CTL-mediated Killing Research Articles

Eradication of B Cell Malignancies by Anti-3rd Party CTLs: A Novel Role for MHC Class I.

Induction of donor type chimerism under reduced intensity conditioning in the absence of GVHD represents a major challenge in transplantation biology. Despite their remarkable potent veto activity, CD8 CTLs could not be used to enhance engraftment due to their GVH reactivity. To address this problem we developed a new approach for the generation of human host-nonreactive CTLs, based on stimulation of donor CD8 T cells against 3rd party stimulators under IL-2 deprivation. A major attribute of such anti-3rd party CTLs reported recently by our group is their ability to eradicate pathological B-CLL cells. This B-CLL killing was shown to be independent of TCR recognition and it was found to be mediated both by autologous and by allogeneic anti-3rd party CTLs. In the present study we demonstrate that anti-3rd party CTLs can also efficiently eradicate primary mantle lymphoma cells and Burkitt and mantle cell-lines. Furthermore, the use of lymphoma cell lines has enabled to define several aspects of the mechanism leading to their apoptosis. Thus, we found that anti-3rd party CTLs avidly bind to target lymphoma cells and form stable aggregates within the first minutes of contact. Conjugate formation as well as the actual killing of the tumor cells were effectively inhibited by blocking antibodies against LFA1 or ICAM1 but not against ICAM3. Moreover, effective inhibition was attained only when applying anti-LFA1 to the CTLs and anti-ICAM1 to the tumor cells (100%±10% and 72%±7% inhibition, respectively) but not vice-versa. Given the role of CD8-MHC class I interaction in veto mechanisms, we sought to evaluate its role in the killing of lymphoma cells by human anti-3rd party CTLs. Indeed, in 10 experiments survival of target lymphoma cells was increased from 50%±5% to 82%±1% upon addition of anti HLA-ABC Ab to the incubation with anti 3rd party CTLs. A similar survival increase to 77.5%±1% was found upon the addition of anti-CD8 Ab, suggesting that CD8 binding to MHC class I constant region is required for lymphoma cell killing. The important role of MHC class I was further established by comparing CTL-mediated killing of Daudi lymphoma cells lacking cell surface MHC class I expression and Daudi cells with reconstituted surface MHC class I due to stable β2-microglobulin overexpression (β2m-Daudi). Thus in contrast to the poor killing of Daudi cells which did not exceed 20%, the β2m-Daudi cell killing was comparable to that exhibited with burkitt lymphoma line expressing MHC class1 (56%±1% and 63%±3%, respectively in 4 experiments). Importantly, FACS analysis showed that, MHC I expression did not influence conjugate formation, but was rather involved in the induction of apoptosis (annexin V). Furthermore, a similar pattern was also found in vivo when β2m-Daudi or Daudi cells were infused into the peritoneum of SCID mice in the presence or absence of a 2.5-fold excess of anti 3rd party CTLs. In 3 experiments, FACS analysis of the cells recovered from the peritoneum showed that the CTLs killed 91% of the β2m-Daudi cells as opposed to 13% killing of the Daudi cells. Taken together, our results suggest that TCR independent killing of B cell malignancies by anti-3rd party CTLs is mediated via a rapid adhesion through ICAM1-LFA1 binding, followed by slow induction of apoptosis upon a critical interaction between CD8 on the CTL and MHC class I on the tumor cell.

Viral vector-based prime-boost immunization regimens: a possible involvement of T-cell competition

