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1.
Br J Haematol ; 197(6): 697-708, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35254660

RESUMO

B-cell depletion induced by anti-cluster of differentiation 20 (CD20) monoclonal antibody (mAb) therapy of patients with lymphoma is expected to impair humoral responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination, but effects on CD8 T-cell responses are unknown. Here, we investigated humoral and CD8 T-cell responses following two vaccinations in patients with lymphoma undergoing anti-CD20-mAb therapy as single agent or in combination with chemotherapy or other anti-neoplastic agents during the last 9 months prior to inclusion, and in healthy age-matched blood donors. Antibody measurements showed that seven of 110 patients had antibodies to the receptor-binding domain of the SARS-CoV-2 Spike protein 3-6 weeks after the second dose of vaccination. Peripheral blood CD8 T-cell responses against prevalent human leucocyte antigen (HLA) class I SARS-CoV-2 epitopes were determined by peptide-HLA multimer analysis. Strong CD8 T-cell responses were observed in samples from 20/29 patients (69%) and 12/16 (75%) controls, with similar median response magnitudes in the groups and some of the strongest responses observed in patients. We conclude that despite the absence of humoral immune responses in fully SARS-CoV-2-vaccinated, anti-CD20-treated patients with lymphoma, their CD8 T-cell responses reach similar frequencies and magnitudes as for controls. Patients with lymphoma on B-cell depleting therapies are thus likely to benefit from current coronavirus disease 2019 (COVID-19) vaccines, and development of vaccines aimed at eliciting T-cell responses to non-Spike epitopes might provide improved protection.


Assuntos
Linfócitos T CD8-Positivos , Vacinas contra COVID-19 , COVID-19 , Linfoma , Rituximab , Anticorpos Antivirais , Linfócitos T CD8-Positivos/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Epitopos , Humanos , Linfoma/tratamento farmacológico , Rituximab/uso terapêutico , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinação
2.
J Immunol ; 200(8): 2847-2859, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29531166

RESUMO

Early studies indicate that rats may have a repertoire of MHC class Ib-reactive Ly49 stimulatory receptors capable of mounting memory-like NK cell alloresponses. In this article, we provide molecular and functional evidence for this assumption. Pairs of Ly49 receptors with sequence similarities in the lectin-like domains, but with opposing signaling functions, showed specificity for ligands with class Ia-like structural features encoded from the first telomeric MHC class Ib gene cluster, RT1-CE, which is syntenic with the H2-D/H2-L/H2-Q cluster in mice. The activating Ly49s4 receptor and its inhibitory counterparts, Ly49i4 and Ly49i3, reacted with all allelic variants of RT1-U, whereas Ly49s5 and Ly49i5 were specific for RT1-Eu NK cell cytolytic responses were predictably activated and inhibited, and potent in vivo NK alloresponses were induced by repeated MHC class Ib alloimmunizations. Additional Ly49-class Ib interactions, including RT1-Cl with the Ly49s4/Ly49i4/Ly49i3 group of receptors, were characterized using overexpressed receptor/ligand pairs, in vitro functional assays, and limited mutational analyses. Obvious, as well as subtle, Ly49-class Ib interactions led to ligand-induced receptor calibration and NK subset expansions in vivo. Together, these studies suggest that in vivo NK alloresponses are controlled by pleomorphic Ly49-class Ib interactions, some of which may not be easily detectable in vitro.


Assuntos
Antígenos de Histocompatibilidade/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Antígenos de Histocompatibilidade Classe I , Ligantes , Ratos
3.
J Immunol ; 189(11): 5178-84, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23100519

RESUMO

Natural killer cells are able to recognize and kill target cells according to differences in MHC class I expression. In rodents, the Ly49 receptors are primarily responsible for this MHC differentiation. We previously described the cloning of a novel C-type lectin-like receptor, KLRH1, encoded in the NK complex adjacent to the Ly49 genes and expressed by subsets of NK and NKT cells. MHC influence on selection of KLRH1(+) NK cells in congenic strains suggested that KLRH1 may have an MHC ligand, although we were unable to identify any such ligand. In this study, we have used a sensitive reporter system and Fc fusion protein to demonstrate that KLRH1 binds specifically to the classical MHC class I molecule RT1-A2 of the RT1(n) haplotype. Cytolytic activity of KLRH1-transfected RNK-16 cells was also inhibited by target cells expressing RT1-A2(n). Thus, KLRH1 represents a novel family of MHC allele-specific inhibitory receptors expressed by NK cells.


