Purpose: To determine the expression level, associations, and biological role of PD-L1, IDO-1, and B7-H4 in non–small cell lung cancer (NSCLC).

Experimental Design: Using multiplexed quantitative immunofluorescence (QIF), we measured the levels of PD-L1, IDO-1, B7-H4, and different tumor-infiltrating lymphoycte (TIL) subsets in 552 stages I–IV lung carcinomas from two independent populations. Associations between the marker levels, TILs, and major clinicopathologic variables were determined. Validation of findings was performed using mRNA expression data from The Cancer Genome Atlas (TCGA) and in vitro stimulation of lung adenocarcinoma A549 cells with IFNγ and IL10.

Results: PD-L1 was detected in 16.9% and 21.8% of cases in each population. IDO-1 was expressed in 42.6% and 49.8%; and B7-H4 in 12.8% and 22.6% of cases, respectively. Elevated PD-L1 and IDO-1 were consistently associated with prominent B- and T-cell infiltrates, but B7-H4 was not. Coexpression of the three protein markers was infrequent, and comparable results were seen in the lung cancer TCGA dataset. Levels of PD-L1 and IDO-1 (but not B7-H4) were increased by IFNγ stimulation in A549 cells. Treatment with IL10 upregulated B7-H4 but did not affect PD-L1 and IDO-1 levels.

Conclusions: PD-L1, IDO-1, and B7-H4 are differentially expressed in human lung carcinomas and show limited co-expression. While PD-L1 and IDO-1 are associated with increased TILs and IFNγ stimulation, B7-H4 is not. The preferential expression of discrete immune evasion pathways in lung cancer could participate in therapeutic resistance and support design of optimal clinical trials. Clin Cancer Res; 23(2); 370–8. ©2016 AACR.

This article is featured in Highlights of This Issue, p. 325

Translational Relevance

Blockade of the PD-1 axis reinvigorates the antitumor immune response and induces clinical benefit in nearly 20% of patients with advanced lung cancer. The presence of PD-L1 (or B7-H1) in the tumor is associated with increased benefit to these therapies. The association and biological role of additional and potentially actionable immune inhibitory targets in lung cancer is not well understood. By quantitatively measuring PD-L1, B7-H4, IDO-1 and different immune cell subsets, we show that lung carcinomas display limited co-expression of these immunosuppressive markers. The presence of the markers in lung tumors is also differentially associated with immune infiltration and specific tumor features. Our data suggest that lung malignancies use preferentially discrete and nonoverlapping routes to evade immunity that could participate in immunotherapy resistance. These results could support the design of clinical studies using biomarker-driven immunotherapies.

Despite progress in the understanding of the molecular basis and development of targeted therapies, lung cancer is still the leading cause of cancer-related morbidity and mortality worldwide (1, 2). Targeted therapies using tyrosine kinase inhibitors (TKI) are safe and effective, but they can be used in a relatively small proportion of patients with advanced disease harboring key actionable mutations such as EGFR, ALK, and ROS1 (3). In addition, response to TKIs is inevitably followed by acquired resistance, typically only months after treatment onset.

Upregulation of immune inhibitory mechanisms such as co-regulatory ligands/receptors and tolerogenic enzymes by cancer cells allow tumors escape from immune attack. Novel anticancer immunotherapies blocking immune coinhibitory pathways (also referred to as "immune checkpoints") such as PD-1 and CTLA-4 have shown prominent and durable responses in diverse malignancies. In particular, blockade of the PD-1 receptor or its ligand PD-L1 induces objective responses in about 20% of patients with heavily pretreated non–small cell lung cancer (NSCLC) with median duration >12 months and some cases with ongoing responses lasting over 2 to 3 years (4–7). The success of these therapies uncovered the power of blocking immune inhibitory pathways. Diverse studies have found that expression of PD-L1 by immunohistochemistry (IHC), pre-existence of tumor-infiltrating lymphocytes (TILs), and increased nonsynonymous mutations or predicted neoantigens are associated with benefit to PD-1 axis blockers (8–11). These findings support the notion that adaptive tumor PD-L1 upregulation, secondary to antitumor immune pressure is associated with lymphocyte reinvigoration using these therapies. Because only a fraction of patients show objective benefit from PD-1 pathway blockade, development of therapeutic strategies to effectively treat primary resistant tumors lacking PD-L1 expression and immune infiltration are needed. Efforts are now focused on combination strategies to block additional immunosuppressive signals and activate costimulatory receptors to increase response rates, prolong responses, and counteract resistance to monotherapy regimens. The expression and biological role of additional potentially actionable immune targets beyond PD-L1 in lung cancer are not well understood.