Vaccination with recombinant viral vectors may be impeded by preexisting vector-specific immunity or by vector-specific immunity induced during the priming immunization. It is assumed that virus-neutralizing antibodies represent the principal effector mechanism of vector-specific immunity, while killing of infected cells by vector-specific cytotoxic T lymphocytes (CTLs) has also been suggested. Using recombinant Semliki Forest virus (rSFV) expressing E6E7 antigen from human papillomavirus, we demonstrate that secondary immune responses against E6E7 are neither affected by vector-specific antibodies nor by CTL-mediated killing of infected cells. Instead, the presence of the antigen during the prime immunization appeared to be the main determinant for the boosting efficacy. After priming with rSFVeE6,7, a homologous booster stimulated the primed E6E7-specific CTL response and induced long-lasting memory. Passively transferred SFV-neutralizing antibodies did not inhibit E6E7-specific CTL responses, although transgene expression was strongly reduced under these conditions. Conversely, in mice primed with irrelevant rSFV, induction of E6E7-specific CTLs was inhibited presumably due to vector-specific responses induced by the priming immunization. When during the priming with irrelevant rSFV, E7-protein was co-administered, the inhibitory effect of vector-specific immunity was abolished. These results suggest that, apart from vector-specific antibodies or killing of infected cells, T-cell competition may be involved in determining the efficacy of viral vector-based prime-boost immunization regimens.

Increased Sensitivity of Radiated Murine Cervical Cancer Tumors to E7 Subunit Vaccine–driven CTL-mediated Killing Induces Synergistic Anti-tumor Activity

The development of therapeutic vaccines has important implications for the treatment of cancer patients. Here we investigate whether human papillomavirus (HPV) E7 subunit vaccines can enhance tumor radioresponse using an established cervical cancer animal model. Radiation plus E7 subunit vaccines improved complete response, cure, and recurrence rates of tumors dramatically compared with single therapy alone. In particular, both components of the E7 subunit vaccines (E7 protein and CpG-oligodeoxynucleotide) were required for the induction of antigen (Ag)-specific cytotoxic T-lymphocyte (CTL) responses and for therapeutic synergy with radiotherapy. Moreover, with combined therapy the radiation dose could be reduced by 16 Gy to achieve an equivalent anti-tumor efficacy to radiation treatment alone. This therapeutic synergy was found to be mediated by CD8(+) CTLs and was concomitant with histological changes (presence of apoptotic bodies and multinucleated giant cells; heavy infiltration of lymphocytes), as determined by in vivo T-cell depletion and histological analysis. Finally, phenotypic changes of radiated tumors and their increased sensitivity to CTL-mediated killing appeared to be responsible for therapeutic synergy. These results show that E7 subunit vaccines act as a potent enhancer of tumor radioresponse and that this is mediated by increased sensitivity of radiated tumors to CTL-mediated killing. This study further suggests that E7-targeted therapeutic vaccines have the potential to improve radiotherapy in patients with cervical cancer.

Dormant Tumor Cells Develop Cross-Resistance to Apoptosis Induced by CTLs or Imatinib Mesylate via Methylation of Suppressor of Cytokine Signaling 1

In the BCR/ABL DA1-3b mouse model of acute myelogenous leukemia, dormant tumor cells may persist in the host in a state of equilibrium with the CD8(+) CTL-mediated immune response by actively inhibiting T cells. Dormant tumor cells also show a progressive decrease of suppressor of cytokine signaling 1 (SOCS1) gene expression and a deregulation of the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway due to methylation of the SOCS1 gene. Dormant tumor cells were more resistant to apoptosis induced by specific CTLs, but resistance decreased when SOCS1 expression was restored via demethylation or gene transfer. AG490 JAK2 inhibitor decreased the resistance of dormant tumor cells to CTLs, but MG132 proteasome inhibitor was effective only in SOCS1-transfected cells. Thus, SOCS1 regulation of the JAK/STAT pathways contributes to the resistance of tumor cells to CTL-mediated killing. Resistance of dormant tumor cells to apoptosis was also observed when induced by irradiation, cytarabine, or imatinib mesylate, but was reduced by SOCS1 gene transfer. This cross-resistance to apoptosis was induced by interleukin 3 (IL-3) overproduction by dormant tumor cells and was reversed with an anti-IL-3 antibody. Thus, tumor cells that remain dormant for long periods in the host in spite of a specific CTL immune response may deregulate their JAK/STAT pathways and develop cross-resistance to various treatments through an IL-3 autocrine loop. These data suggest possible new therapeutic targets to eradicate dormant tumor cells.

