Immunohistological characterisation of tumour infiltrating lymphocytes in melanocytic skin lesions

BACKGROUND: Although the presence of tumour infiltrating lymphocytes (TIL) is a constant feature in melanomas, their immunophenotypic characterisation is still incomplete. We hypothesise that the transition from normal skin to benign naevi (BN) to melanocytic dysplastic naevi (MDN) to radial growth phase cutaneous malignant melanoma (RGP-CMM) to vertical growth phase cutaneous malignant melanoma (VGP-CMM) is associated with alterations in TIL. This study attempted to test this hypothesis and to characterise TIL in the melanocytic skin lesions. METHODS: In total, 74 lesions (12 BN, 12 MDN, 13 RGP-CMM, 26 VGP-CMM, and 11 metastatic melanomas) were examined using immunoperoxidase staining methods and antibodies targeting leukocyte common antigen (LCA+), T (CD3+) and B (CD20+) lymphocytes, and resting cytotoxic T cells (TIA-1+). RESULTS: Histologically, the transitions from normal skin to BN to MDN to RGP-CMM to VGP-CMM was associated with a gradual increase in the numbers of TIL (total, parenchymal, stromal, perivascular, and epidermal TIL, as well as TIL at the base of the lesions). The numbers of TIL were higher at the stroma than at the parenchyma. Similarly, immunostaining revealed that these transitions were associated with a gradual increase in the staining values (staining intensity, percentage of positive cells, and immunoreactivity score) for LCA+, CD20+, CD3+, and TIA-1+cells. The number of CD3+ cells was higher than that of CD20+ cells. All these differences between the normal skin and the lesional ones reached statistical significance (p<0.01). The majority of CD3+ cells were TIA-1+ T cells with cytotoxic potential. Compared with primary melanomas, there was a decrease in TIL in metastatic melanomas. CONCLUSIONS: The gradual increase in TIL during melanoma tumorigenesis may reflect increased antigenicity of the tumour cells. Although both humoral and cell mediated immunity are involved in melanomagenesis, the latter seems to have the major role. The immune profile of MDN suggests their intermediacy between BN and CMM.     

Immunohistochemical analysis on normal nephrogenesis and Wilms' tumour using monoclonal antibodies reactive with lymphohaemopoietic antigens

Summary Adult and fetal human kidneys were investigated for the reactvities of monoclonal antibodies, BA-1, BA-2 and BA-3 against human leukocytes. In developing metanephros, their reactivities changed reflecting the stage of nephrogenesis. Thus BA-1 stained both metanephric blastema and ureteric bud. Glomerular and proximal tubular development was characterized by the disappearance of BA-1 reactive antigen and the appearance of CALLA defined by BA-3. Immuno-electron-microscopically CALLA was solely located on the glomerular epithelial membrane and on the microvilli of the proximal tubules. BA-2 constantly stained ureteric bud-derived tissues. These observations were applied for the analysis of histogenesis of Wilms' tumour. Tumour blastema of the classical type reacted only with BA-1. Epithelial components in the classical as well as the epithelial type reacted both with BA-I and BA-2. CALLA was only detectable in glomeruloid and the connecting tubular structures, while these were unstained by BA-1. Stromal components in the classical and the sarcomatous type did not express any of these antigens. Staining patterns were identical in surgically removed and xenotransplanted tumours. These studies establish that each component of Wilms' tumour can clearly be interpreted regarding its histogenesis and that epithelial components corresponding to proximal tubules are identified in Wilms' tumour.     

Melanocortin 1 Receptor-derived peptides are efficiently recognized by cytotoxic T lymphocytes from melanoma patients

Background

Melanocortin 1 Receptor (MC1R) is expressed in a majority of melanoma biopsies and cell lines. We previously demonstrated that three hydrophobic low-affinity HLA-A2-restricted MC1R-derived peptides: MC1R_291–298, MC1R_244–252 and MC1R_283–291 can elicit cytotoxic T-lymphocytes (CTL) responses from normal donor peripheral blood lymphocytes (PBL). Moreover, peptide-specific CTL recognized a panel of MHC-matched melanomas, demonstrating that human melanoma cell lines naturally present MC1R epitopes. However, the natural presence of MC1R-specific T cells in melanoma patient's tumour and blood remains unknown.

Methods

The presence of anti-MC1R specific CD8⁺ T cells was established in a population of melanoma-specific T cells derived from peripheral blood mononuclear cells (PBMC) and tumour-infiltrating lymphocytes (TIL) from HLA-A2⁺ melanoma patients.