Indoleamine-2,3-dioxygenase 1 (IDO-1) is the rate-limiting enzyme in tryptophan catabolism and exerts a potent immunosuppressive effect through local inhibition of T lymphocytes and other immune cells (12, 13). T cells exposed to tryptophan depletion activate GCN2 kinase, a sensor of low amino acid content that induces a stress response, resulting in impaired T-cell proliferation and effector functions. In addition, tryptophan catabolites such as kynurenine reduce survival of CD4 T-helper cells and promote regulatory T-cell differentiation (12, 13). IDO-1 has been shown to induce immunosuppressive effects and favor tumor progression in animal models of lung cancer (14). Variable levels of IDO-1 have been found in human solid tumors including melanomas, gliomas, and carcinomas from different locations (15, 16). Blockade of IDO-1 using small-molecule inhibitors in combination with immune checkpoint blockade induces prominent antitumor responses in mouse models (17–19) and reversal of tumor-associated immunosuppression by 1-methyl-d-tryptophan [1-MDT (indoximod, NLG8189)] appears to be dependent on host IDO-1 expression (12). Diverse early-phase trials investigating IDO inhibition with 1-MDT and other compounds alone or in combination are underway.

B7-H4 is one of the most recently identified members of the B7 homologue family of immune coregulatory molecules that also encompasses PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273; refs. 20–22). Although the receptor for B7-H4 remains unknown, in vitro studies indicate a potent immunosuppressive role, and its expression has been found with variable levels in diverse human malignancies, including lung cancer (23–25). Blockade of B7-H4 using recombinant monoclonal antibodies enhances antigen-specific T-cell activation and reduces tumor growth in a humanized mice model of ovarian cancer (26) and therapeutic antibody conjugates targeting human B7-H4 are under development (27).

Despite their known expression in human lung cancer and clear therapeutic potential, the association between PD-L1, IDO-1, and B7-H4 expression remains unclear. Here, we measured the levels of these immune targets and study their association with immune cell infiltration in two lung cancer populations using objective methods and validated antibodies. To explore their biological role, we also studied the association between the markers, clinicopathologic characteristics, and outcome. Finally, we confirmed the relationship between the targets in the TCGA lung cancer dataset and their modulation by cytokines in cultured lung cancer cells.

Patients, cohorts, and tissue microarrays

Samples from two previously described retrospective collections of lung cancer, one from Yale University (cohort No. 1) and another other from Greece (cohort No. 2), represented in tissue microarrays (TMA) were used (28, 29). The first cohort includes 202 samples, and the second set includes 350 lung carcinomas. Detailed clinicopathologic characteristics of the cohorts were recently communicated (28–29) and are shown in Supplementary Table S1. TMAs were prepared using 0.6-mm tissue cores, each in twofold redundancy using standard procedures. The actual number of samples analyzed for each study is lower, due to unavoidable loss of tissue or the absence or limited tumor cells in some spots as is commonly seen in TMA studies. All tissue was used after approval from the Yale Human Investigation Committee protocol #9505008219, which approved the patient consent forms or in some cases waiver of consent.

Quantitative immunofluorescence

We measured the levels of PD-L1 (E1L3N, Cell Signaling), IDO-1 (1F8.2, Millipore), B7-H4 (D1M8I, Cell Signaling), CD3 (clone E272, Novus Biologicals), CD8 (clone C8/144B, DAKO), CD20 (clone L26, DAKO), and pancytokeratin (AE1/AE3, DAKO) using QIF in TMA slides containing the cohort cases.

PD-L1, IDO-1, and B7-H4 were stained in serial sections from the TMA blocks, using a previously described protocol with simultaneous detection of cytokeratin and DAPI (29). Briefly, antigen retrieval was with citrate buffer (pH 6.0) for 20 minutes at 97°C in a pressure-boiling container, and blocking was performed with 0.3% BSA in 0.05% Tween solution for 30 minutes. Primary antibodies were incubated overnight at a titer of 1:1,600 for PD-L1, 1:200 for B7-H4, and 1:250 for IDO-1. Secondary antibody for cytokeratin was Alexa 546–conjugated goat anti-mouse or anti-rabbit (Invitrogen Molecular Probes). Cyanine 5 (Cy5) directly conjugated to tyramide (FP1117; Perkin-Elmer) at a 1:50 dilution was used for target antibody detection (29). In selected experiments, PD-L1 and IDO-1 or B7-H4 were simultaneously stained in NSCLC specimens.

