CD8-positive Cytotoxic T-cells Research Articles

CD8 cytotoxic T-cell infiltrates and cellular damage in the hypothalamus in human obesity

Rare cases of paraneoplastic obesity in children suggest sporadic obesity might also arise from an adaptive immune cell-mediated mechanism. Since the hypothalamus is a central regulator of feeding behavior and energy expenditure, we quantified lymphocytic inflammation in this region in a cohort of obese and non-obese human post-mortem brains. We report that CD8-positive cytotoxic T-cells are increased in hypothalamic median eminence/arcuate nucleus (ME/Arc) and bed nucleus of the stria terminalis in 40% of obese compared to non-obese patients, but not in other hypothalamic nuclei or brain regions. CD8 T-cells were most abundant in individuals with concurrent obesity and diabetes. Markers of cytotoxic T-cell induced damage, activated caspase 3 and poly-ADP ribose, were also elevated in the ME/Arc of obese patients. To provoke CD8 cytotoxic T-cell infiltrates in ventromedial region of hypothalamus in mice we performed stereotactic injections of an adeno-associated virus expressing immunogenic green fluorescent protein or saline. AAV but not saline injections triggered hypothalamic CD8 T-cell infiltrates associated with a rapid weight gain in mice recapitulating the findings in human obesity. This is the first description of the neuropathology of human obesity and when combined with its reconstitution in a mouse model suggests adaptive immunity may drive as much as 40% of the human condition.

Tumor-TME Bipartite Landscape of PD-1/PD-L1 in Endometrial Cancers.

The bipartite landscape of tumor cells and stromal cells determines a tumor's response to treatment during disease management. In endometrial cancers (ECs), the mechanistic contribution of PD-L1/L2 and PD-1 signaling of the host's tumor microenvironment (TME) (CAF and immune cells) in the context of the tumor cells is elusive. To understand the tumor-stroma-immune crosstalk, we studied the compartmental pattern of PD-L1/L2 and PD-1 expression in EC tissues and their matched CAFs. Over 116 surgically resected tumors (T) and the tumor-adjacent normal tissues (N) were obtained from consented unselected consecutive patients. IHC was performed in T, N-epi-thelium, and the stromal mesenchymal environment (SME; mesenchyme) in the T and N tissues. The staining intensity and distribution patterns of PD-L1/L2 and PD-1 in the FFPE sections of T and N were evaluated by a pathologist using a standard scoring system of TPS and CPS. We tested the PD-L1/L2 and PD-1 immune landscape of tumor-TME pair and normal epithelial-stromal mesenchyme pairs from patients with different grades of disease vis-à-vis their CAF PD-L1 levels. We used qRT-PCR to determine the expressions of mRNAs, while the flow cytometry and ICC determined the level of expression of proteins. We observed higher levels of PD-L1 mRNA and protein expression in primary CAFs from the resected tumor tissue compared to the tumor-adjacent normal tissues. We also determined the expression of patients' soluble PD-L1/L2 as peripheral readouts of PD-L1/L2 and PD-1. As we evaluated the results in the context of their pathological parameters, such as grades, stages, lymphovascular invasion, percentage of myometrial invasion, and dMMR in patients, the dominance of PD-L1 expression in TME was positively correlated to the higher pathological grades of tumors, and its relationship with the dMMR. Since the neutralization of CD8-positive cytotoxic T-cells is PD-L1-dependent, our data indicate that irrespective of the PD-L1 positivity of tumor cells, the PD-L1-positive CAFs can play a critical role in bringing out an additional load of PD-L1 for an effective engagement of PD-1 within a tumor mass.

Kikuchi disease with an exuberant proliferation of large T-cells: a study of 25 cases that can mimic T-Cell lymphoma.

