BACKGROUND:Persistent immune activation is linked to elevated cardiovascular diseases in people with HIV on antiretroviral therapy. The fat attenuation index (FAI) is a measure of peri-coronary inflammation that independently predicts cardiovascular disease risk in people without HIV. Whether FAI is associated with immune activation is unknown. METHODS:Peripheral blood T-cell activation and homing phenotypes were measured in people with HIV (n=58) and people without HIV (n=16) without known cardiovascular disease who underwent coronary computed tomography angiography and had FAI measurements. A cross-sectional analysis of an observational cohort was performed. The primary aim was to evaluate associations of T-cell activation and phenotypes with the outcome variables, FAI values of the right coronary artery and left anterior descending artery, which were assessed using multivariable regression models adjusted for age, natal sex, race, low-density lipoprotein cholesterol, body mass index, and use of lipid-lowering medication. RESULTS:T cells from people with HIV showed greater activation, as measured by cluster of differentiation (CD) 38/human leukocyte antigen - DR isotype coexpression on CD4 central memory and terminally-differentiated effector memory subsets and on CD8 effector memory (TEM), than did cells from people without HIV. Expression of the chemokine receptor C-C Chemokine Receptor 2 was reduced on CD4 central memory and TEM and CD8 TEM and terminally-differentiated effector memory subsets in people with HIV. Among all participants, PD-1 (programmed cell death 1) in CD8 central memory was associated with worsened peri-coronary inflammation of the right coronary artery, whereas perforin/granzyme B on CD8 TEM was associated with improved peri-coronary inflammation of the right coronary artery and left anterior descending artery in adjusted analyses. When accounting for HIV serostatus, CD38/human leukocyte antigen - DR isotype coexpression on CD8 central memory, TEM, and terminally-differentiated effector memory cells was associated with more peri-coronary inflammation of the left anterior descending artery. CONCLUSIONS:The associations between T-cell activation with FAI are novel and suggest that T-cell activation may be an important driver of peri-coronary inflammation, occurring at an early stage of atherosclerosis, even before the development of clinical disease.

Background: Optimal control of microbial infections requires mucosal-associated invariant T (MAIT) cells. People living with HIV (PWH) on antiretroviral therapy (ART) can be divided into 2 groups: immune responders (IR) who recover or retain CD4 T cell numbers, and immune non-responders (INR) who do not. Compared to IR, INR have fewer MAIT cells and increased systemic inflammation and microbial translocation, but how these factors affect MAIT cells is unknown. Methods: MAIT cells from IR, INR, and from controls without HIV were enumerated and characterized by flow cytometry. To determine the links among MAIT cells, inflammation, and microbial translocation, the correlations of MAIT cell numbers to previously published soluble inflammatory markers and plasma microbial genetic sequences were assessed by Spearman analysis. In vitro assays were used to support our findings. Results: MAIT cell numbers were significantly negatively correlated with levels of IL-6 and IP-10 (markers of inflammation); CD14, LPS, and FABP2 (markers of microbial translocation); and with abundance of Serratia and other Proteobacteria genetic sequences in plasma. In a separate analysis of PWH on ART receiving the IL-6 receptor antagonist tocilizumab (TCZ), we found that blocking IL-6 signaling with TCZ increased IL-7 receptor expression on MAIT cells and reduced plasma IL-7 levels, consistent with improved uptake of IL-7 in vivo. Conclusions: Our findings suggest inflammation and microbial translocation in PWH on ART lead to a loss of MAIT cells via impaired IL-7 responsiveness, resulting in further increased microbial translocation and inflammation.

CD8 T cells are emerging as important mediators in atherosclerosis and cardiovascular disease (CVD). Immune activation may play a particular role in people with HIV (PWH) who are at an increased risk of CVD, even after controlling for known CVD risk factors. Latent CMV infection is associated with increased CVD risk for both PWH and people without HIV, and human CMV-specific CD4 and CD8 T cells are enriched for an immunosenescent phenotype. We previously showed that CMV coinfection in PWH promotes vascular homing and activation of inflammatory CD4 T cells through the CD2-LFA-3 axis. However, the role of CD2/LFA3 costimulation of CD8 T cells in PWH with CMV has yet to be described. In the present study, we demonstrate that CD2 expression on CX3CR1+CD57+CD28- inflammescent CD8 T cells is increased on cells from CMV-seropositive PWH. In vitro CD2/LFA-3 costimulation enhances TCR-mediated activation of these inflammatory CD8 memory T cells. Finally, we show that LFA-3 is highly expressed in aortas of SIV-infected rhesus macaques and in atherosclerotic plaques of people without HIV. Our findings are consistent with a model in which CMV infection enhances CD2 expression on highly proinflammatory CD8 T cells that can then be stimulated by LFA-3 expressed in the vasculature, even in the absence of CD28 costimulation. This model, in which CMV infection exacerbates toxic cytokine and granzyme production by CD8 T cells within the vasculature, highlights a potential therapeutic target in atherosclerosis development and progression, especially for PWH.

