Depletion of CD4+/CD25high regulatory T cells may enhance or uncover factor VIII-specific T-cell responses in healthy individuals.
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Cell Proliferation↗
Biomedical subjects
T Mutis
Publications and source records attributed to T Mutis.
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.
DNA-Binding Proteins↗
Susceptibility of malignant plasma cells to HA-1(H) specific lysis suggests a role for the minor histocompatibility antigen HA-1 in the graft-versus-myeloma effect.
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Disease Susceptibility↗
Targeting alloreactive T cells to hematopoietic system specific minor histocompatibility antigens for cellular immunotherapy of hematological malignancies after stem cell transplantation.
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Hematopoietic Stem Cells↗
The HA-2 minor histocompatibility antigen is derived from a diallelic gene encoding a novel human class I myosin protein.
Human minor histocompatibility Ags (mHag) present significant barriers to successful bone marrow transplantation. However, the structure of human mHag and the basis for antigenic disparities are still largely unknown. Here we report the identification of the gene encoding the human mHag HA-2 as a previously unknown member of the class I myosin family, which we have designated MYO1G. The gene is located on the short arm of chromosome 7. Expression of this gene is limited to cells of hemopoietic origin, in keeping with the previously defined tissue expression of the HA-2 Ag. RT-PCR amplification of MYO1G from different individuals led to the identification of two genetic variants, designated MYO1G(V) and MYO1G(M). The former encodes the peptide sequence previously shown to be the HA-2 epitope (YIGEVLVSV), whereas the latter shows a single amino acid change in this peptide (YIGEVLVSM). This change has only a modest effect on peptide binding to the class I MHC-restricted element HLA-A*0201, and a minimal impact on recognition by T cells when added exogenously to target cells. Nonetheless, as detected using either T cells or mass spectrometry, this amino acid change results in a failure of the latter peptide to be presented at the surface of cells that express MYO1G(M) endogenously. These studies have thus identified a new mHag-encoding gene, and thereby provide additional information about both the genetic origins of human mHag as well as the underlying basis of an Ag-positive vs Ag-negative state.
Alleles↗
HLA class I-minor histocompatibility antigen tetramers select cytotoxic T cells with high avidity to the natural ligand.
INTRODUCTION: Cytotoxic T cells specific for the hematopoietic system-restricted minor histocompatibility antigens HA-1 and HA-2 are potential tools for the treatment of relapsed leukemia after minor histocompatibility antigen mismatched bone marrow transplantation. HA-1/HA-2-specific cytotoxic T cells with strong cytotoxic activity against HA-1/HA-2 positive target cells can be generated in vitro using HA-1 and HA-2 peptide-pulsed dendritic cells as antigen presenting cells. MATERIAL AND METHODS: We used HLA-A2 HA-1/HA-2 tetramers (HA-1(A2)/HA-2(A2) tetramers) to monitor the in vitro generation of HA-1- or HA-2-specific cytotoxic T cells. RESULTS: We show that the intensity of the tetramer-staining of the HA-1/HA-2-specific cytotoxic T cells strongly correlates with their capability to recognize mHag positive target cells. The bright tetramer-staining cytotoxic T cells lyse target cells expressing the natural ligand. The dim tetramer-staining cytotoxic T cells fail to lyse natural ligand positive target cells and lyse peptide-pulsed target cells only. The frequency of bright tetramer-staining, high avidity minor histocompatibility antigen-specific CTLs increases significantly upon appropriate antigen-specific restimulations. CONCLUSION: Our results demonstrate that HLA class I-minor histocompatibility antigen tetramers are useful tools for monitoring and selection of high avidity HA-1- and HA-2-specific cytotoxic T cells for adoptive immunotherapy.
Antigen Presentation↗
Polyriboinosinic polyribocytidylic acid (poly(I:C)) induces stable maturation of functionally active human dendritic cells.
