Immune checkpoint expression on HIV-specific CD4+ T cells and response to their blockade are dependent on lineage and function.

BACKGROUND: Immune checkpoint blockade (ICB) partially reverses the dysfunctional state of antigen-specific T cell in chronic infections. However, its impact on the diverse subsets of CD4+ T cells in humans is largely unknown. METHODS: We examined immune checkpoint (IC) expression and function in HIV-specific CD4+ T cells of viremic individuals (≥5000 vRNA cp/ml, n = 17) prior to ART and persons with spontaneous (n = 11) or therapy-induced (n = 16) viral suppression (<40 cp/ml). We investigated IC patterns associated with exhaustion-related transcription factors and chemokine receptors using activation-induced marker assays. We determined effector functions representative of TFH, TH1, and TH17/TH22 using RNA flow cytometric fluorescence in situ hybridization (FISH). We compared increase in cytokine expression upon ICB across functions and patient status. FINDINGS: Expression of dysfunction-related molecules, such as transcription factors and ICs PD-1, TIGIT, and CD200, followed a hierarchy associated with infection status and effector profile. In vitro responsiveness to PD-L1 blockade varied with defined functions rather than IC levels: frequencies of cells with TH1- and TH17/TH22-, but not TFH-related functions, increased. Cells co-expressing TH1 and TFH functions showed response to ICB, suggesting that the cell's state rather than function dictates responsiveness to PD-L1 blockade. Response to PD-L1 blockade was strongest in viremic participants and reduced after ART initiation. INTERPRETATION: Our data highlight a polarization-specific regulation of IC expression and differing sensitivities of antigen-specific T helper subsets to PD-1-mediated inhibition. This heterogeneity may direct and constrain ICB efficacy in restoring CD4+ T cell function in HIV infection and other diseases. FUNDING: NIH, CIHR, CFI, FRQS.

Combined single-cell transcriptional, translational, and genomic profiling reveals HIV-1 reservoir diversity.

Although understanding the diversity of HIV-1 reservoirs is key to achieving a cure, their study at the single-cell level in primary samples remains challenging. We combine flow cytometric multiplexed fluorescent in situ RNA hybridization for different viral genes with HIV-1 p24 protein detection, cell phenotyping, and downstream near-full-length single-cell vDNA sequencing. Stimulation-induced viral RNA-positive (vRNA+) cells from viremic and antiretroviral-therapy (ART)-suppressed individuals differ in their ability to produce p24. In participants on ART, latency-reversing agents (LRAs) induce a wide variety of viral gene transcription and translation patterns with LRA class-specific differences in reactivation potency. Reactivated proviruses, including in p24+ cells, are mostly defective. Although LRAs efficiently induce transcription in all memory cell subsets, we observe induction of translation mostly in effector memory cells, rather than in the long-lived central memory pool. We identify HIV-1 clones with diverse transcriptional and translational patterns between individual cells, and this finding suggests that cell-intrinsic factors influence reservoir persistence and heterogeneity.

Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease.

Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.

CD73-A2a adenosine receptor axis promotes innate B cell antibody responses to pneumococcal polysaccharide vaccination.

Many individuals at risk of streptococcal infection respond poorly to the pneumococcal polysaccharide vaccine Pneumovax 23. Identification of actionable pathways able to enhance Pneumovax responsiveness is highly relevant. We investigated the contribution of the extracellular adenosine pathway regulated by the ecto-nucleotidase CD73 in Pneumovax-induced antibody responses. Using gene-targeted mice, we demonstrated that CD73-or A2a adenosine receptor deficiency significantly delayed isotype switching. Nevertheless, CD73- or A2aR- deficient adult mice ultimately produced antigen-specific IgG3 and controlled Streptococcus pneumoniae infection as efficiently as wild type (WT) mice. Compared to adults, young WT mice failed to control S. pneumoniae infection after vaccination and this was associated with lower levels of CD73 on innate B cells. We hypothesized that pharmacological activation of A2a receptor may improve Pneumovax 23 immunization in young WT mice. Remarkably, administration of the A2a adenosine receptor agonist CGS 21680 significantly increased IgG3 responses and significantly enhanced survival after S. pneumoniae challenge. Our study thus suggests that pharmacological activation of the A2a adenosine receptor could improve the efficacy of Pneumovax 23 vaccination in individuals at risk of streptococcal infection.

Envelope glycoproteins sampling states 2/3 are susceptible to ADCC by sera from HIV-1-infected individuals.

Recent analysis of HIV-1 envelope glycoproteins (Env) dynamics showed that the unliganded Env trimer can potentially sample three conformations: a metastable "closed" conformation (State 1), an "open" CD4-bound conformation (State 3), and an intermediate "partially open" conformation (State 2). HIV-1 evolved several mechanisms to avoid "opening" its Env in order to evade immune responses such as antibody-dependent cellular cytotoxicity (ADCC), which preferentially targets Envs in the CD4-bound conformation on the surface of infected cells. Here we took advantage of a well-characterized single-residue change in the gp120 trimer association domain to modify Env conformation and evaluate its impact on ADCC responses. We found that cells infected with viruses expressing Env stabilized in States 2/3 become highly susceptible to ADCC responses by sera from HIV-1-infected individuals. Our results indicate that the conformations spontaneously sampled by the Env trimer at the surface of infected cells has a significant impact on ADCC responses.

