CD8 TILs Research Articles

Abstract CT028: Atezolizumab (atezo) + nab-paclitaxel (nab-pac) in metastatic triple-negative breast cancer (mTNBC): 2-year update from a ph Ib trial

Abstract Introduction: Outcomes for patients (pts) with mTNBC remain poor, with chemotherapy as a historic mainstay treatment. Atezo (anti-PD-L1) demonstrated single-agent activity in mTNBC, and initial data suggested the addition of chemotherapy was safe with encouraging activity (Adams, 2016). Here we report long-term outcomes, with OS, for atezo + nab-pac in mTNBC. Methods: Arm F of multi-cohort Ph Ib study (NCT01633970) enrolled pts with locally advanced unresectable/stage IV mTNBC and 0 to 2 prior regimens for advanced disease. Atezo 800 mg IV q2w and nab-pac 125 mg/m2 IV qw (3 weeks on/1 week off) were given concurrently (with nab-pac run-in for biopsy cohort); maintenance atezo was permitted until loss of clinical benefit. In-tumor PD-L1 expression (VENTANA SP142 assay), CD8 and stromal TILs were assessed by IHC; blood-based CD8+Ki67+ via FACS, and CXCL10 cytokine RNA by NanoString. Results: Of 33 evaluable pts, 82% had ECOG PS 1, 58% had visceral mets, and median age was 55 years (data cutoff, April 30, 2017). Grade 3-4 treatment-related AEs (TRAE) occurred in 73%, with no Grade 5 TRAEs. Atezo-related AEs ≥ 20% included fatigue, pyrexia and decreased neutrophil count. Five pts discontinued nab-pac due to typical chemotherapy-related toxicities, and 3 withdrew from atezo due to TRAEs. Responses occurred across all subgroups (Table), with numerically higher ORR and longer OS/PFS in pts with less pre-treatment and higher PD-L1 status. Serial biopsy samples (n = 14) had elevated PD-L1+ TILs after combination treatment (but not after nab-pac alone). Peripheral blood analyses found transient on-treatment increases in CD8+Ki67+ and CXCL10, which were independent of efficacy. Conclusions: In this single-arm study cohort, atezo + nab-pac was well tolerated with promising efficacy, especially for treatment-naive pts. Ongoing randomized Ph III trial IMpassion130 (NCT02425891) is investigating this regimen in untreated mTNBC. Table. EfficacySubgroup/OutcomeCurrent Line of TherapyPD-L1 IC StatusaAll Ptsb (N = 33)1L (n = 13)2L+c (n = 20)IC1/2/3 (n = 12)IC0 (n = 12)Confirmed ORR (95% CI)54% (25, 81)30% (12, 54)42% (15, 72)33% (10, 65)39% (23, 58)mDOR (range)7.8 mo (2.9-11.5+)10.9 mo (3.7-20.9+)9.1 mo (2.9-16.2)10.2 mo (3.7-20.9+)9.1 mo (2.9-20.9+)mPFS (95% CI)8.6 mo (5.2, 11.5)5.1 mo (3.3, 7.3)6.9 mo (5.2, 11.0)5.1 mo (3.5, 6.8)5.5 mo (5.1, 7.7)mOS (95% CI)24.2 mo (11.5, NE)12.4 mo (7.5, 21.9)21.9 mo (13.1, NE)11.4 mo (7.5, NE)14.7 mo (10.1, NE)IC, tumor-infiltrating immune cells; mDOR, median duration of response; mPFS, median progression-free survival; mOS, median overall survival; mTNBC, metastatic triple-negative breast cancer; NE, not estimable; ORR, objective response rate; PD-L1, programmed death-ligand 1.a Excludes 9 pts with unknown PD-L1 status.b Includes 25 pts enrolled in the serial biopsy cohort and 8 in the safety cohort. Median durations of atezolizumab exposure, safety follow-up and efficacy follow-up were 5.6 mo (range, 0-30), 6.9 mo (range, 1.7-30.3) and 24.4 mo (95% CI: 22.1, 28.8), respectively.cIncludes 1 pt who received 3 prior therapies for mTNBC.Citation Format: Paula R. Pohlmann, Jennifer R. Diamond, Erika Hamilton, Sara M. Tolaney, Wei Zhang, Koho Iizuka, Paul Foster, Luciana Molinero, Roel Funke, Sylvia Adams. Atezolizumab (atezo) + nab-paclitaxel (nab-pac) in metastatic triple-negative breast cancer (mTNBC): 2-year update from a ph Ib trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT028.

