We had previously demonstrated the role of CD103 integrin on lung tumor-infiltrating lymphocyte (TIL) clones in promoting specific TCR-mediated epithelial tumor cell cytotoxicity. However, the contribution of CD103 on intratumoral T cell distribution and functions and the prognosis significance of TIL subpopulations in non–small cell lung carcinoma (NSCLC) have thus far not been systematically addressed. In this study, we show that an enhanced CD103+ TIL subset correlates with improved early stage NSCLC patient survival and increased intraepithelial lymphocyte infiltration. Moreover, our results indicate that CD8+CD103+ TIL, freshly isolated from NSCLC specimens, display transcriptomic and phenotypic signatures characteristic of tissue-resident memory T cells and frequently express PD-1 and Tim-3 checkpoint receptors. This TIL subset also displays increased activation-induced cell death and mediates specific cytolytic activity toward autologous tumor cells upon blockade of the PD-1–PD-L1 interaction. These findings emphasize the role of CD8+CD103+ tissue-resident memory T cells in promoting intratumoral CTL responses and support the rationale for using anti–PD-1 blocking Ab to reverse tumor-induced T cell exhaustion in NSCLC patients.

Cytotoxic T lymphocytes play a major role in antiviral and antitumor immune responses through directional exocytosis of cytotoxic granules, specialized secretory lysosomes containing perforin and granzymes, into the specific target leading to cell death (1). Integrins and their cognate ligands are essential in TCR-mediated target cell killing. Indeed, although TCR engagement is necessary for inducing the cytotoxic activity of specific CD8+ T lymphocytes, integrins, mainly LFA-1 (αLβ2), play a crucial role in triggering CTL effector functions. It is now well established that the interaction of LFA-1 with its ligand ICAM-1 is necessary for effective target cell lysis by the released granules (2). CD103 (αEβ7) integrin also plays a pivotal role in epithelial target cell lysis by specific CD8+ T cells. Indeed, the interaction of CD103 on tumor-specific tumor-infiltrating lymphocytes (TIL) with its ligand, E-cadherin on tumor cells, is necessary for positioning the cytotoxic granules near the interface and their delivery into the target, leading to lysis of the latter cells (3). CD103 is expressed at high levels by mucosal CD8+ T lymphocytes, in particular intestinal intraepithelial lymphocytes (4), but it is also found on mucosal mast cells and dendritic cells (5), CD4+ and CD8+ regulatory T cells (6), and on a large proportion of CD8+ T cells infiltrating epithelial tumors, including pancreatic (7), colorectal (8), lung (3), and ovarian cancers (9). It has been recently reported that CD103 expression on TIL is associated with increased survival in high-grade serous ovarian cancer (10). This integrin is induced on CD8+ T lymphocytes upon TCR engagement and exposure to TGF-β1, abundant within the tumor microenvironment, through binding of NFAT-1 and Smad2/3 transcription factors to the promoter and enhancer elements of the ITGAE gene that encodes CD103 (11).

CD103 ligation to E-cadherin not only promotes Ag recognition by favoring T cell adhesion to specific tumor cells (12), but it also induces costimulation in activated CTL by triggering signals that cooperate with TCR-mediated signaling (13). Emerging evidence points to a role for CD8+CD103+ T cells in transplant rejection and in protecting prevalent infections posttransplantation (1416). This integrin serves as a marker of tissue-resident memory (TRM) T cells, and accumulating evidence indicates that CD103 is directly involved in intraepithelial retention of TRM T cells (17). Remarkably, CD103+ TRM T cells play an essential role in protecting human epithelial tissues against viral infections, and this cell subset has been shown to express inhibitory receptors such as CTLA-4 and PD-1, associated with their capacity to maintain peripheral tolerance (18, 19). However, the presence of TRM T cells in human epithelial tumors and their role in the antitumor immune response have thus far not been systematically addressed. In this study, we sought to determine the prognostic value of CD103+ TIL in early stage non–small cell lung carcinoma (NSCLC) and perform combined phenotypic and functional analyses of freshly isolated CD8+CD103+ T cells infiltrating human lung tumor lesions. Our results indicate that a strong tumor infiltration with CD103+ TIL correlates with NSCLC patient survival and increased intraepithelial lymphocyte infiltration, suggesting that CD103 promotes recruitment of TIL within epithelial tumor islets. Moreover, CD8+CD103+ TIL exhibit TRM T cell features, express PD-1 and Tim-3, but not CTLA-4, and exert specific cytotoxic activity against autologous tumor cells upon neutralization of PD-1–PD-L1 interactions. We propose that this integrin can be used as a unique biomarker of tumor-specific CD8+ T cells infiltrating human epithelial tumors.

Fresh NSCLC tumors were obtained from the Institut Mutualiste Montsouris or the Centre Chirurgical Marie-Lannelongue and immediately dissociated mechanically and enzymatically (dissociation kit from Miltenyi Biotec). Among a total number of 210 recruited tumors, only 60% were infiltrated with a sufficient number of TIL to perform ex vivo studies. TIL were either directly analyzed by multicolor flow cytometry or isolated using a FACSAria cell sorter based on a small-size selection of mononuclear cells. PBMC from distinct consented NSCLC patients were obtained from Institut de Cancérologie Gustave Roussy. All experiments were approved by the Institutional Review Board.

Total RNA from small size–selected TIL and PBMC were extracted using TRIzol (Sigma-Aldrich) and then compared using an Agilent Technologies SurePrint G3 human GE 8 × 60K microarray (AMADID 39494) as described (3). Feature extraction software (Agilent Technologies, version 10.7.3.1) was used to quantify the intensity of fluorescent images. Data were normalized by a quantile method from the limma Bioconductor R package. Differential gene expression analysis was performed with limma’s moderated t test. Primary functional analyses were performed using Ingenuity Pathway Analysis and the Database for Annotation, Visualization andIntegrated Discovery bioinformatics. The microarray data are available at the European Molecular Biology Laboratory European Bioinformatics Institute database (https://www.ebi.ac.uk/arrayexpress) under accession no. E-MTAB-3266.

cDNA were synthesized from 1 μg mRNA from small size–selected TIL, PBMC, or CD103+ TIL using an Applied Biosystems kit. Quantitative RT-PCR (qRT-PCR) analysis was performed using a LightCycler and the LightCycler FastStart DNA Master SYBR Green I mix (Roche Applied Science) according to the manufacturer’s instructions. Expression of target genes was normalized to that of 18S.

Anti-human CD3, CD8, CD4, CD103, CD107a, CD127, Bcl-2, KLRG1, and granzyme B mAb as well as mouse and rabbit isotopic controls were purchased from Ozyme. Anti-human CD69, CD62L, CD45RA, CD45RO, CD27, and CD28 mAb were provided by Invitrogen. Anti-human PD-1, Tim-3, and CTLA-4 mAb were purchased from eBioscience. Phenotypic analyses were performed by direct immunofluorescence using a FACS LSR II flow cytometer. Data were processed using FlowJo or FACSDiva software (BD Biosciences).

Activation-induced cell death (AICD) was measured by flow cytometry using an annexin V apoptosis detection kit (BD Pharmingen). After recovery of TIL and tumor cells by FACS based on cell size selection, they were either kept in medium or stimulated overnight with rIL-2 (5–10 U/ml). TIL were then cultured for 4 h in the presence of autologous tumor cells. After 15 min incubation with 5 μl annexin V, labeling buffer, and 5 μl propidium iodide (PI), cells were analyzed within 1 h by FACS.

The cytotoxic activity of TIL was measured by a conventional 51Cr-release assay (3). Freshly isolated TIL were either kept in medium or stimulated overnight with rIL-2 (5 U/ml). Autologous tumor cells were used as targets at a 10:1 E:T ratio. For cytotoxicity induction, TIL or tumor cells were preincubated for 1 h at 37°C with neutralizing anti–PD-1 or anti–PD-L1 mAb, respectively. Cytotoxicity inhibition was performed by preincubating effector or target cells for 1 h with anti-CD103 or anti–MHC class I (MHC-I) blocking mAb, respectively.