Exuberant large T-cell proliferations in Kikuchi disease can potentially be misdiagnosed as lymphoma. In this study, we explore their clinicopathological features and summarize key points that can be used to distinguish them from T-cell lymphoma. The cohort consisted of 25 cases of Kikuchi disease with an exuberant large T-cell proliferation, which, in part, mimicked lymphoma. The median age was 25 years with a female:male ratio of 4:1. By B-scan ultrasonography,patients presented with either isolated lymphadenopathy (68%) involving the cervical and axillary regions or generalized lymphadenopathy (32%). Histologically, lymph nodes showed paracortical and interfollicular expansion by sheets of large cells associated with karyorrhectic debris. Histiocytes and plasmacytoid dendritic cells were present in the background. No case showed complete effacement of lymph node architecture. The large cells were CD8-positive cytotoxic T-cells with a high proliferation rate. These T-cells showed decreased BCL-2 in 17 (68%) cases. CD5 expression was decreased in 10 (40%) cases. Histiocytes in the background were positive for myeloperoxidase. Clonal TRG and/or TRB rearrangements were detected in 2 of 10 (20%) cases. In conclusion, large T-cell proliferations in Kikuchi disease can be alarming at the morphologic and immunophenotypic levels and need to be distinguished from T-cell lymphoma. Clinical features helpful in the differential diagnosis include young patients and lymphadenopathy involving the cervical and axillary regions. Major pathologic features helpful in this differential diagnosis include partial involvement of the lymph node and the presence of karyorrhectic debris, crescent-shaped histiocytes, and/or loose aggregates of plasmacytoid dendritic cells.

Abstract 1022: Evaluation of potential factors contributing to the exhaustion of T lymphocytes in the tumor microenvironment

Abstract The presence of immunosuppressive molecules and cells in the tumor microenvironment (TME) can lead to T cell dysfunction. CD8-positive cytotoxic T cells (CTLs) are ineffective in killing tumor cells primarily due to upregulated expression of inhibitory checkpoint molecules and decreased production of effective cytokines. In addition, immune suppressive cell types such as regulatory T cells (Tregs) and tumor associated macrophages (TAMs) are recruited to the TME, further establishing a suppressive immune environment. In this study, we evaluated expression profiles of key immunosuppressive molecules and cell types by applying RNAscope® assay, a highly specific and sensitive in situ hybridization (ISH) technology, and dual ISH-IHC staining. First, we evaluated CD8-positive cell infiltration in TME of archived human tissues from non-small cell lung cancer and ovarian cancer. Selected tissues with either high or low CD8-positive cell number (CD8-high or CD8-low) were evaluated for (1) the presence of Tregs (FOXP3+CD4+) and TAMs (CD163+, including IL10 and CCL22), (2) the expression of immune checkpoint molecules including PD1, PD-L1, TIM3, and LAG3, (3) the expression of immune suppressive molecules IDO1 and TGFβ, and (4) IFNγ expression in CD8-positive subsets. Examination of immune inhibitory molecules expressed in single cells in the tumor and stromal microenvironment revealed that in general, CD8-high tissues expressed higher level of immune checkpoint molecules, often co-expressed in the same individual cells in the same TME, while the expression of IDO1 and TGFβ was independent of CD8-positive cell inflammation. Tregs and TAMs often co-existed with IFNγ-positive CTLs in the same TME. Unexpectedly, all investigated inhibitory molecules were expressed in both tumor cells and stromal/immune cells in some tumors. Beyond PD-L1, immune checkpoint molecules PD1, TIM3, and LAG3 were frequently expressed in tumor cells but at lower level than in immune cells. Expression of IDO1 and TGFβ was observed in many cell types, including tumor cells, with various expression levels in each tumor cell. The single-cell tumor expression profiles suggest a potential tumor-intrinsic mechanism of expression for these inhibitory molecules. This exploratory study highlights the potential of RNAscope® ISH to better understand the cellular and molecular suppressive mechanisms associated with T cell dysfunction and exhaustion in the TME. The robust RNAscope® ISH platform is well suited for evaluating critical secreted factors and other key molecules in a highly sensitive and cell type-specific manner. As multiple therapeutic approaches to augment the CTL function are being developed, the presented method may facilitate the identification and development of key biomarkers to stratify patients based on their specific tumor and immune cell states. Citation Format: Annelies Laeremans, Na Li, Jeff Kim, Xiao-Jun Ma, Emily Park. Evaluation of potential factors contributing to the exhaustion of T lymphocytes in the tumor microenvironment [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 1022.

Immune Cell Infiltrates in Hippocampal Sclerosis: Correlation With Neuronal Loss.

