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Diminished Peripheral CD29hi Cytotoxic CD4+ T Cells Are Associated With Deleterious Effects During SIV Infection

Cytotoxic CD4+ T cells (CD4+ CTLs) limit HIV pathogenesis, as evidenced in elite controllers (a subset of individuals who suppress the virus without the need for therapy). CD4+ CTLs have also been shown to kill HIV-infected macrophages. However, little is known about their contribution towards HIV persistence, how they are affected following exposure to immune modulators like morphine, and what factors maintain their frequencies and function. Further, the lack of robust markers to identify CD4+ CTLs in various animal models limits understanding of their role in HIV pathogenesis. We utilized various PBMC samples obtained from SIV infected and cART treated rhesus macaques exposed to morphine or saline and subjected to flow cytometry evaluations. Thereafter, we compared and correlated the expression of CD4+ CTL-specific markers to viral load and viral reservoir estimations in total CD4+ T cells. We found that CD29 could be reliably used as a marker to identify CD4+ CTLs in rhesus macaques since CD29hi CD4+ T cells secrete higher cytotoxic and proinflammatory cytokines following PMA/ionomycin or gag stimulation. In addition, this immune cell subset was depleted during untreated SIV infection. Strikingly, we also observed that early initiation of cART reconstitutes depleted CD29hi CD4+ T cells and restores their function. Furthermore, we noted that morphine exposure reduced the secretion of proinflammatory cytokines/cytotoxic molecules in CD29hi CD4+ T cells. Lastly, increased functionality of CD29hi CD4+ T cells as depicted by elevated levels of either IL-21 or granzyme B hi T Bet+ gag specific responses were linked to limiting the size of the replication-competent reservoir during cART treatment. Collectively, our data suggest that CD4+ CTLs are crucial in limiting SIV pathogenesis and persistence.


