LRIG1 engages ligand VISTA and impairs tumor-specific CD8+ T cell responses

Abstract

Immune checkpoint blockade is a promising approach to activate antitumor immunity and improve the survival of patients with cancer. V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint target; however, the downstream signaling mechanisms are elusive. Here, we identify leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) as a VISTA binding partner, which acts as an inhibitory receptor by engaging VISTA and suppressing T cell receptor signaling pathways. Mice with T cell-specific LRIG1 deletion developed superior antitumor responses because of expansion of tumor-specific cytotoxic T lymphocytes (CTLs) with increased effector function and survival. Sustained tumor control was associated with a reduction of quiescent CTLs (TCF1⁺ CD62L^hi PD-1^low) and a reciprocal increase in progenitor and memory-like CTLs (TCF1⁺ PD-1⁺). In patients with melanoma, elevated LRIG1 expression on tumor-infiltrating CD8⁺ CTLs correlated with resistance to immunotherapies. These results delineate the role of LRIG1 as an inhibitory immune checkpoint receptor and propose a rationale for targeting the VISTA/LRIG1 axis for cancer immunotherapy.

Fig. 1.. The interactions of LRIG1 and VISTA and the expression of LRIG1 on activated T cells.

(A) Western blots of immunoprecipitated biotin-labeled proteins from WT and Vsir^−/− splenic T cells pre-activated with anti-CD3/CD28 antibodies and analyzed by SPPLAT. (B and C) SPR sensorgrams show the binding of VISTA.ECD or IgG-Fc control protein to LRIG1.ECD at neutral pH 7.4 (B) and acidic pH 6.0 (C). (D) Detection of VISTA/LRIG1 cis interactions in CD8 OVA-expressing T cells by bioluminescence emission. Quantification of luminescence intensity is shown. n = 4. Representative from two independent experiments is shown. (E) Detection of trans interactions between VISTA⁺ DCs or tumor cells and LRIG1⁺ T cells. n = 4. Representative of two independent experiments is shown. (F) LRIG1 expression on murine T cells. Splenic T cells were activated with anti-CD3 for 24 hours or cultured under T_H0, T_H1, T_H17, or T_H2 skewing conditions for 96 hours before analysis. (G) LRIG1 and VISTA expression on splenic CD8⁺ T cells that were stimulated with anti-CD3 alone or together with anti-CD28. (H) LRIG1 expression on unstimulated and anti-CD3–activated human peripheral CD4⁺ and CD8⁺ T cells from healthy donors. Results are shown as means ± SEM. All experiments were repeated at least two or three times, and the representative results are shown. Unpaired t test was used for (D) and (E). *P < 0.05, ***P < 0.001, and ****P < 0.0001.

Fig. 2.. Coexpression of LRIG1 and VISTA in T cells intrinsically inhibits multiple TCR signaling pathways.

(A) D10 cells expressing LRIG1 (L), VISTA (V), both (VL), or control vector (−) were stimulated with anti-CD3 for 0, 5, and 30 min. Phosphorylation of LAT, PLC-γ, SLP76, AKT⁴⁷³, and Erk1/2 in cell lysates was examined by Western blotting. The ratios of phosphorylated versus total protein levels were quantified and are shown. (B) D10 cells expressing WT LRIG1 alone (lane 1), WT LRIG1 with VISTA (lane 2), or the cytoplasmic domain truncated LRIG1 together with VISTA (lane 3) were stimulated with anti-CD3 and examined as in (A). (C and D) To examine the TCR signaling in splenic CD8⁺ T cells, WT (Lrig1^fl/fl), Lrig1^−/− CD8⁺ T cells (C), or Vsir^−/− CD8⁺ T cells (D) were stimulated with anti-CD3 and anti-CD28 for the indicated time. Total cell lysates were examined by Western blotting. All experiments were repeated at least three times, and the representative results are shown.

Fig. 3.. LRIG1 impairs T cell expansion, survival, and cytokine production and is required for the T cell inhibitory effects of VISTA.