Plasma cytokine levels were quantified among 30 persons with HIV (PWH) identified as elite controllers (15 transient controllers [studied a median of 1.38 years before losing viral control] and 15 persistent controllers). Thirty antiretroviral therapy (ART)-naive PWH, 30 ART-treated PWH with undetectable viremia, and 30 HIV-uninfected controls also were studied. Higher levels of cytokines were recognized among PWH than among controls, with EC displaying the highest levels. Elevated levels of IP-10 and MIG were identified among transient controllers as predictors of the loss of viral control. These findings offer feasible biomarkers for predicting virologic outcome and loss of control in EC.

People with HIV (PWH) experience endothelial dysfunction (ED) that is aggravated by chronic inflammation and microbial translocation across a damaged gut barrier. Although this paradigm is well-described, downstream pathways that terminate in endothelial dysfunction are only partially understood. This study found increased expression of granulocyte macrophage colony stimulating factor (GM-CSF), toll-like receptor-4 (TLR4), and myeloperoxidase in the aortic endothelium of PWH compared to those without HIV. Bacteria-derived lipopolysaccharide (LPS) heightened glucose uptake and induced GM-CSF expression in primary human endothelial cells. Exposure to sodium-glucose cotransporter-2 (SGLT2) inhibitors reduced glucose uptake, GM-CSF release, and ED in LPS-activated endothelial cells ex vivo, , and PWH treated with SGLT2 inhibitors for diabetes had significantly lower plasma GM-CSF levels than non-diabetic PWH not on this medication. The findings suggest that microbial products trigger glucose uptake and GMCSF expression in the endothelium, contributing to localized inflammation in PWH. Modifying this altered state could offer therapeutic benefits.

People with human immunodeficiency virus (PWH), despite achieving viral suppression through antiretroviral therapy, face increased risk and earlier onset of atherosclerotic cardiovascular diseases than the general population. CD57+ T cells can be readily recovered from atherosclerotic plaques and likely contribute to disease by targeting endothelial cells (ECs); however, the specific mechanisms facilitating the infiltration of these cells into plaques remain elusive. Here, we report the development of a novel assay to quantify T cell adhesion to and transmigration through a primary human vascular EC monolayer and show that CD57+ T cells preferentially adhere to and transmigrate through the monolayer. Moreover, activating the ECs with tumor necrosis factor (TNF) significantly increased the transmigration of CD57+ T cells, supporting the role of TNF in promoting the vascular homing of CD57+ T cells. This model will allow for elucidating the mechanisms of and testing interventions to prevent CD57+ T cell infiltration into plaques.

BACKGROUNDPersons with HIV (PWH) have an increased risk of developing cardiovascular disease (CVD) compared to persons without HIV (PWoH). Lipoprotein a (Lp(a)) is a known atherosclerotic risk factor in PWoH, but there are no studies investigating Lp(a) and peri-coronary inflammation.OBJECTIVETo investigate whether Lp(a) is associated with peri-coronary inflammation as assessed by the fat attenuation index (FAI) and activated monocytes and T lymphocytes in PWH and PWoH.METHODSWe measured plasma levels of Lp(a) at study entry in 58 PWH and 21 PWoH without CVD and who had FAI measurements. Associations of Lp(a) with FAI values of the right coronary artery (RCA) and left anterior descending artery (LAD) were evaluated using multivariable regression models adjusted for potential confounders. Correlations between Lp(a) levels and systemic inflammatory markers and immune cell subsets were examined.RESULTSLp(a) was associated with greater peri-coronary inflammation among PWH compared to PWoH (β=1.73, P=0.019) in the RCA, in adjusted models. Significant correlations were observed with certain inflammatory markers (TNFR-I, b=0.295, P<0.001; TNFR-II, b=0.270, P=0.002; hs-CRP, b=0.195, P=0.028). Significant correlations were found between Lp(a) levels and several markers of monocyte activation: CD16 -CD163+ (b= -0.199, P=0.024), and CD16 -DR+ MFI (b= -0.179, P=0.042) and T cell subset CD38+CD4+ TEMRA (b= 0.177, P= 0.044).CONCLUSIONSLp(a) was associated with greater peri-coronary inflammation in the RCA in PWH compared to PWoH, as well as with select systemic inflammatory markers and specific subsets of immune cells in peripheral circulation.