Differential susceptibility of leukocyte subsets to cytotoxic T cell killing: Implications for HIV immunopathogenesis

Cytotoxic T lymphocytes (CTL) are crucial for the host defense against viral infection. In many cases, this anti-viral immune response contributes to host pathogenesis, through inflammation and tissue destruction. Few studies have explored the relative susceptibility of infected cells to CTL killing, and the range of cell types that may be effectively killed by CTLs in vivo, both of which are key to understanding both immune control of infection and immune-related pathogenesis. We developed and optimized a highly sensitive method to quantify the relative susceptibility of leukocyte subsets to CTL-mediated killing. Maximal sensitivity was achieved by uniquely measuring cell death occurring during the assay culture. We found that leukocyte subsets have a wide range of susceptibility to antigen-specific CTL-mediated lysis. Generally, T cells were more susceptible than B or NK cells, with CD4 T cells being more susceptible than CD8 T cells. In all lymphocyte lineages, susceptibility was greater for more differentiated subsets compared with their naïve counterparts; however, for dendritic cells, immature cells are more susceptible than mature cells. We focused on the susceptibility of T cell subsets, and found that naïve cells are far more resistant than memory cells, and in particular, CCR5+ or HLA-DR+ memory cells are highly susceptible to CTL-mediated killing. These results provide an explanation for the observation that certain subsets of CD4 T cells are ablated during chronic HIV infection, and indicate which subsets are most likely to contain the persistent viral reservoir.

Therapeutic Synergy of Human Papillomavirus E7 Subunit Vaccines plus Cisplatin in an Animal Tumor Model: Causal Involvement of Increased Sensitivity of Cisplatin-Treated Tumors to CTL-Mediated Killing in Therapeutic Synergy

The goal of this study was to investigate the therapeutic potentials of combining chemotherapy with human papillomavirus (HPV) E7 subunit vaccines in an animal tumor model and to determine the underlying therapeutic mechanisms. Animals bearing HPV E6/E7-expressing tumors were treated intratumorally with a selected cytotoxic drug, cisplatin, twice at 1-week interval and s.c. with E7 subunit vaccines thrice at 1-week interval. Tumor chemoimmunoresponse was measured by tumor size. Ag-specific CTL activities and tumor histology were checked in mice under treatments. Apoptosis, in vivo T-cell subset depletion, adoptive CTL transfer, and tumor regression were used to determine the mechanisms for antitumor therapeutic effects. Combined therapy using cisplatin plus E7 subunit vaccines improved cure and recurrence rates of tumors and long-term antitumor immunity dramatically more than single therapy alone. In particular, both components of E7 subunit vaccines were required for induction of Ag-specific CTL as well as therapeutic synergy when combined with cisplatin. This therapeutic synergy was abrogated by depletion of CD8(+) T cells in vivo and was concomitant with histologic changes (such as heavy infiltration of lymphocytes and reduced tumor cell density). Finally, the increased sensitivity of cisplatin-treated tumors to CTL-mediated killing was found to be responsible for therapeutic synergy. E7 subunit vaccines plus cisplatin mediate antitumor therapeutic synergy through the increased sensitivity of cisplatin-treated tumors to CTL-mediated killing. Moreover, E7-based therapeutic vaccines have the potential to improve chemotherapy in patients with cervical cancer.

RPE Cells Resist Bystander Killing by CTLs, but Are Highly Susceptible to Antigen-Dependent CTL Killing

