Editorial: CD4+ T cells in HIV: A Friend or a Foe?

2 inhibitors, ruxolitinib, and baricitinib, were tested as a potential therapeutic strategy to target latent reservoirs. The study used the dual reporter virus HIVGKO to investigate latency establishment and maintenance in lymphoid-derived CD4+ T cells. Single-cell technologies were integrated to evaluate protein expression, host gene expression, and HIV transcript expression, and identify and analyze latently infected cells. The latency establishment and maintenance were validated using the tonsillar CD4+ T cells in vitro method. The results from this study show that CD4+ T cells with latent infection exhibit similar activation pro ﬁ les as productively infected cells. Single-cell RNAseq analysis revealed

The relationship between HIV infection and CD4+ T cell population is intricate, partly because of their important functions and heterogenous nature. CD4 + T cell responses are central in orchestrating the adaptive immune responses to pathogens (8). Moreover, they are critical in inducing long-lasting vaccinemediated protection (9). Their main function is to provide help adaptive immune cells; they support antibody production by B cells, affinity maturation, and the selection and they support the generation of long-lasting effective CD8+ T cells, and their antiviral functions. In the last decade, however, our understanding of CD4+ T cell immunology has grown, and it has become clear that CD4+ T cells function extends well beyond the simplistic view of B and T cell help. Many new CD4+ T cell subsets have been discovered (10) with highly specialized and distinct functions and tissue distribution; these subsets include negative regulators of the immune system, T regulatory cells (T-regs), preferentially mucosal CD4+ T cells Th17 and Th9 cells, and B zone resident T follicular helper (Tfh) cells and T follicular regulatory cells (11,12). All subsets can be defined by specific surface and intracellular markers, including their own unique transcription factor and cytokine profiles. However, many questions remain elusive about their specific role in HIV infection. These subsets are not equally susceptible and each of them may play a unique yet significant role in establishing and maintaining virus reservoirs. Moreover, difference in HIV susceptibility of various CD4 T cells and the loss of CD4+ T cells very early in infection and the imbalance between these subsets is likely to affect the effectiveness of prophylactics vaccines and therapeutic approaches for HIV. Activated CD4+ T cells located at mucosal sites are the main targets for HIV infection. Therefore, interventions aimed at reducing the vulnerability of these mucosal targets, particularly in the genital tract which serves as the portal of entry for HIV, could potentially lower the risk of HIV acquisition. Lajoie et al. investigated the effect of acetylsalicylic acid (ASA), a well-known and safe systemic anti-inflammatory drug, on T cell activation of the vaginal tract. The study was conducted in a cohort of HIVuninfected women from Kenya who were given oral ASA at a low dose of 81mg daily for six weeks. Participants were followed for one month to establish a baseline immune activation in the blood and the female genital tract (FGT). Changes to T cell immune activation were measured both systemically and in the mucosal compartment, relative to baseline levels. The authors found that while concentration of ASA in the blood was 58% higher than the level measured in the FGT changes where noticeable in both sites. The blood level of ASA was correlated with lower levels of Th17 cells, CCR5 expressing target CD4+ T cells, and a specialized subset of CD8+ cells called Tc17. Importantly, in the FGT, low-dose ASA resulted in decreased levels of activated CD4+ T cells. Activation was measured by expression of the CCR5, CD95, and CD161+, all of which are markers associate with increased HIV replication (13,14). This study suggests that ASA may be used to decrease activated FGT CD4+ T cells, thereby potentially reducing the risk of HIV vaginal transmission.
Recent studies showed that CD4+ T cells expressing the surface receptor glycoprotein CD32 are more susceptible to infection, and virological analyses revealed that CD32+ CD4+ T cells express high levels of inflammatory markers (HIV co-receptor CCR5, and PD-1, CXCR3) and confirming their heightened susceptibility to HIV infection and serve as a HIV reservoir. Overall, the study characterized a new susceptible CD4+ T cell subset in monkeys that exhibits an activated profile and higher expression of markers associated with HIV-infected and/or reservoir cells, and thus may represent a novel therapeutic target (Huot et al.).
