CD8-positive Cytotoxic T Lymphocytes Research Articles

High membrane expression of CMTM6 in hepatocellular carcinoma is associated with tumor recurrence

CKLF‐like MARVEL transmembrane domain–containing protein 6 (CMTM6) maintains membrane PD‐L1 expression by controlling its endosomal recycling. However, in patients with hepatocellular carcinoma (HCC), the correlation among CMTM6, B7 family ligands, and CD8‐positive cytotoxic T lymphocytes (CTLs), and the molecular function of CMTM6 in HCC have not been established. We performed immunohistochemistry to evaluate the relationships among CMTM6 expression, clinicopathological factors, B7 family ligands expression, and CTL infiltration in HCC samples. Moreover, we established CMTM6‐knockout human HCC cell lines to evaluate the function of human CMTM6 in immune regulation and tumor viability. CMTM6 expression was positively associated with membrane B7 family ligands expression and CTL infiltration in HCC samples. High CMTM6 expression in HCC tissues was associated with the expression of the proliferation marker Ki‐67 and shorter recurrence‐free survival. In vitro analysis showed the downregulation of membrane B7 family ligands and proliferation potency in the CMTM6‐knockout human HCC cell line. High membrane CMTM6 expression was associated with tumor recurrence and proliferation via the regulation of membranous B7 family ligands expression. Thus, CMTM6 might be a biomarker to predict the risk of HCC recurrence and a therapeutic target to suppress tumor growth and increase CTL activity.

Prognostic and Predictive Value of Tumor-infiltrating Leukocytes and of Immune Checkpoint Molecules PD1 and PDL1 in Clear Cell Renal Cell Carcinoma

INTRODUCTION: Immune checkpoint inhibitors (ICI) have been approved for patients with clear cell renal cell carcinoma (ccRCC), but not all patients benefit from ICI. One reason is the tumor microenvironment (TME) that has substantial influence on patient's prognosis and therapy response. Thus, we comprehensively analyzed the TME of ccRCC regarding prognostic and predictive properties. METHODS: Tumor-infiltrating CD3-positive T-cells, CD8-positive cytotoxic T-lymphocytes (CTLs), regulatory T-cells, B-cells, plasma cells, macrophages, granulocytes, programmed cell death receptor 1 (PD-1), and its ligand PD-L1 were examined in a large hospital-based series of ccRCC with long-term follow-up information (n = 756) and in another patient collective with information on response to nivolumab therapy (n = 8). Tissue microarray technique and digital image analysis were used. Relationship between immune cell infiltration and tumor characteristics, cancer-specific survival (CSS), or response to ICI was examined. RESULTS: Univariate survival analysis revealed that increased tumor-infiltrating B-cells, T-cells, and PD-1-positive cells were significantly associated with favorable CSS and high levels of intratumoral granulocytes, macrophages, cytotoxic T-cells, and PD-L1 significantly with poor CSS. High CTL or B-cell infiltration and high PD-L1 expression of ccRCC tumor cells qualified as independent prognostic biomarkers for patients' CSS. Significantly higher densities of intratumoral T-cells, CTLs, and PD-1-positive immune cells were observed in ccRCC with response to ICI compared with patients with mixed or no response (CD3: p = 0.003; CD8: p = 0.006; PD-1: p = 0.01). DISCUSSION: This study shows that subsets of tumor-infiltrating leukocytes in the TME and also PD-1/PD-L1 provide prognostic and predictive information for patients with ccRCC.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

Abstract PD9-08: Breast cancer liver metastases vascularize by vessel co-option, not angiogenesis, and have a desert immune phenotype: A histopathological and gene expression study

