Abstract: which is known to persist, albeit at

Abstract:Background:Recent findings point to a role for Checkpoint Inhibitor (CPI) receptors attissue level in immune homeostasis and of increase in CPI molecules on immunecells in HIV+ persons.  Known enrichmentof LAG3 receptors in the heart prompted this investigation.

Methods:Participants recruited in Chennai, India included HIV+ ART naive viremic (Gp1,n= 102), HIV+ ART experienced aviremic (Gp2, n=172) and HIV negative healthyvolunteers (Gp , n= 64). A cross-sectional analysis of cardiac function,arterial resistance and immunologic assessment was performed. Results:Patients in Gp1 exhibited impaired cardiac function and greater arterialstiffness in comparison to other groups; Gp2 had lower stroke volume indexcompared to Gp3. Frequencies of CD4+ T cells expressing LAG-3 and PD1 werehigher in Gp1 compared to other groups while TIGIT and TIM3 did not differ.

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LAG-3+,PD1+ and dual LAG-3+PD1+ CD4 T cells were inversely correlated with cardiacfunction and arterial stiffness in Gp1 and arterial elasticity in Gp2. Conclusions:HIV induced upregulation of LAG-3 singly or in combination with PD1 in immunecells may regulate cardiac health and warrants mechanistic investigations, asit has implications for potential utility of anti-LAG-3 immunotherapy forcardiac dysfunction in HIV infection. Keywords: HIV, LAG3, CVD andLAG3, Checkpoint inhibitors and HIV, Checkpoint inhibitors and CVD IntroductionCardiovasculardisease (CVD) is a major contributor to mortality and morbidity in HIVinfection, and is largely attributed to underlying inflammation and immuneactivation ( IA) which is known to persist, albeit at a lower level followingantiretrovral therapy (ART) 1, 2. Early in the era of ART, the drugs themselves were found to becardiotoxic, but this issue is now considered of less relevance with newerdrugs that have minimal or no cardiac toxicity 3.  Persistent T cell activation in chronic HIVinfection leads to a chronic inflammatory environment that has multipledeleterious effects at the tissue level, directly or indirectly inflictingdamage to different organ systems, the mechanisms of which are not well understood.  Immune activation at the cellular level thatinvolves CD4 and CD8 cells, results in T cell proliferation and dysfunction 4, 5. Intrinsic mechanisms thatmaintain T cell numbers at a constant level do so by balancing immuneactivation and homeostatic proliferation.

These mechanisms include regulationof cell death molecules such as Fas/FasL 6, 7 and immune check point inhibitor(CPI) molecules such as PD-1, LAG-3, TIM3, TIGIT and CTLA-4 8-10.   In lymphocytes, the CPI have critical rolesin the maintenance of immune homeostasis by ensuring contraction of effector Tcell responses 11, 12. The expression of LAG-3, TIGITand CTLA4 on T regulatory cells (Tregs) enable the Tregs to suppress effector Tcell function 13-17. Elevated expression of CPImolecules on effector Tcells is associated with dampened immunity which on the one hand protects thehost from exuberant anti-microbial responses but on the other, it can lead tofunctional unresponsiveness of the immune system with reduced proliferation andcytokine release 18, 19 and reduced effector function ofCD4 and CD8 T cells eg.

in virus infections and cancer 8, 20, 21.  While all CPIare considered in general terms as having an immunoregulatory role, they mayhave unique properties and distinct mechanisms of action 8, 22-24. For example co-inhibitoryreceptors CTLA4 and PD-1 are primarily responsible for maintaining self toleranceby restricting T cell clonal proliferation in lymphoid organs while LAG-3, TIM3,and TIGIT have been assigned specific roles related to regulation of tissueinflammation 8, 16, 25-31.

Specificity for the regulatoryactivity of the CPI resides at the tissue level, based on the ligands expressedon tissues that maintain tissue tolerance and inhibit immunopathology. Recent evidencesupporting distinct control mechanisms for organ immune homeostasis points tothe selective enrichment of LAG-3 receptors in the heart 8. The present study investigated theexpression of CPI molecules on CD4 T cells in HIV+ subjects who were startingART and an aviremic group on ART for their relationship to measures of cardiacfunction and arterial stiffness. Additionally, healthy individuals wereinvestigated and served as controls.