Paraffin-embedded primary tumor samples were obtained from patients diagnosed with early stage NSCLC and who underwent curatively intended surgical resection between 1995 and 2002. A total of 101 tumor samples were included in this study and analyzed for CD3, CD8, and CD103 expression on TIL, located either in epithelial or stromal regions, and their association with patient survival outcomes. All were in pathological stage I (33.7% were pT1N0 and 66.3% pT2N0) and none of them had received neoadjuvant therapy at the time of surgery. Of these, 44.5% were adenocarcinomas (ADC), 42.6% were squamous cell carcinomas (SCC), 12.9% were mixed or large cell carcinoma, and 3% were other types. Median (range) age at diagnosis was 66.1 y (40.5–83.6 y old); 68.3% of the patients were males; 89.1% were tobacco users; and median (range) tobacco consumption was 50 (5–100) pack years.

Serial sections from paraffin-embedded tumors were stained with H&E with saffron (HES), anti-CD3, anti-CD8 (Thermo Fisher), or anti-CD103 (Abcam) mAb. Briefly, 4-μm-thick sections were mounted on poly-l-lysine–coated slides, deparaffinized, and rehydrated through graded alcohol to water. Primary Ab were incubated for 1 h at room temperature. Immunostaining was visualized following incubation with goat anti-rabbit HRP for 30 min at room temperature and then 3,3′-diaminobenzidine substrate was added (PowerVision, Leica Biosystems). The slides were counterstained with Mayer’s hematoxylin (VWR International). For all staining, whole slides were digitized using a slide scanner (VS120, Olympus). For each slide, three areas of 0.88 mm2 were selected as representative of the potential heterogeneity of immune infiltration within the tumor. Quantification of the number of CD3+, CD8+, and CD103+ cells was performed manually in each of the areas on serial sections for both intraepithelial and stromal compartments. For each slide, data were summarized over the three areas giving a score number of stained cells per square millimeter.

Distributions of CD3, CD8, and CD103 biomarkers according to each clinical factor (age, histological type, pathological stage, smoking status, and gender) were described separately by providing medians and ranges, and comparison of distribution between groups was performed using the Wilcoxon rank or Kruskal–Wallis test when appropriate. Disease-free survival (DFS) was defined by the time from diagnosis to death or relapse, whichever occurred first, or to last follow-up date. Overall survival (OS) was defined as the time from diagnosis to death or date of last follow-up. Considering CD3, CD8, and CD103 as continuous variables, their association with DFS and OS was evaluated using univariate and multivariate Cox regression models adjusted for age, histological type, gender, stage, and smoking status. The required assumptions of proportionality in multivariate survival analysis were checked by Schoenfeld’s test.

For all other experiments, data were compared using the two-tailed Student t test. Two groups were considered as significantly different when p < 0.05.

To exert their lytic function, CTL must infiltrate the tumor tissue and then interact with the target cell to finally trigger their functional activities. Thus, we first investigated the frequency and distribution of CD3+, CD8+, and CD103+ TIL in epithelial tumor and stromal regions from tumor samples of 101 stage I NSCLC patients. Serial tumor sections were stained with HES, anti-CD3, anti-CD8, or anti-CD103 mAb, and intratumoral and stromal cell localization was then quantified (Fig. 1A). Results indicated that the density of CD3+, CD8+, and CD103+ lymphocytes varied from one tumor to another with a median range of 234 (5–1184), 135 (8–685), and 113 (2–697) infiltrations, respectively. Moreover, results showed that the large majority of CD3+, CD8+, and CD103+ TIL were located within the stroma (Fig. 1B, 1C). Indeed 89, 83, and 66% of patients had more than half of anti-CD3–, anti-CD8–, and anti-CD103–stained cells in the stroma, respectively (p < 0.002, see Table I). An increased intraepithelial lymphocyte infiltration was particularly observed in tumors with a high density of CD103+ TIL (Fig. 1C), which was less frequently detected in older patients (p = 0.02) and in women compared with men (p = 0.01; Table I). Moreover, intraepithelial CD103+ infiltration was more present in ever-smokers who are generally male (p = 0.01; Table I). The latter result was also observed for CD8+ TIL (p = 0.04), which might also express CD103. In contrast, CD3+ TIL were not associated with patient clinical information.

FIGURE 1.

Distribution of CD3+, CD8+, and CD103+ cells in intraepithelial tumoral and stromal regions of NSCLC tumors. (A) Tumor sections from 101 early stage NSCLC patients were stained with anti-CD3, anti-CD8, or anti-CD103 mAb. Representative images of CD3, CD8, and CD103 immunostaining of the same tumor area and counterstained with HES (original magnification ×200) are shown. (B) CD3, CD8, and CD103 expression scatter plots in intraepithelial tumor and stromal regions among the 101 NSCLC tumors. Right panels, Bar plots of the total number (blue) and the percentage (yellow) of CD3+, CD8+, and CD103+ cells present in the tumor. (C) Mean percentages of CD3+, CD8+, and CD103+ lymphocytes within tumoral and stromal regions of the 101 NSCLC tumors. **p < 0.001.

FIGURE 1.

Distribution of CD3+, CD8+, and CD103+ cells in intraepithelial tumoral and stromal regions of NSCLC tumors. (A) Tumor sections from 101 early stage NSCLC patients were stained with anti-CD3, anti-CD8, or anti-CD103 mAb. Representative images of CD3, CD8, and CD103 immunostaining of the same tumor area and counterstained with HES (original magnification ×200) are shown. (B) CD3, CD8, and CD103 expression scatter plots in intraepithelial tumor and stromal regions among the 101 NSCLC tumors. Right panels, Bar plots of the total number (blue) and the percentage (yellow) of CD3+, CD8+, and CD103+ cells present in the tumor. (C) Mean percentages of CD3+, CD8+, and CD103+ lymphocytes within tumoral and stromal regions of the 101 NSCLC tumors. **p < 0.001.

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Table I.
CD3+, CD8+, and CD103+ TIL association with clinical characteristics in stage I NSCLC patients
Total
Tumoral
Stromal
Number (%)
Median (Q1–Q3)
p Value
Median (Q1–Q3)
p Value
Median (Q1–Q3)
p Value
CD3+        
 Patient
 
93
 
234.7 (147.7–442.7)
 

 
35.7 (12.3–96.7)
 

 
185.7 (127.0–355.3)
 
