Abstract: which is known to persist, albeit at

Abstract:

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

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Methods:
Participants recruited in Chennai, India included HIV+ ART naive viremic (Gp1,
n= 102), HIV+ ART experienced aviremic (Gp2, n=172) and HIV negative healthy
volunteers (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 arterial
stiffness in comparison to other groups; Gp2 had lower stroke volume index
compared to Gp3. Frequencies of CD4+ T cells expressing LAG-3 and PD1 were
higher in Gp1 compared to other groups while TIGIT and TIM3 did not differ. LAG-3+,
PD1+ and dual LAG-3+PD1+ CD4 T cells were inversely correlated with cardiac
function and arterial stiffness in Gp1 and arterial elasticity in Gp2.

Conclusions:
HIV induced upregulation of LAG-3 singly or in combination with PD1 in immune
cells may regulate cardiac health and warrants mechanistic investigations, as
it has implications for potential utility of anti-LAG-3 immunotherapy for
cardiac dysfunction in HIV infection.

 

Key
words: HIV, LAG3, CVD and
LAG3, Checkpoint inhibitors and HIV, Checkpoint inhibitors and CVD

 

Introduction

Cardiovascular
disease (CVD) is a major contributor to mortality and morbidity in HIV
infection, and is largely attributed to underlying inflammation and immune
activation ( IA) which is known to persist, albeit at a lower level following
antiretrovral therapy (ART) 1, 2. 
Early in the era of ART, the drugs themselves were found to be
cardiotoxic, but this issue is now considered of less relevance with newer
drugs that have minimal or no cardiac toxicity 3.  Persistent T cell activation in chronic HIV
infection leads to a chronic inflammatory environment that has multiple
deleterious effects at the tissue level, directly or indirectly inflicting
damage to different organ systems, the mechanisms of which are not well understood.  Immune activation at the cellular level that
involves CD4 and CD8 cells, results in T cell proliferation and dysfunction 4, 5. Intrinsic mechanisms that
maintain T cell numbers at a constant level do so by balancing immune
activation and homeostatic proliferation. These mechanisms include regulation
of 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 roles
in the maintenance of immune homeostasis by ensuring contraction of effector T
cell responses 11, 12. The expression of LAG-3, TIGIT
and CTLA4 on T regulatory cells (Tregs) enable the Tregs to suppress effector T
cell function 13-17. Elevated expression of CPI
molecules on effector T
cells is associated with dampened immunity which on the one hand protects the
host from exuberant anti-microbial responses but on the other, it can lead to
functional unresponsiveness of the immune system with reduced proliferation and
cytokine release 18, 19 and reduced effector function of
CD4 and CD8 T cells eg. in virus infections and cancer 8, 20, 21.  

While all CPI
are considered in general terms as having an immunoregulatory role, they may
have unique properties and distinct mechanisms of action 8, 22-24. For example co-inhibitory
receptors CTLA4 and PD-1 are primarily responsible for maintaining self tolerance
by restricting T cell clonal proliferation in lymphoid organs while LAG-3, TIM3,
and TIGIT have been assigned specific roles related to regulation of tissue
inflammation 8, 16, 25-31. Specificity for the regulatory
activity of the CPI resides at the tissue level, based on the ligands expressed
on tissues that maintain tissue tolerance and inhibit immunopathology. Recent evidence
supporting distinct control mechanisms for organ immune homeostasis points to
the selective enrichment of LAG-3 receptors in the heart 8. The present study investigated the
expression of CPI molecules on CD4 T cells in HIV+ subjects who were starting
ART and an aviremic group on ART for their relationship to measures of cardiac
function and arterial stiffness. Additionally, healthy individuals were
investigated and served as controls.  

 

 

 

 

 

 

 

 

 

Methods:

Study
setting and subjects: This study was conducted at YRG
CARE, a tertiary care center located at Chennai in South India, and enrolled
274 male and female subjects with chronic HIV infection (HIV+) and 64
HIV-uninfected healthy controls (HC) in the age range >18 yr-50 yr (Table 1). Among the HIV+, 102 subjects were ART naïve (group 1) and
172 subjects were on ART for >12 months (group 2) with viral suppression as
determined by two consecutive plasma viral load values of <40 copies/mL. 63 subjects were healthy controls and were categorized as group 3. Patients with pre-exposure or post-exposure ART prophylaxis and pregnant women were excluded. The study was approved by both YRG CARE and University of Miami institutional review boards.  Informed consent was obtained from all enrolled participants. A detailed interview was conducted at the time of enrolment to collect demographic information. All participants had a one time blood draw for collection of plasma and peripheral blood mononuclear cells (PBMC). Blood samples were processed within an hour of collection according to guidelines of the ACTG.  Plasma was stored in aliquots at -800C and PBMC were cryopreserved in liquid nitrogen in aliquots of 5 million cells/mL. A group of 24 normal age and sex matched healthy adult volunteers from the US was included for the analysis of checkpoint inhibitors on different immune cell subsets. Measures of cardiac function and arterial stiffness: Pulse rate, stroke volume, stroke volume index, cardiac output, cardiac index and cardiac ejection time were determined to ascertain cardiac functioning. Arterial stiffness was estimated by pulse-wave velocity (PWV) using the HDI/PulseWave CR-2000 (Hypertension Diagnostics, Inc., Eagan, MN), a diagnostic tool that was previously applied in the INSIGHT Strategic Timing of Anti Retroviral Treatment arterial stiffness sub-study.41 Along with Large Artery Elasticity index (LAE) and Small Artery Elasticity index (SAE) measures, systemic vascular resistance (SVR) and total vascular impedance (TVI) were measured as arterial stiffness parameters.  Flow cytometry for analysis of checkpoint inhibitors (CPI): Markers of checkpoint inhibitors were analyzed on CD4 T cells. Thawed PBMC were rested overnight and 1x106 cells were stained with LIVE/DEAD® Aqua followed by staining for surface markers CD3, CD4, CD8, and checkpoint inhibitors PD1, TIGIT, TIM3, and LAG-3. Cells were then fixed, and acquired on a Flow cytometer (BD LSRFortessa, San Jose, CA) and analyzed by FlowJo V10 (Treestar, Ashland, OR). Frequencies of check point inhibitor molecules either alone or in combinations were analyzed on live (Aqua-) CD3+CD4+ T cells. A separate antibody panel containing markers for monocytes (CD14, CD16) and NK cells (CD56) along with HLA-DR and T cell specific markers for CD4 and CD8 T cells was used for analyzing CPI molecules in US healthy donors. Monocytes were gated as CD45+CD3-CD56-HLA-DR+CD14+ and NK cells as CD45+CD3-CD14-CD56+ populations.    Statistical analysis: Descriptive statistics such as percentages means and standard deviation, and median were used to describe the demographic characteristics of the study population. For unpaired data, Levene's test was used firstly to check variance heterogeneity followed by Wilcoxon rank-sum test (also called 'Mann-Whitney' U test) was performed using R stats package. For Correlation analyses, Shapiro test was used to check if data is normally distributed using R 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 coefficient
was performed using R stats package.  Linear
regression analysis (OLS) was performed and the data are presented as scatter
plots with regression lines. Pearson product-moment was performed and Pearson correlation
coefficients (r) with P values are present on top
of the plot. All analysis are performed using R stats package. A p value of <0.05 was considered as significant." The data are presented as scatter plots with regression lines and correlation coefficients with P values.                                   Results Demographic characteristics of study population: Demographic characteristics of the study population are shown in Table 1. The mean ages were similar among the study groups. Of the patients on ART, 140 patients were 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 of study enrolment. Absolute CD4 counts were significantly lower in the Gp1 compared to Gp 2. Expression of checkpoint inhibitor molecules 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 T cells in the study groups. Flow cytometry gating strategy for analysis of the CPI is depicted in Fig 1A. Expression of LAG-3 (Fig 1B) on CD4 T cells was significantly higher in Gp1 compared to Gp2 and Gp3.  PD1 (Fig 1C) 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. Absolute numbers of CD4 T cells at study entry inversely correlated with LAG-3 (Fig 2A), PD1 (Fig 2B) and dual LAG-3+PD1+ (Fig 2C) CD4 T cells in Gp1. In Gp2 no correlation was observed of CD4 T cell counts 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 F respectively). Cardiac function and arterial stiffness are impaired in Group 1: Measures of cardiac function and arterial stiffness in the study groups are shown in Fig S1.  