PD-L1, B7-H3, and PD-1 expression in immunocompetent vs. immunosuppressed patients with cutaneous squamous cell carcinoma.

To characterize the expression of co-signaling molecules PD-L1, PD-1, and B7-H3 in cutaneous squamous cell carcinoma (cSCC) by immune status. We retrospectively analyzed 66 cases of cSCC treated with surgical resection from 2012 to 2015. Immunostained tumor sections were analyzed for percent of tumor cells expressing PD-L1 (Tum-PD-L1%), B7-H3 (Tum-B7-H3%), density of peri and intratumoral CD8 T cells (CD8 density), proportion of CD8 T cells expressing PD-1 (CD8-PD-1%) and of tumor-infiltrating immune cells (TII) expressing PD-L1 (TII-PD-L1%). Of 66 cases, 42 were immunocompetent, 24 immunosuppressed (13 organ transplant, 8 HIV+, 3 other). Defining positive expression at > 5%, 26% of tumors were positive for PD-L1, 85% for B7-H3, 80% had CD8 T cells that expressed PD-1 and 55% had TII that expressed PD-L1. Tum-B7-H3% was significantly higher (median 60 vs. 28%, p = 0.025) in immunocompetent vs. immunosuppressed patients, including when factoring in cause of immunosuppression. No significant difference in Tum-PD-L1%, TII-PD-L1%, CD8 density, or CD8-PD-1% was observed. Tumors from HIV+ patients lacked PD-L1 expression, and had lower B7-H3% (median 2.5 vs. 60%, p = 0.007), and higher CD8 density (median 75% vs. 40%, p = 0.04) compared to immunocompetent patients. Higher tumor grade (Rs = 0.34, p = 0.006) and LVI (Rs = 0.61, p < 0.001) were both associated with higher Tum-PD-L1%. cSCC showed expression of PD-L1 on tumor in 26% of cases, and high tumor B7-H3 expression (85%) and PD-1 expression on CD8 TILs (80%). Tumor B7-H3 expression was significantly higher in immunocompetent vs. immunosuppressed patients, largely driven by very low expression in HIV+ patients.

Prognostic role of tumor infiltrating lymphocytes in EBV positive and EBV negative nasopharyngeal carcinoma

ObjectivesTumor infiltrating lymphocytes (TILs) correlate with both better and worse prognosis in solid tumors. As therapeutic modalities for nasopharyngeal carcinoma (NPC) are limited, immunotherapy could be a potential alternative. Up till now there is limited prognostic data on the role of TILs in NPC, so we assessed the prognostic role of TILs in Epstein-Barr-virus (EBV) positive and negative NPC. MethodsTissue of 92 NPCs was assessed for CD3, CD4, CD8, PD1 and PDL1 expression in the tumor’s micro-environment. Correlations between clinicopathological characteristics was assessed using the Pearson X2 test, Fisher’s exact test and ANOVA. Survival was analyzed with the Kaplan-Meier method and Cox regression. Differences in CD3, CD4, CD8, PD1, PDL1 counts/(co)expression between EBV positive and negative NPCs were evaluated using the Mann-Whitney U test. Two-tailed P values below 0.05 were considered statistically significant. ResultsEBV positive NPC contains significantly more CD3, CD4 and CD8 TILs than EBV negative NPC. In the whole NPC group, increased CD8 count is associated with better overall survival (OS) (HR 0.219 (95%CI 0.075–0.640)), but also in cases with PDL1 co-expression (HR 0.073 (95%CI 0.010–0.556)). In EBV positive NPC co-expression of CD8 and PDL1 showed better disease free survival (HR 0.407 (95%CI 0.195–0.850)) and OS (HR 0.170 (95%CI 0.037–0.787)). ConclusionsAlthough TILs are significantly different between EBV positive and negative NPCs, it is especially composition of the infiltrate which determines prognosis. Effects of PD1 and CD8 need more study, because these findings show much potential in using immunotherapeutic modalities in NPC treatment.