Assuntos
Antígenos de Histocompatibilidade/imunologia , Células Matadoras Naturais/imunologia , Receptores Imunológicos/imunologia , Alelos , Animais , Células CHO , Linhagem Celular , Cricetinae , Expressão Gênica , Genes Reporter , Haplótipos , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Ligantes , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Ratos , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
4.
Cell Rep ; 42(1): 111995, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36656713

RESUMO

The emergence of SARS-CoV-2 variants of concern (VOC) is driven by mutations that mediate escape from neutralizing antibodies. There is also evidence that mutations can cause loss of T cell epitopes. However, studies on viral escape from T cell immunity have been hampered by uncertain estimates of epitope prevalence. Here, we map and quantify CD8 T cell responses to SARS-CoV-2-specific minimal epitopes in blood drawn from April to June 2020 from 83 COVID-19 convalescents. Among 37 HLA ligands eluted from five prevalent alleles and an additional 86 predicted binders, we identify 29 epitopes with an immunoprevalence ranging from 3% to 100% among individuals expressing the relevant HLA allele. Mutations in VOC are reported in 10.3% of the epitopes, while 20.6% of the non-immunogenic peptides are mutated in VOC. The nine most prevalent epitopes are conserved in VOC. Thus, comprehensive mapping of epitope prevalence does not provide evidence that mutations in VOC are driven by escape of T cell immunity.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Linfócitos T CD8-Positivos , COVID-19/imunologia , Epitopos de Linfócito T/genética , Epitopos Imunodominantes/genética , SARS-CoV-2/genética
5.
Eur J Immunol ; 40(12): 3535-43, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21108473

RESUMO

We have investigated whether rat Ly49 receptors can monitor Listeria-infected intestinal epithelial cells through altered expression of MHC class I molecules. The rat colon carcinoma epithelial cell line CC531 infected with Listeria expressed higher levels of both classical and nonclassical MHC-I molecules. Reporter cells expressing the activating Ly49s5 receptor displayed increased stimulatory responses when incubated with Listeria-infected CC531 cells in vitro, which could be blocked with mAb 8G10 specific for nonclassical MHC-I molecules of the RT1(u) haplotype, but not with mAb OX18 reacting with classical MHC-I molecules in this haplotype. Similar responses were observed against IFN-γ-treated cells that also upregulated their expression of MHC-I molecules. Thus, the Ly49s5 receptor can respond to increased levels of nonclassical MHC-I molecules induced on target cells by either bacterial infection or cytokine stimulation. We furthermore found that splenic NK and NKT cells produced IFN-γ in response to Listeria-infected CC531 cells, and that this was not limited to Ly49-expressing cells, since similar levels of IFN-γ production were observed in Ly49(+) and Ly49(-) NK cell subsets. Therefore, NK cells may recognize Listeria-infected cells through both MHC-I-dependent and -independent innate immune receptor systems.


Assuntos
Células Epiteliais/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Imunidade Adaptativa/efeitos dos fármacos , Animais , Anticorpos Bloqueadores/farmacologia , Linhagem Celular Tumoral , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Imunidade Inata/efeitos dos fármacos , Interferon gama/imunologia , Interferon gama/metabolismo , Intestinos/patologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Listeria monocytogenes/patogenicidade , Ativação Linfocitária/efeitos dos fármacos , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/patologia , Ratos , Ratos Endogâmicos , Regulação para Cima/efeitos dos fármacos
6.
Immunogenetics ; 63(7): 429-36, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21409442

RESUMO

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3(+) NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3(+) and NKR-P1B(+) NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D(-) and NKR-P1D(+) NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.


Assuntos
Receptores Imunológicos/metabolismo , Animais , Linhagem Celular Tumoral , Sequência Conservada , Camundongos , Camundongos Endogâmicos C57BL , Filogenia , Ratos , Receptores Imunológicos/classificação , Receptores Imunológicos/genética
7.
Eur J Immunol ; 39(2): 541-51, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19130483

RESUMO

A major subset of non-alloreactive NK cells in PVG strain rats is generally low in Ly49 receptors, but expresses the rat NKR-P1B(PVG) receptor (previously termed NKR-P1C). The NKR-P1B(+) NK subset is inhibited by a non-polymorphic target cell ligand, which we have shown here to be a C-type lectin-related molecule (Clr). Clr11 ligates two divergent NKR-P1B alleles as judged by an NFAT-driven reporter assay, and inhibits NK-cell cytotoxicity of NKR-P1B(+) NK cells. Clr11 also interacts with the prototypic NKR-P1A receptor and exerts a stimulatory influence on NK lysis. NKR-P1A and B are encoded by adjacent genes in the proximal part of the NK gene complex and show close sequence homology in their extracellular region. They diverge from another pair, NKR-P1F and -G, which is encoded by a second, distal Nkrp1 gene cluster. NKR-P1F and -G bind an overlapping panel of Clr ligands, but not Clr11. Rat Clr molecules appear to be constitutively expressed by hematopoietic cells; expression in tumor cell lines is more variable. The data show the existence of two phylogenetic groups of NKR-P1 molecules, which demonstrate conservation of ligand-binding properties independent of signaling function.


