ABSTRACT
Pluripotent stem cells are promising candidates for cell-based regenerative therapies. To avoid rejection of transplanted cells, several approaches are being pursued to reduce immunogenicity of the cells or modulate the recipient's immune response. These include gene editing to reduce the antigenicity of cell products, immunosuppression of the host, or using major histocompatibility complex-matched cells from cell banks. In this context, we have investigated the antigenicity of H-Y antigens, a class of minor histocompatibility antigens encoded by the Y chromosome, to assess whether the gender of the donor affects the cell's antigenicity. In a murine transplant model, we show that the H-Y antigen in undifferentiated embryonic stem cells (ESCs), as well as ESC-derived endothelial cells, provokes T- and B cell responses in female recipients.
Subject(s)
Embryonic Stem Cells/metabolism , Graft Rejection/immunology , H-Y Antigen/metabolism , Animals , Animals, Newborn , B-Lymphocytes/immunology , Female , Immune Tolerance , Immunity , Male , Mice , Mice, Inbred BALB C , Stem Cell Transplantation , Survival Analysis , T-Lymphocytes/immunologyABSTRACT
The zinc finger transcription factor ThPOK plays a crucial role in CD4 T-cell development and CD4/CD8 lineage decision. In ThPOK-deficient mice, developing T cells expressing MHC class II-restricted T-cell receptors are redirected into the CD8 T-cell lineage. In this study, we investigated whether the ThPOK transgene affected the development and function of two additional types of T cells, namely self-specific CD8 T cells and CD4(+) FoxP3(+) T regulatory cells. Self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11 and proliferation in response to either IL-2 or IL-15. The ThPOK transgene converted these self-specific CD8 T cells into CD4 T cells. The converted CD4(+) T cells are no longer self-reactive, lose the characteristics of self-specific CD8 T cells, acquire the properties of conventional CD4 T cells and survive poorly in peripheral lymphoid organs. By contrast, the ThPOK transgene promoted the development of CD4(+) FoxP3(+) regulatory T cells resulting in an increased recovery of CD4(+) FoxP3(+) regulatory T cells that expressed higher transforming growth factor-ß-dependent suppressor activity. These studies indicate that the ThPOK transcription factor differentially affects the development and function of self-specific CD8 T cells and CD4(+) FoxP3(+) regulatory T cells.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation , Cell Lineage , Self Tolerance , T-Lymphocytes, Regulatory/metabolism , Transcription Factors/metabolism , Animals , Antigens, Ly/metabolism , Biomarkers/metabolism , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , Cells, Cultured , Female , Forkhead Transcription Factors/metabolism , H-Y Antigen/metabolism , Hyaluronan Receptors/metabolism , Interleukin-15/metabolism , Interleukin-2/metabolism , Interleukin-2 Receptor beta Subunit/metabolism , Male , Mice , Mice, Transgenic , Receptors, Antigen/metabolism , Sex Factors , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , T-Lymphocytes, Regulatory/immunology , Time Factors , Transcription Factors/genetics , Transforming Growth Factor beta/metabolismABSTRACT
In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8(+) T cells, hematopoietic cells (HCs) select innate CD8(+) T cells whose Ag specificity is not fully understood. Here we show that CD8(+) T cells expressing an H-Y Ag-specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H-Y Ag. These HC-selected self-specific CD8(+) T cells resemble innate CD8(+) T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus-independent CD8(+) T-cell population. The peripheral maintenance of H-Y-specific CD8(+) T cells required presentation of the self-Ag and IL-15 on HCs. HC-selected CD8(+) T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self-Ag-specific CD8(+) T cells in TCR non-Tg mice could develop via HC-induced positive selection, supporting results obtained from H-Y TCR Tg mice. These findings indicate the presence of self-specific CD8(+) T cells that are positively selected by HCs in the peripheral T-cell repertoire.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , H-Y Antigen/immunology , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen/biosynthesis , Adoptive Transfer , Animals , Autoantigens/immunology , Autoantigens/metabolism , Cell Differentiation , Female , H-Y Antigen/metabolism , Histocompatibility Antigens Class I/immunology , Interleukin-15/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Antigen, T-Cell/metabolismABSTRACT
