Tumor infiltrating lymphocytes in gastric cancer:Unraveling complex interactions for precision medicine

This editorial will focus on tumor immunity and the factors that alter the tumor immune micro-environment.The role of tumor infiltrating lymphocytes(TILs)will also be discussed in detail,including the types,mechanism of action,and role.Gastric cancer(GC)often presents in the advanced stage and has various factors predicting the outcomes.The interplay of these factors and their correlation with the TILs is discussed.A literature review revealed high intratumoral TILs associated with higher grade,HER2-,and Helicobacter pylori negativity.Moreover,stromal(ST)TILs correlated with lower grade and lesser recurrence risk in GC.High TILs in ST and invasive border also correlated with mismatch repair deficiency status.Further characterization of the CD3+,CD8+,and other cells is also warranted.In the future,this complex correlation of cancer cells with the immune system can be explored for therapeutic avenues.

Ex vivo expansion of tumor-infiltrating lymphocytes from nasopharyngeal carcinoma patients for adoptive immunotherapy

Establishing Epstein-Barr virus (EBV)-specific cytolytic T lymphocytes (EBV-CTLs) from peripheral blood mononuclear cells (PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC). In the current study,we performed ex vivo expansion of tumor-infiltrating lymphocytes (TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody (OKT3), recombinant human interleukin (IL)-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+T cells, a variable percentage of CD3+CD8+and CD3+CD4+T cells, and less than 10% of CD3-CD16+natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α), and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.

Clinical significance of tumor-infiltrating lymphocytes for gastric cancer in the era of immunology

Immunotherapy has begun to revolutionize cancer treatment, by introducing therapies that target the host immune system instead of the tumor, therapies that possess unique adverse event profiles, and therapies that may cure certain types of cancer. The immune microenvironment of tumors is emerging as the most important means of understanding the relationship between a patient' immune system and their cancer, informing prognosis, and guiding immunotherapy, such as an antibody blockade of immune checkpoints. For some solid tumors, simple quantitation of lymphocyte infiltration would seem to have prognostic significance, suggesting that lymphocyte infiltration is not passive but may actively promote or inhibit tumor growth. For gastric cancers, several studies have provided strong evidence that immune cells contribute to determining prognosis. However, the exact role of immune cells in gastric cancer remains unclear. Therefore, this review focuses on the clinical significance of immune cells, especially tumor-infiltrating lymphocytes, in gastric cancer.

Association between Up-regulation of Fas Ligand Expression and Apoptosis of Tumor-infiltrating Lymphocytes in Human Breast Cancer

In order to study the significance of FasL expression in immune escape of breast cancer, FasL protein expression and the number of tumor-infiltrating lymphocytes （TILs） in 40 specimens of breast cancer were detected by immunohistochemitry. The expression of FasL mRNA was measured by in situ hybridization in the consecutive tissue slices of 40 breast cancers respectively. By using terminal deoxynucleotidyl transferase-mediaed dUTP nick end labeling （TUNEL）, apoptotic cells were detected in 40 specimens of breast cancer. The expression of FasL was detected in all 40 specimens to varying degrees. In the consecutive tissue slices, the location of expression of FasL protein corresponded with that of FasL mRNA. In those with FasL extensive expression, the number of TILs was less （P〈0.05）, the apoptotic index （AI） of TILs was higher and the AI of tumor cells was lower （P〈0.01） than those with FasL weak expression respectively. The AI of TILs was correlated with that of tumor cells （r=-0.629, P〈0.01）. In conclusion, breast cancer cells can induce the apoptosis of TILs through the expression of FasL, which can counterattack the immune system. This may be a mechanism of immune evasion in breast cancer.

ISOLATION AND CULTURE OF TUMOR-INFILTRATING LYMPHOCYTES FROM MOUSE HEPATOMA

By uaing enzyme digestion and Flcoll- Hypaque or Percoll discontinuous density methods, we have successfully obtained tumor-infiltrating lymphocytes (TIL) from mouse hepatoma. When analyzing the purity of TIL after separation. It was found that Percoll was more effective than Flcoll (P<0. 01). TIL could be activated In the presence of recombinant lL-2 (rIL-2) and begin to expand after culturing for 5-7 days, the tumor cells tend to decrease and disappeared after 14 days or so. TIL increased 105-fold over 40 days. Conditioned medium containing supernatant of PHA and rIL- 2 stimulated syngeneic spleen cell culture could promote the expansion of TIL.

