Resistance to PD1 blockade in the absence of metalloprotease-mediated LAG3 shedding

Abstract

Mechanisms of resistance to cancer immunotherapy remain poorly understood. Lymphocyte activation gene-3 (LAG3) signaling is regulated by a disintegrin and metalloprotease domain-containing protein-10 (ADAM10)- and ADAM17-mediated cell surface shedding. Here, we show that mice expressing a metalloprotease-resistant, noncleavable LAG3 mutant (LAG3^NC) are resistant to PD1 blockade and fail to mount an effective antitumor immune response. Expression of LAG3^NC intrinsically perturbs CD4⁺ T conventional cells (T_convs), limiting their capacity to provide CD8⁺ T cell help. Furthermore, the translational relevance for these observations is highlighted with an inverse correlation between high LAG3 and low ADAM10 expression on CD4⁺ T_convs in the peripheral blood of patients with head and neck squamous cell carcinoma, which corresponded with poor prognosis. This correlation was also observed in a cohort of patients with skin cancers and was associated with increased disease progression after standard-of-care immunotherapy. These data suggest that subtle changes in LAG3 inhibitory receptor signaling can act as a resistance mechanism with a substantive effect on patient responsiveness to immunotherapy.

Figure 1:. LAG3^NC restricts effective anti-tumor immune responses in vivo.

(A) Schematic of LAG3^NC conditional knock-in mouse. (B) LAG3 and Ametrine expression on CD4⁺ Foxp3⁻, CD4⁺ Foxp3⁺ and CD8⁺ TIL isolated from Lag3^NC.L/L and Lag3^NC.L/LCD4^Cre mice that received 5X10⁵ MC38 adenocarcinoma cells subcutaneously. (C) Individual tumor growth curves and (D) survival plot of Lag3^NC.L/L and Lag3^NC.L/LCD4^Cre mice receiving 5×10⁵ MC38 adenocarcinoma cells subcutaneously and anti-PD1 or IgG (200μg) on d6, d9 and d12 by intraperitoneal injection. (E) Kaplan-Meir curve showing tumor-free animals following secondary MC38 injection (2.5X10⁵ cells subcutaneous) of Lag3^NC.L/L and Lag3^NC.L/LCD4^Cre mice following primary MC38 injection (5X10⁵ cells subcutaneous) and resection (d12), or sham control animals. (F) Mean tumor growth curves (left) and survival plot (right) of Lag3^NC.L/L and Lag3^NC.L/LCD4^Cre mice receiving 1.25X10⁵ B16-gp100 melanoma cells intradermally and immunized with Amph-gp100 or Amph-E7 vaccine subcutaneously on d4 and d11 (20μg) with anti-PD1 or IgG as in (C). Results represent the mean of three (B-D) or two (E and F) independent experiments. *p<0.05, **p<0.01, ***p<0.001, n.s. not significant by (D-F) Log-Rank (Mantel-Cox) and (F) two-way ANOVA. Error bars represent the mean ± s.e.m.

Figure 2:. Single-cell RNAseq analysis of LAG3^NC-expressing TIL.

Single-cell RNAseq analysis of T cells isolated from tumors pooled from Lag3^NC.L/L and Lag3^NC.L/LCD4^Cre mice at d14 injected with 5X10⁵ MC38 adenocarcinoma cells subcutaneously and anti-PD1 or IgG (200μg) on d6, d9 and d12 by intraperitoneal injection. (A) FltSNE visualization and DRAGON clustering of all single cells identified CD4⁺ T_reg (red), CD4⁺ T_conv (blue) and CD8⁺ T cells (green). (B) Quantification of differences by Bhattacharyya distance (BD) between CD4⁺ T_conv, CD4⁺ T_reg and CD8⁺ T cells in Lag3^NC.L/L and Lag3^NC.L/LCD4^Cre mice, receiving anti-PD1 or IgG. (C) Clustering of CD4⁺ T_conv by DRAGON revealed a total of seven clusters across all samples. (D) Scaled sample enrichment in clusters identified in (C). (E) Gene set enrichment analysis revealed signature genes associated with each cluster identified in (C).

Figure 3:. LAG3^NC intrinsically impacts CD4⁺ T cell functionality.