Olwenyi et al. examined a subset of cytotoxic (CTLs) CD4+ T cells and investigated their contribution to HIV persistence. CD4+ CTLs T cells can reduce viral replication and have been shown to kill infected macrophages, by secreting granzyme B and perforin and to kill the target cells in an MHC class II-restricted fashion. While they may play important roles in antiviral immunity, the lack of robust markers in various animal models limits understanding of their role in HIV immunopathology. The group found that CD4+ T cells expressing high levels of the integrin beta CD29 are highly cytotoxic in vitro, and they corroborated the use of CD29 as a marker to detect CD4+ CTLs T cells ex vivo, using PBMCs obtained from SIV infected macaques. Interestingly, CD29+ CD4+ T cells are depleted during untreated SIV infection and reconstituted after early initiation of ART. Moreover, functional characterization of these cells showed that they produce IL-21 and granzyme B and that their ability to secrete anti-viral cytokines is reduced by morphine, an opioid drug. These findings are relevant because they suggest that CD4+ CTLs play a crucial role in limiting HIV pathogenesis and persistence (15).
Despite the remarkable effectiveness of combination antiretroviral therapy (cART) in suppressing HIV replication in the bloodstream over long periods of time, achieving sustained virologic remission in HIV-infected individuals continues to be a formidable challenge. One of the primary reasons for the challenge in achieving sustained virologic remission is the presence of latent HIV reservoirs. These reservoirs consist of HIV-infected cells that are in a dormant or inactive state, allowing them to evade the effects of antiretroviral therapy. While cART effectively suppresses active viral replication, it is unable to eliminate these latent reservoirs. As a result, even with long-term treatment, the potential for viral rebound and the need for ongoing therapy persist. Efforts to target and eliminate these latent reservoirs are a critical focus of HIV cure research. Several attempts have been made to clear viral reservoirs including cART (16), latency-reversing agents (LRA) (17), immune based therapies (15), gene editing and gene therapy (18), stem cell transplantation (19) and combination therapies (20). In the study by de Armas et al., FDA-approved JAK1/2 inhibitors, ruxolitinib, and baricitinib, were tested as a potential therapeutic strategy to target latent reservoirs. The study used the dual reporter virus HIVGKO to investigate latency establishment and maintenance in lymphoid-derived CD4+ T cells. Single-cell technologies were integrated to evaluate protein expression, host gene expression, and HIV transcript expression, and identify and analyze latently infected cells. The latency establishment and maintenance were validated using the tonsillar CD4+ T cells in vitro method. The results from this study show that CD4+ T cells with latent infection exhibit similar activation profiles as productively infected cells. Single-cell RNAseq analysis revealed that Sirtuin signaling, Oxidative Phosphorylation, Mitochondrial Dysfunction, and EIF2 Signaling were enriched when comparing latent cells versus productively infected cells. In addition, molecular JAK1/2 inhibition resulted in dose-dependent downregulation of activation markers on CD4+ T cells. Moreover, the expression of activation markers such as CD25 and CD69 was affected by both drugs. Finally, HIV reactivation above the level of spontaneous reactivation was observed in the presence of a LRA, but pretreatment of cells with baricitinib abrogated the response, with the greatest effect occurring at the highest concentration of the drug. It is therefore, plausible that JAK1/2 inhibition may block HIVinduced IFN and thus lead to a reduction in viral reservoirs.
The crucial involvement of T follicular helper (Tfh) cells in B cell responses, including against HIV is widely recognized (10,21,22). However, while harnessing Tfh may be desirable, they also harbor the virus (23). Moreover, there remains uncertainty regarding which specific subset of Tfh cells, either from lymph nodes or circulating in the blood, harbors a higher reservoir burden.

Article Description Citation
Acetylsalicylic acid (ASA) use was associated with a decrease in activated CD4+ T cells CD4+CCR5+CD161+ and CD4+CCR5+CD95+ cells, both at the systemic and female genital tract.
Lajoie et al.
The CXCR5+ and CXCR4+ CD4 T cell subsets are preferentially killed during HIV infection, while those that are preferentially infected exhibit an activated and exhausted effector memory cell phenotype. Single-cell RNA-seq analysis showed that the preferentially killed subsets express genes that favor abortive infection and pyroptosis, and exhibits increased mRNA expression of inflammatory caspases (caspase 1 and caspase 4) as well as the pyroptotic executioner, gasdermin D.
Luo et al.

The CD32+CD4+ T cells exhibit an activated profile and higher expression of markers associated with HIV-infected and/or reservoir cells.
Huot et al.