For vaccination strategies and adoptive immunotherapy purposes, immature dendritic cells (DC) can be generated from adherent monocytes using GM-CSF and IL-4. Presently, the only clinically applicable method to induce stable maturation of DC is the use of supernatants of activated monocytes (monocyte-conditioned medium (MCM)). MCM contains an undefined mixture of cytokines and is difficult to standardize. Here we report that stable maturation of DC can be simply induced by the addition of polyriboinosinic polyribocytidylic acid (poly(I:C)), a synthetic dsRNA clinically applied as an immunomodulator. Poly(I:C)-treated DC show a mature phenotype with high expression levels of HLA-DR, CD86, and the DC maturation marker CD83. This mature phenotype is retained for 48 h after cytokine withdrawal. In contrast to untreated DC, poly(I:C)-treated DC down-regulate pinocytosis, produce high levels of IL-12 and low levels of IL-10, induce strong T cell proliferation in a primary allo MLR, and effectively present peptide Ags to HLA class I-restricted CTL. In conclusion, we present a simple methodology for the preparation of clinically applicable mature DC.
Antigen Presentation↗
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.
Antigens, Neoplasm↗
Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease.
Graft-versus-host disease (GvHD) is a chief complication of allogeneic bone marrow transplantation. In HLA-identical bone marrow transplantation, GvHD may be induced by disparities in minor histocompatibility antigens (mHags) between the donor and the recipient, with the antigen being present in the recipient and not in the donor. Cytotoxic T lymphocytes (CTLs) specific for mHags of the recipients can be isolated from the blood of recipients with severe GvHD (ref. 3). A retrospective study demonstrated an association between mismatch for mHags HA-1, -2, -4 and -5 and the occurrence of GvHD in adult recipients of bone marrow from HLA genotypically identical donors. Tetrameric HLA-peptide complexes have been used to visualize and quantitate antigen-specific CTLs in HIV-infected individuals and during Epstein-Barr virus and lymphocytic choriomeningitis virus infections. Here we show the direct ex vivo visualization of mHag-specific CTLs during GvHD using tetrameric HLA-class and I-mHag HA-1 and HY peptide complexes. In the peripheral blood of 17 HA-1 or HY mismatched marrow recipients, HA-1- and HY-specific CTLs were detected as early as 14 days after bone marrow transplantation. The tetrameric complexes demonstrated a significant increase in HA-1- and HY-specific CTLs during acute and chronic GvHD, which decreased after successful GvHD treatment. HLA class I-mHag peptide tetramers may serve as clinical tools for the diagnosis and monitoring of GvHD patients.
Adult↗
CD80-Transfected acute myeloid leukemia cells induce primary allogeneic T-cell responses directed at patient specific minor histocompatibility antigens and leukemia-associated antigens.
Despite sufficient levels of HLA class I and class II expression, acute myeloid leukemia (AML) cells usually fail to induce a significant T-cell response in vitro. Therefore, we investigated whether in vitro modifications could enhance the T-cell stimulatory properties of AML cells. AML cells were either cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-alpha (TNF-alpha), or transfected with the CD80 (B7.1) gene and used as stimulator cells for primed and unprimed allogeneic T cells. Cytokine treatment increased HLA class I and II expression, but did not induce CD80 on AML cells. Cytokine-treated AML cells efficiently presented nominal and allo-antigens to primed T-cell clones, induced strong T-cell proliferation in HLA mismatched mixed lymphocyte reactions (MLR), but failed to induce primary T-cell responses from an HLA identical bone marrow donor in MLR. In contrast, CD80-transfected AML cells induced T-cell proliferation of HLA-identical bone marrow donor peripheral blood mononuclear cell (PBMC) in primary MLR, allowing the generation of leukemia reactive CD4(+) T-cell lines and clones. The majority of the generated oligoclonal (25 of 35) T-cell cultures showed patient specific reactivity that did not discriminate between patient's leukemic cells and Epstein-Barr virus (EBV)-transformed B cells (EBV-LCL). The remaining 10 oligoclonal T-cell cultures recognized only leukemic cells. One of these latter leukemia reactive oligoclonal T cells was cloned. The majority of the clones (25 of 29) reacted against both leukemic cells and patient's EBV-LCL. A minority of the T-cell clones with the CD4 phenotype (four of 29) showed strong HLA-DP restricted reactivity against leukemic cells, but not against patient's EBV-LCL or against HLA-matched nonleukemic cells, indicating that their target antigens are preferentially expressed by leukemic cells. In conclusion, our study shows that the in vitro allogeneic T-cell response induced by CD80-transfected AML cells is mainly directed against patient's specific minor histocompatibility antigens, while antigens preferentially expressed by leukemic cells can also trigger T-cell responses.
