Cytotoxic T Lymphocytes Cell Lines Research Articles

Expression of B7-H1 in Mantle Cell Lymphoma Leads to Inhibition of T Cell Response to Tumor Cells

Abstract 2643Mantle cell lymphoma (MCL) is a unique subtype of incurable B-cell, non-Hodgkin lymphoma, and its overall survival currently remains only 4–5 years. Management of relapsed or refractory MCL patients is still challenging. Immunotherapy may provide an alternative treatment for patients with MCL. Recent studies demonstrated that PD-1/B7-H1 signaling plays a crucial role in T-cell regulation in various immune responses and is involved in peripheral tolerance, autoimmunity, infection, and antitumor immunity. In the present study, we examined whether B7-H1 plays an important role in immune evasion in MCL. We demonstrated that B7-H1 gene and protein were expressed in most MCL cell lines and primary MCL cells from all patients examined. CD3+ T cells were cultured with irradiated MCL cell lines and primary cells, which were pre-incubated with or without anti-B7-H1 monoclonal antibody or control antibody. The presence of anti-B7-H1 blocking antibody, but not control antibody, increased CD3+ T cell proliferation. We confirmed the effect of B7-H1 in suppression of T cell proliferation by knockdown of B7-H1 gene expression using B7-H1 specific and non-specific control shRNA lentiviral particles. Upon transfection, the B7-H1-specific shRNA reduced both B7-H1 gene and surface protein expression, while the control shRNA did not. The B7-H1 specific shRNA, but not control shRNA, augmented CD3+ T cell proliferation. To address whether B7-H1 contributed to the suppression of host antitumor immunity in MCL, allogeneic CD3+ T cells isolated from normal donors were cocultured with irradiated MCL cell line SP53, control shRNA SP53 (SP53-ctl), or B7-H1 targeted shRNA SP53 (SP53-kd), respectively. After 7 days of coculture, CD3+ T cells were harvested and restimulated with newly irradiated SP53, SP53-ctl or SP53-kd cells. After at least 3 repeated cycles of in vitro restimulation, three cytotoxic T lymphocyte (CTL) lines were generated, and named CTL-SP53, CTL-SP53-ctl and CTL-SP53-kd. CTL-SP53-kd showed increased killing of target cells as compared with CTL-SP53 (P <.01) or CTL-SP53-ctl (P <.01). We further showed that B7-H1 targeted shRNA MCL cell line (SP53-kd cells) displayed more specific lysis than SP53 (P <.01) or SP53-trl (P <.01). When SP53 cells were pre-incubated with a blocking anti-B7-H1 monoclonal antibody, it also showed more specific lysis as compared to the control antibody pre-treated cells. In these experiments, purified autologous blood B cells and PBMCs were used as target cells to demonstrate whether T cell lines were cytolytic to normal cells. The results showed that the three CTL cell lines did not kill B cells or PBMCs. Intracellular cytokine staining and ELISA assay demonstrated that T-cell lines express IFN-γ, but not IL-4, IL-6, IL-10 or IL-17, and were thus type I T cells. Moreover, T cell lines stimulated by SP53-kd cells express more IFN-γ than SP53 and SP53-ctl. The T cells also expressed CD45RO, CD28, CD44, but not CD45RA, CD27 or CD62L, indicating that they were memory effector cells. In conclusion, B7-H1 expression may be involved in immune evasion mechanism of MCL. Therefore, B7-H1 may be a promising target for immunotherapy in MCL. Disclosures:No relevant conflicts of interest to declare.

Identification of HLA-A24-restricted CD8+ cytotoxic T-cell epitopes derived from mammaglobin-A, a human breast cancer–associated antigen

