Sharon Germana - Academia.edu (original) (raw)

Papers by Sharon Germana

Cell Reports, Oct 1, 2018

CD4 + Foxp3 + regulatory T cells (Tregs) are unique in that the majority of Tregs constitutively ... more CD4 + Foxp3 + regulatory T cells (Tregs) are unique in that the majority of Tregs constitutively express high levels of CD25, the main ligand binding subunit of the IL-2 receptor. Germline knockout models demonstrate that IL-2 is needed during Treg development to induce Foxp3, but the precise roles of IL-2 after Treg development are incompletely understood. We therefore generated mice in which CD25 can be inducibly deleted from Tregs after thymic development. In contrast to Treg development, we find that IL-2 is dispensable for maintaining lineage stability in mature Tregs. Continuous IL-2 signaling was needed for long term Treg

Immunogenetics, 1990

II. Molecular identification and characterization of B (/3) genes from the S L A c haplotype.

Journal of Immunology, May 1, 2013

T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regul... more T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regulatory T cells (Tregs) that mature in the medullar compartment. We have achieved Treg-dependent tolerance of fully allogeneic transplants via the transfer of donor MHC class II genes (MHCII) that induced newly made Tregs specific of donor MHCII peptides. Given that gene therapy for tolerance was effective with MHCII but not MHCI genes, the goal of the study was to identify natural MHCII peptides able to impact Treg development and function. Using experimental models that either recapitulated MHCII peptide presentation - the IEα 52-68 peptide presented on I-Ab molecules - or promoted Treg-mediated tolerance to heart grafts via MHCII gene transfer, we confirmed that high amounts of IEα peptide were presented by MHCII from most if not all I-Ab+ CD11c+ dendritic cells of the thymic medulla, suggesting its role in Treg maturation. This hypothesis was confirmed by demonstrating that the introduction of the IEα peptide through hematopoietic chimerism converted host IEα-specific CD4 T cells into CD25hiFoxp3+ suppressive Tregs. Treg suppression for tolerance to transplants required prior activation by cognate pMHCII complexes as MHCII-treated recipients rejected MHCII-deficient while accepting MHCII-sufficient grafts. These data are consistent with a prominent role of “MHCII self-presentation” in shaping thymic Treg TCR specificities and controlling Treg function in the periphery.

Immunogenetics, Jul 1, 1988

II. Molecular identification and characterization of B (/3) genes from the S L A c haplotype.

Scandinavian Journal of Immunology, 1990

The Journal of Immunology

T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regul... more T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regulatory T cells (Tregs) that mature in the medullar compartment. We have achieved Treg-dependent tolerance of fully allogeneic transplants via the transfer of donor MHC class II genes (MHCII) that induced newly made Tregs specific of donor MHCII peptides. Given that gene therapy for tolerance was effective with MHCII but not MHCI genes, the goal of the study was to identify natural MHCII peptides able to impact Treg development and function. Using experimental models that either recapitulated MHCII peptide presentation - the IEα 52-68 peptide presented on I-Ab molecules - or promoted Treg-mediated tolerance to heart grafts via MHCII gene transfer, we confirmed that high amounts of IEα peptide were presented by MHCII from most if not all I-Ab+ CD11c+ dendritic cells of the thymic medulla, suggesting its role in Treg maturation. This hypothesis was confirmed by demonstrating that the introdu...

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2014

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applie... more Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable c...

American Journal of Transplantation, 2012

Antibody rejection is often accompanied by non-donor HLA specific antibodies (NDSA) and selfreact... more Antibody rejection is often accompanied by non-donor HLA specific antibodies (NDSA) and selfreactive antibodies that develop alongside donor-specific antibodies (DSA). To determine the source of these antibodies, we immortalized 107 B cell clones from a kidney transplant recipient with humoral rejection. Two of these clones reacted to HLA class I or MICA. Both clones were also reactive to self antigens and a lysate of a kidney cell line, hence revealing a pattern of polyreactivity. Monoclonality was verified by the identification of a single rearranged immunoglobulin heavy chain variable region (VH) sequence for each clone. By tracking their unique CDR3 sequence, we found that one such polyreactive clone was highly expanded in the patient blood, representing ~0.2% of circulating B cells. The VH sequence of this clone showed evidence of somatic mutations that were consistent with its memory phenotype and its expansion. Lastly, the reactivity of the expanded polyreactive B cell clone was found in the patient serum at time of rejection. In conclusion, we provide here proof of principle at the clonal level that human antibodies can cross-react to HLA and self. Our findings strongly suggest that polyreactive antibodies contribute to DSA, NDSA as well as autoantibodies, in transplant recipients.