(A) Individual tumor growth curves and (B) survival plot of Lag3^NC.L/L and Lag3^NC.L/LThPOK^CreERT2 mice receiving 5X10⁵ MC38 adenocarcinoma cells subcutaneously and anti-PD1 or IgG (200μg) on d6, d9 and d12 by intraperitoneal injection, as well as five consecutive intraperitoneal injections of tamoxifen (1mg in 5% EtOH/sunflower oil) from d0 to d4. TIL was harvested at d14 from Lag3^NC.L/L or Lag3^NC.L/LCD4^Cre mice injected with 5X10⁵ MC38 adenocarcinoma cells subcutaneously receiving anti-PD1 or IgG (200μg) on d6, d9 and d12 by intraperitoneal injection. (C) IFNγ and TNFα, as well as (D) IL-2 from CD4⁺ Foxp3⁻ TIL was measured following re-stimulation with phorbol myristate acetate (PMA) and ionomycin for 4 hours in the presence of brefeldin A. Mice that received anti-PD1 were stratified into non-responders (N) and responders (R) to treatment. (E) BrdU and Ki67 staining was assessed in CD4⁺ Foxp3⁻ TIL, by intraperitoneal injection of BrdU 12 hours before harvest. (F) Mean clinical scores following EAE induction in Lag3^NC.L/L, Lag3^NC.L/LCD4^Cre and Lag3^NC.L/LE8I^Cre.GFP mice. (G) Lymphocytes were isolated from the brain at d14 post immunization and stimulated with MOG_35–55 peptide for 20 hours and the last 4 hours with Brefeldin A. IL17A, IFNγ and GMCSF was assessed from CD4⁺ Foxp3⁻ T cells. (H) Ki67 was assessed in CD4⁺ Foxp3⁻, CD4⁺ Foxp3⁺ and CD8⁺ T cells isolated in (G). Results represent the mean of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. not significant by (B) Log-Rank (Mantel-Cox), (C-E) Mann Whitney Test, (F) two-way ANOVA and (G and H) unpaired t-test. Error bars represent the mean ± s.e.m.

Figure 4:. LAG3^NC extrinsically restricts CD8⁺ T cell functionality.

(A) Individual tumor growth curves and (B) survival plot of Lag3^NC.L/L and Lag3^NC.L/LE8I^Cre.GFP mice receiving 5X10⁵ MC38 adenocarcinoma cells subcutaneously and anti-PD1 or IgG on d6, d9 and d12 (200μg) by intraperitoneal injection. (C) TIL was harvested at d14 from Lag3^NC.L/L, Lag3^NC.L/LCD4^Cre or Lag3^NC.L/LE8I^Cre.GFP mice injected with 5X10⁵ MC38 adenocarcinoma cells subcutaneously receiving anti-PD1 or IgG (200μg) on d6, d9 and d12 by intraperitoneal injection. IFNγ and TNFα from CD8⁺ TIL was measured following re-stimulation with phorbol myristate acetate (PMA) and ionomycin for 4 hours in the presence of brefeldin A. Mice that received anti-PD1 were stratified into non-responders (N) and responders (R) to treatment. (D) Lag3^NC.L/L or Lag3^NC.L/LCD4^Cre mice (Thy1.2⁺) received an adoptive transfer (AT) of 1×10⁵ pmel (Thy1.1⁺) cells the day before inoculation with 1.25X10⁵ B16-gp100 melanoma cells intradermally. Mice received anti-PD1 (200μg) intraperitoneally on d6, d9 and d12 and tumor volume was measured when sacrificed on d15 post inoculation. (E) IFNγ was measured on both Thy1.1⁺ (pmel) and Thy1.2⁺ (endogenous) CD8⁺ T cells from (D) following re-stimulation as in (C). Results represent the mean of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. not significant by (B) Log-Rank (Mantel-Cox) and (C-E) Mann Whitney Test.

Figure 5:. Low LAG3 and reciprocal high ADAM10 expression on conventional CD4⁺ T cells is indicative of patient survival and responsiveness to PD1 blockade.

(A) Lymphocytes were isolated from peripheral blood of advanced metastatic melanoma patients prior (pre) or following (post) treatment with standard of care anti-PD1+/−anti-CTLA4 (n=37; Cohort B [Table 3]). The change in LAG3 and ADAM10 expression was assessed for CD4⁺Foxp3⁻ T cells and patients were stratified by responsiveness to treatment. (B) Paired analysis of LAG3 and ADAM10 expression on CD4⁺Foxp3⁻ T cells isolated from patients in (A). (C) LAG3:ADAM10 ratio for CD4⁺Foxp3⁻ T cells of patients in (A). (D) Lymphocytes were isolated from peripheral blood of HNSCC patients (n=50; Cohort D [Table 5]) and LAG3 expression on CD4⁺Foxp3⁻, CD4⁺Foxp3⁺ and CD8⁺ T cells was assessed by stage of disease. (E) Paired analysis of LAG3 and ADAM10 expression on CD4⁺Foxp3⁻ T cells. (F) Kaplan-Meier survival curve of advanced disease stage HNSCC patients (n=25) with high LAG3:ADAM10 ratio (±0.3865) or low LAG3:ADAM10 ratio (<0.3865) expressed on CD4⁺Foxp3⁻ T cells. *p<0.05, **p<0.01, ****p<0.0001, n.s. not significant by (A and D) unpaired t-test, (C) Wilcoxon Test and (F) Log-Rank (Mantel-Cox).