Human breast cancer-associated antigen, mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as a breast cancer vaccine. In this study, we define the CD8(+) cytotoxic T lymphocyte (CTL) response to Mam-A-derived candidate epitopes presented in the context of HLA-A24 (A*2402). HLA-A24 has a frequency of 72% in Japanese, 27% in Asian Indian, and 18% in Caucasian populations. Using a human leukocyte antigen (HLA)-binding prediction algorithm we identified 7 HLA-A24-restricted Mam-A-derived candidate epitopes (MAA24.1-7). Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-A2402 (T2.A24) indicated that MAA24.2 (CYAGSGCPL) and MAA24.4 (ETLSNVEVF) have the highest HLA-A24 binding affinity. Further, 2 CD8(+) CTL cell lines generated in vitro against T2.A24 cells individually loaded with Mam-A-derived candidate epitopes demonstrated significant cytotoxic activity against MAA24.2 and MAA24.4. In addition, the same CD8(+) CTL lines lysed the HLA-A24(+)/Mam-A(+) stable transfected human breast cancer cell lines AU565 and MDA-MB-361. However, these CTLs had no cytotoxicity against HLA-A24(-)/Mam-A(+) and HLA-A24(+)/Mam-A(-) breast cancer cell lines. In summary, our results define HLA-A24-restricted, Mam-A-derived, CD8(+) CTL epitopes that can potentially be employed for Mam-A-based breast cancer vaccine therapy to breast cancer patients with HLA-A24 phenotype.

Identification of immunodominant HLA-B7-restricted CD8+ cytotoxic T cell epitopes derived from mammaglobin-A expressed on human breast cancers

Mammaglobin-A (MGBA), a 10-kD protein, is over expressed in 80% of primary and metastatic human breast cancers. Breast cancer patients demonstrate high frequencies of CD8(+) cytotoxic T lymphocytes (CTL) specific to MGBA. Defining CD8(+) CTL responses to HLA class I-restricted MGBA-derived epitopes assumes significance in the context of our ongoing efforts to clinically translate vaccine strategies targeting MGBA for prevention and/or treatment of human breast cancers. In this study, we define the CD8(+) CTL response to MGBA-derived candidate epitopes presented in the context of HLA-B7, which has a frequency of 17.7% in Caucasian and 15.5% in African American populations. We identified seven MGBA-derived candidate epitopes with high predicted binding scores for HLA-B7 using a computer algorithm. Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-B7 indicated that MGBA B7.3 (VSKTEYKEL), B7.6 (KLLMVLMLA), B7.7 (NPQVSKTEY), and B7.1 (YAGSGCPLL) have the highest HLA-B7 binding affinities. Further, two CD8(+) CTL cell lines generated in vitro against T2.B7 cells individually loaded with MGBA-derived candidate epitopes showed significant cytotoxic activity against MGBA B7.1, B7.3, B7.6, and B7.7. In addition, the same CD8(+) CTL lines lysed the HLA-B7(+)/MGBA(+) human breast cancer cell line DU-4475 but had no significant cytotoxicity against HLA-B7(-) or MGBA(-) breast cancer cell lines. Cold-target inhibition studies strongly suggest that MGBA B7.3 is an immunodominant epitope. In summary, our results define HLA-B7-restriced, MGBA-derived, CD8(+) CTL epitopes with all of the necessary features for developing novel vaccine strategies against HLA-B7 expressing breast cancer patients.

Identification of CS1 Peptides for Induction of Antigen-Specific CTLs in Multiple Myeloma.

