Generation of melanoma-specific cytotoxic T lymphocytes for allogeneic immunotherapy

To exploit alloreactive T-cell responses known as the graft-versus-tumor effect, allogeneic hematopoietic stem cell transplantation is being explored as experimental therapy in selected solid tumors, including metastatic melanoma. However, donor T-cell responses are often delayed and associated with severe graft-versus-host disease. Posttransplant adoptive immunotherapy using tumor-specific cytotoxic T lymphocytes (CTL) of donor origin might provide immediate graft-versus-tumor effects but not graft-versus-host disease. Therefore, the aim of the current study was to define in vitro conditions for the expansion of allogeneic major histocompatibility complex-matched CTLs targeting melanoma-associated antigens (MAA). The CTLs were generated from peripheral blood mononuclear cells (PBMCs) of HLA-A*0201+ healthy donors by repetitive stimulations with HLA-A*0201-restricted MAA-derived peptides. Melanoma reactivity, as determined by lysis of peptide-pulsed T2 cells and HLA-A2+/Ag+ melanoma cells, was detected using in vitro expanded CTL targeting MAA peptides AAGIGILTV(MT(27-35)), IMDQVPFSV(G(209-2M)), and YMDGTMSQV(T(368-376)). In contrast, FLWGPRALV(MAGE3(271)-(279)) and VLPDVFIRCV(GnT-V(nt38-67)) induced peptide-specific recognition of T2 target cells only, whereas ITDQVPFSV(G(209-217)), KTWGQYWQV(G9(154)), MLLAVLYCL(T(1-9)), and tumor lysate could not induce specific CTLs. Specific cytolytic activity was accompanied by interferon-gamma secretion. Peptide-pulsed dendritic cells were required only for the initial stimulation of CTLs and could be substituted by PBMCs during restimulations. The median expansion rate of CTL was five to six times, regardless of whether dendritic cells or PBMCs were used after the initial stimulation. The results delineate the conditions for effective ex vivo expansion of melanoma-specific CTLs from PBMCs of healthy donors to be used as an adjunct in allogeneic cell therapy of metastatic melanoma.     

Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV- induced B cell lymphoproliferations in xenografted C.B-17 scid/scid mice [published erratum appears in J Exp Med 1996 Sep 1;184(3):1199]

C.B-17 scid/scid (severe combined immunodeficiency [SCID]) mice inoculated with peripheral blood lymphocytes from Epstein-Barr virus (EBV)-seropositive donors, or with EBV-transformed lymphoblastoid B cell lines (EBV-LCL), develop lethal human EBV+ B cell lymphoproliferative disorders (EBV-LPD) with characteristics similar to those arising in immunodeficient patients. Using this model, we examined the capacity of human effector cells to control human EBV-LPD. SCID mice received rabbit anti-asialo GM1 antiserum to abrogate endogenous natural killer-cell function. Preliminary experiments showed that adoptive transfer of peripheral blood mononuclear cells (PBMC), purified T cells, interleukin (IL) 2-activated PBMC or anti-CD3- activated T cells derived from EBV-seropositive donors did not result in improved survival of treated mice (in vivo effector/target ratio 2:1 to 1:1). In contrast, EBV-specific cytotoxic T lymphocytes (CTL), derived from EBV-seropositive donors and expanded in vitro, exhibited strong EBV-specific and HLA-restricted activity both in vitro and in vivo. SCID mice inoculated intraperitoneally with autologous but not with HLA-mismatched EBV-LCL had significantly improved survival relative to untreated mice after inoculation of EBV-specific CTL either intraperitoneally (P<0.001) or intravenously (P<0.001) (in vivo effector/target ratio 1:1). SCID mice bearing large subcutaneous EBV+ tumors and treated intravenously with 10(7) EBV-specific CTL achieved complete tumor regression. Both CTL- and CTL-plus-IL-2-treated mice survived significantly longer than untreated animals or animals treated with IL-2 alone (P = 0.0004 and P<0.02, respectively). SCID mice bearing two subcutaneous EBV+ tumors, one autologous and the other HLA mismatched to the EBV-specific CTL donor, had regression of only the autologous tumor after intravenous infusion of 10(7) EBV-specific CTL. Moreover, we could demonstrate preferential homing of PKH26-labeled EBV- specific CTL to autologous but not to HLA-mismatched EBV+ tumors as early as 24 h after intravenous adoptive transfer. Immunophenotypic analyses also demonstrated preferential infiltration of T cells into the autologous EBV+ tumor in SCID mice bearing both the autologous and either fully HLA-mismatched or genotypically related haplotype-sharing EBV+ tumors. The human T cells infiltrating EBV+ tumors were CD3+ and, predominantly, CD8+CD4-. Our results indicate that EBV-specific CTL preferentially localize to and infiltrate EBV+ tumors bearing the appropriate HLA antigens and thereafter induce targeted regressions of disease.     