(A to D) WT and Lrig1^−/− OT1 splenic T cells were stimulated with varying amounts of plate-bound anti-CD3/CD28 antibodies. (A) OT1 T cell numbers were enumerated after 96 hours. n = 4. (B) Secreted cytokines [(IFN-γ and interleukin-2 (IL-2)] at 48 hours were examined by ELISA. n = 4. (C) Apoptosis in OT1 T cells was examined by annexin V and PI staining. n = 5. (D) Expression of Bcl-xL and XIAP was examined by Western blotting and quantified using ratios over laminin B. (E) WT and Lrig1^−/− OT1 T cells were stimulated with splenic DCs and OVA257–264 peptides. OT1 cell expansion and IFN-γ production after 96 hours were examined. n = 4. (F) WT and Lrig1^−/− OT1 splenocytes were stimulated with OVA257–264 peptides for 48 hours. CD28 expression was examined, and the mean fluorescence intensity (MFI) was quantified. n = 3. (G) Graphic illustration describes the trans and cis interaction between VISTA and LRIG1. For simplicity, WT T cells were shown as expressing both VISTA and LRIG1. In TILs, the expression of VISTA and LRIG1 may be heterogeneous, and the trans or cis interactions may not occur simultaneously on the same T cells. (H) WT, Lrig1^−/−, and Vsir^−/− OT1 T cells were cocultured with VISTA-expressing (V^OE) or parental (V^neg) DC2.4 cells and peptides. OT1 T cell numbers were enumerated after 96 hours. n = 4. (I) Cell death in OT1 T cells was examined by staining with a viability dye at 96 hours as in (H). n = 5. (J) WT and Lrig1^−/− OT1 T cells were labeled with a CellTrace Violet dye and stimulated with DC2.4 cells and peptides as indicated. Cell division was analyzed after 72 hours. (K) Congenically marked WT (Thy1.1⁺) and Lrig1^−/− (CD45.1⁺) OT1 T cells were cotransferred at a 1:1 ratio (1000 cells each) into naïve mice (CD45.2⁺ Thy1.2⁺). Mice were inoculated with either MC38OVA control tumor (V^neg) or VISTA-expressing (V^OE) tumor cells (500,000) the next day. Tumor-infiltrating OT1 cells were analyzed by flow cytometry on day 9 and are shown as the percentage of host CD8⁺ TILs. n = 6 or 7. The cartoon describes the experimental design. Representative from at least two independent experiments is shown for all panels. Unpaired t test was used for (A) to (F). One-way ANOVA was used for (H), (I), and (K). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

Fig. 4.. LRIG1 expression is induced early on tumor-specific T cells and impairs T cell persistence by inhibiting cell expansion.

(A) Congenically marked WT OT1 cells were transferred into mice bearing B16OVA tumors. Tumor tissues and TDLNs were harvested on day 2 and day 6. The expression of LRIG1 and VISTA on OT1 T cells was examined by flow cytometry. n.d indicates “not detected.” Representative from two independent experiments is shown. (B) Co-adoptive transfer experimental design: Congenically marked WT (Thy1.1) and Lrig1^−/− (CD45.1) OT1 T cells were co-transferred at a 1:1 ratio (500 cells each) into mice (CD45.2). B16OVA tumor cells were inoculated the next day. Tumor tissues and TDLNs were analyzed at indicated time points. (C) Representative flow cytometry plots show the identification of WT versus Lrig1^−/− OT1 cells. (D and E) Percentages of OT1 cells among CD8⁺ T cells in tumor tissues and tumor-draining lymph node (LN) are shown. Representative from two independent experiments is shown. (F) Gene expression analysis of WT and Lrig1^−/− OT1 TILs isolated on day 7. Shown are the volcano plot of differentially expressed genes (P value cut-off of 0.01), the enrichment scores of up-regulated pathways, and a graphic view of GSEA leading edge analysis. (G) Genes involved in cell cycle regulation were up-regulated in Lrig1^−/− OT1 T cells. (H) Cell death, measured as percentage of cells stained positive by the viability dye, was reduced in Lrig1^−/− OT1 TILs on day 7. Representative of two independent experiments is shown. (I) Levels of phosphorylated AKT⁴⁷³ and AKT³⁰⁸ in OT1 TILs on day 7. Unpaired t test was used for (D), (E), and (H). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NES, normalized enrichment score.