Chronic rejection is the major cause of long-term heart allograft failure, characterized by tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signaling, regulating both T cell activation and migration of primed T cells to non-lymphoid antigen-rich tissue. We investigated the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model in which HY-mismatched male hearts were transplanted into female recipients. We show that suppression of p110δ activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment by impairing the localization of antigen-specific T cells to the grafts, while not inducing specific T cell tolerance. p110δ pharmacologic inactivation is effective when initiated after transplantation. Targeting p110δ activity might be a viable strategy for the treatment of heart chronic rejection in humans.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/genetics , Graft Rejection/genetics , Graft Rejection/prevention & control , Heart Transplantation/adverse effects , Phosphoinositide-3 Kinase Inhibitors , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Cells, Cultured , Chronic Disease , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Female , Flow Cytometry , Graft Rejection/etiology , H-Y Antigen/metabolism , Heart Transplantation/methods , Humans , Immune Tolerance/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Quinazolines/pharmacology , Skin Transplantation/adverse effects , Skin Transplantation/methods , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Transplantation, HomologousABSTRACT
Although polyclonal regulatory T cells (Tregs) that once expressed Foxp3 (ex-Tregs) derived from Foxp3(+) Tregs have been described in homeostatic and autoimmune settings, little is known regarding the influence of the tumor environment on ex-Treg development. After adoptive transfer of HY-specific green Tregs (peripheral or thymic) to Rag2(-/-) B6 female mice bearing syngeneic HY-expressing MB49 tumors, a significant fraction rapidly lost expression of Foxp3. On the second transfer to a Rag2(-/-) B6 male environment, these ex-Tregs expanded strongly, whereas Tregs that maintained expression of Foxp3 expression did not. Both FACS and quantitative real-time-PCR analysis revealed that ex-Tregs upregulated genes characteristic of a Th1 effector-memory phenotype including IFN-γ and downregulated a panel of Treg-specific genes. Peripheral HY-specific green Tregs were adoptively transferred to Rag2(-/-) B6 male mice, to dissect the factors regulating ex-Treg differentiation. Development of ex-Tregs was more efficient in the mesenteric lymph node (mLN) than peripheral lymph node environment, correlating with a much greater level of IL-6 mRNA in mLN. In addition, the preferential development of ex-Tregs in mLN was significantly impaired by cotransfer of HY-specific naive CD4 T cells. Collectively, our study not only demonstrates the plasticity of Ag-specific Tregs in the context of the tumor environment, but also defines key molecular and cellular events that modulate ex-Treg differentiation.
Subject(s)
Antigens/immunology , Forkhead Transcription Factors/immunology , Neoplasms, Experimental/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Antigens/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation/immunology , Cell Line, Tumor , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Female , Flow Cytometry , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Profiling , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , H-Y Antigen/immunology , H-Y Antigen/metabolism , Interleukin-2/genetics , Interleukin-2/metabolism , Male , Mice , Mice, Knockout , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes, Regulatory/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: Estrogen as well as CD4(+)Foxp3(+) regulatory T cells were shown to have a protective role not only in maintaining maternal-fetal tolerance but also against autoimmune diseases. We aimed to investigate whether the pregnancy levels of estrogen are enough to induce transplant tolerance as to maintain fetal-maternal tolerance. METHODS: We established H-Y skin graft transplantation in C57BL/6 ovariectomized mice that reconstituted with estrogen. Subsequently, consecutive daily estrogen injection was administrated. Tregs and the cytokines in the peripheral blood were detected by flow cytometry and ELISA pre- and post-transplant. RESULTS: The results indicated that pregnancy levels of estrogen could promote Tregs in secondary lymphoid organs and peripheral blood (P < 0.05) but not thymus (P > 0.05). The estrogen-treated recipients accepted H-Y skin grafts for more than 35 days (median survival time (MST): (44.0 ± 1.2) days) compared with estrogen-untreated mice (MST: (23.0 ± 1.6) days) (P < 0.05). It was also observed that estrogen up-regulated the expression of Foxp3, but did not affect CD3(+)CD8(+) effector T-cells in non-transplant mice. While in the presence of H-Y antigens, the expression of Foxp3 was more significant and CD3(+)CD8(+) effector T cells were decreased significantly (P < 0.05). Meanwhile, the up-regulated IL-10 and IL-4, and down-regulated IFN-γ could be observed (P < 0.05). CONCLUSIONS: Pregnancy levels of estrogen may promote the conversion of peripheral Tregs in secondary lymphoid organs, but show no effect on the natural Tregs production, differentiation and maturity in central lymphoid organs. Furthermore, pregnancy levels of estrogen could significantly prolong the survivals of H-Y skin grafts by the expansion of Tregs, suppression of CD3(+)CD8(+) effector T-cells and immune shift towards Th2 cytokines.