 
 Histological subtype        
  ADC 42 (45.16) 209.8 (151.9–402.0)  26.7 (7.9–81.5)  178.7 (132.3–353.9)  
  SCC 40 (43.01) 198.3 (117.6–437.2)  40.8 (16.8–98.8)  158.7 (78.5–325.9)  
  Other 11 (11.83) 474.0 (331.2–598.2) 0.09 85.7 (37.5–145.0) 0.07 303.7 (247.7–475.7) 0.07 
 Stage        
  Ia 30 (32.26) 194.3 (159–391.5)  25.5 (6.2–79.5)  172.6 (129.5–320.8)  
  Ib 63 (67.74) 277.7 (143.8–477.5) 0.45 49 (17.5–108.8) 0.06 199.7 (118.7–377.7) 0.69 
 Gender        
  Male 30 (32.26) 190.5 (137.2–398.4)  32.2 (8.3–84.8)  169.8 (96.3–381.2)  
  Female 63 (67.74) 266.3 (151.8–458.3) 0.48 39.7 (16.8–110.2) 0.27 202.7 (133.7–346.8) 0.67 
 Smoking status        
  Ever 83 (89.25) 257.0 (145.8–442.2)  41 (18.5–103.5)  185.7 (118.7–352.5)  
  Never 10 (10.75) 202.8 (154.9–502.0) 0.92 15.2 (1.3–68.2) 0.06 178.1 (136.7–429.6) 0.53 
 Age (y)        
  <65 63 (62.4) 277.7 (168.3–442.7)  47.3 (20–106)  209 (143.3–355.3)  
  ≥65 38 (37.6) 187.3 (117.6–439.7) 0.07 28 (8.4–87.2) 0.09 158.7 (68.0–327.2) 0.16 
CD8+        
 Patient 98 135.0 (86.8–249.2)  24.3 (7.4–80.1)  106.8 (53.6–169.9)  
 Histological subtype        
  ADC 45 (45.92) 132.0 (83.0–225.7)  12.3 (2–48.7)  111.7 (65.7–170.7)  
  SCC 40 (40.82) 115.3 (87.6–247.2)  27.6 (12.5–71.1)  82.7 (46.3–140.9)  
  Other 13 (13.27) 331.3 (124.0–411.7) 0.03 85 (27.5–148.7) 0.02 173.7 (116.7–263.0) 0.01 
 Stage        
  Ia 32 (32.65) 124.0 (54.67–248.7)  16 (4.3–56.8)  111.5 (53.3–150.9)  
  Ib 66 (67.35) 138.7 (91.08–249.3) 0.53 27.6 (8.6–80.1) 0.24 103.5 (60.9–174.4) 0.76 
 Gender 31 (31.63) 124.0 (84.17–209.3)  22.7 (3.2–50.5)  103.7 (57.4–170.2)  
  Male 67 (68.37) 139.3 (91.5–251.5) 0.50 27.5 (7.83–90.5) 0.18 107.0 (52.0–168.7) 0.87 
  Female        
 Smoking status 87 (88.78) 139.3 (85.83–255.3)  27.5 (7.8–87.7)  106.7 (50.7–170.3)  
  Ever 11 (11.22) 124.0 (107–138.8) 0.50 8.7 (0–26.5) 0.04 108.3 (80.8 –123.5) 0.91 
  Never        
 Age (y)        
  <65 63 (62.4) 144.7 (88.3–311.4)  27.8 (7.1–113.9)  111.5 (53.2–174.2)  
  ≥65 38 (37.6) 128.0 (82.3–194.6) 0.17 18.7 (7.4–52.7) 0.11 105.2 (56.5–167.9) 0.48 
CD103+        
 Patient 98 112.5 (65.7–230.6)  36.8 (15.4–103.9)  70.1 (37.1–135.1)  
 Histological subtype        
  ADC 45 (45.92) 111.2 (63.4–204.3)  23.9 (1.0–72.0)  77.1 (48.1–143.93)  
  SCC 40 (40.82) 99.4 (67.0–192.5)  46.0 (24.3–98.0)  46.3 (29.3–85.5)  
  Other 13 (13.27) 264.3 (87.3–374.8) 0.13 90 (44–166.1) 0.01 155.2 (43.3–181.8) 0.02 
 Stage        
  Ia 32 (32.65) 107.6 (61.0–203.8)  29.5 (8.5–95.7)  61.8 (36.7–104.7)  
  Ib 66 (67.35) 127.6 (69.5–250.8) 0.24 40.7 (17.0–103.9) 0.35 76.4 (37.9–147.0) 0.36 
 Gender        
  Female 31 (31.63) 104.1 (63.3–157.9)  23.9 (7.2–51.9)  71.3 (39.9–125.2)  
  Male 67 (68.37) 128.2 (67.0–274.2) 0.25 55.2 (22.7–146.7) 0.01 69.6 (36.5–142.2) 0.86 
 Smoking status        
  Ever 87 (88.78) 126.9 (67.0–254.8)  41.5 (21.0–121.3)  71.3 (37.4–140.2)  
  Never 11 (11.22) 87.3 (62.4–118.3) 0.12 15.7 (0–35.7) 0.01 48.1 (32.9–90.6) 0.34 
 Age (y)        
  <65 63 (62.4) 136.1 (76.1–273.0)  51.9 (20.5–160.5)  81.4 (47.5–144.9)  
  ≥65 32 (37.6) 97.4 (52.4–145.7) 0.02 26.6 (9.9–65.5) 0.02 64.1 (28.9–119.6) 0.02 
Total
Tumoral
Stromal
Number (%)
Median (Q1–Q3)
p Value
Median (Q1–Q3)
p Value
Median (Q1–Q3)
p Value
CD3+        
 Patient
 
93
 
234.7 (147.7–442.7)
 

 
35.7 (12.3–96.7)
 

 
185.7 (127.0–355.3)
 
 
 Histological subtype        
  ADC 42 (45.16) 209.8 (151.9–402.0)  26.7 (7.9–81.5)  178.7 (132.3–353.9)  
  SCC 40 (43.01) 198.3 (117.6–437.2)  40.8 (16.8–98.8)  158.7 (78.5–325.9)  
  Other 11 (11.83) 474.0 (331.2–598.2) 0.09 85.7 (37.5–145.0) 0.07 303.7 (247.7–475.7) 0.07 
 Stage        
  Ia 30 (32.26) 194.3 (159–391.5)  25.5 (6.2–79.5)  172.6 (129.5–320.8)  
  Ib 63 (67.74) 277.7 (143.8–477.5) 0.45 49 (17.5–108.8) 0.06 199.7 (118.7–377.7) 0.69 
 Gender        
  Male 30 (32.26) 190.5 (137.2–398.4)  32.2 (8.3–84.8)  169.8 (96.3–381.2)  
  Female 63 (67.74) 266.3 (151.8–458.3) 0.48 39.7 (16.8–110.2) 0.27 202.7 (133.7–346.8) 0.67 
 Smoking status        
  Ever 83 (89.25) 257.0 (145.8–442.2)  41 (18.5–103.5)  185.7 (118.7–352.5)  
  Never 10 (10.75) 202.8 (154.9–502.0) 0.92 15.2 (1.3–68.2) 0.06 178.1 (136.7–429.6) 0.53 
 Age (y)        
  <65 63 (62.4) 277.7 (168.3–442.7)  47.3 (20–106)  209 (143.3–355.3)  
  ≥65 38 (37.6) 187.3 (117.6–439.7) 0.07 28 (8.4–87.2) 0.09 158.7 (68.0–327.2) 0.16 
CD8+        
 Patient 98 135.0 (86.8–249.2)  24.3 (7.4–80.1)  106.8 (53.6–169.9)  
 Histological subtype        
  ADC 45 (45.92) 132.0 (83.0–225.7)  12.3 (2–48.7)  111.7 (65.7–170.7)  
  SCC 40 (40.82) 115.3 (87.6–247.2)  27.6 (12.5–71.1)  82.7 (46.3–140.9)  
  Other 13 (13.27) 331.3 (124.0–411.7) 0.03 85 (27.5–148.7) 0.02 173.7 (116.7–263.0) 0.01 
 Stage        
  Ia 32 (32.65) 124.0 (54.67–248.7)  16 (4.3–56.8)  111.5 (53.3–150.9)  
  Ib 66 (67.35) 138.7 (91.08–249.3) 0.53 27.6 (8.6–80.1) 0.24 103.5 (60.9–174.4) 0.76 
 Gender 31 (31.63) 124.0 (84.17–209.3)  22.7 (3.2–50.5)  103.7 (57.4–170.2)  
  Male 67 (68.37) 139.3 (91.5–251.5) 0.50 27.5 (7.83–90.5) 0.18 107.0 (52.0–168.7) 0.87 
  Female        
 Smoking status 87 (88.78) 139.3 (85.83–255.3)  27.5 (7.8–87.7)  106.7 (50.7–170.3)  
  Ever 11 (11.22) 124.0 (107–138.8) 0.50 8.7 (0–26.5) 0.04 108.3 (80.8 –123.5) 0.91 
  Never        
 Age (y)        
  <65 63 (62.4) 144.7 (88.3–311.4)  27.8 (7.1–113.9)  111.5 (53.2–174.2)  
  ≥65 38 (37.6) 128.0 (82.3–194.6) 0.17 18.7 (7.4–52.7) 0.11 105.2 (56.5–167.9) 0.48 
CD103+        
 Patient 98 112.5 (65.7–230.6)  36.8 (15.4–103.9)  70.1 (37.1–135.1)  
 Histological subtype        
  ADC 45 (45.92) 111.2 (63.4–204.3)  23.9 (1.0–72.0)  77.1 (48.1–143.93)  
  SCC 40 (40.82) 99.4 (67.0–192.5)  46.0 (24.3–98.0)  46.3 (29.3–85.5)  
  Other 13 (13.27) 264.3 (87.3–374.8) 0.13 90 (44–166.1) 0.01 155.2 (43.3–181.8) 0.02 
 Stage        
  Ia 32 (32.65) 107.6 (61.0–203.8)  29.5 (8.5–95.7)  61.8 (36.7–104.7)  
  Ib 66 (67.35) 127.6 (69.5–250.8) 0.24 40.7 (17.0–103.9) 0.35 76.4 (37.9–147.0) 0.36 
 Gender        
  Female 31 (31.63) 104.1 (63.3–157.9)  23.9 (7.2–51.9)  71.3 (39.9–125.2)  
  Male 67 (68.37) 128.2 (67.0–274.2) 0.25 55.2 (22.7–146.7) 0.01 69.6 (36.5–142.2) 0.86 
 Smoking status        
  Ever 87 (88.78) 126.9 (67.0–254.8)  41.5 (21.0–121.3)  71.3 (37.4–140.2)  
  Never 11 (11.22) 87.3 (62.4–118.3) 0.12 15.7 (0–35.7) 0.01 48.1 (32.9–90.6) 0.34 
 Age (y)        
  <65 63 (62.4) 136.1 (76.1–273.0)  51.9 (20.5–160.5)  81.4 (47.5–144.9)  
  ≥65 32 (37.6) 97.4 (52.4–145.7) 0.02 26.6 (9.9–65.5) 0.02 64.1 (28.9–119.6) 0.02 

