Most people with HIV (PWH) develop progressive immune deficiency unless antiretroviral treatment (ART) is initiated. However, a small percentage of PWH, known as HIV controllers (HIC), are able to spontaneously control HIV infection without the use of ART (1). Based on their plasma viral load and CD4⁺T cell counts, HIC are classified as elite controllers (EC) or viremic controllers (2). HIV viral control has been linked to host genetic traits, particularly those related to the HLA region and CCR5 expression (3, 4), as well as to the integration of proviruses into transcriptional silenced regions of the genome, leading to smaller HIV reservoir (5). Moreover, spontaneous control is attributed to the activity of CD8+ T cells known to target HIV-infected cells through cytolytic and non-cytolytic mechanisms (6–8). Recent studies have also highlighted the role of innate immune cells in effective immune responses among HIC, as reviewed by Thobakgale et al. (9). Notably, certain phenotypes of natural killer (NK) exhibiting enhanced effector and cytotoxic functions are more abundant in EC (10). Additionally, plasmacytoid and myeloid dendritic cells (DC) of EC demonstrate heightened type I interferon responses and increased expression of co-stimulatory molecules, facilitating robust interactions with HIV-1-specific CD8+ T-cells (11, 12). Although these studies began to identify and describe the characteristics of different immune cells associated to HIV control, a comprehensive study profiling of both innate and adaptive immune cells is lacking.

The present study aimed to characterize the circulating immune cell landscape and the functional properties of immune cells associated with spontaneous HIV control using high-dimensional flow cytometry in participants from the 2000HIV cohort, including 54 HIV controllers (HIC) and 1044 non-controllers (non-HIC). We first performed a broad assessment of immunological features in all HIC—regardless of current ART status—to capture immune signatures linked to a history of spontaneous control. Thereafter, we focused specifically on ART-naïve elite controllers (EC), representing the most stringently defined controller phenotype, to better elucidate the unconfounded immune characteristics underlying durable HIV control. EC are widely regarded as a model for a functional HIV cure, underscoring the importance of identifying the host immune factors that enable viral control in the absence of ART. To this end, we accounted for all covariates influencing immune cell population variance to identify cellular signatures linked to spontaneous control of HIV.

Overall, we observed that HIC and EC display distinct patterns of immune cell activation, exhaustion, and chemokine receptor profiles compared to non-HIC, highlighting unique immune subsets and underlying mechanisms of spontaneous HIV control. The immune profiles hereby reported contribute to the elucidation of the signatures behind this natural control that are crucial for strategies aimed at achieving a functional HIV control, and to providing a high dimensional phenotyping data resource to explore immune cell subsets and their association with various clinical phenotypes in a large cohort of PWH.

The primary aim of this study was to characterize the circulating immune cell landscape in HIV controllers compared to ART-exposed normal progressors. After identifying sex at birth and other variables as confounders in the association between immune cell composition and HIV control phenotype; we adjusted for these covariates and observed that both innate and adaptive immune compartments in HIV controllers display features that may contribute to spontaneous viral control.

In the innate immune compartment, HIC exhibited reduced frequencies of CCR5⁺ NKT (CD3⁺CD56⁺) cells and CCR5⁺ TCRγδ1 cells compared to non-HIC, suggesting a less activated innate profile. EC, however, demonstrated a distinct innate signature, with elevated frequencies of neutrophils, TCRγδ2 cells, and CXCR4⁺ CD56+bright NK cells, along with reduced eosinophils, TCRγδ1 cells, and HLA-DR⁺ NK cells.