Antigens, Neoplasm↗
Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin.
Celiac disease is a common severe intestinal disease resulting from intolerance to dietary wheat gluten and related proteins. The large majority of patients expresses the HLA-DQ2 and/or DQ8 molecules, and gluten-specific HLA-DQ-restricted T cells have been found at the site of the lesion in the gut. The nature of peptides that are recognized by such T cells, however, has been unclear so far. We now report the identification of a gliadin-derived epitope that dominantly is recognized by intestinal gluten-specific HLA-DQ8-restricted T cells. The characterization of such epitopes is a key step toward the development of strategies to interfere in mechanisms involved in the pathogenesis of celiac disease.
Amino Acid Sequence↗
HLA class II restricted T-cell reactivity to a developmentally regulated antigen shared by leukemic cells and CD34+ early progenitor cells.
After allogeneic bone marrow transplantation (BMT), the beneficial graft-versus-leukemia (GVL) effect but also the life-threatening graft-versus-host disease (GVHD) are mediated by T cells of the grafted marrow. The identification of leukemia cell-reactive T cells and their ligands are, therefore, crucial for the development of new anti leukemia strategies. Here we describe a leukemia-reactive allo-HLA class II restricted CD4+ T-cell clone, 6.2, isolated from a healthy individual after stimulation with allogeneic leukemic cells. Clone 6.2 recognizes leukemic cells from several AML patients without showing reactivity to unfractioned peripheral blood mononuclear cells, monocytes, B cells, T-cell blasts, and proximal tubulus epithelial cells. Interestingly, clone 6.2 also recognizes BM cells derived from healthy individuals and inhibits the colony formation of myeloid and erythroid cell lineages. In the BM, clone 6.2 recognizes only CD34+ early precursor cells but not CD34-, more differentiated cells. Thus, the target antigen of clone 6.2 is developmentally regulated and expressed only by leukemic cells and CD34+ early progenitor cells in the hematopoietic system. We suggest that targeting the T-cell immune response to leukemia-associated, developmentally regulated antigens of the hematopoietic system can provide a basis for the separation of GVL from GVHD, and may lead to new therapeutic approaches for residual and relapsed leukemia.
Acute Disease↗
Role of cytotoxic cells in the protective immunity against and immunopathology of intracellular infections.
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Animals↗
Definition of a human suppressor T-cell epitope.
The quality of the response produced by regulatory or helper T (Th) cells presently receives much attention because of its possible implications for vaccine development and immunomodulation. Apart from cytokines and so-called costimulatory signals, antigens and the presenting major histocompatibility complex (MHC) molecules may play a role in determining the type of T-cell response generated toward antigens. To examine the role of antigen and/or HLA in control of T-cell subset activation, we have studied a special case, namely CD4+ suppressor T (Ts) cells in leprosy. Mycobacterium leprae-induced Ts cell clones have been previously isolated from peripheral blood and skin lesions of lepromatous leprosy patients and were shown to specifically down-regulate mycobacterium-specific Th cell responses. Despite considerable effort, the antigens recognized by these Ts cells have thus far not been identified. Here we report that all HLA-DR2-restricted CD4+ Ts cell clones derived from a lepromatous leprosy patient recognize an epitope that maps between the amino acid residues 439 and 448 of the mycobacterial hsp65. The peptide was presented to these Ts cells by HLA-DRB1*1503, a recently discovered HLA-DR2 variant. Non-suppressor T-cell clones derived from the same patient recognized antigens other than the hsp65 and were also stimulated by other HLA-DR2 variants. In independent cloning experiments peptide 435-449 and recombinant hsp65 induced exclusively Ts cells in this lepromatous leprosy patient. The Ts clones recognizing this particular epitope were derived from at least seven different progenitors, as they expressed different T-cell receptor alpha and beta chains. Thus, our data indicate that a specific peptide-HLA class II combination may exclusively activate Ts cells.