Immune mechanisms have been increasingly recognized in the pathogenesis of hippocampal sclerosis (HS), but infiltration of cytotoxic T-cells and its pathological significance in patients with HS has not been explored. We examined 30 cases of surgically resected hippocampi, including 16 International League Against Epilepsy (ILAE) type 1, 9 ILAE type 2, 1 ILAE type 3 HS, and 4 ILAE No-HS, as well as 6 autopsy No-HS hippocampi. The HS hippocampi showed sparse to scattered CD8-positive T-cells, rare CD4-positive T-cells, and a modest increase in CD68-positive microglia/macrophages, which were significantly more numerous than those in the No-HS controls. The infiltration of CD8-positive T-cells was significantly greater in the CA1 subfield than other subfields of type 1 and type 2 HS. The numbers of CD8-positive T-cells positively correlated with those of CD4-positive T-cells; there was a lower ratio of CD4/CD8-positive T-cells. There were positive correlations between these cells and scores of neuronal loss but no significant correlation between the infiltration of these cells and epilepsy disease duration or age of epilepsy onset. These findings suggest that an autoimmune process may be involved in the pathogenesis of HS and infiltration of immune cells, particularly CD8-positive cytotoxic T-cells, may contribute to neuronal loss in HS.

Transformed aggressive γδ-variant T-cell large granular lymphocytic leukemia with acquired copy neutral loss of heterozygosity at 17q11.2q25.3 and additional aberrations

T-cell large granular lymphocytic leukemia (T-LGLL) is a rare indolent lymphoproliferative disorder characterized by cytopenias, splenomegaly, and various degrees of T-cell lymphocytosis, due to a clonal expansion of CD8-positive cytotoxic T-cells. Phenotypic variants of T-LGLL include CD4(+) /CD8(-) T-cells, with dual CD4(-) /CD8(-) /γδ(+) T-cells being even rarer. Cytogenetic abnormalities in T-LGLL have rarely been reported, and there is scientific debate regarding the existence of aggressive or transformed variants of T-LGLL. We report a patient with T-LGLL, γδ variant, with nearly 20-year-long duration of cytopenias before transformation to an unusual clinical scenario, manifesting with marked lymphocytosis >100 × 10(9) /L and infiltration of lymph nodes, tonsils, and subcutaneous tissue. Single-nucleotide polymorphism assays revealed acquired copy neutral loss of heterozygosity at 17q and deletion of 3p21.31, in addition to trisomy 5, monosomy X, and monosomy 21. These genetic abnormalities provided a better understanding of the molecular nature and the potentiality of disease transformation.

Expression of Master Regulators of Helper T-Cell Differentiation in Peripheral T-Cell Lymphoma, Not Otherwise Specified by Immunohistochemical Analysis.

