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1.
Transfusion ; 61(3): 687-691, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33336414

RESUMO

BACKGROUND: Platelet transfusions remain a mainstay of treatment for many patients with thrombocytopenia, but can lead to alloantibodies to Human Leukocyte Antigens (anti-HLA) resulting in inadequate responses to subsequent platelet transfusions (refractoriness), as well as complicate transplantation. Despite substantial decreases in alloimmunization with the implementation of leukoreduction, a significant percentage of patients still become alloimmunized following platelet transfusions. It remains unclear why some patients make anti-HLA antibodies, but others do not make anti-HLA antibodies even with chronic transfusion. Antecedent pregnancy correlates with risk of alloimmunization due to platelet transfusion in humans - however, isolation of pregnancy as a single variable is not possible in human populations. STUDY DESIGN AND METHODS: A tractable murine model of pregnancy and transfusion was engineered by breeding C57BL/6 (H-2b ) dames with BALB/c (H-2d ) sires. After pregnancy, female mice were transfused with leukoreduced platelets from F1 (H-2b/d ) donors that expressed the same paternal major histocompatibility complex (MHC) H-2d alloantigens as the sires. Control groups allowed isolation of pregnancy or transfusion alone as independent variables. Alloimmunization was determined by testing serum for antibodies to H-2d MHC alloantigens. RESULTS: No alloantibodies were detected after pregnancy alone, or in response to transfusion of platelets alone; however, significant levels of alloantibodies were detected when pregnancy was followed by transfusion. CONCLUSIONS: These findings isolate antecedent pregnancy as a causal contribution to increased frequencies of alloimmunization by subsequent platelet transfusion in mice and provide a platform for ongoing mechanistic investigation.


Assuntos
Antígenos HLA/imunologia , Isoanticorpos/sangue , Isoanticorpos/imunologia , Isoantígenos/sangue , Isoantígenos/imunologia , Transfusão de Plaquetas/efeitos adversos , Animais , Plaquetas/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Gravidez
2.
Front Immunol ; 11: 1516, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32765523

RESUMO

It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system.


Assuntos
Eritrócitos/imunologia , Imunidade Humoral , Imunoglobulina G/imunologia , Imunomodulação , Isoanticorpos/imunologia , Isoantígenos/imunologia , Receptores Fc/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Eritrócitos/metabolismo , Imunização Passiva , Camundongos , Camundongos Knockout
3.
Transfusion ; 60(5): 1060-1068, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32369193

RESUMO

BACKGROUND: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings. STUDY DESIGN AND METHODS: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry. RESULTS: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity. CONCLUSION: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Imunoglobulinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Eritrócitos/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Testes Imunológicos , Isoanticorpos/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Análise de Sequência de Proteína
4.
Transfusion ; 59(2): 744-753, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30681727

RESUMO

BACKGROUND: Humoral alloimmunization to human leukocyte antigen (HLA) can represent a barrier to solid-organ transplantation, can lead to a refractory state in patients requiring platelet transfusion, and can also contribute to transfusion-related acute lung injury (TRALI). While exposure to HLA-mismatched cells/tissues are generally required for HLA alloimmunization, the effect of the extent of major histocompatibility complex (MHC) mismatch between donor and recipient is poorly understood. STUDY DESIGN AND METHODS: A novel mouse was generated that allows the expression of a single MHC Class I alloantigen, Kd . Alloimmune responses to Kd were studied in C57BL/6 mice transfused with splenocytes from different donor mice, allowing the analysis of responses to Kd as an isolated alloantigen, or in the context of additional mismatched MHC molecules. Advanced tools were utilized to study responses to Kd , including T-cell receptor transgenic mice that recognize the immunodominant Kd peptide presented by C57BL/6 mice to CD4+ T cells. RESULTS: A single MHC Class I alloantigen mismatch is less immunogenic than when the same alloantigen is encountered in the context of additional mismatched MHC alloantigens. This difference is due, at least in part, to induction of CD4+ helper T cells, as the effect is overcome by increasing either mature CD4+ T-cell help through immunization or by increasing the precursor frequency of naïve CD4+ T cells by adoptive transfer from T-cell receptor transgenic donors. CONCLUSION: These findings indicate that the immunogenicity of a single alloantigen can be affected by the context in which it is encountered, demonstrating the potential for cooperative effects between different mismatched MHC alloantigens.


