MA11.02 Increased CD3+ TIL Infiltration and Low FOXP3+/CD8+ TIL Ratio Can Predict Anti-PD-1 Therapeutic Response in Non-Small Cell Lung Cancer Patients
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Tumor-infiltrating lymphocytes
To study the CD4+ and CD8+ tumor infiltrating lymphocytes (TIL) in the antitumor response, we propagated these subsets directly from tumor tissues with anti-CD3:anti-CD8 (CD3,8) and anti-CD3:anti-CD4 (CD3,4) bispecific mAb (BSMAB). CD3,8 BSMAB cause selective cytolysis of CD8+ lymphocytes by bridging the CD8 molecules of target lymphocytes to the CD3 molecular complex of cytolytic T lymphocytes with concurrent activation and proliferation of residual CD3+CD4+ T lymphocytes. Similarly, CD3,4 BSMAB cause selective lysis of CD4+ lymphocytes whereas concurrently activating the residual CD3+CD8+ T cells. Small tumor fragments from four malignant melanoma and three renal cell carcinoma patients were cultured in medium containing CD3,8 + IL-2, CD3,4 + IL-2, or IL-2 alone. CD3,8 led to selective propagation of the CD4+ TIL whereas CD3,4 led to selective propagation of the CD8+ TIL from each of the tumors. The phenotypes of the TIL subset cultures were generally stable when assayed over a 1 to 3 months period and after further expansion with anti-CD3 mAb or lectins. Specific 51Cr release of labeled target cells that were bridged to the CD3 molecular complexes of TIL suggested that both CD4+ and CD8+ TIL cultures have the capacity of mediating cytolysis via their Ti/CD3 TCR complexes. In addition, both CD4+ and CD8+ TIL cultures from most patients caused substantial (greater than 20%) lysis of the NK-sensitive K562 cell line. The majority of CD4+ but not CD8+ TIL cultures also produced substantial lysis of the NK-resistant Daudi cell line. Lysis of the autologous tumor by the TIL subsets was assessed in two patients with malignant melanoma. The CD8+ TIL from one tumor demonstrated cytotoxic activity against the autologous tumor but negligible lysis of allogeneic melanoma targets. In conclusion, immunocompetent CD4+ and CD8+ TIL subsets can be isolated and expanded directly from small tumor fragments of malignant melanoma and renal cell carcinoma using BSMAB. The resultant TIL subsets can be further expanded for detailed studies or for adoptive immunotherapy.
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The expression of the CD3zeta subunit was investigated in fresh (uncultured) tumor-infiltrating lymphocytes (TILs) isolated from either solid tumor (ST) specimens or ascites (ASC) from patients with epithelial ovarian carcinoma (EOC). Western blot analysis of CD3zeta immunoprecipitates using anti-CD3zeta rabbit serum revealed that in 6 out of 6 patients with EOC, the CD3zeta protein was absent from ST-TILs. Immunoprecipitation with anti-phosphotyrosine monoclonal antibody (anti-PY20) from ST-TILs from one patient revealed bands co-migrating with the phosphorylated CD3zeta. CD3zeta protein was found to be expressed in only 1 out of 7 ST-TILs from patients with EOC. ASC-TILs were available in 5 of these patients and immunoprecipitation/Western blotting experiments using anti-CD3zeta rabbit serum revealed that CD3zeta protein was expressed in all 5. In addition, CD3zeta protein was expressed in 3 additional ASC-TIL specimens for which ST-TILs were not available. Therefore, the CD3zeta protein was expressed in ASC-TIL isolated from 8 out of 8 patients with EOC. CD3zeta protein was also expressed on peripheral blood mononuclear cells (PBMCs) from patients with EOC and from normal donors. RT-PCR studies of fresh ST-TIL specimens, using CD3zeta-specific primers, revealed that CD3zeta transcripts were absent from 13 out of 21 patients with EOC, down-regulated in 4 patients and present at levels comparable to those found in PBMCs in 4 other patients. In contrast, CD3delta transcripts were present at comparable levels in all specimens. Treatment with recombinant interleukin-2 (rIL-2) (600 IU/ml) restored the expression of CD3zeta protein and transcripts in cultured ST-TILs, whereas fresh ST-TILs did not express CD3zeta, in contrast to fresh ASC-TILs. These results demonstrate differential expression of CD3zeta in ST-TILs versus ASC-TILs in patients with EOC. CD3zeta transcripts and protein were found to be absent from most ST-TILs from patients with EOC, whereas they were expressed in ASC-TILs and PBMCs from such patients.
