Lentivirus-transduced human monocyte-derived dendritic cells efficiently stimulate antigen-specific cytotoxic T lymphocytes

Dendritic cells (DCs) are professional antigen-presentingcells that are highly effective adjuvants for immunizing against pathogens and tumor antigens. The potential merit of genetic approaches to loading DCs with antigens is to express high and sustained levels ofproteins that can be subsequently processed and presented to Tlymphocytes. Replication-defective oncoretroviruses are able toefficiently transduce CD34⁺ progenitor-derived DCs but notmonocyte-derived DCs. Here, it is shown that efficient gene transfer isobtained using a human immunodeficiency virus-1-derived lentiviralvector deleted of all structural and accessory genes. Infection ofimmature DCs with the lentiviral vector at a multiplicity of infectionof 20 resulted in stable gene expression in 30% to 40% of the matured DCs. Proviral DNA was detectable by Alu polymerase chain reaction forthe lentiviral but not the oncoretroviral vector. Most importantly, itis demonstrated that lentivirus-transduced DCs were fully functional and effectively activated autologous HLA A2.1⁺ peripheralblood cytotoxic T lymphocytes (CTLs). DCs expressing lentiviralvector-encoded Flu peptide were at least as efficient as DCs pulsedwith the same peptide in stimulating specific CTLs. The efficacy of thelentivirus-transduced DCs was further demonstrated by their ability todirectly activate freshly harvested peripheral blood Flu-specific CTLsin the absence of CD4⁺ T-cell help and exogenous cytokines.The availability of a stable gene delivery system based on a multiplyattenuated lentivirus that does not encode any viral protein and thatallows sustained antigen presentation by DCs derived from bloodmonocytes will be very useful for the biologic investigation of DCs andthe improvement of immunotherapeutic strategies involving DCs.     

Pregnancy induces minor histocompatibility antigen-specific cytotoxic T cells: implications for stem cell transplantation and immunotherapy

Recipients of HLA-identical stem cell transplants have a poorer transplant outcome if the donor is female rather than male. We analyzed whether pregnancy primes for minor histocompatibility (H) antigens. Peripheral blood mononuclear cells (PBMCs) from healthy multiparous female blood donors were depleted for CD4+, CD14+, CD16+, and CD19+ cells, stained with minor H antigen-specific HLA-A2 tetramers, sorted by fluorescence-activated cell sorting, and tested for cytotoxic activity. Minor H antigens HY-, HA-1-, and HA-2-specific cytotoxic T cells (CD8+, CD45RA-) were present in PBMCs from 4 of 7 female donors up to 22 years after the last delivery. Interestingly, in 2 of the 4 cases microchimerism of the putative immunizing minor H antigen was observed. Thus, pregnancy can lead to alloimmune responses against the infant's paternal minor H antigens. The minor H antigen immunization status of female donors raises important questions for the clinical practice of stem cell transplantation.     

Escherichia coli-nitroreductase suicide gene control of human telomerase reverse transcriptase-transduced minor histocompatibility antigen-specific cytotoxic T cells

Adoptive immunotherapy with ex vivo generated cytotoxic T lymphocytes (CTLs) is applied for the treatment of leukemia relapses or viral infections after allogeneic stem cell transplantation. A common problem of adoptive immunotherapy strategies is the ex vivo expansion of the generated T cells to sufficient numbers. CTLs can be efficiently expanded by ectopic expression of the human telomerase gene (hTert). However, hTert transduction may also increase the chance for malignant transformation. Therefore, we explored the feasibility of suicide gene control of ex vivo generated CTLs expanded through the ectopic expression of hTert. To this end, we compared the efficacy of the new Escherichia coli-nitroreductase (E. coli-Ntr) suicide gene with the well-known herpes simplex virus-thymidine kinase (HSV-Tk). Introduction of hTert provided the transduced CTLs with a distinct growth advantage over the nontransduced CTLs. The hTert-E. coli-Ntr double-transduced CTLs retained their antigen-specific functions. Treatment of hTert-E. coli-Ntr double-transduced CTLs with metronidazole significantly inhibited the proliferation to a similar extent to the treatment of hTert-HSV-Tk double-transduced CTLs with ganciclovir. This is the first application of the E. coli-nitroreductase gene for the elimination of human T cells with metronidazole.     