Coronavirus disease 2019 (COVID-19) has had profound effects on the health of individuals and on healthcare systems worldwide. While healthcare workers on the frontlines have fought to quell multiple waves of infection, the efforts of the larger research community have changed the arch of this pandemic as well. This review will focus on biomarker discovery and other efforts to identify features that predict outcomes, and in so doing, identify possible effector and passenger mechanisms of adverse outcomes. Identifying measurable soluble factors, cell-types, and clinical parameters that predict a patient's disease course will have a legacy for the study of immunologic responses, especially stimuli, which induce an overactive, yet ineffectual immune system. As prognostic biomarkers were identified, some have served to represent pathways of therapeutic interest in clinical trials. The pandemic conditions have created urgency for accelerated target identification and validation. Collectively, these COVID-19 studies of biomarkers, disease outcomes, and therapeutic efficacy have revealed that immunologic systems and responses to stimuli are more heterogeneous than previously assumed. Understanding the genetic and acquired features that mediate divergent immunologic outcomes in response to this global exposure is ongoing and will ultimately improve our preparedness for future pandemics, as well as impact preventive approaches to other immunologic diseases.

Abstract Background Latent cytomegalovirus (CMV) infection is immunomodulatory and could affect mRNA vaccine responsiveness. We sought to determine the association of CMV serostatus and prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with antibody (Ab) titers after primary and booster BNT162b2 mRNA vaccinations in healthcare workers (HCWs) and nursing home (NH) residents. Methods Nursing home residents (N = 143) and HCWs (N = 107) were vaccinated and serological responses monitored by serum neutralization activity against Wuhan and Omicron (BA.1) strain spike proteins, and by bead-multiplex immunoglobulin G immunoassay to Wuhan spike protein and its receptor-binding domain (RBD). Cytomegalovirus serology and levels of inflammatory biomarkers were also measured. Results Severe acute respiratory syndrome coronavirus 2-naive CMV seropositive (CMV+) HCWs had significantly reduced Wuhan-neutralizing Ab (P = .013), anti-spike (P = .017), and anti-RBD (P = .011) responses 2 weeks after primary vaccination series compared with responses among CMV seronegative (CMV−) HCWs, adjusting for age, sex, and race. Among NH residents without prior SARS-CoV-2 infection, Wuhan-neutralizing Ab titers were similar 2 weeks after primary series but were reduced 6 months later (P = .012) between CMV+ and CMV− subjects. Wuhan-neutralizing Ab titers from CMV+ NH residents who had prior SARS-CoV-2 infection consistently trended lower than titers from SARS-CoV-2 experienced CMV− donors. These impaired Ab responses in CMV+ versus CMV− individuals were not observed after booster vaccination or with prior SARS-CoV-2 infection. Conclusions Latent CMV infection adversely affects vaccine-induced responsiveness to SARS-CoV-2 spike protein, a neoantigen not previously encountered, in both HCWs and NH residents. Multiple antigenic challenges may be required for optimal mRNA vaccine immunogenicity in CMV+ adults.