Evaluating tumor-infiltrating lymphocytes in hepatocellular carcinoma using hematoxylin and eosin-stained tumor sections

BACKGROUND Tumor-infiltrating lymphocytes(TILs)constitute a prognostic factor in hepatocellular carcinoma(HCC).However,different methods of assessing TILs have various pre-analytical,analytical,and post-analytical challenges.The evaluation of TILs in hematoxylin and eosin(H&E)-stained tumor sections proposed by the International Immuno-Oncology Biomarker Working Group was demonstrated to be a reproducible,affordable and easily applied method in many tumors.AIM To evaluate the prognostic significance of TILs in H&E-stained slides of HCCs.METHODS This was a retrospective study performed in the hospital.HCC patients who underwent liver resection between 2015 and 2017 in Zhongshan Hospital were enrolled in this study.Patients who experienced recurrence or received therapy in addition to antiviral therapy before surgery at this time were excluded.A total of 204 patients were enrolled in the study.The ILs were counted manually in tumor sections stained with H&E under an optical microscope at 400×.The ILs were assessed separately in the center of the tumor(TILs^(CT)),the invasive front(TILs^(IF)),and peritumor(PILs)areas.Univariate and multivariate survival analyses were performed using a Cox regression model.P<0.05 was considered statistically significant and all P-values were two-sided.RESULTS Among the 204 patients,univariate analysis indicated that macrovascular invasion(MaVI)(P=0.001),microvascular invasion(MVI)(P=0.012),multiple tumors(P=0.008),large tumors(>10 cm)(P=0.001),absence of a tumor capsule(P=0.026),macrotrabecular histological subtype(P=0.001),low density of TILs^(CT)(P=0.039),TILs^(IF)(P=0.014),and PILs(P=0.010)were predictors of progressionfree survival(PFS).Cox multivariate analysis indicated that MaVI(P=0.009),absence of a tumor capsule(P=0.031),low-density of TILs^(IF)(P=0.047)and PILs(P=0.0495)were independent predictors of PFS.A three-category analysis was carried out by combining TILs^(CT),TILs^(IF),and PILs,after which HCCs were classified into immune^(high)[(TILs^(CT))^(high),(TILs^(IF))^(high),and PILs^(high),83 cases],immune^(mod)(tumors other than immune^(high) and immune^(low) subtypes,94 cases),and immune^(low)[(TILs^(CT))^(low),(TILs^(IF))^(low),and PILs^(low),27 cases]subtypes.The immune^(high) subtype had a lower rate of MVI(40.96%)than the immune^(mod)(61.70%,P=0.017)and immune^(low)(66.67%,P=0.020)subtypes.The recurrence rates of the immune^(high),immune^(mod) and immune^(low) subtypes were 10.8%,25.5%and 33.3%,respectively.CONCLUSION HCC patients with high infiltrating lymphocytes tend to have a lower recurrence rate and less MVI.The evaluation of TILs in H&E-stained specimens could be a prognostic parameter for HCC.

Prognostic value of tertiary lymphoid structure and tumour infiltrating lymphocytes in oral squamous cell carcinoma