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Abstract Objective: The aim of the study was to investigate the localization, amount and type of lymphocytic infiltrate, found in different molecular subtypes of breast cancer (BC). Methods: Retrospectively, 100 cases of invasive BC were analyzed and immunohistochemicaly (IHC) stratified in four subtypes (Luminal A and Luminal B-like, HER2-positive, and triple negative (TN). The percentage of stromal areas occupied by tumor-infiltrating lymphocytes (TILs) was assessed for H&E slides. The samples were IHC-stained for CD3, CD4, CD8, CD20 and FoxP3. The immunophenotyped lymphocytes - intratumoural and stromal, were separately counted, semi-quantitatively graded and further analyzed. Results: A total of 10% of all tumors were lymphocyte-predominant BC. Intratumoral and stromal TILs were predominantly CD3+T-lymphocytes. High counts of all subtypes TILs - intratumoral and stromal, were most common for TNBC and HER2-positive BC. In TNBC, the intratumoral CD3+TILs are significantly related to CD8+ (p=0.002) and FoxP3+ phenotype (p=0.010). In HER2 BC, the intratumoral and stromal CD3+ TILs were significantly related to FoxP3+ phenotype (p = 0,035 and p= 0.011, respectively). Conclusion:CD3+T-cell mediated immunity, especially the one related to CD8+ and FoxP3+ lymphocytes was the leading one in antitumor response in BC, and high count of intratumoral and stromal lymphocytes predominated in TN and HER2-positive BC. Key words: Breast cancer, tumor-infiltrating lymphocytes, CD3, CD4, CD8, CD20, FoxP3. Citation Format: Popovska SL, Dimitrova PD, Dineva TB. A study on amount, localization and immune phenotype of tumor-infiltrating lymphocytes in different subtypes of breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-20.
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Objective To study the anti-tumor effect of Huai Er,a Chinese prepared medicine,on the cellular immune function.Methods Forty cases of post-operative gastric cancer patients were divided into treatment group and control group randomly.Huai Er and 5-Fu were used respectively.Peripheral blood samples in the 40 patients were collected to detect the lymphocyte subsets during pre-operatively,2 weeks,6 weeks and 10 weeks after operation,respectively.The data were statistically analyzed by paired samples.Results The CD3+,CD3+CD4+,CD3+CD4+/CD3+CD8+,and NK cells increased remarkably 6 weeks and 10 weeks post-operatively compared with 2 weeks post-operatively(P0.05)in treatment group.Conclusions Huai Er may produce some anti-tumor effect by improving cellular immune function.
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Objective: To investigate the changes of tumor infiltrating T lymphocyte subsets in HCC patients.Methods: Obtained fresh liver tissues from thirty patients with primary liver cancer and thirty patients who underwent hepatectomy because of benign lesions,then prepared infiltrating lymphocytes from these tissues.Used the method of flow cytometry to detect the cell surface molecules of T lymphocytes: CD3,CD4 and CD8.Results: 1)The proportions of CD3+CD4+T lymphocytes in infiltrating lymphocytes in HCC tissues were(22.31±3.68)%,which were significantly higher than those in adjacent liver tissues(10.69±2.47)% and in normal liver tissues(4.21±4.26)%(P=0.000,P=0.001).The proportions of CD3+CD4+T lymphocytes in adjacent liver tissues were significantly higher than those in normal liver tissues too(P=0.019).2)The proportions of CD3+CD8+T lymphocytes in infiltrating lymphocytes in HCC tissues and in adjacent liver tissues were(26.10± 5.82)% and(21.82±2.7)%,which were significantly lower than those in normal liver tissues(41.31± 14.01)%(P=0.014,P=0.004).The proportions of CD3+CD8+T lymphocytes were similar between those in HCC tissues and in adjacent liver tissues(P=0.651).3)The ratio of CD3+CD4+T lymphocytes and CD3+CD8+T lymphocytes in infiltrating lymphocytes in HCC tissues(0.91±0.30)were significantly higher than those in adjacent liver tissues(0.47±0.11,P=0.003)and in normal liver tissues(0.11±0.13,P=0.000).Conclusion: The proportions of CD3+CD4+T lymphocytes in tumor infiltrating lymphocytes of HCC tissues increased evidently,while the proportions of CD3+CD8+T lymphocytes decreased markedly.The ratios of CD3+CD4+/CD3+CD8+T lymphocytes were evidently raised.
