Impairment of the PD-1 pathway increases atherosclerotic lesion development and inflammation

Abstract

OBJECTIVE

Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals upon interaction with its two ligands, PD-L1 or PD-L2. We studied the contribution of PD-1 pathway in regulating T cells that promote atherosclerotic lesion formation and inflammation.

METHODS AND RESULTS

We show that compared to Ldlr^−/− control mice, Pd1^−/−Ldlr^−/− mice developed larger lesions with more abundant CD4⁺ and CD8⁺ T cells and macrophages, accompanied by higher levels of serum TNF-α. Iliac lymph node T cells from Pd1^−/−Ldlr^−/− mice proliferated more to αCD3 or oxidized LDL stimulation compared to controls. CD8⁺ T cells from Pd1^−/−Ldlr^−/− mice display more cytotoxic activity, compared to controls in vivo and in vitro. Administration of a blocking anti-PD-1 antibody increased lesional inflammation in hypercholesterolemic Ldlr^−/− mice with more lesional T cells, and more activated T cells in paraaortic lymph nodes. The changes in lesional T cell content when PD-1 was absent or blocked were also observed in bone marrow chimeric Ldlr^−/− mice lacking PD-L1 and PD-L2 on hematopoietic cells.

CONCLUSIONS

PD-1 has an important role in down-regulating proatherogenic T cell responses, and blockade of this molecule for treatment of viral infections or cancer may increase risk for cardiovascular complications.

INTRODUCTION

CD4⁺ Th1 cells contribute significantly to the progression of atherosclerosis ¹. Previous work from our laboratory has demonstrated the importance of the B7/CD28 costimulatory pathway in the priming of pro-atherogenic Th1 responses ². Negative regulatory (“co-inhibitory”) members of the B7/CD28 family are also important modulators of T cell responses in a variety of disease conditions. The programmed cell death–1 (PD-1) receptor binds programmed death–ligand 1 (PD-L1) and PD-L2 (also known as B7-H1 and B7-DC, respectively)³. PD-1 is inducibly expressed on T cells, B cells, macrophages and some types of dendritic cells (DCs). PD-L1 is expressed on both hematopoietic and nonhematopoietic cells types. In contrast, PD-L2 expression is restricted mainly to DCs and macrophages⁴. PD-1 and PD-L1 interactions have been implicated in controlling immune tolerance, autoimmunity, and immune responses to viral infections and tumors ^5-8. Persistent antigenic stimulation in the setting of chronic viral infection leads to sustained upregulation of PD-1 expression on viral-specific CD8⁺ CTL and contributes to T cell exhaustion; therefore antibody mediated blockade of PD-1 or PD-L1 is being studied as a new therapeutic approach for chronic viral infections and cancer^{5, 9, 10}.

We have shown that PD-L1 is expressed on both DC and macrophages in aortic lesions in hypercholesterolemic low-density lipoprotein receptor knockout (Ldlr^−/−) mice; and hypercholesterolemic Ldlr^−/− mice that lack PD-L1 and PD-L2 develop significantly increased atherosclerosis with more lesional T cells when compared with Ldlr^−/− controls¹¹. However, the role of PD-1 in regulating pro-atherogenic T cell responses has not been directly examined. PD-1 is known to inhibit T cell activation by binding to B7-1 as well to PD-L1 and PD-L2 ¹², and it is possible that other receptors for PD-L1 exist¹³. Because PD-1, and not PD-L1 or PD-L2, is the target of new therapies being developed for cancer and chronic viral infections, it is important to know if PD-1 is required to regulate pro-atherogenic T cell responses in the arterial wall. Here we describe in vivo studies that directly address this question.

MATERIALS AND METHODS

An expanded Methods section is available in the Online Data Supplement.