The Journal of Immunology

Besides their ability to present antigenic peptides and stimulate antigen-specific T lymphocytes,... more Besides their ability to present antigenic peptides and stimulate antigen-specific T lymphocytes, MHC class II (MHCII) molecules have also been implicated in the regulation of ensuing T cell responses. However, the substance of MHCII regulation remains elusive to this day. Defining the variables of this phenomenon appears to be critical for developing rational therapeutic approaches. Using models in which MHCII regulation has been demonstrated, we have revisited the concept by evaluating the impact of MHCII-restricted CD4 regulatory T cells (Tregs) on T cell reactivity to either allogeneic or self-antigens. Studies performed in vitro and in vivo showed that graft host Tregs specific to donor MHCII peptides / host MHCII complexes promoted T cell tolerance of fully allogeneic grafts. These pMHCII complexes - hereafter called TLo - were also involved in Treg-mediated suppression and in initial host Treg activation by graft MHCII. Extending these observations to Treg biology of normal a...

PLOS ONE, 2019

S5 Fig. The effect of 2DG treatment at day 3 on functional molecules. 0.5mM 2-deoxy-Dglucose (2DG... more S5 Fig. The effect of 2DG treatment at day 3 on functional molecules. 0.5mM 2-deoxy-Dglucose (2DG) was added from 3 to 7 days post activation in tTreg (red) and iTreg (blue). Flow analyses for CD25, CTLA-4 and ICOS were performed gated on live CD4+ cells. Data are shown as non-treated control and 2DG treated as solid and dot lines in representative histograms with isotype control staining (filled gray). Solid (non-treated, Cont) and checked (2DG treated, 2DG) boxes with bar graph indicates the mean fluorescence intensity (MFI) ±s.d.. Representative data is shown from at least 3 independent experiments of total 6 individual donors. Statistical analyses were performed by Student's t-test.

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1992

A polymorphism of CD4 in miniature swine has been identified by failure of cells from some animal... more A polymorphism of CD4 in miniature swine has been identified by failure of cells from some animals to react with mAb 74-12-4. The phenotypic, molecular genetic, and functional characteristics of these animals have been defined. Cells from heterozygous animals bear approximately 50% the number of 74-12-4-reactive molecules on their surface as do cells from animals homozygous for the wild type. Animals of both phenotypes demonstrate similar flow cytometric profiles for CD8+ T cells. Northern blot analysis confirms the presence of mRNA for CD4 among PBL of animals failing to stain with 74-12-4. CD4 allelism is confirmed by Southern blot analysis, revealing RFLP. Function of the CD4 subset in vivo, as demonstrated by antibody production against a T cell-dependent Ag, is similar between animals of both phenotypes. Proliferative responses to PHA and alloantigen stimulation by a full haplotype mismatch or a class II mismatch alone are equivalent for animals of both phenotypes. These data s...