Transfer of EBV-specific CTL to prevent EBV lymphoma post bone marrow transplant

EBV-associated lymphoproliferative disorders (EBV-LPD) are a significant problem after hemopoietic stem cell transplantation from unrelated donors or mismatched family members. Risk factors include T-cell depletion, MHC mismatch, and intensity of immunosuppression. New therapeutic strategies involve cellular immunotherapy approaches and both donor T-cells and EBV-specific cytotoxic T lymphocytes (CTLs) have proven to be effective therapies. EBV-specific CTL has also proved to have a major impact on the incidence of this complication when used prophylactically. J. Clin. Apheresis 14:154–156, 1999. © 1999 Wiley-Liss, Inc.     

T否决细胞促进造血干细胞植入的研究

否决效应是一种能特异性抑制识别否决细胞自身表面抗原的细胞毒性T细胞前体细胞(CTL-p)的攻击,而CTL-p对识别第三方抗原无抑制作用。具有否决效应的细胞称为否决细胞。细胞毒性T淋巴细胞(cytotoxic Tlymphocyte,CD8+CTL)是现知否决活性最强的细胞。在异基因造血干细胞移植中,输注供者源的CD8+CTL否决细胞清除宿主同种异体反应细胞可以促进供者干细胞的植入。本文就近年来关于CD8+CTL否决效应的机制、GVH效应、抗肿瘤效应、活体内的应用及与药物和细胞之间的协同作用的研究进展做一综述。     

Rapid generation of CMV pp65-specific T cells for immunotherapy

Adoptive immunotherapy with cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) has been shown to be an effective means of restoring cellular immunity to this virus and preventing CMV infection after allogeneic stem cell transplantation. Problems with current strategies include requirements for generating dendritic cells or other antigen presenting cells for stimulating CTL and the time needed for cell culture. The adherent cell fraction of peripheral blood mononuclear cells from 6 CMV seropositive donors were pulsed with pooled CMV pp65 peptides and incubated with nonadherent peripheral blood lymphocytes. CTL lacking specific cytotoxicity to pp65 were restimulated at day 10 of culture using peptide pulsed adherent cells. Of the 6 CMV seropositive donors tested, 5 had specific cytotoxicity to CMV pp65 (range 31% to 75%), with no alloreactivity. The resulting pp65-specific CTL consisted of a mixture of CD4 and CD8 cells, with 1% to 29% of CD8 cells and 0.5% to 10% CD4 cells making interferon-gamma (IFN-gamma) in response to pp65. The donor from whom we could not detect CMV-specific cytotoxicity had detectable CD4 and CD8 CMV pp65 CTL by intracellular cytokine analysis for IFN-gamma. Using this simplified strategy for expanding CMV pp65 CTL, adoptive immunotherapy with pp65-specific CTL could be made available in a more timely manner for patients who have persistent or therapy refractory CMV infections.     

Selected amino acid change encoding Epstein-Barr virus-specific T cell epitope of the LMP2A gene in Japanese nasal NK/T cell lymphoma patients

Nasal natural killer (NK)/T cell lymphoma is a peculiar lymphoma with a unique immunophenotype. Etiologically, in 1990, the authors first demonstrated the presence of Epstein-Barr virus (EBV) genomes and their products in this lymphoma. EBV-specific cytotoxic T lymphocytes (CTL) are very important in controlling the long-term persistence of EBV infection. Amino acid changes encoding the CTL epitope on the lymphoma cells may result in a reduced CTL response. We focused on two major CTL epitopes SSCSSCPLSK (codon 340 to 349) and FLYALALLLL (codon 356 to 364) of the LMP2A gene and determined the sequence isolated from nasal NK/T cell lymphoma tissues. All isolates from 7 nasal NK/T cell lymphomas showed the same amino acid change from serine to threonine at codon 348 in the CTL epitope SSCSSCPLSK. Threonine or serine substitution at codon 348 was almost equally observed in peripheral blood EBV isolates from healthy individuals in various ethnic origins. The predominant threonine substitution of nasal NK/T cell lymphoma patients may represent disease-associated polymorphism rather than a geographic or race-associated polymorphism. The LMP2A strain including threonine substitution at codon 348 may be selected within tumors and play a role for tumor genesis in Japanese patients with nasal NK/T cell lymphoma through reduced immune recognition.     