Retinal pigmented epithelial (RPE) cells maintain the blood-retinal barrier, sustain retinal photoreceptor cell health and function, and may play a role in ocular immune privilege. If RPE immunomodulatory activities were antigen specific, their expression would require antigen presentation. In a study of antigen processing and major histocompatibility complex (MHC) class I-restricted presentation by RPE cells, the cells' sensitivity to the activity of cytotoxic T lymphocytes (CTLs) was determined. RPE was cultured, with and without proinflammatory cytokines and antigen, followed by the addition of beta-galactosidase (beta-gal)-specific CTLs. Cytotoxic activity was measured by the CTL-dependent activation of caspase-3 in the RPE. Sensitivity to the CTLs was used to evaluate the activity of pathways of antigen processing and presentation using an antigen (beta-gal) that was either applied to or expressed in RPE. RPE cells were sensitive targets for activated CTL-mediated killing in vitro only if prepulsed with cognate peptide, or if beta-gal-expressing RPE was pretreated to induce upregulation of immunoproteasome. Activated CTLs induced apoptosis in RPE within 3 hours of coculture with antigen-positive RPE monolayers. Application of CTLs in a resting state to antigen-positive RPE led to their activation in the absence of exogenous antigen-presenting cells (APCs). This antigen-dependent activation and killing required 24 hours of co-incubation of RPE with resting CD8 T cells specific for beta-gal. Although RPE cells are highly phagocytic, functional evidence for processing that allowed phagocytosed antigens to load into class I MHC was not detected. RPE was minimally sensitive to bystander killing by activated CTLs. Although there are many reports of T-cell inhibition by RPE, we found that CTLs efficiently killed RPE cells by induction of apoptosis in an antigen-dependent manner. The survival of RPE in the face of extensive CTL destruction of adjacent photoreceptor cells in vivo appears to be based on their insensitivity to injury via bystander mechanisms.

Human CD34+ Myeloid Leukemic Progenitor Cells Are Susceptible to Lysis by Minor Histocompatibility Antigen LRH-1-Specific Cytotoxic T Lymphocytes.

Allogeneic stem cell transplantation (SCT) is a specialized form of immunotherapy for treating patients with hematological malignancies. The curative potential is attributed to the graft-versus-tumor (GVT) response during which donor-derived cytotoxic T lymphocytes (CTL) eliminate malignant cells of the recipient. Minor histocompatibility antigens (MiHA) are the major targets of the GVT response, and expansion of MiHA-specific CTL has been shown to coincide with tumor remission following SCT. Recently, we identified a novel hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest. 2005: 115:3506–3516). Interestingly, tetramer analysis showed a direct association between in vivo expansion of LRH-1-specific CD8+ T cells and the disappearance of Bcr-Abl positive tumor cells in the CML patient from whom LRH-1-specific CTL was originally isolated. In addition, we detected in vivo expansion of LRH-1-specific CTL in an AML patient who was in clinical remission without GVHD. Furthermore, we demonstrated that P2X5 mRNA is significantly expressed in leukemic CD34+ progenitor cells from most CML as well as AML patients. These findings indicate a role for LRH-1 in inducing GVT immunity against myeloid leukemic progenitor cells. Here, we investigated the ex vivo responsiveness of myeloid leukemic CD34+ progenitor cells to LRH-1-specific CTL. First, we addressed this question using the CD34+ KG1 cell line which is positive for LRH-1. By using a CFSE-based survival assay we demonstrated that KG1 cells stably transfected with HLA-B7 could be efficiently lysed by LRH-1-specific CTL. Next, we determined responsiveness of purified CD34+ progenitor cells from HLA-B7+ CML patients to LRH-1 CTL-mediated killing. In the CFSE-based survival assay as well as a hematopoietic progenitor cell inhibition assay we showed that LRH-1-specific CTL efficiently recognize and kill CD34+ progenitor cells from LRH-1+ CML patients. In contrast, LRH-1-specific CTL did not inhibit the proliferation of CD34+ progenitor cells from LRH-1- CML patients. These findings illustrate that the P2X5-encoded LRH-1 antigen is an attractive target for adequate eradication of myeloid leukemic progenitor cells after allogeneic SCT.

XIAP targeting sensitizes Hodgkin lymphoma cells for cytolytic T-cell attack

AbstractThe immunosurveillance of Hodgkin lymphoma (HL) by cytotoxic T lymphocytes (CTLs) is insufficient, and the clinical experience with adoptive transfer of CTLs is limited. We have previously reported that defects in mitochondrial apoptotic pathways and elevated XIAP expression confer resistance to different apoptotic stimuli in HL cells. Here, we aimed to develop molecular strategies to overcome the resistance of HL cells against CTL-mediated killing via granzyme B (grzB). In HL cells, grzB-induced mitochondrial release of proapoptotic Smac is blocked, which results in complete abrogation of cytotoxicity mediated by CTLs. Cytosolic expression of recombinant mature Smac enhanced caspase activity induced by grzB and restored the apoptotic response of HL cells. Similarly, down-regulation of XIAP by RNA interference markedly enhanced the susceptibility of HL cells for CTL-mediated cytotoxicity. XIAP gene knockdown sensitized HL cells for killing by antigen-specific CTLs redirected by grafting with a chimeric anti-CD30scFv-CD3zeta immunoreceptor. The results suggest that XIAP targeting by Smac agonists or XIAP-siRNA can be used as a synergistic strategy for cellular immunotherapy of Hodgkin lymphoma.