Animal studies

Pd1^−/− mice on a C57BL/6 background, derived by targeted mutation in C57BL/6 ES cells which results in deletion of the IgV domain as described ¹⁴, were cross bred with the Ldlr^−/− mice on a C57BL/6 background (Jackson laboratories) to establish a Pd1^−/− Ldlr^−/− double knockout mice. Pd-l1/2^−/− mice, derived as described ¹⁵ or C57BL/6 mice (Jackson laboratories) were used as bone marrow donors, to produce Pd-l1/2^−/− →Ldlr^−/− or Pd-l1/2^+/+ →Ldlr^−/− radiation chimeras, as described¹⁶. Some Ldlr^−/− mice were injected i.p, with anti mouse PD-1 antibody (29F.1A12.D5, prepared in house), or rat IgG2a (Bio X Cell, Cat No BE0089), 200μg/mouse, twice a week, for three weeks. Pd1^−/− OT-1 transgenic mice were generated by backcrossing Pd1^−/− mice with OT-1 TCR transgenic mice ¹⁷. All the mice were fed water ad libitum and were maintained on a 12:12-h light-dark cycle under pathogen-free conditions in the Harvard new research building animal facility according to institutional and National Institutes of Health guidelines.

Serum lipid analysis

Mice lipid profiles were measured on the c501 module of the Cobas 6000 analyzer (Roche Diagnostics, Indianapolis, IN) using the assays developed for human use.

Multiplexed Cytokine assays

Sera and culture supernatants were analyzed for cytokine concentrations using luminex bead-based multiplex assays.

Atherosclerotic Lesion Assessment

Atherosclerotic lesions were analyzed in the aortic root, aortic arch, and descending aorta as previously described ^{11, 16}.

Immunohistochemistry and double immunefluorescence staining of aortic lesions

Frozen aortic root sections were stained with antibodies specific for CD4, CD8, F4/80 for macrophages, and smooth muscle cell α actin (SMC-α actin) for smooth muscle cells (SMC), as described ^{11, 16}. Double immunofluorescent staining for CD3 and CD4 or CD8 was performed in the aortic sinus sections, as well as for SMC (SMC-α actin, FITC, Sigma) and annexin V (Alexa Fluor® 568, Invitrogen). Nuclei were stained with DAPI.

Cell immunostaining and flow cytometry

Splenocytes, iliac node lymphocytes and aortic digests were stained for CD3, CD4, CD8, CD62L, CD25, CD44 and PD-1. Aortas from the ascending aorta to iliac bifurcation were cleaned of peri-adventitial connective tissue and subjected to enzymatic digestion, as described¹⁸.

CTL killing Assay

Mouse aortic smooth muscle cells (SMC) and mouse heart endothelial cells (EC) were prepared as previously described^{19, 20}, co-cultured with mouse Pd1^−/− OT-1 or Pd1^+/+ CD8⁺ OT-1 CTL plus SIINFKL peptide antigen for 2h, and apoptotic cells were then quantified by annexinV/7-aminoactinomycin D (7-AAD) staining and flow cytometry.

CD4⁺ and CD8⁺ T cell purifications and quantitative RT-PCR (qRT-PCR) analyses

Splenic CD4⁺ or CD8⁺ T cells were purified by immunomagnetic beads, and RNA was isolated, reverse transcribed, and subjected to qRT-PCR analyses for the genes listed in Table SIV.

In vitro cell proliferation assay

CD4⁺ and CD8⁺ T cells were purified from spleen and lymph node of mice, stimulated with αCD3 or oxidized low-density lipoprotein (oxLDL) in vitro, and proliferation was measured by ³H-thymidine incorporation.

Statistics

All statistical analyses were performed using Prism software. Differences between two groups of mice were analyzed by Student’s t test and expressed as mean ± SEM or by the Mann-Whitney test (for nonparametric data), and ANOVA with Tukey’s Multiple Comparison post test, for three or more group experiments. A value of p < 0.05 was considered to be significant.