Results

CTLs specific for the three MC1R-derived peptides that lysed allogeneic HLA-A2⁺MC1R⁺ melanomas were elicited from PBMC, demonstrating the existence of an anti-MC1R T cell repertoire in melanoma patients. Moreover, TILs also recognized MC1R epitopes and HLA-A2⁺ melanoma cell lines. Finally, HLA-A2/MC1R₂₄₄-specific CD8⁺ T cell clones derived from TILs and a subset of MC1R₂₉₁ specific TILs were identified using HLA-A2/MC1R tetramers.

Conclusion

Our results demonstrate that MC1R-derived peptides are common immunogenic epitopes for melanoma-specific CTLs and TILs, and may thus be useful for the development of anti-melanoma immunotherapy.     

ALDH1 and tumor infiltrating lymphocytes as predictors for neoadjuvant chemotherapy response in breast cancer

Many studies have reported that Aldehyde dehydrogenase 1 (ALDH1) and tumor-infiltrating lymphocytes (TIL) are related to breast cancer prognosis. However, the clinical significance of ALDH1 and tumor-infiltrating immune cells in breast cancer has not been fully investigated in patients who received neoadjuvant chemotherapy (NAC). We studied the significance of the expression of ALDH1 and the population of TIL for predicting the prognosis and chemotherapeutic response of patients with breast cancer who had received NAC. Forty patients who underwent NAC were enrolled in this study. ALDH1 and TIL (T cells and tumor associated macrophages) were evaluated before and after NAC. The influences of ALDH1 expression status and TIL populations on both prognosis and chemotherapeutic response were evaluated. ALDH1 positivity was related to estrogen receptor (p?=?0.026) and progesterone receptor negativity (p?=?0.025). Positive change of ALDH1 after NAC tended to be associated with a poor NAC response (p?=?0.078). Patients with more CD8+ T cells before NAC and fewer CD68 (+) macrophages after NAC tended to have better OS, respectively (p?=?0.086, p?=?0.096). The chemotherapeutic response and prognosis of patients with breast cancer who received NAC are thought to be determined by the tumor microenvironment. Further research with more patients and a longer study period is needed.     

Activated status of tumour-infiltrating lymphocytes and apoptosis in testicular seminoma.

Testicular seminoma is characterized by a prominent lymphoid infiltrate and an excellent prognosis. Cytotoxic T-lymphocytes (CTLs) infiltrating seminoma tumour nests constitute a major subset of the lymphoid infiltrate. The objective of this study was to determine whether CTLs express markers of cytotoxic potential and activity and whether the number of activated CTLs correlates with the extent of apoptosis in testicular seminomas, as opposed to non-seminomatous testicular germ cell tumours (NSTGCTs). Twenty cases of pure seminoma as well as 20 cases of NSTGCTs including 16 mixed germ cell tumours (MGCTs) were studied. Immunohistochemistry for the cytotoxic markers TIA-1 (cytotoxic potential) and granzyme B (cytotoxic activity) and the T-cell markers CD3 and CD8 was performed on formalin-fixed, paraffin-embedded sections. The apoptotic index (AI) was determined by the TUNEL method. The number of CD3(+), CD8(+), TIA-1(+), and granzyme B(+) cells in tumour cell nests was markedly increased in testicular seminomas, compared with NSTGCTs (p<0.01). Activated granzyme B(+) cells numbered 25.6+/-5.2 per high power field in seminomas and 8.9+/-3.2, 8.1+/-3.9, and 0.4+/-0.2 for embryonal carcinomas, yolk sac tumours, and immature teratomas, respectively. Double immunohistochemical staining for granzyme B and CD8 revealed that 82.6+/-8.5% of granzyme B-expressing cells were CD8(+). The tumour cell AI was significantly increased in embryonal carcinoma, compared with the seminoma, yolk sac tumour, and immature teratoma subgroups (6.7+/-1.3, 2.3+/-0.3, 3.0+/-1.1, and 2.3+/-1.1, respectively, p<0.001). TUNEL/CD3 double immunostaining revealed that a significant proportion of the apoptotic seminomatous tumour cells were in direct contact with one or more CD3(+) lymphocytes (47.2+/-6.2%). The number of activated granzyme B(+) CTLs showed a strong linear correlation with the AI in the seminoma group (r=0.71, p<0.0001) but not in other subgroups. TUNEL/granzyme B double immunolabelling revealed that a proportion of activated granzyme B(+) lymphocytes (20%) were often seen in close contact with apoptotic tumour cells. The presence of increased numbers of activated cytotoxic lymphocytes in testicular seminomas suggests that apoptotic tumour cell death in this neoplasm may be triggered by cytotoxic granule effectors. This phenomenon may be one of the key host immune mechanisms leading to the excellent prognosis in this tumour.     

