Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade

Abstract

Programmed death-1 (PD-1) regulates T cell exhaustion during chronic infections. Blocking the PD-1:PD-ligand (PD-L) pathway reinvigorates exhausted CD8 T cells. Exactly how blocking PD-1:PD-L interactions improves T cell immunity, however, remains unclear. PD-1:PD-L blockade could reprogram all exhausted T cells to become antiviral effectors. Alternatively, this blockade might selectively expand a subset of exhausted T cells. We have identified two subpopulations of exhausted CD8 T cells during chronic viral infection in mice. One subset of exhausted CD8 T cells is rescued by alphaPD-L1 blockade, whereas the other subset appears more terminally differentiated and responds poorly to PD-1:PD-L blockade. Blocking PD-1:PD-L interactions reduces spontaneous apoptosis and enhances expansion and protective immunity of the rescuable subset, but not the more terminally differentiated subset of exhausted CD8 T cells. These results have implications for predicting clinical responses to PD-1-based therapeutic interventions and for understanding T cell dynamics during persisting infections.

Fig. 1.

Identification of subsets of exhausted CD8 T cells. (A) CD8⁺ CD44⁺ T cells from Arm vs. clone 13-infected mice (day 30 after infection) were stained with either the J43 clone or the RPM1–30 clone of αPD-1. (B) Splenocytes from either LCMV Arm or clone 13-infected mice (day 30 after infection) were stained with αPD-1 clone RPM1–30 and gated on CD8⁺ T cells. PD-1^Hi (red) and PD-1^Int (blue) DbGP33⁺ CD8⁺ T cells were overlaid on total CD8 T cells (black; Right) from the same mice to assess overlap of these populations and to illustrate differences in CD44 expression. (C) Lymphocytes from SP or BM of clone 13-infected mice (day 90 after infection) were stained with αPD-1 clone RPM1–30. Plots are gated on CD8⁺ T cells. DbGP33⁺ CD8⁺ T cells from the SP or BM. DbGP33⁺ CD8⁺ T cells from SP (blue) or BM (red) were overlaid total CD8⁺ splenocytes (black; Right).

Fig. 2.

In vivo PD-L1 blockade enhanced expansion of splenic PD-1^Int, but not the PD-1^Hi subset of exhausted CD8 T cells. (A) PD-1^Hi and PD-1^Int splenocytes from clone 13-infected (day 30 after infection) Ly5.2⁺ mice were column-purified. Each fraction yielded >90% purity for PD-1^Hi and PD-1^Int. Note that the PD-1^Int fraction contained a small percentage of PD-1^Lo cells, but these cells expressed higher PD-1 levels compared with memory CD8 T cells. DbGP33⁺ CD8⁺ T cells (6 × 10⁴) were adoptively transferred to Ly5.1 naïve recipient mice followed by rechallenge with LCMV clone 13 with or without αPD-L1 antibody treatment (200 μg i.p. on days 0, 3, and 6 after infection). As a control, LCMV-specific memory CD8 T cells from Ly5.2⁺ LCMV Arm immune mice were transferred to Ly5.1⁺ naïve recipients and rechallenged. (B–D) On day 7.5 after rechallenge, recipient mice were killed, and total donor Ly5.2⁺ or DbGP33⁺ Ly5.2⁺ CD8⁺ T cells were identified by flow cytometry (n = 3–5 mice per group, data are representative of three similar experiments).

Fig. 3.

In vivo PD-L1 blockade enhanced expansion of tissue-defined PD-1^Int (SP-derived), but not the PD-1^Hi (BM-derived) subset of exhausted CD8 T cells. (A) PD-1^Hi CD8⁺ T cells were derived from the BM and PD-1^Int CD8⁺ T cells from the SP of clone 13-infected (day 90 after infection) Ly5.2⁺ mice. CD8 T cells from BM and SP were column-purified, CD8⁺ T cell populations were normalized to contain 6 × 10⁴ DbGP33⁺ CD8⁺T cells, and cells were adoptively transferred to Ly5.1 naïve recipient mice followed by rechallenge with LMGP33 (3 × 10⁴ cfu) with or without αPD-L1 antibody treatment (200 μg i.p. on days 0, 3, and 6 after infection). (B) Ly5.2⁺ DbGP33⁺ CD8⁺ T cells were monitored in the blood of recipient mice after LMGP33 rechallenge (n = 3–5 mice per group, data are representative of five experiments). (C) Total donor DbGP33⁺ Ly5.2⁺ CD8⁺ T cell number was assessed in recipient SP and BM on day 18 after challenge.

Fig. 4.

Protective immunity by subsets of exhausted CD8 T cells in the absence or presence PD-L1 blockade. (A) PD-1^Hi and PD-1^Int splenocytes were purified as in Fig. 2, and 1 × 10⁶ LCMV-specific CD8⁺ T cells [normalized based on responsiveness via ICS to a pool of 20 known LCMV CD8 T cell epitopes described by Kotturi et al. (24)] were transferred to Ly5.1 naïve recipient mice followed by rechallenge with LCMV clone 13 (2 × 10⁶pfu i.v.) with or without αPD-L1 antibody treatment as in Fig. 2. As a control, Ly5.1⁺ mice that received no donors cells were also infected. Mice were killed on day 3 after infection. and LCMV quantified in the SP. (B) Adoptive transfers were performed as described in Fig. 3. Recipients were then rechallenged with LMGP33 (1.5 × 10⁴ cfu i.v.) with or without αPD-L1 antibody (200 μg i.p. on days 0 and 3 after infection). Mice were killed on day 4 after infection, and LMGP33 was quantified in the SP.

Fig. 5.

Differential inhibition of apoptosis of exhausted CD8 T cell subsets by PD-1:PD-L blockade. (A) Splenocytes from clone 13-infected mice (day 30 after infection) were isolated and stained with 7-AAD and annexin V directly ex vivo. (B) ACasp3 was used as an indicator of apoptosis in live cells. Dead cells were excluded by using an amine-reactive dye (vivid red), and ACasp3 was detected by intracellular staining. (C) Splenocytes from clone 13-infected mice were incubated at 37°C for 5 h in medium. ACasp3 was detected in live DbGP33⁺ CD8 T cells. (D and E) PD-1^Hi, PD-1^Int, and PD-1^Lo exhausted CD8 T cells subsets were sorted by flow cytometry and incubated at 37°C for 5 h with or without αPD-L1 (10 μg/ml), and ACasp3 was detected in the live cell subset by flow cytometry (data are representative of three independent experiments). Note that PD-1 expression did not change in 5 h in vitro (data not shown). Similar results were observed in the presence of added peptide (data not shown).