Background People with human immunodeficiency virus (PWH) are at increased risk for comorbidities, and plasma interleukin 6 (IL-6) levels are among the most robust predictors of these outcomes. Tocilizumab (TCZ) blocks the receptor for IL-6, inhibiting functions of this cytokine. Methods This was a 40-week, placebo-controlled, crossover trial (NCT02049437) where PWH on stable antiretroviral therapy (ART) were randomized to receive 3 monthly doses of TCZ or matching placebo intravenously. Following a 10-week treatment period and a 12-week washout, participants were switched to the opposite treatment. The primary endpoints were safety and posttreatment levels of C-reactive protein (CRP) and CD4+ T-cell cycling. Secondary endpoints included changes in inflammatory indices and lipid levels. Results There were 9 treatment-related toxicities of grade 2 or greater during TCZ administration (mostly neutropenia) and 2 during placebo administration. Thirty-one of 34 participants completed the study and were included in a modified intent-to-treat analysis. TCZ reduced levels of CRP (median decrease, 1819.9 ng/mL, P < .0001; effect size, 0.87) and reduced inflammatory markers in PWH, including D-dimer, soluble CD14, and tumor necrosis factor receptors. T-cell cycling tended to decrease in all maturation subsets after TCZ administration, but was only significant among naive CD4 T cells. Lipid levels, including lipid classes that have been related to cardiovascular disease risk, increased during TCZ treatment. Conclusions TCZ is safe and decreases inflammation in PWH; IL-6 is a key driver of the inflammatory environment that predicts morbidity and mortality in ART-treated PWH. The clinical significance of lipid elevations during TCZ treatment requires further study. Clinical Trials Registration. NCT02049437.

Direct recognition of Mycobacterium tuberculosis (Mtb)-infected cells is required for protection by CD4(+) T cells. While impaired T cell recognition of Mtb-infected macrophages was demonstrated in mice, data are lacking for humans. Using T cells and monocyte-derived macrophages (MDMs) from individuals with latent Mtb infection (LTBI), we quantified the frequency of memory CD4(+) T cell activation in response to autologous MDMs infected with virulent Mtb. We observed robust T cell activation in response to Mtb infection of M1-like macrophages differentiated using GM-CSF, while M2-like macrophages differentiated using M-CSF were poorly recognized. However, non-infected GM-CSF and M-CSF MDMs loaded with exogenous antigens elicited similar CD4(+) T cell activation. IL-10 was preferentially secreted by infected M-CSF MDMs, and neutralization improved T cell activation. These results suggest that preferential infection of macrophages with an M2-like phenotype limits T cell-mediated protection against Mtb. Vaccine development should focus on T cell recognition of Mtb-infected macrophages.

Background. Inflammation is linked to elevated cardiovascular disease (CVD) risk in people with HIV (PWH) on antiretroviral therapy (ART). Fat attenuation index (FAI) is a measure of peri-coronary inflammation that independently predicts CVD risk in HIV-uninfected persons. Whether FAI is associated with soluble inflammatory markers is unknown. Methods. Plasma levels of inflammatory markers were measured in 58 PWH and 16 controls without current symptoms or prior known CVD who underwent coronary computed tomography angiography and had FAI measurements. A cross-sectional analysis was performed, and associations of markers with FAI values of the right coronary artery (RCA) and left anterior descending artery (LAD) were assessed using multivariable regression models adjusted for the potential confounders age, sex, race, low-density lipoprotein cholesterol, body mass index, and use of lipid-lowering medication. Results. Several inflammatory markers had significant associations with RCA or LAD FAI in adjusted models, including sCD14, sCD163, TNFR-I, and TNFR-II, CCL5, CX3CL1, IP-10. Conclusions. The associations between indices of systemic and peri-coronary inflammation are novel and suggest that these systemic markers and FAI together are promising noninvasive biomarkers that can be applied to assess asymptomatic CVD in people with and without HIV; they also may be useful tools to evaluate effects of anti-inflammatory interventions.

Plasma extracellular vesicle (EV)-associated cytokines were quantified in people with HIV (PWH) with different virological control status, including elite controllers (EC) who maintain persistent control (PC) or not (TC). Cytokine signatures and pathways were determined for each group. Median EV-associated cytokine levels were higher among PWH than HIV-uninfected. EC showed the highest levels of EV-associated cytokines among PWH with PC levels higher than TC levels. IL-18 levels best distinguished PWH from uninfected controls, and EC from ART-treated, and IL-3 distinguished PC from TC. The role of EV-cytokines in intercellular communication and endogenous control of HIV expression should be investigated further.

Antiretroviral therapy (ART) is not curative due to the persistence of a reservoir of HIV-infected cells, particularly in tissues such as lymph nodes, with the potential to cause viral rebound after treatment cessation. In this study, fingolimod (FTY720), a lysophospholipid sphingosine-1-phosphate receptor modulator is administered to SIV-infected rhesus macaques at initiation of ART to block the egress from lymphoid tissues of natural killer and T-cells, thereby promoting proximity between cytolytic cells and infected CD4+ T-cells. When compared with the ART-only controls, FTY720 treatment during the initial weeks of ART induces a profound lymphopenia and increases frequencies of CD8+ T-cells expressing perforin in lymph nodes, but not their killing capacity; FTY720 also increases frequencies of cytolytic NK cells in lymph nodes. This increase of cytolytic cells, however, does not limit measures of viral persistence during ART, including intact proviral genomes. After ART interruption, a subset of animals that initially receives FTY720 displays a modest delay in viral rebound, with reduced plasma viremia and frequencies of infected T follicular helper cells. Further research is needed to optimize the potential utility of FTY720 when coupled with strategies that boost the antiviral function of T-cells in lymphoid tissues.