Tertiary lymphoid structures(TLS)are ectopic lymphoid structures in cancers that are largely associated with favourable prognosis.However,the prognostic value of TLSs in oral squamous cell carcinoma(OSCC)is largely unknown,and the association between tumour infiltrating lymphocytes(TILs)and TLSs has been rarely explored in OSCC.In this study,associated markers of TLS,including peripheral node address(PNAd)in high endothelial venules,CD20 in B cells and CD3 in T cells,were examined in 168 OSCC patients,and survival analysis was performed between TLS-positive and TLS-negative cohorts.We detected the presence of TILs by staining CD8+cytotoxic T cells and CD57+NK cells as well.TLSs appeared as highly organized structures in 45(26.8%)cases.TLSpositive patients had a better 5-year overall survival(OS)rate(88.9%vs.56.1%,P<0.001)and relapse-free survival(RFS)rate(88.9%vs.63.4%,P=0.002).Moreover,the presence of TLS was an independent prognostic factor for both the 5-year OS rate(hazard ratio[HR]=3.784;95%confidence interval[CI],1.498–9.562)and RFS rate(HR=3.296;95%CI,1.279–8.490)in multivariate analysis.Furthermore,a higher density of CD8+T cells and CD57+NK cells was found in TLS-positive sections than in TLS-negative counterparts(P<0.001),and their combination provided a higher predictive accuracy(AUC=0.730;95%CI,0.654–0.805).In conclusion,our results suggest that TLS is an independent positive prognostic factor for OSCC patients.These findings provide a theoretical basis for the future diagnostic and therapeutic value of TLSs in OSCC treatment.

Tumor infiltrating lymphocytes in triple negative breast cancer receiving neoadjuvant chemotherapy

AIM To determine influence of neoadjuvant-chemotherapy(NAC) over tumor-infiltrating-lymphocytes(TIL) intriple-negative-breast-cancer(TNBC).METHODS TILs were evaluated in 98 TNBC cases who came to Instituto Nacional de Enfermedades Neoplasicas from 2005 to 2010. Immunohistochemistry staining for CD3, CD4, CD8 and FOXP3 was performed in tissue microarrays(TMA) sections. Evaluation of H/E in full-face and immunohistochemistry in TMA sections was performed in pre and post-NAC samples. STATA software was used and P value < 0.05 was considered statistically significant. RESULTS Higher TIL evaluated in full-face sections from pre-NAC tumors was associated to pathologic-complete-response(pCR)(P = 0.0251) and outcome(P = 0.0334). TIL evaluated in TMA sections showed low level of agreement with full-face sections(ICC = 0.017-0.20) and was not associated to pCR or outcome. TIL in post-NAC samples were not associated to response or outcome. PostNAC lesions with pC R had similar TIL levels than those without pCR(P = 0.6331). NAC produced a TIL decrease in full-face sections(P < 0.0001). Percentage of TIL subpopulations was correlated with their absolute counts. Higher counts of CD3, CD4, CD8 and FOXP3 in pre-NAC samples had longer disease-free-survival(DFS). Higher counts of CD3 in pre-NAC samples had longer overallsurvival. Higher ratio of CD8/CD4 counts in pre-NAC was associated with pCR. Higher ratio of CD4/FOXP3 counts in pre-NAC was associated with longer DFS. Higher counts of CD4 in post-NAC samples were associated with pCR.CONCLUSION TIL in pre-NAC full-face sections in TNBC are correlated to longer survival. TIL in full-face differ from TMA sections, absolute count and percentage analysis of TIL subpopulation closely related.

TREATMENT OF 23 PATIENTS WITH ADVANCED GASTRIC CANCER BY INTRAVENOUSLY TRANSFER OF AUTOLOGOUS TUMOR－INFILTRATING LYMPHOCYTES

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Effect of Tumor Infiltrating Lymphocyte on Local Control of Rectal Cancer after Preoperative Radiotherapy

Objective： To study the effect of tumor infiltrating lymphocytes at cancer nest on local control of rectal cancer after preoperative radiotherapy. Methods： From Jan. 1999 to Oct. 2007, a total of 107 patients with rectal cancer were reviewed. They were treated by preoperative radiotherapy, 30 Gy/10 fractions/12 days. Two weeks later, the patient underwent a surgical operation. Their pathological samples were kept in our hospital before and after radiotherapy. Lymphocyte infiltration （LI） degree, pathologic degradation and fibrosis degree after radiotherapy in paraffin section were evaluated under microscope. Results： After followed-up of 21 months （2-86 months）, a total of 107 patients were reviewed. Univariate analysis showed that lymphocyte infiltration （LI）, fibrosis and pathologic changes after radiotherapy were significant factors on local control. Logistic regression analysis showed that LI after radiotherapy was a significant effect factor on local control. Conclusion： LI, fibrosis and pathologic degradation after radiotherapy are significant for local control of rectal cancer after preoperative radiotherapy. LI after radiotherapy was a significantly prognostic index for local control of rectal cancer after preoperative radiotherapy.