T cell receptor–targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice

To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8⁺ T cells, we designed synTacs, infusible biologics that recapitulate antigen-specific T cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB–specific ligands to HLA-A2 MHC molecules covalently tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8⁺ T cells, respectively, with potent antiviral activities. Intravenous injection of HIV- or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8⁺ T cells populating their spleens. Notably, these expanded HIV- or CMV-specific CD8⁺ T cells directed potent in vivo suppression of HIV or CMV infections in the humanized mice, providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8⁺ T cells, enabling optimization of individualized therapies, including HIV cure strategies.     

Rapid,large-scale generation of highly pure cytomegalovirus-specific cytotoxic T cells for adoptive immunotherapy

Adoptive transfer of donor-derived cytomegalovirus (CMV)-specific cytotoxic T cell (CTL) clones can restore immunity in allogeneic stem cell transplant recipients, providing protection against CMV disease. Current methods for selecting and expanding CMV-specific T cell clones are technically difficult, making adoptive T cell therapy impractical for routine clinical use. In this study, we describe a method for ex vivo generation and expansion of high-purity CMV-specific CTL using peptide-pulsed dendritic cells as antigen-presenting cells. Generation of CMV-specific CTL in numbers sufficient for clinical use in the time span of 4 weeks was accomplished in 6 of 8 CMV-seropositive donors. Examination of pp65 specificity by HLA/peptide tetramer staining demonstrated that a purity of greater than 95% peptide-specific cells could be obtained after two weekly stimulations and retained after further expansion for 3-4 weeks. Median expansion of total cell number was greater than 500-fold and expansion of peptide-specific CTL by tetramer staining was greater than 1.7 x 10(5)-fold. Four weeks after initiating CTL culture, we were able to generate greater than 10(9) total cells that specifically lysed target cells loaded with CMV peptide and cells infected with CMV. This simple and rapid method for generating high-purity CMV-specific CTL for adoptive immunotherapy is currently being examined for routine clinical use for allogeneic stem cell transplantation.     

HLA identical leukemia cells and T cell growth factor activate cytotoxic T cell recognition of minor locus histocompatibility antigens in vitro.

Lymphocytes from a healthy HLA-identical bone marrow transplant donor were tested for their ability to destroy her brother's acute myelogenous leukemia blasts in vitro. Primary mixed lymphocyte culture (MLC) and cell-mediated lysis (CML) responses between the patient's remission (pretransplant) and donor's lymphocytes were negative. Stimulation of donor lymphocytes for 7 d in vitro with irradiated leukemia cells, leukemia cells plus allogeneic irradiated lymphocytes, or a pool of irradiated lymphocytes from 10 donors, did not activate any cytotoxic cells able to destroy the HLA identical leukemic blasts. Further culturing for 7 additional d in T cell growth factor (TCGF) generated lymphocytes that induced effective cytotoxicity against the leukemic blasts, but not against autologous lymphocytes. Effective killing against the leukemia was observed only in cultures initially stimulated with the irradiated leukemia cells. These cytotoxic cells were maintained in TCGF and mediated persistent killing against the leukemic target cells. They were also able to destroy lymphocytes from the patient's mother and father, but not from an unrelated cell donor. This suggested specific recognition of non-HLA antigens inherited by the patient, that were foreign to the HLA identical bone marrow donor. These lymphocytes were cloned by a limiting dilution technique and one clone maintained cytotoxicity to the AML blasts and the father's lymphocytes, but not lymphocytes from the mother or an HLA-identical donor. This cytotoxicity was inhibited by a monoclonal anti-HLA antibody. Thus, in vitro sensitization of this sibling's lymphocytes with AML blasts followed by TCGF expansion, and cloning, enabled the detection of HLA-restricted cytotoxic cells that recognize minor locus histocompatibility antigens. This immune recognition may be relevant to the "graft vs. leukemia" effect that has been observed in leukemic animals and patients following histocompatible hematopoietic transplants.     