RESULTS

Atherosclerotic lesion development and phenotype in Pd1^−/−Ldlr^−/− mice

We generated Pd1^−/−Ldlr^−/− mice by crossbreeding the relevant parent strains, and compared lesion development in Pd1^−/−Ldlr^−/− and Ldlr^−/− mice fed a cholesterol diet for 5 and 10 weeks. Serum lipids did not differ significantly between the two groups of mice after either 5 or 10 weeks of diet (Supplemental Table I). At 5 weeks, lesion size was similar in both groups (Fig 1a and 1c), but the lesions of the PD-1 deficient mice had markedly more inflammatory cells including CD4⁺ and CD8⁺ T cells and macrophages (Fig. 2a and 2b). After 10 weeks of diet, the Pd1^−/−Ldlr^−/− mice had increased lesion size compared to Ldlr^−/− controls (Fig. 1a and 1c). The percent of the total cross-sectional vessel wall area was increased, and there was an outward remodeling resulting in an increase in total aortic root cross sectional area in Pd1^−/−Ldlr^−/−mice compared to Ldlr^−/− controls (Fig 1a). Additionally, en face lesional analysis of the entire aorta distal to the aortic root revealed more lesions in the Pd1^−/−Ldlr^−/− mice than in the Ldlr^−/− controls (Fig. 1b and 1d). Immunohistochemical staining revealed more abundant CD4⁺ and CD8⁺ T cells and macrophages in the lesion of Pd1^−/−Ldlr^−/− mice compared to Ldlr^−/− mice (Fig. 2a and 2b). SMC content was similar in the lesions of both groups of mice (Fig 2a, 2b, bottom panels). All these data indicate that PD-1 acts to limit proatherogenic immune responses.

Figure 1. Effects of Programmed death–1 (PD-1) deficiency on atherosclerotic lesion development.

a. Representative cross sections of the ORO-stained aortic sinuses are shown for Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 5 and 10 weeks of cholesterol diet (original magnification, ×40). b. Representative en face lesion of aortic arch and descending aortas are shown for Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 10 weeks of cholesterol diet (original magnification, ×40). c–d Quantitative analysis of lesion area in aortic sinus sections taken after 5 and 10 weeks of high-cholesterol diet (c), and on aortic arch and descending aorta en face lesions (d), after 10 weeks of high-cholesterol diet. Each data point represents the mean value obtained from multiple sections from each mouse (c) or total area for each mouse (d); horizontal bars represent the mean value for each group.

Figure 2. Effects of PD-1 deficiency on atherosclerotic lesion phenotype.

a. Representative cross sections of immunohistochemistry for CD4, CD8, macrophage (F4/80) and smooth muscle cell (SMC-α actin) in aortic sinus section from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 5 and 10 weeks of cholesterol diet (original magnification ×200). b. Quantitative analysis of immunohistochemical stains for each experimental group. Each data point represents the mean value determined for each mouse; horizontal bars represent the mean value for each group. *P < 0.05 (ANOVA with Tukey’s Multiple Comparison post test).

Effects of PD-1 deficiency on lesional cell death and CTL cytotoxicity

The increased number of CD8⁺ T cells in lesions of Pd1^−/− mice, and the lack of increased SMC content despite an overall increase in lesion size suggested that PD-1 deficiency may cause more CD8+ CTL cytotoxicity and SMC death in the lesions. We therefore performed double immunefluorescent staining of aortic sinus of the Pd1^−/−Ldlr^−/− and Ldlr^−/− mice for annexin V, to detect apoptotic cells, and SMC-α actin, to detect SMC. We found more apoptotic cells including SMC and non-SMC (Fig 3a and 3b) in the lesion of PD-1 deficient mice after 10 weeks of diet, compared to the lesions from control mice. We did not detect any annexin V positive lesional SMC at 5 weeks of diet (data not shown). We also performed an in vitro CTL killing assays by co-culturing mouse aortic SMC or heart endothelial cells with Pd1^−/− or control CD8⁺ CTL from OT-1 TCR transgenic mice. FACS analyses of annexin V/7-AAD stained cells demonstrated that effector Pd1^−/−OT-1 cells kill more SMC or ECs, compared to Pd1^+/+OT-1 (Fig 3c).