Subject(s)
Cytokines/metabolism , Graft Survival/drug effects , H-Y Antigen/immunology , Skin Transplantation/immunology , T-Lymphocyte Subsets/immunology , Animals , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Forkhead Transcription Factors/metabolism , H-Y Antigen/metabolism , Immunohistochemistry , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-4/metabolism , Mice , Mice, Inbred C57BL , Ovariectomy , PregnancyABSTRACT
We have dissected the role of the primary cytotoxic pathways Fas-FasL and perforin-granzyme in host-versus-graft reactions after allogeneic bone marrow transplantation. Sublethally irradiated female recipient mice deficient for FasL (B6.gld) or perforin (B6.prf-/-) were transplanted with bone marrow from B6 male donors. Donor engraftment in B6.prf-/- recipients was higher when compared with B6.gld, particularly when assessed by in vivo killing, demonstrating the importance of the perforin pathway over the Fas-FasL pathway. In the absence of both pathways, however, donor bone marrow engraftment was not fully restored identifying a role for an additional pathway(s) in the host-versus-graft response.
Subject(s)
Bone Marrow Transplantation/immunology , Bone Marrow Transplantation/physiology , Fas Ligand Protein/metabolism , Host vs Graft Reaction/physiology , Perforin/metabolism , Signal Transduction/physiology , fas Receptor/metabolism , Animals , Cytotoxicity, Immunologic , Fas Ligand Protein/genetics , Female , Gene Expression Regulation/radiation effects , H-Y Antigen/metabolism , Hematopoietic Stem Cell Transplantation , Host vs Graft Reaction/genetics , Male , Mice , Mice, Inbred C57BL , Perforin/genetics , Signal Transduction/genetics , Spleen/cytology , Spleen/transplantation , fas Receptor/geneticsABSTRACT
The Ag-specific CD4(+) regulatory T (Tr) cells play an important role in immune suppression in autoimmune diseases and antitumor immunity. However, the molecular mechanism for Ag-specificity acquisition of adoptive CD4(+) Tr cells is unclear. In this study, we generated IL-10- and IFN-gamma-expressing type 1 CD4(+) Tr (Tr1) cells by stimulation of transgenic OT II mouse-derived naive CD4(+) T cells with IL-10-expressing adenovirus (AdV(IL-10))-transfected and OVA-pulsed dendritic cells (DC(OVA/IL-10)). We demonstrated that both in vitro and in vivo DC(OVA/IL-10)-stimulated CD4(+) Tr1 cells acquired OVA peptide MHC class (pMHC) I which targets CD4(+) Tr1 cells suppressive effect via an IL-10-mediated mechanism onto CD8(+) T cells, leading to an enhanced suppression of DC(OVA)-induced CD8(+) T cell responses and antitumor immunity against OVA-expressing murine B16 melanoma cells by approximately 700% relative to analogous CD4(+) Tr1 cells without acquired pMHC I. Interestingly, the nonspecific CD4(+)25(+) Tr cells can also become OVA Ag specific and more immunosuppressive in inhibition of OVA-specific CD8(+) T cell responses and antitumor immunity after uptake of DC(OVA)-released exosomal pMHC I complexes. Taken together, the Ag-specificity acquisition of CD4(+) Tr cells via acquiring DC's pMHC I may be an important mean in augmenting CD4(+) Tr cell suppression.