Abstract 5004 BackgroundPeripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) comprises nodal and extranodal mature T-cell lymphomas, which do not correspond to any of the specifically defined entities of mature T-cell lymphoma in the current classification.During T-cell maturation, naïve T-cells include two distinct cell types, CD4-positive helper T-cells (Th) and CD8-positive cytotoxic T-cells. Following antigen stimulation, naïve Th cells differentiate into several subtypes of effector Th cells, including helper T-cells type1 (Th1), helper T-cells type 2 (Th2), follicular helper T-cells (TFH), regulatory T-cells (Treg), IL-17 producing T-cells (Th17), central memory T-cells (TCM), and effector memory T-cells (TEM).Previously, these subtypes of effector Th cells have been classified according to their pattern of cytokine secretion and expression of surface antigens, including chemokine receptors. More recent immunological researches have identified several transcription factors responsible for T-cell differentiation. The master regulators associated with Th, Th1, Th2, Th17, and Treg differentiation are reported to be Th-POK, T-Bet, GATA3, RORγt, and FOXP3, respectively.The origin of the neoplastic cells of PTCL-NOS is thought to be effector Th cells because the tumor cells usually express specific surface antigens such as pan-T-cell markers (CD2 and CD3), an effector T-cell marker (CD45RO), and a Th marker (CD4). However, the normal cellular derivation of PTCL-NOS remains unclear because the immunophenotypic profiles of PTCL-NOS are heterogeneous and not entirely disease specific.To presume the normal counterparts of PTCL-NOS, we used immunohistochemical analysis to examine the expression of the master regulators of T-cell differentiation and surface antigens, including chemokine receptors associated with the subtypes of Th cells. Patients and MethodsThe study population included ten Japanese patients who were diagnosed histopathologically with PTCL-NOS at the hospital of Kyoto Prefectural University of Medicine from 1993 to 2008. There were six male and four female patients who ranged in age from 26 to 78 years (mean 56 years) at diagnosis.Immunohistochemical staining was applied to 4-μm-thick paraffin-embedded sections of diagnostic lymph node, skin or liver tumor biopsy specimens. Sections were stained using a standard indirect avidin-biotin horseradish peroxidase method and diaminobenzidine (DAB) color development. Cases were considered positive if 25% or more of the tumor cells were stained with an antibody. For negative and positive staining controls, we used paraffin-embedded tissue sections of tonsillitis, nonspecific lymphadenitis, AILT, follicular lymphoma and diffuse large B-cell lymphoma. ResultsFirst, we examined the expression of the master regulator of helper T-cells (Th-POK) and the marker of effector T-cells (CD45RO), and all cases were positive for both.Next, we examined the expression of the master regulators of differentiation to subpopulations within Th cells (Th1, Th2, Treg and Th17). Three cases each were positive for T-Bet, and GATA3, but none of the cases showed FOXP3 or RORγt.Among the four cases not expressing these master regulators, two cases were positive for the surface antigens of central memory (TCM) (CCR7 and CD62L), and one case was positive for follicular helper T-cell (TFH) phenotype (BCL6, CXCL13 and PD-1). The remaining case was negative for all markers of effector Th subtypes. ConclusionsAll ten PTCL-NOS cases exhibited CD45RO and Th-POK, which characterized effector helper T-cells. Furthermore, the lymphoma cells of nine of the ten cases exhibited the expression of master regulators or surface antigens which characterized Th1, Th2, TCM and TFH. DisclosuresNo relevant conflicts of interest to declare.

Islet Autoreactive CD8 T-cells in Type 1 Diabetes

While differentiating T-cell autoreactivity against pancreatic islets between type 1 diabetic patients and nondiabetic control subjects has, over time, proven to be a difficult task, the ultimate challenge for cellular markers of this disease will involve the definition of immunological measures associated with disease progression and intervention. With the notion that type 1 diabetes is a T-cell–mediated autoimmune disease, it is therefore logical for investigators to direct research efforts toward studies of T-cells. However, relatively few studies associating T-cell autoreactivity with disease progression have been attempted (i.e., in comparison with efforts monitoring autoantibodies), and those that have been performed are largely limited to CD4 T-cells (1). Ever since the demonstration of their presence in insulitic lesions in the pancreas of patients at clinical onset of type 1 diabetes, CD8-positive cytotoxic T-cells have been implicated in the disease process (2). This association and its potential importance for disease was corroborated by the genetic predisposition of HLA class I loci for type 1 diabetes that is independent of linkage disequilibrium with HLA class II (3); a setting that implied the role of HLA class I with type 1 diabetes is more than guilt by genetic association alone (4). Theoretically, HLA class I–restricted CD8 T-cells could be the sole candidate …

Detection of Significantly Increased Frequencies of Proteinase-3 (Myeloblastin) - Specific CD8+ T-Cells in Patients with De Novo Diagnosed Hepatitis C and Infiltration of Liver Tissue with Proteinase 3 Overexpressing Mononuclear Cells.