SOCS-1 protects from virally-induced CD8 T cell mediated type 1 diabetes

CD8 + cytotoxic T lymphocytes (CTL) can rapidly kill β-cells and therefore contribute to the development of type 1 diabetes (T1D). CTL-mediated β-cell killing can occur via perforin-mediated lysis, Fas–Fas-L interaction, and the secretion of TNF-α or IFN-γ. The secretion of IFN-γ can contribute to β-cell death directly by eliciting nitric oxide production, and indirectly by upregulating MHC class I and ‘unmasking’ β-cells for recognition by CTL. Earlier studies in the RIP-LCMV mouse model of diabetes showed that disruption of β-cell IFN-γ signaling alone abolished the direct detrimental effects of IFN-γ, but not MHC class I upregulation. Suppressor of cytokine signaling-1 (SOCS-1) represses several crucial cytokine signaling pathways simultaneously, among them IFN-γ and IL-1-β. We therefore evaluated the protective capacity of islet cell SOCS-1 expression in the CD8 + mediated RIP-LCMV diabetes model. Clinical disease was prevented in over 90% of the mice. Not only absence of MHC-I and Fas upregulation, but also resistance to cytokine-induced killing of β-cells and a complete lack of CXCL-10 (IP10) production in islets led to a lack of islet infiltration and impaired activation of autoaggressive CD4 + and CD8 + T-cells in these mice. Thus, SOCS expression renders β-cells resistant to CTL attack in a mouse model of T1D.

The 26th International Symposium of Sapporo Cancer Seminar: Innate immunity in cancer and infectious diseases

T 26th International Symposium of the Sapporo Cancer Seminar was held at Hokkaido University, Sapporo, Japan, on July 21–23, 2006. More than 120 researchers from various countries participated in the meeting (Fig. 1). The weather was fine every day, and the participants enjoyed walks around the university campus and dining at Chara Restaurant and Kirin Beer Garden. The symposium consisted of seminars presented by leading researchers from Japan and overseas in the field of innate immunity. Oral presentations were given by 12 researchers and poster presentations were given by 21 researchers. The symposium was organized by Dr Tsukasa Seya (Hokkaido University, Japan), as the chairman, and committee members Drs Misako Matsumoto, Hirofumi Sawa, Tokiyoshi Ayabe, and Roberto Cattaneo. Ms Saoko Kume was particularly involved in the organization of this meeting.

Significant Inhibition of Human CD8+ Cytotoxic T Lymphocyte-Mediated Xenocytotoxicity by Overexpression of the Human Decoy Fas Antigen

Human CD8(+) CTL-mediated killing may be important for xenograft rejection. The purpose of this study was to explore the preventing methods for CTL-mediated xenocytotoxicity by overexpression of human decoy Fas, which lacks a death domain in its cytoplasmic region, by binding competition with endogenous pig Fas. Moreover, the cytoprotective effect of this CTL-killing of membrane-bound human FasL, which is resistant to metalloproteolytic cleavage, was also assessed. Human CTL were generated by the stimulation of human PBMC with swine endothelial cells (SEC) and human IL-2, subsequently a CD8(+) population were selected by magnetic beads and employed as the effector cells. Stable SEC transfectants expressing either decoy Fas or membrane-bound FasL were established. Double-transfectants were also created. The amelioration of cytotoxicity to these transfectants was examined with Cr release assay. RESULTS.: Human CD8(+) CTL were highly detrimental against parental SEC. This CTL-killing was strongly inhibited by anti-FasL mAb treatment, however partial suppression was observed by Concanamycin A treatment. The overexpression of either decoy Fas or membrane-bound FasL in SEC markedly inhibited CTL-xenocytotoxicity. The double expressions of these molecules also significantly reduced this xenocytotoxicity despite the low levels of expression of either decoy Fas or membrane-bound FasL. These findings indicate that the strong xenocytotoxicity of human CD8(+) CTL is mediated mainly by the Fas/FasL pathway. The overexpression of either decoy Fas or membrane-bound FasL were quite effective in preventing CTL-killing. Furthermore, the combined expression of both molecules in pig cells may create a window of opportunity for prolonging xenograft survival.