INTRODUCTION
The proposed excision of latently infected HIV cells in a second person with HIV infection offers renewed optimism towards a cure that would usher an end to the AIDS crisis (1,2). Although great strides have been undertaken, the cellular responses associated with sustaining persistence versus viral eradication remain poorly understood (3)(4)(5). HIV-infected individuals worldwide live different lifestyles stemming from varied cultural and sexual practices, co-exposures to other pathogens, and comorbid substance abuse not limited to the illicit use of drugs like morphine, cocaine, and heroin (6)(7)(8)(9)(10)(11). Collectively, these behaviors differentially impact the body's immune response, alter disease pathogenesis and need to be considered during the development of a universal HIV cure (6,12).
The body's immune system is built around the activities of CD4+ T cells (13). CD4+ T cells are a significant immune cell subset, which coordinates the immune system and also aid diverse immune (B, CD8 + T, and NK) cell function (14)(15)(16). In addition, they are crucial in generating prompt and protective memory responses against recalled pathogens (17,18). CD4+ T cells are highly plastic and exist as numerous phenotypes like T helper (Th) 1, Th 2, and Th 17, cytotoxic (CD4+ CTL), T follicular helper (Tfh), and T regulatory (T regs) (19). These diverse CD4+ T cell phenotypes express different transcriptional profiles during health and disease, have distinct fates, and carry various functions, including immune regulation (CD+ T regs) (20). Beyond the simplistic provision of help, CD4+ T cells have also been shown to play other roles like regulation of immune responses (CD4+ T regs). They could also directly target infected cells through CD4+ CTLs (13). In supercentenarians, it was suggested that CD4+ CTLs were crucial in healthy aging, where they were found to be expanded while offering long-lasting protection (21).
The hallmark of HIV infection is the progressive loss of total CD4+ T cells and dysregulation of homeostasis, later culminating in acquired immune deficiency syndrome (AIDS) (22,23). Early during infection, HIV causes massive gut damage accompanied by leakage of microbial biproducts into the periphery (24,25). Synchronously, gut dysbiosis occurs as the landscape of the gut microbiome shifts towards more proinflammatory and pathogenic bacterial communities such as Prevotella and Enterobacteriaceae (26)(27)(28). As a result of microbial translocation and gut dysbiosis, chronic immune activation and systemic inflammation later ensue (29,30).
Progressively, enhanced CD4+ T cell activation leads to immense CD4+ T cell loss by apoptosis and exaggerated viral cytolysis (31,32). Following combined antiretroviral therapy (cART) initiation, the recovery of absolute CD4+ T-cell counts is often viewed as the benchmark for the immune system reconstitution (33,34). However, ongoing dysregulation of cellular function limits the implementation of robust antiviral responses (35). Furthermore, specific CD4+ T-cell lineages like Tfh have been extensively reported to harbor viral reservoirs within the periphery and lymphoid tissues (36,37). Although CD4+ T cells are targeted during virus replication and persistence, considerable evidence supports a crucial role of cytotoxic CD4+ T-cell phenotypes in HIV control and slowing disease progression (38,39). During acute HIV infection, a robust HIV-specific CD4+ CTL response comprised of elevated granzyme A, interferon-gamma (IFN g+), and CD40 ligand (CD40L) has been documented to lower viral load set points (40). Johnson et al. also made similar observations and noticed that CD4+ CTLs predominantly expressed perforin, granzyme B, and Eomes during acute HIV infection (38). Subsequently, HIV Nef-specific CD4+ CTLS has been documented to suppress viral replication in macrophages ex-vivo (41).
The lack of a specific CD4+ CTL surface biomarker with consensus across different animal models limits the proper follow-up and interrogation of these cells. Recently, studies by Johnson et al., and Phetsouphanh et al., together used CD57 to identify and track this cell phenotype in different HIV-1 infection conditions (38,44). Despite this, the lack of crossreactive antibodies raised against the CD57 carbohydrate epitope in non-human primate (NHP) models limits its use (45,46). However, recently, in a pre-print article, Nicolet et al. suggested that CD29 enriches human CD4+ CTLs (47). Nonetheless, the utility of this biomarker as a surface marker for CD4+ CTLs remains to be evaluated in NHP models. Although various efforts have been undertaken to explore the effects of CD4+ CTL with HIV disease progression (39,48), the impact of virus-specific functionality of this cell phenotype on the size of replicationcompetent reservoirs remains to be studied. Moreover, the modulation of this cell lineage following exposure to substance abuse such as morphine remains unevaluated.
To address some of above-mentioned questions, we utilized samples obtained from SIV-infected rhesus macaques that were exposed to either morphine or saline. Of these animals, a subset was also treated with cART to suppress viral replication. We found that enhanced CD29+ expression on the surface of CD4+ T cells could be used to identify CD4+ CTLs. Thus, CD29hi CD4+ T cells express higher cytotoxic molecules like CD107a and granzyme B plus proinflammatory cytokines like IFN-g and TNF-a compared to their CD29lo counterparts. In addition, increased gagspecific secretion of cytokines like IL-21 and granzyme B was accompanied by smaller sizes of replication-competent viral reservoirs suggesting that CD4+ CTLs contribute towards limiting SIV persistence in cART-treated rhesus macaques.

Ethics Statement
This study was approved by the University of Nebraska Medical Center (UNMC) Institutional Animal Care and Use Committee (IACUC) as designated by assigned protocol numbers. For this study, we used: (1) 16-073-07-FC named "The Effect of cART and Drugs of Abuse and the Establishment of CNS Viral Reservoirs".
(2) 15-113-01-FC referred to as "The Combinatorial effects of Opiates and Promoter Variant Strains of HIV-1 subtype C on Neuropathogenesis and Latency".

