Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB) infection, is the most common opportunistic infection and the major cause of death among people living with HIV (PLWH) (UNAIDS, 2019). TB harms the immune response to HIV infection, accelerating the progression from HIV infection to AIDS. HIV is also recognized as a crucial risk factor for developing active TB. Therefore, understanding the immunological characteristics in the process of HIV/TB co-infection is crucial for disease control.

γδ T cells are non-conventional T cells that express a T cell receptor (TCR) consisting of a γ and a δ chain with diverse structural and functional heterogeneity. Although the proportion of γδ T cells in total peripheral T lymphocytes is only 1-5%, their rapid cytokine production following activation plays an essential role in patients with tumors, autoimmunity, and infectious diseases (Halary et al., 2005; Zhang et al., 2006; Wu et al., 2014). Depending on the difference in δ chain, γδ T cells can be defined into two major subsets, including Vδ₁ and Vδ₂. Vδ₁ subset, rare in peripheral blood, mainly resides in the epithelium of mucosal tissue and participates in respiratory and intestinal mucosal immunity (Wu et al., 2014). Vδ₂ subset is predominately distributed in peripheral blood and represents a significant portion of γδ T cells (accounting for 90%), and the Vγ₂Vδ₂ (also known as Vγ₉Vδ₂) subtype is the primary circulating Vδ₂ subset (Wu et al., 2014).

Both Vδ₁ and Vδ₂ subsets are altered after HIV infection. Previous reports have described an increased level of Vδ₁ subset and decreased level and function of Vδ₂ subset in chronically HIV-infected patients (Li et al., 2008; Zheng et al., 2011). Furthermore, Vδ₂ subset is associated with disease progression in HIV infection and can inhibit active viral replication (Poccia et al., 1999; Li et al., 2008). γδ T cells can also enhance human immunity against MTB by secreting cytokines and promoting anti-TB immune response caused by other immune cells such as macrophages (Kawakami et al., 2004; Zhang et al., 2006). Similar to HIV infection, the frequency and function of Vδ₂ subset is also decreased in TB patients (Agrati et al., 2011; Negash et al., 2018). Few evidences suggested that HIV/TB co-infection had additive effects on peripheral Vδ₂ subset depletion and dysfunction (Rojas et al., 2005; Shao et al., 2008).

The frequency and function of γδ T cells are still poorly understood, especially in HIV/TB co-infection. By directed intracellular cytokine stain (ICS) (Yao et al., 2014; Zou et al., 2022) and flow cytometry, this study was performed to evaluate the phenotypes and function of γδ T cells in peripheral blood of HIV/TB co-infection patients (uninfected controls, patients with HIV or TB infection as control group).

γδ T cells have been proven to have anti-viral capability by lysing HIV-infected cells in vitro (Gan and Malkovsky, 1996). They can also enhance human immunity against HIV or MTB by secreting cytokines with the stimulation of phosphoantigens (Poccia et al., 1999; Zhang et al., 2006). However, the phenotype and function of γδ T cells during HIV/TB co-infection were poorly understood. The aim of this study was to evaluate the roles of γδ T cells in the peripheral blood of patients with HIV/TB co-infection, thus offering new insights into the role played by these cells in immunity against MTB and HIV.