Inflammation associated with increased risk of comorbidities persists in people living with HIV (PWH) on combination antiretroviral therapy (ART). A recent placebo-controlled trial of low-dose methotrexate (MTX) in PWH found that numbers of total CD4 and CD8 T cells decreased in the low-dose MTX arm. In this report we analyzed T cell phenotypes and additional plasma inflammatory indices in samples from the trial. We found that cycling (Ki67+) T cells lacking Bcl-2 were reduced by MTX but plasma inflammatory cytokines were largely unaffected. In a series of in vitro experiments to further investigate the mechanisms of MTX activity, we found that MTX did not inhibit effector cytokine production but inhibited T cell proliferation downstream of mTOR activation, mitochondrial function, and cell cycle entry. This inhibitory effect was reversible with folinic acid, suggesting low-dose MTX exerts anti-inflammatory effects in vivo in PWH largely by blocking T cell proliferation via dihydrofolate reductase inhibition, yet daily administration of folic acid did not rescue this effect in trial participants. Our findings identify the main mechanism of action of this widely used anti-inflammatory medicine in PWH and may provide insight into how MTX works in the setting of other inflammatory conditions.

PURPOSE OF REVIEW:Systemic inflammation increases as a consequence of aging (inflammaging) and contributes to age-related morbidities. Inflammation in people living with HIV is elevated compared with the general population even after prolonged suppression of viremia with anti-retroviral therapy. Mechanisms that contribute to inflammation during aging and in treated HIV disease are potentially interactive, leading to an exaggerated inflammatory phenotype in people with HIV.RECENT FINDINGS:Recent studies highlight roles for anti-retroviral therapy, co-infections, immune system alterations, and microbiome perturbations as important contributors to HIV-associated inflammation. These factors likely contribute to increased risk of age-related morbidities in people living with HIV. Understanding mechanisms that exaggerate the inflammaging process in people with HIV may lead to improved intervention strategies, ultimately, extending both lifespan and healthspan.

SARS-CoV-2 causes complex effects on microvascular homeostasis that potentially contribute to organ dysfunction and coagulopathies. SARS-CoV-2 binds to, and causes downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects.

Background: Immune non-responders (INR) are HIV+, ART-controlled (>2 yrs) people who fail to reconstitute their CD4 T cell numbers. Systemic inflammation and markers of T cell senescence and exhaustion are observed in INR. This study aims to investigate T cell senescence and exhaustion and their possible association with soluble immune mediators and to understand the immune profile of HIV-infected INR. Selected participants were <50 years old to control for the confounder of older age.Methods: Plasma levels of IL-6, IP10, sCD14, sCD163, and TGF-β and markers of T cell exhaustion (PD-1, TIGIT) and senescence (CD57, KLRG-1) were measured in ART-treated, HIV+ participants grouped by CD4 T cell counts (n = 63). Immune parameters were also measured in HIV-uninfected, age distribution-matched controls (HC; n = 30). Associations between T cell markers of exhaustion and senescence and plasma levels of immune mediators were examined by Spearman rank order statistics.Results: Proportions of CD4 T cell subsets expressing markers of exhaustion (PD-1, TIGIT) and senescence (CD57, KLRG-1) were elevated in HIV+ participants. When comparing proportions between INR and IR, INR had higher proportions of CD4 memory PD-1+, EM CD57+, TEM TIGIT+ and CD8 EM and TEM TIGIT+ cells. Plasma levels of IL-6, IP10, and sCD14 were elevated during HIV infection. IP10 was higher in INR. Plasma TGF-β levels and CD4 cycling proportions of T regulatory cells were lower in INR. Proportions of CD4 T cells expressing TIGIT, PD-1, and CD57 positively correlated with plasma levels of IL-6. Plasma levels of TGF-β negatively correlated with proportions of TIGIT+ and PD-1+ T cell subsets.Conclusions: INR have lower levels of TGF-β and decreased proportions of cycling CD4 T regulatory cells and may have difficulty controlling inflammation. IP10 is elevated in INR and is linked to higher proportions of T cell exhaustion and senescence seen in INR.