Subject(s)
Adoptive Transfer , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , H-2 Antigens/metabolism , H-Y Antigen/immunology , Models, Immunological , Peptides/immunology , T-Lymphocytes, Regulatory/transplantation , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation/immunology , Cross-Priming/immunology , Dendritic Cells/immunology , Female , Graft vs Host Disease/immunology , H-2 Antigens/immunology , H-Y Antigen/administration & dosage , H-Y Antigen/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Minor Histocompatibility Antigens , Peptides/administration & dosage , Peptides/metabolism , Proteins/immunology , Proteins/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Time FactorsABSTRACT
During thymocyte development, the T-cell receptor (TCR) can discriminate major histocompatibility complex (MHC)/peptide ligands over a narrow range of affinities and translate subtle differences into functional fate decisions. How small differences in TCR input are translated into absolute differences in functional output is unclear. We examined the effects of galectin-1 ablation in the context of class-I-restricted thymocyte development. Galectin-1 expression opposed TCR partial agonist-driven positive selection, but promoted TCR agonist-driven negative selection of conventional CD8(+) T cells. Galectin-1 expression also promoted TCR agonist-driven CD8alphaalpha intestinal intraepithelial lymphocytes (IEL) development. Recombinant galectin-1 enhanced TCR binding to agonist/MHC complexes and promoted a negative-selection-signaling signature, reflected in intensified rapid and transient extracellular signal-regulated kinase (ERK) activation. In contrast, galectin-1 expression antagonized ERK activity in thymocytes undergoing positive selection. We propose that galectin-1 aids in discriminating TCR-directed fate decisions by promoting TCR binding to agonist/MHC complexes and enforcing agonist-driven signals, while opposing partial-agonist signals. In this way, galectin-1 widens the distinction between TCR-directed functional fate cues.
Subject(s)
Galectin 1/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Base Sequence , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation , DNA Primers/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Galectin 1/genetics , Galectin 1/metabolism , Galectin 1/pharmacology , Gene Expression , H-Y Antigen/genetics , H-Y Antigen/metabolism , Histocompatibility Antigens Class I/metabolism , MAP Kinase Signaling System , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Recombinant Proteins/pharmacology , T-Lymphocytes/cytologyABSTRACT
The death or survival of double positive (DP) thymocytes is determined by the strength of their TCR signaling. Of the three Runx family proteins, the DP cells only express the Runx1 transcription factor. We introduced and expressed in murine thymocytes the Runt domain of Runx1, which antagonizes the activity of endogenous Runx1. The Runt transgenic DP thymocytes expressed higher levels of the proapoptotic molecules Fas and Bim compared with the wild-type cells. Furthermore, the Runt transgenic cells were more susceptible to apoptosis induced by the artificial cross-linking of the TCR by the anti-CD3 Ab. This susceptibility was partially abrogated by the lpr/lpr background. In addition, Runx1:HY-TCR-double transgenic DP thymocytes were resistant to the apoptosis induced by the endogenously presented HY Ag. We propose that Runx1 functions to suppress the apoptotic sensitivity of DP thymocytes in the context of TCR signaling.