Abstract Phase 3 trials of bevacizumab combined with chemotherapy in metastatic breast cancer have consistently failed to demonstrate a survival benefit for the addition of bevacizumab. When cancers metastasize to highly vascular organs (including the liver), they can utilize vessel co-option, instead of angiogenesis, as a mechanism to obtain a vascular supply (1). We have repeatedly shown by histopathological analyses that almost all (95%, 2 cohorts) breast cancer liver metastases utilize vessel co-option instead of angiogenesis to vascularize (2,3). The prevalence of vessel co-option in breast cancer could explain, at least in part, why anti-angiogenic therapy has been a disappointing therapeutic approach in metastatic breast cancer. Animal models of non-angiogenic liver and lung metastases also displayed resistance to anti-VEGF treatment (3,4). We have now undertaken a gene expression study (mRNA sequencing) of targeted samples at the tumor-liver interface to discover gene expression patterns and signaling pathways that are associated with non-angiogenic growth of metastatic cancer in the liver (n = 70). A network to detect biological themes of non-angiogenic growth was built by gene set enrichment analysis. Key components of this network are: cancer cell motility and invasion, epithelial-to-mesenchymal transition, stemness and proliferation. This contrasts with the network of angiogenic liver metastases of which the most important components are inflammation and ECM remodeling. Semi-automated image analyses of CD8-immunostained section of liver metastases confirms that non-angiogenic liver metastases have a significantly lower density of CD8-positive cytotoxic T-lymphocytes at the tumor-liver interface when compared with angiogenic liver metastases (300 cells/mm2 and 1000 cells/mm2, respectively (p<0.0001)). In addition, a clear CXCL13-driven B-cell gene expression signature is associated with angiogenic growth of liver mets but is absent in non-angiogenic growth of breast cancer liver metastases. Gene expression patterns that may be play a role in vessel co-option are the up-regulation of LAMA3, LAMB3, LAMC2, coding for the 3 subunits of laminin-5, and of ITGA3, ITGB1, ITGA6 and ITGB4, coding for both integrin-receptors of laminin-5. This supports the concept of 'adhesive' vessel co-option during which cancer cells use the basement membrane of the co-opted blood vessels as a soil (5). In addition, the claudin-2 gene (CLDN2) is significantly overexpressed in non-angiogenic liver metastases which is consistent with earlier reports on the role of claudin-2 during breast cancer metastasis to the liver (6). In conclusion, we provide evidence, based on morphology and gene expression, for the almost exclusive non-angiogenic growth of breast cancer liver metastases. In addition, non-angiogenic breast cancer liver metastases are characterized by a desert immune phenotype. Both observations can have an impact on the treatment strategy of patients with metastatic breast cancer.

Positive correlation between the density of macrophages and T-cells in undifferentiated sarcoma.

Undifferentiated sarcoma (US) is a frequent soft tissue sarcoma. Although the 10-year survival rate is around 60%, advanced US is highly resistant to chemo/radiotherapy. The tumor microenvironment (TME) is closely associated with tumor progression. However, few studies of infiltrated immune cells in US have been published. In this study, we evaluated tumor-associated macrophages (TAMs) and CD8-positive cytotoxic T lymphocytes (CTLs) in 28 cases of US. Iba1, CD163, and CD204 were used as markers for TAMs. The density of CTLs was positively correlated with the density of TAMs. However, a negative correlation was seen between the density of CTLs and the percentage of CD204-positive TAMs. We found no significant association between the density of Iba1-/CD204-/CD8-positive cells and clinicopathological factors. No significant correlation between immune cell infiltration and clinical outcome was observed. Although we found no significant association between immune cells and clinicopathological factors, these findings may provide new insight into the characterization of immune cells in the TME of US.

Microglia retard dengue virus-induced acute viral encephalitis.

Patients with dengue virus (DENV) infection may also present acute viral encephalitis through an unknown mechanism. Here, we report that encephalitic DENV-infected mice exhibited progressive hunchback posture, limbic seizures, limbic weakness, paralysis, and lethality 7 days post-infection. These symptoms were accompanied by CNS inflammation, neurotoxicity, and blood-brain barrier destruction. Microglial cells surrounding the blood vessels and injured hippocampus regions were activated by DENV infection. Pharmacologically depleting microglia unexpectedly increased viral replication, neuropathy, and mortality in DENV-infected mice. In microglia-depleted mice, the DENV infection-mediated expression of antiviral cytokines and the infiltration of CD8-positive cytotoxic T lymphocytes (CTLs) was abolished. DENV infection prompted the antigen-presenting cell-like differentiation of microglia, which in turn stimulated CTL proliferation and activation. These results suggest that microglial cells play a key role in facilitating antiviral immune responses against DENV infection and acute viral encephalitis.

