Faculty Bibliography

HIV-1 infection of resting CD4 T cells plays a crucial and numerically dominant role during virus transmission at mucosal sites and during subsequent acute replication and T cell depletion. Resveratrol and pterostilbene are plant stilbenoids associated with several health promoting benefits. Resveratrol has been shown to inhibit replication of several viruses, including herpes simplex 1 and 2, papillomaviruses, SARS virus and influenza virus. Alone, resveratrol does not inhibit HIV-1 infection of activated T cells, but it does synergize with nucleoside reverse transcriptase inhibitors in these cells to inhibit reverse transcription. Here, we demonstrate that resveratrol and pterostilbene completely block HIV-1 infection at low micromolar dose in resting CD4 T cells, primarily at the reverse transcription step. The anti-HIV effect was fully reversed by exogenous deoxynucleosides and Vpx, a simian immunodeficiency virus protein that increases dNTP levels. These findings are consistent with the reported ability of resveratrol to inhibit ribonucleotide reductase and to lower dNTP levels in cells. This study supports the potential use of resveratrol, pterostilbene or related compounds as adjuvants in anti-HIV pre-exposure prophylaxis (PrEP) formulations.

A long-lived reservoir of latently infected T cells prevents antiretroviral therapy from eliminating HIV-1 infection. Furthering our understanding of the dynamics of latency generation and maintenance is therefore vital to improve treatment outcome. Using mathematical models and experiments, we suggest that the death of latently infected cells brought about by pyroptosis, or to a lesser extent by superinfection, might be key mechanisms to account for the size and composition of the latent reservoir. Pyroptosis is a form of cell death that occurs in a resting (and thus latently infected) T cell when a productively infected cell attempts cell-to-cell transmission of virus. Superinfection of latently infected cells by productive virus could similarly remove those cells through active virus replication and resulting cytopathicity. The mathematical models presented can explain a number of previously published clinical observations including latent reservoir size and the relationships to viral load in acute HIV infection, measurements of the latent reservoir in chronic infection, and the replacement of wild-type virus by CTL escape mutants within the latent reservoir. Basic virus dynamics models of latency that do not take into account pyroptosis, superinfection, or other potential complexities cannot account for the data.

Background: HIV replication is concentrated within follicular T cells in B cell follicles (F) during asymptomatic disease. Whether T follicular regulatory cells (TFR) are more permissive than other subsets and what factors drive permissivity are not understood.
Method(s): Disaggregated HIV- human tonsil cells were spinoculated with R5-tropic GFP reporter virus (NLYUV3-GFP), 3 R5-tropic transmitted/founder viruses (T/F) (CH58, CH470, CH40) or mock spinoculated at day 0 and cultured for 2 days. Some cells were cultured for 2 days alone or with imatinib, a T cell proliferation inhibitor, prior to spinoculation. GFP or p24 expression were determined by flow cytometry. Due to CD4 downregulation by HIV, we gated on CD3+CD8- cells, and defined subsets as: extrafollicular (EF; CXCR5-CD25- CD127+/-), EF Treg (CXCR5-CD25+CD127-), TFH (CXC5+CD25-CD127+/-) and TFR (CXCR5+CD25+CD127-). CCR5, HLA-DR/CD38, CD69 and Ki67 expression were determined prior to spinoculation. Lymph node cells (LN) from HIV+ untreated subjects were sorted into EF, EF Treg, TFH and TFR, and RNA quantified by RT PCR. Data were log transformed. Mixedeffects models and repeated-measures ANOVA were used with pairwise tests if overall p<0.05. Geometric means and 95% CIs are reported.
Result(s): GFP expression differed by cell type (p<=0.001) with %GFP+ cells in TFR (2.0%; 1.3, 2.8) > EF Treg (0.6%; 0.4, 0.9), TFH (0.3%; 0.2, 0.5) > EF (0.09%; 0.04, 0.18), and TFR > TFH (n=6; p<0.01). The same pattern was observed for p24 Ag expression in T/F infections (n=6; overall p<0.01). %Ki67 expression differences between cell subsets at day 0 (n=6; p<0.001) paralleled differences in %GFP and %p24 expression, but CCR5, CD95, and CD69 expression did not. At day 0 for each 1 log10 increase in %Ki67 expression, there was a 0.8 (0.6, 1.1) log10 increase in %GFP expression (p<0.001). Compared to untreated cells, imatinib reduced Ki67 expression by 34% (6, 53; p=0.02) and reduced GFP expression by 77% (61, 87; p<0. 001), without affecting viability. In sorted HIV+ LN, TFR harbored more (1167; 207, 6577) HIV RNA copies/ng total RNA than TFH (585; 113, 3020), EF Treg (516; 163, 1641), and EF (71; 10, 507) (n=6; overall p=0.04; pairwise p<0.05). LN Ki67 expression was higher in TFR (20%; 13, 30) than TFH (10%; 7, 15) and higher in EF Treg (19%; 15, 24) than EF (4.7%; 2, 10) (n=6; overall p=0.01; pairwise p<0.05).
Conclusion(s): TFR are highly permissive to HIV, likely due to heightened proliferation, and harbor the highest concentrations of HIV RNA in vivo