Rhesus Macaques Used for This Study
For protocol 16-073-07-FC, 6 animals were escalated to 6mg/kg twice daily injections of morphine alongside five rhesus macaques Olwenyi et al. Peripheral Loss of CD29 hi Cytotoxic CD4+ T Cells Favors SIV Pathogenesis Frontiers in Immunology | www.frontiersin.org October 2021 | Volume 12 | Article 734871 exposed to saline (controls). Following this, all the animals were infected with 200 TCID 50 of SIVmac251 and later treated with a cART regimen comprising of 40 mg/ml emtricitabine (FTC), 20 mg/ml tenofovir (TFV), and 2.5 mg/ml dolutegravir (DTG). Alternatively, protocol 15-113-01-FC involved 8 rhesus macaques separately exposed to equivalent doses of either morphine (n = 4) or saline (n = 4). Similarly, this was followed with SIVmac251infection at an equivalent dose. However, no subsequent cART was administered. All animal characteristics and treatment regimen details were previously described (10). The experimental schema utilized for the study included a total of 19 rhesus macaques. Briefly, in the untreated group of eight animals, 6-mg/kg intramuscular (i.m.) injections of morphine were administered twice daily for 2 weeks to four rhesus macaques. Then, continued morphine dosing was maintained for 7 weeks while the remaining four animals received normal saline (control group). After 9 weeks, 200 TCID 50 of SIVmac251 was intravenously administered to the rhesus macaques and continued with morphine/saline treatments until the end of the study. (B) In the separate group of 11 animals exposed to cART, six were given intramuscular morphine for a total of 9 weeks while five received equal doses of saline (control group). After 9 weeks, 200 TCID 50 of SIVmac251 was intravenously administered to all animals. Five weeks later, cART was initiated and given daily at 1 ml/kg body weight up to the termination end of the study. Saline was given to the controls at a similar dose. Lastly, to evaluate the effects of cART on CD29hi CD4+ T cells, we utilized eight rhesus macaques exposed to either four drugs (FTC, tenofovir alafenamide (TAF), DTG and maraviroc (MVC); n = 4) or two drugs (DTG and FTC; n = 4) that were treated four weeks post SIV inoculation (protocol #16-001-02-FC, Assessment of Antiretroviral Pharmacology in Lymphoid Tissues using the SIV Macaque Model).

Isolation and Cryopreservation of PBMCs
Femoral blood, collected in K2-EDTA vacutainer tubes (BD, 367841), was layered above Lymphoprep ™ Density Gradient Medium from STEMCELL Technologies following plasma separation. PBMCs were then later isolated using the density gradient centrifugal separation method described previously (49).

Phenotype Analysis of Immune Cells (Flow Cytometry)
Cryopreserved PBMCs processed from the blood of rhesus macaques included in this study were utilized for flow cytometry.
Post-thaw viabilities of greater than 80% were considered for further experimentation. Briefly, this involved a brief 12-18h rest, and surface staining protocols proceeded at this step. First, Zombie Aqua fixable viability dye was added to discriminate dead cells.
After that, all surface markers included in the lineage and integrin/ chemokine receptor panels (Supplementary Table 1 Table 1) involving Eomes and T-bet transcription factors, the fix/perm solution (Tonbo Biosciences) was used for fixation and permeabilization. The remaining antibody cocktail was dissolved in FoxP3 transcription factor buffer (Tonbo biosciences) and added following permeabilization. Alternatively, in the cytokine secretion panel (Supplementary Table 1), 2% PFA was added for fixation, followed by permeabilization using BD perm wash buffer. Then, cells were incubated with a previously prepared cocktail of antibodies. Following incubation and later washes, the acquisition was performed on the Becton Dickson Fortessa X450 flow cytometer, and analysis was carried out using Flowjo version 10.6 (Trees Star Inc., Ashland, Oregon, USA). For ICS quantification, all values were reported following background subtraction.

Quantification of Plasma Viral Load in Peripheral Blood
Quantitative real-time PCR (qRT-PCR) was utilized to estimate levels of SIV RNA in EDTA plasma (50). Briefly, RNA was extracted from EDTA plasma using a QIA amp viral RNA mini kit (Qiagen, Germantown, MD, USA; Cat no: 52906). Additional information on primers and probes with details of PCR conditions were described previously (10).

Quantitative Viral Outgrowth Assay (QVOA)
CD4+ T cells were enriched from PBMCs using the non-human primate microbead CD4+ T cell isolation kit, (STEMCELL Technologies Inc, Seattle, WA, USA) and later resuspended in complete media (RPMI1640, 10% FBS, 2 mM glutamine, Penstrep (100 U/mL penicillin and 100 mg/mL streptomycin)), 10 U/mL IL-2 and 300 nM efavirenz (EFV; NIH AIDS Reagent Program). The resultant purified CD4+ T cells were then co-stimulated with CD3/CD28 beads (Dynabeads, Life Technologies, Waltham, MA, USA) and 10-fold limiting dilutions ranging from 10 2 to 10 5 cells/mL were generated. Following 72 h of incubation, 10 5 CEMx174 cells were added to aid in viral expansion. This time point was noted as day 0 of incubation and was later prospectively followed for 21 days. SIV RNA measures were then performed using qRT-PCR. Levels of replication-competent viruses were denoted as infectious units per million (IUPM) and were estimated using the IUPMStats v1.0 infection frequency calculator (10,51).