CD3, CD8, CD20, and cytokeratin were simultaneously stained using a sequential staining protocol, as previously described (28). Briefly, freshly cut TMA sections were deparaffinized and subjected to antigen retrieval using EDTA buffer (pH 8.0; Sigma-Aldrich) and boiled for 20 minutes at 97°C in a pressure-boiling container (PT module, Lab Vision, Thermo Scientific). Slides were then incubated with dual endogenous peroxidase block (DAKO #S2003) for 10 minutes at room temperature and subsequently with a blocking solution containing 0.3% BSA in 0.05% Tween solution for 30 minutes. Residual horseradish peroxidase activity between incubations with secondary antibodies was eliminated by exposing the slides twice for 7 minutes to a solution containing benzoic hydrazide (100 mmol/L) and hydrogen peroxide (50 mmol/L) in 10 mL of PBS. Isotype-specific, fluorophore-conjugated antibodies were used for signal detection, and nuclei were highlighted using DAPI (28).

Fluorescence signal quantification and cases stratification

Quantitative measurement of the fluorescent signal was performed using the AQUA method of QIF, as previously described (28–30). Briefly, the QIF score of each fluorescence channel was calculated by dividing the target marker pixel intensities by the area of the desired compartment. Scores were normalized to the exposure time and bit depth at which the images were captured, allowing scores collected at different exposure times to be comparable. A pathologist (K.A. Schalper) visually examined the stained slides and cases with artifacts were excluded. The immune target scores considered the signal detected in the tumor (cytokeratin-positive) or stromal (cytokeratin-negative) compartment and cases with less than 5% tumor were excluded from the analysis. Measurement of TIL markers considered the levels detected in the whole tumor sample (e.g., tumor and stroma). For stratification purposes, cases were considered as target expressers when the QIF score was above the signal detection threshold determined using the negative control preparations and visual inspection (28–30). The levels of CD3, CD8, and CD20 were classified as high/low using the median score as cutpoint.

Cell culture and cytokine treatment

Human A549 lung carcinoma cells were plated in a 100-mm2 petri dish and allowed to reach 70% to 80% confluence, as previously reported (30). Cells were washed 3× with PBS and incubated for 24 hours in serum-free RPMI-1640. Cells were treated with recombinant IFNγ (15 ng/mL) and/or IL10 (50 ng/mL) for 24 hours in serum-free RPMI-1640 and harvested for protein isolation. Controls included both normal serum concentration and serum-starved cells.

Protein measurement by Western blotting

Protein extraction and immunoblotting of cultured cells was performed as previously reported (30). Briefly, harvested cells were lysed in RIPA buffer supplemented with 1× protease inhibitors and 1 mmol/L sodium orthovanadate. Centrifugation was performed at 12,000 rpm for 10 minutes at 4°C to remove cellular debris. Protein quantification was performed using the bicinchoninic acid (BCA) assay (Pierce). Isolated protein in sample buffer was heated to 95°C for 5 minutes, and proteins were resolved on 4% to 12% Bis–Tris gels (Invitrogen) at 45 mA maximum V for 1.5 hours. Protein was transferred to nitrocellulose membrane using 1× NuPage Transfer Buffer at 50 V, maximum mA for 2 hours. The membrane was blocked with 5% milk in 0.05% TBST at room temperature for 1 hour and then incubated overnight at 4°C in blocking solution with 1:500 antibody dilution of anti-PD-L1, 1:1,000 anti-B7-H4, and 1:1,000 anti-IDO-1. Membranes were washed 3× in 0.05% TBST and incubated with anti-rabbit/mouse as appropriate for 1 hour at room temperature. Detection of resolved protein was performed using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific).

Statistical analyses

QIF signal differences between groups were analyzed using t test for continuous variables and χ2 test for categorical variables. Linear regression coefficients were calculated to determine the association between continuous scores. Survival analysis based on the markers expression was performed using Kaplan–Meier analyses with log-rank test and overall survival as endpoint. Statistical significance was considered at P < 0.05, and analyses were performed using JMP Pro software (version 9.0.0, 2010, SAS Institute Inc.) and GraphPad Prism v6.0 for Windows (GraphPad Software, Inc). All statistical tests were two-sided.

Assay validation and performance

Validation of the PD-L1 protein assay was reported in a previous publication from our group (30). Stringent validation of the IDO-1 and B7-H4 assays using parental cells, cell line transfectants, and endogenous tissue controls is shown in the Supplementary Fig. S1.

