CD8+ T cells promote HIV latency by remodeling CD4+ T cell metabolism to enhance their survival, quiescence, and stemness.

HIV infection persists during antiretroviral therapy (ART) due to a reservoir of latently infected cells that harbor replication-competent virus and evade immunity. Previous ex vivo studies suggested that CD8+ T cells from people with HIV may suppress HIV expression via non-cytolytic mechanisms, but the mechanisms responsible for this effect remain unclear. Here, we used a primary cell-based in vitro latency model and demonstrated that co-culture of autologous activated CD8+ T cells with HIV-infected memory CD4+ T cells promoted specific changes in metabolic and/or signaling pathways resulting in increased CD4+ T cell survival, quiescence, and stemness. Collectively, these pathways negatively regulated HIV expression and ultimately promoted the establishment of latency. As shown previously, we observed that macrophages, but not B cells, promoted latency in CD4+ T cells. The identification of CD8-specific mechanisms of pro-latency activity may favor the development of approaches to eliminate the viral reservoir in people with HIV.

CD8+ lymphocytes do not impact SIV reservoir establishment under ART.

Persistence of the human immunodeficiency virus type-1 (HIV-1) latent reservoir in infected individuals remains a problem despite fully suppressive antiretroviral therapy (ART). While reservoir formation begins during acute infection, the mechanisms responsible for its establishment remain unclear. CD8+ T cells are important during the initial control of viral replication. Here we examined the effect of CD8+ T cells on formation of the latent reservoir in simian immunodeficiency virus (SIV)-infected macaques by performing experimental CD8+ depletion either before infection or before early (that is, day 14 post-infection) ART initiation. We found that CD8+ depletion resulted in slower decline of viremia, indicating that CD8+ lymphocytes reduce the average lifespan of productively infected cells during acute infection and early ART, presumably through SIV-specific cytotoxic T lymphocyte (CTL) activity. However, CD8+ depletion did not change the frequency of infected CD4+ T cells in the blood or lymph node as measured by the total cell-associated viral DNA or intact provirus DNA assay. In addition, the size of the persistent reservoir remained the same when measuring the kinetics of virus rebound after ART interruption. These data indicate that during early SIV infection, the viral reservoir that persists under ART is established largely independent of CTL control.

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 α4ß7 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.

Author Correction: Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells.

Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1-4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection.

Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming.

Natural killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C+FcεRIγ- long-lived "memory" NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV-seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterized yet. Here, we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansions. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy.

Peripheral blood T cell alterations in newly diagnosed diffuse large B cell lymphoma patients and their long-term dynamics upon rituximab-based chemoimmunotherapy.

The importance of T cell-dependent immune responses in achieving long-term cure of chemoimmunotherapy-treated cancer patients is underscored by the recently described "vaccinal effect" exerted by therapeutic mAbs. In accordance, pre- and post-therapy peripheral blood lymphopenia represents a well-established negative prognostic factor in DLBCL. We analyzed the phenotypic and functional (IFNγ production, and Granzyme B (GrzB) cytotoxic granule marker expression) profile of peripheral blood T lymphocyte subsets ("conventional" CD4+ and CD8+, FOXP3+CD25bright Treg, and "innate-like" CD56+) in DLBCL patients at diagnosis, and assessed the long-term impact of R-CHOP chemoimmunotherapy, in a prospective study. At diagnosis, DLBCL patients showed lower lymphocyte counts, due to selective decrement of CD4+ T (including Treg) and B lymphocytes. While all T cell subsets transiently decreased during therapy, CD4+ T cell and Treg remained significantly lower than controls, up to 1 year after R-CHOP. Phenotypically skewed profile of CD4+ and CD8+ T cell subsets associated with higher frequencies of IFNγ+ and GrzB+ cells at diagnosis, that transiently decreased during therapy, and re-attained persistently elevated levels, till up to 1 year after therapy. Differently, the pre-therapy elevated levels of circulating monocytes, and of plasma IL-6 and IL-10 rapidly normalized upon R-CHOP. In sum, we describe a quantitatively and functionally altered status of the peripheral blood T cell compartment in DLBCL patients at diagnosis, that persists long-term after tumor eradication, and it is only transiently perturbed by R-CHOP chemoimmunotherapy. Moreover, data suggest the association of selected T cell functional features with DLBCL phenotype, and with therapy outcome.