In recent decades, extensive use of antiretroviral therapy (ART) in people living with HIV (PLWH) has made continued progress in controlling viral replication and restoring CD4⁺ T cell count (1). However, part of patients (from 15% to 30%) remained insufficient in immune recovery, leading to a high risk of AIDS-related and non-AIDS complications. These patients were classified as poor immunological responders (PIRs), which had higher rates of morbidity and mortality than immunological responders (IRs) (2–5). Thus, it is a major clinical challenge and urgently needed to identify PIRs early and improve their immune homeostasis in time.

Persistent immune activation played a prominent role in the loss of CD4⁺ T cells and disease progression during HIV infection (6, 7). High levels of immune activation occurred early in primary HIV infection and still existed in PLWH who were receiving effective ART (8, 9). Indeed, immune activation is a key factor that leads to impaired immune reconstitution (10, 11). The successful control of immune activation could be a target in clinical therapy for improving damaged immune recovery. Double negative T cells (DNT cells) were characterized to express CD3 and TCRαβ, but not NK cell markers (12, 13). Although DNT cells comprised only 1–3% of total T cells in peripheral blood and lymphoid organs of humans and mice, these cells were crucial for maintaining immune homeostasis (14–17). A series of previous studies indicated that DNT cells were predominately memory phenotype in SIV infection, and have a T helper function, secreting CD4-like cytokines (IL-4, IL-17, IFN-γ and TNF-α) in SIV-infected or healthy monkeys such as sooty mangabeys (18–20). However, most studies identified these cells as similar to regulatory T cells (Tregs), which could suppress T cells, B cells, dendritic cells, and NK cells in graft-versus-host disease, autoimmune diseases, and infectious disease (21–23). Elevated DNT cells were reported to associate with the control of immune activation in both primary HIV-1 infection and IRs, suggesting the role of DNT cells in disease progression and immune reconstitution of PLWH (23, 24).

However, the precise mechanisms by which DNT cells exert their suppressive capacity during HIV infection remained puzzled. DNT cells with distinct phenotype and function can mediate inhibition via different mechanisms, including secretion of TGF-β, IL-10, perforin, and interaction of CTLA-4, which also involved in Treg-mediated immune suppression (25–28). Nevertheless, extracellular adenosine (ADO), which participated in the suppressive activity of Tregs, has never been noticed in DN T cells (29). ADO is a critical regulator of innate and adaptive immune responses, inhibiting T cell proliferation and the secretion of inflammatory cytokines, including IL-2, TNFα, and IFN-γ (30, 31). The ADO production is generally regulated by two enzymes sequentially, CD39 and CD73, which were expressed by many different cell types including immune cells (32–34). CD39 firstly catalyzes the dephosphorylation of circulating ATP and ADP to 5’-AMP. After that, CD73, an ecto-5’-nucleotidase existing in a soluble or membrane-bound form, converts AMP to ADO (35, 36). Several studies have confirmed the absence of CD73 on Tregs in human despite the key role of CD39 and ADO in immune suppression of Tregs (35). Thus, whether DNT expressed CD73 and mediated immune suppression through CD73- ADO pathway needs to be investigated.

In the present study, we found that CD73⁺ DNT cells in PLWH were associated with the control of immune activation, disease progression and immune reconstitution after ART. Of interest, soluble CD73 (sCD73) was also involved in immune suppression during HIV infection. Importantly, we provided evidence that elevated baseline levels of sCD73 could predict poor immune recovery in PLWH after long-term ART.

Previous studies have identified the crucial role of CD39/ADO pathway in immune suppression of Tregs during chronic HIV infection (37–39). Due to the lack of CD73 in Tregs, the source of CD73 and its essential role in AIDS progression remained puzzled and needed to be well investigated. In the present study, we demonstrated the involvement of CD73⁺ DNT cells in chronic HIV infection and disease progression. We found that the frequency of CD73⁺ DNT cells in PLWH, including TNs and those who experienced long-term ART was associated with CD4⁺ T cell count and immune activation. Additionally, the serum concentration of sCD73 showed a negative correlation with CD73⁺ DNT cells in PLWH who experienced ART. More importantly, the level of sCD73 at the baseline could predict CD4⁺ T cell recovery in PLWH. Patients with higher baseline levels of sCD73 had a higher risk of poor immune reconstitution. To our knowledge, this is the first evidence for a predictive role of sCD73 in immune reconstitution after long-term ART.