Figure 3. Cytotoxic effects of PD-1 deficiency on lesional cells in vivo and on mouse endothelial cells and smooth muscle cells in vitro.

a. Representative imgaes of double immunofluorescent staining for annexin V (red) and SMC-α actin (green) and mounted with DAPI mounting medium (blue) in aortic sinus section from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 10 weeks of cholesterol diet. Arrows indicate triple positive stained cells (yellow and orange) in top panels and annexin V (red) positive but SMC negative cells in bottom panels; scale bars: 20μm. b. Quantitative analysis of double positive staining for annexin V and SMC-α actin (upper panel) and of annexin positive but SMC-α actin negative staining (lower panel) for each experimental group. Each data point represents the mean value determined from three aortic sinus sections from each mouse; horizontal bars represent the mean value for all the mice in each group. *P < 0.05 (ANOVA with Tukey’s Multiple Comparison post test). c: FACS analyses for CTL killing of mouse aortic SMC (left panel) and mouse heart endothelial cells (EC) (right panel). Mouse effector CD8⁺ T cells prepared from Pd1^−/− OT-1 and Pd1^+/+ OT-1 transgenic mouse (see details in Supplemental Materials), were cocultured with mouse aortic SMC or mouse heart EC, respectively for one hour before stained for CD90 and annexin V and 7-AAD. AnnexinV and 7-AAD were characterized as being in late apoptosis, and cells that were positive for 7-AAD but not annexinV were categorized as dead. Data shown are mean ± SD, are one of representative from two different sets of experiments with similar results. Differences between two groups of mice were analyzed by the Mann-Whitney test. * p<0.05 indicates significant difference, compared to counterpart Pd1^+/+ T cell group.

Effects of PD-1 deficiency on T cell gene expression and immune responses in Ldlr^−/− mice

In order to determine why CD4⁺ and CD8⁺ T cell infiltration was greater in the lesions of hypercholesterolemic Pd1^−/−Ldlr^−/− mice, we analyzed expression of selected genes by qRT-PCR of RNA from purified splenic CD4⁺ and CD8⁺ T cells, taken after 10 weeks of cholesterol diet. The results demonstrated that both PD-1 deficient CD4⁺ and CD8⁺ T cells expressed higher levels of pro-inflammatory cytokine genes Ifng and Tnf, as well as increased levels of chemokine receptor genes Ccr5, Ccr6 and Cxcr3, compared to T cells from Ldlr^−/− mice (Fig. 4a and 4b). These data suggested that PD-1 deficient T cells may be more competent at migrating to inflammatory sites compared to wild type T cells, and that they produce more inflammatory cytokines.

Figure 4. Effects of PD-1 deficiency on T cell gene expression and immune responses of the atherosclerotic mice.

a and b qRT-PCR analyses of Ifng, Tnf, Ccr5, Ccr6, and Cxcr3 were performed on unstimulated splenic CD8⁺ (a) and CD4⁺ T cell (b) RNA isolated from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 10 weeks of cholesterol diet. n=8 from each group. Data shown are mean±SEM. c. FACS analyses for intracellular IFNγ expression by immunofluorescence staining on splenic CD8⁺ T cells in response to αCD3 for 48h. Splenic CD8⁺ T cells were purified from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 5 weeks of cholesterol diet. d and e. Iliac lymphocyte proliferation measured by ³H-thymidine incorporation during the final 16 hours of 72 hr culture with αCD3 (d) or oxLDL (e). f and g. TNFα secretion measured by Luminex cytokine assays (see Materials and Methods) from supernatants of iliac lymphocytes cultured with αCD3 for 48h from Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 5 weeks of cholesterol diet (f) and from sera of Pd1^−/− Ldlr^−/− and Ldlr^−/− mice after 10 weeks of cholesterol diet (g). c to f, n= 6 from each group. g, n=10 from each group. Data shown are mean ± SEM. Differences between two groups of mice were analyzed by the Mann-Whitney test.