CD8+ minor histocompatibility antigen-specific cytotoxic T lymphocyte clones eliminate human acute myeloid leukemia stem cells

Effective immunotherapy for human leukemia based on infusions of T lymphocytes requires the identification of effector T cells that target the leukemic stem cell. The transplantation of human acute myeloid leukemia into nonobese diabetic/severe combined immune deficient (SCID) mice has identified a rare leukemic progenitor termed the SCID leukemia-initiating cell, which is present in low frequency in the leukemic population and is essential for establishing leukemic hematopoiesis. Thus, this transplant model may be ideally suited to identify effector T cells with antileukemic activity. We report that CD8(+) cytotoxic T lymphocyte (CTL) clones specific for minor histocompatibility antigens inhibit the engraftment of human acute myeloid leukemia cells in nonobese diabetic/SCID mice and demonstrate that this inhibition is mediated by direct CTL recognition of SCID leukemia-initiating cells. These results indicate that CD8(+) minor histocompatibility antigen-specific CTL may be mediators of the graft-versus-leukemia effect associated with allogeneic hematopoietic cell transplantation and provide an experimental model to identify and select T cell clones for immunotherapy to prevent or treat relapse after allogeneic hematopoietic cell transplantation.     

A novel minor histocompatibility antigen recognized by HLA-A31 restricted cytotoxic T lymphocytes generated from HLA-identical bone marrow donor lymphocytes.

Bulk cytotoxic T lymphocytes (CTL) were generated by in vitro stimulation of BMT donor lymphocytes with Philadelphia chromosome (Ph)-positive leukemic cells from an HLA-identical sibling patient. CTL were cytotoxic against the patient's leukemic cells as well as the EBV-lymphoblastoid cell line (EBV-LCL) generated from the patient's cells, suggesting that they recognize a minor histocompatibility antigen (mHAg). Subsequently, several CTL lines were established by a limiting dilution method and analyzed. One of these CTL lines, 16C12 CTL which used a single TCRbetaV3S1 for CD8 cells, lysed HLA-A31-positive leukemic cells and EBV-LCL, but not fibroblasts. The cytotoxicity against the patient's leukemic cells and EBV-LCL was blocked by anti-HLA-A31 moAb, anti-HLA-class I moAb, and anti-CD8 moAb, suggesting that this mHAg was presented with HLA-A31. The antigen recognized by 16C12 CTL seemed to be a novel mHAg, since HLA-A31 restricted antigen has not been reported to date and 16C12 CTL showed no cytotoxicity against EBV-LCL which probably express known mHAgs. CTL detecting this mHAg may play an important role in the GVL effect in HLA-A31-positive BMT patients.     

Semi-allogeneic cell hybrids stimulate HIV-1 envelope-specific cytotoxic T lymphocytes

OBJECTIVE: The present study was designed to determine whether the HLA allogeneic T helper response stimulated by semi-allogeneic cell lines could be used as an in vitro model of immune-based therapy to stimulate HIV-specific cytotoxic T lymphocytes. DESIGN AND METHODS: Semi-allogeneic cell hybrids were obtained by the fusion of peripheral blood mononuclear cells from HIV-infected patients with the allogeneic beta2-microglobulin-deficient FO1-12 melanoma cell line. These hybrids were used as antigen presenting cells for HIV envelope peptide (env)-specific cytotoxic assays. RESULTS: The hybrid cell lines express HLA class I and II antigens from both parental cells, as well as the CD86 costimulatory molecule. HIV-specific cytotoxic T lymphocyte activity was obtained when patients' peripheral blood mononuclear cells were costimulated with env peptides plus semi-allogeneic hybrids, in contrast with stimulation with either env or hybrid cells alone. Thus, the semi-allogeneic hybrids enhanced HIV-specific killing of target cells. CONCLUSIONS: Irradiated, semi-allogeneic cell hybrids engineered for individual AIDS patients provide efficient and simultaneous co-recognition of HLA allogeneic determinants and viral antigenic determinants presented by self-HLA molecules on the same antigen presenting cells and results in the generation of enhanced HIV-specific cytotoxic T lymphocyte activity.     

Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+ CD28+ T lymphocyte responses

ObjectiveTo develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8⁺ CD28⁺ cytotoxic T lymphocyte (CTL) responses.MethodsCell-sized Dynabeads® M-450 Epoxy beads coated with H-2K^b: Ig-TRP2_180-188and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8⁺CD28⁺ CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs.ResultsDimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8⁺CD28⁺CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- γ under the stimulation of H-2K^b: Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells.ConclusionsThe new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8⁺ CD28⁺ CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.     

HBsAg-serum protein complexes stimulate immune T lymphocytes more efficiently than do pure HBsAg

HBsAg from plasma of chronic hepatitis B carriers was purified by affinity chromatography using a mouse monoclonal antibody specific for HBsAg. Elution with buffer at two different pH values separated HBsAg into two fractions: one contained high amounts of immune complexes associated with HBsAg; the other contained larger quantities of the HBsAg polypeptides P24 and GP27 and only small amounts of immunoglobulin. When compared for effects on stimulating the proliferative response of freshly isolated lymphocytes and an HBsAg-specific T cell clone, the HBsAg fraction containing a high proportion of immunoglobulin was much more potent than HBsAg with low amounts of immunoglobulins or pure HBsAg, which was isolated from the culture supernatant of the human hepatoma cell line (PLC/PRF/5). The plasma-derived HBsAg with low amounts of complexed immunoglobulins became more immunogenic in the presence of an anti-HBsAg monoclonal IgG. The present results, combined with earlier findings, suggest that HBsAg associated with immune complexes is a more potent stimulator of T cells than purer HBsAg preparations due to an increase in the efficiency of monocytes to capture the antigen through binding to immune complexes for subsequent processing and presentation of the antigen. These observations could be of relevance for the preparation of effective hepatitis B vaccines from recombinant DNA and peptide synthesis technologies.     

Feasibility of immunotherapy of relapsed leukemia with ex vivo-generated cytotoxic T lymphocytes specific for hematopoietic system-restricted minor histocompatibility antigens

Allogeneic bone marrow transplantation (BMT) is a common treatment of hematologic malignancies. Recurrence of the underlying malignancy is a major cause of treatment failure. Donor-derived cytotoxic T lymphocytes (CTLs) specific for patients' minor histocompatibility antigens (mHags) play an important role in both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) reactivities. mHags HA-1 and HA-2 induce HLA-A*0201-restricted CTLs in vivo and are exclusively expressed on hematopoietic cells, including leukemic cells and leukemic precursors, but not on fibroblasts, keratinocytes, or liver cells. The chemical nature of the mHags HA-1 and HA-2 is known. We investigated the feasibility of ex vivo generation of mHag HA-1- and HA-2-specific CTLs from unprimed mHag HA-1- and/or HA-2-negative healthy blood donors. HA-1 and HA-2 synthetic peptide-pulsed dendritic cells (DCs) were used as antigen-presenting cells (APC) to stimulate autologous unprimed CD8(+) T cells. The ex vivo-generated HA-1- and HA-2-specific CTLs efficiently lyse leukemic cells derived from acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) patients. No lytic reactivity was detected against nonhematopoietic cells. Sufficient numbers of the CTLs can be obtained for the adoptive immunotherapy purposes. In conclusion, we present a feasible, novel therapy for the treatment for relapsed leukemia after BMT with a low risk of GVHD.     