          Methods:Studysetting and subjects: This study was conducted at YRGCARE, a tertiary care center located at Chennai in South India, and enrolled274 male and female subjects with chronic HIV infection (HIV+) and 64HIV-uninfected healthy controls (HC) in the age range >18 yr-50 yr (Table 1). Among the HIV+, 102 subjects were ART naïve (group 1) and172 subjects were on ART for >12 months (group 2) with viral suppression asdetermined by two consecutive plasma viral load values of <40 copies/mL. 63 subjects were healthycontrols and were categorized as group 3. Patients with pre-exposure orpost-exposure ART prophylaxis and pregnant women were excluded. The study wasapproved by both YRG CARE and University of Miami institutional review boards.  Informed consent was obtained from allenrolled participants. A detailed interview was conducted at the time ofenrolment to collect demographic information.

All participants had a one timeblood draw for collection of plasma and peripheral blood mononuclear cells(PBMC). Blood samples were processed within an hour of collection according toguidelines of the ACTG.  Plasma wasstored in aliquots at -800C and PBMC were cryopreserved inliquid nitrogen in aliquots of 5 million cells/mL. A group of 24 normal age andsex matched healthy adult volunteers from the US was included for the analysisof checkpoint inhibitors on different immune cell subsets. Measuresof cardiac function and arterial stiffness: Pulse rate,stroke volume, stroke volume index, cardiac output, cardiac index and cardiacejection time were determined to ascertain cardiac functioning. Arterial stiffnesswas estimated by pulse-wave velocity (PWV) using the HDI/PulseWave CR-2000(Hypertension Diagnostics, Inc., Eagan, MN), a diagnostic tool that waspreviously applied in the INSIGHT Strategic Timing of Anti Retroviral Treatmentarterial stiffness sub-study.41 Along with Large Artery Elasticityindex (LAE) and Small Artery Elasticity index (SAE) measures, systemic vascularresistance (SVR) and total vascular impedance (TVI) were measured as arterialstiffness parameters.

 Flow cytometry for analysisof checkpoint inhibitors (CPI): Markers of checkpoint inhibitors wereanalyzed on CD4 T cells. Thawed PBMCwere rested overnight and 1×106 cells were stained with LIVE/DEAD®Aqua followed by staining for surface markers CD3, CD4, CD8, and checkpointinhibitors PD1, TIGIT, TIM3, and LAG-3. Cells were then fixed, and acquired ona Flow cytometer (BD LSRFortessa, San Jose, CA) and analyzed by FlowJo V10(Treestar, Ashland, OR). Frequencies of check point inhibitor molecules eitheralone or in combinations were analyzed on live (Aqua-) CD3+CD4+ T cells.

Aseparate antibody panel containing markers for monocytes (CD14, CD16) and NKcells (CD56) along with HLA-DR and T cell specific markers for CD4 and CD8 Tcells was used for analyzing CPI molecules in US healthy donors. Monocytes weregated as CD45+CD3-CD56-HLA-DR+CD14+ and NK cells as CD45+CD3-CD14-CD56+populations.   Statistical analysis: Descriptivestatistics such as percentages means and standard deviation, and median wereused to describe the demographic characteristics of the study population.

Forunpaired data, Levene’s test was used firstly to check variance heterogeneityfollowed by Wilcoxon rank-sum test (also called ‘Mann-Whitney’ U test) wasperformed using R stats package. For Correlationanalyses, Shapiro test was used to check if data is normally distributed usingR stats package. If Shapiro test showed p>0.05,Pearson correlation coefficient was performed using R stats package; in other instances Spearman’s rank correlation coefficientwas performed using R stats package.  Linearregression analysis (OLS) was performed and the data are presented as scatterplots with regression lines. Pearson product-moment was performed and Pearson correlationcoefficients (r) with P values are present on topof the plot. All analysis are performed using R stats package. A p value of <0.