Subject(s)
Apoptosis Regulatory Proteins/biosynthesis , Apoptosis/immunology , CD4 Antigens/biosynthesis , CD8 Antigens/biosynthesis , Membrane Proteins/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Receptors, Tumor Necrosis Factor/biosynthesis , Thymus Gland/metabolism , Up-Regulation/immunology , Animals , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , H-Y Antigen/metabolism , Humans , Membrane Proteins/genetics , Mice , Mice, Transgenic , Protein Structure, Tertiary/genetics , Proto-Oncogene Proteins/genetics , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/physiology , Receptors, Tumor Necrosis Factor/genetics , Signal Transduction/immunology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism , Thymus Gland/cytology , Thymus Gland/immunology , fas ReceptorABSTRACT
In vivo studies suggest that vascular endothelial cells (ECs) can acquire and cross-present exogenous Ag on MHC-I but the cellular mechanisms underlying this observation remain unknown. We tested whether primary female mouse aortic ECs could cross-present exogenous male Ag to the T cell hybridoma, MHH, specific for HYUty plus D(b). MHC-I-deficient male spleen cells provided a source of male Ag that could not directly stimulate the MHH cells. Addition of male but not female MHC-I-deficient spleen cells to wild-type syngeneic female EC induced MHH stimulation, demonstrating EC cross-presentation. Lactacystin treatment of the donor male MHC-I-deficient spleen cells, to inhibit proteasome function, markedly enhanced EC cross-presentation showing that the process is most efficient for intact proteins rather than degraded peptide fragments. Additional experiments revealed that this EC Ag-processing pathway is both proteasome and TAP1 dependent. These studies demonstrate that cultured murine aortic ECs can process and present MHC-I-restricted Ag derived from a separate, live cell, and they offer insight into the molecular requirements involved in this EC Ag presentation process. Through this pathway, ECs expressing cross-presented peptides can participate in the effector phase of T cell-mediated inflammatory responses such as autoimmunity, anti-tumor immunity, and transplant rejection.
Subject(s)
ATP-Binding Cassette Transporters/physiology , Cross-Priming/immunology , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , H-Y Antigen/metabolism , Histocompatibility Antigens Class I/metabolism , Proteasome Endopeptidase Complex/physiology , Signal Transduction/immunology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , Animals , Antigen Presentation , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Female , H-Y Antigen/genetics , H-Y Antigen/immunology , Histocompatibility Antigens Class I/immunology , Hybridomas , Intracellular Fluid/immunology , Intracellular Fluid/metabolism , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Minor Histocompatibility Antigens , Proteins/genetics , Proteins/immunologyABSTRACT
A characteristic of the secondary response of CD8(+) T cells that distinguishes it from the primary response is the generation of greater numbers of effector cells. Because effector CD8(+) T cells are derived from a pool of less differentiated, replicating cells in secondary lymphoid organs, and because IL-2 mediates effector differentiation, the enhanced secondary response may reflect the enlargement of this generative pool by the transient repression of IL-2-mediated differentiation. We have examined for this function the transcriptional repressor BCL6b, a homologue of BCL6 that represses IL-2-induced B cell differentiation. BCL6b is expressed in a small subset of antigen-experienced CD8(+) T cells. Ectopic expression of BCL6b in CD8(+) T cells diminishes their growth in response to IL-2 in vitro. Female mice in which the BCL6b gene has been interrupted have normal primary responses of CD8(+) T cells to infection with vaccinia expressing the H-Y epitope, Uty, but Uty-specific, BCL6b(-/-), memory CD8(+) T cells have diminished recall proliferative responses to this epitope in vitro. BCL6b(-/-) mice also have normal primary CD8(+) T cell responses to influenza infection, but nucleoprotein peptide-specific, BCL6b(-/-), memory CD8(+) T cells have a cell autonomous defect in the number of effector cells generated in response to reinfection. Therefore, BCL6b is required for the enhanced magnitude of the secondary response of memory CD8(+) T cells.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Immunologic Memory , Repressor Proteins/metabolism , Silencer Elements, Transcriptional/immunology , Animals , CD8-Positive T-Lymphocytes/metabolism , DNA Primers , Epitopes/metabolism , Female , Gene Components , Gene Targeting , Genetic Vectors , H-Y Antigen/metabolism , Humans , Interferon-gamma/metabolism , Interleukin-2/immunology , Interleukin-2/metabolism , Mice , Mice, Knockout , Moloney murine leukemia virus , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Vaccinia/immunologyABSTRACT
