Generation Of CTL Research Articles

Role of IFN-g in Allograft Rejection

Interferon (IFN)-gamma is a cytokine produced mostly by activated T cells and NK cells that has complex effects on immune and nonimmune cells. IFN-gamma plays important roles in inflammation, usually in synergy with other cytokines, such as IL-1beta and TNF-alpha. The uniqueness of IFN-gamma lies in its ability to induce major histocompatibility complex (MHC) expression in many tissues, making it particularly relevant to transplantation. The results of graft rejection in the absence of IFN-gamma show that IFN-gamma modulates but is not essential for the allogeneic responses, suppressing generation of CTL. In vivo IFN-gamma has a protective role early in the response to vascularized organ allografts: transplants in mice have a tendency to develop necrosis when IFN-gamma is not available, apparently by failure of the microcirculation. The lack of IFN-gamma greatly reduces the induction of MHC in organ allografts, and it is possible that this is indirectly related to the protective effect of IFN-gamma. Nevertheless IFN-gamma also promotes graft vessel disease later in the course ofthe transplant. Thus IFN-gamma has diverse and potentially contradictory effects on organ allograft survival, acting both on the immune system and on the graft itself, the net effect depending on the graft type and the time post-transplant.

Interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) are necessary in the early stages of induction of CD4 and CD8 cytotoxic T cells by Mycobacterium leprae heat shock protein (hsp) 65 kD.

Cytotoxic T cells (CTL) may play an important role in host defence against mycobacterial infections. CD4 CTL are preferentially induced by mycobacteria, but both CD4 and CD8 CTL may be necessary components of a protective immune response. The 65-kD mycobacterium heat shock protein (hsp65) is a poor inducer of CTL in multibacillary leprosy (MB) patients. In this study we evaluate the possible role of cytokines in modulating the cytotoxic activity of CTL from leprosy patients and normal individuals (N) against autologous macrophages presenting Mycobacterium leprae hsp65. Our results show that hsp65-specific CTL were generated from both CD4 and CD8 lymphocytes. In N, individual cytokines as well as the combination of them were able to modify the hsp65-induced cytotoxic activity. The effect of cytokines on leprosy patients' lymphocytes was different in MB and paucibacillary (PB) patients. Thus, IL-6, IL-2, IFN-gamma or TNF-alpha did not modify the generation of hsp65-CTL from either MB (with or without an erythema nodosum episode (ENL)) or PB. In all the patients the simultaneous addition of two cytokines was required in order to increase CTL generation. In MB, IL-6 plus IFN-gamma or IL-2 increased both CD4 and CD8 CTL, while TNF-alpha plus IFN-gamma up-regulated only CD4 CTL. In PB, CD8 CTL were prominent with IL-6 plus IFN-gamma, while the increase was significant in CD4 CTL with IL-6 plus IL-2. Down-regulation of CTL was observed by addition of IL-4, IL-10, anti-IFN-gamma or anti-TNF-alpha in N controls. Our data demonstrate that IFN-gamma and TNF-alpha must be present for at least the first 60 h of the induction stage in order to generate full hsp65 CTL. Hence, IFN-gamma and TNF-alpha would be key factors in the generation of hsp65 CTL.

IL-2 during in vitro priming promotes subsequent engraftment and successful adoptive tumor immunotherapy by persistent memory phenotypic CD8(+) T cells.

Adoptive T cell tumor immunotherapy potentially consists of two protective components by the transferred effector cells, the immediate immune response and the subsequent development of memory T cells. The extent by which adoptively transferred CD8(+) CTL are destined to become memory T cells is ambiguous as most studies focus on the acute effects on tumor shortly following adoptive transfer. In this study we show that a substantial fraction of the input CTL develop into memory cells that reject a s.c. tumor challenge. The use of exogenous IL-2 or a combination of IL-2 and IL-4, but not solely IL-4, during the ex vivo culture for the CTL inoculation was necessary for efficient development of CD8(+) memory T cells. Thus, an important component of adoptive immunotherapy using CTL is the production of CD8(+) Ag-specific memory cells which is primarily favored by IL-2 receptor signaling during ex vivo generation of the effector CTL.

Th1 cytokine-conditioned bone marrow-derived dendritic cells can bypass the requirement for Th functions during the generation of CD8+ CTL.