In agreement with previous reports that the destruction of γδ T cells was in accordance with HIV infection progression (Zheng et al., 2011; Li et al., 2015), the present study confirmed that HIV infection caused an increased percentage of Vδ₁ subset and a decreased percentage of Vδ₂ subset. Several studies proved that Vδ₂ subset could inhibit and directly kill the intracellular MTB, thus preventing disease progression (Li et al., 1996; Dieli et al., 2001). In the present study, a lower proportion of Vδ₂ subset was observed in TB group compared with HC group, which was consistent with other studies (Li et al., 1996; Carvalho et al., 2002). Whether the reduction of Vδ₂ subset is a predisposing factor to the development of TB or is a consequence of MTB infection itself is still unknown. These results suggested HIV or TB infection could alter the balance of γδ T cells subsets (especially the decrease of Vδ₂ subset), which may deteriorate the status of patients with HIV or MTB infection. Moreover, in HIV/TB co-infection patients, the proportion of Vδ₁ and Vδ₂ subset had a further increased and decrease, respectively. Vδ₁ expansion may be linked to microbial translocation and may impact coinfection with several herpesviruses. Vδ₂ subset plays an important role in controlling HIV replication and disease progression (Poccia et al., 1999; Li et al., 2008). The decrease of Vδ₂ in the HIV/TB group may indicate a decrease in the body's ability to control HIV (Halary et al., 2005; Harris et al., 2010). The reduction in Vδ₂ subset observed in our study may attribute to the presence of specific HIV and MTB ligands inducing a sustained activation of Vδ₂ T cells, followed by a reduction in this cell subset by spontaneous and activation-induced apoptosis (Poccia et al., 1996; Li et al., 1998). Our results also indicated that HIV/TB co-infection might synergistically affect escaping the immune attack. In addition, some metabolites may influence the phenotype and function of γδ T cells. Various manipulations affecting isoprenoid metabolism can lead to the stimulation of Vγ₂Vδ₂ T cells (Wang et al., 2011). In vitro, the metabolic conversion of isopentenyl pyrophosphate (IPP) was blocked by zoledronate, resulting in the selective activation and expansion of Vγ₂Vδ₂ T cells (Roelofs et al., 2009; Wang et al., 2011). Moreover, mycobacterial metabolites, such as 3-Formyl-1-butyl-PP, were demonstrated to activate human γδ T cells (Belmant et al., 1999). How HIV/TB co-infection affects the metabolism is still unclear. Further studies on the function of Vδ₂ subset need to explain why the HIV/TB co-infection progresses more quickly and is much more complicated to control than single pathogen infection from the immunological and metabolic aspect.

Most γδ T cells are CD4 and CD8 double negative, while only a tiny part express CD4 or CD8 molecules (Zheng et al., 2011). Our study revealed characteristics of CD4 or CD8 expression on γδ T cells in patients with HIV infection and TB. As is known, CD4⁺ T lymphocytes are attacked and destroyed by HIV. In our study, the proportion of CD4⁺CD8^- αβ T cells was significantly lower in HIV and HIV/TB group than that in HC and TB groups, respectively. However, the comparison between HIV and HC groups in our study revealed no significant difference in the percentage of CD4⁺CD8^- Vδ₂ T cells. This result might raise doubt about that HIV possibly infecting γδ T cells by CD4 molecules and co-receptors (CXCR4 and CCR5) to cause the depletion of Vδ₂ subset (Imlach et al., 2003; Spencer et al., 2013). This result illustrated that CD4⁺CD8^- Vδ₂ T cells could survive during HIV infection and might be able to resist HIV infection but not HIV/TB co-infection. Recent studies suggested that double-positive cells (DP) T cells which accounted for only about 1%-2% of circulating humans are mature memory cells and may participate in the adaptive immune response against some infections (Overgaard et al., 2015; Zou et al., 2022). Our study described a significant increase in the percentage of DP T cells among Vδ₂ subset and αβ T cells in the peripheral blood of HIV and TB group and incredibly high DP cells in HIV/TB co-infection group. These data showed that DP cells might play a certain role in the immune response of HIV or TB infection, particularly the co-infection of two pathogens.

Activated γδ T cells can secrete a variety of cytokines, including TNF-α, interleukin, IFN-γ, perforin, granzyme, and so on, which function as effector molecules in controlling infectious microorganisms (Li et al., 2008; Spencer et al., 2013). In this study, we investigated the function of γδ T cells in HIV and/or TB infection by analyzing cytokines produced by Vδ₂ subset. TNF-α is the main component of the natural defense mechanism of the host. TNF-α can recruit monocytes, granulocytes and other innate immune cells to the location of infection and helps to form tuberculous granulomas in the infected tissues, which is beneficial to prevent the dissemination of TB bacteria in the body (Saunders et al., 2004). The levels of TNF-α also positively correlate with the progression of HIV infection (Benjamin et al., 2013; Vaidya et al., 2014), suggesting the critical role of TNF-α in HIV or/and TB. In the present study, we showed that the percentage of TNF-α producing γδ T cells was significantly lower in HIV/TB group compared to HIV group. However, there were no significant differences between the HC and TB group. Our data implied that HIV infection impaired the ability of γδ T cells to secrete TNF-α in active TB. Some studies also showed lower TNF production and cellular proliferation in MTB-specific peripheral T cells in HIV/MTB co-infection individuals than in individuals with HIV infection (Mendonca et al., 2007; Geldmacher et al., 2008), and HIV/MTB co-infected macrophages released fewer TNF-α than macrophages infected with only MTB (Kumawat et al., 2016). The reason might be that the expression of TNF-α in cells was down-regulated by HIV/MTB co-infection through some mechanism. Or the TNF-α-secreting cells may die through certain pathways in the case of HIV/MTB co-infection.