In Epstein-Barr virus-associated gastric carcinoma a high density of CD66b-positive tumor-associated neutrophils is associated with intestinal-type histology and low frequency of lymph node metastasis.

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is characterized by prominent intratumoral lymphocyte infiltration, with some infiltration by other types of inflammatory cells, such as neutrophils. In this study, the significance of tumor-associated neutrophils (TANs) and cytotoxic T lymphocytes (CTLs) was investigated in EBVaGC. CD66b-positive TANs and CD8-positive CTLs in surgically resected EBVaGC tissues were evaluated by immunohistochemistry and digital imaging analysis. Cut-off values were determined with receiver operating characteristic curve analyses. Forty-two of 77 cases (55%) had some infiltration of TANs (CD66b-positive areas >0.5%, TAN(+)) and 35 (45%) had scant infiltration (TAN(-)). Thirty-five cases (45%) had more CTLs (CD8-positive area >18%, CTL-high) and 42 (55%) had fewer (CTL-low). There was no correlation between CD66b- and CD8-positive areas in the tumor (P=0.453). TAN(+) correlated with intestinal-type histology (P=0.048) and low frequency of lymph node metastasis (P=0.023), while CTL-low with upper location (P=0.030) and advanced invasion depth (pT2 or greater) (P=0.006). Disease-specific survival was not significantly correlated with TANs or CTLs. Multivariate logistic regression analysis revealed TAN(-) to be independently associated with lymph node metastasis (P=0.036). None of the cases of TAN(+) early EBVaGC with submucosal invasion showed lymph node metastasis (95% confidence interval 0-13.3%). When compared with 24 EBV-negative gastric carcinomas, EBVaGC had significantly more CTLs (P<0.001), while there was no difference in the number of TANs. TANs in EBVaGC may suppress lymph node metastasis, and absence of TANs might suggest careful follow-up or additional therapy in cases of post-endoscopic resection.

Visualization of HTLV-1-specific cytotoxic T lymphocytes in the spinal cords of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis.

Activated human T-lymphotropic virus type-1 (HTLV-1)–specific CD8-positive cytotoxic T lymphocytes (CTLs) are markedly increased in the periphery of patients with HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP), an HTLV-1–induced inflammatory disease of the CNS. Although virus-specific CTLs play a pivotal role to eliminate virus-infected cells, the potential role of HTLV-1–specific CTLs in the pathogenesis of HAM/TSP remains unclear. To address this issue, we evaluated the infiltration of HTLV-1–specific CTLs and the expression of HTLV-1 proteins in the spinal cords of 3 patients with HAM/TSP. Confocal laser scanning microscopy with our unique staining procedure made it possible to visualize HTLV-1–specific CTLs infiltrating the CNS of the HAM/TSP patients. The frequency of HTLV-1–specific CTLs was more than 20% of CD8-positive cells infiltrating the CNS. In addition, HTLV-1 proteins were detected in CD4-positive infiltrating T lymphocytes but not CNS resident cells. Although neurons were generally preserved, apoptotic oligodendrocytes were frequently in contact with CD8-positive cells; this likely resulted in demyelination. These findings suggest that the immune responses of the CTLs against HTLV-1–infected CD4-positive lymphocytes migrating into the CNS resulted in bystander neural damage.