Bone marrow-derived dendritic cell (BMDC) subsets have distinct immunoregulatory functions. Th1 cytokine-induced BMDC (BMDC1), compared with Th2 cytokine-induced BMDC2, have superior activities for the differentiation and expansion of CTL. To evaluate the cellular interactions between dendritic cells and CD8+ T cells for the induction of CTL, BALB/c-derived BMDC subsets were cocultured with purified CD8+ T cells from C57BL/6 mice. Our results demonstrate that BMDC1 support the generation of allogeneic CD8+ CTL in the absence of CD4+ Th cells. In contrast, BMDC0 (GM-CSF- plus IL-3-induced BMDC) and BMDC2 failed to promote the differentiation of CD8+ CTL. Using Ab-blocking experiments and studies with gene knockout mice, IL-2 and LFA-1 are demonstrated to be critical for BMDC1-induced CTL differentiation. Unexpectedly, BMDC1 were able to induce CTL from CD8+ T cells isolated from IFN-gamma-/- and IFN-gamma receptor-/- mice. However, BMDC1 produced higher levels of IFN-beta than other BMDC subsets, and anti-IFN-beta mAb blocked BMDC1-dependent CTL generation. These results indicated an indispensable role of IFN-beta, but not IFN-gamma, during BMDC1-induced CTL differentiation. We conclude that Th1-cytokine-conditioned BMDC1 can bypass Th cell function for the differentiation of naive CD8+ T cells into CTL.

The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells.

Induction of antitumor immunity involves the presence of both CD8(+) CTLs and CD4(+) Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34(+) hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8(+) CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4(+) Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4(+) T cells were cloned that released IFN-gamma upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8(+) and CD4(+) T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.

Immune recovery and immunotherapy after stem cell transplantation in children.

Careful longitudinal studies of the lymphoid cell recovery after stem cell transplantation with other than HLA-identical sibling donors illustrated the prolonged T- and B-cellular immunodeficiency post-SCT, whereas NK-cell recovery was fast. Only low numbers of CD45RO memory T-cells, with a restricted TCR-repertoire, are present in the first 6 months post-SCT. The consequence is an increased risk of viral infections and possibly of leukemia relapse. The latter complication can be prevented by enhancing the anti-leukemic immune reactivity shortly after SCT. Different technical approaches were presented, the majority of them still being in the pre-clinical phase. NK-cell reactivity based on KIR-epitope mismatches between donor and recipient are promising for AML- and CML-, not for ALL-patients. The ALL-blasts may be killed by an antibody-dependent cellular cytotoxicity, using anti-CD19 antibodies, as was shown to be effective in vitro. Also the generation of leukemia-specific CTL's, making use of differences in minor histocompatibility antigens between donor and recipient, is now operational and may be a highly effective approach in a number of leukemic graft recipients.

Sperm protein 17 (Sp17) in multiple myeloma: opportunity for myeloma-specific donor T cell infusion to enhance graft-versus-myeloma effect without increasing graft-versus-host disease risk.

We recently found that sperm protein 17 (Sp17), a spermatozoa-restricted protein, is aberrantly expressed on the tumor cells in patients with multiple myeloma (MM). It may therefore be possible to generate donor-derived Sp17-specific CTL for administration following allogeneic stem cell transplant to augment graft-versus-myeloma (GVM) effect without inducing a global GVHD. To assess this approach, we have produced recombinant Sp17 protein and used Sp17 protein-pulsed dendritic cells to generate HLA class I-restricted Sp17-specific CTL from a previously unimmunized healthy donor. These CTL were able to lyse autologous Epstein-Barr virus-transformed lymphoblastoid cells in a Sp17-dependent manner. Target lysis was HLA-A1 and HLA-B27 restricted. Cytotoxicity could be blocked by antibodies against monomorphic HLA class I, HLA-A1 and HLA-B27 molecules but not HLA class II molecules. Most importantly, the CTL lysed HLA class I-matched Sp17-positive tumor cells, suggesting that Sp17 is processed and presented in association with the HLA class I molecules in Sp17-positive tumor cells in a concentration and configuration that could be recognized by recombinant protein-primed CTL. Analysis by flow cytometry of the CTL indicated that they were predominantly CD8 in phenotype and they produced IFN-gamma and very little IL-4. Our results suggest the potential for the generation and administration of donor-derived Sp17-specific CTL to augment GVM without inducing GVHD following allogeneic stem cell transplant for MM.