Xenotransplantation, 2002

We recently established that molecular chimeras of major histocompatibility complex (MHC) class I... more We recently established that molecular chimeras of major histocompatibility complex (MHC) class II molecules, created via retroviral transfer of allogeneic class II cDNAs into bone marrow cells (BMCs), alleviated complications associated with mixed BMC chimeras while leading to T cell tolerance to renal grafts sharing the transferred class II. Initially demonstrated for allogeneic transplants in miniature swine, this concept was extended to T-dependent antibody (Ab) responses to xenogeneic antigens (Ags) in the pig ® baboon combination. Successful down-regulation of T cell responses appeared, however, to be contingent on a tight lineage-speci®c expression of transferred class II molecules. The present studies were, therefore, designed to evaluate the in¯uence of construct design and cellular environment on expression of retrovirally transferred xenogeneic class II cDNAs. Proviral genomes for pig class II SLA-DR expression, diering only at the marker neo(r) or enhanced green¯uorescent protein (EGFP) gene, showed increased membrane SLA-DR density on HLA-DR ± ®broblasts as well as HLA-DR + , TF-1 erythroleukemia cells. More importantly, HLA-DR + human B cell lines, although ef®ciently transduced with pig DR retroviruses, exhibited unstable surface pig DR. Surface pig DR ± B cells, nevertheless, stimulated autologous human T cells pre-sensitized to pig Ags, a proliferation likely occurring through presentation of class II-derived peptides. Collectively, these data suggest that surface expression of transferred class II molecules is not related to the ability of recipient cells to synthesize xenogeneic class II molecules but rather to their Ag processing capacities.

Transplantation, 1997

Transfer of MHC class II genes, through allogeneic bone marrow (BM) transplantation, induced long... more Transfer of MHC class II genes, through allogeneic bone marrow (BM) transplantation, induced long-lasting acceptance of renal allografts in miniature swine. To adapt this approach to the clinic, we have now examined whether somatic transfer of allogeneic class II DR genes, into otherwise autologous bone marrow cells (BMC), can provide the matching required for inducing immune tolerance. Autologous BMC were transduced ex vivo with recombinant retroviruses for allogeneic DRB followed by BM transplantation. The recipients were then challenged with kidney allografts solely matched to the DRB transgene. Five miniature swine received autologous BMC conditioned with growth factors and transduced with recombinant retrovirus vectors containing allogeneic (n=4) or syngeneic (n=1) class II DRB genes and a drug-resistance marker. Expression of retrovirus-derived products in BM-derived cells was demonstrated by the detection of drug-resistant colony-forming progenitors and the presence of DRB retrovirus transcripts in peripheral cells. Analysis of selective mixed lymphocyte reaction responses to DR or DQ antigens indicated decreased reactivity toward the transduced DR gene product. Among all of the animals receiving fully mismatched kidney allografts, but with DRB matched to the transduced DRB, the one with the highest gene transduction rate showed stable allograft function and essentially normal renal histology for 2.5 years. A control animal, which received a syngeneic DRB gene, rejected its kidney allograft in 120 days after an earlier rejection crisis. These studies demonstrate that allogeneic MHC gene transfer into BM provides a new strategy for inducing tolerance across MHC barriers.

Proceedings of the National Academy of Sciences, 1990

As part of our studies of the class II genes of miniature swine, we have isolated and characteriz... more As part of our studies of the class II genes of miniature swine, we have isolated and characterized cDNA clones corresponding to DRB genes from two major histocompatibility complex homozygous strains. Comparison of the sequences of these clones to those of human DRB genes revealed a striking amino acid homology between the hypervariable residues of SLA-DRBc and the human DRB1-0101 allele. The percentage of differences in these residues between the pig DRBc allele and the human DRB1-0101 allele was significantly lower (29%) than that between the DRB1-0101 allele and all other human alleles (average, 66.2%). This similarity was not seen in a comparison of the number of silent substitutions, by which the swine DRBc and the human DRB-0101 differed. Since phenotypic selection operates at the level of protein products rather than nucleotide sequences, these data suggest the existence of selective mechanisms that have resulted in similar hypervariable regions in certain alleles even in the...

Proceedings of the National Academy of Sciences, 1995

Clones encoding porcine interleukin 10 (IL-10) were isolated from a cDNA library produced from ph... more Clones encoding porcine interleukin 10 (IL-10) were isolated from a cDNA library produced from phytohemagglutinin-activated pig peripheral blood mononuclear cells. The porcine IL-10 nucleotide sequence was found to be highly homologous to the rat, mouse, and human IL-10 counterparts and to one of the open reading frames from the Epstein-Barr virus. In addition, pig IL-10 caused inhibition of gamma-interferon gene transcription as determined by a bioassay. To investigate the possible immunomodulatory role of IL-10, its expression during the induction of tolerance to kidney allografts by cyclosporin A in miniature swine was also investigated. Delayed expression and higher levels of IL-10 were observed in tolerant animals compared with animals rejecting their allografts. Since tolerance is achieved by a short course of cyclosporin A, we have also studied the in vitro effect of this drug on IL-10 gene transcription in blood mononuclear cells and have found that cyclosporin A inhibits IL...