Assuntos
Plaquetas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Isoantígenos/imunologia , Transfusão de Plaquetas , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Plaquetas/patologia , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Isoantígenos/genética , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Auxiliares-Indutores/patologia , Reação Transfusional/genética , Reação Transfusional/patologia
5.
J Clin Pathol ; 71(6): 559-561, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29550761

RESUMO

There are four subtypes of human IgG with different effector functions. Quantifying the relative amount of each IgG subtype is important for laboratory diagnosis in multiple settings. However, in an evolving landscape of the appreciation of human variability and the need for precision/personalised laboratory diagnosis, it has also been shown that there are numerous natural variants of IgG subtypes, with at least 29 having been described. It has recently been reported that commercially available polyclonal antisera to IgG3 cross react with variants of other IgG subtypes, while available monoclonal anti-IgG3 have a blind-spot for the IgG3-04 variant. Herein, we report that IgG3-04 contains an epitope in common with all known IgG3 variants and absent in other subtypes. A novel monoclonal anti-IgG3 is described that is specific to IgG3 but without any blind-spots for known IgG3 variants, providing a remedy to the problem of genetic variability of IgG3.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/genética , Epitopos/imunologia , Variação Genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Animais , Especificidade de Anticorpos , Reações Cruzadas , Humanos , Imunoglobulina G/classificação , Testes Imunológicos , Camundongos
6.
J Immunol Methods ; 455: 10-13, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29395164

RESUMO

Methods designed to monitor humoral immune responses, in a variety of settings, typically use a broadly reactive detection reagent (e.g. polyclonal anti-Ig (immunoglobulin)) in order to characterize antibody responses. In the context of murine models of immunity, which are widely used, this would typically be antisera to mouse Ig or mouse IgG. However, there are 4 different subtypes of mouse IgG; thus, the validity of the above approach, as a general screen for humoral immune responses, depends upon the assumption that the antisera recognize all IgG subtypes. This seems like a reasonable assumption, since polyclonal antisera recognize multiple epitopes; however, herein we report that two commercial sources of goat anti-mouse Ig are hyporeactive with IgG3. Given that relative IgG3 levels are different in distinct types of immune response, these findings demonstrate a potential for misinterpretation, and suggest a need to modify immunological methods in this context.


Assuntos
Epitopos/metabolismo , Imunoglobulina G/metabolismo , Animais , Cabras , Humanos , Soros Imunes/imunologia , Imunidade Humoral , Switching de Imunoglobulina , Testes Imunológicos , Camundongos
7.
Front Immunol ; 8: 1366, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163471

RESUMO

Autoimmune hemolytic anemia (AIHA) results from breakdown of humoral tolerance to RBC antigens. Past analyses of B-cell receptor transgenic (BCR-Tg) mice that recognize RBC autoantigens led to a paradigm in which autoreactive conventional B-2 B cells are deleted whereas extramedullary B-1 B cells escape deletion due to lack of exposure to RBCs. However, BCR-Tg mice utilized to shape the current paradigm were unable to undergo receptor editing or class-switching. Given the importance of receptor editing as mechanism to tolerize autoreactive B cells during central tolerance, we hypothesized that expansion of autoreactive B-1 B cells is a consequence of the inability of the autoreactive BCR to receptor edit. To test this hypothesis, we crossed two separate strains of BCR-Tg mice with transgenic mice expressing the BCR target on RBCs. Both BCR-Tg mice express the same immunoglobulin and, thus, secrete antibodies with identical specificity, but one strain (SwHEL) has normal receptor editing, whereas the other (IgHEL) does not. Similar to other AIHA models, the autoreactive IgHEL strain showed decreased B-2 B cells, an enrichment of B-1 B cells, and detectable anti-RBC autoantibodies and decreased RBC hematocrit and hemoglobin values. However, autoreactive SwHEL mice had induction of tolerance in both B-2 and B-1 B cells with anti-RBC autoantibody production without anemia. These data generate new understanding and challenge the existing paradigm of B cell tolerance to RBC autoantigens. Furthermore, these findings demonstrate that immune responses vary when BCR-Tg do not retain BCR editing and class-switching functions.