A viral protein that selectively downregulates ICAM-1 and B7-2 and modulates T cell costimulation.

Kaposi's sarcoma-associated (KS-associated) herpesvirus (KSHV) is a B-lymphotropic agent linked to AIDS-related lymphoproliferative disorders and KS. We and others have earlier identified two viral genes, K3 and K5, that encode endoplasmic reticulum proteins that downregulate surface MHC-I chains by enhancing their endocytosis. Here we have examined the ability of these proteins to influence the disposition of other host surface proteins implicated in immune recognition and activation. We report that K5, but not K3, expression in BJAB cells dramatically reduces ICAM-1 and B7-2 surface expression; B7-1 expression is unaffected. This K5-induced reduction can be reversed by coexpression of a dominant negative mutant of dynamin, indicating that the loss of ICAM and B7-2 surface expression is due to their enhanced endocytosis. This downregulation is functionally significant, because K5-transfected B cells show substantial impairment in their ability to induce T cell activation. K5 is thus the first example of a viral modulator of immunological synapse formation and T cell costimulation. We propose that its expression reduces T cell responses to KSHV-infected B cells early in infection, thereby diminishing antiviral cytokine release and the production of stimulatory signals for CTL generation.

Immune enhancing effect of a growth hormone secretagogue.

Growth hormone (GH) has been known to enhance immune responses, whether directly or through the insulin like growth factor-1, induced by GH. Recently a nonpeptidyl small m.w. compound, a GH secretagogue (GHS), was found to induce the production of GH by the pituitary gland. In this study, we examined the effect of GHS in immunological functions of 5- to 6-wk-old and 16- to 24-month-old mice. In young mice, we observed a significant increase in PBLs, but T and B cell-proliferative responses were not consistently enhanced. The old mice, treated with GHS for 3 wk, did not show increases in peripheral lymphocytes, but they exhibited a statistically significant increase in thymic cellularity and differentiation. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect, particularly in the old mice. We have also found that GHS promoted better thymic engraftment in bone marrow transplant of SCID mice. We found more cycling cells in the spleens of treated mice, suggesting that GHS may exert its immune enhancing effect by promoting cell division in lymphoid cells. These observations ascribe to GHS a novel therapy possible for aging, AIDS, and transplant individuals, whose immune functions are compromised.

Use of autologous dendritic cells loaded with apoptotic LCL for ex vivo generation of specific CTL from the PBMC of EBV− individuals

Use of autologous dendritic cells loaded with apoptotic LCL for ex vivo generation of specific CTL from the PBMC of EBV− individuals

In vivo cross-presentation of a soluble protein antigen: kinetics, distribution, and generation of effector CTL recognizing dominant and subdominant epitopes.

Cross-presentation of exogenous Ags via the MHC class I pathway is now recognized for its role in self-tolerance, tumor immunity, and vaccine development. However, little is known about the in vivo distribution and kinetics of cross-presented protein Ags, nor the subsequent development of CTL effector responses to dominant or subdominant epitopes. We examined the location and duration of cross-presented Ag by using 5,6-carboxy-succinimidyl-fluorescein ester-labeled T cells from class I-restricted Ag-specific TCR mice. Comparisons of results from an in vitro (51)Cr release CTL assay with an in vivo CTL assay provided physiologically relevant insights into the functional capacities of CTL specific for epitopes with differing affinities. These data demonstrate that efficient cross-presentation of a dominant class I-restricted Ag is dose related and remains largely localized, but not limited to the draining lymph nodes for up to 3 wk following a single injection of soluble protein. Within this period, dominant peptide-specific CTL are fully functional in vivo throughout the secondary lymphoid system. However, no in vivo responses are seen to a subdominant or cryptic epitope. Prolonging Ag cross-presentation via use of IFA promoted persisting in vivo dominant epitope-specific CTL activity and revealed dose-responsive precursor CTL to the subdominant, but not to a cryptic epitope. Analysis of functional in vivo CTL responses demonstrated that, in the presence of strong ongoing responses to the dominant peptide, lytic activity of CTL directed at weaker epitopes is undetectable.