A Role for Phosphatidylinositol 3-Kinase in TCR-Stimulated ERK Activation Leading to Paxillin Phosphorylation and CTL Degranulation

PI3K is an important regulator of a number of cellular processes. We examined the contribution of PI3K to mouse CTL signaling, leading to degranulation. We show that TCR-triggered, but not phorbol ester and calcium ionophore-induced, CTL degranulation is dependent on PI3K activity. Although PI3K activity is required for optimal LFA-1-mediated adhesion and cell spreading, this most likely does not account for its full contribution to degranulation. We demonstrate that PI3K is required for TCR-stimulated ERK activation in CTL, which we have shown previously to be required for CTL degranulation. We thus define a pathway through which PI3K most likely regulates degranulation and in which ERK appears to be a key signaling molecule. Furthermore, we identified the cytoskeletal adaptor paxillin as a target of ERK downstream of TCR stimulation. Consistent with a role in degranulation, we demonstrate that paxillin is localized to the microtubule organizing center in resting cells and upon target cell binding is recruited to the contact point with the target cell. These studies demonstrate that PI3K regulates ERK activity leading to CTL degranulation, and identify paxillin as a target of ERK downstream of the TCR. That paxillin is independently phosphorylated by both tyrosine kinase(s) and ERK downstream of the TCR and localized both at the microtubule organizing center and at the target cell contact point suggests an important role for paxillin in CTL-mediated killing.

Synergistic Effects on the Inhibition of Human CD8 + Cytotoxic T Lymphocytes-Mediated Killing Against Xenograft Cells by Coexpression of Membrane-Bound Human FasL and Decoy Fas Antigen

The principal barrier to the use of pigs as donors to humans is hyperacute rejection mediated by the interaction of α-gal abundantly expressed on pig cells and the natural anti-Gal antibody, abundantly produced in humans. This antibody-mediated hyperacute rejection may be overcome by an α1, 3 galactosyltransferase gene-knockout pig. However, xenograft cells could be rejected by T cells, especially CD8 + cytotoxic T lymphocytes (CTL)-mediated response, because these elements show great cytotoxicity against xenografted cells. We previously demonstrated that the Fas/FasL pathway is a major contributor to CD8 + CTL function. Furthermore, we sought to prevent this cytotoxicity by overexpression of membrane-bound FasL carrying the deletion at the metalloproteinase cleavage site or by decoy Fas antigen that does not contain the death domain in its cytoplasmic region. To investigate the effects of coexpression of these molecules, we cotransfected both genes into swine endothelial cells (SEC). The double-overexpression effectively prevented CD8 + CTL-mediated killing. Although cotransfectants and single transfectants of either membrane-bound FasL or decoy Fas gene showed similar inhibition of cytotoxicity, the expression levels of decoy Fas in SEC cotransfectants were much lower than those of decoy Fas single transfectants. These data suggest that beneficial effects for prevention of CTL-mediated xenocytotoxicity may be produced by the double expression of these molecules. The overexpression of both molecules on xenografted cells may decrease the innate cellular response to xenografts creating a window of opportunity to facilitate xenograft survival.