Cyclophosphamide-sensitive T lymphocytes suppress the in vivo generation of antigen-specific cytotoxic T lymphocytes

Murine T lymphocytes sensitized in vitro against either allogeneic lymphocytes or syngeneic hapten-conjugated lymphocytes do differentiate into highly effective cytotoxic T lymphocytes (CTL) (1-3). In vivo immunization of T lymphocytes to the same antigens, however, results in the generation of only marginal cytotoxic activity (1,4,5). Recently we found that the weakness of in vivo generated cytotoxicity is not due to a failure of antigen-induced T-cell sensitization but rather due to suppression of the in vivo differentiation of sensitized CTL precursors into effective CTL(6). In keeping with this finding it was postulated that suppressor cells may regulate the in vivo differentiation of CTL. We now report, that cyclophosphamide-sensitive T cells suppress the in vivo differentiation of antigen-specific CTL. Thus, pretreatment of mice with a single dose of cyclophosphamide (100 mg/kg) converts their state of low responsiveness to a state of high responsiveness.     

A phase I-II trial to examine the toxicity of CMV- and EBV-specific cytotoxic T lymphocytes when used for prophylaxis against EBV and CMV disease in recipients of CD34-selected/T cell-depleted stem cell transplants

Epstein-Barr virus (EBV)-induced lymphoproliferative disease and cytomegalovirus (CMV) infection are major causes of morbidity and mortality in individuals with compromised cellular immunity. Although anti-viral pharmacological agents exist, severe side effects such as myelosuppression often limit the application of these medications. Infusion of ex vivo-expanded, virus-specific cytotoxic T-lymphocytes (CTL) has been proven to be safe and efficacious for the prophylaxis and treatment of EBV and CMV complications. While EBV-specific CTL can be readily and reliably produced with EBV-immortalized B-lymphoblastoid cell lines (BLCL) as stimulators, current protocols for CMV-specific CTL, which use CMV-infected fibroblasts as stimulators, may be associated with alloreactivity and the need for cloning, as well as the potential for exposure to human blood-born infectious agents. Our laboratory has developed a novel system to generate EBV/CMV-bi-specific CTL by co-culturing PBMC with autologous BLCL expressing a CMV protein pp65 (BLCLpp65) (Sun et al., 1999). pp65, an immunodominant CMV antigen, is transduced into BLCL by a recombinant retrovirus MSCVpp65. While low in alloreactivity, BLCLpp65-stimulated CTL are cytolytic to autologous cells infected with EBV or CMV, and this cytotoxicity is mediated by polyclonal, CD8+, MHC Class I-restricted T-cells. Further experiments revealed that retroviral transduction and expression of pp65 do not compromise the capacity of presenting EBV antigens, and T cells stimulated by BLCLpp65 recognize clinical strains of CMV (Sun et al., 2000). These data indicated that BLCLpp65 could substitute for BLCL as antigen presenting cells in adoptive immunotherapy against EBV-LPD, with the benefit of providing protection against CMV reactivation. This protocol is a Phase I/II study to examine the toxicity associated with and the immunologic effects of ex vivo simultaneously expanded EBV- and CMV-specific CTL for prophylaxis against EBV and CMV complications in recipients of CD34 selected/T-cell depleted stem cell transplants (SCT). EBV/CMV-specific CTL will be generated from peripheral blood mononuclear cells (PBMC) of EBV/CMV-seropositive donors in a course of from 21-28 days by weekly stimulation with autologous BLCLpp65. Qualified CTL will be administered to consenting patients at 40, 60, and 80 days post-transpOFF criteria of molecular virology and immunological reconstitution, which include blood levels of pp65 antigen and EBV viral DNA, and virus-specific CTL precursor frequency. Patients will also be tested for replication-competent retrovirus at 3, 6, and 12 month intervals post-transplant to ensure bio-safety.     

Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development.

