Blockade of PD-1/PD-L1 promotes adoptive T-cell immunotherapy in a tolerogenic environment

Abstract

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Blockade of PD-1/PD-L1 impairs induction and partially reverses tolerance in naïve CD8+ T cells.

A) CD45.1+ OT-I T cells were transferred to 11c.OVA or C57BL/6 (non-Tg) mice treated with αPD-1, αPD-L1 or isotype control mAb at transfer and every subsequent three days. At the indicated time points after transfer spleens were collected and OT-I T cell number determined by a flow-cytometric counting assay. B-E): CD45.1+ OT-I T cells were transferred to C57BL/6 (non-Tg) or 11c.OVA mice and 21 days later mice were treated with αPD-1, αPD-L1 or isotype control mAb every three days. Six days after commencement of mAb treatment some mice were challenged with OVA/QuilA immunization and a further five days later mice were euthanized and tissues collected for analysis using a flow-cytometric counting assay and intracellular cytokine staining. (A) ***: for 11c.OVA recipients, αPD-L1 significantly greater than isotype control at day 3, 7, 14 (p<0.001), αPD-L1 significantly greater than αPD-1 at d7 (p<0.001) and d14 (p<0.05), αPD-1 greater than isotype at day 3 (p<0.001) and day 7, 14 (p<0.05). Data comprise: (A) four to seven mice for each time point (mean ± SEM) pooled from two or more experiments with 2–3 mice per group, (B-E) data pooled from 2 or 3 individual experiments with 1–2 mice per group (n = 4 for all groups) with values for individual mice shown.

Fig 2. Blockade of PD-1/PD-L1 impairs tolerance in memory CD8+ T cells.

A, B) In vitro generated CD45.1+ OT-I memory T cells were transferred to 11c.OVA or C57BL/6 (non-Tg) mice treated with αPD-1, αPD-L1 or isotype control mAb at transfer and every subsequent three days. At the indicated time points spleens were collected and OT-I T cell number (A) and total IFN-γ-producing OT-I number (B) determined by a flow-cytometric counting assay and intracellular cytokine staining. C, D) In vitro generated CD45.1+ OT-I Tcm-phenotype cells were transferred to 11c.OVA or C57BL/6 (non-Tg) mice treated with αPD-1, αPD-L1 or isotype control mAb at transfer and every subsequent 3 days and seven (C) or 14 (D) days later CTL activity determined in vivo. Data comprise: (A) 4–6 mice for each time point (mean ± SEM) pooled from two or more experiments of 2–3 mice per group, ** 11c.OVA + αPD-L1 significantly greater than 11c.OVA + isotype and 11c.OVA + αPD-1 at day 7 (p<0.01), (B) 4–6 mice for each time point (mean ± SEM) pooled from more than two experiments of 2 mice per group, * 11c.OVA + αPD-L1 significantly greater than 11c.OVA + isotype and 11c.OVA + αPD-1 at day 3 (p<0.05), ** 11c.OVA + αPD-L1 significantly greater than 11c.OVA + isotype and 11c.OVA + αPD-1 at day 7 (p<0.01), (C, D) data pooled from 2 individual experiments of 2 mice per group with values for individual mice shown.

Fig 3. Blockade of PD-1/PD-L1 partially reverses non-responsiveness of tolerised memory CD8+ T cells.

A, B) In vitro generated memory CD45.1+ OT-I T cells were transferred to 11c.OVA or C57BL/6 mice. Twenty eight days later mice were treated with αPD-1, αPD-L1 or isotype control mAb every subsequent three days. Nine days after commencement of mAb treatment some mice were challenged with OVA/QuilA immunization and a further five days later mice were euthanized and tissues collected for analysis using a flow-cytometric counting assay and intracellular cytokine staining. Data comprise: (A) four mice pooled from 4 experiments with n = 1 for each treatment set. Values for individual mice shown.

Fig 4. Anti-PD-1 and anti-PD-L1 synergises with Tcm-phenotype cell transfer to a tolerogenic environment.

A) B16.mOVA cells were analysed for expression of PD-1 and PD-1 ligands. Histogram plots show specific antibody (solid line) and isotype control (dashed line) stained cells. Data are from a single analysis representative of 3 separate analyses. B) B16.mOVA cells (105) were injected s.c. to 11c.OVA mice. Mice were left untreated (●) or 3 days later OT-I Tcm-phenotype cells (5 x 106) transferred i.v. OT-I recipients were injected on the day of OT-I transfer and every subsequent 3 days with isotype control (◯), αPD-1 (▲) or αPD-L1 (▽) mAb. C-E) B16.mOVA cells (105, C; 0.5 x 105, D; 0.25 x 105, E) were injected s.c. to C57BL/6 (C) or 11c.OVA mice (D, E). Mice were left untreated (●) or 3 days later OT-I Tcm-phenotype cells (5 x 106) transferred i.v. OT-I recipients were injected on the day of OT-I transfer and every subsequent 3 days with isotype control (◯), αPD-1 (▲) or αPD-L1 (▽) mAb. Data show survival curves (left) or cumulative mean tumour area ± SEM (right).

Fig 5. Anti-PD-L1 treatment increases tumour-specific CD8+ T cell infiltration and promotes effector function within tumours.

A-G) B16.mOVA cells (105) were injected s.c. to 11c.OVA mice and 3 days later OT-I Tcm-phenotype cells (5 x 106) transferred i.v. OT-I recipients were injected on the day of OT-I transfer and every subsequent 3 days with isotype control or αPD-L1 mAb as indicated. Twelve days after OT-I Tcm-phenotype cell transfer, spleen (A, E), tumour draining LN (TDLN) (B), and tumour (C, D, F, G) were harvested and OT-I (CD45.1+/CD8+/Vα2+) number and cytokine production determined using a flow-cytometric counting assay (A, B, C, D) and intracellular cytokine staining (E, F) or a combination of both. Data represent individual mice pooled from 2 experiments of 2–3 mice per group.

Fig 6. Anti-PD-L1 treatment reduces exhaustion marker expression on tumour-specific CD8+ T cells.

A-G) B16.mOVA cells (105) were injected s.c. to 11c.OVA mice and 3 days later OT-I Tcm-phenotype cells (5 x 106) transferred i.v. OT-I recipients were injected on the day of OT-I transfer and every subsequent 3 days with isotype control or αPD-L1 mAb as indicated. Twelve days after OT-I Tcm-phenotype cells transfer, spleen, tumour draining LN (TDLN), and tumour sites were harvested for flow-cytometric analysis. A) PD-L1 expression was determined in tumour, spleen and TDLN (A) gated on the total bulk population for isotype control mAb-injected (solid line) mice. Isotype-control staining of tumour is shown (dashed line). B, C) the proportion of DC expressing PD-L1 (B) and PD-L1 expression level on DC (C) was determined (gated on CD11c+I-Ab+ DC). CD62L and CD44 (D), PD-1 (E, F) and LAG-3 (G) expression was determined on OT-I T cells in spleen, TDLN and tumour. Data are representative of 3 mice/group in 2 separate experiments (A) or individual mice pooled from 2 separate experiments of 2–3 mice per group (B-G) except TDLN in (B, C) which is 3 mice from a single experiment of the 2 performed.