Severe necroinflammatory reaction caused by natural killer cell-mediated Fas/Fas ligand interaction and dendritic cells in human hepatocyte chimeric mouse

The necroinflammatory reaction plays a central role in hepatitis B virus (HBV) elimination. Cluster of differentiation (CD)8-positive cytotoxic T lymphocytes (CTLs) are thought to be a main player in the elimination of infected cells, and a recent report suggests that natural killer (NK) cells also play an important role. Here, we demonstrate the elimination of HBV-infected hepatocytes by NK cells and dendritic cells (DCs) using urokinase-type plasminogen activator/severe combined immunodeficiency mice, in which the livers were highly repopulated with human hepatocytes. After establishing HBV infection, we injected human peripheral blood mononuclear cells (PBMCs) into the mice and analyzed liver pathology and infiltrating human immune cells with flow cytometry. Severe hepatocyte degeneration was observed only in HBV-infected mice transplanted with human PBMCs. We provide the first direct evidence that massive liver cell death can be caused by Fas/Fas ligand (FasL) interaction provided by NK cells activated by DCs. Treatment of mice with anti-Fas antibody completely prevented severe hepatocyte degeneration. Furthermore, severe hepatocyte death can be prevented by depletion of DCs, whereas depletion of CD8-positive CTLs did not disturb the development of massive liver cell apoptosis. Our findings provide the first direct evidence that DC-activated NK cells induce massive HBV-infected hepatocyte degeneration through the Fas/FasL system and may indicate new therapeutic implications for acute severe/fulminant hepatitis B.

P1-01-13: Prognostic Impact of CD8 in Node-Negative Breast Cancer.

Abstract Background: Infiltration of cytotoxic T lymphocytes (CTL) is a common feature in breast cancer. We examined the prognostic impact of CD8 positive CTL in formalin-fixed-paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) in node-negative breast cancer and validated our findings in previously published cohorts using RNA expression. Methods: CD8 was evaluated in FFPE tissue of 339 medically untreated node-negative breast cancer patients utilizing IHC. Results were validated utilizing microarray based gene-expression data of four cohorts of medically untreated node-negative breast cancer patients (n=824). Impact of CD8 on metastasis-free survival (MFS) was analyzed with univariate and multivariate Cox regression. Meta-analysis of previously published cohorts was performed using a random effects model. Prognostic significance was examined in the whole cohort and in different molecular subtypes (ER+/HER2−, ER−/HER2−, HER2). Correlation between RNA expression and IHC was analyzed according to Spearman. Results: Immunohistochemical detection of CD8+ CTL was associated with MFS in univariate (hazard ratio [HR] 0.76, 95% confidence interval [95% CI] 0.59−0.98, P=0.037) as well as in multivatiate analysis (HR 0.61, 95% CI 0.46−0.81, P=0.001). Protein levels of CD8 had a good correlation with RNA expression (r=0.488). Higher RNA expression of CD8 was related to better MFS in a meta-analysis of the whole cohort (HR 0.74, 95% CI 0.60−0.90, P=0.0023). However, prognostic significance was confined to ER+/HER2− (HR 0.71, 95% CI 0.51−0.99, P=0.0201) and HER2+ (HR 0.60, 95% CI 0.38−0.97, P=0.037) but not to ER−/HER2− molecular subtypes (HR 0.77, 95% CI 0.52−1.14, P=0.1965). Conclusion: CD8 positive CTL have independent prognostic significance in node-negative breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-13.

Characterization of Cytotoxic T Lymphocyte Clones Isolated from Bone Marrow Transplant Recipient with HLA-Cw-Mismatched Donor