The p value was determined using the Wilcoxon rank or Kruskal–Wallis test when appropriate. Bold type indicates significant (p <0.05).

Q1 and Q3, the first and the third quartile, respectively; stage Ia, pT1N0; stage Ib, pT2N0.

We then tested whether a high density of CD103+ TIL correlated with a favorable clinical outcome. The median follow-up was 6.3 y. Results revealed that CD103+ TIL significantly correlated with favorable clinical outcome as a continuous variable in DFS and OS in univariate analysis (Table II). For each 50 CD103+ increment, the risk of relapse or death was reduced by 16% (unadjusted hazard ratio [HR] = 0.84, 95% confidence interval [CI], p = 0.01) and a 12% reduction in risk of death (unadjusted HR = 0.88, 95% CI, p = 0.04). Notably, age, stage, and histology at diagnosis were significantly associated with patient outcome, whereas for gender, a trend toward a significant association was observed. Multivariate analysis was then carried out, including these four factors together with CD103+ TIL. As summarized in Table II, CD103+ lymphocytes maintained a strong prognostic value in the multivariate model for DFS, but not for OS (HR = 0.85, 95% CI, p = 0.02 and HR = 0.89, 95% CI, p = 0.09 for DFS and OS, respectively). Notably, none of the other parameters that were independent predictors of outcome remained significantly associated with DFS (Table III). Similar results were obtained when we analyzed the association of CD103+ TIL in tumor regions with patient OS (Table II, Supplemental Table I). With regard to CD8+ TIL, no correlation with OS was found. However, an association with DFS was observed in univariate analyses only for either CD8+ total or for CD8+ lymphocytes within tumor islets (Table II, Supplemental Table I). CD8+ TIL within epithelial tumor regions remained significantly associated with DFS in multivariate analysis (Table II). In contrast, the CD3 marker was never associated with patient outcome. These results suggested that CD103 promoted T cell infiltration within epithelial tumor islets and indicated that a high density of CD103+ TIL was associated with stage I NSCLC patient survival.

Table II.
Association of CD3, CD8, and CD103 with prognosis in univariate and multivariate analyses

No. of Patients
No. of Events
UnivariateMultivariate
HR (95% CI) for 50 Increment Increase
p Value
HR (95% CI) for 50 Increment Increase
p Value
OS       
 Total       
  CD3 93 44 0.97 (0.91–1.04) 0.4   
  CD8 98 45 0.9 (0.80–1.02) 0.1   
  CD103 98 45 0.88 (0.77–0.99) 0.04 (0.78–1.02) 0.09 
 Epithelial tumor islets       
  CD3 93 44 0.94 (0.76–1.15) 0.5   
  CD8 98 45 0.83 (0.63–1.09) 0.2   
  CD103 98 45 0.84 (0.67–1.06) 0.09 (0.66–1.05) 0.13 
 Stromal region       
  CD3 93 44 0.97 (0.88–1.06) 0.5   
  CD8 98 45 0.88 (0.74–1.05) 0.2   
  CD103 98 45 0.78 (0.62–0.98) 0.03 (0.67–1.06) 0.14 
DFS       
 Total       
  CD3 92 48 0.96 (0.89–1.02) 0.2   
  CD8 97 50 0.88 (0.78–0.99) 0.04 (0.78–1.02) 0.09 
  CD103 97 50 0.84 (0.74–0.96) 0.01 (0.74–0.97) 0.02 
 Epithelial tumor islets       
  CD3 92 48 0.82 (0.65–1.03) 0.09   
  CD8 97 50 0.72 (0.54–0.96) 0.02 (0.51–0.97) 0.03 
  CD103 97 50 0.78 (0.63–0.96) 0.02 (0.61–0.96) 0.02 
 Stromal region       
  CD3 92 48 0.96 (0.89–1.05) 0.4   
  CD8 97 50 0.88 (0.74–1.04) 0.1   
  CD103 97 50 0.76 (0.61–0.94) 0.01 (0.64–1.01) 0.06 

No. of Patients
No. of Events
UnivariateMultivariate
HR (95% CI) for 50 Increment Increase
p Value
HR (95% CI) for 50 Increment Increase
p Value
OS       
 Total       
  CD3 93 44 0.97 (0.91–1.04) 0.4   
  CD8 98 45 0.9 (0.80–1.02) 0.1   
  CD103 98 45 0.88 (0.77–0.99) 0.04 (0.78–1.02) 0.09 
 Epithelial tumor islets       
  CD3 93 44 0.94 (0.76–1.15) 0.5   
  CD8 98 45 0.83 (0.63–1.09) 0.2   
  CD103 98 45 0.84 (0.67–1.06) 0.09 (0.66–1.05) 0.13 
 Stromal region       
  CD3 93 44 0.97 (0.88–1.06) 0.5   
  CD8 98 45 0.88 (0.74–1.05) 0.2   
  CD103 98 45 0.78 (0.62–0.98) 0.03 (0.67–1.06) 0.14 
DFS       
 Total       
  CD3 92 48 0.96 (0.89–1.02) 0.2   
  CD8 97 50 0.88 (0.78–0.99) 0.04 (0.78–1.02) 0.09 
  CD103 97 50 0.84 (0.74–0.96) 0.01 (0.74–0.97) 0.02 
 Epithelial tumor islets       
  CD3 92 48 0.82 (0.65–1.03) 0.09   
  CD8 97 50 0.72 (0.54–0.96) 0.02 (0.51–0.97) 0.03 
  CD103 97 50 0.78 (0.63–0.96) 0.02 (0.61–0.96) 0.02 
 Stromal region       
  CD3 92 48 0.96 (0.89–1.05) 0.4   
  CD8 97 50 0.88 (0.74–1.04) 0.1   
  CD103 97 50 0.76 (0.61–0.94) 0.01 (0.64–1.01) 0.06 

HR (95% CI) was calculated for an increment of 50 for CD3, CD8, and CD103.