05was considered as significant.” Thedata are presented as scatter plots with regression lines and correlationcoefficients with P values.                 ResultsDemographiccharacteristics of study population: Demographiccharacteristics of the study population are shown in Table 1. The meanages were similar among the study groups. Of the patients on ART, 140 patientswere on first-line reverse transcriptase inhibitors (RTI; AZT/D4T/TDF+3TC/FTC+EFV/NVP)while 32 were on PI (RTV-boosted LPV) based second-line therapy at the time ofstudy enrolment. Absolute CD4 counts were significantly lower in the Gp1compared to Gp 2.

Expression of checkpoint inhibitormolecules LAG-3 and PD1 is increased on CD4 T cells of treatment naïve HIV+ individuals:We analyzed the expression of CPI molecules LAG-3, PD1, TIGIT and TIM3 on CD4 Tcells in the study groups. Flow cytometry gating strategy for analysis of theCPI is depicted in Fig 1A. Expressionof LAG-3 (Fig 1B) on CD4 T cells wassignificantly higher in Gp1 compared to Gp2 and Gp3.  PD1 (Fig1C) also showed a similar pattern as LAG-3, but the expression of TIGIT (Fig 1D) and TIM3 (Fig 1E) were not different between the study groups. Absolutenumbers of CD4 T cells at study entry inversely correlated with LAG-3 (Fig 2A), PD1 (Fig 2B) and dual LAG-3+PD1+ (Fig2C) CD4 T cells in Gp1.

In Gp2 no correlation was observed of CD4 T cellcounts at study entry or of nadir CD4 T cell counts with LAG-3 or PD1 or dual LAG-3+PD1+CD4 T cells (Figs 2D, E and Frespectively). Cardiac function and arterialstiffness are impaired in Group 1: Measures of cardiac function and arterialstiffness in the study groups are shown in FigS1.  Gp1had lower cardiac ejection time (Fig S1A), lower stroke volume (Fig S1B)and lower stroke volume index (Fig S1C) than Gps 2 and 3 while cardiacoutput (Fig S1D) did not differ between study groups. Cardiac index waslower in Gp1 compared to Gp 3 (Fig S1E). Measures of arterial stiffnessshowed higher systemic vascular resistance (Fig S1F) with lower largeand small artery elasticity (Fig S1G and S1H respectively) in Gp 1compared to Gp 2.

Cardiac function and arterial stiffness parameters were not differentbetween Gp2 and Gp3. These data support an HIV induced effect on multiplecardiac functions and on arterial stiffness in treatment naïve HIV+ group. InGp 2, all of these parameters are comparable to the general population (Gp3)implying ART induced improvement.Correlations of cardiac function andarterial stiffness with LAG3+ CD4 T cells (Fig 3; Table 2).Linearregression plots for association of cardiac function measures and Pearson correlation analysis (Figs 3A, 3B) showed that in Gp1, singleLAG-3+ or LAG-3+ PD1+ CD4 T cell subsetsrespectively were inversely correlated with measures of cardiac ejectiontime, cardiac output, cardiac index, stroke volume, and stroke volume index.

In Gp 2 and Gp 3, no association of LAG-3or LAG-3 and PD1 expressing CD4 T cells with measures of cardiac function wasevident. Arterial stiffness measure of systemicvascular resistance directly correlated with LAG-3 (Fig 3C) or LAG-3+PD1+ CD4 T cells in Gp1 and Gp2 (Fig 3D). Large artery elasticity indexinversely correlated with LAG-3+PD1+ CD4 T cells in Gp1 but with LAG-3 alone,or in combination with PD1 in Gp2.

Inverse correlations of small arteryelasticity index and single LAG-3 or LAG3 coexpressed with PD1 on CD4 T cellswere only found in Gp2. Single PD1 expressing CD4 T cells followed a similarpattern but were not consistent. A summary of all the correlations are depictedin a heatmap shown in Fig S2. Noassociations were found between arterial stiffness measures and CPI in Gp3 (Fig 3C, 3D).Checkpoint molecule distribution inimmune cells: Since we found the association of CD4T cells expressing LAG-3 and PD1 with cardiac function and vascular resistance,we examined expression of various CPI on different immune cell subsets in agroup of 24 age matched healthy adults (Fig4).  Monocytes exhibited highestfrequency of LAG-3 and PD1 expression followed by LAG-3 on CD4 T cells, CD8 Tcells and NK cells respectively (Fig 4A)and PD-1 on CD8, NK cells and CD4 T cells (Fig4B). TIGIT expression was highest on NK cells followed by CD8 and  CD4 T cells with lowest expression onmonocytes (Fig 4C) while TIM3expression was highest on NK cells followed by monocytes, CD4 T cells and CD8 Tcells respectively (Fig 4D). Thesedata suggest that pattern of expression of CPI may vary on different innate andadaptive immune cells.