Statistical Analysis
Prism V9.0 (GraphPad Software) was used for Spearman's rank correlation and paired non-parametric tests with the aid of the Wilcoxon test. In addition, comparisons between morphine and saline groups were also performed using the Mann Whitney U test. Using R version 3.4.3, a correlation matrix was generated following multiple correlations. Resultant P values were then corrected for type 1 error with the aid of Holm's correction. Lastly, following Boolean gating in FlowJo, combinatorial expression of multiple cytokines was evaluated and graphed using SPICE ("Simplified Presentation of Incredibly Complex Evaluations") (52).

Loss of CD29hi CD4+ T Cells Is Positively Interrelated to Declining Immune Status and Increasing Viral Loads During SIV Infection
To gain insight into cellular and virological interactions of the peripheral immune system, changes in CD29hi CD4+ T cells alongside viral load and various immune cell subsets during baseline, acute (Day 14), and chronic (Day 245) phases of SIV disease progression (Supplementary Figure 1). Following SIV infection, viral load levels peaked during the acute phase and were maintained through the chronic time point ( Figure 1A). Compared to baseline, there was a progressive loss in %CD4+ T cell frequencies across the acute phase of infection (P = 0.0078) and the latter chronic phase of infection (P = 0.0003) ( Figure 1B). Subsequently, this was accompanied by an expansion of CD8+ T cells during both the acute (P = 0.0078) and chronic (P = 0.0002) phases of untreated SIV infection ( Figure 1C). Unsurprisingly, there was a progressive loss in the CD4/CD8 ratio across the acute (P = 0.0156) and chronic (P = 0.0156) phases of infection ( Figure 1D). The frequencies of %CD29hi CD4+ T cells were reduced during the acute (P = 0.0469) and chronic (P = 0.0078) phases of untreated infection ( Figure 1E). Similar trends were observed with absolute CD29hi CD4+ T cells (Supplementary Figure 2A) and absolute CD4+ T cells (Supplementary Figure 2B), while absolute CD8+ T cells were elevated during SIV progression (Supplementary Figure 2C). Remarkably, % CD8+/CD29+ T cells remained stabilized during the acute and chronic phases of untreated SIV infection despite the concurrent expansion of CD8+ T cells ( Figure 1F). The percent of both CD29hi CD4+ T cells and CD29+/CD8+ T cells exhibited an increase in activation shown by increased expression of HLA-DR (P < 0.05) ( Figures 1G, H). No changes were observed in innatelike CD8+ T cells that express CD16 ( Figure 1I). A progressive non-significant reduction of NK cells occurs through the acute to chronic phases of untreated SIV infection ( Figure 1J). However, NK cell subset analysis revealed that there was a reduction in % CD16+CD56-NK cells, (P = 0.024) ( Figure 1K) together with concurrent expansions of %CD16-CD56+ NK cells, (P = 0.039) ( Figure 1L) and %CD16-CD56-NK cells (P = 0.039) that are later maintained during chronic infection ( Figure 1M). Multiple Spearman rank correlation analysis revealed that reduced % CD29hi CD4+ T cells were positively associated with expanding %CD8+ T cells (rho = -0.532, P = 0.0044), CD16-CD56+ NK cells (rho = -0.647, P = 0.0006) while a positive correlation was seen with improved immune status (CD4/CD8 ratio) (rho = 0.42, P = 0.044). Additionally, negative associations were noticed with CD16-CD56-NK cells (rho = -0.7278374) and CD16-CD56+ NK cells (rho = -0.3928776), (all P < 0.05) ( Figure 1N). Furthermore, CD16+CD56-NK cells were positively correlated with absolute CD29hi CD4+ T cells (rho = 0.7510 and P = 0.00001) and absolute CD4+ T cells (rho = 0.7206 and P = 0.0002) respectively (Supplementary Figures 2D, G). In turn, CD16-CD56+ NK cells were associated with loss of CD29 hi CD4+ T cells (Supplementary Figure 2E)