In this study, we suggest that CD8 levels on T cells are not static, but can change and, as a result, modulate CD8(+) T cell responses. We describe three models of CD8 modulation using novel weak-agonist (K1A) and super-agonist (C2A) altered peptide ligands of the HY smcy peptide. First, we used peripheral nonresponsive CD8(low) T cells produced after peripheral HY-D(b) MHC class I tetramer stimulation of female HY TCR transgenic and wild-type mice. Second, we used genetically lowered CD8(int) T cells from heterozygote CD8(+/0) mice. Finally, we used pre-existing nonresponsive CD8(low) T cells from male HY TCR transgenic mice. In CD8(low) and CD8(high) mice, presence of a lower level of CD8 greatly decreased the avidity of the peptide-MHC for HY TCR as reflected by avidity (K(D)) and dissociation constant (T(1/2)) measurements. All three models demonstrated that lowering CD8 levels resulted in the requirement for a higher avidity peptide-MHC interaction with the TCR to respond equivalently to unmanipulated CD8(high) T cells of the same specificity. Additionally, direct injections of wild-type HY-D(b) and C2A-D(b) tetramers into female HY TCR or female B6 mice induced a high frequency of peripheral nonresponsive CD8(low) T cells, yet C2A-D(b) was superior in inducing a primed CD8(+)CD44(+) memory population. The ability to dynamically modulate the size and responsiveness of an Ag-specific T cell pool by "CD8 tuning" of the T cell during the early phases of an immune response has important implications for the balance of responsiveness, memory, and tolerance.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation/immunology , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , Animals , CD8 Antigens/biosynthesis , CD8-Positive T-Lymphocytes/cytology , Cytotoxicity, Immunologic/genetics , Down-Regulation/genetics , Down-Regulation/immunology , Epitopes, T-Lymphocyte/administration & dosage , Female , H-Y Antigen/administration & dosage , H-Y Antigen/immunology , H-Y Antigen/metabolism , Ligands , Lymphocyte Activation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptides/administration & dosage , Peptides/immunology , Peptides/metabolism , Protein Binding/genetics , Protein Binding/immunology , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Sex Characteristics , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Transplantation Tolerance/geneticsABSTRACT
Female-to-male hemopoietic stem cell transplantation (HSCT) elicits T cell responses against male-specific minor histocompatibility (H-Y) Ags encoded by the Y chromosome. All previously identified H-Y Ags are encoded by conventional open reading frames, but we report in this study the identification of a novel H-Y Ag encoded in the 5'-untranslated region of the TMSB4Y gene. An HLA-A*3303-restricted CD8(+) CTL clone was isolated from a male patient after an HSCT from his HLA-identical sister. Using a panel of cell lines carrying Y chromosome terminal deletions, a narrow region controlling the susceptibility of these target cells to CTL recognition was localized. Minigene transfection and epitope reconstitution assays identified an 11-mer peptide, EVLLRPGLHFR, designated TMSB4Y/A33, whose first amino acid was located 405 bp upstream of the TMSB4Y initiation codon. Analysis of the precursor frequency of CTL specific for recipient minor histocompatibility Ags in post-HSCT peripheral blood T cells revealed that a significant fraction of the total donor CTL response in this patient was directed against the TMSB4Y epitope. Tetramer analysis continued to detect TMSB4Y/A33-specific CD8(+) T cells at least up to 700 days post-HSCT. This finding underscores the in vivo immunological relevance of minor histocompatibility Ags derived from unconventional open reading frame products.