PolyI:C and CpG Synergize with Anti-ErbB2 mAb for Treatment of Breast Tumors Resistant to Immune Checkpoint Inhibitors.

Innate and adaptive immune cells play an important role in the therapeutic activity of anti-ErbB2 mAbs, such as trastuzumab. In the clinic, breast tumors poorly infiltrated with immune cells are more resistant to trastuzumab, and patients have a worse prognosis. Because type I and II IFNs are critical to the immune-mediated activity of anti-ErbB2 mAb, we investigated the effect of combining polyI:C and CpG with trastuzumab-like therapy in immunocompetent mouse models of ErbB2+ breast cancer. We demonstrated that in situ delivery of polyI:C and CpG combined to systemic anti-ErbB2 mAb triggered a potent inflammatory response in breast tumors able to induce long-lasting CD8+ T cell-dependent antitumor immunity. Remarkably, polyI:C and CpG was superior to combined PD-1/CTLA-4 blockade in sensitizing tumors to anti-ErbB2 mAb therapy. Local injection of CpG and polyI:C in a primary tumor significantly enhanced the activity of systemic anti-ErbB2 mAb against a distant untreated tumor. Type I and II IFNs, as well as natural killer cells and CD8+ T cells, were indispensible to the synergistic activity of the combination treatment. Because synthetic RNA analogues and CpG oligodeoxynucleotides have been safely used in clinical trials, our study supports combination treatments with anti-ErbB2 mAbs. Cancer Res; 77(2); 312-9. ©2016 AACR.

Single-Cell Characterization of Viral Translation-Competent Reservoirs in HIV-Infected Individuals.

HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5-1 gag-pol mRNA(+)/Gag protein(+)-infected cells per million. In the peripheral blood of untreated persons, active HIV replication correlated with viremia and occurred in CD4 T cells expressing T follicular helper cell markers and inhibitory co-receptors. In virally suppressed subjects, the approach identified latently infected cells capable of producing HIV mRNA and protein after stimulation with PMA/ionomycin and latency-reversing agents (LRAs). While ingenol-induced reactivation mirrored the effector and central/transitional memory CD4 T cell contribution to the pool of integrated HIV DNA, bryostatin-induced reactivation occurred predominantly in cells expressing effector memory markers. This indicates that CD4 T cell differentiation status differentially affects LRA effectiveness.

CD73 Expression Is an Independent Prognostic Factor in Prostate Cancer.

PURPOSE: CD73 is an adenosine-generating ecto-enzyme that suppresses antitumor immunity in mouse models of cancer, including prostate cancer. Although high levels of CD73 are associated with poor prognosis in various types of cancer, the clinical impact of CD73 in prostate cancer remains unclear. EXPERIMENTAL DESIGN: We evaluated the prognostic value of CD73 protein expression and CD8(+) cell density in 285 cases of prostate cancer on tissue microarray (TMA). Normal adjacent and tumor tissues were evaluated in duplicates. RESULTS: Univariate and multivariate analyses revealed that high levels of CD73 in normal adjacent prostate epithelium were significantly associated with shorter biochemical recurrence (BCR)-free survival. Notably, CD73 expression in normal epithelium conferred a negative prognostic value to prostate-infiltrating CD8(+) cells. Surprisingly, high levels of CD73 in the tumor stroma were associated with longer BCR-free survival in univariate analysis. In vitro studies revealed that adenosine signaling inhibited NF-κB activity in human prostate cancer cells via A2B adenosine receptors. Consistent with these results, CD73 expression in the prostate tumor stroma negatively correlated with p65 expression in the nuclei of prostate tumor cells. CONCLUSIONS: Our study revealed that CD73 is an independent prognostic factor in prostate cancer. Our data support a model in which CD73 expression in the prostate epithelium suppresses immunosurveillance by CD8(+) T cells, whereas CD73 expression in the tumor stroma reduces NF-κB signaling in tumor cells via A2B adenosine receptor signaling. CD73 expression, including in normal adjacent prostate epithelium, can thus effectively discriminate between aggressive and indolent forms of prostate cancer.

CD73 plays a protective role in collagen-induced arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease with significant morbidity and mortality. Recent studies suggest that modulation of adenosine signaling, a potent immunosuppressive pathway, is a promising approach for treatment of RA. Extracellular adenosine can come from two sources: transport of intracellular adenosine and hydrolysis of extracellular adenine nucleotides by CD73. In this study, we investigated the susceptibility of CD73-deficient C57BL/6 mice to collagen-induced arthritis (CIA), a well-established mouse model of RA. Our data demonstrated that CD73-deficient mice are significantly more susceptible to CIA than wild-type mice. CD73 deficiency resulted in an increased production of proinflammatory cytokines in the joints, increased Th1 T cell responses, and increased joint destruction. Surprisingly, this was accompanied by delayed anticollagen IgG responses, suggesting defective isotype class switching in CD73-deficient mice. Using bone marrow chimera mice, we demonstrated that CD73 expression on nonhematopoietic cells, but not on hematopoietic cells, was important for protection from CIA. We further demonstrated that administration of a selective A2A adenosine receptor agonist to CD73-deficient mice resulted in arthritis incidence similar to wild-type mice in support of a protective role for A2A signaling. Taken together, our study identifies CD73 as an important regulator of CIA in mice. It also strengthens the notion that CD73-generated adenosine by nonhematopoietic cells plays a protective role in RA and suggests that strategies able to enhance CD73 activity or expression levels may be a valid therapeutic option.