PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in regulating T cell responses during chronic infection in mice (original) (raw)
PD-L1 expression on hematopoietic cells regulates CTL responses during viral infection. Functional exhaustion and deletion of responding T cells occur during infection of mice with LCMV CL-13. Cytotoxic T cells progressively lose the ability to produce the cytokines IL-2, TNF-α, and IFN-γ as they become exhausted (8–10). The PD-1/PD-L1 pathway has an important role in regulating T cell exhaustion, since blocking this interaction leads to enhanced CTL number and function in chronically infected individuals (1, 11–13). PD-L1 is expressed by BM-derived cells to varying degrees in uninfected mice (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI40040DS1). PD-L1 expression is rapidly upregulated on B and T lymphocytes, NK cells, DCs, macrophages, and granulocytes within 1 day after infection with LCMV CL-13 and remains increased for at least 1 week. Interestingly, DCs expressed very high levels of PD-L1 within 1 day of infection, suggesting that this expression may be important for controlling immune responses.
To better understand the source of PD-L1 signals for T cells, in particular the roles of PD-L1 expression by hematopoietic cells (such as DCs and macrophages) and nonhematopoietic cells (such as stromal cells and other supportive cells in the tissues) during infection, we generated mice lacking PD-L1 on either BM and/or non-BM–derived cells. These BM chimeric mice were made by lethally irradiating WT C57BL/6 mice or Pdl1–/– mice and reconstituting them with either WT or Pdl1–/– BM (Figure 1A). The resulting chimeras consisted of 4 groups: WT BM into WT recipients (W-W), WT into Pdl1–/– (W-P), Pdl1–/– into WT (P-W), and Pdl1–/– into Pdl1–/– (P-P). Expression of PD-L1, or lack thereof, was confirmed on PBMCs and splenocytes 8 weeks after reconstitution (Figure 1A).
PD-L1 expression on hematopoietic cells inhibits virus-specific CD8+ T cell responses. (A) Lethally irradiated Pdl1–/– or WT mice were reconstituted with Pdl1–/– BM (P-P or P-W) or WT BM (W-P or W-W) and infected with LCMV CL-13 8 weeks later. Expression of PD-L1 on splenic hematopoietic cells was confirmed by flow cytometry. (B) GP33- and GP276-specific responses in the spleen 8 days after CL-13 infection were measured using MHC class I tetramers. Numbers above gates represent the percentage of CD8+ T cells staining positive for GP33 tetramer and PD-1. (C) The numbers of tetramer-positive cells in the spleen of chimeric mice 8 days after infection. (D) Expression of PD-1 on tetramer-positive cells from the spleen. n = 4–8 mice per group. Mean + SEM of data from 1 of 4 representative experiments are shown.
We examined antigen-specific T cell responses in the spleen 8 days after infection with LCMV CL-13 (Figure 1, B and C). LCMV DbGP33-41–specific and DbGP276-286–specific CD8+ T cell populations were significantly increased in chimeric mice lacking PD-L1 on BM-derived cells (P-P and P-W mice). However, mice with PD-L1–sufficient BM had fewer antigen-specific CD8+ T cells, irrespective of whether PD-L1 was expressed on nonhematopoietic cells (W-W and W-P). Numbers of annexin V+ DbGP33–specific and DbGP276–specific CD8+ T cells were slightly decreased in Pdl1–/– mice (Supplemental Figure 2), suggesting that increased survival might also contribute to increased responses in the absence of PD-L1 signals, in addition to increased proliferation. Together, these data show that PD-L1 expression on hematopoietic cells, and not on nonhematopoietic cells, significantly influenced CD8+ T cell responses soon after infection. Interestingly, antigen-specific T cells also expressed increased levels of PD-1 when PD-L1 was lacking on either BM- or non-BM–derived cells (Figure 1D).
PD-L1 expression on hematopoietic cells influences CTL function during chronic viral infection. Since blockade of the PD-1/PD-L1 pathway enhances CTL function during chronic infection (1, 11–13), we next determined whether CTL primed in mice lacking expression of PD-L1 on either BM-derived cells or parenchymal cells were capable of producing cytokines upon antigen stimulation. Mice lacking PD-L1 on BM-derived cells (P-P and P-W) demonstrated an increase in GP33- and GP276-specific CD8+ T cells producing IFN-γ after infection (Figure 2A). Moreover, increased numbers of IFN-γ+ virus–specific T cells responding to multiple epitopes were induced in these mice in comparison with mice expressing PD-L1 on hematopoietic cells (W-W and W-P; Figure 2B). Mice lacking PD-L1 on BM-derived cells also demonstrated an increase in polyfunctional CD8+ T cells capable of producing both IFN-γ and TNF-α after stimulation (Figure 2, A and C). In contrast, mice lacking PD-L1 expression on nonhematopoietic cells (W-P) displayed no such increase in antigen-specific IFN-γ+TNF-α+CD8+ T cells after CL-13 infection when compared with W-W mice. Together, these data demonstrate that PD-L1 expression on BM-derived cells regulates T cell numbers and function early during viral infection. These results also indicate that PD-L1 expression on nonhematopoietic cells does not significantly influence the induction of virus-specific T cell responses during chronic infection.