8.
Front Immunol ; 8: 1425, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163500

RESUMO

Red blood cells (RBCs) have a well-defined lifespan, indicating a mechanism by which senescent cells of a certain age are removed from circulation. However, the specifics by which senescent cells are recognized and removed are poorly understood. There are multiple competing hypotheses for this process, perhaps the most commonly cited is that senescent RBCs expose neoantigens [or senescent antigen(s)] that are then recognized by naturally occurring antibodies, which opsonize the senescent cells and result in clearance from circulation. While there are a large volume of published data to indicate that older RBCs accumulate increased levels of antibody on their surface, to the best of our knowledge, the causal role of such antibodies in clearance has not been rigorously assessed. In the current report, we demonstrate that RBC lifespan and clearance patterns are not altered in mice deficient in antibodies, in C3 protein, or missing both. These data demonstrate that neither antibody nor C3 is required for clearance of senescent RBCs, and questions if they are even involved, in a murine model of RBC lifespan.

9.
JCI Insight ; 2(13)2017 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-28679954

RESUMO

In recent years, the extent of our vulnerability to misinterpretation due to poorly characterized reagents has become an issue of great concern. Antibody reagents have been identified as a major source of error, contributing to the "reproducibility crisis." In the current report, we define an additional dimension of the crisis; in particular, we define variation of the targets being analyzed. We report that natural variation in the immunoglobulin "constant" region alters the reactivity with commonly used subtype-specific anti-IgG reagents, resulting in cross-reactivity of polyclonal regents with inappropriate targets and blind spots of monoclonal reagents for desired targets. This raises the practical concern that numerous studies characterizing IgG subtypes in human disease may contain errors due to such previously unappreciated defects. These studies also focus attention on the broader concern that genetic variation may affect the performance of any laboratory or research test that uses antibodies for detection.

10.
Front Immunol ; 7: 348, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27698653

RESUMO

Autoimmune hemolytic anemia (AIHA) occurs when pathogenic autoantibodies against red blood cell (RBC) antigens are generated. While the basic disease pathology of AIHA is well studied, the underlying mechanism(s) behind the failure in tolerance to RBC autoantigens are poorly understood. Thus, to investigate the tolerance mechanisms required for the establishment and maintenance of tolerance to RBC antigens, we developed a novel murine model. With this model, we evaluated the role of regulatory T cells (Tregs) in tolerance to RBC-specific antigens. Herein, we show that neither sustained depletion of Tregs nor immunization with RBC-specific proteins in conjunction with Treg depletion led to RBC-specific autoantibody generation. Thus, these studies demonstrate that Tregs are not required to prevent autoantibodies to RBCs and suggest that other tolerance mechanisms are likely involved.

11.
Transfusion ; 56(12): 2953-2962, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27632931

RESUMO

BACKGROUND: Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty-nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized. STUDY DESIGN AND METHODS: A novel monoclonal anti-K antibody (PugetSound Monoclonal Antibody 1 [PUMA1]) was isolated and sequenced, and a panel of PUMA1 variants was expressed, consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was preincubated with K-positive red blood cells (RBCs) and then subjected to testing with currently approved anti-IgG by flow cytometry, solid phase systems, gel cards, and tube testing. RESULTS: A US Food and Drug Administration (FDA)-approved monoclonal anti-IgG (gamma-clone) failed to recognize 2 of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 of 3 IgG4 isoallotypes (IgG4-01, IgG4-02, and IgG4-03). In contrast, an FDA-approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes. CONCLUSION: These findings demonstrate "blind spots" in isoalloantibody detection by a monoclonal anti-IgG. If a patient has anti-RBC antibodies predominantly of an IgG3 subtype (the IgG3-03 and/or IgG3-13 variety), then it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 have an estimated frequency of 1% to 3% in Caucasian populations and 20% to 30% in certain African populations. Nonreactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported.


Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Anticorpos Monoclonais/imunologia , Alótipos de Imunoglobulina/imunologia , Imunoglobulina G/análise , Animais , Anticorpos Anti-Idiotípicos/imunologia , Erros de Diagnóstico , Variação Genética , Humanos , Imunoglobulina G/genética , Coelhos , Grupos Raciais
12.
Transfusion ; 56(8): 1974-83, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27158944

RESUMO

BACKGROUND: Transfusion of platelets (PLTs) is a common therapy in a number of clinical settings. However, it is well understood that there is substantial donor-to-donor variation in how well PLTs store and thus the quality of the products that are transfused. The basis of such variation is poorly understood, and there are limited metrics by which units of PLTs can be assessed for their posttransfusion performance. It has repeatedly been demonstrated that myriad biologic changes take place during PLT storage; however, which of the changes correlate with quality of the stored PLTs and/or are mechanistically involved in PLT function remains undetermined. STUDY DESIGN AND METHODS: The current study tested stored PLTs from 21 normal subjects, combining high-resolution metabolomics of stored PLTs with in vivo PLT recoveries and survivals. Both individual analytes and metabolic pathways that correlate with posttransfusion PLT viability were identified. RESULTS: Caffeine metabolites were associated with poor PLT recovery; caffeine metabolism was not ongoing in the PLT bag and remained at prestorage levels. Acylcarnitines, particular fatty acid metabolites, and oxidized fatty acids were associated with poor PLT survivals. Of the myriad metabolic changes during PLT storage, these are the first reported metabolic findings to begin distinguishing which changes are of functional importance regarding posttransfusion PLT performance. CONCLUSIONS: Together, these findings provide novel mechanistic insights into the functional biology of the PLT storage lesion as well as identifying potential targets for modifying donor environment (e.g., caffeine consumption) and also metrics of quality assessment for stored human PLTs.


Assuntos
Plaquetas/metabolismo , Plaquetas/fisiologia , Preservação de Sangue/métodos , Cafeína/análise , Ácidos Graxos/análise , Humanos , Metabolômica/métodos , Transfusão de Plaquetas/métodos , Fatores de Tempo
13.
Transfusion ; 56(1): 91-100, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26400622

RESUMO

BACKGROUND: Platelet (PLT) transfusions can be an essential therapy for patients with thrombocytopenia to maintain hemostasis. However, some patients become alloimmunized to antigens on PLTs (typically HLA), which can prevent efficacy of PLT transfusion due to antibody-mediated clearance. In extreme cases, patients with alloimmunization to multiple HLAs can become "refractory" to PLT transfusion, such that insufficient compatible PLT units can be found to meet transfusion needs. MATERIALS AND METHODS: An in vivo murine model of PLT-induced alloimmunization was refined so as to include both transfusion with allogeneic leukoreduced PLTs and studies of posttransfusion PLT recoveries, allowing assessment of alloimmunization and refractoriness. Basic mechanisms of antibody-mediated PLT clearance were investigated using recipients missing either the C3 complement gene or the common gamma chain for Fc receptors. In addition, the efficacy of using costimulatory blockade as a therapeutic intervention was assessed by testing CTLA4-Ig administration before PLT transfusion. RESULTS: Fcγ receptors (but not complement C3) are required for alloantibody-mediated PLT refractoriness. In addition, levels of anti-MHC predict the extent of refractoriness in a given animal. Finally, costimulatory blockade as a therapeutic modality prevents transfusion-induced PLT refractoriness. CONCLUSIONS: Together these findings introduce new experimental methods, basic mechanistic understanding, and a potential therapeutic intervention for alloimmunization to MHC-based antigens on transfused PLTs.