CD29hi CD4+ T Cells Express Elevated Levels of Pro-Inflammatory and Cytolytic Mediators in Rhesus Macaques Exposed to cART and Morphine During Chronic Infection
Upon noticing that CD29hi CTLs were depleted during progressive untreated SIV infection, we next turned our efforts to characterizing these cells. Utilizing PBMCs collected from cART treated rhesus macaques, we assessed the extent to which CD4+ CD29hi T cells express diverse integrins (CD11a and CD11b) and the chemokine receptor CX3CR1 (Supplementary Figure 4). In addition, after PMA/ionomycin stimulation, we assessed transcription factors T bet and Eomes, the cytotoxic mediators granzyme B and CD107a, IL-21, T helper (Th) 1 cytokines TNF-a and IFN-g plus the Th2 cytokine IL-4 (Supplementary Figure 5). Surprisingly, there were no significant differences between CD11a expression in CD29hi vs. CD29lo CD4+ T cell subsets ( Figure 3A). However, we noticed a three-fold increment of surface CD11b expression on CD29hi versus CD29lo CD4+ T cells (P = 0.0020) ( Figure 3B). Similarly, we observed a close to two-fold elevation in the expression of CX3CR1 in CD29hi CD4+ T cells (P = 0.0010) ( Figure 3C). Unexpectedly, the CD29hi CD4+ T cell subset had lower levels of Eomes (P = 0.0020) ( Figure 3D) while expressing over two-fold levels of T bet in comparison to the CD29lo CD4+ T cell subset (P = 0.0020) ( Figure 3E). Moreover, elevated granzyme B expression was observed in the CD29hi CD4+ T cell subset versus the CD29lo CD4+ T cell subset ( Figure 3F). Similarly, the level of granzyme B+ hi expression was elevated in the CD29hi CD4+ T cell subset (P = 0.0010) ( Figure 3G), which remained augmented even after testing for co-expression of granzyme B hi and Tbet+ (P = 0.0010) ( Figure 3H). The level of IL-4 was similar between CD29hi vs CD29lo CD4+ T cells ( Figure 3I) whilst elevated expression of diverse cytokines such as: CD107a (P = 0.0010) ( Figure 3J), IL-21 (P = 0.0010) ( Figure 3K), IFN-g (P = 0.0010) ( Figure 3L) and TNF-a (P = 0.0010) ( Figure 3M) were observed. Similarly, there were higher frequencies of % IFN-g+ and TNF-a + co-expressing CD29hi CD4+ T cells in comparison to CD29lo CD4+ T cells (P = 0.0087) ( Figure 3N). Likewise, IL-21+ and IFN g+ co-expressing cells are elevated in CD29hi CD4+ T cells in comparison to CD29lo CD4+ T cells (P = 0.0010) ( Figure 3O). Next, we observed that morphine causes downregulation of cytokine secretion ranging from IL-21, TNF-a, IFN-g, IFN-g, and TNF-a dual positive plus IL-21 and IFNg co-expressing CD29hi CD4+ T cells (all P < 0.01) (Figures 3P-U). Morphine treatment was also observed to similarly downmodulate the expression of inflammatory cytokines and cytotoxic molecules in the CD29lo CD4+ T cell subset (Supplementary Figure 6). By evaluating polyfunctionality of CD29hi CD4+ T cells revealed that the majority of cytokine-producing cells were mainly TNF-a + cells and demonstrated greater frequencies of multiple cytokines secreting cells such as combinations with TNF-a + and CD107a cells, TNF-a + and IL-21 and TNF-a + and IFN-g+ co-expressing cells, (all P <0.05) ( Figure 3V).