Amino Acid Sequence↗
A novel, antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders.
It is now well established that cultured human melanocytes are capable of expressing immunologically important cell surface molecules and that they can produce cytokines. Not all cells with the ability to express MHC class II molecules are capable of effective Ag presentation. However, the dendritic nature of melanocytes, their strategic position within the skin, and their phagocytic capacity seem to suggest a role for these cells in processing and presenting Ag. This study demonstrates that cultured normal human skin melanocytes can present peptide Ag, and process and present the mycobacterial HSP65 kDa protein and whole Mycobacterium leprae sonicate to CD4+ cytotoxic proliferative T cell clones in an Ag-specific and HLA-class II-restricted manner. T cell stimulation was dependent on costimulatory signals, i.e., LFA-3/CD2 and LFA-1/ICAM-1. Besides eliciting a T cell proliferative response, our studies further demonstrate that melanocytes can function as target cells for T cell-mediated cytotoxicity. The described Ag-processing and -presenting functions of melanocytes, taken together with in vivo behavior of melanocytes in hypopigmentation, provide new clues for the etiopathogenesis of melanin pigmentary disorders.
Antigen Presentation↗
Analysis of cytokine production by Mycobacterium-reactive T cells. Failure to explain Mycobacterium leprae-specific nonresponsiveness of peripheral blood T cells from lepromatous leprosy patients.
Recent analyses of antimycobacterial T cells clones from a small number of individuals indicate that mycobacteria preferentially induce Th cells that produce high levels of IFN-gamma and no or little IL-4 in Mycobacterium leprae-resistant tuberculoid leprosy (TT) patients and healthy subjects, whereas in one study M. leprae-induced Ts clones from polar lepromatous leprosy (LL) patients showed a reciprocal cytokine secretion profile and mediated their suppressive activity via the release of high levels of IL-4. We have evaluated these findings in peripheral blood T cells from a larger panel of TT and LL patients as well as healthy individuals. Mycobacterium-reactive T cell lines generated from the PBMC of these individuals were tested for cytokine secretion and proliferative capacity in response to M. leprae, Mycobacterium tuberculosis, and various individual mycobacterial Ag. The lepromatous pole of the leprosy spectrum was additionally investigated by analyzing the cytokine-secretion profile of M. leprae-induced (suppressor) T cell clones as well as primary ex vivo PBMC. All T cell lines from healthy individuals and TT patients responding to M. leprae, M. tuberculosis, or individual Ag, produced high levels of IFN-gamma and TNF-alpha but little or no IL-4 and IL-6. At the lepromatous pole, T cell lines failed to proliferate upon stimulation with M. leprae but in some cases produced significant levels of IFN-gamma. No IL-4 or IL-6 secretion was observed in response to M. leprae. These lines displayed strong proliferation and Th1-like cytokine production upon stimulation with M. tuberculosis. Similarly, stimulation of primary PBMC from LL patients with M. leprae or M. tuberculosis resulted in the release of IFN-gamma but no detectable IL-4 production. Control tetanus toxoid-reactive T cell lines from the same individuals instead produced large amounts of IL-4 and low levels of IFN-gamma. The analysis of M. leprae-induced T cell clones, including those with known suppressive activity, revealed that all lepromatous T cell clones produced large amounts of IFN-gamma. Most of these clones released no or little IL-4, but some clones produced higher levels of IL-4 in addition to IFN-gamma. Most clones tested produced IL-10 as well. The suppressor activity of suppressor T cell clones could not be inhibited by a neutralizing anti-IL-4 antibody and only in one case by neutralizing anti-IL-10 antibody. Anti-IL-4 and anti-IL-10 could not overcome the M. leprae-specific unresponsiveness observed in primary PBMC from LL patients.(ABSTRACT TRUNCATED AT 400 WORDS)
Antigen-Presenting Cells↗
Mycobacteria induce CD4+ T cells that are cytotoxic and display Th1-like cytokine secretion profile: heterogeneity in cytotoxic activity and cytokine secretion levels.