Allorecognition of purified major histocompatibility complex glycoproteins by cytotoxic T lymphocytes

To investigate how T cells recognize allogeneic class I proteins encoded by the major histocompatibility complex (MHC), we examined the human cytotoxic T lymphocytes (CTL) elicited in a mixed lymphocyte reaction against a lymphoblastoid B-cell line (JY) whose MHC-class I proteins are HLA-A2 and -B7. By panning the responding T cells on plates that were coated with purified HLA-A2, an essentially pure population of CD8+ anti-HLA-A2 CTL was isolated in a single step and established as a cell line designated A2p. In addition to lysing HLA-A2+ target cells, the A2p cells lysed HLA-A2- cells, including mouse cells (P815), when purified native HLA-A2 was attached to them, but not when denatured HLA-A2 was attached. Thus, contrary to the general rule that T cells recognize sequential antigenic determinants in denatured protein antigens, the alloreactive CTLs appear to recognize determinants that depend upon the native configuration of HLA-A2; however, the possibility that these T cells recognize a peptide adduct persistently associated with purified, soluble HLA-A2 has not been ruled out.     

Recognition of interspecies hybrid class I histocompatibility antigens by antigen-specific cytolytic T lymphocytes.

Two reciprocal interspecies hybrid class I histocompatibility genes have been constructed between genomic clones of human HLA-A2 and murine H-2Kb. The proteins encoded by these genes have been designated A21+2/Kb, where the polymorphic domains, alpha 1 and alpha 2, of HLA-A2 are linked to the carboxyl-terminal domains (alpha 3, transmembrane, and intracytoplasmic domains) of H-2Kb, and Kb1+2/A2, where the alpha 1 and alpha 2 domains of the H-2Kb antigen are linked to the carboxyl-terminal domains of HLA-A2. These genes have been transfected and expressed in recipient mouse L cells and human RD (rhabdomyosarcoma) cells. Both hybrid antigens were found to be serologically intact when tested with a panel of antigen-specific monoclonal antibodies. The monoclonal antibody W6/32, which recognizes a monomorphic determinant on all HLA-A, -B, and -C antigens, recognizes the alpha 1 and/or the alpha 2 domain, rather than the more conserved alpha 3 domain. Human cytolytic T lymphocytes (CTL) specific for the HLA-A2 antigen recognized the A2 and A21+2/Kb proteins only when expressed in human cells and not when expressed in mouse cells, even when surface antigen levels were 10-fold greater on the mouse cells than on the human cells. In contrast, a long-term, murine anti-H-2b CTL line not only lysed mouse L-cell lines that expressed the parental Kb and hybrid Kb1+2/A2 antigens but also lysed the Kb and Kb1+2/A2 human cell RD transformants as well. In both cases, the level of CTL recognition and lysis of the transformants that expressed the native antigen Kb was greater than of those transformants that expressed the hybrid antigen Kb1+2/A2. These data suggest that the carboxyl-terminal domains play some role in CTL allorecognition. The lack of human CTL recognition of HLA molecules expressed in mouse L cells, however, cannot be explained by the presence of a xenogeneic carboxyl terminus. Since murine CTL can recognize their target antigen when expressed on the surface of human cells, the possibility remains either that a ligand necessary for other molecular interactions of human CTL may be absent on mouse target cells or that murine and human CTL differ in affinity of binding to target antigens in the absence of accessory-molecule interactions.     