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The present study on tumor infiltrating lymphocytes (TILs) from tumors of cattle has revealed 13% MHC-II+ve cells in TILs from horn cancer while those from nasal granuloma had 18% cells expressing MHC-II. In case of horn cancer, 54% TlLs were CD4+ve and 18% were CD8+ve whereas in case of nasal granuloma, 43% of TlLs were CD4+ve and 19% were CD8+ve. This indicates that TILs comprise mostly of T-cells with a predominance of helper cells in these tumors of cattle. The substantial percentage of MHC-ll+ve cells in TILs may indicate the presence of activated lymphocytes.
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Aim To investigate the effect of Panax Quinquefolium Saponin (PQS) on the cell immune system of the patients with Chronic Pulmonary Heart Disease (CPHD) and then to find out the relationship between the immune system and the mechanism of CPHD. Methods The T lymph subgroup and NK (CD3 -/CD16+56 +) cells in patients’ peripheral blood were detected by flow cytometry, and the expression of IL 2, IFNγ mRNA were analyzed by RT PCR. Results CD3 +, CD4 + and CD4/CD8 in patients with CPHD were significantly lower than those in the control, while CD8 + higher ( P 0 01). CD3 +, CD4 +, CD4/CD8 and NK (CD3 -/CD16+56 +) cells in the patients were increased after PQS treated, and CD3 +, CD4 + were significantly different from pre treated ( P 0 05), while CD8 + cells were significantly lower than those in pre treated ( P 0 05). PQS stimulated the expression of IL 2, IFNγ mRNA excreted by T lymph cells. Conclusions The results showed that PQS can improve the cell immune function, especially in patients with CPHD whose immune function are low.
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Alloreactive CTL clones and naive CTL precursor cells (CTLp) from TCR-transgenic mice were analyzed for their response in total and in TCR-associated kinase activation upon stimulation with the relevant class I allo-APCs. The responses were found to be stronger and more sustained for the CTL clone and CTLp expressing a TCR previously characterized as CD8 coreceptor independent than for the CTL clone and CTLp expressing a TCR characterized as CD8 dependent. Unexpectedly, it was found that also in response to CD3 engagement, total and TCR-associated kinase activation were stronger and more sustained in the CTL clone and CTLp expressing the CD8-independent TCR. In both types of CTL clones, p56(lck) was found associated with the TCR complex, and CD3 components were found associated with CD8 before CD3 engagement. Upon CD3 engagement, ZAP-70 was also found associated with the TCR complex and the kinase activity (p56(lck)) associated with CD8 increased. This increase was more pronounced for the CD8-independent than for the CD8-dependent clone. An increased association of CD3zeta with CD8 was also detected after CD3 engagement for each clone. These data indicate that signals resulting from exclusive CD3 engagement can influence CD8 molecular associations and activate CD8-bound p56(lck). They further suggest that clonal differences exist that influence the efficiency of signaling upon binding of the same CD3 ligand. The observation that this property was shared between independently derived CTL clone and CTLp expressing the same TCR suggests that it may be acquired during repertoire selection.
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In 93 persons aged 90 to 103 years old the subpopulation compositon of lymphocytes and the level of thymic serum activity (TSA) was studied. In the presence of polymorbidity the low level of TSA was observed in 81% of examinees, with the quantitative deficiency in the relative and absolute number of CD3+CD8+ T-lymphocytes (62% and 78% respectively) and the excess in the number CD3+CD4-CD8- T-lymphocytes (42% and 57% respectively). Among CD3+CD4-CD8- T-lymphocytes cells with phenotypes CD3+/-CD4-CD8-CD16- and CD3+CD4-CD16+ were determined. In prolonged imunomonitoring the reciprocacity of changes in the amount of CD3+/-CD4-CD8-CD16- T-lymphocytes was established in relation to the level of TCA and the number of CD3+CD8+ T-lymphocytes. The reversibility of these changes is indicative of the potential of reactivity of the immune system in longlivers. The positive result of laser therapy in persons with quantitative deficiency of CD3+CD8+ T-lymphocytes and the presence of cells with phenotype CD3+/-CD4-CD8-CD16- was associated with the restoration of the size of CD3+CD8+ subpopulation and a decrease in an amount of CD3+/-CD4-CD8-CD16-. The unfavorable prognostic sign was, seemingly, the quantitative deficiency of CD3+CD8+ T-lymphocytes with the low level of TCA in the absence of CD3+/-CD4-CD8-CD16- T-lymphocytes.
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