Subject(s)
HLA-A Antigens/genetics , Minor Histocompatibility Antigens/genetics , Open Reading Frames/immunology , Thymosin/genetics , Thymosin/immunology , 5' Untranslated Regions/chemistry , 5' Untranslated Regions/genetics , 5' Untranslated Regions/immunology , Amino Acid Sequence , Antigen Presentation/immunology , Base Sequence , Cell Line , Cell Line, Transformed , Chromosome Mapping , Chromosomes, Human, Y , Clone Cells , Cytotoxicity Tests, Immunologic , Female , H-Y Antigen/immunology , H-Y Antigen/metabolism , HLA-A Antigens/immunology , HLA-A Antigens/metabolism , Hematopoietic Stem Cell Transplantation , Humans , Male , Middle Aged , Minor Histocompatibility Antigens/immunology , Minor Histocompatibility Antigens/metabolism , Molecular Sequence Data , Organ Specificity/genetics , Organ Specificity/immunology , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/immunology , RNA, Messenger/biosynthesis , Sequence Deletion/immunology , T-Lymphocytes, Cytotoxic/chemistry , T-Lymphocytes, Cytotoxic/immunology , Thymosin/biosynthesisABSTRACT
The physiologic significance of MHC-peptide complex presentation by endothelial cells (ECs) to trafficking T lymphocytes remains unresolved. On the basis of our observation that cognate recognition of ECs enhanced transendothelial migration of antigen-specific T lymphocytes in vitro, we have proposed that by displaying antigenic peptides from the underlying tissue, ECs promote the recruitment of antigen-specific T cells. In this study, we have tested this hypothesis by comparing the trafficking of HY-specific T lymphocytes into antigenic and nonantigenic tissue using in vivo models of T-cell recruitment. Up-regulated expression of H2 molecules presenting endogenous antigen in the peritoneal mesothelium and vessels led to the local recruitment of HY-specific T cells in male, but not female, mice. Intravital microscopy experiments analyzing EC-HY-specific T-cell interactions in the cremasteric vascular bed revealed that cognate recognition of the endothelium results in enhanced diapedesis of T cells into the tissue, while not affecting rolling and adhesion. Our results are consistent with the hypothesis that, under inflammatory conditions, antigen presentation by the endothelium contributes to the development and specificity of T-cell-mediated inflammation by favoring the selective migration of antigen-specific T cells.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/physiology , Endothelium, Vascular/immunology , H-Y Antigen/metabolism , Animals , Cell Movement , Cells, Cultured , Female , In Vitro Techniques , Inflammation/immunology , Inflammation/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Receptors, Antigen, T-Cell/metabolismABSTRACT
To evaluate the priming and trafficking of male Ag-reactive CD4(+) T cells in vivo, we developed an adoptive transfer model, using Marilyn (Mar) TCR transgenic T cells that are specific for the H-Y minor transplantation Ag plus I-A(b). By manipulating donor and recipient strain combinations, we permitted the Mar CD4(+) T cells to respond to the H-Y Ag after processing and presentation by recipient APCs (indirect pathway), or to the male Ag as expressed on donor APCs (direct pathway). Mar CD4(+) T cells responding through the indirect pathway specifically proliferated and expressed activation markers between days 2 and 4 posttransplant, migrated to the graft 2-3 days before cessation of graft heartbeat, and were detected in close proximity to transplant-infiltrating recipient APCs. Intriguingly, adoptively transferred Mar T cells did not respond to male heart or skin grafts placed onto syngeneic MHC class II-deficient female recipients, demonstrating that activation of Mar T cell preferentially occurs through cognate interactions with processed male Ag expressed on recipient APCs. The data highlight the potency of indirect processing and presentation pathways in vivo and underscore the importance of indirectly primed CD4(+) T cells as relevant participants in both the priming and effector phases of acute graft rejection.
Subject(s)
Antigen Presentation/immunology , CD4-Positive T-Lymphocytes/immunology , H-Y Antigen/physiology , Lymphocyte Activation/immunology , Signal Transduction/immunology , Amino Acid Sequence , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , Cell Movement/genetics , Cell Movement/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , Female , H-Y Antigen/genetics , H-Y Antigen/immunology , H-Y Antigen/metabolism , Heart Transplantation/immunology , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/immunology , Peptide Fragments/metabolism , Signal Transduction/geneticsABSTRACT
Negative selection serves as a major mechanism to maintain self-tolerance. We previously reported that LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor), a TNF family member, plays an important role in thymocyte development via promoting apoptosis of double-positive thymocytes. Here, we demonstrated that LIGHT-mediated deletion of thymocyte requires the strong interaction of TCR with MHC/self-peptide. Transgenic mice overexpressing LIGHT in thymocytes were bred with a transgenic mouse line expressing a TCR recognizing the H-Y male Ag in the context of H-2b class I MHC molecules. In male H-Y/LIGHT double-transgenic mice, more efficient negative selection of H-Y T cells occurred, and total thymocyte number was further reduced compared with H-Y/negative littermates. In contrast, the presence of LIGHT transgene had no evident impact on the thymocyte development of female H-Y/LIGHT double-transgenic mice. Taken together, LIGHT plays a role in negative selection of thymocytes via inducing the apoptosis of thymocytes bearing high affinity TCR during negative selection.