 DiscussionCardiovasculardisease is a major contributor to mortality and morbidity in HIV infection 3, 32-34. Recent evidence points to expression of receptors for CPI molecules atthe tissue level for controlling organ immune homeostasis, with the heartshowing selective enriched for LAG 3 receptors 8. This cross sectional study wasaimed at defining relationship of CPI molecules and CVD in HIV infection. Weconducted the study in treatment naïve viremic patients (Gp1) as well as inpatients on ART with viral suppression (Gp2) and healthy age-matched controls(Gp3). Cardiac function was decreased with increased vascular resistance in Gp1compared to Gps 2 and 3 and exhibited an inverse correlation with LAG-3, PD-1or LAG-3 plus PD1 expressing CD4 T cells but without association of TIGIT or TIM3. Patients in Gp 2, despite having startedART at CD4  nadirs similar to the Gp 1 hadlittle evidence of impaired cardiac function but had residual increasedvascular resistance involving large and small vessel elasticity which was alsoinversely correlated with LAG-3+CD4 T cells and maximally with cellsco-expressing LAG-3 and PD1.  Theseobservations, together with the high LAG-3 expression in monocytes from healthydonors, imply a dominant role of cells expressing LAG-3 alone or in combinationwith PD1 in regulating cardiac health particularly in viremic patients who havenot started ART.  The benefits of ART onCVD and on expression of CPI on CD4 T cells were clearly manifest in this studysubjects based on comparisons with Gp2 and Gp 3.

  Investigations to understand the potentialrole of LAG-3 have implications for immunotherapy targeting specific CPImolecules such as LAG-3, currently in clinical trials in cancer, for preventionor treatment of cardiac dysfunction in HIV.  LAG-3is a 498-amino acid Transmembrane protein identified on activated human NK andT cell line 35. In humans, theLAG-3 gene is located adjacent to CD4 on chromosome 12 and is structurallyhomologous to CD4 with four extracellular immunoglobulin superfamily likedomains D1, D2, D3, and D4 36-38. In T cells, LAG-3 mRNA levels increase 10 fold upon cell activation 39 which is highly relevant as LAG-3function is mediated by modulation of LAG-3 expression at the transcriptionallevel 38.

Tian et al 40 demonstrated that LAG-3 expressionin T cells was significantly upregulated in HIV infected subjects and wascorrelated with disease progression. Further analysis of LAG-3 expression inrelationship to immune activation based on CD38 and HLA-DR expression showedLAG-3+ cells were largely coexpressed with CD38 on 70% of CD4 T cells and 74%of CD8+ T cells in 28 HIV infected subjects, while the percentage of cellsco-expressing LAG-3 and DR was less. In that study MFI of LAG-3 expression declinedin both CD4+ and CD8+ T cells after more than one year of ART treatment. Ourfindings are in agreement with these observations as we found that both immuneactivation (not shown) and LAG 3 and PD1 on CD4 T cells were greater in Group 1than in Group 2 where the lower LAG-3 is most likely a consequence of ART. Intracellularstorage in lysosomal compartments, may also serve to facilitate rapid LAG-3cell surface expression following T cell activation (11, 12, 41, 42. Riskfactors for CVD are under intense investigation and involve both traditionaland non-traditional factors.