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.

HIV-1 replication is concentrated within CD4+ T cells in B cell follicles of secondary lymphoid tissues during asymptomatic disease. Limited data suggest that a subset of T follicular helper cells (TFH) within germinal centers (GC) is highly permissive to HIV-1. Whether GC TFH are the major HIV-1 virus-producing cells in vivo has not been established. In this study, we investigated TFH permissivity to HIV-1 ex vivo by spinoculating and culturing tonsil cells with HIV-1 GFP reporter viruses. Using flow cytometry, higher percentages of GC TFH (CXCR5highPD-1high) and CXCR5+programmed cell death-1 (PD-1)low cells were GFP+ than non-GC TFH (CXCR5+PD-1intermediate) or extrafollicular (EF) (CXCR5-) cells. When sorted prior to spinoculation, however, GC TFH were substantially more permissive than CXCR5+PD-1low or EF cells, suggesting that many GC TFH transition to a CXCR5+PD-1low phenotype during productive infection. In situ hybridization on inguinal lymph node sections from untreated HIV-1-infected individuals without AIDS revealed higher frequencies of HIV-1 RNA+ cells in GC than non-GC regions of follicle or EF regions. Superinfection of HIV-1-infected individuals' lymph node cells with GFP reporter virus confirmed the permissivity of follicular cells ex vivo. Lymph node immunostaining revealed 96% of CXCR5+CD4+ cells were located in follicles. Within sorted lymph node cells from four HIV-infected individuals, CXCR5+ subsets harbored 11-66-fold more HIV-1 RNA than CXCR5- subsets, as determined by RT PCR. Thus, GC TFH are highly permissive to HIV-1, but downregulate PD-1 and, to a lesser extent, CXCR5 during HIV-1 replication. These data further implicate GC TFH as the major HIV-1-producing cells in chronic asymptomatic HIV-1 infection.

HIV-1 infection leads to the progressive depletion of the CD4 T cell compartment by various known and unknown mechanisms. In vivo, HIV-1 infects both activated and resting CD4 T cells, but in vitro, in the absence of any stimuli, resting CD4 T cells from peripheral blood are resistant to infection. This resistance is generally attributed to an intracellular environment that does not efficiently support processes such as reverse transcription, resulting in abortive infection. Here, we show that in vitro HIV-1 infection of resting CD4 T cells induces substantial cell death, leading to abortive infection. In vivo, however, various micro-environmental stimuli in lymphoid and mucosal tissues provide support for HIV-1 replication. For example, common gamma chain cytokines (CGCC) such as IL-7 render resting CD4 T cell permissible to HIV-1 infection without inducing T cell activation. Here we find that CGCC primarily allow productive infection by preventing HIV-1 triggering of apoptosis, as evidence by early release of cytochrome C and caspase 3/7 activation. Cell death is triggered by both products of reverse transcription and by virion-borne Vpr protein, and CGCC block both mechanisms. When HIV-1 RT efficiency was enhanced by SIVmac239 Vpx protein, cell death was still observed, indicating that the speed of reverse transcription and the efficiency of its completion contributed little to HIV-1 induced cell death in this system. These results show that a major restriction to HIV-1 infection in resting CD4 T cells resides in the capacity of these cells to survive the early steps of HIV-1 infection. IMPORTANCE: A major consequence of HIV-1 infection is the destruction of CD4 T cells. Here we show that delivery of virion-associated Vpr protein and the process of reverse transcription are each sufficient to trigger apoptosis of resting CD4 T cells isolated from peripheral blood. While these 2 mechanisms have been previously described in various cell types, we show for the first time their concerted effect in inducing resting CD4 T cell depletion. Importantly, we found that cytokines such as IL-7 or IL-4, which are particularly active in sites of HIV-1 replication, protect resting CD4 T cells from these cytopathic effects, and primarily through this protection, rather than through enhancement of specific replicative steps, they promote productive infection. This study provides important new insights for the understanding of the early steps of HIV-1 infection and T cell depletion.