Fig. 5.. T cell–specific deletion of LRIG1 boosts antitumor T cell responses and long-term tumor control in combination with therapeutic vaccine treatment.

(A to E) Antitumor T cell responses in the B16OVA model: Lrig1^−/− mice and WT littermates (Lrig1^fl/fl) were inoculated with B16OVA tumor cells (100,000). Tumor growth was monitored by a caliper. Tumor tissues from separate cohorts of mice were examined by flow cytometry. (A) Tumor growth curve. n = 9 (Lrig1^fl/fl) and 12 (Lrig1^−/−). (B) Viability of CD8⁺ TILs. n = 6. (C) Ki67 expression. n = 6. (D) IFN-γ expression in CD8⁺ TILs. n = 6. (E) MFI of CD28 expression on CD8⁺ TILs. n = 6. (F to I) Tumor growth in the B16bl6 melanoma model. Lrig1^−/− mice and WT littermates (Lrig1^fl/fl) were inoculated with B16bl6 melanoma cells. On day +3, mice were treated with peptide vaccine (F) or vaccine together with anti–CTLA-4 antibodies (I). Tumor growth curves (F and G) and tumor-free survival after combined therapy (H) were shown. (I) Survived Lrig1^−/− mice were rechallenged with B16bl6 tumor cells and monitored for secondary tumor growth. Naïve Lrig1^−/− mice were analyzed as parallel control. n = 15 (naïve) and 17 (surviving mice). (J to Q) Antitumor T cell responses. Tumor tissues were harvested on day 18 after vaccine treatment, and CD8⁺ CTLs were examined by flow cytometry. (J) Percentages of CD8⁺ CTLs among CD45⁺ immune infiltrates and the numbers of CD8⁺ CTLs normalized to total tumor cells. n = 13 (Lrig1^fl/fl) and n = 9 (Lrig1^−/−). (K) Ratio of CD8⁺ CTLs to Foxp3⁺ T_regs. n = 9 (Lrig1^fl/fl) and n = 9 (Lrig1^−/−). (L) Cell death in PD-1 ⁺CD8⁺ CTLs. n = 13 (Lrig1^fl/fl) and n = 9 (Lrig1^−/−). (M) Ki67 expression in progenitor/memory-like (TCF1⁺ TIM3⁻) and exhausted (TCF1⁻ TIM3⁺) CTLs. n = 11 (Lrig1^fl/fl) and n = 10 (Lrig1^−/−). (N) Proportion of TIM3⁺ exhausted CTLs among PD1⁺CD8⁺ TILs. n = 6 (Lrig1^fl/fl) and n = 8 (Lrig1^−/−). (O) Expression of IFN-γ, TNF-α, and CD107a in CD8⁺ TILs after ex vivo stimulation with TRP2 peptides. n = 8 (Lrig1^fl/fl) and n =7 (Lrig1^−/−). (P) Enumeration of tumor-specific CD8⁺ CTLs that were identified by TRP2/MHC dextramer staining, compared with fluorescence minus one (FMO) control. n = 5 (Lrig1^fl/fl) and n = 5 (Lrig1^−/−). (Q) CD28 expression on TRP2-specific CD8⁺ CTLs. n = 7 (Lrig1^fl/fl) and n = 6 (Lrig1^−/−). All experiments were repeated at least two times. Unpaired t test was used for (A) to (E) and (J) to (Q). Mann-Whitney test was used for (F) and (G). Log-rank test was used for (H). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.. Loss of LRIG1 reduces the quiescence of tumor-specific CD8⁺ CTLs and increases the abundance of progenitor/memory-like subsets.