PD-L1 expression on hematopoietic cells influences CTL function. (A) IFN-γ and TNF-α production by GP33- and GP276-specific CD8+ T cells in the spleen 8 days after infection. Numbers in the upper quadrants represent the proportion of total CD8+ T cells expressing IFN-γ (left) or IFN-γ and TNF-α (right) after GP33 or GP276 stimulation. (B) The number of IFN-γ+ and (C) IFN-γ+TNF-α+ cells responding to NP396, GP33, GP276, GP118, and NP235 epitopes. n = 4–8 mice per group. Mean + SEM of data from 1 of 4 representative experiments are shown.
Increased T cell responses in nonlymphoid tissues in mice lacking PD-L1 expression on hematopoietic cells. Chronic LCMV infection involves systemic dissemination of virus to lymphoid (both primary and secondary) and nonlymphoid tissues. Effector T lymphocytes are found in all tissues, though the relative distribution of cells and their function can vary depending on the tissue and time since infection (8). We examined DbGP33-41–specific and DbGP276-286–specific CD8+ T cell populations in the liver and lung in chimeric mice 8 days after LCMV CL-13 infection. Mice lacking PD-L1 expression on BM-derived cells displayed increased numbers of antigen-specific CTL in the nonlymphoid tissues (Figure 3A). The liver and lungs from chimeric mice lacking PD-L1 on BM cells (P-P and P-W) demonstrated a significant increase in the number of IFN-γ+ virus–specific CD8+ T cells responding to multiple epitopes, similar to that observed in the spleen (Figure 3B). We also observed increased numbers of polyfunctional IFN-γ+TNF-α+CD8+ T cells in the liver and lungs of P-P and P-W mice after CL-13 infection (not shown). In contrast, no significant increase in virus-specific cells was observed in mice that only lacked PD-L1 expression on parenchymal cells (W-P).
PD-L1 expression on hematopoietic cells controls CD8+ and CD4+ T cell responses in lymphoid and nonlymphoid tissues. (A) GP33- and GP276-specific responses in the liver and lungs 8 days after CL-13 infection were measured using MHC class I tetramers. (B) The number of CD8+ IFN-γ+ cells responding to 6 LCMV epitopes (GP33, GP276, NP396, NP235, NP205, and GP118) in the spleen, liver, and lung 8 days after infection. Responses to individual epitopes were obtained for each sample and then pooled for display. (C) The number of CD4+IFN-γ+GP61–specific T cells in the spleen, liver, and lungs 8 days after LCMV CL-13 infection. n = 3–4 mice per group. Mean + SEM of data from 1 of 3 representative experiments are shown. *P < 0.05; **P = 0.01; ***P < 0.005.
We also examined virus-specific CD4+ T cell responses in the spleen, liver, and lungs. LCMV GP61-80–specific CD4+ T cells producing IFN-γ were significantly increased in all tissues in mice lacking PD-L1 on hematopoietic cells (P-P and P-W), yet not in W-P mice (Figure 3C). Together, these data demonstrate that CD8+ and CD4+ effector T cells are increased in both lymphoid and nonlymphoid tissues in mice deficient in PD-L1 on BM-derived cells. Thus, T cell numbers and function are controlled by BM-derived PD-L1 but not nonhematopoietic PD-L1 during chronic LCMV infection.
PD-L1 expression on nonhematopoietic cells controls viral clearance and tissue immunopathology. The preceding experiments show that the pool of antigen-specific T cells in LCMV-infected mice lacking PD-L1 expression on BM-derived cells was significantly increased as compared with WT mice or mice lacking PD-L1 expression only on nonhematopoietic cells. Since PD-1 signals can influence effector T cell responses in tissues such as the liver (14), we wanted to ascertain whether PD-L1 expression on either the BM-derived or parenchymal cells controlled the clearance of virus in different organs. We measured viral titers in the blood, spleen, liver, and lungs. Viral titers in the blood and tissues of mice lacking PD-L1 on all cells (P-P) were reduced in comparison to those in normal (W-W) mice (Figure 4). Surprisingly, mice with PD-L1–deficient BM and PD-L1–sufficient parenchyma (P-W) did not display enhanced viral clearance in the blood and many tissues, despite increased numbers of functional CTL in these tissues (see Figures 1–3). These mice, however, did display improved viral clearance in the liver in comparison with W-W mice. In contrast, mice lacking PD-L1 expression on parenchymal cells (W-P) demonstrated significantly greater viral clearance in the blood and all tissues examined, similar to that observed in P-P mice. Thus, despite displaying virus-specific T cell numbers similar to that in W-W mice, W-P mice were able to clear virus more efficiently. These data demonstrate that PD-L1 on parenchymal cells significantly influenced viral clearance from the tissues during LCMV CL-13 infection.