Assuntos
Células Matadoras Naturais/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/classificação , Subfamília B de Receptores Semelhantes a Lectina de Células NK/genética , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Citotoxicidade Imunológica , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/imunologia , Ligantes , Dados de Sequência Molecular , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Filogenia , Ratos , Alinhamento de Sequência
8.
Mol Immunol ; 45(8): 2380-90, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18160104

RESUMO

The T-cell specific adapter protein (TSAd) encoded by the SH2D2A gene is up-regulated in activated human CD4+ T-cells in a cAMP-dependent manner. Expression of SH2D2A is important for proper activation of T-cells. Here, we show that SH2D2A expression is regulated both at the transcriptional and translational level. cAMP signaling alone induces TSAd-mRNA expression but fails to induce increased TSAd protein levels. By contrast, TCR engagement provides signals for both TSAd transcription and translation. We further show that cAMP signaling can prime T-cells for a more prompt expression of TSAd protein upon TCR stimulation. Our study thus points to a novel mechanism for how cAMP signaling may modulate T-cell activation through transcriptional priming of resting cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Linfócitos T CD4-Positivos/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , Transcrição Gênica , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Apresentação Cruzada/efeitos dos fármacos , Apresentação Cruzada/imunologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Isoquinolinas/farmacologia , Modelos Imunológicos , Biossíntese de Proteínas/efeitos dos fármacos , Transporte de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Transcrição Gênica/efeitos dos fármacos
9.
Transplantation ; 85(1): 102-11, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-18192919

RESUMO

BACKGROUND: We have employed a rat model for human acute myeloid leukemia, a promyelocytic leukemia in the BN rat strain (BNML), to develop new protocols for immunotherapy in combination with allogeneic bone marrow transplantation (alloBMT). The status of mixed chimerism in allotransplanted rats provided an opportunity for immunotherapy using alloreactive donor cells. In addition to T or natural killer (NK) cells, we introduced a second infusion of bone marrow cells as prophylactic donor lymphocyte infusions (DLI) to test whether an effective graft-versus-leukemia (GVL) response could be obtained without clinical graft-versus-host disease (GVHD). METHODS: BN rats were sublethally irradiated and transplanted with T-cell depleted bone marrow cells from either fully major histocompatibility complex (MHC)-mismatched (PVG) donor rats or MHC-matched (PVG.1N) as controls. Seven days after transplantation, rats were given 500 leukemic cells to mimic minimal residual disease. Additional cellular therapy was given at day +7. The efficiency of DLI was monitored by chimerism analysis in peripheral blood. RESULTS: Rats receiving infusions of NK cells succumbed to leukemia. T-DLI induced complete donor T-cell chimerism and lethal GVHD. A second alloBMT protected against leukemia. This effect was dependent on an MHC incompatibility between the donor and host and also on the presence of alloreactive T cells in the second bone marrow inoculum, resulting in an increased, mixed donor T-cell chimerism. CONCLUSION: A second prophylactic transplantation influenced the degree of T-cell chimerism to balance favorably between GVL and GVHD. If applicable to humans, repeated alloBMT may provide a novel approach to leukemia therapy.


Assuntos
Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/métodos , Doença Enxerto-Hospedeiro/prevenção & controle , Leucemia Mieloide Aguda/terapia , Complexo Principal de Histocompatibilidade/imunologia , Animais , Transplante de Medula Óssea/patologia , Quimerismo , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Efeito Enxerto vs Leucemia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Células Matadoras Naturais/fisiologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Masculino , Neoplasia Residual/imunologia , Ratos , Ratos Endogâmicos BN , Ratos Nus , Linfócitos T/imunologia , Linfócitos T/patologia , Linfócitos T/fisiologia
10.
BMC Immunol ; 7: 15, 2006 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-16839418

RESUMO

BACKGROUND: The activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd. RESULTS: TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239-256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239-334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239-334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells. CONCLUSION: These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239-334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos T CD4-Positivos/imunologia , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/enzimologia , Núcleo Celular/metabolismo , Éxons , Expressão Gênica , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Dados de Sequência Molecular , Fosforilação , Prolina/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Tirosina/metabolismo , Domínios de Homologia de src
11.
Front Immunol ; 7: 572, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28003815

