GITR activation ex vivo impairs CD8 T cell function in people with HIV on antiretroviral therapy.

Glucocorticoid-induced tumor necrosis factor related protein (GITR) is a co-stimulatory immune checkpoint molecule constitutively expressed on regulatory T cells (Tregs) and on activated T conventional cells (Tconv). In blood collected from PWH on suppressive ART, GITR expression was reduced in multiple activated CD4 and CD8 T cell subsets but was increased in Tregs. HIV specific CD8 T cells expressed higher levels of GITR and programmed cell death protein 1 (PD-1) compared to total CD8 T cells. Following stimulation with HIV peptides and GITR-ligand (L), we demonstrated a significant decrease in killing by HIV specific CD8 T cells and an increased exhausted profile. T cell receptor co-stimulation with GITR-L abrogated Treg suppression and induced expansion of CD4 Tconv. We conclude that GITR activation is an additional factor contributing to an impaired HIV immune response in PWH on ART and that GITR agonist antibodies should not be pursued for HIV cure strategies.

Memory CD4+ T cells that co-express PD1 and CTLA4 have reduced response to activating stimuli facilitating HIV latency.

Programmed cell death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) suppress CD4+ T cell activation and may promote latent HIV infection. By performing leukapheresis (n = 21) and lymph node biopsies (n = 8) in people with HIV on antiretroviral therapy (ART) and sorting memory CD4+ T cells into subsets based on PD1/CTLA4 expression, we investigate the role of PD1 and CTLA 4 in HIV persistence. We show that double-positive (PD1+CTLA4+) cells in blood contain more HIV DNA compared with double-negative (PD1-CTLA4-) cells but still have a lower proportion of cells producing multiply spliced HIV RNA after stimulation as well as reduced upregulation of T cell activation and proliferation markers. Transcriptomics analyses identify differential expression of key genes regulating T cell activation and proliferation with MAF, KLRB1, and TIGIT being upregulated in double-positive compared with double-negative cells, whereas FOS is downregulated. We conclude that, in addition to being enriched for HIV DNA, double-positive cells are characterized by negative signaling and a reduced capacity to respond to stimulation, favoring HIV latency.

Immune checkpoint blockade in HIV.

Antiretroviral therapy (ART) has dramatically improved life expectancy for people with HIV (PWH) and helps to restore immune function but is not curative and must be taken lifelong. Achieving long term control of HIV in the absence of ART will likely require potent T cell function, but chronic HIV infection is associated with immune exhaustion that persists even on ART. This is driven by elevated expression of immune checkpoints that provide negative signalling to T cells. In individuals with cancer, immune checkpoint blockade augments tumour-directed T-cell responses resulting in significant clinical cures. There is therefore high interest if ICB can contribute to HIV cure or remission by reversing HIV-latency and/or drive recovery of HIV-specific T-cells. We here review recent evidence on the role of immune checkpoints in persistent HIV infection and discuss the potential for employing immune checkpoint blockade as a therapeutic approach to target HIV persistence on ART.

Combination Immune Checkpoint Blockade Enhances IL-2 and CD107a Production from HIV-Specific T Cells Ex Vivo in People Living with HIV on Antiretroviral Therapy.

In people with HIV (PWH) on antiretroviral therapy (ART), immune dysfunction persists, including elevated expression of immune checkpoint (IC) proteins on total and HIV-specific T cells. Reversing immune exhaustion is one strategy to enhance the elimination of HIV-infected cells that persist in PWH on ART. We aimed to evaluate whether blocking CTL-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell Ig domain and mucin domain 3 (TIM-3), T cell Ig and ITIM domain (TIGIT) and lymphocyte activation gene-3 (LAG-3) alone or in combination would enhance HIV-specific CD4+ and CD8+ T cell function ex vivo. Intracellular cytokine staining was performed using human PBMCs from PWH on ART (n = 11) and expression of CD107a, IFN-γ, TNF-α, and IL-2 was quantified with HIV peptides and Abs to IC. We found the following: 1) IC blockade enhanced the induction of CD107a and IL-2 but not IFN-γ and TNF-α in response to Gag and Nef peptides; 2) the induction of CD107a and IL-2 was greatest with multiple combinations of two Abs; and 3) Abs to LAG-3, CTLA-4, and TIGIT in combinations showed synergistic induction of IL-2 in HIV-specific CD8+ and CD107a and IL-2 production in HIV-specific CD4+ and CD8+ T cells. These results demonstrate that the combination of Abs to LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2 that associated with cytotoxicity and survival of HIV-specific CD4+ and CD8+ T cells in PWH on ART. These combinations should be further explored for an HIV cure.

The impact of immune checkpoint therapy on the latent reservoir in HIV-infected individuals with cancer on antiretroviral therapy.

