found that within follicles, they were entirely absent in 60% of the germinal centers (GCs) examined. Furthermore, levels of SIV-specific CD8+ T cells in follicular but not extrafollicular areas significantly correlated inversely with levels of viral RNA+ cells. In addition, subsets of follicular SIV-specific CD8+ T cells were activated and proliferating and expressed the cytolytic protein perforin. These studies suggest that a paucity of SIV-specific CD8+ T cells in follicles and complete absence within GCs during early infection may set the stage for the establishment of persistent chronic infection

CD8 T_FR induces T_FH apoptosis via HLA-E.

<p>Human tonsil cells were sorted to isolate CD8 T_FR, CD8 conv, and T_FH, spinoculated with R5-tropic HIV, and cultured for 2 days. (A) Representative flow plots showing the percent of Annexin-V+ T_FH in co-culture at 1:1 ratio with CD8 T_FR or CD8 conv 2 days after R5-spinoculatoin. (B) Results from a total of 6 tonsil for mock-, X4-, and R5-spinoculation (isotype n = 3) in A. Co-cultures were also performed with HLA-E blocking antibody or isotype controls (500 ng/μl). (C) Number of T_FH per microliter on day 0 and day 2 when cultured alone (circle, triangle), 1:1 with CD8 T_FR (square), or 1:1 with conventional CD8 T cells (upside down triangle) (n = 6). Statistical significance was determined by Wilcoxon matched-pairs tests and is displayed as * = p<0.05 and ** = p<0.01.</p

Human tonsil CD8 T_FR inhibit T_FH and GC B cell function.

<p>Tonsil cells were sorted to isolate CD8 T_FR, CD8 conv, CD3+CD8-CXCR5+ T_FH, and CD19+CD38+ GC B cells and X4- or R5-spinoculated. Cells were then co-cultured at indicated ratios for 2 days and analyzed. (A) IL-21 production by T_FH with increasing (left to right) number of CD8 T_FR (n = 4). (B) Representative examples from X4- and R5-spinoculations showing IL-21 production by T_FH alone, 1:1 with CD8 T_FR, 1:1 with CD8 T_FR and anti-Tim3 antibody (500 ng/μl; right panels), and 1:1 with CD8 T_FR and an isotype control antibody (500 ng/μl). (C) Results from a total of 6 tonsils (isotype n = 3) as described in B. (D) IgG production in X4-spinoculated cultures with 2.5 μg/mL CpG-B stimulation in CD8 T_FR, T_FH, and B cell co-cultures as measured by ELISA. All co-cultures are 1:1 (n = 7). Statistical significance was determined by non-parametric Wilcoxon matched-pairs tests (B) or one-way ANOVA (Friedman test, C) and is displayed as * = p<0.05, ** = p<0.01 and *** = p<0.001.</p

CD8 T_FR suppress IL-21 production in T_FH via Tim-3 and induce apoptosis via HLA-E in SIV-infected rhesus macaques.

<p>Disaggregated cells from lymphoid tissues of SIV-infected rhesus macaques (n = 2) were sorted for T_FH and CD8 T_FR and co-cultured at a 1:1 ratio for 2 days with or without HLA-E blocking antibody and analyzed by flow cytometry. (A) Flow gating showing the percent T_FH producing IL-21, and (B) percent T_FH expressing Annexin-V (n = 2).</p