The association and prognostic significance of programmed death-ligand 1 (PD-L1) and isocitrate dehydrogenase (IDH-1) mutation in glioblastome multiforme(GBM) patients.

e13536 Background: We aimed to assessed thePD-L1, PD-1 expression, TILs and IDH-1 mutation associations and their prognostic importance in GBM. Methods: Newly diagnosed and all were operated GBM patients who folllowed between February 2006 to February 2017 were included retrospectively in the study. We assessed the PD-L1, PD-1 expression, CD4(+) ve CD8(+)TILs, IDH-1 mutation in initial tumor specimens. Immunohistochemistry was performed using Ventana® antibody (clon-SP263, ROCHE) for PD-L1 and PD-1, real time PCR used for IDH-1 mutation analysis. Intensity was graded as low, moderate, dense for PD-1, CD4, CD8 TILs. PD-L1 evaluated in percents. The cutoff value assumed as ≥5% for PD-L1 positivity. We used Kaplan Meier, Cox regression tests and SPSS 23. Results: Fourty eight patients included in the study. The mean age was 54± 14,4 and 34 (70%) patients were male. Thirty (70 %) patients were received chemoradiotherapy with temozolamide and 20 (41%) of them also received chemotherapy.We identified two different staining pattern of PD-L1 expression as diffuse fibrillary [13(27%)] and membranous staining [14(29%)]. IDH-1 mutation was found in 30 (62%) patients. We found that TILs were more intensely located in the perivascular area than intratumoral area and in that TILs, PD-1 intensity were dense. We also found a correlation between the percentages of PD-L1 positivity and the density of TILs in the intratumoral and perivascular areas (p &lt; 0,002, r = 0,46). The intensity of both TILs in perivascular areas were significantly lower in PD-L1 (-) tumors than in PD-L1 (+) tumors (p &lt; 0,001). We didn’t find any association between IDH-1 and PD-L1 or TILs. According to survival and multivariate analysis; age, sex, PD-L1 positivity and IDH-1 mutation status did not appear to have a significant effect on survival. (log rank 0,08; 0,30; 0,89; 0,14 respectively) The presence of dense intratumoral CD8(+) and PD-1 (+)TILs were found as positive; advanced age and membranous PD-L1 staining was found as negative independent prognostic factors. Conclusions: Different staining pattern of PD-L1 in tumor tissue and PD-1 positivity in TILs may be a prognostic importance in GBM

MTE08.01 Immunotherapy in Early and Locally Advanced NSCLC: Challenges and Perspectives

MTE08.01 Immunotherapy in Early and Locally Advanced NSCLC: Challenges and Perspectives

443O EGFR mutations are associated with unfavourable efficacy to anti-PD1 therapy in NSCLC

Background Along with the development of targeted therapies and immunotherapy, the management of NSCLC is facing a tremendous changing. However, only 20% patients could benefit from PD-1 inhibitors. The precise biomarker in immunotherapy remains exclusive now. Furthermore, unlike targeted therapies the role of genetic alterations in oncogenic drivers remains unclear in anti-PD1 therapy. Here we conducted this data analysis to find out whether EGFR mutant status would have an influence on immunotherapy and how it works in NSCLCs. Methods A meta-analysis was conducted from data of EGFR mutant patients enrolled in CheckMate 057 and Keynote 010. Furthermore we analyzed 462 patients with lung adenocarcinoma from TCGA to investigate the mechanisms, such as nonsynonymous mutation counts, incidence of MMR (mis-match repair)-related gene mutations and the content of CD8 TILs. Results 1543 NSCLC patients were enrolled in our meta-analysis, of which 168 harbored EGFR mutations. We can see EGFR mutant patients showed worse efficacy of anti-PD1 therapy compared with chemotherapy on PFS (hazard ratio [HR] 1.57, 95% CI 1.07 – 2.31, p=0.02), while EGFR-wild patients treated with anti-PD1 therapy had significantly longer PFS (hazard ratio [HR] 0.83, 95% CI 0.73 – 0.94, p=0.004) (Fig. 1). Moreover for OS, EGFR mutant patients received anti-PD1 therapy presented a slightly increase in the relative risk of death (HR 1.05, 95% CI 0.69 – 1.60, p=0.81), while a 33% decrease was observed in EGFR-wild patients (HR 0.67, 95% CI 0.58 – 0.77, p<0.001) (Fig. 2). As for variations represented immunogenicity, we figured out that EGFR mutant patients had significantly less nonsynonymous mutation counts (median: 66 vs. 211, p<0.001), lower rate of transversion/transition (Tv/Ti) (p<0.001) and less incidence of MMR-related gene (MSH2, MSH6, MLH1 and PMS2) mutations (5.5% vs. 11.5%). Furthermore, CD8A mRNA expression was also significantly lower in EGFR mutant patients (TCGA RNA Seq V2: mean: 251 vs. 425.7, p=0.01) (Fig. 3) Conclusions Our study indicated that EGFR mutant patients may not be an appropriate subtype to receive immunotherapy partly because of weak immunogenicity and lower content of CD8þ CTL. Further prospective studies to confirm these observations are necessary Legal entity responsible for the study N/A Funding Guangdong Provincial Key Laboratory of Lung Cancer translational Medicine (Grant No. 2012A061400006) Disclosure All authors have declared no conflicts of interest.