Of particular interest, EC showed enrichment of TCRγδ2 and a reduction of TCRγδ1 cells, consistent with previous reports comparing elite controllers to untreated participants and ART-treated non-controllers (20, 21). TCRγδ2 cells are known to be depleted during HIV infection through HIV envelope-mediated CCR5-dependent cell death, and their recovery remains incomplete even after ART (22). In contrast, EC maintain these cells at levels comparable to uninfected participants, implicating them in natural HIV control. Our unsupervised analysis revealed that TCRγδ2 cells express higher levels of CCR5 than TCRγδ1 cells, consistent with previous findings (23). However, EC had reduced frequencies of CCR5- expressing TCRγδ2 cells, supporting the hypothesis that early protection of this subset aids their long-term preservation. Furthermore, TCRγδ2 cells from EC showed signs of activation, evidenced by elevated γδTCR and CD11c expression. CD11c is not typically expressed on T cells, but may be induced under certain conditions. A previous study in chlamydia infection demonstrated that circulating CD11c⁺ γδT cells were highly activated, with enhanced IFNγ secretion capabilities compared to CD11c- γδ T cells (19). In our study, CD11c expression levels (MFI) on TCRγδ2 cells correlated positively with IFNγ production upon PBMC stimulation in EC, suggesting enhanced antiviral functionality.

Additionally, we observed elevated CD1c expression on both TCRγδ1 and TCRγδ2 cells. Although CD1c is an Antigen-presenting cell (APC) marker involved in lipid antigen presentation, it is not normally expressed on γδT cells. This CD1c expression may be explained by trogocytosis—a process in which lymphocytes acquire membrane fragments, including surface molecules, from APCs—a phenomenon previously reported in γδT cells (24). CD1c expression in EC may endow γδT cells with APC-like properties, further amplifying their effector functions (25). Altogether, these phenotypic signatures highlight the need for deeper functional studies on γδT cells from ECs, including assessments of proliferative capacity, cytokine production (e.g., IFN-γ, TNF-α, IL-17), cytotoxic granule release (e.g., granzyme B, perforin), and the expression of maturation, activation and exhaustion markers such as CD45RA, CCR7, CD38 and PD-1. Understanding which functional phenotypes of γδ T cells are most conducive to effective viral control could provide essential mechanistic insights and support the development of γδ T cell-based immunotherapeutic strategies (26).

In the adaptive compartment, HIC exhibit elevated CD4+ naïve and Th1/17 cells, naïve Tregs, and CD8+ TEMRA cells, with reduced CD8+ memory subsets. This profile suggests preserved immune homeostasis, improved responsiveness to new antigens, and robust effector responses. EC share trends but with fewer significant changes, including higher CD4+ naïve and Th1/17 cells and reduced Th17 cells.

CD4⁺ T cells from both HIC and EC exhibited reduced signs of exhaustion and dysregulated activation, as indicated by lower frequencies of PD-1⁺ and HLA-DR⁺ cells. As reported by Nojan et al., reduced exhaustion in CD4⁺ T cells contributes to sustained viral control by preserving their proliferative capacity, cytokine production, and ability to support both CD8⁺ T cell and B cell responses (27). Moreover, PD-1⁺ memory CD4⁺ T cells have been shown to preferentially harbor latent, replication-competent HIV, suggesting that elevated PD-1 expression may promote reservoir persistence (28, 29). The observed reduction in PD-1 expression among elite controllers may therefore play a role in limiting the size of the viral reservoir, although the precise mechanisms remain incompletely understood.

HIC display unique features such as increased CD38+ and CXCR4+ Tregs and reduced CCR5+ subsets, reflecting controlled immune activation. The metabolites produced by CD38 activity contribute to adenosine generation, which can suppress effector T cell activity and inflammation. CD38+ Tregs could play a role in controlling excessive inflammation while potentially limiting viral replication through immune suppression (30).

In contrast, EC exhibit an uniform reduction in CD38⁺ CD4⁺ T cell subsets and increased frequencies of CCR5⁺ and CXCR4⁺ CD4⁺ T cells, highlighting distinct mechanisms of HIV control. Insights from cancer immunology suggest that CCR5 expression on CD4⁺ T cells enhance their capacity to support anti-tumor immunity by promoting antigen-presenting cell (APC) maturation and effective CD8⁺ T cell priming, ultimately leading to stronger cytotoxic responses (31). Translating this to the context of HIV elite control, it is plausible that CCR5⁺ CD4⁺ T cells traffic to lymph nodes, where CCR5-mediated signaling enhances their secretion of agonists and CD40L. This, in turn, facilitates full APC maturation with optimal expression of MHC-II, CD80, and CD86, thereby improving the cross-priming of CCR5⁺ CD8⁺ T cells and boosting virus-specific cytotoxic T lymphocyte (CTL) responses to maintain viral suppression, as previously proposed (31).