CD29hi Gag Specific CD4+ T Cells Also Express Elevated Levels of Pro-Inflammatory and Cytolytic Mediators in Rhesus Macaques Exposed to cART and Morphine
In synchrony with earlier observations seen in Figure 2, CD29 hi CD4+ T cells expressed higher levels of IFN-g ( Figure 4A), CD107a ( Figure 4B), IL-21 ( Figure 4C), TNF-a ( Figure 4D) (all P<0.05) (Supplementary Figure 6). The same trend was seen  Figure 4J) in comparison to CD29lo CD4+ T cells, (all P<0.05). Notably, no significant differences were observed with levels of IL-4 ( Figure 4E), (P > 0.05). In congruence with earlier observations noticed, morphine similarly led to downregulation of various gag-specific cytokines such as IL-21, TNF-a, and granzyme B hi T Bet+ in CD29hi CD4+ T cells (all P < 0.05) (Figures 4K-O). Next, we investigated whether there was a relationship between either IL-21 or granzyme B hi T Bet+ gag specific release on CD29hi CD4+ T cells with the size of the replication-competent viral reservoir as measured by quantitative viral outgrowth assay (QVOA) ( Figure 4P). Negative correlations were observed between QVOA and either IL-21 gag specific CD4+/CD29+T cells or granzyme B hi T bet+ gag specific CD29 hi CD4+ T cells. The association between granzyme B hi T Bet+ CD29hi CD4+ (Figures 4Q, R). Paired comparisons between CD29lo and CD29hi CD4+ T cells were performed using the non-parametric Wilcoxon test. Comparisons between morphine and saline were also performed in CD29+ CD4+ T cells using the Mann Whitney U test. For SPICE analyses, pie charts showing cytokine secretion patterns together with arc plots show the predominant cytokine being secreted. Bar graphs denote median cytokine expressing cells. Simultaneously, the extent of cytokine polyfunctionality was compared within CD29lo vs CD29 hi CD4+ T cell subsets with aid of the Wilcoxon test. For all comparisons, * shows p < 0.05, ** indicates p < 0.01 while *** denotes p < 0.001. ns represents non significant. Red dots denote morphine treated while blue dots represent rhesus macaques exposed to saline. individuals capable of suppressing HIV without therapy, possess greater frequencies of circulating virus-specific CD4+ CTLs (44). Remarkedly, the development of CD4+ CTL escape SIV mutants is accompanied by loss of elite control in rhesus macaques (53). Thus, there is no surprise that declining immune status occurred simultaneously with the loss of proposed CD4+ CTLs and increased viral loads suggesting this cell subset is crucial in limiting HIV pathogenesis. Similarly, the loss of HIV-specific CD4+ CTL function has been reported in chronic progressors (44). Predictably, during progressive infection, CD29hi CD4+ T cells exhibited increased HLA-DR activation levels, hinting that this cell phenotype could be targeted and depleted by the virus (54). Furthermore, the expansion of cytokine secreting (CD16-CD56+) and double negative CD16-CD56-NK cells are crucial for limiting viremia post-peak viral loads (55,56). However, the loss of CD16 expression on prevailing NK cell subsets followed by the decrease of both CD29hi CD4+ T cells and absolute CD4+ T cells further highlights SIV-induced NK cell and CD4+ CTL dysfunction during disease progression (57).