It is acknowledged that a significant percentage of ART-treated patients, classified as PIRs, failed to achieve optimal CD4⁺ T cell recovery, despite the HIV RNA being undetectable after several years of ART (4). These PIRs had a higher risk of AIDS-related and non-AIDS complications, such as cardiovascular disease, bone, and renal disease, neurocognitive decline, and premature aging (40–43). Notably, PIRs had higher rates of morbidity and mortality than IRs (5). Thus, there is an urgent need to identify PIRs early and provide effective treatment as soon as possible. Most prior studies indicated that the potential baseline predictors of poor immune reconstitution contained several clinical features (such as age, co-infection, CD4⁺ T cell count, CD8 count, CD4/CD8 ratio, and viral load), immune activation (CD38⁺HLA-DR⁺), genetic factors (CCR5 polymorphisms, IL7RA polymorphisms and mitochondrial haplogroups), and thymic function (44–52). In fact, the baseline CD4⁺ T cell count is the most recognized method for predicting the immune recovery after ART, however, it remains a considerable challenge to acquire this data in resource-limiting settings. Additionally, in contrast to stable serological biomarkers, CD4⁺ T cell count is susceptible to several factors, including physiology, emotion, drugs, age, and even the skill of lab technicians. Indeed, a series of studies indicated that several plasma biomarkers of systemic inflammation after ART initiation are potentially more predictive for future non-AIDS complications or CD4⁺ T cell recovery than cellular markers of immune activation (53–55).

Here, we took sCD73 and eleven other cytokines, which were indicated to associate with disease progression or clinical outcomes in PLWH, into the present study (56–64). Based on a series of rigorous statistical analysis, only the baseline concentration of sCD73 was identified as the most important predictive biomarker for immune reconstitution after ART. However, Prebensen et al. pointed out that elevated baseline levels of MIP-1β identified long-term PIRs who started ART at CD4⁺ T cell counts<200 cells/μl, which was not consistent with our study (64). Several possibilities were considered to explain the contradiction. On one hand, our cohort included PLWH with a baseline CD4⁺ T cell count<350 cells/μl significantly higher than their baseline CD4⁺ T cell count. Distinct immune characteristics and responses in these two cohorts could contribute to the heterogeneity of the results. On the other hand, they did not perform accurate predictive analysis, but only binary logistic regression, which was not enough to conclude a predictive biomarker.

More importantly, we evaluated the clinical outcomes not only by CD4⁺ T cell count but also by using CD4/CD8 ratio, which has been increasingly recognized as a biomarker of immune reconstitution during HIV treatment (65, 66). Indeed, CD4/CD8 ratio has been recommended as a stronger predictor of poor outcomes than the CD4⁺ T cell count in some clinical guidelines (67). It was gradually recognized that CD4⁺ T cell count does not reflect immune activation and risk of non-AIDS-defined events, despite its crucial role in monitoring immune recovery (65). Instead, CD4/CD8 ratio was found to independently associate with immune activation and serious non-AIDS events (68–71). In fact, severe immune defects were also identified in some PLWH with virological suppression who had CD4⁺ T cell count of more than 500 cells/μl but a low CD4/CD8 ratio (65, 72). Therefore, immune reconstitution and prognosis after ART were well monitored by both CD4⁺ T cell count and CD4/CD8 ratio. In the present study, we found that sCD73 instead of TNF-α could predict CD4⁺ T cell recovery as well as the restoration of CD4/CD8 ratio, further demonstrating a predictive value of sCD73 for the clinical outcome.

