Selective loss of CD107a TIGIT+ memory HIV-1-specific CD8+ T cells in PLWH over a decade of ART

  1. Oscar Blanch-Lombarte
  2. Dan Ouchi
  3. Esther Jimenez-Moyano
  4. Julieta Carabelli
  5. Miguel Angel Marin
  6. Ruth Peña
  7. Adam Pelletier
  8. Aarthi Talla
  9. Ashish Sharma
  10. Judith Dalmau
  11. José Ramón Santos
  12. Rafick-Pierre Sékaly
  13. Bonaventura Clotet
  14. Julia G Prado

  • Curated by eLife

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    eLife assessment

    The present study shows that the expression of some inhibitory receptors (IRGs) on CD8 T cells is increased in people living with HIV (PLWH) and remain elevated even after years of viral suppression by antiretroviral therapy. The authors further report that inhibition of TGIT partially restores the ability of CD8 T cells to produce CD107a but not the other functions. Altogether, the results provide some valuable insights into our understanding of inhibitory receptor expression in the HIV infected individuals but some evidence seems incomplete.

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Abstract

The co-expression of inhibitory receptors (IRs) is a hallmark of CD8+ T-cell exhaustion (Tex) in people living with HIV-1 (PLWH). Understanding alterations of IRs expression in PLWH on long-term antiretroviral treatment (ART) remains elusive but is critical to overcoming CD8+ Tex and designing novel HIV-1 cure immunotherapies. To address this, we combine high-dimensional supervised and unsupervised analysis of IRs concomitant with functional markers across the CD8+ T-cell landscape on 24 PLWH over a decade on ART. We define irreversible alterations of IRs co-expression patterns in CD8+ T cells not mitigated by ART and identify negative associations between the frequency of TIGIT+ and TIGIT+ TIM-3+ and CD4+ T-cell levels. Moreover, changes in total, SEB-activated, and HIV-1-specific CD8+ T cells delineate a complex reshaping of memory and effector-like cellular clusters on ART. Indeed, we identify a selective reduction of HIV-1 specific-CD8+ T-cell memory-like clusters sharing TIGIT expression and low CD107a that can be recovered by mAb TIGIT blockade independently of IFNγ and IL-2. Collectively, these data characterize with unprecedented detail the patterns of IRs expression and functions across the CD8+ T-cell landscape and indicate the potential of TIGIT as a target for Tex precision immunotherapies in PLWH at all ART stages.

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  1. Version published to 10.7554/elife.83737 on eLife
  2. Version published to 10.1101/2022.07.13.499924v2 on bioRxiv
  3. eLife

    eLife assessment

    The present study shows that the expression of some inhibitory receptors (IRGs) on CD8 T cells is increased in people living with HIV (PLWH) and remain elevated even after years of viral suppression by antiretroviral therapy. The authors further report that inhibition of TGIT partially restores the ability of CD8 T cells to produce CD107a but not the other functions. Altogether, the results provide some valuable insights into our understanding of inhibitory receptor expression in the HIV infected individuals but some evidence seems incomplete.

  4. eLife

    Reviewer #1 (Public Review):

    Blanch-Lombarte and colleagues demonstrated that the expression of certain inhibitory receptors (IRGs) on CD8 T cells is elevated in people living with HIV (PLWH) and they remain elevated despite years of viral suppression on antiretroviral therapy (ART). A comprehensive single-cell analysis by multiparametric flow cytometry demonstrated that TGIT+ CD8 T cells have a skewed phenotype following HIV infection. Blocking of TGIT partially restores the ability of CD8 T cells to produce CD107a but not the other functions.

    Strengths of the current study include the comprehensive analysis of IRGs on CD8 T cells of a well-characterized group of individuals with and without HIV. Additionally, they have confirmed that blocking of TGIT should be evaluated further as a potential therapy for PLWH. The conclusions seem well justified from the presented data.

    Weaknesses include the cross-sectional data and minor confusion stemming in part from the lack of clarity and rationale for some of the experiments.

  5. eLife

    Reviewer #2 (Public Review):

    Antiretroviral therapy (ART) can control HIV replication and improve the quality of life for people living with HIV (PLWH); however, it does not cure infection, nor does it revert T cell exhaustion. Inhibitory receptor expression is a characteristic of CD8+ T cell exhaustion and a better understanding of the differences in receptor expression dynamics between healthy donors and PLWH on ART is of interest. In this comparative study, Blanch-Lombarte et al. use single-cell analysis of flow cytometric PBMC profiling to examine inhibitory receptor expression (IR) and functional markers in CD8+ T cells derived from PLWH on ART and healthy donors.

    The authors first perform a mix of cross-sectional and longitudinal characterization of IR expression and memory differentiation markers in donors who are healthy controls, are in the early stages of HIV infection, PLWH on ART for ~ 2 years, and PLWH on ART for ~10 years. They conduct both supervised and unsupervised analyses of the phenotypic results. The authors use three experimental conditions (unstimulated, SEB stimulation, HIV Gag peptide pool stimulation) to perform cluster analyses. The longitudinal paired samples allow determination of the persistence of the alterations observed early after initiation of ART. The analyses show inverse correlations between frequencies of TIGIT+ and TIGIT+ TIM+ CD8 subsets and CD4 counts. However, findings for HIV-specific CD8 were different, with a selective reduction of TIGIT+ clusters whose functionality in terms of CD107 expression was recovered by anti-TIGIT blockade.