There is considerable interest in designing an effective vaccine to the ubiquitous Epstein-Barr virus (EBV). An important role for EBV-specific cytotoxic T lymphocytes (CTLs) in eliminating virus-infected cells is well established. Limited studies using a small number of immune donors have defined target epitopes within the latent antigens of EBV. The present study provides an extensive analysis of the distribution of class I-restricted CTL epitopes within EBV-encoded proteins. Using recombinant vaccinia encoding individual EBV latent antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP, and LMP 1), we have successfully localized target epitopes recognized by CTL clones from a panel of 14 EBV-immune donors. Of the 20 CTL epitopes localized, five were defined at the peptide level. Although CTL clones specific for nine epitopes recognized both type 1 and type 2 transformants, a significant number of epitopes (7/16 epitopes for which EBV type specificity was determined) were detected only on type 1 EBV transformants. Vaccinia recombinants encoding EBNA 3A and EBNA 3C were recognized more frequently than any other vaccinia recombinants used in this study, while no CTL epitopes were localized in EBNA 1. Surprisingly, epitope specificity for a large number of EBV-specific CTL clones could not be localized, although vaccinia recombinants used in this study encoded most of the latent antigens of EBV. These results suggest that any EBV vaccine based on CTL epitopes designed to provide widespread protection will need to include not only latent antigen sequences but also other regions of the genome. The apparent inability of human CTLs to recognize EBNA 1 as a target antigen, often the only latent antigen expressed in Burkitt's lymphoma and nasopharyngeal carcinoma, suggests that EBV-specific CTL control of these tumors will not be feasible unless the down-regulation of latent antigens can be reversed.     

Ex vivo purging of stem cell autografts using cytotoxic cells

Autologous stem cell transplantation (SCT) is the treatment alternative offered to patients that cannot benefit from allogeneic transplantation due to lack of suitable donor or age limitations. However, the outcome of autologous SCT is largely hindered by the high relapse rate. Two major factors can account for relapse after autologous SCT: the persistence of residual malignant cells resistant to chemo/radiotherapy left either in the body or in the autograft. Therefore, the rationale for purging autografts of residual malignant cells comes from the limitations of conventional high-dose chemo/radiotherapy in achieving a complete eradication of residual tumor cells in the marrow. To date, different purging modalities have been exploited. Immunological methods of purging present the advantage of being non-cross-reactive with conventional chemotherapy. These immunologic methods include depletion using antibody targeting of the malignant cells, ex vivo activation/generation of the autologous cytotoxic cells, in particular that of natural killer/lymphokine-activated killer (NK/LAK) and cytokine-induced killer (CIK) cells, and ex vivo purging of autografts using cytotoxic cell lines. The generation of ex vivo-expanded and activated autologous cytotoxic cells (CTL or NK) has generated increasing interest for the treatment of different malignancies. Unfortunately, the isolation and expansion of these cells have proven to be technically difficult. As an alternative, the use of cytotoxic cell lines as immune effectors has been proposed. The two available human cytotoxic cell lines TALL104 and NK-92 are currently in clinical trials and a number of studies have suggested their effectiveness as an immunotherapeutic agent including for ex vivo purging of autografts.     

Therapy of disseminated murine leukemia with cyclophosphamide and immune Lyt-1+,2- T cells. Tumor eradication does not require participation of cytotoxic T cells

The ability of noncytolytic Lyt-1+,2- T cells immune to FBL-3 leukemia to effect eradication of disseminated FBL-3 was studied. Adult thymectomized, irradiated, and T-depleted bone marrow-reconstituted (ATXBM) B6 hosts were cured of disseminated FBL-3 by treatment with 180 mg/kg cyclophosphamide (CY) and adoptively transferred Lyt-1+,2- T cells obtained from congenic B6/Thy-1.1 donors immune to FBL-3. Analysis of the T cell compartment of ATXBM hosts treated and rendered tumor-free by this therapy revealed that the only T cells present in the mice were donor-derived Lyt-1+,2- T cells. In vitro stimulation of these T cells with FBL-3 tumor cells, which express class I but no class II major histocompatibility complex antigens, induced lymphokine secretion, but did not result in the generation of cytotoxic T lymphocytes (CTL). Thus, in a setting in which mice lack Lyt-2+ T cells, and in which no CTL of either host or donor origin could be detected, immune Lyt-1+,2- T cells, in conjunction with CY, mediated eradication of a disseminated leukemia. The results suggest that delayed-type hypersensitivity responses induced by immune T cells represent a potentially useful effector mechanism for in vivo elimination of disseminated tumor cells.     