T-cell accumulation in atherosclerotic plaques contributes to plaque destabilization. We found that several chemokine receptors are differentially expressed on peripheral blood compared to plaque-resident T cells and corresponding ligands are upregulated in plaques. These data indicate that T-cell migration into human atherosclerotic plaques may predominantly occur via CCR5-CCL3 and CX3CR1-CX3CL1 interactions. The linkage of atherosclerosis to immune activation can be traced back as far as to Rudolf Virchow [1]. Cytokine release and alteration of surface marker expression on immune cells reflect this activation. Disorders characterized by a proinflammatory environment have been associated with an increased risk of atherosclerosis and many works implicate adaptive immunity in this risk [2, 3]. Advanced atherosclerotic plaques are enriched for activated T cells, thus pointing to their potential role in plaque progression and destabilization [4]. Nonetheless, the mechanisms of T-cell accumulation in plaques remain to be elucidated. To gain insight into these mechanisms, we analyzed the surface phenotypes of T cells isolated from human peripheral blood and autologous atherosclerotic plaques. Generally, immune cell phenotypes found in the plaques in our study are in agreement with those recently reported in mass cytometry (CyTOF) and single-cell RNA sequencing studies [5, 6]. Specifically, among plaque CD4+ and CD8+ T cells, the predominant population had a CD45RO+CD27- effector memory phenotype (Fig. 1A and C), which is associated with viral infection and/or other chronic inflammatory states that trigger T-cell activation. Accordingly, proportions of plaque-resident T cells expressing the early activation marker CD69 were increased (Fig. 1B and D). CD69 blocks tissue egress of T cells to blood according to a sphingosine-1-phosphate gradient. Thus, elevated surface CD69 expression may provide a mechanism for T-cell retention within the atherosclerotic plaque by blocking their exit. But what drives T cells to the plaques? Unlike most previous works on human atherosclerotic plaques that focused on T-cell heterogeneity or activation/exhaustion status, we addressed the potential migratory mechanisms that may drive T cells from the bloodstream into the vessel wall. Thus, we analyzed the chemokine receptors that are differentially expressed on T cells in peripheral blood and atherosclerotic plaques. CCR2 (where CCR is CC (cysteine-cysteine) chemokine receptor) was not found at substantial levels on CD4+ and CD8+ T cells, while T cells expressing CCR4, CCR5, CCR7, and CX3CR1 (where CX3CR is CC (cysteine-XXX-cysteine) chemokine receptor) were differentially represented in plaques and blood (Fig. 1B and D). The fraction of CCR5+ cells in plaques was significantly higher, indicating the possible involvement of this surface molecule in T-cell migration into the plaques. In contrast, fractions of CCR4+, CX3CR1+, and CCR7+ cells were lower in plaques than in blood (Supporting Information Tables S3 and S4). Low expression of CCR7 on plaque T cells was expected since this receptor is not expressed by tissue-resident cells. However, CCR4 and CX3CR1 decrease could result from their rapid internalization upon binding to corresponding ligands. Indeed, CCL2 (where CCL is CC (cysteine-cysteine) chemokine ligand), CCL3, and CX3CL1 mRNAs (where CX3CL is CC (cysteine-XXX-cysteine) chemokine ligand) that encode known ligands to CCR4, CCR5, and CX3CR1, respectively, were increased in plaques compared to their levels in blood leucocytes (Fig. 2). Next, we analyzed chemokine receptor turnover upon binding to their plate-bound ligands (Supporting Information Fig. S4). Exposure to CCL2 did not result in CCR4 removal from the T-cell surface, whereas CX3CL1 exposure did lead to CX3CR1 removal. Importantly, in contrast to CCL4 and CCL5 exposure that diminished surface CCR5 levels, CCL3 exposure did not alter CCR5 surface levels. In contrast to above-mentioned experiments, data on surface chemokine receptor clearance driven by soluble chemokines (e.g., [7]), confirmed in our study (data not shown), indicate that soluble CCL3 can drive CCR5 clearance. However, we suppose that plate-bound experiments reflect the in vivo situation more faithfully since in plaques chemokine receptors on T cells seem to interact with ECM-bound rather than with soluble chemokines (discussed in [8, 9]). Our in vitro surface chemokine receptor clearance experiments indicated that the low expression of CCR4 on