A study by Golden et al 43 found that the lipoproteinscavenger receptor class B type 1 (SCARBI) rs10846744 noncoding variant issignificantly associated with atherosclerotic disease independently oftraditional cardiovascular risk factors. They identified a connection betweenrs10846744 with LAG-3 in plasma and concluded that plasma LAG-3 is anindependent predictor of HDL-cholesterol levels and CVD risk. Besidestranscriptional modulation, LAG-3 function is also mediated by cell surfaceshedding which releases soluble LAG-3 (sLAG-3). LAG-3 cleavage is mediated bythe metalloproteinase ADAM10 and ADAM17, which cleave a wide range oftransmembrane proteins including CD62L, TIM3 and TNFa 44 with ADAM17 action becomingevident following cell activation 11, 12, 41, 42. It is possible that sLAG-3 isshed off from circulating cells and mediates tissue damage to the heart bytargeting its receptors.

 Itis important to understand the physiological role of individual CPI moleculesin terms of their independent effects and dependence upon each other. Althoughthey belong to the same class of receptors, they act in a tier fashion, withCTLA-4 and PD1 in the first tier for maintaining self-tolerance and LAG-3, TIM3and TIGIT in a second tier with distinct roles in regulating immune responsesparticularly at sites of tissue inflammation 8, 16, 25-31. Upregulation of LAG-3 on T cells alsodefines a subpopulation with functional exhaustion and as noted above, correlateswith disease progression in HIV positive subjects 40. As LAG-3 is a natural high-affinity ligand for MHC class II molecules 8, 36, 37 it has an inhibitory role inregulating T cell immune responses 8, 38 and also acts synergistically withPD1 to regulate T cell function 38, 45. In a murine transplant tumor model, it was observed that PD-1 and LAG-3were coexpressed on tumor-infiltrating CD4+ and CD8+ T cells and in cancerpatients 42, 46-48. In HIV, blockade of LAG-3enhanced HIV-specific T cell responses 40.

In our study PD-1 and LAG-3coexpression was highly correlated with cardiac measures and significance wasoften greater than that of independent molecules implicating their involvement inregulation of organ systems at tissue levels.PD1expression on CD4 T cells could also be involved in influencing cardiacfunction, based on the higher PD1 expression observed in Gp1 than in the othergroups, and results of the correlation analyses. Upregulation of PD-1 and PDL-1have been demonstrated in cardiac injury models of ischemic-reperfused andcryoinjured hearts 49 in which cardiomyocytes were foundto be a major source of inflammatory cytokine generation in isolatedischemic-reperfused hearts 50-52; importantly, PD-1 and PDL-1 were expressedon cardiomyocytes as well as on different populations of cardiac cells leadingthe authors to postulate a role of the cardiac PD-1/PDL-1 pathway in cardiacinjury 49. These authors ascribed thecardiac injury to an increase in GADD153, a regulator of the inflammatoryresponse that leads to pro-inflammatory changes in the ischemic refused hearts 49 with a marked increase of IL-17+cells and only a mild increase in IL-10+ cells.

Furthermore they demonstratedthat PDL-1 blocking antibody treatment reduced cardiac GADD153 expression withreduction in expression of inflammatory cytokines. As PD-L1 is also expressedin human heart myocytes 53, LAG-3 plus PD1 coexpressingimmune cells are expected to have the maximal impact on cardiac damage. Monocytesare strongly implicated in cardias damage 54. Although we did not directlyinvestigate the role of CPI on monocytes in our study population we conductedan independent analysis on healthy volunteers to examine expression of LAG-3 andPD1 on different cell populations. The extremely high expression of LAG-3 inmonocytes suggests the possibility that LAG-3 or PD1 expressing monocytes couldinteract with T cells to act at the tissue level in the heart muscle and influencecardiac function, opening up new avenues for future investigation.  In conclusion, we found a novel association ofLAG-3 expressing CD4 T cells either alone or in combination with PD1 withcardiac dysfunction in treatment naïve HIV+ patients, and with cardiac arterystiffness in both treatment naïve and virally suppressed subjects on ART.

Keyquestions to investigate are 1) mechanism of action of LAG-3 affecting thecardiac function and arterial structure, and 2) the basis that underliessynergies of LAG-3 with PD-1 in regulating cardiac function. This understandingcould provide insight into potential role of LAG-3 immunotherapy (which is inearly clinical trials in cancer) in prevention or treatment of cardiacdysfunction in HIV.