PD-L1 expression on nonhematopoietic cells influences viral clearance. Viral titers in the blood (serum), spleen, liver, and lungs 8 days after LCMV CL-13 infection. n = 3–4 mice per group. Mean + SEM of data from 1 of 4 representative experiments are shown. *P ≤ 0.05; **P ≤ 0.005; ***P < 0.0001.
To further understand the biological significance of BM versus non-BM–derived PD-L1 signals, we compared the survival of groups of chimeric mice after CL-13 infection. Mice lacking PD-L1 on non-BM cells (P-P and W-P) demonstrated early mortality after infection (Figure 5A). These mice succumbed to infection after approximately 7–10 days. This correlated with enhanced viral clearance in these mice. Interestingly, mice lacking PD-L1 on hematopoietic cells (P-W) also succumbed to infection, although they were consistently found to survive a few days longer than P-P or W-P mice (11–13 days after infection). This correlated with reduced viral clearance in these mice (see Figure 4), potentially reflecting less severe immune damage to the tissues than in mice lacking PD-L1 on nonhematopoietic cells. Mice lacking PD-L1 expression also displayed higher levels of the cytokines IL-6 and TNF-α in the circulation compared with W-W mice (data not shown), which may have contributed to their demise. These cytokines were found to be slightly higher in the serum of mice lacking PD-L1 expression on parenchymal cells when compared with P-W mice. Together, these results suggest that the inhibitory PD-1/PD-L1 pathway may be critical for controlling virus-induced immunopathology.
PD-L1 expression on nonhematopoietic cells influences survival and immunopathology after infection. (A) Chimeric mice were infected with LCMV CL-13 and survival monitored daily. n = 7–10 mice; 1 representative experiment of 3 is shown. (B) Severe pathology in the BM compartment in chimeric mice lacking PD-L1 on nonhematopoietic cells. Representative photomicrographs of BM from CL-13–infected chimeric mice 8 days after infection. Pdl1–/– mice reconstituted with WT or Pdl1–/– BM (P-P or W-P) demonstrated significant necrosis of the BM compartment in contrast with W-W and P-W groups, which did not show necrosis. Original magnification, ×400.
To further examine how PD-L1 expression on hematopoietic or nonhematopoietic cells influenced immunopathology during LCMV CL-13 infection, we examined tissues from chimeric mice after infection. No significant histopathological changes were observed in the heart or kidney 8 days after LCMV CL-13 infection. Examination of the central nervous system also revealed no significant differences among groups, and there was no meningeal inflammation. Spleens from all groups of mice were highly activated, with expanded white-pulp regions and apoptotic cells. This was most striking in mice lacking PD-L1 on both hematopoietic and nonhematopoietic cells. By immunohistochemistry, there was secondary follicle formation in all groups and no significant differences in the numbers or distribution of T cells, B cells, and macrophages. There was a slight yet noticeable increase in apoptosis in the thymus of P-P and W-P mice compared with WT recipients.
The lungs of W-W chimeric mice displayed minimal histopathological changes, and this was only slightly increased in P-W mice after infection. However, P-P and W-P mice showed increased interstitial inflammation with some alveolar edema in the lungs of 25% of the W-P mice and 100% of P-P mice. The livers from all groups showed changes typically seen in viral hepatitis. There was scattered inflammation affecting both the portal areas and the hepatic lobules, as well as significant hepatocellular necrosis in all groups with dying hepatocytes identified singly and occasionally in small clusters scattered throughout the liver. The hepatocellular necrosis was greatest in chimeric mice lacking PD-L1 on nonhematopoietic cells (W-P). Immunohistochemical staining revealed that the inflammation in the portal areas and hepatic lobules consisted of both T and B cells. Measurement of liver enzymes (serum aspartate aminotransferase [AST]; and alanine aminotransferase [ALT]) demonstrated that acute hepatocyte damage was increased in mice lacking PD-L1 on BM cells (Supplemental Figure 3). In comparison, serum bilirubin levels were increased in mice lacking PD-L1 on non-BM cells. The similar viral control (Figure 4) yet differential immunopathology in the livers of W-P and P-W mice suggests that PD-L1 on both the BM- and non-BM–derived cells in the liver plays a significant role in disease during chronic viral infection.
BM from mice after CL-13 infection also showed pronounced changes. Both groups of mice with PD-L1–sufficient parenchyma (W-W and P-W) displayed hypercellularity in the BM compartment, with a predominance of myeloid cells consistent with a systemic inflammatory response (Figure 5B). In contrast, mice lacking expression of PD-L1 on all cells (P-P) showed extensive necrosis of the BM, and the accelerated mortality in P-P mice may relate, in part, to the extensive marrow necrosis seen in this group. Mice lacking PD-L1 only on parenchymal cells (W-P) also displayed considerable necrosis of hematopoietic elements in the marrow. However, no marrow necrosis was seen in any of the W-W or P-W groups, suggesting that PD-L1 expression on nonhematopoietic cells is protective of fulminant marrow failure in LCMV CL-13 infection. In conclusion, our data show that expression of PD-L1 on nonhematopoietic cells is critical for controlling viral clearance and immunopathological damage during LCMV CL-13 infection, while expression on BM-derived cells controls the priming of T cell responses.