By inducing the expression of chemokines, recruiting monocytes and granulocytes and promoting the formation of granuloma, several studies demonstrated that IL-17 contributes to protective immunity in TB infection (Khader and Cooper, 2008; Torrado and Cooper, 2010). Nevertheless, some studies found that TB patients resistant to multiple drugs express high levels of IL-17 accompanied by severe tissue damage (Basile et al., 2011). Agree with another study (Peng et al., 2008), in the present study, we found that the percentage of IL-17A (referred to as IL-17) producing γδ T cells in TB patients was significantly higher than that in HC group, and there was a similar feature of change between HIV/TB and HIV patients. Our data not only further proved that γδ T cells might be involved in the immunity to MTB infection by secreting IL-17, but also suggests that HIV infection does not reduce the function of γδ T cells in producing IL-17. Considering the double-edged effect of IL-17, it is critical to elucidate further the mechanisms of IL-17 producing γδ T cells in the aspect of protective immunity and immunopathology during tuberculosis.

More studies show that effective cytotoxic T lymphocytes (CTL) responses require a coordinated expression of perforin and granzymes (Andersson et al., 1999; Chattopadhyay et al., 2009; Wu et al., 2014). A study describes that in TB, MTB-specific CD8⁺T cells express a low level of perforin but a relatively high level of granzyme A and B (Rozot et al., 2013). It is also reported that perforin^-granzyme A⁺ CD8⁺ T cells had a weak capacity to kill target cells (Takata and Takiguchi, 2006). In our study, there was no significant difference in the percentage of γδ T cells secreting granzyme A among the four groups. However, the percentage of γδ T cells secreting perforin in HIV/TB group was significantly lower than that in HIV and TB group, and there was no significant difference between HIV and TB group and HC group. In addition, granzyme A secreted by Vδ₂ subset has been found to induce TNF-α production in MTB-infected macrophages and inhibit the growth of intracellular MTB independently of perforin (Spencer et al., 2013). These results suggested that the anti-HIV and anti-TB effect of granzyme A and perforin secreted by Vδ₂ subset was independent. Moreover, HIV/TB co-infection but not HIV or TB mono-infection could influence the ability of γδ T cells to secrete perforin.

This study has several limitations. First, the sample size was small. In addition, antigen-specific assays were not performed in our study. Although direct ICS represents the actual immune response in the human body, there is seldom such a high concentration of antigens in blood, and our results could not be compared with those of other studies using antigen-specific stimulation.

In conclusion, our results demonstrated a significant decrease in Vδ₂ T cells in HIV/TB co-infection. Further studies on CD4⁺CD8^- Vδ₂ T cells will be needed to clarify their ability to resist HIV infection. The influence of HIV and /or MTB on the function of γδ T cells to produce cytokines is very complicated, which will shed light on further investigations on the mechanisms of the immune response against HIV and/or MTB infection.

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 Research and Ethics Committee of Zhongnan Hospital, Wuhan University, P. R. China (2016009-1K). The patients/participants provided their written informed consent to participate in this study.

LS, YF, and KL conceived and designed this investigation. SZ, YX, and WG helped to design the scheme of the investigation and collected the original data. YT and QZ performed the experiments. YY and SW analyzed the data. SZ, YX, and KL contributed to the writing of the paper. All authors contributed to the article and approved the submitted version.