Despite progress in the treatment of multiple myeloma (MM), the disease remains incurable. Therefore novel therapeutic approaches are required to achieve better outcome in patients. Development of peptide-based immunotherapy against tumor specific or associated antigens offers an attractive approach that may lead to tumor-targeted cellular therapy in MM patients. Recently, CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family, was found to be highly expressed by the tumor cells of the majority of MM patients but not other normal tissues. Importantly, a novel anti-CS1 HuLuc63 mAb induced significant anti-myeloma killing in vitro and in vivo (Abstract #950059). In addition, CS1 may play a role in MM cell survival (Abstract #953659). In this study, we asked whether CS1 is a suitable myeloma-associated antigen for cellular therapy in MM. We wanted to identify potential immunogenic peptides derived from CS1 and tested whether these peptides evoke MM-specific cytotoxic T lymphocytes (CTLs). First, we predicted the potential peptide sequences of CS1 that could bind to HLA-A*0201 molecule using three databases (RANKPEP, BIMAS and NetMHC). A total of four peptides were selected and synthesized according to the high binding scores among all the databases. Next, peptide-T2 cells binding assay confirmed these peptides with high binding affinity to HLA-A*0201 molecule (Fluorescence index: 2.19, 3.28, 3.44 and 3.49). To generate the peptide-specific CTLs, HLA-A*0201-positive normal human CD8+ T lymphocytes were stimulated with autologuous dendritic cells (DCs) or T2 cells pulsed with candidate CS1-peptides. The CTL cell lines were established after several rounds restimulation. We have evaluated the immunogenicity of the expanded CTLs by the stimulation with peptide-pulsed or unpulsed T2 cells. Our data demonstrated that one peptide-induced CTLs (CS1-CTLs) possessed high antigen-specific immune responses with the HLA-A*0201-restriction relative to a certain level of immune responses displayed by another three peptide-induced CTLs. We further evaluated the proliferative activity of CS1-CTLs by 3H-TdR incorporation assay. Upon stimulation by peptide-pulsed T2 cells, the cell proliferation of CS1-specific CTLs increased approximately 55-fold, compared with unstimulated cells (pulsed vs. unpulsed: 22894 vs. 423 cpm). The activation of CS1-CTLs was monitored by the expression of CD25 (IL-2Rα chain) on peptide-stimulated CS1-CTLs (%CD25+ cells: pulsed vs. unpulsed: 98% vs. 13%). CS1-CTLs significantly secreted 14-fold higher levels of IFN- γ in response to peptide-pulsed T2 cells (pulsed vs. unpulsed: 615 vs. 46 pg/ml). Importantly, the expanded CS1-CTLs generated high cytotoxicity (93%) in response to peptide pulsed T2 cells. Furthermore, more than 30% of killing activity by CS1-CTLs against HLA-A*0201+CS1+ MCCAR cells was observed at the effector: target ratio of 20:1. In contrast, these CS1-CLTs did not kill HLA-A*0201+/CS1− U266 and HLA-A*0201−/CS1+ MM1S target cells. These results support CS1 as an antigenic target for the induction of peptide-specific CTLs against MM cells. Further functional studies are underway to confirm potential CS1-specific cellular therapy to treat MM.

Viral-Specific Cytotoxic T Lymphocytes Lyse Human Immunodeficiency Virus–Infected Primary T Lymphocytes by the Granule Exocytosis Pathway

Cytotoxic T lymphocytes (CTL) lyse antigen-bearing target cells by two distinct pathways. Whereas granule exocytosis targets any antigen-bearing cell, fas-mediated cytotoxicity kills only fas-expressing cells and does not require antigen expression. Fas pathway activation can potentially lead to lysis of uninfected bystander cells. We examined the relative usage of the two pathways by CTL clones and cell lines directed against four different human immunodeficiency virus (HIV) proteins in lysing primary HIV-infected targets. Although fas was expressed on HIV-infected primary CD4+ T cells, their lysis by antigen-specific CD8+ CTL was only by the granule pathway. Fas ligand (fasL) was not detectable on antigen-specific CD8 clones, T-cell lines, or circulating HIV-specific CD8 T cells from HIV-infected donors, stained with a tetrameric HLA-A2-HIV-peptide complex. FasL expression by HIV-specific CTL clones was not activated by exposure to HIV-presenting cells, but was after unphysiological stimulation with phorbol myristate acetate (PMA). CTL clones did not lyse bystander Jurkat cells, but HIV-infected primary CD4+ T cells lysed uninfected bystander cells by the fas-mediated pathway. These results suggest that HIV-specific CD8+ CTL do not cause HIV immunopathology by lysing bystander cells. On the contrary, fas-mediated lysis of uninfected cells by HIV-infected cells may contribute to CD4 decline.

CD4+Human Immunodeficiency Virus Type 1 (HIV-1) Envelope-Specific Cytotoxic T Lymphocytes Derived from the Peripheral Blood Cells of an HIV-1-Infected Individual

Virus-specific cytotoxic T lymphocytes (CTL) are frequently of the CD8+ surface phenotype, although CTL of the CD4+ surface phenotype have also been described. Published reports of CTL derived from peripheral blood mononuclear cells (PBMC) of individuals infected with human immunodeficiency virus type 1 (HIV-1) have described primarily cells of the CD8+ surface phenotype. However, CD4+ HIV-1 envelope-specific CTL have been reported after in vitro stimulation with HIV-1 envelope protein of peripheral blood cells obtained from HIV-1-seronegative donors, in peripheral blood cells after vaccination of HIV-1-seronegative persons with HIV-1 envelope proteins, and in cerebrospinal fluid cells of HIV-1-infected individuals. Recently, CD4+ HIV-1 gag-specific CTL were also reported. We now report a patient from whom we derived HIV-1 envelope-specific CTL cell lines of the CD4+ surface phenotype. Our cell culture technique did not employ exogenous viral antigenic stimulation, and may therefore yield cells that more closely reflect those in the underlying populations from which they were derived. These CTL did not appear to have the clear human leukocyte antigen (HLA) class II restriction pattern typically seen in CD4-expressing cells and were not functionally inhibited by anti-CD3 antibody. Further work will be required to define the role of CD4+ CTL in the pathogenesis of HIV-1 disease.