Immunohistological analysis of human mononuclear phagocytes and dendritic cells by using monoclonal antibodies

Mononuclear phagocytes and dendritic cells were analyzed in detail with a panel of monoclonal antibodies by immunoalkaline phosphatase and immunofluorescence techniques. The panel included two antibodies (KB90 and SHCL3) against the two chain membrane constituent, P150,95, an antibody (Mo1) against C3bi receptor, an antibody (To5) against C3b receptor, and two antibodies (Mo2 and UCHM1) against a single chain glycoprotein with a molecular weight of 55 to 60 previously found on monocyte/macrophages. In addition, the panel contained several antibodies including EBM11, LeuM3 and FMC32 whose molecular specificities are not known at present. Frozen sections of spleen, bone marrow, thymus, lymph node, liver, brain, lung, kidney, colon, and skin were examined. Several populations of mononuclear phagocytes and dendritic cells could be distinguished on the basis of their reactivity with the antibody panel. Populations of macrophages resident in parenchymal organs had reaction patterns distinct from those of circulating macrophages. Many of the antibodies in the panel reacted with both macrophages and dendritic cells suggesting a close cytogenic, if not functional, relationship between these cells. There was wide variability in the reactions of macrophages and dendritic cells in different tissue and less variability among cells within individual anatomic locations.     

Cytoplasmic expression of high mobility group B1 (HMGB1) is associated with tumor‐infiltrating lymphocytes (TILs) in breast cancer

High mobility group box 1 (HMGB1) is a prototypic alarmin or damage‐associated molecule inducing inflammatory mediator release and immune response. Several studies have revealed the prognostic and predictive importance of tumor‐infiltrating lymphocytes (TILs) in breast cancer. The present study analyzed the expression of HMGB1 in each breast cancer subtype and the relationship between the expression level of HMGB1 and pathologic parameters including TILs. Two cohorts were studied: 575 consecutive breast cancer patients who underwent surgery between 1995 and 1998; and 767 triple negative breast cancer (TNBC) patients who underwent surgery between 2004 and 2010. The immunohistochemical expression level of HMGB1 in cytoplasm and nucleus was evaluated using tissue microarrays. High HMGB1 expression in cytoplasm was associated with high histologic grade, pT stage, and abundant TILs in the consecutive breast cancer cohort. Cytoplasmic HMGB1 expression was higher in TNBCs and HER2‐positive tumors than in hormone receptor‐positive tumors. In the TNBC cohort, high cytoplasmic HMGB1 expression was significantly associated with high histologic grade, abundant TILs, and high numbers of CD8+ cells. However, nuclear HMGB1 expression was not associated with histologic grade or TIL levels. Neither cytoplasmic nor nuclear expression of HMGB1 showed prognostic significance in TNBC. Cytoplasmic HMGB1 expression is associated with TIL levels in breast cancer.     

人肝癌组织肿瘤浸润淋巴细胞穿孔素,Fas配体表达的研究

目的 研究肝癌组织肿瘤浸润淋巴细胞（ＴＩＬ）的穿孔素及Ｆａｓ配体（Ｆａｓ－Ｌ）表达情况，方法 应用原位杂交及免疫组化ＡＢＣ方法检测２０例病人原发性肝细胞癌组织的ＴＩＬ中穿孔素及Ｆａｓ－Ｌ的ｍＲＮＡ及其蛋白的表达情况。结果 ２０例肝癌病人中１６例，肝癌组织中ＴＩＬ表达穿孔素及Ｆａｓ－１，其中１例病人的绝大多数ＴＩＬ表达穿孔素及Ｆａｓ－１，该例病人手术后１年６个月仍未复发，除该例以外，其余１５例肝癌组     

卵巢癌浸润淋巴细胞的生物学特性研究

目的 :研究卵巢癌浸润淋巴细胞 (TIL)在体外扩增后生物学特性 ,评估TIL用于晚期卵巢癌治疗的前景。方法 :利用流式细胞仪分析TIL的细胞表型 ,使用分子生物学和免疫学方法研究TIL分泌细胞因子的能力和杀伤肿瘤细胞的活性。结果 :TIL细胞表型的差异可能与肿瘤的种类、性质、取材部位有关 ,结缔组织、基质中来源的TIL以CD3 CD4 为主 ,肿瘤组织和小血管周围以CD3 CD8 为主 ,体外IL 2的浓度对TIL的免疫学特性有很大的影响。经rIL 2扩增后 ,TIL分泌产生IL 2、TNF α、IFN γ等多种细胞因子能力及杀伤肿瘤细胞的活性均有明显提高。再加入抗CD3单抗或PHA(合适浓度 ) ,TIL细胞因子表达可进一步增强。TIL联合磷酰胺或IL 2治疗晚期卵巢癌患者 ,可显著改善患者外周血细胞表型 ,具有一定的疗效。结论 :TIL在体外经rIL 2扩增后 ,免疫活性有明显提高。体内肿瘤细胞的消退可能主要并不是通过输入的TIL直接杀伤肿瘤细胞 ,而是在很大程度上依靠其分泌多种细胞因子增强了机体细胞免疫活性和免疫调节能力。TIL辅助其它疗法可成为晚期卵巢癌患者的一种有效治疗手段。     