Correlation of metabolic information on FDG-PET with tissue expression of immune markers in patients with non-small cell lung cancer (NSCLC) who are candidates for upfront surgery

Eliciting antitumor T-cell response by targeting the PD-1/PD-L1 axis with checkpoint inhibitors has emerged as a novel therapeutic strategy in non-small cell lung cancer (NSCLC). The identification of predictors for sensitivity or resistance to these agents is, therefore, needed. Herein, we investigate the correlation of metabolic information on FDG-PET with tissue expression of immune-checkpoints and other markers of tumor-related immunity in resected NSCLC patients. All patients referred to our institution for upfront surgical resection of NSCLC, who were investigated with FDG-PET prior to surgery, were consecutively included in the study. From January 2010 to May 2014, 55 patients (stage IA-IIIB; M:F = 42:13; mean age 68.9years) were investigated. Sampled surgical tumor specimens were analyzed by immunohistochemistry (IHC) for CD68-TAMs (tumor-associated macrophages), CD8-TILs (tumor infiltrating lymphocytes), PD-1-TILs, and PD-L1 tumor expression. Immunoreactivity was evaluated, and scores were compared with imaging findings. FDG-PET images were analyzed to define semi-quantitative parameters: SUVmax and SUVmean. Metabolic information on FDG-PET was correlated with tissue markers expression and disease-free survival (DFS) considering a median follow-up of 16.2months. Thirty-six adenocarcinomas (ADC), 18 squamous cell carcinomas (SCC), and one sarcomatoid carcinoma were analyzed. All tumors resulted positive at FDG-PET: median SUVmax 11.3 (range: 2.3-32.5) and SUVmean 6.4 (range: 1.5-13) both resulted significantly higher in SCC compared to other NSCLC histotypes (p = 0.007 and 0.048, respectively). IHC demonstrated a median immunoreactive surface covered by CD68-TAMs of 5.41% (range: 0.84-14.01%), CD8-TILs of 2.9% (range: 0.11-11.92%), PD-1 of 0.65% (range: 0.02-5.87%), and PD-L1 of 0.7% (range: 0.03-10.29%). We found a statistically significant correlation between SUVmax and SUVmean with the expression of CD8 TILs (rho = 0.31; p = 0.027) and PD-1 (rho = 0.33; p = 0.017 and rho = 0.36; p = 0.009, respectively). The other tissue markers correlated as follows: CD8 TILs and PD-1 (rho = 0.45; p = 0.001), CD8 TILs and PD-L1 (rho = 0.41; p = 0.003), CD68-TAMs and PD-L1 (rho = 0.30; p = 0.027), PD-1 and PD-L1 (rho = 0.26; p = 0.059). With respect to patients' outcome, SUVmax, SUVmean, and disease stage showed a statistically significant correlation with DFS (p = 0.002, 0.004, and <0.001, respectively). The present study shows a direct association between metabolic parameters on FDG-PET and the expression of tumor-related immunity markers, suggesting a potential role for FDG-PET to characterize the tumor microenvironment and select NSCLC patients candidate to checkpoint inhibitors.

PD-1 and CD103 Are Widely Coexpressed on Prognostically Favorable Intraepithelial CD8 T Cells in Human Ovarian Cancer.