Bone marrow cells promote TH2 polarization and inhibit virus-specific CTL generation

This laboratory recently reported that human bone marrow cells (BMC) inhibit the generation of virus-specific CTL in culture. The culture supernatants contained increased levels of prostaglandin E 2 (PGE 2) (shown to favor TH2 cell development) and also inhibited EBV-CTL effector cell development. In this study, we obtained PBL from Epstein-Barr virus (EBV) IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) one year after renal transplantation. EBV-specific CTL were then generated in vitro by stimulating PBL with autologous EBV-transformed B cells (EBV-B) in the presence or absence of autologous BMC. The addition of BMC to the EBV-CTL generation cultures increased the intracellular expression in CD3+ cells of IL-4,-5,-6,-10, and -13. These CD3+ cells also expressed increased levels of the TH2 associated receptor CCR3. Inhibition was even observed by preparing EBV-CTL generating cultures in trans-wells that separated the autologous BMC from the PBL + EBV-B. It was then observed that CD3+ cells obtained after 7 days of culture in the presence of autologous BMC could be used as inhibitors of EBV-CTL generation. Protein Kinase A (PKA), a cAMP kinase that is involved in the upregulation of TH2 cytokine activity, was increased in EBV-CTL cultures by the presence of BMC. Additionally, IL-4-mediated signal transduction and activation of transcription (STAT-6) phosphorylation was slightly increased. These results show that the BMC inhibition is mediated by soluble factors (cytokines) and that cell-cell contact in this autologous system is not required, so that BMC (at least partially, via cytokine production) promote TH2 polarization in culture. Moreover, TH2 cells induced by culturing with autologous BMC directly inhibit EBV-CTL generation, and TH2 associated PKA, CCR3, and STAT-6 phosphorylation are enhanced by BMC.

Enhancement of antitumor immunity against B16 melanoma tumor using genetically modified dendritic cells to produce cytokines

Dendritic cells (DC) that have been genetically modified to express cytokine genes may be novel tools for inducing antitumor immune responses. In the present study, the pMX retroviral vector was modified to express the mouse IL-2 (mIL-2pMX) and mouse IL-12 (mIL-12pMX) genes. Supernatants from 293 cells transfected with pMX retroviral vectors were harvested and used to transduce mouse lin- bone marrow (BM) progenitor cells. After 48 h co-culture with pseudotype retrovirus, BM cells were cultured for 12 days in the presence of mGM-CSF, mSCF and mTNF-alpha to obtain a DC-enriched fraction. Flow cytometric analysis showed that GFP protein expression in these cultures was 20-40% and that 40-50% of the cultured BM cells were positive for the DC marker, DEC205. About 60% of cells sorted for DEC205 also expressed GFP. The supernatants of DC-mIL-2 and DC-mIL-12 cultured for 48 h contained 5.2 +/- 0.15 and 33.9 +/- 2.6 ng cytokine protein per milliliter, respectively. Intratumoral injection of DC-mIL-2 or DC-mIL-12 on days 8 and 15 after the intradermal injection of 1 x 105 B16F10 cells, resulted in a significant reduction in tumor size by day 21, as compared with mice treated with unmodified DC or DC-GFP. Longer term analysis as assessed at day 42 revealed that B16 tumor-bearing mice treated with cytokine gene-modified DC survived significantly longer than mice from other groups. Spleen cells obtained from DC-treated mice were specifically sensitized for the generation of CTL by subsequent restimulation with gene-modified DC. These results suggested that DC genetically modified to express IL-2 or IL-12 can induce potent antitumor responses against well-established, poorly immunogenic B16F10 tumors. Gene Therapy (2000) 7, 2113-2121.

Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells.

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.

Either B7 costimulation or IL-2 can elicit generation of primary alloreactive CTL.