HLA-Cw disparity in a donor that can occur even in recipient/donor pairs that are matched for HLA-A, B and DRB1, increases the risk of severe acute graft-versus-host disease (GVHD) after bone marrow transplantation. Acute GVHD is mediated by donor cytotoxic T lymphocytes (CTLs). However, the immunogenicity of HLA-Cw antigens is considered to be low as a consequence of their low level of cell surface expression. In fact, HLA-Cw-specific CTLs generated in post-transplant recipients who develop acute GVHD have not been characterized in detail. Here, we characterized CTL clones isolated from a recipient at the onset of grade II acute GVHD who was transplanted from an HLA-A, B and DRB1-matched, HLA-Cw-mismatched (recipient, Cw*0303/Cw*0702; donor, Cw*0801/Cw*0702) and killer immunoglobulin-like receptor ligand-matched unrelated donor. All of seven isolated CTLs lysed Epstein Barr virus-transformed lymphoblastoid cells (B-LCL) from the recipient, but not B-LCL from the donor. Four CTLs were CD8- positive, one was CD4-positive, and two were CD4/CD8-double positive. Three of those including a CD8-positive CTL, a CD4-positive CTL and a CD4/CD8-double positive CTL had exactly the same nucleotide sequences in the CDR3 region of their T cell receptors, suggesting these three CTLs with variable phenotypes originated from a single clone. To identify the genes encoding the antigen recognized by the isolated CTLs, we initially focused on the CD8-positive CTLs. Two CD8-positive clones lysed all of 15 B-LCL lines from unrelated individuals that shared HLA-Cw*0303 with the recipient, but not B-LCL from 17 unrelated individuals that shared class I HLA molecules other than HLA-Cw* 0303. These data indicated that both CTL clones recognize HLA-Cw*0303 molecule as an alloantigen. We transfected donor B-LCL with a full-length HLA-Cw*0303 cDNA construct and examined their cytotoxicity. Both CTL clones lysed donor B-LCL transfected with the HLA-Cw*0303 cDNA as well as recipient B-LCL. Five other CTLs also lysed HLA-Cw*0303-transfected donor B-LCL. Thus, all CTLs recognized HLA-Cw*0303 as an alloantigen. The sequences of Cw*0303 and Cw*0801 differ by 16 amino acids. To determine which residue affects recognition of the Cw*0303 molecule by Cw*0303- specific Cw*0801-positive CTLs, we generated 16 Cw*0303 mutants in which individual amino acids were substituted with the corresponding amino acid in Cw*0801. COS cells transfected with Cw*0303 mutants in which amino acids constituting peptide-binding pockets (aa position: 114, 116, 152, or 163) were substituted with Cw*0801-amino acids could not stimulate IFN-γ production by any CTLs, whereas COS cells transfected with Cw*0303 mutants bearing Cw*0801-amino acids outside the positions constituting peptidebinding pockets stimulated all CTLs as well as the wild-type Cw*0303 construct. These data indicated that peptides bound to Cw*0303 molecules influence the allorecognition of Cw*0303-specific CTLs. Taken together, our findings suggest HLA-Cw-specific CTL clones with a variable phenotype can naturally arise in post-transplant recipients and cause acute GVHD through recognition of the peptides bound to Cw molecules. This is consistent with a recent statistical study showing a significant association between some specific amino acid substitutions at positions constituting peptide-binding pockets of the HLA-Cw molecule and the occurrence of severe acute GVHD after unrelated bone marrow transplantation (Blood . 2007; 110:2235–2241). Further efforts to identify the peptides recognized by isolated CTLs should help to elucidate the mechanism of GVHD development in recipients with HLA-Cw-mismatched donors.

Escherichia Coli Expressing Recombinant WT1 Protein and Listeriolysin O Stimulate Antigen-Specific T Cells Following Vaccination in C57BL/6 Mice.

The Wilm's Tumor (WT1) gene is expressed at high levels in most types of leukemia, but also in various types of solid tumors. We tested the efficacy of recombinant Escherichia Coli (E. Coli) expressing Listeriolysin O (LLO) as an antigen delivery vector for tumor specific immunotherapy targeting WT1 gene products. C57BL/6 (B6) mice were immunised with E. Coli expressing a form of WT1 protein truncated of 70 amino acids at the N-Terminus portion, and then challenged subcutaneously with 5X106 WT1-expressing TRAMP-C tumor cells. A significant inhibitory effect on TRAMP-C tumor growth was observed in vivo in vaccinated mice, with no signs of toxicity in tissues such as liver and kidney on histopathlogic examination. Furthermore, splenocytes from immunised mice were stimulated in vitro either with peptides that have been shown to contain major histocompatibility (MHC) class I binding anchor motifs (P126: RMFPNAPYL and P330: CNKRYFKL), or with irradiated TRAMP-C tumor cells. Specific CD8-positive cytotoxic T lymphocytes (CTLs) responses against TRAMP-C tumor cells were seen in cultures stimulated with irradiated tumor cells. However, no CTL activity was found in cultures stimulated with the relevant WT1 peptides that specifically lyse TRAMP-C tumor cells or the leukemia cell line RMA-S cells pulsed with each of the peptides. These results suggest that P126 and P330 might not be naturally processed WT1 epitopes for B6 mice and supports the possibility of other B6 CTLs epitopes. We are currently using a WT1 peptide library to screen immunised animals to look for novel peptides. Immunisation with E. Coli expressing WT1 protein but not LLO did not show any effect on TRAMP-C tumor growth in vivo. In this latter group, western blot analysis revealed WT1 specific antibodies directed against the N-terminus portion of the WT1 protein in the sera of immunised mice. Altogether the results presented here showed that E. Coli expressing WT1 could stimulated antigen-specific T cells dependant on LLO co-expression and suggest possible vaccine strategies for generating CD8+ T cell responses against tumors.