Assuntos
Plaquetas/imunologia , Antígenos de Histocompatibilidade/imunologia , Isoanticorpos/imunologia , Transfusão de Plaquetas/efeitos adversos , Reação Transfusional/imunologia , Animais , Biomarcadores/sangue , Isoanticorpos/sangue , Modelos Lineares , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
14.
J Immunol ; 179(4): 2105-14, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17675469

RESUMO

CD4(+)CD25(+) regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred K(d)-specific, CD4(+) TCR (TCR75) transgenic mice to Foxp3(gfp) knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3(gfp).TCR75 mice did not express significant numbers of natural FoxP3(+) Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with K(d) plus TGF-beta. The resulting GFP(+) TCR75 cells were anergic, whereas the GFP(-) TCR75 cells proliferated upon restimulation with K(d) peptide. Yet both exhibited severely reduced expression of intracellular IFN-gamma and TNF-alpha upon restimulation. GFP(+), but not GFP(-), TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP(+) TCR75 cells also inhibited polyclonal C57BL/6 anti-K(d) CTL responses if the APC expressed K(d) and both MHC class I and class II, and responses by OT1 T cells to B6.K(d).OVA but not B6.K(d) plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.


Assuntos
Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Forkhead/imunologia , Tolerância Imunológica/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/imunologia , Regulação para Cima/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Células Apresentadoras de Antígenos/imunologia , Antígenos/genética , Complexo CD3/imunologia , Proliferação de Células , Anergia Clonal/efeitos dos fármacos , Anergia Clonal/genética , Anergia Clonal/imunologia , Fatores de Transcrição Forkhead/genética , Genes Reporter/genética , Genes Reporter/imunologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/genética , Interferon gama/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
15.
Int Immunol ; 18(11): 1549-62, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16966495

RESUMO

Although CD4+CD25+FoxP3+ regulatory T cells play a role in allograft tolerance, the role of CD8+ cells with immunosuppressive function is less clear. To address this issue, spleen cells from Rag-1-deficient TCR transgenic (Tg) mice expressing a receptor for ovalbumin (OVA) in the context of MHC class I (OT1) were activated with OVA expressing antigen-presenting cell (APC) in the presence or absence of exogenous transforming growth factor beta (TGFbeta). TGFbeta inhibited the expression of IFN-gamma, granzyme B and the lytic activity of the OT1 T cells while inducing FoxP3 expression in 5-15% of the cells. By contrast, FoxP3 expression was not detected in naive OT-1 T cells or OT-1 T cells activated without exogenous TGFbeta. TGFbeta-activated OT1 cells inhibited the activation of Kd-specific CD8+ CTL responses by normal B6 T cells and the proliferation by Kd-specific CD4+ TCR Tg T cells, but only if the OVA epitope was co-expressed by Kd+ APC. This antigen-specific inhibitory activity, referred to as linked suppression, was neither mediated by residual lytic activity within the activated OT1 T cells nor did it depend upon IL-10 or TGFbeta. Suppression correlated with inhibition of CD86 expression on CD11c+ APC. TGFbeta-activated OT1 T cells also delayed the rejection of heterotopic, vascularized cardiac allografts mediated by anti-Kd-specific CD4+ TCR Tg T cells, but only if the cardiac allograft expressed both OVA and Kd as transgenes. Prolonged survival of allografts was associated with rapid migration of the FoxP3+ OT1 T cells into the donor heart raising the possibility that suppression may be mediated within the allograft. These data show that TGFbeta-activated CD8+ T cells mediate antigen-specific, APC-focused patterns of suppression in vitro and in vivo.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Forkhead/metabolismo , Rejeição de Enxerto/imunologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígeno B7-2/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/efeitos dos fármacos , Transplante de Coração/imunologia , Tolerância Imunológica/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
16.
J Neuroimmunol ; 164(1-2): 66-75, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15913791