Immunohistological phenotyping of thyroid infiltrating lymphocytes in Graves'' disease and Hashimoto''s thyroiditis.

Subsets of lymphocytes in the thyroid were immunophenotyped by their surface antigens in frozen tissues of Hashimoto's thyroiditis and Graves' disease. Using triple layer immunoperoxidase staining (IP), monoclonal antibodies (T3, Leu 3, T8, anti-Tac and Leu 7) were employed to detect markers of T cell subsets, activated T cells, and a natural killer associated antigen. B cells were identified by 2 step IP with anti-IgD antisera. Excluding those cells forming lymphoid follicles, the density of lymphocytes infiltrating between thyroid epithelial cells was much higher in Hashimoto's thyroiditis than in Graves' disease. However, relative proportions of subsets were similar in both diseases. Most of the infiltrating cells were T3 positive T cells (T3+), with more T8+ (suppressor/cytotoxic T) than Leu 3+ (inducer/helper T). Some Leu 7+ were occasionally seen, but surface IgD positive mature B cells (IgD+) were almost absent. In contrast, IgD+ cells were densely aggregated in primary lymphoid follicles and mantle zones of secondary follicles. In these regions, Leu 3+ cells were about twice as frequent as T8+ cells. Some Leu 7+ and scarce Tac+ cells were also found. The present study indicates a major involvement of immunoregulatory T cells in autoimmune thyroid disease, and also suggested intrathyroidal maturation of B cells.     

T cell receptor β‐chain repertoire analysis reveals intratumour heterogeneity of tumour‐infiltrating lymphocytes in oesophageal squamous cell carcinoma

Oesophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, due to the lack of effective treatment methods. Immunotherapeutic approaches based on tumour‐infiltrating lymphocytes (TILs) have demonstrated that durable responses are produced in some patients with solid tumours, which suggests the potential feasibility of clinical application of immunotherapy for ESCC. However, many of the basic characteristics of TILs in ESCC are poorly understood, including clonality, specificity and spatial heterogeneity of the response of TILs, which depends on the interaction between antigens and T cell receptors (TCRs). We used ultra‐deep sequencing of rearranged genes in TCR β‐chain (TCRβ) to profile the basic characteristics of T cells in tumour tissues (four to six regions from each tumour) as well as matched adjacent normal tissue and peripheral blood from seven patients diagnosed with primary ESCC. We found that T cell clones within ESCCs were quite different from those of the peripheral blood and even the adjacent normal tissues in general. Although there was a relatively higher degree of overlap of intratumoural TCRβ repertoires than those between the tumour and other tissues, intratumoural TCRβ repertoires were spatially heterogeneous. Due to the restricted sampling, high‐throughput TCRβ sequencing could characterize the diversity and composition of a limited (compartment‐dependent) fraction of the respective T cell clones in any individual ESCC, expanding our understanding of immune behaviour and immune response and shedding more light on ESCC immunotherapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Prognostic value of tumour-infiltrating lymphocytes (TILs) in colorectal cancer

Clinical follow-up data of 276 colorectal adenocarcinoma patients treated in Kuopio University Hospital between 1976 and 1986 and followed up for a mean of 14 years were analysed. The clinical findings were correlated with tumour-infiltrating lymphocytes (TILs) and with histological and quantitative factors including nuclear parameters and volume-corrected mitotic index. In univariate survival analysis, TNM classification, Dukes' stage, histological grade, and TILs were significant predictors of survival. TNM classification, Dukes' stage, and TILs also predicted recurrence-free survival. In multivariate analysis, TILs were an independent prognostic factor of survival in all cases, as well as in patients with T1-4N0-3M0 and T1-4N1-3M1. TILs also independently predicted recurrence-free survival. TILs can provide important prognostic information in colorectal cancer to be used in evaluating for adjuvant therapy in different tumour stages. © 1997 John Wiley & Sons, Ltd.