Gp1 had lower cardiac ejection time (Fig S1A), lower stroke volume (Fig S1B) and lower stroke volume index (Fig S1C) than Gps 2 and 3 while cardiac output (Fig S1D) did not differ between study groups. Cardiac index was lower in Gp1 compared to Gp 3 (Fig S1E). Measures of arterial stiffness showed higher systemic vascular resistance (Fig S1F) with lower large and small artery elasticity (Fig S1G and S1H respectively) in Gp 1 compared to Gp 2. Cardiac function and arterial stiffness parameters were not different between Gp2 and Gp3. These data support an HIV induced effect on multiple cardiac functions and on arterial stiffness in treatment naïve HIV+ group. In Gp 2, all of these parameters are comparable to the general population (Gp3) implying ART induced improvement. Correlations of cardiac function and arterial stiffness with LAG3+ CD4 T cells (Fig 3; Table 2). Linear regression plots for association of cardiac function measures and Pearson correlation analysis (Figs 3A, 3B) showed that in Gp1, single LAG-3+ or LAG-3+ PD1+ CD4 T cell subsets respectively were inversely correlated with measures of cardiac ejection time, cardiac output, cardiac index, stroke volume, and stroke volume index. In Gp 2 and Gp 3, no association of LAG-3 or LAG-3 and PD1 expressing CD4 T cells with measures of cardiac function was evident. Arterial stiffness measure of systemic vascular resistance directly correlated with LAG-3 (Fig 3C) or LAG-3+PD1+ CD4 T cells in Gp1 and Gp2 (Fig 3D). Large artery elasticity index inversely 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 artery elasticity index and single LAG-3 or LAG3 coexpressed with PD1 on CD4 T cells were only found in Gp2. Single PD1 expressing CD4 T cells followed a similar pattern but were not consistent. A summary of all the correlations are depicted in a heatmap shown in Fig S2. No associations were found between arterial stiffness measures and CPI in Gp3 (Fig 3C, 3D). Checkpoint molecule distribution in immune cells: Since we found the association of CD4 T cells expressing LAG-3 and PD1 with cardiac function and vascular resistance, we examined expression of various CPI on different immune cell subsets in a group of 24 age matched healthy adults (Fig 4).  Monocytes exhibited highest frequency of LAG-3 and PD1 expression followed by LAG-3 on CD4 T cells, CD8 T cells and NK cells respectively (Fig 4A) and PD-1 on CD8, NK cells and CD4 T cells (Fig 4B). TIGIT expression was highest on NK cells followed by CD8 and  CD4 T cells with lowest expression on monocytes (Fig 4C) while TIM3 expression was highest on NK cells followed by monocytes, CD4 T cells and CD8 T cells respectively (Fig 4D). These data suggest that pattern of expression of CPI may vary on different innate and adaptive immune cells.   Discussion Cardiovascular disease is a major contributor to mortality and morbidity in HIV infection 3, 32-34.  Recent evidence points to expression of receptors for CPI molecules at the tissue level for controlling organ immune homeostasis, with the heart showing selective enriched for LAG 3 receptors 8. This cross sectional study was aimed at defining relationship of CPI molecules and CVD in HIV infection. We conducted the study in treatment naïve viremic patients (Gp1) as well as in patients on ART with viral suppression (Gp2) and healthy age-matched controls (Gp3). Cardiac function was decreased with increased vascular resistance in Gp1 compared to Gps 2 and 3 and exhibited an inverse correlation with LAG-3, PD-1 or LAG-3 plus PD1 expressing CD4 T cells but without association of TIGIT or TIM3.  Patients in Gp 2, despite having started ART at CD4  nadirs similar to the Gp 1 had little evidence of impaired cardiac function but had residual increased vascular resistance involving large and small vessel elasticity which was also inversely correlated with LAG-3+CD4 T cells and maximally with cells co-expressing LAG-3 and PD1.  These observations, together with the high LAG-3 expression in monocytes from healthy donors, imply a dominant role of cells expressing LAG-3 alone or in combination with PD1 in regulating cardiac health particularly in viremic patients who have not started ART.  The benefits of ART on CVD and on expression of CPI on CD4 T cells were clearly manifest in this study subjects based on comparisons with Gp2 and Gp 3.  Investigations to understand the potential role of LAG-3 have implications for immunotherapy targeting specific CPI molecules such as LAG-3, currently in clinical trials in cancer, for prevention or treatment of cardiac dysfunction in HIV.   LAG-3 is a 498-amino acid Transmembrane protein identified on activated human NK and T cell line 35. In humans, the LAG-3 gene is located adjacent to CD4 on chromosome 12 and is structurally homologous to CD4 with four extracellular immunoglobulin superfamily like domains 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-3 function is mediated by modulation of LAG-3 expression at the transcriptional level 38. Tian et al 40 demonstrated that LAG-3 expression in T cells was significantly upregulated in HIV infected subjects and was correlated with disease progression. Further analysis of LAG-3 expression in relationship to immune activation based on CD38 and HLA-DR expression showed LAG-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 cells co-expressing LAG-3 and DR was less. In that study MFI of LAG-3 expression declined in both CD4+ and CD8+ T cells after more than one year of ART treatment. Our findings are in agreement with these observations as we found that both immune activation (not shown) and LAG 3 and PD1 on CD4 T cells were greater in Group 1 than in Group 2 where the lower LAG-3 is most likely a consequence of ART. Intracellular storage in lysosomal compartments, may also serve to facilitate rapid LAG-3 cell surface expression following T cell activation (11, 12, 41, 42.   