Expression of PD-L1, IDO-1, B7-H4, and TILs in lung cancer

In lung cancer, PD-L1, IDO-1 and B7-H4 showed distinct staining patterns. As shown in Fig. 1A, PD-L1 and B7-H4 showed a predominant cytoplasmic/membranous distribution. As expected for a cytosolic enzyme, IDO-1 expression showed a predominant perinuclear staining pattern (Fig. 1A, center). The levels of all three markers were significantly higher in the tumor compartment than in stromal cells (Fig. 1B). For PD-L1 and IDO-1, the stromal signal was typically focal and positively associated with the tumor signal [linear correlation coefficient (R2) of 0.64 in the first cohort and 0.66 in the second cohort for PD-L1 and R2 = 0.65 and 0.82 for IDO-1, respectively]. B7-H4 staining was detected almost exclusively in tumor cells (R2 = 0.25 and 0.39 in each cohort, respectively). Using a previously validated multiplex QIF staining panel including the markers DAPI, cytokeratin, CD3, CD8, and CD20 (28), we identified tumors with variable lymphocyte infiltration patterns. As shown in Fig. 1C, some cases displayed low/minimal TILs (Fig. 1C, left), other prominent CD3+ T cells (middle) or mixed inflammation with abundant CD20+ B-cell infiltration (right).

Figure 1.

Detection of PD-L1, IDO-1, B7-H4, and TIL subsets using multiplex QIF in lung cancer. A, Representative fluorescence images showing the simultaneous detection of PD-L1, IDO-1, or B7-H4 (red fluorescence channel) and cytokeratin (green channel) in lung cancer samples using QIF. Top, tumors that are positive for each of the markers; bottom, cytokeratin positivity in the same samples. The target protein is indicated with red colored text in each figure. Green indicates the cytokeratin-positive compartment and blue designated the DAPI-positive nuclei. B, Levels of PD-L1, IDO-1, and B7-H4 in the tumor and stromal compartment of lung carcinomas from the studied cohorts. Each dot indicates the QIF level of the marker in a different tumor sample. ***, P < 0.001. C, Representative fluorescence images showing the detection of TIL subsets in lung cancer samples by simultaneous staining of DAPI, cytokeratin (CK, yellow channel), CD3 (red channel), CD8 (green channel), and CD20 (white channel). Cases with low TILs (left), high TILs with predominant CD3+ T cells (center), and with high CD3+ and CD20+ B lymphocytes (right) are presented. Bar, 100 μm.

Figure 1.

Detection of PD-L1, IDO-1, B7-H4, and TIL subsets using multiplex QIF in lung cancer. A, Representative fluorescence images showing the simultaneous detection of PD-L1, IDO-1, or B7-H4 (red fluorescence channel) and cytokeratin (green channel) in lung cancer samples using QIF. Top, tumors that are positive for each of the markers; bottom, cytokeratin positivity in the same samples. The target protein is indicated with red colored text in each figure. Green indicates the cytokeratin-positive compartment and blue designated the DAPI-positive nuclei. B, Levels of PD-L1, IDO-1, and B7-H4 in the tumor and stromal compartment of lung carcinomas from the studied cohorts. Each dot indicates the QIF level of the marker in a different tumor sample. ***, P < 0.001. C, Representative fluorescence images showing the detection of TIL subsets in lung cancer samples by simultaneous staining of DAPI, cytokeratin (CK, yellow channel), CD3 (red channel), CD8 (green channel), and CD20 (white channel). Cases with low TILs (left), high TILs with predominant CD3+ T cells (center), and with high CD3+ and CD20+ B lymphocytes (right) are presented. Bar, 100 μm.

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The levels of the markers in the lung cohorts showed a continuous distribution. Using a cutpoint established from scores obtained in negative control preparations and the visual detection threshold, PD-L1 signal was identified in 16.9% of cases in the first cohort (Fig. 2A) and 21.8% of cases in the second collection (Fig. 2B). IDO-1 was expressed in 42.6% of cases in cohort No.1 (Fig. 2C) and 49.8% in the validation set (Fig. 2D) and B7-H4 was found in 12.8% and 22.6%, respectively (Fig. 2E and F).

Figure 2.

Levels of PD-L1, IDO-1, and B7-H4 in lung cancer. A–F, Distribution of PD-L1, IDO-1, and B7-H4 QIF scores in lung cancer samples from cohort No. 1 (A, C, E) and cohort No. 2 (B, D, F). Scores are expressed as arbitrary units of fluorescence, and the dashed gray line indicates the signal detection threshold determined as described in Materials and Methods. The proportion of cases with detectable target signal is indicated within each chart.