BACKGROUND: HIV-1 integration is prone to a high rate of failure, resulting in the accumulation of unintegrated viral genomes (uDNA) in vivo and in vitro. uDNA can be transcriptionally active, and circularized uDNA genomes are biochemically stable in non-proliferating cells. Resting, non-proliferating CD4 T cells are prime targets of HIV-1 infection and latently infected resting CD4 T cells are the major barrier to HIV cure. Our prior studies demonstrated that uDNA generates infectious virions when T cell activation follows rather than precedes infection. RESULTS: Here, we characterize in primary resting CD4 T cells the dynamics of integrated and unintegrated virus expression, genome persistence and sensitivity to latency reversing agents. Unintegrated HIV-1 was abundant in directly infected resting CD4 T cells. Maximal gene expression from uDNA was delayed compared with integrated HIV-1 and was less toxic, resulting in uDNA enrichment over time relative to integrated proviruses. Inhibiting integration with raltegravir shunted the generation of durable latency from integrated to unintegrated genomes. Latent uDNA was activated to de novo virus production by latency reversing agents that also activated latent integrated proviruses, including PKC activators, histone deacetylase inhibitors and P-TEFb agonists. However, uDNA responses displayed a wider dynamic range, indicating differential regulation of expression relative to integrated proviruses. Similar to what has recently been demonstrated for latent integrated proviruses, one or two applications of latency reversing agents failed to activate all latent unintegrated genomes. Unlike integrated proviruses, uDNA gene expression did not down modulate expression of HLA Class I on resting CD4 T cells. uDNA did, however, efficiently prime infected cells for killing by HIV-1-specific cytotoxic T cells. CONCLUSIONS: These studies demonstrate that contributions by unintegrated genomes to HIV-1 gene expression, virus production, latency and immune responses are inherent properties of the direct infection of resting CD4 T cells. Experimental models of HIV-1 latency employing directly infected resting CD4 T cells should calibrate the contribution of unintegrated HIV-1.

A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of anti-retroviral therapy (ART), which reseed viremia after treatment is stopped. In general it is assumed that the reservoir consists of CD4+ T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view, and that the cells that contribute to the reservoir may be a diverse population that includes both CD4+ and CD4- cells. Here, we directly infected resting CD4+ T cells and used fluorescence-activated cell sorting (FACS) and Fiber-Optic Array Scanning Technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef and Env-mediated receptor internalization. We also extend our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag+ cells persist during ART and that these cells are often negative for CD4. We propose that these double negative alpha/beta T cells that express HIV protein may be a component of the long-lived reservoir. IMPORTANCE: A reservoir of infected cells persists in HIV-infected patients during anti-retroviral therapy (ART) that leads to rebound of virus if treatment is stopped. Here, we use flow cytometry and cell imaging to characterize protein expression in HIV infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins such as Env and Nef. Gag+CD4- cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs.

Human and simian immunodeficiency viruses (HIV and SIV) exploit follicular lymphoid regions by establishing high levels of viral replication and dysregulating humoral immunity. Follicular regulatory T cells (TFR) are a recently characterized subset of lymphocytes that influence the germinal centre response through interactions with follicular helper T cells (TFH). Here, utilizing both human and rhesus macaque models, we show the impact of HIV and SIV infection on TFR number and function. We find that TFR proportionately and numerically expand during infection through mechanisms involving viral entry and replication, TGF-beta signalling, low apoptosis rates and the presence of regulatory dendritic cells. Further, TFR exhibit elevated regulatory phenotypes and impair TFH functions during HIV infection. Thus, TFR contribute to inefficient germinal centre responses and inhibit HIV and SIV clearance.