Free fatty acid release from human breast cancer tissue inhibits cytotoxic T-lymphocyte-mediated killing

Immune-mediated antitumor activities confront a variety of tumor-mediated defense mechanisms. Here, we describe a new mechanism involving FFA that may allow breast cancer to evade immune clearance. We determined the IC50 at which unbound free fatty acids (FFA(u)) inhibit murine cytotoxic T-lymphocyte (CTL)-mediated killing to assess the physiologic relevance of this phenomenon. We found that the IC50 for unbound oleate is 125 +/- 30 nM, approximately 200-fold greater than normal plasma levels. FFA inhibition, however, may play an important role in breast cancer because we found that large quantities of FFAs are released constitutively into the media surrounding samples of human breast cancer but not normal or benign tissue. FFA(u) concentration ([FFA(u)]) increased to at least 25 nM in 20 of 22 cancer tissue samples and exceeded 100 nM in 11 patients. Media from these samples inhibited CTL-mediated killing. Extrapolation from our in vitro conditions suggests that for tumor interstitial fluid, in vivo [FFA(u)] may be 300-fold greater than we observed in vitro. Although breast cancer release of FFA may suppress effector cell antitumor activity, strategies that reduce interstitial [FFA(u)] may significantly improve antitumor immune therapies.

Chemosensitization of Pancreatic Carcinoma Cells to Enhance T Cell-Mediated Cytotoxicity Induced by Tumor Lysate-Pulsed Dendritic Cells

Dendritic cells (DCs) can induce cytotoxic T-cell (CTL) responses against tumor antigens in vitro and in vivo, yet few cancer patients experience tumor regression after DC-based vaccination. Combination with other treatment modalities, such as radiation or pharmacologic anticancer agents, may reduce tumor cell resistance against immune responses. The authors tested whether treatment with gemcitabine or cyclooxygenase-2 (COX-2) inhibitors increases the sensitivity of pancreatic carcinoma cells to CTL-mediated killing. Monocyte-derived DCs of HLA-A2+ donors were loaded with lysate from the HLA-A2+ pancreatic carcinoma cell line Panc-1 and co-cultured with autologous CD3+ T cells. ELISPOT and cytotoxicity assays performed after two rounds of in vitro stimulation confirmed induction of a tumor-specific CTL response. Changes in the magnitude and the effector mechanism of the CTL response were analyzed after treatment of Panc-1 cells with gemcitabine and COX-2 inhibitors. Compared with gemcitabine, COX-2 inhibitors more effectively sensitized Panc-1 cells to CTL-mediated killing and showed less inhibition of T-cell activation by DCs in vitro. Using anti-CD95 blocking antibody, the authors showed that the increase in CTL-mediated tumor cell killing observed after treatment with COX-2 inhibitors is dependent on CD95/CD95 ligand interaction. Increased apoptosis of Panc-1 cells treated with COX-2 inhibitor was also observed after incubation with agonistic anti-CD95 antibody. Sensitization of cancer cells to CD95-dependent killing by CTLs represents a novel mechanism of action for COX-2 inhibitors and provides a rationale for their concomitant use with immunotherapeutic strategies such as DC-based vaccination.

Impaired Cytolytic Activity in Calreticulin-Deficient CTLs

Calreticulin is an endoplasmic reticulum-resident chaperone that is stored in the cytotoxic granules of CTLs and NK cells and is released with granzymes and perforin upon recognition of target cells. To investigate the role of calreticulin in CTL-mediated killing, we generated CTL lines from crt(+/+) and crt(-/-) mice expressing a constitutively active form of calcineurin in the heart. Crt(-/-) CTLs showed reduced cytotoxic activity toward allogeneic target cells despite normal production, intracellular localization, and activity of granzymes and despite perforin overexpression. Comparable or higher amounts of granzymes were degranulated by crt(-/-) cells in response to immobilized anti-CD3 Abs, indicating that calreticulin is dispensable for the signal transduction that leads to granule exocytosis. The ability to form conjugates with target cells was affected in the crt(-/-) CTLs, explaining the observed reduction in cytotoxicity. Conjugate formation and cytotoxicity were completely restored by treatments that facilitate recognition and contact with target cells, a prerequisite for degranulation and killing. Therefore, we conclude that calreticulin is dispensable for the cytolytic activity of granzymes and perforin, but it is required for efficient CTL-target cell interaction and for the formation of the death synapse.