Computer-Based Design of an HLA-Haplotype and HIV-Clade Independent Cytotoxic T-Lymphocyte (CTL) Assay for Monitoring HIV-Specific Immunity

Human immunodeficiency virus (HIV)- specific CD8-positive cytotoxic T-lymphocytes (CTL) play a key role in controlling HIV infection. Monitoring CTL response could be clinically relevant during structured therapy interruption (STI), HIV exposure, and vaccine trials. However, HLA patients' restriction and HIV variability limited the development of a CTL assay with broad specificity. We designed an HLA-class I/HIV-1 clade independent assay for assessing HIV- specific CTL by using a computer-assisted selection ofthe CTL epitopes. Twenty-eight 15-mers were selected by peptide-binding motifs analysis using different databases (HIV-Immunology Database, SYFPEITHI, BIMAS). Altogether they putatively bind to more than 90% of HLA haplotypes in different populations, with an overall HIV-1 variability below 9%. The peptide pool was used as an antigen in an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CTL response. The test can be performed using both fresh and cryopreserved peripheral blood mononuclear cells (PBMC), whereas GAG protein as antigen works only on fresh PBMC. A significantly higher CTL response with respect to HIV-negative controls was detected in all HIV-1 infected subjects of two groups of patients with different ethnicities (Caucasians and Africans) and coming from areas with different HIV-1 clade prevalences (clade B and A/G, respectively). In Caucasian patients, after month of STI, the number of HIV-1 specific CTL (2,896 +/- 2,780 IFN-gamma specific CD8 cells/ml) was significantly higher than that found at enrolment (2,125 +/- 4,426 IFN-gamma specific CD8 cells/ml, p< 0.05). These data indicate that this CTL assay is broadly specific and could represent a useful clinical tool for HIV immunodiagnostic independent of HLA-haplotype and HIV-clade variabilities.

WT1 as a novel target antigen for cancer immunotherapy.

Wild-type Wilms' tumor gene WT1 is expressed at high levels not only in most of acute myelocytic, acute lymphocytic, and chronic myelocytic leukemia, but also in various types of solid tumors including lung cancer. We tested the ability of the gene product (WT1) to serve as a target antigen for tumor specific immunotherapy both in human in vitro system and mouse in vivo system. In the latter, we can evaluate the efficacy and the side effects of WT1 vaccination in vivo. In the human in vitro system, two WT1 peptides that contain HLA-A2.1 binding anchor motifs were determined to bind to HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from an HLA-A2.1-psitive donor were repeatedly stimulated in vitro with TAP-deficient T2 cells pulsed with each of these two peptides, and CD8-positive cytotoxic T lymphocytes (CTLs) that specifically lyse WT1-expressing, HLA-A2.1-positive tumor cells were induced. Other groups also have succeeded in generating CTLs which specifically lyse WT1-expressing leukemia cells, and which do not inhibit colony-formation of normal hematopoietic cells that express WT1 at physiological levels. In the mouse in vivo system, immunization of C57BL/6 mice with one WT1 peptide with relatively high binding affinity for H-2D(b) molecules, which contain H-2D(b) binding anchor motifs, induced CTLs, which specifically lysed WT1-expressing tumor cells in an H-2D(b)-restricted manner. Furthermore, mice immunized with the WT1 peptide (peptide vaccination) or WT1 cDNA (DNA vaccination) rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to WT1-expressing normal organs by the induced CTLs. The WT1 protein has been identified as a novel tumor antigen and recent investigations provide a rationale for developing WT1-based adoptive T cell therapy and vaccination against various kinds of malignant neoplasms.