The CD29hi CD4 T cells can serve as a reliable marker to identify CD4+ cytotoxic T lymphocytes (CTLs) in rhesus macaques. These CTLs secrete higher levels of cytotoxic and proinflammatory cytokines, and elevated expression of either IL-21 or granzyme B hi T Bet+ upon antigen stimulation. CD4+ CTLs play a crucial role in limiting SIV pathogenesis and persistence. However, their expression is reduced following morphine exposure.
Olwenyi et al. Additionally, they found increased frequencies of CXCR5+ CD8+ T cells and CD8+ Tscm cells, as well as memory B cells responsible for NAb development in HIV-2 infected persons. Interestingly, the frequency of memory CD4+ T cells and memory B cells significantly correlated with neutralizing antibody titers in HIV-2infected individuals. Overall, this study suggests that a more robust CD4+ T cell response that supports B cell differentiation, antibody production, and CD8+ T cell development in HIV-2 infected individuals contributes to better control of the virus and slower disease progression.
Luo et al. conducted a study to explore the diversity of CD4+ T cell subsets in terms of their susceptibility or resistance to HIVassociated killing to understand the pathogenesis of disease progression in CD4+ T cells. The authors used lymphoid cells obtained from human tonsils and employed mass cytometry and single-cell RNA-seq techniques to test their hypothesis. Their results indicated that subsets of CD4+ T cells that are preferentially killed express CXCR4 and CXCR5, a marker used to identify Tfh and the receptor allowing CD4 T cells to migrate to the B cell zone. Further analysis using single-cell RNA-seq showed that the preferentially killed subsets express genes that favor abortive infection and pyroptosis. These findings highlight the complex interplay between HIV and distinct tissue based CD4+ T cell subsets, including Thfs. The study also found that bystander death is more likely to happen when infection involves X4-tropic viruses and is associated with higher expression levels of CXCR4. Additionally, susceptible bystander cells appear to be primed for death by pyroptosis, as indicated by increased mRNA expression of inflammatory caspases (caspase 1 and caspase 4) as well as the pyroptotic executioner, gasdermin D (25).
The review by Onabajo and Mattapallil discusses the effect of HIV infection on Tfh cells and mucosal antibody responses in the gastrointestinal tract (GIT). They highlighted the consequences of such immune deregulations on gut dysbiosis and pathogenesis during chronic HIV infection. Mucosal immunoglobulins A (IgA) play a critical role in protecting the GIT from invasive bacteria. The study also suggests that the dysregulation of Tfh cells leads to compromise B cell responses, especially the secretion of microbespecific IgA, and it is likely to drive gut microbial dysbiosis during chronic HIV infection. Hence, preserving and maintaining Tfh cell responses in the mucosa could potentially restore high-affinity mucosal IgA, aiding in protecting the mucosal epithelial barrier from invasive dysbiotic bacteria (26).
T cell responses are intricately regulated by a complex network of non-coding RNAs, which includes microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) (27,28). These non-coding RNAs play crucial roles in modulating gene expression, influencing various aspects of T cell development, activation, differentiation, and effector functions. MicroRNAs are small RNA molecules that can bind to target messenger RNAs (mRNAs), leading to their degradation or inhibition of translation, thereby regulating the expression of specific genes. On the other hand, long non-coding RNAs are larger RNA molecules that can interact with chromatin, proteins, and other RNA molecules to regulate gene expression at multiple levels (24, 29, 30). Together, these noncoding RNAs contribute to the fine-tuning and coordination of T cell responses in both health and disease. Further research into the functions and mechanisms of non-coding RNAs in T cells continues to expand our understanding of immune regulation and may provide insights into HIV pathogenesis. The study conducted by Nguyen et al. aimed to investigate the role of long non-coding RNAs in the development of CD4+ T cell dysfunction and apoptosis in people living with HIV (PLHIV). The results revealed that the growth arrest-specific transcript 5 (GAS5) negatively regulates miR-21 expression, which controls critical signaling pathways involved in DNA damage and cellular response. Prolonged T cell stimulation reduced GAS5 and increased miR-21, leading to dysfunction and apoptosis in CD4+ T cells. These findings suggest that GAS5 regulates TCR-mediated activation and apoptosis in CD4 T cells during HIV infection through miR-21-mediated signaling pathway (37).
