Cancer Cell-Intrinsic Expression of MHC Class II Regulates the Immune Microenvironment and Response to Anti-PD-1 Therapy in Lung Adenocarcinoma

Abstract

MHC class II (MHCII) expression is usually restricted to APC but can be expressed by cancer cells. We examined the effect of cancer cell-specific MHCII (csMHCII) expression in lung adenocarcinoma on T cell recruitment to tumors and response to anti-PD-1 therapy using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis lung carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, whereas LLC tumors are resistant. RNA sequencing analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, whereas resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss- and gain-of-function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII and converted tumors from anti-PD-1 sensitive to anti-PD-1 resistant. This was associated with lower levels of Th1 cytokines, decreased T cell infiltration, increased B cell numbers, and decreased macrophage recruitment. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, which provided a survival benefit and positively correlated with T cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T cell infiltration and sensitivity to anti-PD-1.

FIGURE 1.

NSCLC cells differentially express an Ag presentation signature. (A) Heatmap of RNA-seq of MHC-associated genes from the Ag processing presentation signature using KEGG pathway analysis of LLC or CMT167 cells derived from in vitro or in vivo conditions. (B) The percentage of MHCII⁺ staining measured by flow cytometry gated on singlets of live cells of CMT167 or LLC cells cultured in vitro and/or CMT167 or LLC tumor cells from C57BL/6 GFP⁺ mice gated on singlets of live cells of GFP⁻ in vivo (n = 5, one-way ANOVA). (C) Ciita mRNA expression in LLC and CMT167 cells measured by qRT-PCR (n = 3, unpaired t test). (D) Percentage of MHCII⁺ expression measured in LLC and CMT167 cells measured by flow cytometry gated on singlets of live cells (n = 3, unpaired t test). (E) Ciita mRNA expression in CMT167 cells expressing an NT-ctrl shRNA or a shCIITA untreated or treated with IFN-γ for 48 h measured by qRT-PCR (n = 3, one-way ANOVA). (F) The percentage of MHCII⁺ staining from live, singlet cells of CMT167 cells expressing an NT-ctrl shRNA or a shCIITA untreated or treated with IFN-γ for 48 h measured by flow cytometry (n = 3, unpaired t test). (G) Western blot of CIITA, p-STAT1, and total STAT1 in cells cultured in the presence or absence of IFN-γ. (H) Flow cytometric analysis of percentage of MCHII⁺ events focused on live, singlet cells that were GFP⁻ (n = 3, unpaired t test). *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 2.

Loss of MHCII decreases CD4⁺ and CD8⁺ tumor infiltration and decreases T cell effector function. (A) Average count of immunofluorescence of CD3⁺, CD4⁺, and CD8⁺ T cells per HPF (original magnification ×40) in CMT167–NT-ctrl tumors and CMT167-shCIITA tumors (n = 6, n = 5, respectively; unpaired t test). (B) Representative pictures of CMT167–NT-ctrl and CMT167-shCIITA tumors immunofluorescence: DAPI (blue), CD3 (red), and CD4 or CD8 (green). qRT-PCR analysis of CMT167–NT-ctrl and CMT167-shCIITA of GFP⁺ cells of (C) Cxcl9, (D) Cxcl10, and (E) Ifnγ (n = 5, unpaired t test). qRT-PCR analysis of CMT167–NT-ctrl and CMT167-shCIITA of GFP⁻ cells of (F) Cxcl9, (G) Cxcl10, and (H) Ifnγ (n = 3, unpaired t test). Flow cytometry on tumor-bearing lung events gated on singlets that are live, CD45⁺, and MHCII⁻ that were either CD4⁺ or CD8⁺ (I) IFN-γ⁺ CD4⁺ T cells and (J) TNF-α⁺ CD4⁺ T cells (n = 3, unpaired t test). Percentage of positive (K) IFN-γ⁺CD8⁺ T cells in CMT167–NT-ctrl and CMT167-shCIITA tumors (n = 3, unpaired t test). *p < 0.05, **p < 0.01.

FIGURE 3.

NSCLC cells expressing MHCII directly activate CD4⁺ T cells in vitro. Flow cytometric analysis of CD4⁺ T cells isolated from a CMT167 tumor-bearing lung and cocultured for 24 h with CMT167–NT-ctrl or CMT167-shCIITA, defined as singlets that are live, CD45⁺, or CD4⁺. (A) Percentage of IFN-γ⁺ CD4⁺ T cells, (B) percentage of TNF-γ⁺ CD4⁺ T cells, (C) percentage of IL-10⁺ CD4⁺ T cells, (D) percentage of Foxp3⁺ CD4⁺ T cells, and (E) percentage of CD69⁺ CD4⁺ T cells (n ≥ 3, one-way ANOVA). ***p < 0.001, **p < 0.01.

FIGURE 4.