DISCUSSION
During our time course studies, our observation that timely initiation of therapy reconstitutes peripheral CD29hi CD4+ T cells and restores IFN-g, TNF-a, and CD95 polyfunctionality, in turn, supports findings from other groups who have reported that early ART preserves T cell function (58,59). Following gag and PMA/ ionomycin stimulation, CD29hi CD4+ T cells display cytotoxic properties through elevated secretion of proinflammatory cytokines such as IFN-g, TNF-a, and IL-21 or cytolytic molecules such as granzyme and CD107a (60). In turn, increased expression of T bet dually favors IFN-g and TNF-a . Scheme for correlation analysis between gag specific IL-21 or Granzyme B hi T bet+ CD29 hi CD4+ T cells and QVOA measures of replication competent reservoirs in total CD4+ T cells (P). Correlation between gag-specific IL-21+ CD29hi CD4+ T cell frequencies and QVOA (Q). Correlation between gag specific Granzyme B+ hi T bet+ CD29hi CD4+ T cell frequencies and QVOA (R). Paired comparisons between CD29 lo and CD29 hi CD4+ T cells were performed using the non-parametric Wilcoxon test. Comparisons between morphine and saline were also performed in CD29+ CD4+ T cells using the Mann Whitney U test. For all comparisons, * shows p < 0.05, ** indicates p < 0.01 while *** denotes p < 0.001. ns signifies non significant. Red dots denote morphine treated while blue dots represent rhesus macaques exposed to saline. secretion while supporting cytotoxicity by binding to downstream promoters of granzyme B and perforin genes (61). Similarly, augmented expression of CX3CR1 also tends to favor CD4+ CTL transmigration into inflamed tissue, as exemplified in Dengue, where virus-specific CX3CR1 CD4+CTLs mediate protective cytotoxicity (62). Similarly, Chen et al. showed that a cytomegalovirus-driven CD57hi CD4+ CTL phenotype uses CX3CR1 to facilitate vascular trafficking by attaching to the fractalkine receptor on the endothelium during HIV infection (63).
Since CD29hi CD4+ T cells exhibit greater polyfunctionality as demonstrated by a distinct functional profile typified by elevated secretion of multiple inflammatory cytokines and cytotoxic molecules, they could serve as robust antiviral mediators that directly target virus-infected cells (59). In agreement with this, we noted that increased ex-vivo secretion of IL-21 in response to gag was negatively associated with the size of the replication-competent viral reservoir within the periphery. In a separate study, IL-21 secretion by CD4+ T cells was noted to support CD8+ cytotoxic T cell responses in chronic progressors, even during the late-stage infection, leading to diminished virus replication (64). Furthermore, therapeutic administration of IL-21 has been documented to reduce the size of viral reservoirs in rhesus macaques (64,65).
The association between granzyme B hi T Bet+ CD29hi CD4+ T cells and diminished replication-competent viral reservoirs mirrors earlier findings by a separate study group found that HIV specific granzyme B and not IFN-g CD8 CTLs were linked to reduced HIV reservoirs during acute HIV infection (42). In addition, increased inflammation, as noted by elevated levels of pro inflammatory cytokines such as IL-18 (data not shown), has been linked to development of AIDS (43,66). The observation that elevated CD29hi CD4+ CTLs were negatively associated with proinflammatory plasma IL-18 during untreated SIV infection, data not shown, corroborates the importance of these cells in fostering better disease outcomes.
The observation that morphine downmodulated the secretion of virus-specific proinflammatory cytokines and the release of granzyme B by CD4+ CD29hi CTLs coincides with several reports that suggest that this drug inhibits CD4+ T cell release of Th1 cytokines (IFN g, TNF-a) while causing a shift towards Th2 phenotypes (67,68). However, future studies are needed to evaluate the mechanisms by which morphine causes immune modulation of this cell subset. Further, larger sample sizes and studies using sorted CD29hi CD4+ CTLs are necessary for further validation of the contribution of this cell phenotype towards targeting persistence.
Additional future strategies aimed at improving the CD4+ CTL functionality, such as utilizing chimeric engineering strategies could reinvigorate CD4+ CTLs fostering improved disease outcomes (69,70). Further, the unexpected transcription profile of lower Eomes expression in CD29hi CD4+ T cells while harnessing augmented levels of Tbet warrants further research in understanding how regulation of transcription factors affects CD4+ CTL functionality and differentiation. In their CD8+ T cell counterparts, Smith et al., revealed that cells expressing Tbet hi and Eomes lo profiles are more efficient in recognizing major histocompatibility complex (MHC) peptides (71). Finally, there is a need to track changes in this cell lineage in body compartments such as lymph nodes, central nervous system, and the gut that provide safe niches for low-level virus replication, evolution, and persistence following effective cART (72).
In conclusion, we demonstrate that CD29 could be reliably used to identify CD4+ CTLs in rhesus macaques. Furthermore, CD29hi CD4+ CTLs are beneficial towards limiting SIV pathogenesis by limiting virus replication and secreting crucial cytokines associated with reducing the size of the viral reservoir within the periphery.

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

ETHICS STATEMENT
The animal study was reviewed and approved by University of Nebraska Medical Center (UNMC) Institutional Animal Care and Use Committee (IACUC).