To evaluate the changes in T cell activation in the absence of PD-1 in the context of atherosclerosis, we examined iliac lymph nodes and spleens from hypercholesterolemic Pd1^−/− Ldlr^−/− mice. After 5 weeks of diet, there was no difference in the number of iliac lymph node cells between these two groups (Supplemental Fig Ia). However, FACS analyses showed that there were more total CD4⁺ T cells, and a higher fraction of activated CD4⁺ and CD8⁺T cells (CD25⁺) compared to T cells from control mice. (Supplemental Fig Ib to Ie), although the number of splenic CD4⁺ and CD8⁺ T cell numbers and the fraction of activated T cells in the spleens were similar between these two groups after 5 or 10 week diet (data not shown). Therefore, we asked if the iliac lymphocyte response to TCR stimulation might be different when PD-1 signaling is absent in the hypercholesterolemic Ldlr^−/− mice. Polyclonal stimulation of the splenic CD4⁺ or CD8⁺ T cells by αCD3 resulted in higher levels of IFNγ production by CD8⁺ T cells (Fig. 4c) but not CD4⁺ T cells (data not shown). Neither splenic CD8⁺ nor CD4⁺ T cells responded to the putative athero-antigen oxLDL in vitro (data not shown). When exposed to αCD3, a T cell –specific stimulus, or oxLDL, which could activate antigen-specific T cells and macrophages, the iliac lymph node cells from Pd1^−/−Ldlr^−/− mice proliferated more than those from control Ldlr^−/− mice (Fig 4d and 4e), and secreted more TNFα (Fig. 4f). It is possible that some of the TNFα we measured in response to anti-CD3 was produced by macrophages secondarily activated by T cell IFNγ. Furthermore, there was a significantly increased level of serum TNFα in the Pd1^−/−Ldlr^−/− mice after 10 weeks of cholesterol diet, compared to Ldlr^−/− mice (Fig 4g).

Effects of PD-1 blocking antibody administration on atherosclerotic lesions in Ldlr^−/− mice

We next evaluated the impact of an anti-PD-1 blocking antibody on atherosclerosis. Six-week-old Ldlr^−/− mice were fed with cholesterol diet for five weeks, and starting from the third week of diet, mice received i.p. injections of PD-1 antibody or rat IgG, 200 μg/mouse, twice a week for three weeks before all the tissue collections. Serum lipids did not differ significantly between the two groups at time of sacrifice (Supplemental Table II).

PD-1 antibody treatment did not change the lesion size development, but significantly increased lesional CD4⁺ and CD8⁺ T cells, compared to mice receiving control IgG at 5 weeks of diet (Fig 5a and 5b). There was similar lesional macrophage and SMC content in both groups (Fig 5a and 5b, bottom panel).

Figure 5. Effects of PD-1 blocking antibody treatment on atherosclerotic lesions in hypercholesterolemic Ldlr^−/− mice.

a. Representative cross sections of ORO staining (original magnification ×40) and of immunohistochemistry for CD4, CD8, macrophage (F4/80) and SMC (SMC-α actin) on aortic sinus from Ldlr^−/− mice after 5 weeks of cholesterol diet. Starting from third week of cholesterol diet, mice were treated with either PD-1 antibody or rat IgG, 200μg/mouse, by i.p injections, twice a week, for three weeks (original magnification ×200). b. Quantitative analysis of each staining for each experimental group. Each data point represents the mean value determined for each mouse; horizontal bars represent the mean value for each group. Differences between two groups of mice were analyzed by the Mann-Whitney test. NS indicates not statistically significant.

Evaluation of iliac lymph nodes showed that mice receiving anti-PD-1 antibody treatment developed larger nodes with more CD4⁺ T cells and CD8⁺ T cells (Fig 6a, 6b and 6e), FACS analyses of activation markers on T cells revealed more CD44⁺ and IFNγ producing CD4⁺ and CD8⁺ T cells (Fig. 6c and 6d; fig 6f and 6g) in the anti-PD-1-treated group. Moreover, the percentage of splenic IFNγ producing CD4⁺ T cells and CD8⁺ T cells was significantly higher in the mice treated with anti-PD-1 antibody, compared to the control IgG treated mice (data not shown), although the total percentage of splenic CD4⁺ T cells and CD8⁺ T cells were similar in both groups of mice.

Figure 6. Effects of PD-1 blocking antibody treatment on immune responses of the hypercholesterolemic Ldlr^−/− mice.

a. Comparison of iliac lymph node cell counts from Ldlr^−/− mice treated with PD-1 antibody or rat IgG. b to g FACS analyses for numbers of total CD4⁺ (b), CD8⁺ (e) T cells and a fraction of CD44 (c and f), as well as IFNγ producing CD4⁺ and CD8⁺ T cells (d and g) in the iliac lymph nodes of Ldlr^−/− mice receiving either PD-1 antibody or rat IgG treatment after 5 week cholesterol diet. Horizontal bars represent the mean value for each group. Differences between two groups of mice were analyzed by the Mann-Whitney test.