Of surprise, we observed reduced CD73⁺ DNT cells accompanied with an accumulation of CD39⁺ Tregs in TNs, despite CD73 being a similar rate-limited enzyme as CD39 in CD39/CD73/ADO pathway (30). In addition, CD73 was also down-regulated in CD8 T cells and B cells during HIV infection (73–75). The distinct dynamics of CD73 and CD39 during HIV infection might be explained by the increased plasma level of sCD73 in PLWH. The notion was further supported by a higher enzymatic activity of CD73 in its soluble form than the membrane-bound variant (76, 77). Thus, we speculated that CD73⁺ immune cells might release sCD73 in an inflammatory environment, which could cooperate with CD39⁺ Treg to produce extracellular ADO for immune suppression. Interestingly, the concentration of sCD73 was related to the frequencies of CD73⁺ CD8 T cells in TNs, while in ART-treated patients, the sCD73 level correlated with CD73⁺ DNT cells. It was implied that the major source of sCD73 might come from different cells in different immune status, which needed to be further explored. Considering that CD73 was widely expressed on various types of immune cells, further investigation was urgently needed to determine the exact origin of sCD73 in the future.

However, the exact mechanisms of increased sCD73 in plasma during HIV infection still remained in puzzle. To our knowledge, soluble form of CD73 can be cleaved from the cellular membrane or secreted by various tumor cells (78–81). Previous studies showed that the decrease of the membrane-bound CD73 activity on activated CD8⁺ T cells was accompanied by an increase in the concentration of soluble counterpart, and paralleled by elevated generation of ADO in the cell culture supernatant of activated cells. Similar results were observed on CD8⁺ T cells when using phosphatidylinositol-specific phospholipase C (PI-PLC) to force shedding of cell surface CD73 (76–78). Thus, considering the inverse trend between cell-bound and soluble forms of CD73 in our study, we preferred to assume that sCD73 were produced by cleavage of membrane-bound counterpart, although the source of secretion could not be completely ruled out.

Given that ADO was a key factor in CD73-mediated immunosuppression, we also detected the concentration of ADO in plasma from PLWH. Unfortunately, no correlation was observed between ADO and sCD73, CD73⁺ DNT, and CD73⁺ CD8⁺ T cells, which could be explained by several possibilities. First, ADO was mainly generated in the local environment of tissue injury, hypoxia, or inflammation where high local concentrations of ATP and ADP would be released and correspondingly exerted its immune effects restricted locally. Thus, the plasma concentration of ADO was hard to reflect their concentration and activity in the local environment owing to dilution and time of circulation (82–84). Second, due to the rapid formation and rapid clearance of ADO in blood, it was challenging to determine accurate concentrations of extracellular ADO in blood samples (84). Finally, the purinergic signaling is a complex network including a number of purinergic ligands, receptors, enzymes, channels, and transporters. In addition to CD73, many other purine-metabolizing enzymes, including adenosine deaminase (ADA), CD38, ATP-degrading enzymes like ENPPs and CD39, as well as ATP-regenerating kinases were also present in human plasma (78, 84–86). Thus, the plasma concentration of ADO could be mediated by multiple pathways, implying a complicated relationship of ADO with CD73-expressing cells or sCD73.

The present study has several limitations. First, due to a minimal number of DNT cells in PLWH, it is difficult to perform in vitro functional experiments to verify the role of CD73 in the inhibition mediated by DNT cells and the origin of sCD73. Second, we did not take the frequency of CD73⁺ DNT cells into the predictive model for clinical outcomes of PLWH, owing to the lack of the baseline flow cytometry data of the retrospective cohort. Third, the confounding factor of therapy initiation was not excluded from the correlation analysis in the present study. Finally, only a single-center cohort study was conducted for the predictive analysis; we need to recruit more cohorts in additional sites to verify our findings.

In summary, our study demonstrated that a higher concentration of sCD73 at the baseline could predict poor CD4⁺ T cells recovery in PLWH after long-term ART, providing a potential biomarker for detecting immune reconstitution early.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Committee of Ethics at Beijing Ditan Hospital. The patients/participants provided their written informed consent to participate in this study.

XW, LZ, and JD performed the experiments and analyzed the data. YW, DW, CS, DC, BL, MJ, and MZ collected samples, and performed the experiments. HZ participated in the critical review of the manuscript and revised the manuscript. YK designed the experiments, analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by National Natural Science Foundation of China (82171548, 81971307), Beijing Municipal Natural Science Foundation for Distinguished Young Scholars (JQ21023), Beijing Municipal Administration of Hospitals’ Ascent Plan (DFL20191802), and Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX202126).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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