    The authors conclude that TIGIT could be a therapeutic target to revive exhausted T cells (Tex) at all ART stages.

    Strengths:
    - The study addressed relevant questions for the field.
    - The is a logical sequence of experiments and analyses.
    - The authors investigate interesting samples - longitudinal time points on ART several years apart are a significant asset.
    - Assessment of CD8 T cell populations as bulk unstimulated cells, broad stimulation with a superantigen (SEB), and HIV-specific responses (Gag peptide pool stimulation).
    - Complementary use of supervised and detailed unsupervised analyses of flow cytometry data.
    - The analyses are overall detailed and carefully presented, except for minor issues in color coding and font size.
    - Functional assays to assess the functional impact of TIGIT upregulation on CD8 T cell function.
    -
    Weaknesses:
    - While the paper reads overall well, the hypotheses and concepts should be clarified in several instances. For example, the authors speak of T cell exhaustion, which in principle is understood as antigen-specific T dysfunction associated with antigen persistence. However, a good part of the paper is focused on total CD8 T cells, and the links between findings in the different populations of CD8 examined (total, SEB-stimulated, Gag-stimulated) are hard to understand.
    - Upregulation of IRs can be associated with the state of T cell differentiation and also modulated by chronic inflammation independently of TCR signaling (eg, common gamma-chain cytokines upregulate PD-1), so defining these cells as univocally Tex is not correct.
    - The study mostly focuses on descriptive phenotypic analyses of CD8 T cells rather than dynamics studies which would imply more in-depth investigations of T cell evolution and fate.
    - HIV-specific CD8 T cells can be both quantitatively and quantitatively impaired, but the quantitative aspects are not considered, nor shifts in phenotype. For example, HIV-specific TIGIT+ CD8 responding to blockade proportionally over time - It is unclear if this is compensated by other subsets.
    - The functional assays with TIGIT blockade are limited and do not include other markers of cytotoxic cells (perforin, granzyme B expression...). It is not clear how do these subsets compare to the other clusters in terms of CD107 expression.
    - The statistical analyses do apparently not include correction for multiple comparisons.

  6. eLife

    Reviewer #3 (Public Review):

    Here the authors use high-parameter flow cytometry to address expression patterns of inhibitory receptors and concordant functional responses in CD8+ T cells from people living with HIV (PLWH) during early vs. long-term ART treatment in order to understand the potential evolution of exhausted T cells in HIV infection. High-dimensional bioinformatic analysis is employed to uncover different subsets of CD8+ T cells expressing TIM-3, TIGIT, PD1, LAG3, and CD39. Stimulation assays were further conducted to assess polyclonal T cell responses (superantigen) or HIV-gag-specific CD8+ T cells, and whether the responding cells displayed inhibitory receptors. Finally, inhibitory receptor blockade was used (focusing on TIGIT and TIM-3 only) to examine the potential reversal of exhaustion. The authors found that CD107a+ degranulating central memory T cells apparently were sensitive to TIGIT blockade, yielding increased responses in cells from ART-treated PLWH.

    Methods and Results Major Strengths: Sample size and data density. Longitudinal samples from long-term treated PLWH. Mechanistic studies to assess inhibitory receptor blockade.

    Methods and Results Major Weaknesses: Lack of clarity on flow cytometric analysis and statistical methodology, including correction for multiple comparisons. Clustering density in tSNE analysis is unjustified, leading to potentially spurious outcomes. Insufficient raw flow cytometry data presented on inhibitory receptor expression in the various contexts of the study to allow determination of whether the subsequent bioinformatic analysis was merited due to the very low expression of 3/5 markers examined. Unclear whether differences observed are biologically meaningful (despite statistical differences). Finally, although the longitudinal samples are a distinct strength of the study, changes over time within individuals are unfortunately not assessed.

    Aims and conclusions: The authors do find differences between the cohorts as described in the manuscript; however, the biological relevance of the findings is questionable due to an absence of direct studies on the cell populations found to be different. The use of unbiased clustering analysis is both a strength and a weakness. Specifically, the algorithm uncovers potential cell clusters that might be missed; however, the clustering program requires pre-set inputs on the expected number of clusters to be found, leading to possible irrelevant subsets being identified. The conclusions of the study are appropriately limited in scope.

    Impact: There have been numerous studies of CD8+ T cell inhibitory receptor expression and T cell exhaustion in the context of HIV infection. It is well-accepted that T-cell exhaustion is a hallmark of progressive infection. This study contributes to the current knowledge in this area specifically through the examination of very long-term ART-treated PLWH. Unfortunately, it is not clear that several of the examined inhibitory receptors could be adequately detected, limiting the interpretation of the findings. Finally, it is unclear that this study justifies the potential use of TIGIT blockade to improve T cell function given the unclear biological relevance of the differential populations of CD8+ T cells observed.

  7. Version published to 10.1101/2022.07.13.499924v1 on bioRxiv