PD-L1/2 deficiency in bone marrow derived cells results in enhanced atherosclerotic lesion inflammation

We have previously reported that cholesterol diet-fed Pd-l1/2^−/−Ldlr^−/− mice had more lesion development and more lesion inflammation than Ldlr^−/− controls ¹¹. In order to assess if PD-L1 and PD-L2 expression on bone marrow-derived cells was responsible for regulation of pro-atherogenic T cell responses, we prepared Pd-l1/2^−/−Ldlr^+/+ →Ldlr^−/− and control Pd-l1/2^+/+Ldlr^+/+ →Ldlr^−/− bone marrow chimeras. Hematopoietic reconstitution with donor cells was confirmed by PCR detection of the wildtype Ldlr allele in an aliquot of blood collected 4 weeks after BMT (Supplemental Fig IIa). The rest of blood was lysed and cultured with or without IFNγ stimulation overnight to induce PD-L1 expression. FACS analysis showed that PD-L1 was expressed to the similar level in all the wild type to Ldlr^−/− chimeras (WT chimeras) after IFNγ treatment, while no PD-L1 was detected in all Pd-l1/2^−/− →Ldlr^−/− chimeras (Pd-L1/2^−/− chimeras) (Supplemental Fig IIb). All these data indicated a successful reconstitution. Therefore, we started feeding the chimeras with cholesterol diet at this point.

After 10 weeks of cholesterol diet feeding, the bone marrow chimeras were sacrificed and aortic root lesions were analyzed. Serum lipids did not differ significantly between the two groups at this time (Supplemental Table III). Lesion size did not differ between recipients of Pd-l1/2^+/+ or Pd-l1/2^−/− marrow (Supplemental Fig. IIIa and IIIb, top panels). However, there were significantly more CD4⁺ T cells, CD8⁺ T cells, and macrophages in the lesions of the Pd-l1/2^−/− marrow recipients (Supplemental Fig. IIIa and IIIb), while SMC content was equivalent in both group of chimeras (Supplemental Fig. IIIa and IIIb, bottom panel). Strikingly, there were abundant CD8⁺ T cells in the lesion of Pd-l1/2^−/− marrow recipients, while there were scarce CD8⁺ T cells in the WT chimeras. This result is consistent with our previous findings in the hypercholesterolemic Pd-l1/2^−/− Ldlr^−/− mice ¹¹. There was no difference in the expression of activation markers on splenic or iliac node T cells between the two groups, and the T cells from spleen and nodes of both groups proliferated the same amount and secreted comparable amounts of cytokines in response to in vitro reactivation by αCD3 or oxLDL. Furthermore, there was no evidence of increased systemic inflammation in either group, since plasma cytokines were mostly undetectable (data not shown).

Marked increases in both CD4⁺ and CD8⁺ T cells are consistently seen in atherosclerotic lesions of Ldlr^−/− mice with impaired PD-L/PD-1 signaling

To further characterize the lesional infiltrating CD4⁺ cells and CD8⁺ cells, we performed double immunofluorescent staining for CD3 and CD4 or CD8 in aortic sinus lesions from Pd1^−/− Ldlr^−/− mice, anti-PD-1 treated mice, and Pd-l1/2^−/− bone marrow chimeras, as well as appropriate controls. Confocal analyses of these stained lesions showed marked increases in both CD4⁺ and CD8⁺ T cells in mice with deficient or blocked PD-1 pathway (Supplemental Fig IV).