B7-1 and B7-2 are important costimulatory molecules in the activation of T cell immunity. We have used mice made genetically deficient in either or both B7 molecules to determine the role of B7 molecules in activation of primary alloreactive CTL. The absence of either B7-1 or B7-2 did not alter generation of CTL from unfractionated lymphocytes, but the absence of B7-2 greatly decreased CTL generation from purified CD8+ responder cells. However, if B7-1 was induced on the stimulating cells then CTL generation was restored to wild-type levels. Absence of both B7-1 and B7-2 from MLR using whole splenocytes resulted in a profound reduction in generation of CTL. This could completely be reversed by the addition of IL-2. B7 molecules could directly costimulate CD8+ cells, as purified CD8+ cells developed into mature CTL when stimulated with wild-type APC, but not with B7-deficient APC. Again, IL-2 could drive CTL generation from purified CD8+ cells, even in the absence of B7 molecules. Taken together, these results demonstrate an important role for B7 costimulation in CTL generation.

CpG-DNA-mediated transient lymphadenopathy is associated with a state of Th1 predisposition to antigen-driven responses.

Infections can influence concurrent and subsequent Th1 vs Th2 immune responses to Ags. Through pattern recognition of foreign unmethylated CpG dinucleotides, the vertebrate innate immune system can sense infectious danger and typically replies with a Th1-polarized adaptive immune response. We examined whether CpG-DNA exposure would influence subsequent responses to infection and soluble Ags. CpG-DNA injection led to local lymphadenopathy characterized by maintenance of cellular composition with some biasing toward elevated dendritic cell composition. Sustained local production of IL-12 and IFN-gamma from dendritic cells and T cells was shown. Prior injection by up to 2 wk with CpG-DNA protected BALB/c mice from Th2 driven lethal leishmaniasis. CpG-DNA injection by up to 5 wk before soluble Ag challenge resulted in the generation of Ag-specific CTL, Th1 recall responses to Ag, and Th1-polarized Ag-specific Abs. Thus, CpG-DNA instigated a local predisposition for intense CTL responses and Th1-polarized immune responses to subsequent infections or Ag challenge. The induction by the innate immune system of a locally contained hypersensitivity could represent a capacitating immune reaction yielding rapid conditioned responses to secondary infections.

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Inhibit T Cell-Mediated Cytotoxicity by Inhibiting Fas Ligand Expression

We reported recently that the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) protect CD4+ T cells against Ag-induced apoptosis by down-regulating the expression of Fas ligand (FasL). Because the cytotoxic activity of CD8+ CTLs is mediated through two mechanisms, which involve the perforin/granzyme and the FasL/Fas pathways, in this study we investigated the effects of VIP/PACAP on the generation and activity of allogeneic CTLs, of CD8+ T1 and T2 effector cells and of alloreactive peritoneal exudate cytotoxic T cells (PEL) generated in vivo. VIP/PACAP did not affect perforin/granzyme-mediated cytotoxicity, perforin gene expression, or granzyme B enzymatic activity, but drastically inhibited FasL/Fas-mediated cytotoxicity against allogeneic or syngeneic Fas-bearing targets. VIP/PACAP inhibit CTL generation, but not the activity of competent CTLs. The inhibition is associated with a profound down-regulation of FasL expression, and these effects are mediated through both VPAC1 and VPAC2 receptors. VIP/PACAP inhibit the FasL/Fas-mediated cytotoxicity of T1 effectors and do not affect T2 cytotoxicity, which is entirely perforin/granzyme mediated. Similar effects were observed in vivo. Both the FasL/Fas-mediated cytotoxicity and FasL expression of cytotoxic allogeneic PELs generated in vivo in the presence of VIP or PACAP were significantly reduced. We conclude that, similar to their effect on CD4+ T cells, the two structurally related neuropeptides inhibit FasL expression in CD8+ cytotoxic T cells and the subsequent lysis of Fas-bearing target cells.