Generation of an HLA-restricted cytotoxic T cell line reactive against cultured tumor cells from a patient infected with human T cell leukemia/lymphoma virus

Lymphocytes from a patient who had an unusually long survival after therapy for a human T cell leukemia/lymphoma virus (HTLV)-associated T cell lymphoma were stimulated in vitro with an autologous tumor cell line, and the generation of cytotoxic T lymphocytes (CTL) was studied. CTL generated were directed against autologous (HTLV-associated tumor cells. These propagated CTL were OKT3+, OKT4-, and OKT8+. The cytotoxic activity required target tumor cells that were infected with HTLV and also expressed histocompatibility antigens in common with the patient, suggesting a major histocompatibility complex-restricted associative recognition of target antigens expressed on the tumor cell membrane.     

Intestinal graft-versus-host disease is initiated by donor T cells distinct from classic cytotoxic T lymphocytes

In these studies, the role of T helper and T cytotoxic cells in generating intestinal graft-vs.-host disease (GVHD) was examined. Treatment of C57BL/6J (B6) splenocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes natural killer cells, cytotoxic T lymphocyte (CTL) precursors, and the capacity to cause lethal GVHD in irradiated B6xDBA/2 F1 (B6D2F1) mice while preserving T helper cell function. Neither control nor Leu-Leu-OMe-treated DBA/2 donor spleen and bone marrow cells were found to induce lethal GVHD in B6D2F1 recipients. However, extensive colonic GVHD developed in B6D2F1 recipients of DBA/2 bone marrow and spleen cells. Enteropathic GVHD in DBA/2----B6D2F1 mice was reduced in severity after anti-L3T4 + C treatment of donor cells, and was eliminated by anti-Thy1.2 + C or the combination of anti-L3T4 and anti-Lyt2 + C treatment of the donor cell inoculum. However, neither anti-Lyt2 + C, Leu-Leu-OMe, nor anti-Lyt2 + C and Leu-Leu-OMe treatment of donor cells significantly decreased severity of gut GVHD. Leu-Leu-OMe treatment of DBA/2 or B6 SpC was comparably effective in preventing in vitro or in vivo generation of B6D2F1-specific CTL. These findings, therefore, demonstrate that histologically severe enteropathic GVHD does not require participation of CTL and is not always associated with high mortality rates.     

Epitope focusing in the primary cytotoxic T cell response to Epstein- Barr virus and its relationship to T cell memory

The relationship between primary and memory cytotoxic T lymphocyte (CTL) responses, and the factors influencing entry into memory, are poorly understood. Here we address this in the context of Epstein-Barr virus (EBV), a persistent human herpesvirus in which memory CTL responses in long-term virus carriers are highly focused on epitopes preferentially drawn from just three of the eight available virus latent proteins, EBNAs 3A, 3B, and 3C. To determine whether this unusual level of focusing is a consequence of long-term virus challenge, we carried out a detailed analysis of EBV antigen/epitope specificities in the primary virus-induced CTL response in 10 infectious mononucleosis (IM) patients of different HLA types. Primary effectors, studied in ex vivo assays and by limiting dilution cloning in vitro, were again highly skewed toward a small number of viral epitopes, almost all derived from the EBNA3 proteins, with CTL to the immunodominant epitope accounting for at least 1% of the circulating CD8+ IM T cell pool. This is the first unequivocal demonstration of an EBV-specific CD8+ CTL response in IM. Prospective studies on individual patients showed that, whereas all of the EBV reactivities found in CTL memory had been detectable earlier during primary infection, the memory population was not simply a scaled down version of the primary response. In particular (a) differences in the relative frequencies of CTL to immunodominant versus subdominant epitopes appeared to be much less marked in memory than in primary populations, and (b) we found at least one clear example in which a significant virus-specific reactivity within the primary response was never detectable in memory.     