plaque-resident T cells is not a consequence of its removal upon ligand exposure. In contrast, sustained surface expression of CCR5 and loss of CX3CR1 observed after exposure to CCL3 and CX3CL1, respectively, are consistent with the observed phenotypes of plaque-resident T cells. This suggests that the CCL3-CCR5 and CX3CL1- CX3CR1 axes are involved in the homing of circulating T cells to atherosclerotic plaques. These data support our recent report on the importance of the CX3CL1-CX3CR1 axis for CD4+ T-cell migration into plaques [10]. We found that inhibition of CX3CL1-CX3CR1 interactions reduced cell migration toward activated endothelial cells in vitro in some but not all donors, suggesting that a second signal could also govern CD4+ T-cell traffic to endothelium. Our current findings suggest that the CCL3-CCR5 axis might provide a second migratory signal that could differentially drive T-cell subpopulations to infiltrate atherosclerotic plaques. Still, other CCR5 ligands, CCL4 and CCL5, might be involved in T-cell recruitment to atherosclerotic plaques since their expression was detected in all plaques. However, we suspect that their contribution to the total T-cell recruitment is likely to be less important than that of CCL3. Thus, among CCR5 ligands CCL3 may be a key, but not the only, driver of T-cell migration. Taken together, CCR4, CCR5, and CX3CR1 chemokine receptors were differentially expressed on human T cells from atherosclerotic plaques and peripheral blood. We showed that plaques were enriched for mRNAs encoding CCL2, CCL3, and CX3CL1, the ligands to these receptors. Our experiments on receptor turnover confirmed that the CCR4-CCR5+CX3CR1- phenotype of plaque-infiltrating T-cells may originate from the surface receptor clearance in case of CX3CR1 but not CCR4 and that the accumulation of CCR5+ T-cells in plaque could be related to heightened plaque expression of CCL3 that does not efficiently promote CCR5 removal. Thus, T-cell migration from peripheral blood into atherosclerotic plaques in humans may occur predominantly through the CCL3-CCR5 and CX3CL1-CX3CR1 interaction axes. Pharmacological disruption of these interactions may represent a useful strategy to prevent atherosclerotic plaque progression. This study was funded by Russian Science Foundation #20-75-10085 (to A.K. and D.P.); Intramural Program of National Institute of Child Health and Human Development (to L.M.); and Richard J. Fasenmeyer Foundation and the National Institutes of Health AI 068636 (to M.L.F. and M.M.L.). The authors declare no commercial or financial conflict of interest. The peer review history for this article is available at https://publons.com/publon/10.1002/eji.202049004. Data are available in article Supporting Information Materials. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Cytotoxic CD4 T cells are linked to cardiovascular morbidities and accumulate in both HIV and CMV infections, both of which are associated with increased risk of cardiovascular disease (CVD). In this study, we identify CMV coinfection as a major driver of the cytotoxic phenotype, characterized by elevated CD57 expression and reduced CD28 expression, in circulating CD4 T cells from people living with HIV infection, and investigate potential mechanisms linking this cell population to CVD. We find that human CD57(+) CD4 T cells express high levels of the costimulatory receptor CD2 and that CD2/LFA-3 costimulation results in a more robust and polyfunctional effector response to TCR signals, compared with CD28-mediated costimulation. CD57(+) CD4 T cells also express the vascular endothelium-homing receptor CX3CR1 and migrate toward CX3CL1-expressing endothelial cells in vitro. IL-15 promotes the cytotoxic phenotype, elevates CX3CR1 expression, and enhances the trafficking of CD57(+) CD4 T cells to endothelium and may therefore be important in linking these cells to cardiovascular complications. Finally, we demonstrate the presence of activated CD57(+) CD4 T cells and expression of CX3CL1 and LFA-3 in atherosclerotic plaque tissues from HIV-uninfected donors. Our findings are consistent with a model in which cytotoxic CD4 T cells contribute to CVD in HIV/CMV coinfection and in atherosclerosis via CX3CR1-mediated trafficking and CD2/LFA-3-mediated costimulation. This study identifies several targets for therapeutic interventions and may help bridge the gap in understanding how CMV infection and immunity are linked to increased cardiovascular risk in people living with HIV infection.