Protective immunity against mycobacteria is dependent on antigen-specific T cells. Current evidence suggests that not only helper T cells that activate infected macrophages but also cytotoxic T cells (CTL) that lyse infected macrophages are involved in protection. Mycobacterium-specific CD4+ CTL are readily detectable among primary peripheral T cells but what proportion of CD4+ T cells display cytotoxic activity is not known. Whether the cytotoxic CD4+ T cells are identical to or distinct from those that produce interferon (IFN)-gamma is also unknown. In addition, studies on CTL in mycobacterial infections have focused primarily on selected antigens like hsp65 but have not analyzed systematically whether other mycobacterial antigens can activate CTL as well. These issues are relevant not only to a further understanding of protective immunity and immunopathology but also may have implications for the design of effective vaccines. To start addressing these issues, we have studied a large panel of CD4+ T cell clones specific for a broad range of mycobacterial antigens, and analyzed their ability to lyse mycobacterium-pulsed target cells and to release IFN-gamma and interleukin (IL)-4. Our results show that the vast majority of CD4+ T cell clones are able to lyse mycobacterial antigen-pulsed target cells, and that those CTL can be triggered by a wide variety of mycobacterial antigens. CD4+ CTL released high levels of IFN-gamma, but low or nondetectable levels of IL-4. In contrast, control tetanus toxoid-specific T cell clones or lines displayed poor or weak cytotoxic activity and released high levels of IL-4. The antimycobacterial clones appeared to be heterogeneous in their levels of cytotoxic activity and IFN-gamma release. Interestingly one T cell clone was able to lyse only mycobacterium-pulsed macrophages but not B cells suggesting possible selectivity in target cell recognition for some CTL. These in vitro data have to be interpreted with some caution. Nevertheless they confirm and significantly extend previous observations and suggest that mycobacteria preferentially induce CD4+ T helper type 1 (Th1)-like cells that display cytotoxic activity, and release high levels of IFN-gamma but no or little IL-4. The induction of such Th1 like cells is specific for mycobacteria since tetanus toxoid induced T cells that were poorly or not cytolytic and secreted high levels of IL-4.
Antigens, Bacterial↗
HLA class II+ human keratinocytes present Mycobacterium leprae antigens to CD4+ Th1-like cells.
In a variety of inflammatory skin diseases like leprosy, keratinocytes (KC) are induced to express MHC class II molecules and may therefore serve as antigen-presenting cells (APC) for MHC class II restricted T cells infiltrating the lesions. However, KC have been thought to be improper APC for MHC class II restricted T cells and to drive T cells into an anergic rather than into an activation state. We evaluated this issue in relation to leprosy and tested whether HLA-DR+ KC could present M. leprae antigens to well-defined, CD4+, cytotoxic as well as proliferative, Th1-like cell clones. Using a recently developed sensitive assay system which employs intact layers of basal KC as APC we found that most T-cell clones (6/8) lysed HLA-DR+ KC pulsed with M. leprae antigens. KC were only recognized after induction of HLA-DR expression by IFN-gamma, in an antigen-specific and HLA class II restricted manner. All T-cell clones tested also showed significant proliferation and IFN-gamma production in response to M. leprae antigens presented by HLA-DR+ KC, arguing against a KC dependent anergizing effect on T cells. Thus, HLA class II+ KC can function as proper APC for HLA class II restricted CD4+ Th 1-like cells. It seems therefore possible that antigen presentation by KC contributes to the local cell-mediated immune responses in DTH lesions.
3T3 Cells↗