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop. Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial. Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection. We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes. This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development.

Quantitative analysis of T lymphocyte subsets in pregnant and nonpregnant human endometrium : Vassiliadou N., Bulmer J.N. Royal Victoria Infirmary, University Department of Pathology, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, UK [Biology of Reproduction 55/5, 1017–1022; 1996

In order to characterize directly the T-cell repertoire utilized in human renal allograft rejection, we analyzed 20 long-term T-cell lines established from lymphocytic infiltrates present in renal tissue obtained by needle biopsy from transplant patients with rejection indications. All cell lines are strongly cytotoxic against one or more of the HLA antigens for which the kidney donor and the recipient were mismatched. Traditionally, cytotoxicity in allograft rejection has been attributed to CD8-positive cytotoxic T lymphocytes (CTLs) directed against class I alloantigens. Cell lines described here are mixtures of distinct CD4-antigen-positive and CD8-antigen-positive subpopulations, and exhibit class I- as well as class II-directed killing. Using a cell line that demonstrates class II-directed cytoxicity and a set of class II deletion mutants as target cells, we show that class II antigens, in particular HLA-DP, can serve as targets in renal allograft rejection. The role of CD4-positive CTLs was shown by analysis of clonal populations or sorted CD8-positive and CD4-positive subpopulations. In several instances we have obtained cell lines from serial biopsies performed on the same patient at distinct time points during an ongoing rejection. Comparisons of the phenotype, function, and specificities allow for speculation regarding T-cell population dynamics within the rejection response.

Recognition of Human Renal Cell Carcinoma and Melanoma by HLA‐A2‐Restricted Cytotoxic T Lymphocytes is Mediated by Shared Peptide Epitopes and Up‐Regulated by Interferon‐γ

Cytotoxic T lymphocytes (CTL) have previously been isolated from peripheral blood of patients with renal cell carcinoma (RCC). The CD8-positive CTL line MZ1257-CTL-5 (CTL-5) has been shown to lyse autologous cultured RCC cells in an HLA-A2 restricted fashion. Allogeneic, HLA-A2-matched RCC and melanoma cell lines were also lysed by CTL-5, suggesting that melanoma and renal cancer share antigenic determinants. The aim of the study was to determine whether RCC and melanoma share peptide epitopes that are recognized by CTL-5 in the context of HLA-A2 molecules. Peptides were acideulated from various cell lines, separated by reversed phase high performance liquid chromatography (RP-HPLC), and assessed for their ability to reconstitute the CTL-5-defined epitope by pulsing the peptides on HLA-A2 positive antigen-processing mutant cell line CEM x 721.174.T2 (T2). Peptides eluted from allogeneic HLA-A2-matched RCC and melanoma cell lines exhibited the CTL-5-defined epitope in the same HPLC fractions as peptides derived from the autologous RCC line. Renal cancer and melanoma cells preincubated with interferon-gamma (IFN-gamma) resulted in an additional peak of reconstitution activity in both cell types. This second lytic peak was also observed when high amounts of autologous RCC cells were used for peptide preparation without IFN-gamma pretreatment, indicating that IFN-gamma increases the amount of MHC class I/peptide complexes per cell, rather than inducing a neo-epitope.

Herpes simplex virus-1-specific human cytotoxic T lymphocytes are induced in vitro by autologous virus-infected mononuclear cells.