Figure 2.

Levels of PD-L1, IDO-1, and B7-H4 in lung cancer. A–F, Distribution of PD-L1, IDO-1, and B7-H4 QIF scores in lung cancer samples from cohort No. 1 (A, C, E) and cohort No. 2 (B, D, F). Scores are expressed as arbitrary units of fluorescence, and the dashed gray line indicates the signal detection threshold determined as described in Materials and Methods. The proportion of cases with detectable target signal is indicated within each chart.

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Association between PD-L1, IDO-1, B7-H4, and TILs in lung cancer

As previously reported by our group using a different PD-L1 assay targeting the extracellular protein domain (antibody clone 5H1; ref. 29), tumor PD-L1 expression was positively associated with lymphocyte infiltration. In particular, the PD-L1 signal was independently associated with elevated CD3-, CD8-, or CD20-positive lymphocytes in both cohorts (Tables 1 and 2). No consistent association was seen between PD-L1 expression and sex, age, clinical stage, and histology. IDO-1 was also positively associated with all three TIL markers in both cohorts, but not with other clinicopathologic variables (Tables 1 and 2). Tumor expression of B7-H4 was consistently associated with squamous cell histology, but not with the presence of lymphocyte infiltration or other variables. Despite their association with TILs, expression of the markers was not consistently associated with 5-year overall survival, suggesting a limited prognostic effect (Supplementary Fig. S2).

Table 1.

Association between PD-L1, IDO-1, B7-H4, TILs and clinicopathologic variables in cohort No. 1

ParameterStratificationPD-L1 lowPD-L1 highPIDO-1 lowIDO-1 highPB7-H4 lowB7-H4 highP
Sex Male 73 18 0.329 50 38 0.981 75 15 0.136 
 Female 83 14  53 40  87  
Age <70 y 106 22 0.891 76 57 0.168 115 13 0.116 
 >70 y 51 10  38 21  48 11  
Stage I–II 108 25 0.302 69 58 0.281 118 13 0.071 
 III–IV 48  34 20  44 11  
Histology Adenocarcinoma 112 16 0.006 75 50 0.111 114 12 0.041 
 Squamous cell carcinoma 26  17 14  24  
 Other 12  13  19  
CD3 Low 86 10 0.011 62 31 0.009 82 14 0.444 
 High 72 22  43 47  82 10  
CD8 Low 85 10 0.017 60 32 0.031 79 16 0.085 
 High 73 22  45 46  85  
CD20 Low 89 0.004 63 34 0.013 87 13 0.894 
 High 72 23  45 50  85 12  
ParameterStratificationPD-L1 lowPD-L1 highPIDO-1 lowIDO-1 highPB7-H4 lowB7-H4 highP
Sex Male 73 18 0.329 50 38 0.981 75 15 0.136 
 Female 83 14  53 40  87  
Age <70 y 106 22 0.891 76 57 0.168 115 13 0.116 
 >70 y 51 10  38 21  48 11  
Stage I–II 108 25 0.302 69 58 0.281 118 13 0.071 
 III–IV 48  34 20  44 11  
Histology Adenocarcinoma 112 16 0.006 75 50 0.111 114 12 0.041 
 Squamous cell carcinoma 26  17 14  24  
 Other 12  13  19  
CD3 Low 86 10 0.011 62 31 0.009 82 14 0.444 
 High 72 22  43 47  82 10  
CD8 Low 85 10 0.017 60 32 0.031 79 16 0.085 
 High 73 22  45 46  85  
CD20 Low 89 0.004 63 34 0.013 87 13 0.894 
 High 72 23  45 50  85 12  
Table 2.