Telomerase mRNA-Transfected Dendritic Cells Stimulate Antigen-Specific CD8+ and CD4+ T Cell Responses in Patients with Metastatic Prostate Cancer

Telomerase reverse transcriptase (hTERT) represents an attractive target for cancer immunotherapy because hTERT is reactivated in most human tumors. A clinical trial was initiated in which hTERT mRNA-transfected dendritic cells (DC) were administered to 20 patients with metastatic prostate cancer. Nine of these subjects received DC transfected with mRNA encoding a chimeric lysosome-associated membrane protein-1 (LAMP) hTERT protein, allowing for concomitant induction of hTERT-specific CD8+ and CD4+ T cell responses. Treatment was well tolerated. Intense infiltrates of hTERT-specific T cells were noted at intradermal injection sites after repeated vaccination. In 19 of 20 subjects, expansion of hTERT-specific CD8+ T cells was measured in the peripheral blood of study subjects, with 0.9-1.8% of CD8+ T cells exhibiting Ag specificity. Patients immunized with the chimeric LAMP hTERT vaccine developed significantly higher frequencies of hTERT-specific CD4+ T cells than subjects receiving DC transfected with the unmodified hTERT template. Moreover, CTL-mediated killing of hTERT targets was enhanced in the LAMP hTERT group, suggesting that an improved CD4+ response could augment a CTL response. Vaccination was further associated with a reduction of prostate-specific Ag velocity and molecular clearance of circulating micrometastases. Our findings provide a rationale for further development of hTERT-transfected DC vaccines in the treatment of prostate and other cancers.

Vascular Endothelial Cells Have Impaired Capacity to Present Immunodominant, Antigenic Peptides: A Mechanism of Cell Type-Specific Immune Escape

Vascular endothelial cells (EC) are an exposed target tissue in the course of CTL-mediated alloimmune diseases such as graft-vs-host disease (GVHD) or solid organ transplant rejection. The outcome of an interaction between CTL and target cells is determined by the amount of Ag presented and the costimulatory signals delivered by the target cells. We compared human EC with leukocytes and epithelial cells as targets for peptide-specific, MHC class I-restricted CTL clones. EC were poor targets for immunodominant CTL. Both endogenously processed antigenic proteins and exogenously added antigenic peptides are presented at 50- to 5000-fold lower levels on EC compared with any other target cell analyzed. This quantitative difference fully explained the poor CTL-mediated killing of EC. There was no evidence that lack of costimulation would contribute significantly to this cell type-specific difference in CTL activation. An HLA-A2-specific CTL clone that killed a broad selection of HLA A2-positive target cells equally well, killed EC less efficiently. Our data suggest that EC present a different Ag repertoire compared with other cell types. By this mechanism, these cells may escape an attack by effector CTL, which have been educated by professional APCs and are specific for immunodominant antigenic peptides.

Preventing human CD8 + cytotoxic T lymphocyte–mediated cytotoxicity against swine endothelial cells by overexpression of human decoy Fas antigen

Although the birth of homozygous α1, 3 galactosyltransferase gene-knockout pigs raised hopes for an imminent breakthrough in the prevention in the antibody-mediated rejection of pig to human discordant xenotransplants, human CD8 + cytotoxic T lymphocyte (CTL)–mediated killing may represent a new immunological barrier to long-term survival in xenograft recipients. In this study, we demonstrated that the cytotoxicity of human CD8 + CTL against swine endothelial cells (SEC) is highly detrimental and mediated at least in part by the Fas/FasL pathway. To prevent this CTL-mediated xenocytotoxicity, we overexpressed the human decoy Fas antigen, which does not contain a death domain in its cytoplasmic region, by means of binding competition with endogenous pig Fas antigen on SEC for the common ligand, human FasL. Furthermore, we generated a membrane-bound form of human FasL that cannot be cleaved by a putative metalloproteinase to produce a soluble form, which was assessed as an inhibitor of CTL cytotoxicity. Both human decoy Fas and membrane-bound FasL were effective to prevent CTL-mediated killing, suggesting that these novel molecules may represent a step forward toward preventing CD8 + CTL-mediated xenograft rejection. The combined expression of both molecules may be more beneficial to protect xenograft cells.