RESUMO

In this report, we provide evidence that muscarinic receptors play a role in the generation of CD8+ cytolytic T lymphocytes. Analysis of mice with targeted deletions of each of the known muscarinic receptors (M1-M5) showed that CD8+ T cells from M1 receptor-deficient mice had a defect in the ability to differentiate into cytolytic T lymphocytes. Additional pharmacological experiments support the role of muscarinic receptors in wild type mice and suggest that acetylcholine may be involved. Together, these findings suggest that the M1 muscarinic receptor is involved in CTL development, thus providing novel insights into CD8+ T cell biology and the potential role of cholinergic signaling in immune regulation.


Assuntos
Acetilcolina/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Antagonistas Muscarínicos/farmacologia , Receptor Muscarínico M1/fisiologia , Animais , Northern Blotting/métodos , Complexo CD3/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/fisiologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/biossíntese , Receptor Muscarínico M1/deficiência , Receptores Muscarínicos/classificação , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo
17.
Transplantation ; 79(4): 409-18, 2005 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-15729166

RESUMO

BACKGROUND: Transplantation of human islets has been successful clinically. Since human islets are scarce, we are studying microencapsulated porcine islet xenografts in nonobese diabetic (NOD) mice. We have evaluated the cellular immune response in NOD mice with and without dual costimulatory blockade. METHODS: Alginate-poly-L-lysine-encapsulated adult porcine islets were transplanted i.p. in untreated diabetic NODs and NODs treated with CTLA4-Ig to block CD28/B7 and with anti-CD154 mAb to inhibit CD40/CD40-ligand interactions. Groups of mice were sacrificed on subsequent days; microcapsules were evaluated by histology; peritoneal cells were analyzed by FACS; and peritoneal cytokines were quantified by ELISA. Controls included immunoincompetent NOD-Scids and diabetic NODs given sham surgery or empty microcapsules. RESULTS: Within 20 days, encapsulated porcine islets induced accumulation of large numbers of macrophages, eosinophils, and significant numbers of CD4 and CD8 T cells at the graft site, and all grafts were rejected. During rejection, IFNgamma, IL-12 and IL-5 were significantly elevated over sham-operated controls, whereas IL-2, TNFalpha, IL-4, IL-6, IL-10, IL-1beta and TGFbeta were unchanged. Treatment with CTLA4-Ig and anti-CD154 prevented graft destruction in all animals during the 26 days of the experiment, dramatically inhibited recruitment of host inflammatory cells, and inhibited peritoneal IFNgamma and IL-5 concentrations while delaying IL-12 production. CONCLUSIONS: When two different pathways of T cell costimulation were blocked, T cell-dependent inflammatory responses were inhibited, and survival of encapsulated islet xenografts was significantly prolonged. These findings suggest synergy between encapsulation of donor islets and simultaneous blockade of two host costimulatory pathways in prolonging xenoislet transplant survival.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno B7-1/fisiologia , Antígenos CD28/fisiologia , Antígenos CD40/fisiologia , Ligante de CD40/fisiologia , Imunoconjugados/farmacologia , Terapia de Imunossupressão , Transplante das Ilhotas Pancreáticas/imunologia , Transplante Heterólogo/imunologia , Abatacepte , Animais , Citocinas/biossíntese , Feminino , Sobrevivência de Enxerto , Masculino , Camundongos , Camundongos Endogâmicos NOD , Suínos
18.
Immunol Res ; 29(1-3): 93-102, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15181273

RESUMO

Gammadelta T cells were discovered in the mid-1980s, but the antigens they recognize and the biological functions they mediate are poorly understood. Although gammadelta T cells have the capacity to augment immunity to certain infections and kill certain tumor cells, they are generally not required for development of antibody responses, for graft rejection, or for development of autoimmune diseases. Nevertheless, gammadelta T cells accumulate at sites of inflammation induced by infection, tumor growth, and autoimmune lesions, where they have been shown to reduce the inflammatory reaction and tissue damage. In this review, we summarize the evidence that gammadelta T cells play an important role in the induction of various forms of tolerance.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica , Receptores de Antígenos de Linfócitos T gama-delta/análise , Subpopulações de Linfócitos T/imunologia , Animais , Humanos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia
19.
Immunology ; 111(2): 155-64, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15027900