Association between PD-L1, IDO-1, B7-H4, TILs and clinicopathologic variables in cohort No. 2

ParameterStratificationPD-L1 lowPD-L1 highPIDO-1 lowIDO-1 highPB7-H4 lowB7-H4 highP
Sex Male 168 57 0.005 146 139 0.289 190 59 0.261 
 Female 31  16 22  32  
Age <70 y 157 50 0.377 131 132 0.791 186 50 0.271 
 >70 y 40  30 28  35 14  
Stage I–II 112 42 0.031 92 100 0.293 135 37 0.527 
 III–IV 85 16  69 59  82 27  
Histology Adenocarcinoma 83 19 0.068 62 64 0.323 102 12 <0.0001 
 Squamous cell carcinoma 99 29  87 76  95 46  
 Other 16 11  13 20  25  
CD3 Low 115 18 0.0003 98 66 0.0004 109 39 0.125 
 High 90 42  69 101  120 28  
CD8 Low 121 16 <0.0001 102 63 <0.0001 108 39 0.111 
 High 84 44  65 104  121 28  
CD20 Low 114 23 0.012 109 62 <0.0001 115 37 0.6842 
 High 91 37  61 111  118 34  
ParameterStratificationPD-L1 lowPD-L1 highPIDO-1 lowIDO-1 highPB7-H4 lowB7-H4 highP
Sex Male 168 57 0.005 146 139 0.289 190 59 0.261 
 Female 31  16 22  32  
Age <70 y 157 50 0.377 131 132 0.791 186 50 0.271 
 >70 y 40  30 28  35 14  
Stage I–II 112 42 0.031 92 100 0.293 135 37 0.527 
 III–IV 85 16  69 59  82 27  
Histology Adenocarcinoma 83 19 0.068 62 64 0.323 102 12 <0.0001 
 Squamous cell carcinoma 99 29  87 76  95 46  
 Other 16 11  13 20  25  
CD3 Low 115 18 0.0003 98 66 0.0004 109 39 0.125 
 High 90 42  69 101  120 28  
CD8 Low 121 16 <0.0001 102 63 <0.0001 108 39 0.111 
 High 84 44  65 104  121 28  
CD20 Low 114 23 0.012 109 62 <0.0001 115 37 0.6842 
 High 91 37  61 111  118 34  

PD-L1, IDO-1, and B7-H4 are infrequently coexpressed in lung cancer

Samples from lung carcinomas with prominent levels of one of the markers had typically low levels of the other. Representative examples of three tumors with mutually exclusive expression of PD-L1, IDO-1, and B7-H4 are shown in Fig. 3A–C. Consistent with the visual assessment and using the continuous QIF scores for each marker, we identified only a few cases with concomitant expression of PD-L1 and IDO-1 (Fig. 3D and E). Overall, the proportion of cases coexpressing PD-L1 and IDO-1 was 7.1% in the first cohort and 10.9% in the second group. Cases showing coexpression of PD-L1 and B7-H4 were only 3.2 and 3.4% in each cohort, respectively; and cases with detectable levels of IDO-1 and B7-H4 were 6.1 and 9.8% of tumors. Comparable results were seen by simultaneous costaining of PD-L1 and IDO-1 or B7-H4 in lung carcinomas (Supplementary Fig. S3).

Figure 3.

PD-L1, IDO-1, and B7-H4 are infrequently coexpressed in lung cancer. A–C, Representative fluorescence pictures showing lung carcinomas with mutually exclusive expression of PD-L1, IDO-1, and B7-H4. The same tumors were stained for the different markers and the results are shown in the top and bottom. The target protein is indicated in the red fluorescence channel and tumor cells are highlighted with cytokeratin (CK, green fluorescence channel). Nuclei are stained with DAPI. D and E, Histograms showing the levels of PD-L1, IDO-1, and B7-H4 protein in lung carcinomas from cohort No. 1 (D) and cohort No. 2 (E). The proportion of cases showing coexpression of the markers based on the signal detection threshold (see methods section) is indicated within each chart.

Figure 3.

PD-L1, IDO-1, and B7-H4 are infrequently coexpressed in lung cancer. A–C, Representative fluorescence pictures showing lung carcinomas with mutually exclusive expression of PD-L1, IDO-1, and B7-H4. The same tumors were stained for the different markers and the results are shown in the top and bottom. The target protein is indicated in the red fluorescence channel and tumor cells are highlighted with cytokeratin (CK, green fluorescence channel). Nuclei are stained with DAPI. D and E, Histograms showing the levels of PD-L1, IDO-1, and B7-H4 protein in lung carcinomas from cohort No. 1 (D) and cohort No. 2 (E). The proportion of cases showing coexpression of the markers based on the signal detection threshold (see methods section) is indicated within each chart.

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To rule out possible bias induced by limited tumor representation using TMAs and/or the effect of marker heterogeneity, we analyzed the levels of PD-L1, IDO-1, and B7-H4 mRNA from the TCGA lung cancer dataset obtained by RNA sequencing from whole-tissue section tumor samples (31). As shown in Fig. 4, a similarly restricted coexpression between the markers was observed in both squamous carcinomas (Fig. 4A, n = 178) and primary lung adenocarcinomas (Fig. 4B, n = 230).