Subject(s)
Autoantigens/metabolism , Clonal Deletion/immunology , H-Y Antigen/metabolism , Membrane Proteins/physiology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocyte Subsets/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Tumor Necrosis Factor-alpha/physiology , Animals , Apoptosis/genetics , Apoptosis/immunology , Autoantigens/biosynthesis , Autoantigens/genetics , Cell Differentiation/genetics , Cell Differentiation/immunology , Epitopes, T-Lymphocyte/metabolism , Female , H-Y Antigen/biosynthesis , H-Y Antigen/genetics , Ligands , Lymphotoxin beta Receptor , Male , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor/physiology , Receptors, Tumor Necrosis Factor, Member 14 , Receptors, Virus/metabolism , Sex Characteristics , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism , Thymus Gland/metabolism , Tumor Necrosis Factor Ligand Superfamily Member 14 , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y-specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y-specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.
Subject(s)
CD28 Antigens/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Immune Tolerance , Animals , CD8-Positive T-Lymphocytes/cytology , Cell Division , Female , Flow Cytometry , H-Y Antigen/immunology , H-Y Antigen/metabolism , Interferon-gamma/immunology , Interferon-gamma/metabolism , Lymphocyte Activation , Male , Mice , Mice, Knockout , Mice, Transgenic , Pregnancy , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
Minor histocompatibility antigens (mHags) are immunogenic peptides from polymorphic cellular proteins that induce strong T-cell responses after human leukocyte antigen (HLA)-matched, mHag-mismatched stem-cell transplantation. mHags with broad or limited tissue expression are target antigens for graft-versus-host (GvH) and graft-versus-leukemia (GvL) reactivities. Separation of these activities is crucial for adoptive immunotherapy of leukemia without GvH disease. Therefore, using a skin-explant assay we investigated the in situ activities of cytotoxic T lymphocytes (CTLs) specific for the ubiquitously expressed mHag H-Y and for the hematopoietic-restricted mHags HA-1 and HA-2. H-Y-specific CTLs, visualized by tetrameric HLA-mHag peptide complexes, infiltrated male skin sections within 24 hours, induced severe GvH reactions of grade III-IV and produced high levels of IFN-gamma. In contrast, CTLs specific for the hematopoietic system-specific mHags HA-1 and HA-2 induced no or low GvH reactions above background and produced little or no interferon-gamma, unless the skin sections were preincubated with HA-1/HA-2 synthetic peptides. These results provide the first in situ dissection of GvH effects by mHag-specific CTLs and show that ubiquitously expressed mHags are the prime targets of GvH disease.
Subject(s)
Graft vs Host Reaction/immunology , Minor Histocompatibility Antigens/metabolism , T-Lymphocytes, Cytotoxic/immunology , Graft vs Host Disease/etiology , Graft vs Host Disease/immunology , Graft vs Host Disease/pathology , H-Y Antigen/metabolism , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , In Vitro Techniques , Neoplasm Proteins/metabolism , Oligopeptides/metabolism , Skin/immunology , Skin/pathology , T-Lymphocytes, Cytotoxic/pathologyABSTRACT
The development of TCR alphabeta(+), CD8alphabeta(+) intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8alphaalpha(+) IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8(+) T cells in RAG 2(-/-) mice expressing the H-2D(b)-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D(b)) and nonrestricting (H-2D(d)) MHC class I alleles (MHC F(1)) were composed of a mixture of CD8alphabeta(+) and CD8alphaalpha(+) T cells, while T cells in the spleen were mostly CD8alphabeta(+). This was unlike IEL in male mice homozygous for H-2D(b), which had predominantly CD8alphaalpha(+) IEL and few mostly CD8(-) T cells in the spleen. Our results demonstrate that deletion of CD8alphabeta(+) cells in H-Y TCR male mice is dependent on two copies of H-2D(b), whereas the generation of CD8alphaalpha(+) IEL requires only one copy. The existence of CD8alphabeta(+) and CD8alphaalpha(+) IEL in MHC F(1) mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8alphabeta(+) T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8alphaalpha(+) IEL is dependent on, but less sensitive to, this MHC class I allele.