Effects of silencing csMHCII on the TME. Flow cytometric analysis of percentage of PD-L1 of (A) GFP⁻ and (B) GFP⁺ cells from CMT167–NT-ctrl and CMT167-shCIITA tumors injected into GFP⁺ mice and FACs sorted into GFP⁻ and GFP⁺ populations (n = 3, unpaired t test). (C) t-Distributed stochastic neighbor embedding (tsne) plot of unsupervised clustering analysis of spectral flow cytometry–based analysis of CMT167–NT-ctrl and CMT167-shCIITA. Biaxial-based analysis of spectral flow cytometry–based analysis of singlets that are live to identify (D) B cells (percentage of CD45⁺, CD19⁺), (E) percentage of B cells (percentage of CD45⁺, CD19⁺) that are MHCII⁺, (F) percentage of B cells (percentage of CD45⁺, CD19⁺) that are PD-L1⁺, (G) alveolar macrophages (CD45⁺, SiglecF⁺, Ly6G⁻, CD11c⁺), (H) recruited macrophages (CD45⁺, SiglecF⁻, Ly6G⁻, CD11b⁺), (I) percentage of recruited macrophages (CD45⁺, SiglecF⁻, Ly6G⁻, CD11b⁺) that are MHCII⁺, (J) percentage of recruited macrophages (CD45⁺, SiglecF⁻, Ly6G⁻, CD11b⁺) that are PD-L1⁺, (K) dendritic cells (CD45⁺, MHCII⁺, SiglecF⁻, Ly6G⁻, CD11c⁺), (L) percentage of dendritic cells (CD45⁺, MHCII⁺, SiglecF⁻, Ly6G⁻, CD11c⁺) that are MHCII⁺, (M) percentage of dendritic cells (CD45⁺, MHCII⁺, SiglecF⁻, Ly6G², CD11c+) that are PD-L1+, and (N) neutrophils (CD45+, SiglecF², Ly6G+) (n = 3, unpaired t test). *p < 0.05.

FIGURE 5.

Overexpression of CIITA in LLC cells induces MHCII and increases CD4⁺ and CD8⁺ tumor infiltration. (A) Fold change of Ciita mRNA expression for LLC–EV-ctrl and LLC-CIITAOE (n = 3, unpaired t test). (B) Flow cytometry analysis of MHCII on LLC–EV-ctrl and LLC-CIITAOE cells gated on live singlets (n = 3, unpaired t test). (C) Average count of immunofluorescence of CD3⁺, CD4⁺, and CD8⁺ T cells per HPF (original magnification ×40) in LLC–EV-ctrl tumors and LLC-CIITAOE tumors (n = 3, n = 4, respectively; unpaired t test). (D) Representative pictures of LLC–EV-ctrl and LLC-CIITAOE tumors immunofluorescence: DAPI (blue), CD3 (red), and CD4 or CD8 (green). *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 6.

MHCII mediates the tumor progression and response to checkpoints. (A) Primary tumor volume of CMT167–NT-ctrl or CMT167-shCIITA tumors at 4 wk (n ≥ 17, unpaired t test). (B) Primary tumor volume of CMT167–NT-ctrl or CMT167-shCIITA 4.5 wk postimplantation treated with IgG Ab or PD-1 Ab (n ≥ 11, two-way ANOVA). (C) Primary tumor volume of LLC–EV-ctrl or LLC-CIITAOE treated with IgG or PD-1 Ab at 3 wk (n = 5, two-way ANOVA). *p < 0.05, **p < 0.01.

FIGURE 7.

Adenocarcinoma lung cancer expresses HLA-DR (MHCII) and is correlated with enhanced CD3⁺, CD4⁺, and CD8⁺ tumor infiltrating lymphocytes. (A) Overall 5-y survival curve stratified by HLA-DR–low (<10%) and HLA-DR–high (≥10%) patients (n = 90, log-rank (Mantel–Cox) test). (B) The correlation of the percentage of the number of counts of HLA-DR⁺ on cancer cells and the percentage of the number of counts of CD8 (r² = 0.0069, p = 0.439) and CD4 (CD3⁺, CD8⁻) (r² = 0.1227) in the tumor. (C) Percentage of CD4⁺ and (D) CD8⁺ T cell infiltration in the tumor in patients stratified by HLA-DR low (<10%) and HLA-DR high (≥10%) (n = 90, unpaired t test). Representative images of the composite image after spectral unmixing. DAPI nuclear marker (pseudocolored blue), CK (membrane, Cy3, pseudocolored magenta), HLA-DR (membrane, Cy5, pseudocolored yellow), CD3 (membrane, FITC, pseudocolored green), and CD8 (membrane, Texas Red, pseudocolored red) of (E) HLA-DR–low tumor of patient 28 and (F) HLA-DR–high tumor of patient 3. *p < 0.05, **p < 0.001, ***p < 0.0001.