CD3⁺ TILs were sorted from tumor tissues (pooled from 10 mice for each genotype) on day 18 after vaccine treatment and analyzed by scRNA-seq. (A) Uniform Manifold Approximation and Projection (UMAP) clusters of CD8⁺ TILs. (B) Expression of marker genes enriched in naïve-like and progenitor/memory-like T cells is illustrated in UMAP space. (C) Expression of genes Tcf7, Sell, and Pdcd1 distinguishes the quiescent C1 cluster from progenitor/memory-like clusters (i.e., C0, C2, C8, and C9). (D) Percentages of quiescent naïve-like C1 cluster versus progenitor/memory-like cells (combined clusters C0/C2/C8/C9) among total CD8⁺ TILs. (E to G) Flow cytometry analyses to quantify the subsets of CD8⁺ TILs. Shown are the percentages and numbers of TCF1⁺CD8⁺ TILs (n = 9) (E), percentages of quiescent (TCF1⁺ CD62L^hi PD-1⁻) cells among TCF1⁺ CD8⁺ TILs or total CD8⁺ TILs (n = 10 and 9) (F), and percentages of quiescent cells (CD62L^hi PD-1⁻) among TRP2/MHC dextramer-specific CTLs (n = 8 and 6) (G). (H to K) Studies of adoptively transferred PMEL T cells. Pre-activated WT (Thy1.1⁺ Thy1.2⁻) and Lrig1^−/− (Thy1.1⁺ Thy1.2⁺) PMEL T cells were mixed at a 1:1 ratio (200,000 each) and adoptively transferred into mice (Thy1.1⁻Thy1.2⁺) bearing established B16bl6 tumors. Tumor tissues were harvested on day 7 after transfer and examined by flow cytometry. (H) Total TCF1 expression. n = 9. (I) Representative plots show the quiescent (TCF1⁺ CD62L^hi PD-1^neg) and Tpex (TCF1⁺ PD-1⁺) subsets among TCF1⁺ PMEL cells. Percentages of quiescent and Tpex subsets were shown in (J) and (K), respectively. n = 9. (L) Polyclonal quiescent and Tpex subsets of CD8⁺ TILs were sorted from B16bl6 tumor tissues and restimulated ex vivo by anti-CD3/CD28 for 16 hours. IFN-γ expression was examined by flow cytometry. The Ifng gene expression from CD8⁺ TILs is shown as a comparison. All experiments were repeated at least two times. Unpaired t test used for (E) to (H) and (J) and (K). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 7.. Expression of LRIG1 in human melanoma tumor infiltrating CD8⁺ T cells is associated with resistance to immunotherapy.

(A) LRIG1 and VISTA expression in human melanoma infiltrating CD8⁺ TILs. Single-cell suspensions from melanoma tissues were stimulated with anti-CD3 and CD28 antibodies for 24 hours. LRIG1 and VISTA expression on subsets of CD8⁺ TILs was analyzed by flow cytometry and shown. (B) LRIG1 and VISTA expression on TILs from human endometrial cancer and lung cancer specimens following stimulation with anti-CD3/CD28. (C to F) Analysis of a scRNA-seq dataset from studies by Sade-Feldman et al. (6). CD8⁺ TILs were clustered, visualized with UMAP, and shown in (C). Clusters were named according to the original author’s definition. Expression of LRIG1 and VSIR genes were visualized and shown in (D). Percentages of LRIG1⁺ cells in each CTL cluster in post-ICI tissues were shown in (E). Percentages of CD8⁺ TILs that express LRIG1, TCF1, VSIR (VISTA), and TOX genes were enumerated in pre-ICI and post-ICI biopsies and shown in (F). Games-Howell’s multiple comparison statistical analysis test was used. *P < 0.05 and **P < 0.01. R, responder; NR, nonresponder.