The p value was determined using the Wilcoxon rank or Kruskal–Wallis test when appropriate. Bold type indicates significant (p <0.05).

Table III.
Complete results of the univariate and the multivariate analyses for the DFS outcome

Univariate
Multivariatea
Multivariateb
Multivariate
c
Multivariate
d
Multivariatee
HR95% CI
p Value
HR
95% CI
p Value
HR
95% CI
p Value
HR
95% CI
p Value
HR
95% CI
p Value
HR
95% CI
p Value
For an increase of 50 U                  
 CD3 total 0.96 (0.89–1.02) 0.2                
 CD3 tumor 0.82 (0.65–1.03) 0.09                
 CD3 stroma 0.96 (0.89–1.05) 0.4                
 CD8 total 0.88 (0.78–0.99) 0.04 0.89 (0.78–1.02) 0.09             
 CD8 tumor 0.72 (0.54–0.96) 0.02    0.70 (0.51–0.97) 0.03          
 CD8 stroma 0.88 (0.74–1.04) 0.1                
 CD103 total 0.84 (0.74–0.96) 0.01       0.85 (0.74–0.97) 0.02       
 CD103 tumor 0.78 (0.63–0.96) 0.02          0.76 (0.61–0.96) 0.02    
 CD103 Stroma 0.76 (0.61–0.94) 0.01             0.80 (0.64–1.01) 0.06 
Age (y)                   
 <65 1.00   1.00   1.00   1.00   1.00   1.00   
 ≥65 2.48 (1.38–4.44) 0.002 1.88 (1.01–3.5) 0.05 1.74 (0.93–3.24) 0.08 1.59 (0.84–3) 0.15 1.57 (0.83–2.98) 0.16 1.81 (0.96–3.38) 0.07 
Gender                   
 Female 1.00   1.00   1.00   1.00   1.00   1.00   
 Male 1.34 (0.72–2.51) 0.4 1.59 (0.78–3.2) 0.20 1.55 (0.76–3.13) 0.23 1.63 (0.81–3.25) 0.17 1.71 (0.85–3.44) 0.13 1.56 (0.78–3.13) 0.21 
Smoking status                   
 Ever 1.00                  
 Never 1.09 (0.46–2.56) 0.8                
Stage                   
 Ia 1.00   1.00   1.00   1.00   1.00   1.00   
 Ib 1.54 (0.81–2.92) 0.2 1.54 (0.75–3.16) 0.24 1.62 (0.79–3.32) 0.19 1.69 (0.82–3.47) 0.15 1.64 (0.8–3.35) 0.18 1.58 (0.77–3.24) 0.21 
Histology                   
 ADC 1.00   1.00   1.00   1.00   1.00   1.00   
 SCC 1.89 (1.04–3.45) 0.04 1.38 (0.69–2.76) 0.36 1.53 (0.76–3.08) 0.24 1.40 (0.7–2.78) 0.34 1.55 (0.77–3.1) 0.22 1.29 (0.65–2.59) 0.47 
 Others 0.82 (0.28–2.42) 0.7 0.89 (0.31–2.58) 0.83 1.05 (0.36–3.08) 0.93 1.02 (0.35–2.95) 0.97 1.10 (0.38–3.24) 0.86 0.85 (0.3–2.42) 0.76 

Univariate
Multivariatea
Multivariateb
Multivariate
c
Multivariate
d
Multivariatee
HR95% CI
p Value
HR
95% CI
p Value
HR
95% CI
p Value
HR
95% CI
p Value
HR
95% CI
p Value
HR
95% CI
p Value
For an increase of 50 U                  
 CD3 total 0.96 (0.89–1.02) 0.2                
 CD3 tumor 0.82 (0.65–1.03) 0.09                
 CD3 stroma 0.96 (0.89–1.05) 0.4                
 CD8 total 0.88 (0.78–0.99) 0.04 0.89 (0.78–1.02) 0.09             
 CD8 tumor 0.72 (0.54–0.96) 0.02    0.70 (0.51–0.97) 0.03          
 CD8 stroma 0.88 (0.74–1.04) 0.1                
 CD103 total 0.84 (0.74–0.96) 0.01       0.85 (0.74–0.97) 0.02       
 CD103 tumor 0.78 (0.63–0.96) 0.02          0.76 (0.61–0.96) 0.02    
 CD103 Stroma 0.76 (0.61–0.94) 0.01             0.80 (0.64–1.01) 0.06 
Age (y)                   
 <65 1.00   1.00   1.00   1.00   1.00   1.00   
 ≥65 2.48 (1.38–4.44) 0.002 1.88 (1.01–3.5) 0.05 1.74 (0.93–3.24) 0.08 1.59 (0.84–3) 0.15 1.57 (0.83–2.98) 0.16 1.81 (0.96–3.38) 0.07 
Gender                   
 Female 1.00   1.00   1.00   1.00   1.00   1.00   
 Male 1.34 (0.72–2.51) 0.4 1.59 (0.78–3.2) 0.20 1.55 (0.76–3.13) 0.23 1.63 (0.81–3.25) 0.17 1.71 (0.85–3.44) 0.13 1.56 (0.78–3.13) 0.21 
Smoking status                   
 Ever 1.00                  
 Never 1.09 (0.46–2.56) 0.8                
Stage                   
 Ia 1.00   1.00   1.00   1.00   1.00   1.00   
 Ib 1.54 (0.81–2.92) 0.2 1.54 (0.75–3.16) 0.24 1.62 (0.79–3.32) 0.19 1.69 (0.82–3.47) 0.15 1.64 (0.8–3.35) 0.18 1.58 (0.77–3.24) 0.21 
Histology                   
 ADC 1.00   1.00   1.00   1.00   1.00   1.00   
 SCC 1.89 (1.04–3.45) 0.04 1.38 (0.69–2.76) 0.36 1.53 (0.76–3.08) 0.24 1.40 (0.7–2.78) 0.34 1.55 (0.77–3.1) 0.22 1.29 (0.65–2.59) 0.47 
 Others 0.82 (0.28–2.42) 0.7 0.89 (0.31–2.58) 0.83 1.05 (0.36–3.08) 0.93 1.02 (0.35–2.95) 0.97 1.10 (0.38–3.24) 0.86 0.85 (0.3–2.42) 0.76 

HR (95% CI) was calculated for an increment of 50 for CD3, CD8, and CD103. The p value was determined using the Wald test when appropriate. Bold type indicates significant (p <0.05).

ae

Five multivariate Cox regression models adjusted on the clinical factors (age, gender, smoking status, stage, and histology), and including CD8 totala, CD8 tumorb, CD103 totalc, CD103 tumord, and CD103 stromae.

Stage Ia, pT1N0; stage Ib, pT2N0.

Little has been known about the phenotypic and functional features of human epithelial TIL and their contribution in controlling tumor progression. Experiments were therefore conducted to determine the transcriptional profiles of freshly isolated NSCLC TIL, which include 85% (mean, 82 ± 5%) of CD3+, 51% (mean, 51 ± 7%) of CD4+, and 37% (mean, 37 ± 11%) of CD8+ T lymphocytes, respectively (Supplemental Fig. 1A). For this purpose, we cell sorted TIL from 13 independent NSCLC specimens, including 5 SCC, 1 large cell carcinoma, and 7 ADC, and compared their transcriptional profiles to PBMC from 19 independent NSCLC patients (Supplemental Fig. 1A) by microarray analysis. Differential gene expression analyses performed with a p value of ≤10−10 and a fold change of ≥2 identified 487 genes, 233 of which were less strongly expressed in TIL than in PBMC, and 254 genes that were more strongly expressed in TIL than PBMC (Supplemental Fig. 1B). Further filtering, acquired with an overall TIL signal intensity ≥1000, identified an expression profile of 103 genes (Supplemental Fig. 2A), including a cluster of 33 genes related to adhesion, exhaustion, activation, immunosuppression, and apoptosis, which were more strongly expressed in TIL than in PBMC (Supplemental Table II). The gene expression profile also included a cluster of 70 genes that, on the contrary, were less strongly expressed in TIL than in PBMC (Supplemental Table II). For adhesion and exhaustion, RGS1, RALGDS and ITGAE, and PDCD1, LAG-3, CTLA-4, TNFRSF-18,and HAVCR2 were the most strongly overexpressed genes in TIL, respectively (Supplemental Table II). These results were further confirmed by qRT-PCR analyses (Supplemental Fig. 2B), which also showed a correlation with gene chip expression profiles (Supplemental Fig. 2C) and did not reveal any differences between SCC and ADC histological types. These results suggest that a subset of TIL expresses higher levels of TRM T cell marker genes than do PBMC.