Proteinase 3 (PR3), also referred to as myeloblastin, is a 26kD serine protease which is overexpressed in precursor cells of chronic myeloid leukaemia (CML). PR3 has previously been identified as the autoantigen of Wegener's granulomatosis, which is characterized by a self-destructive humoral and T-cell response against PR3. However, high avidity PR3-specific cytotoxic T-cells (CTL) from CML patients specifically lysed CML blasts and their presence in vivo correlated with the achievement of a complete cytogenetic remission under interferon alpha (IFNα) therapy. Thus, overcoming tolerance to PR3 contributed to the elimination of leukaemia without causing auto-immunity (Molldrem et al., Nat. Med. 2000). We have previously shown that the induction of PR3 by IFNα in CD14+ monocytes may contribute to elicit a PR3-specific CTL expansion in CML. Since PR3-upregulation has subsequently been reported in monocytes of HIV- and pneumonia patients as well as in hepatitis C liver specimens, we asked, whether the upregulation of PR3 under inflammatory conditions may be linked to the expansion of PR3-specific T-cells. Therefore, we studied the frequency of PR3 specific CD8 positive CTL in patients with de novo diagnosed hepatitis C, a disease known to be associated with various autoimmune phenomenona. Thirty four HLA-A2.1 positive patients were analyzed by means of staining peripheral blood mononuclear cells using PR3-specific tetramers. 17 patients (50%) had PR3 specific CTL either at diagnosis (n=13) or developed them during follow up (n=4). Of the 13 patients, who had PR3-tetramer positive T-cells at diagnosis, six patients had both high and low aviditiy PR3-CTL, whereas seven patients had only low avidity PR3-specific T-cells. The frequency of these CD8 positive CTL at diagnosis ranged between 0.16% to 0.53% of all T-cells for the high avidity population and between 0.12 to 1.58% for the low avidity population. Upon antiviral therapy with IFNα and Ribaverin, the frequency of PR3-CTL became undetectable within less than 5 month of initiation of therapy in five of six patients of the high avidity PR3-CTL group. Only one patient of the patients with low avidity PR3-CTL at diagnosis was available for follow up testing and became negative. Interestingly, in four patients we saw a transient increase of low avidity PR3 specific CTL after initiation of therapy. Immunohistology of four hepatitis C liver sections and four healthy control specimens revealed a strong infiltration of PR3-overexpressing mononuclear cells in hepatitis liver, but PR3-negativity in normal livers. Hepatocytes did not express PR3. In conclusion: Hepatitis C is significantly associated with the induction of PR3-specific CTL and overexpression of PR3 in mononuclear cells may be important to drive their induction. PR3-specific CTL may be responsible for autoimmune phenomena in hepatitis C such as vasculitis and myelosuppression.

Characterization of lymphoid infiltrates in chronic obstructive sialadenitis associated with sialolithiasis.

Chronic obstructive sialadenitis is characterized by acinar atrophy, lymphocytic infiltrates and progressive fibrosis. The immunological mechanisms involved in the pathogenesis of this disease are, for the most part, unknown. The aim of the present study was to characterize the lymphocytic infiltrates in chronic obstructive sialadenitis associated with sialolithiasis. Paraffin-embedded tissue samples from 23 affected submandibular glands were immunostained for T-cells (CD3, CD4, CD8), cytotoxic T-cells (granzyme B), B-cells (CD20), plasma cells (CD38) and macrophages (Ki-M1P). CD4-positive subsets were the predominant cells, and they were located mainly periductally. Isolated intraepithelial CD8-positive cytotoxic T-cells associated with ductal epithelial cell destruction were observed in all cases. B lymphocytes were restricted to lymphoid follicles located periductally and around intralobular ducts. In early stages of the disease, a large number of CD38-positive plasma cells were distributed diffusely in the periacinar area. With progression of the disease, conspicuous clusters of plasma cells were located especially between atrophic acini adjacent to fibrotic tissue. An intimate relation between the lymphocytic infiltrates and the ductal epithelium, the target of the inflammatory process, was observed. The composition and distribution of inflammatory cells suggest that intraepithelial infectious agents may be the cause of the inflammatory reaction and the progressive fibrosis in this disease.

Cytotoxic T cell activity against peptides of Hu protein in anti-Hu syndrome

Half of all patients with limbic encephalitis and small cell lung carcinoma (SCLC) have anti-Hu antibodies that react with all of central and peripheral nervous system neuronal nuclei in immunohistochemical studies and 35- to 40-kDa reactive bands on western blots of extracts from isolated central nervous system neurons. The roles of anti-Hu antibodies in neuronal damage, however, have yet to be shown. Evidence of infiltration of CD8-positive T cells to tumors and affected nervous tissues and limited use of the T cell receptor repertoire in the central nervous system suggests that CD8-positive cytotoxic T cells (CTL) cause neuronal loss. We found the HLA B7 supertype in all of seven Japanese patients with anti-Hu syndrome. We identified HLA class I-restricted, CD 8-positive cytotoxic T cell activity in peripheral blood from three patients with anti-Hu syndrome for five peptides with binding motifs for the HLA B7 supertype in the amino acid sequence of the Hu protein. This study support the involvement of CD8-positive cytotoxic T cells in the development of paraneoplastic neurological syndrome with anti-Hu antibodies.