Figure 4.

PD-L1, IDO-1, and B7-H4 mRNA are infrequently coexpressed in lung cancer. Histograms showing the coexpression (top charts) and levels (bottom charts) of PD-L1, IDO-1, and B7-H4 mRNA transcripts in lung cancer cases from the TCGA lung squamous carcinoma (A,n = 178) and lung adenocarcinoma (B,n = 230) datasets. Levels of target mRNAs were measured using RNA-sequencing in whole-tissue section samples and obtained through the cBioportal analysis platform.

Figure 4.

PD-L1, IDO-1, and B7-H4 mRNA are infrequently coexpressed in lung cancer. Histograms showing the coexpression (top charts) and levels (bottom charts) of PD-L1, IDO-1, and B7-H4 mRNA transcripts in lung cancer cases from the TCGA lung squamous carcinoma (A,n = 178) and lung adenocarcinoma (B,n = 230) datasets. Levels of target mRNAs were measured using RNA-sequencing in whole-tissue section samples and obtained through the cBioportal analysis platform.

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Modulation of PD-L1, IDO-1, and B7-H4 by cytokines in lung cancer cells

In efforts to explain the nearly mutually exclusive pattern of expression at both the protein and mRNA levels, we examined factors inducing their expression. Previous studies have indicated that PD-L1 and IDO-1 expression is induced by IFNγ stimulation (9, 10, 16) and B7-H4 upregulation occurs after IL6 or IL10 stimulation in cultured monocytic cells (24). To confirm the modulation of the targets in human tumor cells, we treated A549 lung adenocarcinoma cells for 24 hours with 15 ng/mL recombinant IFNγ, 50 ng/mL IL10, or the combination. As shown in Supplementary Fig. S4, control serum-starved A549 cells lack detectable levels of PD-L1, IDO-1, and B7-H4 protein by Western blotting. Treatment with IFNγ prominently increases the levels of PD-L1 and IDO-1, but not of B7-H4. Reciprocally, treatment with IL10 increases B7-H4 but does not alter the levels of PD-L1 and IDO-1. Treatment with the combination of IFNγ and IL10 mimics the effect of IFNγ alone. Cell lines used in this study were purchased in ATCC and authentication was performed every 3 to 6 months using the GenePrint 10 System in the Yale University DNA Analysis Facility.

Using multiplexed QIF, we show that PD-L1, IDO-1, and B7-H4 are differentially expressed and infrequently coexpressed in human lung cancer. The markers are located preferentially in tumor cells and show distinct association with lymphocyte infiltration. While PD-L1 and B7-H4 were found in 10% to 25% of cases, IDO-1 expression was detected in more than 40% of tumors in two independent populations. The proportion of cases showing PD-L1 expression reported here is lower than in our previous study using the same retrospective lung cancer collections (29). This difference may be explained by the use of a different PD-L1 monoclonal antibody in this study recognizing the intracellular protein domain (clone E1L3N) and not an extracellular epitope as in our previous report (clone 5H1) or other differences between the two antibodies. In support of this notion, recent work by our group using whole-tissue section specimens confirmed the frequent discordance between validated PD-L1 assays in lung carcinomas (32).

The proportion of lung cancer cases showing IDO-1 and B7-H4 expression is relatively lower than in previous reports. In NSCLC, IDO-1 protein has been detected in 67% to 79% of cases using chromogenic immunohistochemistry (15–16), and B7-H4 was found in 43% of lung cancers (25). The determinants for this apparent discrepancy are uncertain and could be related with the use of relatively small-tissue fragments in our study. However, the rigorous validation of our assays and the objective/quantitative platform used support a precise measurement of the targets. The relative high frequency of IDO-1 expression found in lung cancers supports the possibility of using available (small molecule) IDO inhibitors as anticancer immunostimulatory therapy.

Notably, the markers showed an exclusive pattern with infrequent coexpression, suggesting that most lung tumors use preferentially only one immune evasion mechanism/pathway. Our results also suggest the possibility of alterations of these targets as possible mechanisms of acquired resistance to PD-1 axis therapies.