Next, we purified the CD103+ TIL subpopulation and analyzed the expression of the selected 33 genes by qRT-PCR. Initial phenotypic analyses indicated that >80% of CD103+ cells were CD3+ T lymphocytes, whereas <20% were CD11c+CD83+ dendritic cells (Fig. 2A). Moreover, most CD3+CD103+ T lymphocytes were CD8+, with 8–53% (mean, 27 ± 13; n = 21) of CD8+ T cells and only 1–10% (mean, 7 ± 6; n = 21) of CD4+ T cells (Fig. 2B). qRT-PCR indicated that the CD3+CD8+CD103+ TIL subpopulation, isolated from 10 NSCLC, expressed more strongly the TRM T cell markers PDCD1, CTLA4, and HAVCR2 genes, encoding PD-1, CTLA-4, and Tim-3, respectively, as well as RGS1, HSPA1A, ICOS, NR4A2, EGR1, and BAG-3, also described as markers of TRM T cells (18, 19), than total PBMC (Fig. 2C). In contrast, SIP1 was less strongly expressed in CD3+CD8+CD103+ TIL than in total PBMC. Moreover, all of these TRM T cell markers were more strongly expressed in CD3+CD8+CD103+ TIL than in CD3+CD8+CD103 PBMC (Fig. 2D). These results indicate that lung TIL often include a large subset of CD8+CD103+ T cells and suggest that, along with CD103 expression, this subset exhibits TRM T cell features.

FIGURE 2.

Expression of CD103 integrin on TIL from NSCLC specimens. Freshly resected NSCLC tumors were dissociated and then directly analyzed by flow cytometry for CD103, CD3, CD8, CD4, CD83, and CD11c expression. (A) Surface expression of CD3 (black fill), CD11c, and CD83 on CD103+ TIL. An isotypic control mAb was included (gray fill). Three independent experiments are shown. Lower panel, Mean percentages of CD3+ T cells and CD11c+CD83+ dendritic cells among CD103+ TIL (n = 12). (B) Surface expression of CD103 on CD8+ and CD4+ TIL from three independent NSCLC. Percentages of positive cells are indicated. Numbers in parentheses correspond to mean fluorescence intensity. Right panel, Mean percentages of CD8+CD103+ and CD4+CD103+ cells among CD3+ T cells (n = 21). (C) Genes differentially expressed in CD3+CD8+CD103+ TIL, cell sorted from 10 NSCLC with anti-CD103 mAb, and patient total PBMC. Genes described as markers of TRM T cells are shown in bold type. (D) TRM T cell marker genes differentially expressed in CD3+CD8+CD103+ TIL versus CD3+CD8+CD103 PBMC. ***p < 0.0001.

FIGURE 2.

Expression of CD103 integrin on TIL from NSCLC specimens. Freshly resected NSCLC tumors were dissociated and then directly analyzed by flow cytometry for CD103, CD3, CD8, CD4, CD83, and CD11c expression. (A) Surface expression of CD3 (black fill), CD11c, and CD83 on CD103+ TIL. An isotypic control mAb was included (gray fill). Three independent experiments are shown. Lower panel, Mean percentages of CD3+ T cells and CD11c+CD83+ dendritic cells among CD103+ TIL (n = 12). (B) Surface expression of CD103 on CD8+ and CD4+ TIL from three independent NSCLC. Percentages of positive cells are indicated. Numbers in parentheses correspond to mean fluorescence intensity. Right panel, Mean percentages of CD8+CD103+ and CD4+CD103+ cells among CD3+ T cells (n = 21). (C) Genes differentially expressed in CD3+CD8+CD103+ TIL, cell sorted from 10 NSCLC with anti-CD103 mAb, and patient total PBMC. Genes described as markers of TRM T cells are shown in bold type. (D) TRM T cell marker genes differentially expressed in CD3+CD8+CD103+ TIL versus CD3+CD8+CD103 PBMC. ***p < 0.0001.

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To further characterize CD3+CD8+CD103+ TIL, we performed multicolor immunofluorescent staining. Flow cytometry analyses of TIL from five tumors (two SCC and three ADC) showed a homogeneous T cell subpopulation with a CD69+CD62LCD28CD27+CD45RA+CD45RO+CCR7 phenotype characteristic of memory CD8+ T cells. Moreover, TIL from SCC and ADC tumors displayed similar T cell phenotypes. Results also indicated that these lymphocytes expressed chemokine receptors CCR5 and CCR6 (Fig. 3A), and the prosurvival family member Bcl-2, but not KLRG1, and only low levels of CD127 (Supplemental Fig. 1C). Remarkably, whereas CD3+CD8+CD103+ TIL expressed high levels of PD-1 and Tim-3, only a small subset of CD3+CD8+CD103 TIL expressed these inhibitory receptors, and CTLA-4 was slightly expressed by both subpopulations (Fig. 3B).

FIGURE 3.

Characterization of T cell surface markers of CD3+CD8+CD103+ TIL. (A) TIL from freshly isolated NSCLC tumors were analyzed by flow cytometry using indicated mAb (black fill) or corresponding isotypic controls (gray fill). Two representative experiments from five are shown. (B) Surface expression of PD-1, Tim3, and CTLA-4 among CD103+ and CD103 TIL. Three representative experiments are included. Right panel, Mean percentages of PD-1+, Tim-3+, and CTLA-4+ cells among CD3+CD8+ TIL expressing (or not) CD103 (n = 12). (C) AICD of CD3+CD8+ TIL expressing (or not) CD103. Percentages of apoptotic cells among CD3+CD8+CD103+ and CD3+CD8+CD103 TIL. Size-selected freshly isolated TIL were cultured with cell-sorted autologous tumor cells and then analyzed for apoptosis induction. Three independent NSCLC tumors are included. Annexin V+PI apoptotic cells are shown in the lower right quadrant. Right panel, Mean percentages of annexin V+PI apoptotic lymphocytes among CD3+CD8+ TIL expressing (or not) CD103 (n = 5). *p < 0.05, ***p < 0.0001.

FIGURE 3.

Characterization of T cell surface markers of CD3+CD8+CD103+ TIL. (A) TIL from freshly isolated NSCLC tumors were analyzed by flow cytometry using indicated mAb (black fill) or corresponding isotypic controls (gray fill). Two representative experiments from five are shown. (B) Surface expression of PD-1, Tim3, and CTLA-4 among CD103+ and CD103 TIL. Three representative experiments are included. Right panel, Mean percentages of PD-1+, Tim-3+, and CTLA-4+ cells among CD3+CD8+ TIL expressing (or not) CD103 (n = 12). (C) AICD of CD3+CD8+ TIL expressing (or not) CD103. Percentages of apoptotic cells among CD3+CD8+CD103+ and CD3+CD8+CD103 TIL. Size-selected freshly isolated TIL were cultured with cell-sorted autologous tumor cells and then analyzed for apoptosis induction. Three independent NSCLC tumors are included. Annexin V+PI apoptotic cells are shown in the lower right quadrant. Right panel, Mean percentages of annexin V+PI apoptotic lymphocytes among CD3+CD8+ TIL expressing (or not) CD103 (n = 5). *p < 0.05, ***p < 0.0001.