PREPARATION AND CYTOLYTIC TUMOR ACTIVITY OF OSTEOSARCOMA TUMOR INFILTRATING LYMPHOCYTES IN VITRO

In this study, the isolation, purification and differentiation of tumor-lnflltratlng lymphocytes (TIL) from 6 fresh osteosarcoma specimens were achieved by discontinuous density gradient centrifugation. One specimen of the osteosarcoma TIL were enlarged in IL-2 for long time in vitro, reaching 28 days and their cytolytic activity against different tumor cell lines was Investigated. The experimental results indicated that the preparation of osteosarcoma TIL adopted by the mechanical means was simple, having higher purifity, keeping higher effects on killing NK- sensitive tumor cell lines and NK-insensitive tumor cell lines as well as rapid proliferation in vitro cultured in IL-2.

The Tumor Infiltrating Lymphocytes (TILs): Did We Find the Missed Piece of the Huge Puzzle?

Tumor infiltrating lymphocytes (TILs) are used in evaluating the prognosis and determining treatment of different types of cancer with variable degrees of success. The usage of checkpoint inhibitor immunotherapy as a treatment variety for cancer and Adoptive cell therapy is associated with many complications, severe side effects and high expenses. Recently, in a limited number of metastatic GIT and breast cancers, the identification of T-cell specific against so-called tumor neo-antigens and Adoptive transfer of those lymphocytes resulted in some improvement. In 2020, Detection of a T cell receptor (TCR) in a T cell clone that recognized and killed most human cancer cell lines in vitro via the monomorphic MHC class I-related protein MR1, offers an opportunity for pan-cancer therapy Twenty three years earlier, Moist Heat was used successfully to activate a whole different and new immune response that was able to detect genetic mutation in the affected cancer cells and cured many cases of squamous and basal cell carcinomas. In this commentary review, we aimed to revise the literature for updates of TILs usage in cancer prognosis and treatment.

Effects of“Moxibustion Serum”on Proliferation and Phenotypes of Tumor Infiltrating Lymphocytes

Tumor infiltrating lymphocytes (TIL) were cultured with “moxibustion serum”(MS), and the results were examined by flow cytometry. The results indicated that MS could enhance the proliferation of TIL,accelerate it to reach the exponential growth phase, and assist recombinant interleukin 2 (rIL-2) to enhance successively the percentage of CD3^+ positive cells, maintain the number of CD4^+ positive T cells, promote greatly the percentage of CD8^+ positive T cells among TILs, and reverse the CD4^+/CD8^+ ratio. Such cooperative effects rely on relative specificity of acupoints. It is suggested that MS is beneficial to the growth of TIL both in the aspects of proliferation and phenotypes.

T SUBSETS AND ANTITUMOR ACTIVITY OF LYMPHOCYTES INFILTRATING HUMAN PRIMARY BRAIN GLIOMAS

Glioma-infiltrating lymphocytes (GIL) were isolated from 9 surgical biopsy specimens of primary brain gliomas using mechanical and enzymatic digestion and discontinuous density gradent centrifugation. During cultured in the presence of interleukin-2 (IL-2) for a period of four weeks, GIL were expanded 48. 4-fold on the average, even up to 118-fold. GIL activated by IL- 2 had specific cytolytic activity against autologous glioma cells. Analysis of T subsets of GIL freshly isolated showed that CD3+ cells were 71.0±11.9%, CD4+ cells 34.2±6.1% and CD3+cells 37.0±7.6%. Ability of activated GIL to produce γ-Interferon (γ-IFN) was significantly higher than that of freshly isolated GIL and autologous peripheral blood lymphocytes (PBL). The results suggest that GIL have many advantages for an adoptive immunotherapy of patients with brain gllomas and be a new type of antitumor immune effector.