Consistent with their previously reported induction by TH1/IFNγ signaling (9, 10, 16), elevated PD-L1 and IDO-1 were significantly associated with high CD3- and CD8-positive T-lymphocyte infiltration. Increased PD-L1 and IDO-1 were also associated with high CD20-positive B lymphocytes. Our in vitro results also confirm the adaptive induction of PD-L1 and IDO-1 by IFNγ in cultured adenocarcinoma cells. Therefore, the frequent lack of coexpression of PD-L1 and IDO-1 seen in lung tumors both at the protein and mRNA level in three different populations (e.g., two retrospective cohorts and the TCGA set) could be mediated by additional signaling events occurring in the tumor or tumor microenvironment. For instance, expression of PD-L1 can be modulated by IFNα, IFNβ, granulocyte macrophage colony-stimulating factor (GM-CSF), VEGF, lipopolysaccharide (LPS), IL4, and IL10 (10). IDO-1 levels are sensitive to COX2 signaling through prostaglandin E2, TGFβ, and nitric oxide (33). Future studies will be required to clarify this apparent contradiction. On the contrary and as expected by its previously identified induction by TH2/IL10 signaling (24), B7-H4 was not associated with PD-L1, IDO-1, or increased TILs. The expression of B7-H4 in noninflamed or less inflamed tumors suggests the possibility of using this molecule as therapeutic target in these malignancies (10, 29). In addition, and as previously described (20), B7-H4 levels were higher in squamous cell carcinomas than in the other histologic variants of lung cancer, suggesting a therapeutic opportunity for this aggressive malignancy with limited available therapies. Although previously shown to have marked immunosuppressive effect in cultured cells and animal models of ovarian cancer (23–26), the precise biological role of B7-H4 in lung cancer requires further investigation.

Despite their association with TILs, expression of PD-L1, IDO-1, and B7-H4 was not consistently associated with survival. The absence of prognostic value of the markers may not be a limitation for their possible role as predictive biomarkers. For instance, EGFR mutations and ALK rearrangements are highly predictive of response to targeted therapies in lung adenocarcinomas, but their prognostic value is limited.

Our study has a number of limitations. First, we included retrospectively collected samples from cases with variable follow-up, different treatments, and lacking molecular/genomic annotation. However, we found comparable results in both collections, suggesting that our findings are independent from treatment and molecular subtype. Also, our cases were represented in TMA format, which may induce under- or overrepresentation of the marker levels due to tumor heterogeneity. However, the comparable results seen in the TCGA dataset using mRNA measurements in whole-tissue section tumor samples supports the validity of our findings.

Although a myriad of additional immunosuppressive ligands and receptors have been described and could play a relevant role in lung cancer, our results suggest the opportunity for optimization of immunotherapy through measurement of the drug targets in pretreatment tumor samples.

V. Velcheti is a consultant/advisory board member for Bristol-Myers Squibb, Celgene, Clovis, Foundation Medicine, Genentech, and Merck. L. Chen reports receiving a commercial research grant from Boehringer Ingelheim, NextCure, and Pfizer and is a consultant/advisory board member for AstraZeneca, NextCure, and Pfizer. D. L. Rimm reports receiving a commercial research grant from Genoptix and is a consultant/advisory board member for AstraZeneca, Bristol-Myers Squibb, and Genoptix. No potential conflicts of interest were disclosed by the other authors.

Conception and design: K.A. Schalper, M. Altan, V. Velcheti, L. Chen, R.S. Herbst, D.L. Rimm

Development of methodology: K.A. Schalper, D. Carvajal-Hausdorf, D. McLaughlin, V. Velcheti, L. Chen, R.S. Herbst

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.A. Schalper, D. Carvajal-Hausdorf, M. Altan, P. Gaule, R.S. Herbst

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.A. Schalper, D. Carvajal-Hausdorf, L. Chen, R.S. Herbst

Writing, review, and/or revision of the manuscript: K.A. Schalper, D. Carvajal-Hausdorf, D. McLaughlin, M. Altan, V. Velcheti, M.F. Sanmamed, R.S. Herbst, D.L. Rimm

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K.A. Schalper, D. McLaughlin, D.L. Rimm

Study supervision: K.A. Schalper, D.L. Rimm

We would like to thank Nikita Mani for experimental support and Lori Charette from the Yale Pathology Tissue Services for production of optimal TMAs and tissue slides.

This research was supported by a Stand Up To Cancer - American Cancer Society Lung Cancer Dream Team Translational Research Grant (Grant Number: SU2C-AACR-DT17-15). Stand Up to Cancer is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. This work was also funded by the Yale SPORE in Lung Cancer (P50CA196530) Career Development Program award (to K.A. Schalper), a Lung Cancer Research Foundation grant (to K.A. Schalper), and the Yale Cancer Center Support Grant (P30CA016359).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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