αE(CD103)β7 is a TGFβ-regulated integrin that mediates retention of lymphocytes in peripheral tissues by binding to E-cadherin expressed on epithelial cells. We recently reported that αE(CD103)β7 specifically demarcates intraepithelial CD8(+) tumor-infiltrating lymphocytes (CD8 TIL) in ovarian cancer and that CD103(+) TIL have a surface profile consistent with an active effector phenotype (HLA-DR(+), Ki67(+), and CD127(lo)). These findings led us to hypothesize that, over time, CD103-mediated retention of CD8 TIL within the tumor epithelium might result in chronic stimulation by tumor antigen, which in turn might lead to an exhausted phenotype. To investigate this possibility, we evaluated PD-1 expression in a large cohort of ovarian tumors (N = 489) with known CD103(+) TIL content. PD-1(+) cells were present in 38.5% of high-grade serous carcinomas (HGSC), but were less prevalent in other histologic subtypes. PD-1(+) TIL were strongly associated with increased disease-specific survival in HGSC (HR, 0.4864; P = 0.0007). Multicolor immunohistochemistry and flow cytometry revealed a high degree of PD-1 and CD103 coexpression, specifically within the CD8 TIL compartment. PD-1(+)CD103(+) CD8 TIL were quiescent when assessed directly ex vivo yet were capable of robust cytokine production after pharmacologic stimulation. Moreover, they showed negligible expression of additional exhaustion-associated markers, including TIM-3, CTLA-4, and LAG-3. Thus, as hypothesized, CD103(+) CD8 TIL express PD-1 and appear quiescent in the tumor microenvironment. However, these cells retain functional competence and demonstrate strong prognostic significance. We speculate that, after standard treatment, PD-1(+)CD103(+) CD8 TIL might regain functional antitumor activity, an effect that potentially could be augmented by immune modulation.

CD103+ Tumor Infiltrating Lymphocytes Predict a Favorable Prognosis in Urothelial Cell Carcinoma of the Bladder

CD8(+) TILs at different tumor sites have diverse clinical attributes, which might result from distinct tumor microenvironments that promote differentiation into distinct subsets. However, only a few markers have been identified that can define CD8(+) T-cell subsets. CD103 is a marker of tissue resident memory CD8(+) T cells. In this retrospective study we investigated the cellular source and clinical significance of CD103 expression in urothelial cell carcinoma of bladder tissues in situ. Immunohistochemistry and immunofluorescence were used to identify the cellular source of CD103 in bladder urothelial cell carcinoma tissues. Kaplan-Meier analysis and Cox proportional hazards regression models were applied to estimate overall and recurrence-free survival in 302 patients with bladder urothelial cell carcinoma. CD8(+) T cells but not natural killer cells accounted for most CD103 expressing cells in bladder urothelial cell carcinoma tissues. Notably CD103(+) cells were predominantly located in intratumor regions rather than in associated stroma (p < 0.0001). The density of intratumor CD103(+) TILs was inversely associated with tumor size (p < 0.0001) and could represent a favorable prognostic predictor of overall and recurrence-free survival (p = 0.002 and 0.011, respectively). Moreover, intratumor CD103(+) TILs were positively associated with the expression of cognate ligand E-cadherin in intratumor regions of bladder urothelial cell carcinoma tissues (p = 0.008). Our findings suggest that CD8(+) T cells might have a significant role in tumor immunity by expressing CD103 in intratumor regions of bladder urothelial cell carcinoma tissues. Intratumor CD103(+) TILs could potentially serve as a prognostic marker in patients with bladder urothelial cell carcinoma.

Loss of nuclear prothymosin-α expression is associated with disease progression in human superficial bladder cancer.

In this paper, we report a study on the clinical relevance of prothymosin-α expression and its correlation with intratumoral Foxp3(+) and CD8(+) lymphocytes (Foxp3(+)TIL and CD8(+)TIL) in bladder cancer patients. We used immunohistochemical staining for prothymosin-α, Foxp3, and CD8 on 101 tumor specimens harvested by endoscopic resection. The results were correlated with clinicopathological variables and clinical outcome in bladder cancer patients, particularly in 73 patients with superficial disease, using the log-rank test and Cox proportional hazard model. Overall, of the tumors, 30% were negative, 34% showed nuclear, and 37% showed cytoplasmic prothymosin-α expression. Foxp3(+)TILs were detected in 11% of patients (nonnuclear vs. nuclear, p = 0.096). Patients with a history of urothelial carcinoma have a higher frequency of nonnuclear prothymosin-α expression than those without (p = 0.016, chi-square test). By univariate and multivariate analyses of cases with superficial disease, grade and stage were identified as independent predictors for recurrence-free survival (p = 0.016 and 0.016, respectively). Higher stage and nonnuclear prothymosin-α expression independently predict shorter progression-free survival (p = 0.006 and 0.043, respectively). The presence of Foxp3(+)TILs was significantly associated with disease progression by univariate analysis (p = 0.022), but not by multivariate analysis (p = 0.147). In vitro assays showed that J82 cells which express ectopically nuclear prothymosin-α exhibit higher growth rate and secrete less TGF-β1 than those with cytoplasmic expression or control cells. Altogether, prothymosin-α expression is a determinant of disease progression in superficial bladder cancer. Foxp3(+)TILs tend to be found more often in bladder cancer with nonnuclear prothymosin-α expression. Future study is required to unravel their interaction.