PD-1 is upregulated on aortic T cells in hypercholesterolemic Ldlr^−/− mice

We examined if diet-induced hypercholesterolemia could induce increased PD-1 expression in T cells from aortic lesions. Pooled aortas from Ldlr^−/− mice either fed with cholesterol or control diet for 8 weeks were enzymatically digested and the recovered leukocytes were stained for CD3, CD8 and PD-1. The enzymatic digestion rendered CD4 undetectable; and we therefore regarded that a large majority of the CD3⁺ CD8⁻ cells were CD4⁺ T cells. FACS analyses indicated that cholesterol diet induced an increase in aortic wall T cells including CD3⁺ CD8⁺ T cells and CD3⁺ CD8⁻ T cells (Supplemental Fig.Va -Vc). More T cells from hypercholesterolemic aortas expressed PD-1 than T cells from aortas of Ldlr^−/− mice fed control diet (Supplemental Fig.Vd and Ve).

DISCUSSION

In this report, we establish a clear role for the PD-1 in modulating experimental atherosclerosis in Ldlr^−/− mice. The influence of PD-1 pathway on atherosclerosis was examined directly using two different approaches, including complete genetic loss of PD-1 receptor, and treatment with blocking antibody to PD-1. In addition, we used bone marrow chimeras to study effects of a lack of PD-1 receptors PD-L1/2 only on hematopoietically derived cells. Our data from all three approaches indicate that the PD-1 pathway tightly controls lesional T cell responses, and restrains a potentially robust CD8⁺ T cell response, which is usually minimal in mice when this co-inhibitory pathway is intact.

PD-1 is a well characterized receptor on T cells for PD-L1 and PD-L2; PD-L1 has also another receptor namely B7-1⁴; a second receptor for PD-L2 has been proposed but not yet published ¹³. PD-1 is an activation antigen that is upregulated by T cell activation and returns to basal levels following antigen clearance. In chronic infection and cancer PD-1 remains high. Thus, the PD-1 molecule has been recognized as a hallmark for T cell exhaustion and PD-1 expressing antigen-specific T cells are dysfunctional in cytokine production and proliferation upon antigen re-stimulation in a variety of viral infections^{7, 14, 21}. The role of PD-1 in atherosclerosis has not been previously studied.

Our data comparing hypercholesterolemic Pd1^−/−Ldlr^−/− and Ldlr^−/− mice establish that PD-1 exerts significant anti-inflammatory, atheroprotective effects, and is the likely relevant receptor for the anti-inflammatory atheroprotective effects PD-L1 or PD-L2, which we have previously observed ¹¹. The influence of PD-1 is manifest early in disease progression, since we observed marked increases in both CD4⁺ T cells and CD8⁺ T cells and macrophages in the aortic lesions in the PD-1 deficient mice after only 5 weeks of diet. Analyses of iliac node T cell numbers, phenotype, proliferation, and cytokine secretion at 5 weeks indicate early dysregulation of systemic immune responses to the hypercholesterolemia in the Pd1^−/−Ldlr^−/ mice. These data indicate that PD-1 deficiency results in an exaggerated T cell-mediated immune response in the early stages of hypercholesterolemia and atherosclerosis. After 10 weeks of diet, the Pd1^−/−Ldlr^−/− lesion phenotype was more striking, with larger lesional size, outward aortic root remodeling, and increased inflammatory cell infiltration including a remarkable number of CD8⁺ T cells, as well as more apoptosis in the lesion. At the 10 week time point, splenic CD4⁺ and CD8⁺ T cells from Pd1^−/−Ldlr^−/− mice expressed higher levels than the Ldlr^−/− control mice of mRNAs indicative of functional activation, including Ifng, Tnf, Ccr5, Ccr6 and Cxcr3. Furthermore, the detection of elevated levels of serum TNFα in the Pd1^−/−Ldlr^−/− after 10 weeks of cholesterol diet is also consistent with continued profound dysregulation of the T cell mediated immune response. Based on the cytokine profiles from either sera or supernatant of cultured cells, there was no obvious change in the balance of Th1 and Th2 cells when PD-1 deficient. We also did not detect any difference in numbers of CD4⁺ FOXP3⁺ cells between Pd1^−/−Ldlr^−/− and Ldlr^−/− mice either at 5 or 10 weeks, despite evidence suggesting PD-1 influences regulatory T cell development and functions ²². Overall, it appears that PD-1 plays a direct role in inhibiting activation of pro-atherogenic T cells.