Risk factors for CVD are under intense investigation and involve both traditional and non-traditional factors. A study by Golden et al 43 found that the lipoprotein scavenger receptor class B type 1 (SCARBI) rs10846744 noncoding variant is significantly associated with atherosclerotic disease independently of traditional cardiovascular risk factors. They identified a connection between rs10846744 with LAG-3 in plasma and concluded that plasma LAG-3 is an independent predictor of HDL-cholesterol levels and CVD risk. Besides transcriptional modulation, LAG-3 function is also mediated by cell surface shedding which releases soluble LAG-3 (sLAG-3). LAG-3 cleavage is mediated by the metalloproteinase ADAM10 and ADAM17, which cleave a wide range of transmembrane proteins including CD62L, TIM3 and TNFa 44 with ADAM17 action becoming evident following cell activation 11, 12, 41, 42. It is possible that sLAG-3 is shed off from circulating cells and mediates tissue damage to the heart by targeting its receptors.   It is important to understand the physiological role of individual CPI molecules in terms of their independent effects and dependence upon each other. Although they belong to the same class of receptors, they act in a tier fashion, with CTLA-4 and PD1 in the first tier for maintaining self-tolerance and LAG-3, TIM3 and TIGIT in a second tier with distinct roles in regulating immune responses particularly at sites of tissue inflammation 8, 16, 25-31. Upregulation of LAG-3 on T cells also defines a subpopulation with functional exhaustion and as noted above, correlates with 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 in regulating T cell immune responses 8, 38 and also acts synergistically with PD1 to regulate T cell function 38, 45.  In a murine transplant tumor model, it was observed that PD-1 and LAG-3 were coexpressed on tumor-infiltrating CD4+ and CD8+ T cells and in cancer patients 42, 46-48. In HIV, blockade of LAG-3 enhanced HIV-specific T cell responses 40. In our study PD-1 and LAG-3 coexpression was highly correlated with cardiac measures and significance was often greater than that of independent molecules implicating their involvement in regulation of organ systems at tissue levels. PD1 expression on CD4 T cells could also be involved in influencing cardiac function, based on the higher PD1 expression observed in Gp1 than in the other groups, and results of the correlation analyses. Upregulation of PD-1 and PDL-1 have been demonstrated in cardiac injury models of ischemic-reperfused and cryoinjured hearts 49 in which cardiomyocytes were found to be a major source of inflammatory cytokine generation in isolated ischemic-reperfused hearts 50-52; importantly, PD-1 and PDL-1 were expressed on cardiomyocytes as well as on different populations of cardiac cells leading the authors to postulate a role of the cardiac PD-1/PDL-1 pathway in cardiac injury 49. These authors ascribed the cardiac injury to an increase in GADD153, a regulator of the inflammatory response 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 demonstrated that PDL-1 blocking antibody treatment reduced cardiac GADD153 expression with reduction in expression of inflammatory cytokines. As PD-L1 is also expressed in human heart myocytes 53, LAG-3 plus PD1 coexpressing immune cells are expected to have the maximal impact on cardiac damage. Monocytes are strongly implicated in cardias damage 54. Although we did not directly investigate the role of CPI on monocytes in our study population we conducted an independent analysis on healthy volunteers to examine expression of LAG-3 and PD1 on different cell populations. The extremely high expression of LAG-3 in monocytes suggests the possibility that LAG-3 or PD1 expressing monocytes could interact with T cells to act at the tissue level in the heart muscle and influence cardiac function, opening up new avenues for future investigation.  In conclusion, we found a novel association of LAG-3 expressing CD4 T cells either alone or in combination with PD1 with cardiac dysfunction in treatment naïve HIV+ patients, and with cardiac artery stiffness in both treatment naïve and virally suppressed subjects on ART. Key questions to investigate are 1) mechanism of action of LAG-3 affecting the cardiac function and arterial structure, and 2) the basis that underlies synergies of LAG-3 with PD-1 in regulating cardiac function. This understanding could provide insight into potential role of LAG-3 immunotherapy (which is in early clinical trials in cancer) in prevention or treatment of cardiac dysfunction in HIV.