Intratumoral Expression of IL-17 and Its Prognostic Role in Gastric Adenocarcinoma Patients

In this study, we characterized the intratumoral expression of IL-17 and CD8(+) TILs in gastric adenocarcinoma patients after resection and determined the correlation between the survival probability of gastric adenocarcinoma patients and the expression of IL-17 in tumor. Expression of IL-17 and CD8 was assessed by immunohistochemistry, and the prognostic effects of intratumoral IL-17 expression and CD8(+) TILs were evaluated by Cox regression and Kaplan-Meier analysis. Immunohistochemical detection revealed the presence of IL-17 and CD8(+) cells in gastric adenocarcinoma tissue samples (90.6%, 174 out of 192 patients and 96.9%, 186 out of 192 patients, respectively). We have also found that intratumoral IL-17 expression was significantly correlated with age (p=0.004) and that the number of CD8(+)TILs was significantly correlated with UICC staging (p=0.012) and the depth of tumor invasion (p=0.022). The five-year overall survival probability among patients intratumorally expressing higher levels of IL-17 was significantly better than those expressing lower levels of IL-17 (p=0.036). Multivariate Cox proportional hazard analyses revealed that intratumoral IL-17 expression (HR: 0.521; 95% CI: 0.329-0.823; p=0.005) was an independent factor affecting the five-year overall survival probability. We conclude that low levels of intratumoral IL-17 expression may indicate poor prognosis in gastric adenocarcinoma patients.

Cancer/testis antigen expression and specific cytotoxic T lymphocyte responses in non small cell lung cancer

Non small cell lung cancers (NSCLC) express cancer/testis antigens (CTA) genes and MAGE-A expression correlates with poor prognosis in squamous cell carcinomas. We addressed cytotoxic T lymphocytes (CTL) responses to HLA class I restricted CTA epitopes in TIL from NSCLC in an unselected group of 33 patients consecutively undergoing surgery. Expression of MAGE-A1, -A2, -A3, -A4, -A10, -A12 and NY-ESO-1 CTA genes was tested by quantitative RT-PCR. Monoclonal antibodies (MAb) recognizing MAGE-A and NY-ESO-1 CTA were used to detect CTA by immunohistochemistry. CD8(+) TIL obtained from tumors upon culture with anti CD3 and anti CD28 mAb and IL-2 were stimulated with autologous mature DC (mDC) and HLA-A*0101 restricted MAGE-A1(161-169) or MAGE-A3(168-176) peptides or HLA-A*0201 restricted MAGE-A4(230-239), MAGE-A10(254-262), NY-ESO-1(157-165) or multi-MAGE-A (YLEYRQVPV) peptides or a recombinant vaccinia virus (rVV) encoding MAGE-A and NY-ESO-1 HLA-A*0201 restricted epitopes and CD80 co-stimulatory molecule. Specificity was assessed by (51)Cr release and multimer staining. At least one CTA gene was expressed in tumors from 15/33 patients. In 10 specimens, at least 4 CTA genes were concomitantly expressed. These data were largely confirmed by immunohistochemistry. TIL were expanded from 26/33 specimens and CTA-specific CTL activity was detectable in 7/26 TIL. In 6, however, specific cytotoxicity was weak, (<40% lysis at a 50:1 E:T ratio) and multimer staining was undetectable. In one case, high (>60% lysis at 50:1 E:T ratio) MAGE-A10(254-262) specific, HLA-A*0201 restricted response was observed. Supportive evidence was provided by corresponding multimer staining. Although CTA genes are frequently expressed in NSCLC, detection of CTL reactivity against CTA epitopes in TIL from nonimmunized NSCLC patients represents a rare event.