A new technique for in vitro activation of cytotoxic T lymphocytes (CTLs) specific for herpes simplex virus type 1 (HSV-1) is described. Autologous phytohemagglutinin (PHA)-activated, HSV-1-infected peripheral blood mononuclear cells (PBMC) were used, after fixation with 1% paraformaldehyde, to activate virus-specific CTLs in short-term cultures. The same unfixed PBMC were used as target cells in the cytotoxicity assay. By using this technique high levels of HSV-1-specific cytotoxic activity (50.06 +/- 16.76% at 30:1 effector:target ratio) were repeatedly obtained in 24 experiments using PBMC from 16 HSV-1 antibody-positive healthy donors, while no cytotoxic activity was observed using PBMC from 3 HSV-1 antibody-negative donors. HSV-1-induced CTLs were shown to be virus-specific as they did not lyse autologous, PHA-activated PBMC infected with influenza A virus or autologous Epstein-Barr virus (EBV) lymphoblastoid cell line (LCL), while they were able to lyse both HSV-1-infected, autologous PHA-activated PBMC and EBV-LCL. HSV-1-specific cytotoxicity was mediated by T lymphocytes, since depletion of CD3-positive cells from the effector population completely removed the killing of HSV-1-infected target cells. CD8-positive CTLs were primarily involved in the killing of HSV-1-infected targets since depletion of CD8-positive cells caused a strong reduction of virus-specific cytotoxic activity while elimination of CD4-positive lymphocytes increased killing capacity. Finally, this technique has proven to be highly reproducible, easy to perform, and thus suitable for clinical investigations.

HLA class I-restricted human cytotoxic T cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen.

Knowledge of the immune effector mechanisms responsible for clearance of hepatitis B virus (HBV)-infected cells has been severely limited by the absence of reproducible systems to selectively expand and to characterize HBV-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of patients with viral hepatitis. By using a strategy involving sequential stimulation with HBV nucleocapsid synthetic peptides followed by autologous, or HLA class I-matched, HBV nucleocapsid transfectants, we now report the existence of CTLs able to lyse target cells that express endogenously synthesized HBV nucleocapsid antigen in the peripheral blood of patients with acute viral hepatitis B. The CTL response is HLA-A2 restricted, mediated by CD8-positive T cells, and specific for a single epitope, located between amino acid residues 11 and 27 of HBV core protein; these residues are shared with the secretable precore-derived hepatitis B e antigen. Equivalent lysis of target cells that express each of these proteins suggests that their intracellular trafficking pathways may intersect. The current report provides definitive evidence that HLA class I-restricted, CD8-positive CTLs that recognize endogenously synthesized HBV nucleocapsid antigen are induced during acute HBV infection in humans and establishes a strategy that should permit a detailed analysis of the role played by HBV-specific CTLs in the immunopathogenesis of viral hepatitis.

Human allograft-derived T-cell lines: Donor class I- and class II-directed cytotoxicity and repertoire stability in sequential biopsies

In order to characterize directly the T-cell repertoire utilized in human renal allograft rejection, we analyzed 20 long-term T-cell lines established from lymphocytic infiltrates present in renal tissue obtained by needle biopsy from transplant patients with rejection indications. All cell lines are strongly cytotoxic against one or more of the HLA antigens for which the kidney donor and the recipient were mismatched. Traditionally, cytotoxicity in allograft rejection has been attributed to CD8-positive cytotoxic T lymphocytes (CTLs) directed against class I alloantigens. Cell lines described here are mixtures of distinct CD4-antigen-positive and CD8-antigen-positive subpopulations, and exhibit class I- as well as class II-directed killing. Using a cell line that demonstrates class II-directed cytoxicity and a set of class II deletion mutants as target cells, we show that class II antigens, in particular HLA-DP, can serve as targets in renal allograft rejection. The role of CD4-positive CTLs was shown by analysis of clonal populations or sorted CD8-positive and CD4-positive subpopulations. In several instances we have obtained cell lines from serial biopsies performed on the same patient at distinct time points during an ongoing rejection. Comparisons of the phenotype, function, and specificities allow for speculation regarding T-cell population dynamics within the rejection response.