Biocharacterizations of T lymphocytes infiltrating human primary brain gliomas

Glioma-infiltrating lymphocytes(GIL)were isolated from 9 surgical biopsy specimens of pri-mary brain gliomas using mechanical and enzymatic digestion and discontinuous density gradientcentfifugation.Durng culture in the presence of interleukin-2(IL-2)for a period of four weeks,GIL were expanded by 48.4-fold on the avea-age,even up to 118-fold.GIL activated by IL-2 hadspcific cytolytic activity against autologous glioma cells.Analysis of cell surface phenotypes offreshly isolated GIL showed that CD3^+ cells were 71.0±11.9％,CD4^+ cells 34.2±6.1％ and CD8^+cells 37.0±7.6％.Ability of IL-2-activated GIL to secrete γ-interferon(γ-IFN)was significantlyhigher than that of freshly isolated GIL and autologous peripheral blood lymphocytes(PBL).Theresults suggest that GIL have many advantages as an adoptive immunotherapy for patients withgliomas and as a new type of antitumor immune effector.

IMMUNOLOGIC CHARACTER OF TUMOR INFILTRATING LYMPHOCYTES IN OVARIAN CARCINOMA

Objective: To study immunologic character of tumor-infiltrating lymphocytes (TIL) on postin vitro expansion in ovarian carcinoma, and evaluate the prospects by adopting TIL treatment of ovarian carcinoma at an advanced stage. Methods: Cellular phenotype changes in TIL were analyzed by flow cytometry. By means of molecular biology and immunologic methods, ability to secrete cytokines and anti-tumor activities of in TIL was studied. Results: Difference of cellular phenotypes in TIL was probably related to the type, feature and resource of the tumor. TIL obtained from phoroplast and parenchyma was dominant in CD3+CD4+. TIL obtained from tumor tissues, around microvessels and ascitic fluid was dominant in CD3+CD8 Concentration of rIL-2in vitro played a significant role in immunologic character of TIL. By means of rIL-2 expansionin vitro, TIL has apparently been improved in competence of secreting some cytokines, such as IL-2, TNF-α, IFN-γ, and anti-tumor activities. The activated TIL was more stimulated by further adding anti-CD3 or PHA (suitable concentration), which significantly increased its ability to secrete cytokines. Treatment with TIL+CTX or TIL+ rIL-2, could apparently improve phenotypes in peripheral blood of patients, with definitive effects. Conclusion: Immunologic activities of TILin vitro are apparently improved by rIL2 expansion. Regression of tumor, by means of infusion TIL, is not largely attributed to direct cytotoxicity to tumor cells, but indirectly and partly augmenting cellular activities and abilities of immunomodulation in patients with ovarian carcinoma being dependent on secreting multiple cytokines.

Separation and Expansion of Tumor Infiltrating Lymphocytes of Digestive System in Vitro and Their Cytotoxicity

Primary tumor tissues in the digestive system were harvested form 15 patients. By mincing,enzymatic digestion and gradient density separation, sufficient TILs(>5×106) were obtained from 13 of 15 (88.7%) patients in vitro in the presence of 500 μ/ml of recombinant interleukin-2 and 5% fetal calf serum after one month culture. 92.3% (12/13) of TILs proliferated well in vitro (92.3%). TILs expanded from 102-folds to 103-folds after being cultured for one month. CD25+ cell of the most fresh TILs was more than that of peripheral blood lymphocytes. CD25+ cells of TILs during 4th week of the culture was significantly greater (P<0.01) than that of fresh TILs. CD4+/CD8+ ratio was decreased during four culture weeks because of increase of CD8 cells. By using modified colorimetric MTT assay for measuring activity of TILs against various tumor cells the results showed that cytotoxicity of gastro-intestinal TILs autologous tumor cells is greater than on the other tumor cells.