慢性乙型肝炎患者特异性细胞毒性T淋巴细胞的变化

目的探讨慢性乙型肝炎急性发作及病情严重程度与特异性细胞毒性T淋巴细胞(CTL)水平的关系。方法从29例人白细胞抗原(HLA)-A2阳性慢性乙型肝炎急性发作及免疫耐受慢性乙型肝炎患者外周血中分离外周血单个核细胞,用HLA-A2*HBcAg抗原表位肽-五聚体复合体及CD8单克隆抗体染色后,用流式细胞仪检测乙型肝炎病毒(HBV)特异性的CTL细胞。并对6例急性发作患者进行动态检测HBV特异性的CTL、肝功能和病毒载量。结果19例急性发作慢性乙型肝炎患者五聚体阳性细胞占CD8阳性细胞的比例平均为1．4%±0．8%,而10例免疫耐受患者为0．6%±0．4%,两组间比较,t=2．180,P＜0．05,差异有统计学意义。急性发作导致重型肝炎的患者中,乙型肝炎核心抗原特异性CTL细胞阳性率平均为1．3%±1．0%,而非重型肝炎患者为1．4%±0．8%,两组间比较,差异无统计学意义。在12周的随访期内,患者丙氨酸氨基转移酶和病毒载量逐渐下降,但乙型肝炎核心抗原特异性CTL维持在较高的水平(＞0．7%)。结论慢性乙型肝炎患者急性发作与HBV特异性CTL有关,但主要的肝细胞损害可能并非直接由HBV特异性CTL引起的。     

Generation of primary antigen-specific human cytotoxic T lymphocytes in human/mouse radiation chimera

Severe combined immunodeficient (SCID) mice are increasingly used as hosts for the adoptive transfer of human lymphocytes. Human antibody responses can be obtained in these xenogeneic chimeras, but information about the functionality of the human T cells in SCID mice is limited and controversial. Studies using human peripheral blood lymphocytes (PBL) injected intraperitoneally (IP) into SCID mice (hu-PBL-SCID mice) have shown that human T cells from these chimeras are anergic and have a defective signaling via the T-cell receptor. In addition, their antigenic repertoire is limited to xenoreactive clones. In the present study, we tested the functionality of human T cell in a recently described chimeric model. In this system, BALB/c mice are conditioned by irradiation and then transplanted with SCID bone marrow, followed by IP injection of human PBL. Our experiments demonstrated that human T cells, recovered from these hu-PBL-BALB mice within 1 month posttransplant, proliferated and expressed activation markers upon stimulation with anti-CD3 monoclonal antibody. A vigorous antiallogeneic human cytotoxic T-lymphocyte (CTL) response could be generated in these mice by immunizing them with irradiated allogeneic cells. Moreover, anti-human immunodeficiency virus type 1 (HIV-1) Net- specific human CTLs could be generated in vivo from naive lymphocytes by immunization of mouse-human chimeras with a recombinant vaccinia-nef virus. This model may be used to evaluate potential immunomodulatory drugs or cytokines, and could provide a relevant model for testing HIV vaccines, for production of antiviral T-cell clones for adoptive therapy, and for studying human T-cell responses in vivo.     

Rapid expansion of cytomegalovirus-specific cytotoxic T lymphocytes by artificial antigen-presenting cells expressing a single HLA allele

Cytomegalovirus (CMV) is a major threat in patients undergoing allogeneic bone marrow transplantation. The adoptive transfer of CMV-specific cytotoxic T lymphocytes (CTLs) expanded from the blood of CMV-seropositive donors has been shown to effectively control CMV infection. However, the requirement for safe and effective antigen-presenting cells (APCs) for each patient precludes broad applicability of this successful form of therapy. Here we analyze the ability of artificial APCs (AAPCs) to activate and expand CMV-specific CTLs from peripheral blood of seropositive HLA A2.1+ donors. We demonstrate that AAPCs expressing the CMV P495 peptide or the full-length pp65 protein stimulate P495-specific CTLs at least as effectively as autologous, peptide-pulsed, peripheral blood mononuclear cells or EBV-transformed B cells. Starting from 100 mL of blood, the AAPCs reliably yield clinically relevant CTL numbers after a single stimulation. CTLs activated on AAPCs effectively kill CMV-infected fibroblasts and have a Tc1 memory effector phenotype identical to that of CTLs generated with autologous APCs. AAPCs thus offer a rapid, controlled, convenient, and highly reproducible system for expanding CMV-specific CTLs. Furthermore, the CTL expansion obtained with AAPCs encoding full-length pp65 indicates that AAPCs may be used to present known as well as unknown CTL epitopes in the context of the AAPC's HLA.     