Human Gene Therapy, 2000

Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine ... more Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine transplantation model by retrovirus-mediated transfer of allogeneic MHC class II genes into bone marrow cells (BMCs) of recipient animals. Graft survival correlated with transient expression of the somatic transgene (Tg) in the induction phase of tolerance. With the aim of investigating the effects of timing and threshold levels of Tg expression on induction of hyporesponsiveness to the grafted tissues, two recombinant retrovirus constructs containing the tetracycline binary regulatory system were used to achieve conditional expression of either the green fluorescent protein (tetGFP) as a control, or the porcine MHC class II DRbeta chain (tetDRB). Effective downregulation of GFP gene transcription was demonstrated in transduced murine fibroblasts after doxycycline treatment, leading to a > 90% reduction of GFP fluorescence. Similar diminution of the DRB gene transcription was achieved in transduced pig endothelial cells (ECs). Drug-dependent downregulation of DRBc gene expression in SLAd pig ECs coincided with complete inhibition of allogeneic activation of anti-class IIc-primed SLAd T cells. These in vitro results suggest that the binary tetracycline retrovirus system may also be adequate to regulate MHC class II Tg expression in vivo.

Human Gene Therapy, 1999

Transplantation tolerance to renal allografts can be induced in large animal preclinical models i... more Transplantation tolerance to renal allografts can be induced in large animal preclinical models if the donor and recipient have identical major histocompatibility complex (MHC) class II loci. Such class II matching is, however, not clinically achievable owing to the extreme diversity of class II sequences. With the ultimate goal of creating a somatic class II match in the bone marrow of an allograft recipient, the aim of the study is to develop a double-copy retrovirus construct to express both chains of the MHC class II DQ glycoprotein on a single transduced cell. Analysis of the expression patterns of the retroviral DQ transgenes in both virus producer and transduced fibroblasts revealed correct transcription and stable surface expression of the DQ heterodimers. In addition, we demonstrate that both the DQA and DQB sequences are functional within the same proviral copy, a prerequisite for efficient induction of transplantation tolerance following transduction of bone marrow precursor cells. The DQ double-copy retrovirus vector showed efficient expression of the transferred class II cDNA in murine colony-forming units for the granulocyte-monocyte lineage (CFU-GM), indicating that it is suitable for gene therapy of multimeric proteins in hematopoietic cells.

Biology of Blood and Marrow Transplantation, 2003

Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoieti... more Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoietic cell transplantation that leads to mixed chimerism and allograft tolerance in miniature swine across minor and major histocompatibility disparities. Stable chimerism persisted in most of these animals but was restricted to T cells and confined to peripheral blood. Because of the importance of myeloid and erythroid progenitors for the treatment of hematologic disorders, the objective of this study was to assess whether such cells existed in the bone marrow of these lymphoid chimeras as an indication of functional engraftment. Colony-formation assays were performed on donor inocula before infusion and on bone marrow cells harvested from the transplant recipients. Donor-origin myeloid/erythroid progenitor colonies were detected in bone marrow from 6 of 7 lymphoid chimeric recipients. A delayed donor leukocyte infusion successfully converted a stable lymphoid chimera to full multilineage chimerism within 2 weeks. Donor-origin myeloid/erythroid progenitors could be detected in the bone marrow of a host-matched recipient after myeloablation and adoptive transfer of mobilized cells from one of the engrafted lymphoid chimeras. These data suggest that even when only lymphoid chimerism is readily detected by flow cytometry, dormant myeloid/erythroid progenitors can exist and subsequent conversion to full donor chimerism can be achieved. The ability to establish multilineage engraftment and chimerism without significant toxicity may have important clinical implications for the management of nonmalignant hematopoietic disorders and hematologic malignancies.