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Next, we compared the activation status of CD8+CD103+ TIL to CD8+CD103 TIL. For this purpose, freshly size-selected TIL were stimulated with cell-sorted autologous tumor cells, and CD3+CD8+ lymphocytes were gated and analyzed for CD103, annexin V, and PI staining. As shown in Fig. 3C, annexin V+PI apoptotic cells comprised 19, 17, and 33% of CD103+ T cells from patients 97, 98, and 99, whereas they comprised only 4, 1, and 6% of CD103 T cells, respectively. Higher susceptibility of CD103+ T cells to TCR activation-dependent apoptosis was further confirmed in TIL from two additional patients (Fig. 3C). These results further support the hypothesis that CD8+CD103+ TIL belong to a TRM subset and that they are rapidly activated following ex vivo stimulation with autologous tumor cells.

The above results suggested that the CD8+CD103+ TIL subpopulation was enriched with tumor-reactive T lymphocytes. To test this hypothesis, we examined whether CD8+CD103+ T cells specifically recognized autologous tumor cells. To this end, freshly isolated TIL from 11 of 18 tumors were isolated and included in functional assays. Among these 11 TIL populations, 5 were tested for their capacity to degranulate after stimulation with cell-sorted target cells. Results indicated that CD8+CD103+ TIL expressed much higher levels of the CD107a molecule than did CD8+CD103 TIL following ex vivo stimulation with autologous tumor cells in the presence of low doses of rIL-2 (Fig. 4A). We then examined granzyme B expression in CD8+CD103+ and CD8+CD103 lymphocytes unstimulated or stimulated with low concentrations of rIL-2. Intracytoplasmic immunofluorescence analyses indicated that nonstimulated CD8+ T cell subsets did not express granzyme B, but stimulation of size-selected TIL with rIL-2 induced a much higher increase in serine protease expression in CD103+ than in CD103 T cells (Fig. 4B).

FIGURE 4.

Functional characterization of freshly size-selected TIL. (A) CD107a induction on CD3+CD8+ TIL expressing (or not) CD103. TIL, cultured in medium or in the presence of rIL-2, were stimulated with autologous tumor cells, and then CD3+CD8+CD103+ and CD3+CD8+CD103 lymphocytes were analyzed for surface expression of CD107a. Two representative experiments are included. Right panel, Mean percentages of CD107a+ lymphocytes among CD3+CD8+ T cells expressing (or not) CD103 (n = 5). (B) Expression of granzyme B in CD3+CD8+CD103+ and CD3+CD8+CD103- TIL, unstimulated or stimulated with rIL-2. Two representative experiments are included. Right panel, Mean percentages of granzyme B–expressing CD3+CD8+CD103+ and CD3+CD8+CD103 lymphocytes (n = 3). (C) Cytotoxic activity of freshly isolated TIL toward autologous tumor cells. TIL were preincubated or not with anti–PD-1 blocking mAb and then cultured with autologous tumor cells, untreated or pretreated with anti–PD-L1, in the absence or presence of anti–MHC-I mAb. Anti–MHC-I alone and IgG1 isotypic control were included. (D) Inhibition of TIL-mediated tumor cell killing with neutralizing anti-CD103 mAb. TIL were preincubated with anti-CD103 mAb and then cultured with target cells pretreated with anti–PD-L1 mAb or IgG1 isotypic control. *p < 0.05, **p < 0.001, ***p < 0.0001.

FIGURE 4.

Functional characterization of freshly size-selected TIL. (A) CD107a induction on CD3+CD8+ TIL expressing (or not) CD103. TIL, cultured in medium or in the presence of rIL-2, were stimulated with autologous tumor cells, and then CD3+CD8+CD103+ and CD3+CD8+CD103 lymphocytes were analyzed for surface expression of CD107a. Two representative experiments are included. Right panel, Mean percentages of CD107a+ lymphocytes among CD3+CD8+ T cells expressing (or not) CD103 (n = 5). (B) Expression of granzyme B in CD3+CD8+CD103+ and CD3+CD8+CD103- TIL, unstimulated or stimulated with rIL-2. Two representative experiments are included. Right panel, Mean percentages of granzyme B–expressing CD3+CD8+CD103+ and CD3+CD8+CD103 lymphocytes (n = 3). (C) Cytotoxic activity of freshly isolated TIL toward autologous tumor cells. TIL were preincubated or not with anti–PD-1 blocking mAb and then cultured with autologous tumor cells, untreated or pretreated with anti–PD-L1, in the absence or presence of anti–MHC-I mAb. Anti–MHC-I alone and IgG1 isotypic control were included. (D) Inhibition of TIL-mediated tumor cell killing with neutralizing anti-CD103 mAb. TIL were preincubated with anti-CD103 mAb and then cultured with target cells pretreated with anti–PD-L1 mAb or IgG1 isotypic control. *p < 0.05, **p < 0.001, ***p < 0.0001.

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To assess whether CD8+CD103+granzyme B+ T cells were able to kill autologous tumor cells, which include 93 ± 3% MHC-I+, 85 ± 5% Epcam+, 70 ± 21% E-cadherin+, and 61 ± 18% PD-L1+ cells (Supplemental Fig. 1D), we performed a chromium release assay in the absence or presence of anti–PD-1 mAb combined or not with anti–PD-L1. Results included in Fig. 4C show that TIL, previously stimulated with rIL-2, displayed a weak cytotoxic activity toward autologous size-selected tumor cells. However, blocking of PD-1 or PD-L1 with neutralizing mAb induced a strong increase in target cell lysis. Moreover, anti–MHC-I (Fig. 4C) and anti-CD103 (Fig. 4D) blocking mAb strongly inhibited tumor cell killing by autologous TIL pretreated with anti–PD-1 and/or in the presence of anti–PD-L1 mAb, respectively. These results further emphasized that CD8+CD103+ TIL were exhausted tumor-specific T lymphocytes and that a subset could be rescued by blocking PD-1 signals leading to T cell activation and autologous tumor cell lysis. They also suggest that this T cell subpopulation mediates in situ antitumor CTL response that leads to improved patient survival.

In this study, we show that NSCLC TIL include a homogeneous CD8+ T cell population that displays a phenotypic signature associated with TRM cells, mainly expression of CD103 and CD69 and the absence of CCR7. TRM T cells are highly activated T lymphocytes that reside within a variety of peripheral tissues, including intestine (17, 20), brain (21), skin (22, 23), and lung (24), and provide early responses to viral reinfection (25). Accumulating evidence indicates that TGF-β1 plays an essential role in the formation and maintenance of TRM T cells, at least in part via induction of CD103 and CD69. Although the natural ligand for CD69 has not been clearly defined, evidence for an interaction of CD69 with S1P1, a receptor for sphingosine 1-phosphate that mediates the egress of T cells from lymphoid organs (26), has been suggested to also play a role in retention of TRM T cells by modulating their capacity to exit nonlymphoid tissues (27). Indeed, downregulation of SIP1 was observed in CD103+CD8+ TRM T cells (18) and appeared to be required for their retention in peripheral tissues (28). Our results revealed that CD103+ TIL display a transcriptomic signature characteristic of TRM cells, with downregulation of S1P1 and upregulation of RGS1 and ICOS as well as genes encoding PD-1 and Tim-3, along with Bag-3 and transcription factors EGR1 and Nr4a2 (18, 19). Microarray studies also showed downregulation of the ITGB2 gene in TIL (Supplemental Table II), suggesting that dampening of the αLβ2 integrin, most likely through TGF-β1 secretion, also participates in T cell residency within the tumor. Additionally, CD8+CD103+ TIL are able to mediate tumor-specific cytotoxic activity toward autologous malignant cells after blockade of PD-1. These results further support the notion that CD103 is induced on tumor-specific T cells upon entry into a TGF-β1–rich tumor microenvironment and interaction of TCR with the specific tumor peptide/MHC-I complex (29). Persistent tumor Ag stimulation, together with TGF-β1 secretion, are likely required for maintaining high expression levels of CD103 on TIL. Indeed, frequent stimulation of CD8+CD103+ T cell clones (3) with autologous tumor Ag–expressing feeder cells is required to maintain high surface expression levels of CD103. Consistently, CD103 was maintained on vesicular stomatitis virus-specific CD8+ T cells only as long as the Ag was present (21). Moreover, influenza virus–specific T lymphocytes, but not EBV- and CMV-specific T lymphocytes, express the integrin, further emphasizing that a sufficient Ag amount is needed to maintain CD103 expression (24).