AUTHOR CONTRIBUTIONS
OO and SNB designed the conceptual framework of the study and analyzed the data. OO performed all the flow cytometry experiments, wrote the initial draft of the manuscript. SJ, KP, MT, and AA carried out virological assays. SJB, HF, AP, CF, and SNB supported fundraising for the animal studies and edited the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This study was supported by NIH grants R01DA043164, P30MH062261 to SNB, SJB, and HF, R01DA041751 to SNB and SJB, and R01AI124965 to CF. The funders had no role in designing the study or in the interpretation of the results.

ACKNOWLEDGMENTS
We appreciate the staff and veterinarians of the University of Nebraska Medical Center (UNMC) Comparative Medicine department for housing and animal procedures. We also appreciate other team members like Brenda M. Morsey, Benjamin Lamberty, Shannon Callen, and personnel from Dr. Byrareddy's laboratory who routinely took care of the rhesus macaques included in this study. We are also grateful to members of the flow core section at UNMC. Finally, we thank NIH HIV Reagent Program, Division of AIDS, NIAID, NIH for the provision of Peptide Array, Simian Immunodeficiency Virus (SIV) mac239 Gag Protein, ARP-6204.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. 734871/full#supplementary-material Supplementary Figure 1 | Longitudinal Changes In Diverse Lymphocyte Subsets During Progressive Untreated Siv Infection. Gating strategy based on a representative sample used for delineation of lymphocyte subpopulations within PBMCs obtained from untreated SIV rhesus macaques. Lymphocytes were gated out based on size and complexity (FSC-a vs. SSC-a). NKG2A vs. CD3+ was used to exclude CD3+ T cells, NKT cells, and NK cells. CD3+ T cells were later differentiated based on CD4+ and CD8+ markers and expression of CD29 was used to mark cytotoxicity in either subset. HLA-DR levels were then used to identify activation levels in either CD4+ or CD8+ T cells. MAIT cells were also studied based on CD161 and Va7.2Ja co-expression. CD16+ CD8+ T cells (innate like CD8+ T cells) were also evaluated. NK cell subsets based on CD16 and CD56 expression were also studied for maturity based on CD27 and activation based on HLA-DR expression. During Art Treated Chronic Siv Infection. To achieve this, a time vs FSC-A gate was used to obtain a steady stream of cells during cell acquisition that is devoid of interruptions caused by micro bubbles, clogs or air. The FSC-A vs FSC-H gate and SSC-A vs SSC-H was used to obtain single cells. Dead cells were excluded using positive zombie aqua live dead staining. Lymphocytes were then segregated based on FSC-A vs SSC-A gate based on size and complexity and T cells later gated off using the CD3+ marker. CD4+ T cells were then further gated followed by the extent of their expression of CD29. Comparisons of CD29hi vs CD29lo CD4+ T cells were then carried out to evaluate the expression of CD11a and CD11B integrins together with the chemokine receptor CX3CR1. Fluorescent minus one (FMO) controls were included to set a background for gate placement. Supplementary Figure 6 | evaluation of cytokine secretion and cytotoxic molecule expression in cd29lo cd4+ t cells of cart treated siv infected macaques exposed to morphine or saline. Levels of (A) ifn-g (B) il-4 (C) tnf-a (D) cd107a (E) mfi of granzyme b (F) granzyme b hi (G) ifn-g and tnf-a (H) il-21 and ifn-g expression in cd29lo cd4+ t cells.
Supplementary Figure 7 | evaluation of cytotoxic molecules, transcription factors in cart treated siv infected macaues exposed to morphine or saline. Cd3+ t cells were identified from live cells. Cd4+ t cells were then gated out followed by testing for cd29 expression. In unstimulated, gag or pma/ionomycin conditions, percent expression of: the cytotoxic molecule granzyme together with transcription factors like t bet or eomes were evaluated in cd29 hi vs cd29 lo subsets.
Supplementary Table 1 | various antibody panels used for the study. Exhaustive list of monoclonal antibodies with insights into specificity, fluorochrome tagged, ab clone, vendor/source together with volumes/test used.