A novel immunogenic CS1-specific peptide inducing antigen-specific cytotoxic T lymphocytes targeting multiple myeloma

The CS1 antigen provides a unique target for the development of an immunotherapeutic strategy to treat patients with multiple myeloma (MM). This study aimed to identify HLA‐A2⁺ immunogenic peptides from the CS1 antigen, which induce peptide‐specific cytotoxic T lymphocytes (CTL) against HLA‐A2⁺ MM cells. We identified a novel immunogenic HLA‐A2‐specific CS1_239‐247 (SLFVLGLFL) peptide, which induced CS1‐specific CTL (CS1‐CTL) to MM cells. The CS1‐CTL showed a distinct phenotype, with an increased percentage of effector memory and activated CTL and a decreased percentage of naïve CTL. CS1_239‐247 peptide‐specific CD8⁺ T cells were detected by DimerX analyses and demonstrated functional activities specific to the peptide. The CTL displayed HLA‐A2‐restricted and antigen‐specific cytotoxicity, proliferation, degranulation and γ‐interferon (IFN‐γ) production against both primary MM cells and MM cell lines. In addition, the effector memory cells subset (CD45RO⁺CCR7^?/CD3⁺CD8⁺) within CS1‐CTL showed a higher level of CD107a degranulation and IFN‐γ production as compared to effector cells (CD45RO^?CCR7^?/CD3⁺CD8⁺) against HLA‐A2⁺ primary MM cells or MM cell lines. In conclusion, this study introduced a novel immunogenic HLA‐A2‐specific CS1_239‐247 peptide capable of inducing antigen‐specific CTL against MM cells that will provide a framework for its application as a novel MM immunotherapy.     

Interindividual conservation of T-cell receptor beta chain variable regions by minor histocompatibility antigen-specific HLA-A*0201- restricted cytotoxic T-cell clones

Minor histocompatibility antigens (mHags) are involved in the induction of graft-versus-host disease (GVHD) after HLA-identical bone marrow transplantation. Previously, we isolated a series of HLA-A*0201- restricted cytotoxic T-cell (CTL) clones specific for the same mHag HA- 1 from peripheral blood of three unrelated patients who were suffering from GVHD. We have now analyzed the composition of the T-cell receptor (TCR) V regions of 12 of these mHag HA-1-specific HLA-A*0201-restricted CTL clones by DNA sequencing of the alpha and beta chains. Of these 12 clones, derived from three unrelated individuals, five independent TCR alpha V- and beta V-region sequences were established. The TCR alpha chains were composed of varying TCR alpha V and TCR alpha J genes with no obvious similarities in structure in the N regions. However, the TCR beta chains all used the TCR beta V6S9 gene segment, and showed remarkable similarities within the N-D-N regions; ie, three independent beta-chain sequences (originating from donors Ha and Gy) shared a leucine/valine amino acid pair, whereas the other two (originating from donors Ha and Wi) shared a serine/threonine pair, all at positions 99 and 100 of the TCR beta V region. In conclusion, the TCR analysis of HA- 1 mHag-specific CTL clones has shown that the HA-1 mHag/HLA-A*0201 complex selects for highly similar TCR beta V regions.     

HIV-1 Vpu represents a minor target for cytotoxic T lymphocytes in HIV-1-infection

We have previously shown that Vpu is rarely targeted by HIV-1-specific cytotoxic T lymphocytes (CTL). The present report extends these findings and describes the characterization of the first CTL epitope within HIV-1 Vpu, identified in an individual with long-term non-progressive HIV-1 infection. The epitope was shown to be highly conserved among HIV clade B sequences and is restricted by HLA-A*3303, an HLA allele commonly seen in Asian and west-African populations.