Reconstitution of CD8+ T cells by retroviral transfer of the TCR alpha beta-chain genes isolated from a clonally expanded P815-infiltrating lymphocyte.

Gene transfer of TCR alphabeta-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and Vbeta subfamily usage of CD8(+) TIL in the tumor sites demonstrated that T cells using Vbeta10 efficiently infiltrated and expanded clonally. The TCR alpha- and beta-chain sequences derived from a tumor-infiltrating CD8(+)/Vbeta10(+) single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR alphabeta-chain genes were retrovirally introduced into CD8(+) T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2L(d) mAb. Furthermore, T cells containing both TCR alpha- and beta-chains, but not TCR beta-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8(+) T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.

Association of ovarian tumor epithelium coexpressing HLA-DR and CA-125 antigens with tumor infiltrating cytotoxic T lymphocytes.

Understanding of the relationship between tumor infiltrating cytotoxic T lymphocytes (CD 8(+)TILs) and tumor cells as well as tumor-associated antigens is important and may reflect the extent of immune response of the patient to the tumor, thus providing a useful clue relevant to the prognosis. The purpose of this study was to determine the relationship between the expression of HLA-DR and CA-125 antigens and the presence of CD 8(+)TILs with regard to the established clinicopathological factors of ovarian carcinomas using immunohistochemical methods. Thirty-one ovarian carcinomas consisting of 20 serous, 7 mucinous, and 4 clear cell types were examined. Of these, 18 (58%) and 22 (71%) were positive for HLA-DR and CA-125 antigens respectively, and the overall mean number of the CD 8(+)TILs was 7.2+/-2.9. A significant association was observed between the mean number of CD8(+)TILs and tumor grade (P=0.01), disease stage (P=0.003), and patient outcome (P=0.01). The mean number of CD 8(+)TILs in HLA-DR positive (8.6+/-2.2) or CA-125 positive (8.4+/-2.1) tumors was significantly higher than that in HLA-DR negative (5.2+/-2.5; P=0.0003) or CA-125 negative (4.2+/-2.2; P=0.00002) tumors. These significant levels were further enhanced by one order of magnitude when the mean number of CD 8(+)TILs in tumors positive for both HLA-DR and CA-125 antigens (9.1+/-1.7) was compared to that in HLA-DR negative or CA-125 negative tumors. The frequency of cancer-related deaths in HLA-DR and CA-125 positive tumors was significantly lower than in the negative tumors (P=0.01). These data suggest that concurrent expression of HLA-DR and CA-125 antigens may augment the immune response of the patient to the tumor, thus providing a potential clue relevant to the prognosis.

LIGHT, a novel ligand for lymphotoxin beta receptor and TR2/HVEM induces apoptosis and suppresses in vivo tumor formation via gene transfer.

LIGHT is a new member of tumor necrosis factor (TNF) cytokine family derived from an activated T cell cDNA library. LIGHT mRNA is highly expressed in splenocytes, activated PBL, CD8(+) tumor infiltrating lymphocytes, granulocytes, and monocytes but not in the thymus and the tumor cells examined. Introduction of LIGHT cDNA into MDA-MB-231 human breast carcinoma caused complete tumor suppression in vivo. Histological examination showed marked neutrophil infiltration and necrosis in LIGHT expressing but not in the parental or the Neo-transfected MDA-MB-231 tumors. Interferon gamma (IFNgamma) dramatically enhances LIGHT-mediated apoptosis. LIGHT protein triggers apoptosis of various tumor cells expressing both lymphotoxin beta receptor (LTbetaR) and TR2/HVEM receptors, and its cytotoxicity can be blocked specifically by addition of a LTbetaR-Fc or a TR2/HVEM-Fc fusion protein. However, LIGHT was not cytolytic to the tumor cells that express only the LTbetaR or the TR2/HVEM or hematopoietic cells examined that express only the TR2/HVEM, such as PBL, Jurkat cells, or CD8(+) TIL cells. In contrast, treatment of the activated PBL with LIGHT resulted in release of IFNgamma. Our data suggest that LIGHT triggers distinct biological responses based on the expression patterns of its receptors on the target cells. Thus, LIGHT may play a role in the immune modulation and have a potential value in cancer therapy.