Cell Reports, Oct 1, 2018

CD4 + Foxp3 + regulatory T cells (Tregs) are unique in that the majority of Tregs constitutively ... more CD4 + Foxp3 + regulatory T cells (Tregs) are unique in that the majority of Tregs constitutively express high levels of CD25, the main ligand binding subunit of the IL-2 receptor. Germline knockout models demonstrate that IL-2 is needed during Treg development to induce Foxp3, but the precise roles of IL-2 after Treg development are incompletely understood. We therefore generated mice in which CD25 can be inducibly deleted from Tregs after thymic development. In contrast to Treg development, we find that IL-2 is dispensable for maintaining lineage stability in mature Tregs. Continuous IL-2 signaling was needed for long term Treg

Immunogenetics, 1990

II. Molecular identification and characterization of B (/3) genes from the S L A c haplotype.

Journal of Immunology, May 1, 2013

T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regul... more T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regulatory T cells (Tregs) that mature in the medullar compartment. We have achieved Treg-dependent tolerance of fully allogeneic transplants via the transfer of donor MHC class II genes (MHCII) that induced newly made Tregs specific of donor MHCII peptides. Given that gene therapy for tolerance was effective with MHCII but not MHCI genes, the goal of the study was to identify natural MHCII peptides able to impact Treg development and function. Using experimental models that either recapitulated MHCII peptide presentation - the IEα 52-68 peptide presented on I-Ab molecules - or promoted Treg-mediated tolerance to heart grafts via MHCII gene transfer, we confirmed that high amounts of IEα peptide were presented by MHCII from most if not all I-Ab+ CD11c+ dendritic cells of the thymic medulla, suggesting its role in Treg maturation. This hypothesis was confirmed by demonstrating that the introduction of the IEα peptide through hematopoietic chimerism converted host IEα-specific CD4 T cells into CD25hiFoxp3+ suppressive Tregs. Treg suppression for tolerance to transplants required prior activation by cognate pMHCII complexes as MHCII-treated recipients rejected MHCII-deficient while accepting MHCII-sufficient grafts. These data are consistent with a prominent role of “MHCII self-presentation” in shaping thymic Treg TCR specificities and controlling Treg function in the periphery.

Immunogenetics, Jul 1, 1988

II. Molecular identification and characterization of B (/3) genes from the S L A c haplotype.

Scandinavian Journal of Immunology, 1990

The Journal of Immunology

T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regul... more T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regulatory T cells (Tregs) that mature in the medullar compartment. We have achieved Treg-dependent tolerance of fully allogeneic transplants via the transfer of donor MHC class II genes (MHCII) that induced newly made Tregs specific of donor MHCII peptides. Given that gene therapy for tolerance was effective with MHCII but not MHCI genes, the goal of the study was to identify natural MHCII peptides able to impact Treg development and function. Using experimental models that either recapitulated MHCII peptide presentation - the IEα 52-68 peptide presented on I-Ab molecules - or promoted Treg-mediated tolerance to heart grafts via MHCII gene transfer, we confirmed that high amounts of IEα peptide were presented by MHCII from most if not all I-Ab+ CD11c+ dendritic cells of the thymic medulla, suggesting its role in Treg maturation. This hypothesis was confirmed by demonstrating that the introdu...

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2014

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applie... more Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable c...

American Journal of Transplantation, 2012

Antibody rejection is often accompanied by non-donor HLA specific antibodies (NDSA) and selfreact... more Antibody rejection is often accompanied by non-donor HLA specific antibodies (NDSA) and selfreactive antibodies that develop alongside donor-specific antibodies (DSA). To determine the source of these antibodies, we immortalized 107 B cell clones from a kidney transplant recipient with humoral rejection. Two of these clones reacted to HLA class I or MICA. Both clones were also reactive to self antigens and a lysate of a kidney cell line, hence revealing a pattern of polyreactivity. Monoclonality was verified by the identification of a single rearranged immunoglobulin heavy chain variable region (VH) sequence for each clone. By tracking their unique CDR3 sequence, we found that one such polyreactive clone was highly expanded in the patient blood, representing ~0.2% of circulating B cells. The VH sequence of this clone showed evidence of somatic mutations that were consistent with its memory phenotype and its expansion. Lastly, the reactivity of the expanded polyreactive B cell clone was found in the patient serum at time of rejection. In conclusion, we provide here proof of principle at the clonal level that human antibodies can cross-react to HLA and self. Our findings strongly suggest that polyreactive antibodies contribute to DSA, NDSA as well as autoantibodies, in transplant recipients.