The present study also suggests that CD103 is involved, at least in part, in recruitment of CD8+ T cells in lung tumor islets and provides evidence that a high density of CD103+ TIL is associated with early stage NSCLC patient survival. In our series of NSCLC patients, the density of CD103+ cells ranged from 2 to 697/mm2 of tumor area. The essential cause of this variation is not clearly understood, but it may reflect interpatient variations in the intratumoral level of TGF-β1 or factors involved in its activation, such as αV integrins (30, 31), the number of tumor Ag-specific T cell precursors, or both. Indeed, as mentioned above, engagement of both the TGF-β1 receptor and TCR via an active form of TGF-β1 and the tumor peptide/MHC-I complex, respectively, is required for CD103 induction (11). Thus, TGF-β1, considered as an immunosuppressive cytokine, can control immune responses to viral infections and tumors via induction of CD103 and formation of CD8+CD103+ TRM T cells. The CD103 lymphocytes observed in several NSCLC tumors might represent either nonspecific T cells passing through the lung tumor lesion or recently recruited tumor-specific T cells that do not yet express the integrin. Indeed, a prolonged period (8–10 d) of residency of human tumor-specific T cells within the cognate tumor, engrafted in immunodeficient mice, was needed for CD103 induction (29). The notion that CD103 is a marker of Ag-reactive T lymphocytes is further supported by studies of antiviral and antitumor CTL responses in which CD103 is expressed on CD8+ T cells specific to influenza virus in the lung (24), EBV in the tonsil (32), and cancer testis Ag NY-ESO-1 in ovarian cancer (9). In the latter and present studies, CD103+ T cells expressed high levels of PD-1, which has been defined as a biomarker of tumor-specific T cells (33). More importantly, we show in the present study a correlation between dense tumor infiltration by CD103+ TIL, without the need to discriminate their location in epithelial versus stromal regions, and NSCLC patient DFS. Similar results were obtained in ovarian cancer, and CD103 expression on TIL was also associated with increased patient survival (10). These results suggest that CD103 alone may be used as a prognostic biomarker in epithelial tumors. Conversely, no correlation between total CD8+ TIL and NSCLC patient survival was observed unless they were located in intraepithelial tumor regions, in which case improved DFS outcome was observed. Thus, CD103 identifies a subpopulation of CD8+ T cells that more accurately predicts patient outcome.

Immune checkpoints are important for maintaining self-tolerance and regulating immune responses in peripheral tissues (34). Among these inhibitory receptors, CTLA-4, PD-1, and Tim-3 appeared to be associated with T cell exhaustion in chronic viral infections (3538) and tumor progression (39). Exhausted CD8+ T cells frequently coexpress PD-1 and Tim-3, and triggering of both molecules has been reported to transmit a death signal to T cells (40, 41). It has also been suggested that tumor cells use the PD-1/PD-L1 pathway to escape CD8+ T cell immunity by inducing T cell apoptosis (42). Our data indicate that CD103+ TIL frequently express PD-1 and Tim-3, and that this T cell subset is much more highly susceptible to AICD than CD103 T cells. Moreover, anti–PD-1 neutralizing mAb promotes tumor cell killing by autologous TIL, supporting the conclusion that CD103+ TIL correspond to an activated CD8+ T cell subpopulation, and that blocking PD-1–PD-L1 interactions enhances antitumor effector functions. It is now admitted that binding of PD-1, frequently upregulated on tumor-specific T cells, to its ligand PD-L1 on malignant cells results in inhibition of T lymphocyte activation (41). Targeting of the PD-1/PD-L1 pathway to enhance antitumor immunity is supported by the recent success of anti–PD-1 and anti–PD-L1-based cancer immunotherapy in patients with various types of advanced solid tumors, including NSCLC (43, 44).

We previously reported that TIL entry into the tumor site requires, at least in part, expression of CCR5, and that CCR6 is induced within the tumor and likely participates in intratumoral T cell migration (29). In the present study we show that freshly isolated TIL also express high levels of both chemokine receptors, suggesting that they may be involved in the formation of CD8+CD103+ TRM T cells in human lung tumors. This is consistent with our previous findings demonstrating that CD103-mediated recruitment of CCR5 at the immune synapse between TIL and specific cancer cells contributes, at least in part, to T cell retention within the tumor by inhibiting lymphocyte responsiveness to CCL5 (29). CCR5 ligand CCL5 was one of the major chemokines involved in regulating antitumor immune responses (45). Increased levels of CCR5 ligands have been demonstrated to improve antitumor functions by inducing effector T cell infiltration (46). Indeed, intratumoral production of CCL5 and CCL3 was reported to promote recruitment of CD8+ T cells and, thereby, CTL-dependent control of tumor progression (47). Moreover, CCR5 deficiency was correlated with reduced CD8+ T cell infiltration, poor prognosis (48, 49), and reduced efficacy of cancer immunotherapy (50).

Overall, our results demonstrate a major role for CD8+CD103+ T cells infiltrating human epithelial tumor lesions in the antitumor immune response and identify CD103 as a biomarker of tumor-reactive CD8+ TIL. Indeed, CD103 delineates a highly activated tumor-specific T cell subset that is able to kill malignant cells upon neutralization of the immune checkpoint receptor PD-1. Thus, investigating freshly isolated patient TIL contributes not only to our understanding of the local tumor-specific CTL function in NSCLC, but may also provide insight into the role of TRM T cell populations in antitumor immune response. By characterizing TRM TIL and identifying the mechanisms involved in their retention within the tumor ecosystem and tumor-induced dysfunctions, we hope that manipulation of this tumor-specific T cell subset will lead to enhanced immune protection and improvement in current immunotherapy approaches for cancer treatment.

We thank Yann Lecluse and Philippe Rameau for help with FACS analyses. We also thank Olivia Bawa for help with immunohistochemistry staining. We are grateful to Stéphanie Corgnac for critical reading of the manuscript.

This work was supported by grants from INSERM, the Association pour la Recherche sur le Cancer, the Institut National du Cancer, the Ligue contre le Cancer, and the Site de Recherche Intégrée sur le Cancer SOCRATE (Institut National du Cancer-Direction Générale de l'Offre de Soins Grant 6043). F.D. was supported by the Cancéropôle Ile de France and the Université Paris-Sud. J.A. was supported by the Institut Thématique Multi-Organismes Cancer and the Institut National du Cancer.

The microarray data presented in this article have been submitted to the European Molecular Biology Laboratory European Bioinformatics Institute database (https://www.ebi.ac.uk/arrayexpress) under accession no. E-MTAB-3266.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • ADC

    adenocarcinoma

  •  
  • AICD

    activation-induced cell death

  •  
  • CI

    confidence interval

  •  
  • DFS

    disease-free survival

  •  
  • HES

    H&E with saffron

  •  
  • HR

    hazard ratio

  •  
  • MHC-I

    MHC class I

  •  
  • NSCLC

    non–small cell lung carcinoma

  •  
  • OS

    overall survival

  •  
  • PI

    propidium iodide

  •  
  • qRT-PCR

    quantitative RT-PCR

  •  
  • SCC

    squamous cell carcinoma

  •  
  • TIL

    tumor-infiltrating lymphocyte

  •  
  • TRM

    tissue-resident memory.

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The authors have no financial conflicts of interest.

Supplementary data