Van de Wijer et al. conducted a study aimed to investigate longterm changes in the immune system of people living with HIV (PLWH) who have successfully undergone treatment. Samples obtained from a cohort of 211 PLWH on stable antiretroviral therapy and 56 HIV-uninfected controls were assessed using flow cytometry to analyze 108 white blood cell (WBC) populations. The results indicated significant differences in T cell maturation and differentiation between PLWH and HIV-uninfected controls, with the PLWH exhibiting reduced percentages of CD4+ T cells and naive T cells, and increased percentages of CD8+ T cells, effector T cells, and T helper 17 (Th17) cells. In addition, the Th17/regulatory T cell (T-reg) ratios were found to be increased. PLWH also had altered B cell maturation, with reduced percentages of memory B cells and increased numbers of plasma blasts. Overall, this study suggests that the composition of circulating innate and adaptive immune cells is altered in PLHIV receiving cART for more than six months. The findings further suggest that while some adaptive immune responses such as Th17 are preserved, IFN-g responses are compromised in PLHIV (Van de Wijer et al.).
The manuscript by Zhu et al. conducted a study to investigate the immunological failure seen in immunological non-responders (InRs), HIV-infected individuals treated with ART, although successful in suppressing viral replication, they cannot properly reconstitute circulating CD4+ T-cell numbers to immunocompetent levels. The study found that InRs had platelets containing infectious HIV, which presence was linked to T-cell dysfunctions. The authors showed that platelet-T cell conjugates were more frequent among CD4+ T cells in InRs with HIVcontaining platelets (<350 CD4+ T cells/ml blood for >1 year) compared to healthy donors or IRs (>350 CD4+ T cells/ml). Based on these findings, the authors suggest that targeted therapy aimed at platelets may improve immune reconstitution in InRs (39).
The systematic review by Le Hingrat et al. focused on the dynamics of CD4+ T cells in SIV and HIV. The reviewed literature includes studies on the possible mechanism of CD4+ T cell depletion and restoration in disease progression, and during early ART, respectively. The role of cell death and loss of CD4+ cell subsets in gut health are discussed. Reviewed studies suggest that the loss of CD4+ T cells contributes to mucosal inflammation and enteropathy, which weakens the mucosal barrier, leading to microbial translocation, a major driver of immune activation/ inflammation. Studies also suggest that the loss of CD4+ T cells drives opportunistic infections, cancers, and comorbidities. Additionally, the authors discussed the crucial role of Th17 cells in maintaining gut integrity and protecting against bacterial and fungal infections (40).
In their systematic review, Inderbitzin et al. utilized primary CD4+ T cell models to investigate HIV-1 latency and potential cure strategies. The authors performed a pooled data analysis, comparing transcriptome profiles of latently and reactivated HIV-1 infected cells from five in vitro primary CD4+ T cell models and two ex-vivo studies of reactivated HIV-1 infected primary CD4+ T cells from HIV-1 infected individuals. The authors found that natural ligands CCL4, CCL5 and chemokine receptor CXCR6 were predominantly downregulated in latently infected cells, while genes associated with apoptosis, cell cycle, and HLA class II were upregulated in reactivated infected cells. Furthermore, they identified 5 genes FRY, GCSAM, GNLY, GPR15 and MSRB2 that co-occurred in both latently-and reactivated HIV-1 infected primary CD4+ T cells. The upregulation of MSRB2 in latently HIV-1 infected primary CD4+ T cells could inhibit apoptosis, while downregulation in reactivated HIV-1 infected genes leads to apoptosis due to the cytotoxic response of LRA or infectious virus particle release. This study sheds light on the differentially expressed genes that may contribute to the HIV-1 latency (41).
Both SARS-CoV-2 and HIV can dysregulate the immune system, leading to acute and long lasting inflammation, albeit through different mechanisms. By comparing the immune dysregulation caused by these viruses, we can identify commonalities and differences, which may inform the development of immunomodulatory therapies and approaches to mitigate immune-mediated damage in COVID-19. Peng et al. , with about 6.2% of people living with HIV (PLWH) also co-infected with HCV. PLWH have a six times higher incidence of being infected with HCV compared to those who are HIV-negative. The prevalence of co-infection is highest among individuals who inject drugs (PWID) and men who have sex with men (MSM). When HIV and HCV coexist, HCV's natural history is negatively affected, leading to higher rates of HCV persistence following acute infection, higher viral loads, accelerated progression of liver fibrosis, and development of endstage liver disease compared to HCV infection alone. Similarly, HCV coinfection harms the homeostasis of CD4+ T cell counts and facilitates HIV replication and viral reservoir persistence in PLWH receiving antiretroviral therapies (ART). Moreover, the consequences of Direct Acting antiviral (DAA)-mediated HCV cure on immune reconstitution and HIV reservoir persistence in coinfected patients is also discussed. The author comment on studies where it was shown that HIV reservoir is higher in HCV/ HIV coinfected individuals than in those with HIV mono-infection, and DAA-mediated HCV cure does not reduce HIV persistence or protect against HCV reinfection. Therefore, developing effective treatment and prophylactic anti-HCV and anti-HIV vaccines remains a priority to achieve HCV/HIV elimination (43).