The Journal of Immunology

Besides their ability to present antigenic peptides and stimulate antigen-specific T lymphocytes,... more Besides their ability to present antigenic peptides and stimulate antigen-specific T lymphocytes, MHC class II (MHCII) molecules have also been implicated in the regulation of ensuing T cell responses. However, the substance of MHCII regulation remains elusive to this day. Defining the variables of this phenomenon appears to be critical for developing rational therapeutic approaches. Using models in which MHCII regulation has been demonstrated, we have revisited the concept by evaluating the impact of MHCII-restricted CD4 regulatory T cells (Tregs) on T cell reactivity to either allogeneic or self-antigens. Studies performed in vitro and in vivo showed that graft host Tregs specific to donor MHCII peptides / host MHCII complexes promoted T cell tolerance of fully allogeneic grafts. These pMHCII complexes - hereafter called TLo - were also involved in Treg-mediated suppression and in initial host Treg activation by graft MHCII. Extending these observations to Treg biology of normal a...

PLOS ONE, 2019

S5 Fig. The effect of 2DG treatment at day 3 on functional molecules. 0.5mM 2-deoxy-Dglucose (2DG... more S5 Fig. The effect of 2DG treatment at day 3 on functional molecules. 0.5mM 2-deoxy-Dglucose (2DG) was added from 3 to 7 days post activation in tTreg (red) and iTreg (blue). Flow analyses for CD25, CTLA-4 and ICOS were performed gated on live CD4+ cells. Data are shown as non-treated control and 2DG treated as solid and dot lines in representative histograms with isotype control staining (filled gray). Solid (non-treated, Cont) and checked (2DG treated, 2DG) boxes with bar graph indicates the mean fluorescence intensity (MFI) ±s.d.. Representative data is shown from at least 3 independent experiments of total 6 individual donors. Statistical analyses were performed by Student's t-test.

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1992

A polymorphism of CD4 in miniature swine has been identified by failure of cells from some animal... more A polymorphism of CD4 in miniature swine has been identified by failure of cells from some animals to react with mAb 74-12-4. The phenotypic, molecular genetic, and functional characteristics of these animals have been defined. Cells from heterozygous animals bear approximately 50% the number of 74-12-4-reactive molecules on their surface as do cells from animals homozygous for the wild type. Animals of both phenotypes demonstrate similar flow cytometric profiles for CD8+ T cells. Northern blot analysis confirms the presence of mRNA for CD4 among PBL of animals failing to stain with 74-12-4. CD4 allelism is confirmed by Southern blot analysis, revealing RFLP. Function of the CD4 subset in vivo, as demonstrated by antibody production against a T cell-dependent Ag, is similar between animals of both phenotypes. Proliferative responses to PHA and alloantigen stimulation by a full haplotype mismatch or a class II mismatch alone are equivalent for animals of both phenotypes. These data s...