Expression of MAGE antigens and analysis of the inflammatory T-cell infiltrate in human seminoma.

The MAGE gene family encodes antigens that are recognized by cytotoxic T-cells. The expression of MAGE antigens has been linked to tumor stage, and MAGE peptides are under investigation as possible vaccines. Seminomas are tumors that are typically accompanied by a heavy inflammatory infiltrate, but have not been studied with regard to their MAGE antigen expression and its correlation with the inflammatory infiltrate. We investigated, therefore, MAGE protein expression, the amount of cytotoxic T-cells, clonality of the lymphocytic infiltrate, apoptotic activity and occurrence of necrosis. Specimens of 27 patients with classical seminoma were examined by immunohistochemistry for CD4, CD8, CD56, CD45R0, beta2-microglobulin and HLA-DR. MAGE expression was detected with the monoclonal antibody 57B, reactive with MAGE-1, -3, -4, -6 and -12. Clonality of the inflammatory infiltrate was examined by multiplex polymerase chain reaction (PCR) analysis of the T-cell receptor rearrangement. Apoptotic cells were detected by DNA nick-end labeling of fragmented DNA, and the apoptotic index was determined semi-quantitatively. Expression of 57B was found in 19 (70%) of 27 seminomas. In all cases, more than 70% of T-cells expressed CD45R0. In four cases, a predominant infiltration of CD8-positive cytotoxic T-cells (CD4/CD8 ratio < 1) was present. However, 15 seminomas showed a CD4/CD8 ratio > 1. In all cases, infiltration of CD56-positive natural killer cells was only focal. HLA-DR expression was not detectable in tumor tissue; beta2-microglobulin was only focal in three cases. Analysis of the T-cell clonality revealed a polyclonal population. The apoptotic index was not significantly different in 57B-positive seminomas (4.15%) compared with 57B negative seminomas (3.80%). Also, no correlation between the 57B expression and the occurrence of necrosis was found. MAGE antigens are homogeneously expressed in most seminomas, but their presence does not appear to represent a dominant epitope responsible for the lymphocytic infiltrate.

Apoptosis of cytotoxic T-cells in histiocytic necrotizing lymphadenitis.

Cell death is necrotic or apoptotic. In histiocytic necrotizing lymphadenitis (HNL), apoptosis is the main form of cell death, resulting in the creation of nuclear debris that is one of the characteristic features of HNL. To investigate the cell type of apoptotic cells, 12 cases of HNL were analyzed using the immunohistochemical staining for TIA-1, a cytotoxic granule of either cytotoxic T or NK cells. One quarter to over half of all apoptotic cells were positive for TIA-1, and some of the nuclear debris was also positive. The necrotic lesions of HNL were found to consist of nuclear debris, apoptotic cells, histiocytes and lymphocytes. The lymphocytes were mainly CD8-positive T-cells or CD4-positive cells, while B- and NK cells were only rarely observed. The number of TIA-1-positive lymphocytes was more closely related to the number of CD8-positive cells than to the number of CD4 cells. In double staining, the TIA-1 positive lymphocytes were mainly CD8 positive, but rarely CD4 positive. In HNL, then, CD8-positive cytotoxic T-cells are likely to undergo apoptosis.

Search for immunosuppressive substances produced in the culture of enteric bacteria isolated from marine fish

Culture supernatants of enteric bacteria isolated from the gastrointestinal tracts of two marine fishes were screened for the production of immunosuppressive substances. At least 50% of the 60 bacterial isolates tested were found to produce immunosuppressants that significantly inhibited the induction of cytotoxic T cells (CTL) in vitro. The strains that secreted the highest suppressive activity was identified as Aeromonas caviae. The immunosuppressive substance purified from the A. caviae culture supernatant was a polysaccharide with a molecular weight of about 70 kDa that contained mannose: glucose (4:1). This polysaccharide did not show nonspecific cytotoxicity, and was found to inhibit the proliferation and differentiation of CD8-positive CTL.