Bone marrow cells inhibit the generation of autologous EBV-specific CTL

Recently, we reported that human bone marrow cells (BMC) inhibited the proliferative (recall) response of lymphocytes to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) protein antigens [12]. To clarify further the effect of BMC on the immune response to viral antigens, we obtained PBL from EBV IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) 1 year after renal transplantation and generated EBV-specific CTL in vitro in the presence or absence of autologous BMC. The addition of freshly aspirated autologous iliac crest BMC from either R or LRD caused a significant inhibitory effect on the generation of EBV-specific CTL from CTL precursors, in contrast to the addition of autologous PBL used as controls (62.29 ± 10.85% inhibition using BMC from the kidney transplant recipients; 74.47 ± 15.21% inhibition using BMC from the living-related donors). This inhibitory effect was only exerted during the CTL generation phase; but not in the effector CTL killing phase. The expression of CD94, a component of the killer inhibitory receptor (KIR) on CD3 + cells was elevated in the cultures with BMC, in contrast to the cultures without BMC. The BMC inhibitory effect was partially abrogated by pre-incubation of the CTL effectors with anti-CD94 monoclonal antibody, in contrast with its isotype control. In addition, supernatants obtained from the CTL generating cultures with BMC contained high levels of prostaglandin E 2 (PGE 2), and EBV-specific CTL activity was inhibited by the addition of exogenous PGE 2 in the absence of BMC. The induction of CD40L cell surface expression by anti-CD3 was also decreased on the effector T cell population when BMC were added. There was a concomitant reduction in protein kinase C (PKC) activity. These studies demonstrate that BMC exert an inhibitory effect on T cell-mediated immunity to viral antigens in humans by regulating autologous effector T cell generation and early T cell activation signaling pathways.

Human vascular endothelial cells stimulate memory but not naive CD8+ T cells to differentiate into CTL retaining an early activation phenotype.

Endothelial cell (EC)-selective alloreactive CTL may mediate alloimmune vascular injury. In the present study, EC-selective CTL were generated in cocultures of purified human CD8+ T cells with allogeneic EC and were compared with conventional CTL against corresponding B lymphoblastoid cells (BLC). EC caused activation and expansion of memory but not naive CD8+ T cells, which differentiated into EC-selective CTL that retained high surface expression of CD69, CD25, and CD62L and displayed low intracellular perforin content. In contrast, BLC-stimulated CTL could be generated from naive or memory CD8+ T cells and showed a more mature phenotype (low CD69, CD25, and CD62L with higher levels of perforin). The expansion of alloreactive T cells by EC stimulation was 5- to 20-fold less effective than in corresponding BLC-stimulated cultures, accounting for a reduction in the assayable cytotoxicity of individual microcultures. In these IL-2-supplemented cocultures, no effect on CTL generation or phenotype was observed by mAb blocking of costimulation provided by LFA-3, ICAM-1, or CD40, by addition of comitogenic anti-CD28 mAb, or by preactivation of EC with CD40 ligand. Cyclosporine inhibited CTL expansion and cytotoxicity similarly in both EC- and BLC-stimulated cultures but did not affect the phenotype of those CTL that did emerge. This study extends the characterization of endothelium as an immunoregulatory cell type distinct from conventional APC and may explain why graft rejection within the arterial intima, an anatomic compartment in which EC may be the primary type of APC, is separable from rejection in the graft parenchyma.

Human dendritic cells transfected with RNA encoding prostate-specific antigen stimulate prostate-specific CTL responses in vitro.

Although immunological tolerance to self Ags represents an important mechanism to prevent normal tissue injury, there is growing evidence that tolerance to tumor Ags, which often represent normal peripherally expressed proteins, is not absolute and can be effectively reverted. Prostate-specific Ag (PSA) is a self Ag expressed by both normal and malignant prostatic epithelium, and therefore offers a unique opportunity to examine the ability of self Ags to serve as specific CTL targets. In this study, we investigated the efficacy of autologous dendritic cells (DC) transfected with mRNA encoding PSA to stimulate CTL against PSA Ags in vitro. Ag in form of RNA carries the advantage to encode multiple epitopes for many HLA alleles, thus permitting induction of CTL responses among many cancer patients independent of their HLA repertoire. In this study, we show that PSA mRNA-transfected DC were capable of stimulating primary CTL responses against PSA Ags in vitro. The PSA-specific CTL did not cross-react with kallikrein Ags, a protein, which shares significant homology with PSA, suggesting that harmful autoimmune toxicity may not represent a significant problem with this approach. PSA RNA-transfected DC generated from male or female healthy volunteers or from cancer patients were equally effective in stimulating PSA-specific CTL in vitro, implying that neither natural tolerance to PSA Ags nor tumor-mediated T cell anergy may represent major barriers for CTL generation against the self Ag PSA. This study provides a preclinical rationale for using PSA RNA-transfected DC in active or adoptive immunization protocols.