Similar to CCR5, the enrichment of CXCR4⁺ CD4⁺ and CD8⁺ T cells observed in EC, along with their positive correlation with plasma Macrophage migration inhibitory factor (MIF) levels, may reflect an environment that favors T cell trafficking and functional competence. MIF can bind CXCR4, forming a heteromeric complex with CD74 that promotes T cell recruitment and migration (32, 33). CXCR4 expression is also associated with a less differentiated, naïve or central memory phenotype, and inversely correlated with terminal activation or effector maturation (34). Thus, the increased frequency of CXCR4⁺ T cells in EC may reflect a less exhausted, more plastic T cell compartment capable of efficient antigen sensing and trafficking.

HIC showed an increased frequency of naïve B cells, less switched memory B cells and lower proportions of B cells expressing CD307d/Fc receptor-like 4 (FCRL4), suggesting preservation of B cell responsiveness. Availability of naïve B cells in circulation might provide to HIC an advantage to response to new antigens. In contrast, FCRL4 is a marker related to B cell disfunction and exhaustion specifically in chronic infections such as HIV (35), since it is not present on B cells from healthy subjects (36). In EC it has been proposed that reduced B cell exhaustion is a consequence of the CTL antiviral response, as rapid control of viral load favor the development of efficient HIV-specific memory B-cell responses with cross-neutralization capacity (37, 38). EC exhibit a unique B cell profile characterized by higher abundances of IgA⁺ switched memory B cells. This aligns with previous observations suggesting that HIV-specific IgA responses and affinity maturation of anti-gp41 IgA antibodies occur to a greater extent in EC than in participants on ART (39). These enhanced IgA responses may reflect a more functional and finely tuned B cell compartment in EC, potentially contributing to improved mucosal immunity and viral control. Moreover, IgA is known to bind Fcα receptor I (FcαRI/CD89), which is expressed on myeloid cells such as monocytes, neutrophils, macrophages, and certain dendritic cell subsets (40). Through this interaction, IgA can trigger a range of pro-inflammatory responses, including cytokine and chemokine release, phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs) (41, 42). In our study, elite controllers exhibited higher frequencies of circulating neutrophils. While we did not assess CD89 expression on neutrophils directly, this finding may point to a potential link between elevated IgA responses and enhanced innate immune activation contributing to viral control. However, further in vitro studies are needed to experimentally validate this interaction in EC.

In conclusion, HIC and EC display distinct immune cell activation, exhaustion, and chemokine receptor profiles compared to non-HIC, highlighting unique immune subsets and underlying mechanisms of spontaneous HIV control. These distinct immune profiles underscore tailored strategies in spontaneous HIV suppression.

Finally, our study presents several key strengths. First, we performed high-dimensional flow cytometry profiling of 355 immune cell populations, offering a comprehensive overview of the circulating immune landscape in PWH. This dataset represents a valuable resource for the HIV research community to further explore immune signatures associated with viral control, as well as other clinical phenotypes. Second, we accounted for important confounding variables—including age, sex, COVID-19 vaccination status, and seasonality—thereby increasing the specificity and robustness of the identified immune associations. Third, we employed unsupervised analysis to characterize TCRγδ cells in greater depth, uncovering an activated phenotype linked to long-term viral control.

However, our study also has limitations. Notably, the observational design prevents us from establishing causality, as we report associations without functional validation of the immune profiles. Additionally, the validation cohort included only one HIC participant, which limits the strength of validation for HIC-specific findings and warrants caution in interpreting these results. Of note, since some HIC participants were receiving ART, we cannot exclude a possible influence of treatment on the observed HIC profile. While some trends did not reach statistical significance after adjustment, consistent patterns across both elite controllers (EC) and HIV controllers (HIC) reinforce the potential biological relevance of our findings.

Overall, the extensive immunophenotyping in this study provides a base for hypothesis generation and future mechanistic studies. Importantly, immunophenotyping remains a powerful tool for uncovering immune patterns associated with viral control, which may be translated or further investigated in the context of HIV cure strategies.