The robust increase in lesion CD8⁺ T cells seen in lesions of the PD-1 deficient mice suggests that there may have been more CTL-mediated cytotoxicity in those lesions. Although at 10 weeks, lesion size was greater in the Pd1^−/−Ldlr^−/−mice compared to Ldlr^−/− mice, there was not a concomitant increase in lesional SMC. We did observe more apoptotic lesional SMC in the Pd1^−/−Ldlr^−/−mice compared to Ldlr^−/− controls, which may explain why SMC content did not increase, and this finding is consistent with more CTL-mediated cytotoxicity. In vitro assay also confirmed that PD-1 deficient CD8⁺ T cells are more potent killers of mouse aortic SMC.

Our finding that PD-1 expression is up-regulated in aortic T cells in cholesterol diet-fed Ldlr^−/− mice compared to control diet-fed mice suggests that chronic exposure to athero-antigens may lead to a partial suppression of CD4⁺ and marked suppression of CD8⁺ T cell responses to these antigens. Specific peptides that may drive pro-atherogenic T cell response are not well characterized, and therefore we could not examine PD-1 expression or T cell exhaustion on tetramer-identified T cells, as has been done for viral-specific T cells in mice and humans^{7, 23}. Given the phenotype of the PD-1 deficient Ldlr^−/− mice, we reasoned that antibody-mediated blockade of PD-1 may aggravate atherosclerotic lesion inflammation. Our results from short-term injection of rat anti-mouse PD-1 antibody to hypercholesterolemic Ldlr^−/− mice confirmed our prediction. The antibody treated mice showed similar increases in lesional CD4⁺ and CD8⁺ T cells as we saw in the PD-1 deficient Ldlr^−/− mice. There was also a remarkable effect of this short-term PD-1 blockade on iliac lymph node T cell activation. We do not consider the lack of increase lesion size in the mice treated with anti-PD-1 Ig a significant finding, given the limited time we could treat the mice due to development of mouse anti-rat Ig responses. Furthermore, plaque inflammation may be more clinically relevant than plaque growth in the clinical context of treatment of patients.

We found that Ldlr^−/− chimeras with Pd-l1/2^−/− bone marrow developed aortic lesions with more T cells and macrophages than did control chimeras with Pd-l1/2^+/+ bone marrow, but similar plaque sizes in the two groups. This is in contrast to our previous study ¹¹ in non-irradiated Pd-l1/2^−/−/Ldlr^−/− mice showed both enhanced lesion inflammation and lesion size. This difference between enhanced lesion inflammation but comparable lesion size in the current study may reflect the influence of irradiation on lesion development, which we and others find, is generally reduced compared to non irradiated mice at relatively early time points ²⁴. Since PD-L1 is also expressed on non-bone marrow derived cells, including endothelial cells, it is possible that PD-L1 deficiency on both non-hematopoietic and hematopoietic cells is required to influence lesion size. Remarkably, there were abundant CD8⁺ T cells, almost as many as CD4⁺ T cells in the lesions of the Pd-l1/2^−/− bone marrow chimeras. We believe this is the finding of greatest translational significance in this experiment, given that plaque inflammation is tightly associated with acute coronary syndromes. In this study, the lack of hematopoietic PD-L likely impaired regulatory functions of lesional antigen presenting cells interacting with PD-1⁺ T cells.

In summary, our data demonstrate that PD-1 plays a significant role in regulating both CD4⁺ and CD8⁺ T cell responses in experimental atherosclerosis. Furthermore, CD8⁺ T cells, which are usually difficult to detect in mouse lesions and less frequent than CD4⁺ T cells, become prominent when PD-1 is absent or blocked. The data suggest that PD-1 agonists could have therapeutic benefit for exacerbations of atherosclerotic lesion inflammation, such as in acute coronary syndromes. Conversely, humanized function-blocking anti-PD-1 antibody therapies are being developed to enhance T cell responses in patients with cancers or chronic viral infections, such as HIV and Hepatitis C. HIV patients are at elevated risk for coronary artery disease and myocardial infarction compared to non-infected individuals, and highly active antiretroviral therapy leads to dyslipidemia and insulin resistance ²⁵. It will therefore be important to consider the potential added risk of deregulating plaque-based T cell responses by PD-1 blockade.