Genomic organization of the mouse granzyme A gene. Two mRNAs encode the same mature granzyme A with different leader peptides.

Granzyme A is a serine protease that, together with the other granular components of cytotoxic T lymphocyte (CTL) cells, has been implicated in the cytolysis process. We report here two different messages and the genomic organization of the mouse granzyme A gene. The granzyme A gene is composed of six exons spanning 7 kilobases. Alternative splicing of the second exon results in the two transcripts. The two mRNA species encode the same mature granzyme A protein but with different leader sequences. The first (HF1) encodes a typical leader signal sequence similar to other granzymes, but the second (HF2) putative leader sequence is different and less hydrophobic. Both messages are present in cultured CTL cell lines and in normal lymphoid tissues. They are both induced when CTL cells are activated in vitro or in vivo. Both messages can be translated in vitro, although the HF1 message appears to be much more efficient as a template. The putative 5' promoter region of the HF gene sequenced (500 base pairs of upstream sequences) contains no well defined promoter sequences aside from the TATA box. The results suggest that (a) granzyme A may be produced with putative different leader sequences from two different mRNAs; (b) this may provide a model system for studying alternate splicing and the evolution of a complex enzymatic system in an organelle; and (c) the genomic DNA reported will be useful for studying transcription regulations involved in controlling the specific expression pattern of this gene.

Human Cytotoxic Lymphocytes Reactive with Pancreatic Adenocarcinoma Cells

In this study we describe the establishment of long-term cytotoxic T lymphocyte (CTL) cell lines and clones from lymph nodes of 9 pancreatic cancer patients by stimulation with allogeneic pancreatic tumor cell lines. The CTL cells exhibited strong cytolytic activity against many, but not all, allogeneic pancreatic tumor cell lines, but showed little or no reactivity against most nonpancreatic tumor cells, indicating they were detecting non-HLA antigens. Most of the cells from the established CTL cell lines were CD3+, and the CD8 antigen was also expressed on the majority of the cells. Occasional cultures exhibited a broad spectrum of cytolytic activity, and such CTL cell lines showed high expression of the natural killer (NK) cell markers, Leu-19 and Leu-11b. Seven clones were established from two CTL cell lines (LT and RE). These clones exhibited functional and phenotypic heterogeneity. The cytolytic activity of CTL cell lines and clones was inhibited by antibodies to CD3 antigen. Immunoprecipitation experiments using an anti-CD3 monoclonal antibody (Leu4) or anti-alpha/beta T cell receptor antibody (beta f1) revealed the presence of two bands of Mr 40,000 and Mr 50,000 in clones positive for the alpha/beta T cell receptor. The in vivo effects of the LT cytotoxic clones were studied in the Winn assay using pancreatic tumor xenografts in nude mice. Subcutaneous injections of a mixture of the cytotoxic clones and pancreatic tumor cells resulted in a complete inhibition of tumor development, whereas mice given injections of tumor cells and CTL clones that lacked cytotoxic activity against pancreatic cells developed tumors.

Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.

Resistance of cytotoxic T lymphocytes to the lytic effects of their toxic granules.

A cytotoxic T lymphocyte (CTL) characteristically kills target cells one after the other by releasing toxic granules that contain one or more cytolytic components. To determine how CTLs avoid destroying themselves when they release granules and lyse target cells, 7 murine CD8+ CTL cell lines were compared with 19 other cell lines for susceptibility to lysis by the isolated toxic granules. Murine CD8+ CTLs were clearly the most resistant cells: granules did not lyse them even after they were exposed to azide, cyanide, and 2-deoxyglucose, conditions that were found to enhance the susceptibility of all the other cells tested, including other T cells. Thus, resistance of CD8+ CTLs to cytotoxic granules appears to be independent of cellular ATP. To reconcile these findings with other observations that, under some circumstances, CTLs can be lysed by other CTLs, we suggest a model in which a CTL releases only a limited proportion of its toxic granules at each antigen-specific encounter with a target cell; the amount released is sufficient to kill most target cells but to leave the CTL undamaged and with enough granules to attack other target cells.