LAG-3 expression on tumor-infiltrating T cells in soft tissue sarcoma correlates with poor survival

Objective: To elucidate the role and prognostic significance of lymphocyte activation-gene-3(LAG-3) in soft tissue sarcoma(STS).Methods: The expression of LAG-3 in patient and matched normal blood samples was analyzed by flow cytometry. The localization and prognostic values of LAG-3^+ cells in 163 STS patients were analyzed by immunohistochemistry. In addition, the expression of tumor-infiltrating CD3^+ T, CD4^+ T, and CD8^+ T cells and their role in the prognosis of STS were evaluated by immunohistochemistry. The effect of LAG-3 blockade was evaluated in an immunocompetent MCA205 fibrosarcoma mouse model.Results: Peripheral CD8^+ and CD4^+ T cells from STS patients expressed higher levels of LAG-3 than those from healthy donors.LAG-3 expression in STS was significantly associated with a poor clinical outcome(P = 0.038) and was correlated with high pathological grade(P < 0.001), advanced tumor stage(P = 0.016). Additionally, LAG-3 expression was highly correlated with CD8^+ T-cell infiltration(r = 0.7034, P < 0.001). LAG-3 was expressed in murine tumor-infiltrating lymphocytes, and its blockade decreased tumor growth and enhanced secretion of interferon-gamma by CD8^+ and CD4^+ T cells.Conclusions: LAG-3 blockade may be a promising strategy to improve the effects of targeted therapy in STS.

CXCL16 participates in pathogenesis of immunological liver injury by regulating T lymphocyte infiltration in liver tissue

AIM: To investigate the role of CXCL16 in the pathogenesis of immunological liver injury and to explore the possible mechanism of T lymphocyte infiltration requlated by CXCL16.METHODS: Immunological liver injury in murine model was induced by Bacille Calmette-Guerin and lipopolysaccharide.Expression pattem and distribution of CXCL16 were examined by real-time quantitative RT-PCR and immunohistochemical analysis. Anti-CXCL16 antibody was administrated in vivo to investigate its effect on T-cell recruitment and acute hepatic necrosis. The survival of murine model was also evaluated.RESULTS: The murine immunological liver injury model was successfully established. CXCL16 expression increased and predominantly distributed in periportal areas and vascular endothelia in injured liver tissues. Administration of anti-CXCL16 Ab protected the mice from death and acute liver damage. Approximately 70% of the mice survived for 72 h in the anti-CXCL16 Ab treatment group, whereas 80% died within 72 h in control Ab group. The number of liver-infiltrating T lymphocytes was significantly reduced from 1.01×L07 to 3.52x 106/liver, compared with control Ab treatment.CONCLUSION: CXCL16 is involved in immunological liver injury by regulating T lymphocyte infiltration in liver tissue.

Autoimmune pancreatitis characterized by predominant CD8+ Tlymphocyte infiltration

Autoimmune pancreatitis(AIP)is a rare form of pan-creatitis characterized by prominent lymphocyte inf iltration and pancreatic f ibrosis resulting in organ dysfunc-tion.The pathogenesis and pathology of AIP remain unknown.A 64-year-old Chinese man presented with symptoms and signs of bile duct obstruction diffuse enlargement of the head of pancreas,elevated IgG levels,and negative autoimmune antibody responses.A pylorus-preserving pancreatoduodenectomy was per-formed and a pancreatic tumor was suspected.Howev-er,periductal lymphoplasmacytic inf iltration and f ibrosis were found in the head of pancreas and nearby organs instead of tumor cells.Four months after surgery,the patient was readmitted because of reoccurrence ofsevere jaundice and sustained abdominal distension.Prednisone 30 mg/d was administered orally as an AIP was suspected.One and a half months later,the symp-toms of the patient disappeared,and globulin,amino-transferase and bilirubin levels decreased signif icantly.Over a 9-mo follow-up period,the dose of prednisone was gradually decreased to 10 mg/d and the patient remained in good condition.We further demonstrated dominant CD3+/CD8+ populations,CD20+ cells and a few CD4+ cells in the pancreatic parenchyma,duo-denum and gallbladder wall by immunohistochemical assay.This AIP case presented with signif icant CD8+ T lymphocyte inf iltration in the pancreas and extra-pan-creatic lesions,indicating that this cell population may be more important in mediating AIP pathogenesis than previously known and that AIP might be a poorly defined autoimmune disease with heterogeneous pathogenesis.