RESUMO

Oral administration of antigen induces a state of tolerance that is associated with activation of CD8+ T cells that can transfer unresponsiveness to naïve syngeneic hosts. These T cells are not lytic, but they inhibit development of antibody, CD4+ T helper cell, and CD8+ cytotoxic T lymphocyte (CTL) responses upon adoptive transfer into naïve, syngeneic mice. In addition, we have shown that depletion of gammadelta T cells by injection of the anti-delta chain antibody (GL3) down modulates the expression of gammadelta T-cell receptor (TCR) and inhibits the induction of oral tolerance to ovalbumin. Oral administration of antigen also fails to induce tolerance in TCR delta-chain knockout mice suggesting that gammadelta T cells play a critical, active role in tolerance induced by orally administered antigen. To further study the contribution of gammadelta T cells to tolerance, murine gammadelta T cells were isolated from intraepithelial lymphocytes (IEL) of the small intestine by stimulation with splenic filler cells, concanavalin A and growth factors. gammadelta IEL lines demonstrated lytic activity in a redirected lysis assay. gammadelta T-cell clones express different gammadelta TCR genes and secrete large amounts of interleukin (IL)-10, but little or no IL-2, IL-4, or interferon-gamma. gammadelta IEL clones expressed transforming growth factor-beta1 and macrophage migration inhibitory factor, as well as IL-10, mRNA. Moreover, gammadelta T-cell clones potently inhibited the generation of CTL responses by secreted molecules rather than by direct cell-to-cell contact.


Assuntos
Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/análise , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Linhagem Celular , Células Cultivadas , Células Clonais/imunologia , Citotoxicidade Imunológica/imunologia , Células Epiteliais/imunologia , Tolerância Imunológica , Imunidade nas Mucosas , Imunofenotipagem , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Receptores de Antígenos de Linfócitos T alfa-beta/análise
20.
Int Immunol ; 15(11): 1389-99, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14565937

RESUMO

We have previously described a monoclonal antibody, 984, which specifically recognizes murine CD8(+) suppressor T cells (Ts) but not CD8(+) cytolytic T lymphocytes (CTLs). Removal of 984(+) cells abrogates the suppressive effect of CD8(+) Ts generated either in vivo or in vitro while having no effect upon CTL. In this report, the molecules recognized by 984 are identified as 2-6 sialylated neolacto series gangliosides, which are members of the newly defined CD75s cluster. We proceed to demonstrate that like 984, a separate anti-CD75s antibody (CRIS-4), recognizes primary CD8(+) Ts cells. In addition, the 2,6 sialyltransferase responsible for the synthesis of the 984 epitope is identified, allowing the manipulation and study of the regulation of this epitope. This is the first report of CD75s on murine cells and the first report that delineates lymphocyte function based upon CD75s expression. In addition to contributing to the growing body of evidence that lineage dependent gangliosides are expressed by T lymphocytes, these findings suggest that CD8(+) CD75s(+) T lymphocytes represent a functionally distinct subset of CD8(+) T cells with negative regulatory function.


Assuntos
Antígenos CD/análise , Gangliosídeos/análise , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Amino Açúcares/química , Amino Açúcares/imunologia , Animais , Antígenos CD/química , Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Sequência de Bases , Biomarcadores/análise , Epitopos/biossíntese , Epitopos/química , Feminino , Gangliosídeos/biossíntese , Gangliosídeos/química , Glicolipídeos/biossíntese , Glicolipídeos/química , Hibridomas/imunologia , Ativação Linfocitária , Camundongos , Dados de Sequência Molecular , Linfócitos T Reguladores/classificação
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