OBJECTIVE: The aim of this study was to quantify HIV-specific immunological and virological changes in people with HIV (PWH) on antiretroviral therapy (ART) with malignancy who received immune checkpoint blockade (ICB). DESIGN: An observational cohort study. METHODS: Blood samples were collected before and after four cycles of ICB in HIV-positive adults on ART. Virological assessments performed on CD4+ T cells included cell-associated unspliced HIV RNA, cell-associated HIV DNA, Tat/rev-induced limiting dilution assay (TILDA) and plasma HIV RNA using a single copy assay (SCA). Flow cytometry was used to assess the frequency of precursor exhausted T cells (Tpex) and exhausted T cells (Tex), and Gag-specific CD4+ and CD8+ T cells positive for IFN-γ, TNF-α or CD107a by intracellular cytokine staining (ICS). RESULTS: Participant (P)1 received avelumab (anti-PD-L1) for Merkel cell carcinoma. P2 and P3 received ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) for metastatic melanoma. An increase in CA-US RNA following each infusion was noted in all three participants. There were no consistent changes in HIV DNA or the proportion of cells with inducible MS HIV RNA. P2 demonstrated a striking increase in the frequency of gag-specific central and effector memory CD8+ T cells producing IFN-γ, TNF-α and CD107a following anti-PD1 and anti-CTLA-4. The frequency of CD8+ Tpex cells pre-ICB was also highest in this participant. CONCLUSION: In three PWH with cancer on ART, we found that ICB activated latent HIV and enhanced HIV-specific T cell function but with considerable variation.

Dynamics of the Coreceptor-LCK Interactions during T Cell Development Shape the Self-Reactivity of Peripheral CD4 and CD8 T Cells.

Overtly self-reactive T cells are removed during thymic selection. However, it has been recently established that T cell self-reactivity promotes protective immune responses. Apparently, the level of self-reactivity of mature T cells must be tightly balanced. Our mathematical model and experimental data show that the dynamic regulation of CD4- and CD8-LCK coupling establish the self-reactivity of the peripheral T cell pool. The stoichiometry of the interaction between CD8 and LCK, but not between CD4 and LCK, substantially increases upon T cell maturation. As a result, peripheral CD8+ T cells are more self-reactive than CD4+ T cells. The different levels of self-reactivity of mature CD8+ and CD4+ T cells likely reflect the unique roles of these subsets in immunity. These results indicate that the evolutionary selection pressure tuned the CD4-LCK and CD8-LCK stoichiometries, as they represent the unique parts of the proximal T cell receptor (TCR) signaling pathway, which differ between CD4+ and CD8+ T cells.

Cocapture of cognate and bystander antigens can activate autoreactive B cells.

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are associated with autoimmune central nervous system diseases like acute disseminated encephalomyelitis (ADEM). For ADEM, it is speculated that a preceding infection is the trigger of the autoimmune response, but the mechanism connecting the infection to the production of MOG antibodies remains a mystery. We reasoned that the ability of B cells to capture cognate antigen from cell membranes, along with small quantities of coexpressed "bystander" antigens, might enable B-cell escape from tolerance. We tested this hypothesis using influenza hemagglutinin as a model viral antigen and transgenic, MOG-specific B cells. Using flow cytometry and live and fixed cell microscopy, we show that MOG-specific B cells take up large amounts of MOG from cell membranes. Uptake of the antigen from the membrane leads to a strong activation of the capturing B cell. When influenza hemagglutinin is also present in the membrane of the target cell, it can be cocaptured with MOG by MOG-specific B cells via the B-cell receptor. Hemagglutinin and MOG are both presented to T cells, which in turn are activated and proliferate. As a consequence, MOG-specific B cells get help from hemagglutinin-specific T cells to produce anti-MOG antibodies. In vivo, the transfer of MOG-specific B cells into recipient mice after the cocapture of MOG and hemagglutinin leads to the production of class-switched anti-MOG antibodies, dependent on the presence of hemagglutinin-specific T cells. This mechanism offers a link between infection and autoimmunity.

Monoclonal regulatory T cells provide insights into T cell suppression.

Regulatory T cells (Tregs) have a crucial role in maintaining lymphocyte homeostasis. However an understanding of how Tregs function at a cellular and molecular level has not yet been fully elucidated. Here, we make use of a T cell receptor (TCR) transgenic, Rag(-/-) mouse expressing a Forkhead-Box-Protein P3 (Foxp3) transgene. This mouse provides a source of monoclonal CD4(+) Foxp3(+) T cells with a defined specificity. Here we show that monoclonal B3K506 Tregs are functional in vitro and in vivo and clearly require cognate antigen to be suppressive. We further show that the strength of Treg stimulation determines the strength of Treg mediated suppression. Finally we analysed various suppressive mechanisms used by monoclonal Tregs and found that Treg-Tconv proximity is a parameter, which correlates with enhanced suppression.