Xenotransplantation, 2002

We recently established that molecular chimeras of major histocompatibility complex (MHC) class I... more We recently established that molecular chimeras of major histocompatibility complex (MHC) class II molecules, created via retroviral transfer of allogeneic class II cDNAs into bone marrow cells (BMCs), alleviated complications associated with mixed BMC chimeras while leading to T cell tolerance to renal grafts sharing the transferred class II. Initially demonstrated for allogeneic transplants in miniature swine, this concept was extended to T-dependent antibody (Ab) responses to xenogeneic antigens (Ags) in the pig ® baboon combination. Successful down-regulation of T cell responses appeared, however, to be contingent on a tight lineage-speci®c expression of transferred class II molecules. The present studies were, therefore, designed to evaluate the in¯uence of construct design and cellular environment on expression of retrovirally transferred xenogeneic class II cDNAs. Proviral genomes for pig class II SLA-DR expression, diering only at the marker neo(r) or enhanced green¯uorescent protein (EGFP) gene, showed increased membrane SLA-DR density on HLA-DR ± ®broblasts as well as HLA-DR + , TF-1 erythroleukemia cells. More importantly, HLA-DR + human B cell lines, although ef®ciently transduced with pig DR retroviruses, exhibited unstable surface pig DR. Surface pig DR ± B cells, nevertheless, stimulated autologous human T cells pre-sensitized to pig Ags, a proliferation likely occurring through presentation of class II-derived peptides. Collectively, these data suggest that surface expression of transferred class II molecules is not related to the ability of recipient cells to synthesize xenogeneic class II molecules but rather to their Ag processing capacities.

Transplantation, 1997

Transfer of MHC class II genes, through allogeneic bone marrow (BM) transplantation, induced long... more Transfer of MHC class II genes, through allogeneic bone marrow (BM) transplantation, induced long-lasting acceptance of renal allografts in miniature swine. To adapt this approach to the clinic, we have now examined whether somatic transfer of allogeneic class II DR genes, into otherwise autologous bone marrow cells (BMC), can provide the matching required for inducing immune tolerance. Autologous BMC were transduced ex vivo with recombinant retroviruses for allogeneic DRB followed by BM transplantation. The recipients were then challenged with kidney allografts solely matched to the DRB transgene. Five miniature swine received autologous BMC conditioned with growth factors and transduced with recombinant retrovirus vectors containing allogeneic (n=4) or syngeneic (n=1) class II DRB genes and a drug-resistance marker. Expression of retrovirus-derived products in BM-derived cells was demonstrated by the detection of drug-resistant colony-forming progenitors and the presence of DRB retrovirus transcripts in peripheral cells. Analysis of selective mixed lymphocyte reaction responses to DR or DQ antigens indicated decreased reactivity toward the transduced DR gene product. Among all of the animals receiving fully mismatched kidney allografts, but with DRB matched to the transduced DRB, the one with the highest gene transduction rate showed stable allograft function and essentially normal renal histology for 2.5 years. A control animal, which received a syngeneic DRB gene, rejected its kidney allograft in 120 days after an earlier rejection crisis. These studies demonstrate that allogeneic MHC gene transfer into BM provides a new strategy for inducing tolerance across MHC barriers.

Proceedings of the National Academy of Sciences, 1990

As part of our studies of the class II genes of miniature swine, we have isolated and characteriz... more As part of our studies of the class II genes of miniature swine, we have isolated and characterized cDNA clones corresponding to DRB genes from two major histocompatibility complex homozygous strains. Comparison of the sequences of these clones to those of human DRB genes revealed a striking amino acid homology between the hypervariable residues of SLA-DRBc and the human DRB1-0101 allele. The percentage of differences in these residues between the pig DRBc allele and the human DRB1-0101 allele was significantly lower (29%) than that between the DRB1-0101 allele and all other human alleles (average, 66.2%). This similarity was not seen in a comparison of the number of silent substitutions, by which the swine DRBc and the human DRB-0101 differed. Since phenotypic selection operates at the level of protein products rather than nucleotide sequences, these data suggest the existence of selective mechanisms that have resulted in similar hypervariable regions in certain alleles even in the...

Proceedings of the National Academy of Sciences, 1995

Clones encoding porcine interleukin 10 (IL-10) were isolated from a cDNA library produced from ph... more Clones encoding porcine interleukin 10 (IL-10) were isolated from a cDNA library produced from phytohemagglutinin-activated pig peripheral blood mononuclear cells. The porcine IL-10 nucleotide sequence was found to be highly homologous to the rat, mouse, and human IL-10 counterparts and to one of the open reading frames from the Epstein-Barr virus. In addition, pig IL-10 caused inhibition of gamma-interferon gene transcription as determined by a bioassay. To investigate the possible immunomodulatory role of IL-10, its expression during the induction of tolerance to kidney allografts by cyclosporin A in miniature swine was also investigated. Delayed expression and higher levels of IL-10 were observed in tolerant animals compared with animals rejecting their allografts. Since tolerance is achieved by a short course of cyclosporin A, we have also studied the in vitro effect of this drug on IL-10 gene transcription in blood mononuclear cells and have found that cyclosporin A inhibits IL...