Resistance of cytotoxic T lymphocytes to lysis by a clone of cytotoxic T lymphocytes.

To investigate how cytotoxic T lymphocytes (CTL) avoid killing themselves when they destroy target cells, we compared 20 different cell lines as target cells, including several CTL cell lines, for their susceptibility to lysis by CTL. Variations in recognition of this diverse set of target cells was circumvented by attaching to all of them a monoclonal antibody to the antigen-specific receptor of a cloned CTL cell line (clone 2C) and using the 2C cell line as the standard aggressor or effector cell. All of the nine tumor cell lines and the four noncytolytic T-helper cell lines tested as targets were highly susceptible to lysis by the aggressor CTL, but seven cytotoxic T-cell lines (six CTL and one T-helper cell line with cytotoxic activity) were largely resistant. These results, and the use of the lectin Con A as an alternative means for triggering CTL activity, point clearly to a level of resistance that could enable CTL to avoid their own destruction when they lyse target cells. The resistance of the cytolytic T cells did not appear to be accompanied by a similar resistance to complement-mediated lysis, indicating that mechanisms of CTL-mediated and complement-mediated lysis are not identical.

Fractionation of mouse cytotoxic T cells by use of lectins

Mouse cytotoxic T lymphocytes (CTLs), induced in vivo and in vitro in mixed-lymphocyte cultures, were fractionated into agglutinated and unagglutinated cells by use of various lectins. Cytolytic activity was enriched in the cell fraction agglutinated by various 2-acetamido-2-deoxy-α- d-galactopyranose-specific lectins, namely, Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), and Phaseolus limensis agglutinin (LBA). Only a little cytolytic activity remained in the unagglutinated cell fraction. Furthermore, the relationship between the binding of lectins and the cytolytic activity in various CTL cell lines was investigated with a fluorescence-activated cell sorter, and high-rank correlation was found between the cytolytic activity of these CTL cell-lines and the binding of DBA or HPA to them, but weaker rank correlation in the cases of LBA and peanut agglutinin (PNA). DBA was found to bind to almost all cells of interleukin-2-dependent CTL cell-lines tested. Although the DBA-positive CTL cell-lines have strong cytolytic activity, the DBA-negative cells, which consist of a small proportion of the CTLs, also exhibited cytolytic activity. Thus, the major part of CTLs has binding sites for α- d-GalNAc-specific lectins, particularly for DBA, and this lectin is useful for the enrichment of CTLs. However, the binding sites for DBA cannot be regarded as an exclusive differential marker for CTLs.

Inhibition of human lectin-dependent cell-mediated cytotoxicity, natural killer-like cytotoxicity, and cytotoxic T-lymphocyte-mediated cytolysis by xenoantisera raised against concanavalin A-stimulated human lymphocytes

This report describes the in vitro inhibition of several classes of human lymphocyte-mediated lysis by antisera from mice and rats immunized with concanavalin A-stimulated human T-enriched lymphocytes. The inhibitory antibodies in these antisera recognized molecules on the surface of cytotoxic lymphocytes, and did not bind to B-cell lines. Experiments were performed to demonstrate that these antisera were not inhibiting cytolysis by trivial mechanisms such as (i) masking of target cell antigens by anti-target cell antibodies, (ii) agglutinating effector cells, thereby preventing effector-target cell contact, or (iii) toxicity to the effector cells. Dextran dispersion experiments revealed that these antisera inhibit the post-conjugate formation phase of cytolysis mediated by cytotoxic T lymphocytes. These antisera inhibited natural killer-like cytotoxicity and lectin-dependent cell-mediated cytotoxicity as well as lysis mediated by both mixed cell culture-generated cytotoxic T lymphocytes and cytotoxic T-lymphocyte cell lines. These findings suggest that these in vitro systems of cell-mediated lysis may share a common step; however the different classes of cytolysis may have been inhibited by separate antibodies in the antisera. This rapid reproducible method of generating inhibitory antisera will facilitate the study of the molecules involved in the post-conjugate formation phase of human cytotoxic T-lymphocyte-mediated lysis.