{"doi":"10.1101/2021.09.23.461484","title":"Immune checkpoint expression on HIV-specific CD4+ T cells and response to their blockade are dependent on lineage and function","abstract":"Abstract Background Antigen-specific T cell impairment is observed in chronic infections. CD4+ T cells are diverse in phenotype and function; how their different lineages are impacted by inhibitory immune checkpoints (IC) is unknown. Methods We examined IC expression and function in HIV-specific CD4+ T cells of viremic individuals prior to ART initiation and persons with spontaneous or therapy-induced viral suppression. We investigated IC patterns associated with exhaustion-related transcription factors and chemokine receptors using cytokine-independent activation-induced marker assays. We determined effector functions representative of T FH , T H 1 and T H 17/T H 22 using ultra-sensitive RNA flow cytometric fluorescence in situ hybridization (FISH), and their response to IC blockade. Findings The dysfunction-related transcription factor TOX was elevated in HIV-specific CD4+ T cells of viremic patients, and its expression was associated with lineage differentiation. We observed a hierarchy of PD-1, TIGIT and CD200 expression associated with both infection status and effector profile. In vitro responsiveness to PD-L1 blockade varied with defined CD4+ T cell functions rather than IC expression levels: frequencies of cells with T H 1- and T H 17/T H 22-, but not T FH -related functions, increased. 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 Thelper subsets to PD-1-mediated inhibition. This heterogeneity may direct ICB efficacy on CD4+ T cells in HIV infection. Funding This work was supported by the National Institutes of Health, the Canadian Institutes for Health Research, the Canada Foundation for Innovation and the Fonds de Recherche du Québec-Santé. Research in Context Evidence before this study Combination antiretroviral therapy (ART) is highly effective in controlling HIV but requires life-long medication due to the latent viral reservoir, and does not restore suppressive immune responses. In particular, there is no generation of effective HIV-specific T cell responses, which are thought to play an important role in controlling HIV in the rare individuals who can spontaneously control the virus. Inhibitory immune checkpoints (IC) such as PD-1 contribute to T cell dysfunction and failure to control viral infections, including HIV, and IC blockade (ICB) represents a potential adjuvant to ART through restoration of T cell functions. While most studies have focused on CD8+ T cells, increasing evidence shows that the remarkable impact of ICB therapy in a subset of cancer patients is enhanced by functional CD4+ T cell help, which can be directly affected by ICB. While effective virus-specific CD4+ T cell responses are also thought to be important for immune control of HIV, these cells are highly heterogenous. How IC expression and function differs across CD4+ T cell lineages and the consequences of this diversity for IC blockade (ICB) strategies are still poorly understood. Added value of this study To compare various stages of immune dysfunction, we examined people living with HIV (PLWH) with different levels of viral control pre-ART (including elite controllers who spontaneously control virus) and followed a cohort longitudinally post-ART. We used a panel of assays to characterize HIV-specific CD4+ T cell subsets, including activation-induced marker (AIM) assays and flow cytometric detection of mRNAs coding for a wider variety of HIV-specific CD4+ T cell functions than what is detected by standard procedures. Our experiments indicate a hierarchy of IC (PD-1, TIGIT, CD200) expression on blood HIV-specific CD4+ T cells that depends not only on the person’s infection status but also on expression of lineage differentiation markers and effector functions representative of CD4+ T cell subsets critical for antiviral responses (T FH , T H 1 and T H 1","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":226921,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9608,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":349168,"name":"Antigoni Morou","orcid":null,"position":1,"is_corresponding":false},{"id":254285,"name":"Mathieu Dubé","orcid":"0000-0002-5522-9019","position":2,"is_corresponding":false},{"id":254282,"name":"Julia Niessl","orcid":"0000-0001-8852-1924","position":3,"is_corresponding":false},{"id":105448,"name":"Amy E. Baxter","orcid":"0000-0002-1555-0713","position":4,"is_corresponding":false},{"id":623261,"name":"Olivier Tastet","orcid":"0000-0001-5694-6266","position":5,"is_corresponding":false},{"id":347021,"name":"Nathalie Brassard","orcid":"0000-0002-4320-9416","position":6,"is_corresponding":false},{"id":830761,"name":"Gloria Ortega-Delgado","orcid":null,"position":7,"is_corresponding":false},{"id":657569,"name":"Roxanne Charlebois","orcid":null,"position":8,"is_corresponding":false},{"id":4539,"name":"Gordon J. Freeman","orcid":"0000-0002-7210-5616","position":9,"is_corresponding":false},{"id":17392,"name":"Cécile Tremblay","orcid":"0000-0002-3440-3436","position":10,"is_corresponding":false},{"id":233493,"name":"Jean‐Pierre Routy","orcid":"0000-0001-9897-7589","position":11,"is_corresponding":false},{"id":254288,"name":"Daniel E. Kaufmann","orcid":"0000-0003-4467-136X","position":12,"is_corresponding":false},{"id":347019,"name":"Elsa Brunet‐Ratnasingham","orcid":"0000-0002-3613-1315","position":0,"is_corresponding":true}],"reference_count":54,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:54:38.004707Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}