Human Gene Therapy, 2000

Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine ... more Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine transplantation model by retrovirus-mediated transfer of allogeneic MHC class II genes into bone marrow cells (BMCs) of recipient animals. Graft survival correlated with transient expression of the somatic transgene (Tg) in the induction phase of tolerance. With the aim of investigating the effects of timing and threshold levels of Tg expression on induction of hyporesponsiveness to the grafted tissues, two recombinant retrovirus constructs containing the tetracycline binary regulatory system were used to achieve conditional expression of either the green fluorescent protein (tetGFP) as a control, or the porcine MHC class II DRbeta chain (tetDRB). Effective downregulation of GFP gene transcription was demonstrated in transduced murine fibroblasts after doxycycline treatment, leading to a > 90% reduction of GFP fluorescence. Similar diminution of the DRB gene transcription was achieved in transduced pig endothelial cells (ECs). Drug-dependent downregulation of DRBc gene expression in SLAd pig ECs coincided with complete inhibition of allogeneic activation of anti-class IIc-primed SLAd T cells. These in vitro results suggest that the binary tetracycline retrovirus system may also be adequate to regulate MHC class II Tg expression in vivo.

Human Gene Therapy, 1999

Transplantation tolerance to renal allografts can be induced in large animal preclinical models i... more Transplantation tolerance to renal allografts can be induced in large animal preclinical models if the donor and recipient have identical major histocompatibility complex (MHC) class II loci. Such class II matching is, however, not clinically achievable owing to the extreme diversity of class II sequences. With the ultimate goal of creating a somatic class II match in the bone marrow of an allograft recipient, the aim of the study is to develop a double-copy retrovirus construct to express both chains of the MHC class II DQ glycoprotein on a single transduced cell. Analysis of the expression patterns of the retroviral DQ transgenes in both virus producer and transduced fibroblasts revealed correct transcription and stable surface expression of the DQ heterodimers. In addition, we demonstrate that both the DQA and DQB sequences are functional within the same proviral copy, a prerequisite for efficient induction of transplantation tolerance following transduction of bone marrow precursor cells. The DQ double-copy retrovirus vector showed efficient expression of the transferred class II cDNA in murine colony-forming units for the granulocyte-monocyte lineage (CFU-GM), indicating that it is suitable for gene therapy of multimeric proteins in hematopoietic cells.

Biology of Blood and Marrow Transplantation, 2003

Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoieti... more Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoietic cell transplantation that leads to mixed chimerism and allograft tolerance in miniature swine across minor and major histocompatibility disparities. Stable chimerism persisted in most of these animals but was restricted to T cells and confined to peripheral blood. Because of the importance of myeloid and erythroid progenitors for the treatment of hematologic disorders, the objective of this study was to assess whether such cells existed in the bone marrow of these lymphoid chimeras as an indication of functional engraftment. Colony-formation assays were performed on donor inocula before infusion and on bone marrow cells harvested from the transplant recipients. Donor-origin myeloid/erythroid progenitor colonies were detected in bone marrow from 6 of 7 lymphoid chimeric recipients. A delayed donor leukocyte infusion successfully converted a stable lymphoid chimera to full multilineage chimerism within 2 weeks. Donor-origin myeloid/erythroid progenitors could be detected in the bone marrow of a host-matched recipient after myeloablation and adoptive transfer of mobilized cells from one of the engrafted lymphoid chimeras. These data suggest that even when only lymphoid chimerism is readily detected by flow cytometry, dormant myeloid/erythroid progenitors can exist and subsequent conversion to full donor chimerism can be achieved. The ability to establish multilineage engraftment and chimerism without significant toxicity may have important clinical implications for the management of nonmalignant hematopoietic disorders and hematologic malignancies.