Tetanus toxoid provides efficient T-cell help for the induction of HA-1(H) cytotoxic T cells

BACKGROUND: In vitro generation and expansion of leukemia‐reactive T cells may improve the efficacy and specificity of cellular immunotherapy against hematologic malignancies in the context of allogeneic stem cell transplantation. Since the expression of minor histocompatibility antigen HA‐1^H is limited to hematopoietic cells, ex vivo generated HA‐1^H‐specific CD8+ cytotoxic T lymphocytes (CTLs) can be used for adoptive immunotherapy. STUDY DESIGN AND METHODS: Numerous studies have shown that primary CTL induction from naïve precursors requires professional antigen‐presenting cells. Here, the feasibility of ex vivo induction of HA‐1^H‐specific CD8+ CTLs is demonstrated from unfractionated peripheral blood mononuclear cells (PBMNCs) from healthy blood donors when CD4+ T‐cell help is provided during primary stimulation. As a stimulus for the induction of T‐cell help, tetanus toxoid (TT) was used. RESULTS: After the second restimulation cycle, approximately 1 percent of CD8+ T cells stained positively with the HLA‐A*0201/HA‐1^H pentamer. Positive T cells were further expanded more than 1000‐fold by antigen‐independent stimulation with anti‐CD3/CD28 monoclonal antibodies. HA‐1^H‐induced T cells showed the classical phenotype for CD8+ memory effector cells: the phenotype changed from a mixed CD45RA/RO phenotype to an activated phenotype characterized by high expression of CD45RO and no expression of CCR7. The generated T cells revealed a very potent CTL response, even at low E:T ratios. CONCLUSION: This study demonstrates that TT provides a very potent and cost‐effective tool for the in vitro induction of antigen‐specific CTLs from precursor PBMNCs that can easily be adapted to GMP conditions for translational purposes.     

T cell memory. Long-term persistence of virus-specific cytotoxic T cells

This study documents that virus-specific CTL can persist indefinitely in vivo. This was accomplished by transferring Thy-1.1 T cells into Thy-1.2 recipient mice to specifically identify the donor T cell population and to characterize its antigenic specificity and function by using a virus-specific CTL assay. Thy-1.1+ T cells from mice previously immunized with lymphocytic choriomeningitis virus (LCMV) were transferred into Thy-1.2 mice persistently infected with LCMV. The transferred LCMV-specific CTL (Thy-1.1+ CD8+) eliminate virus from the chronically infected carriers and persist in the recipient mice in small numbers, comprising only a minor fraction of the total T cells. Upon re-exposure to virus, these long-lived "resting" CD8+ T cells proliferate in vivo to become the predominant cell population. These donor CD8+ T cells can be recovered up to a year post-transfer and still retain antigenic specificity and biological function. They kill LCMV infected H-2-matched cells in vitro and can eliminate virus upon transfer into a second infected host. In addition, these long-lived CD8+ T cells appear not to be dependent on help from CD4+ T cells, since depletion of CD4+ T cells has minimal or no effect on their biological properties (proliferation, CTL response, viral clearance). These donor CTL also exhibit an immunodominance over the host-derived LCMV-specific CTL response. When both host and donor T cells are present, the donor CTL response is dominant over the potential CTL response of the cured carrier host. Taken together, these results suggest that virus-specific CTL can persist for the life span of the host as memory cells.     

Analysis of T cell responses in liver allograft recipients. Evidence for deletion of donor-specific cytotoxic T cells in the peripheral circulation.

Analysis of cell-mediated lympholysis in long-term liver allograft recipients indicated that there was a donor-specific unresponsiveness that could not be reversed by the addition of rIL-2 and/or mixed lymphocyte culture supernatant or by nonspecific stimulation of the cultures with PHA. Stimulation of recipient cells with semisyngeneic cells having both donor and third-party HLA antigens failed to reveal the presence of cytotoxic T cells (CTL) specific to the donor, whereas the CTL response to third-party antigens remained normal. Removal of B lymphocytes from the responding cell population did not influence the responses. Furthermore, limiting dilution analysis showed that the liver transplant recipients did not have detectable levels of CTL precursors (CTLp) reactive to the donor antigens, whereas their CTLp to third-party antigens remained normal. Donor-specific CTLp were present before and during the early post-transplant period; these cells were eliminated from the peripheral circulation by 10 mo after transplantation. Taken together, these results indicate that there is a deletion of CTLp specific to donor MHC antigens in the peripheral circulation of long-term liver allograft recipients that may account in part for the success of liver transplantation across MHC barriers.