HIV vaccine and cure strategies aim to enhance immune responses to achieve long-lasting and, ideally, sterilizing immunity against the virus. The presence of antibodies (preferably broadly neutralizing) and cytotoxic T cells at the site of viral entry is crucial in stopping infection and virus dissemination. In contrast, CD4+ T cells are necessary for mounting a robust vaccine response. However, inducing a strong CD4+ T cell response in HIV infection is a double-edged sword. This is because CD4+ T cells also serve as primary targets of HIV infection and activated CD4+ T cells can be preferentially infected during the early stages of infection. High frequencies of vaccineelicited CD4+ T cells may also limit vaccine-mediated protection. The specific lineages of CD4+ T cells have been reported to act as HIV reservoirs during chronic HIV infection (10,25,31). Furthermore, accumulating evidence suggests that some forms of vaccine specific CD4+ T cell responses may increase susceptibility to HIV infection.
While CD4 T cells are important to generate and maintain effective B and CD8 T cells responses, CD4 T cell induction trough vaccination may be a double edge sword. Evidence is mounting that some of vaccine induced CD4+ T cell responses may increase susceptibility to HIV infection. Results from the STEP HIV vaccine trial highlighted a potential role for total activated vaccine induced CD4+ T cells in promoting HIV acquisition (26, 32,33). The trial also revealed that preexisting adenoviral immunity triggered a recall response, leading to the activation of adenovirus specific CD4+ T cells. These activated CD4+ T cells could potentially become additional target cells and may have increased the susceptibility to HIV infection in the vaccine arm of the study (34). Interestingly recent studies in macaques showed that the DNA prime/modified vaccinia Ankara boost vaccine induced interferon-g (IFN-g + ) CD4+ T cells (Th1 cells), which rapidly migrate to multiple tissues including colon, cervix, and vaginal mucosa. These mucosal Th1 cells persisted at higher frequencies and expressed higher density of CCR5, a viral coreceptor, compared to cells in blood. After intravaginal or intrarectal simian immunodeficiency virus (SIV)/simian-human immunodeficiency virus (SHIV) challenges, strong vaccine protection was evident only in animals that had lower frequencies of vaccine-specific Th1 cells but not in animals that had higher frequencies of Th1 cells, despite comparable vaccine-induced humoral and CD8+ T cell immunity in both groups (35). These results corroborate the notion that high and persisting frequencies of HIV vaccine-induced Th1-biased CD4+ T cells in the intestinal and genital mucosa can mitigate beneficial effects of protective antibodies and CD8+ T cells, highlighting the critical need to better understand how to harness protective responses without increasing HIV susceptibility (36). Given the paradoxical role of CD4+ T cells in HIV infection, understanding the interplay between vaccine-induced CD4+ T cells and vaccine-mediated protection is paramount. To tip the balance in favor of vaccine-mediated protection, a rational design and delivery of vaccines in combination with newer immunotherapeutic strategies like engineered T cells (such as CAR/ TCR-T cells) are required. This Research Topic is expected to bring together the current knowledge of CD4+ T cells as important central mediators of adaptive immunity while also being the cellular reservoir and targets for the multiplication and persistence of HIV. Recent research has provided valuable insights into the mechanisms underlying CD4+ T cell depletion and dysfunction in HIV infection, as well as the factors contributing to the natural control of the virus. This Research Topic presents a comprehensive overview (Highlights summarized in Table 1) of these recent advances, focusing on CD4+ T cell dynamics in HIV immunology and pathogenesis. By summarizing the latest findings and highlighting key knowledge gaps, this Research Topic aims to provide a useful resource for researchers and clinicians working on HIV/AIDS, as well as to inspire further research into the discovery of novel therapeutic targets.