RESUMO

Measuring degranulation through CD107a expression has become an advantageous tool for testing the functional capacity of cytotoxic cells. Such functional studies have been hampered in the rat by the lack of a suitable anti-rat CD107a antibody. In this study, we report a novel hybridoma generated by immunizing Armenian inbred hamsters with transfected Chinese hamster ovary cells expressing CD107a. The SIM1 clone exhibited specific reactivity with CD107a and measured degranulation from natural killer (NK) cells stimulated with target cells or mAb crosslinking of their activating receptors. Degranulation in IL-2-activated NK cells could also be measured, when using low effector to target ratios. SIM1 also stained activated CD8, but not CD4 T cells. This report characterizes the degranulation response in cytotoxic rat cells with a new antibody against rat CD107a.

12.
J Leukoc Biol ; 89(4): 617-23, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21248147

RESUMO

Ly49 receptors in rodents, like KIRs in humans, regulate NK cell activity. Although inhibitory Ly49 receptors clearly recognize MHC-I molecules, ligands for the activating Ly49 receptors are less well defined. Here, we show that the activating Ly49s4 and the inhibitory Ly49i4 receptors recognize nonclassical MHC-I molecules on the rat macrophage cell line R2 (RT1(d)). Listeria infection of R2 macrophages led to increased expression of classical and nonclassical MHC-I molecules. Coincubation of these infected cells with reporter cells expressing Ly49i4 or Ly49s4 increased the reporter cell responses. These responses were blocked by mAb OX18 (anti-MHC-I) and AAS1 (anti-nonclassical MHC-I). IFN-γ treatment of normal R2 cells also increased the MHC-I expression and enhanced the reporter cell responses. These results suggest that activating and inhibitory Ly49 receptors monitor MHC-I expression on Listeria-infected cells.


Assuntos
Antígenos Ly/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Listeria monocytogenes/fisiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Receptores de Células Matadoras Naturais/imunologia , Animais , Anticorpos Bloqueadores , Anticorpos Monoclonais/farmacologia , Citometria de Fluxo , Células Matadoras Naturais/microbiologia , Ratos
13.
J Immunol ; 176(7): 4133-40, 2006 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-16547249

RESUMO

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C(+) NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C(+) NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3(+) subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor repertoires.


Assuntos
Antígenos Ly/imunologia , Antígenos de Superfície/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos Ly/genética , Antígenos Ly/metabolismo , Antígenos de Superfície/química , Antígenos de Superfície/metabolismo , Células Cultivadas , Concanavalina A/farmacologia , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Células Matadoras Naturais/imunologia , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Ligantes , Dados de Sequência Molecular , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Filogenia , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Tempo
14.
Immunogenetics ; 58(11): 905-16, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17028855

RESUMO

Natural killer (NK) cells from certain rat strains promptly kill MHC allogeneic lymphocytes in vivo, a rejection phenomenon termed allogeneic lymphocyte cytotoxicity (ALC). ALC can be reproduced in vitro, and is preferentially mediated by a subset of NK cells expressing the Ly49 stimulatory receptor 3 (Ly49s3) in PVG strain rats. Functional studies have suggested that Ly49s3 triggers NK cell alloreactivity, but its importance relative to other Ly49 receptors has not been investigated. In this study, we have characterized three rat Ly49 receptors with close sequence similarity to Ly49s3 in the extracellular region, i.e., Ly49s4, Ly49 inhibitory receptor 3 (Ly49i3), and Ly49i4. Similar to Ly49s3, Ly49s4 mediated cellular activation while Ly49i4 inhibited NK cytolytic function. Ly49s4, -i3, and -i4 all reacted with a previously described anti-Ly49s3 monoclonal antibody (mAb) (DAR13), but not a novel mAb (STOK6), which was shown to be specific for Ly49s3. Expression of these Ly49 receptors varied markedly between inbred strains, in patterns related to their NK gene complex (NKC) haplotype, and ability to mediate ALC. Three major groups of NKC haplotypes could be discerned by restriction fragment length polymorphism analysis. Ly49s3 was present in strains from one of the groups, which corresponded with the "high" ALC responders. Ly49s3 surface expression was also markedly reduced in the presence of its putative MHC class Ib ligand(s) in MHC congenic strains. These data support the notion that Ly49s3 functions as a triggering MHC receptor both in vitro and in vivo. MHC ligands for the other three Ly49 receptors remain to be determined.


Assuntos
Antígenos Ly/análise , Antígenos Ly/genética , Haplótipos , Células Matadoras Naturais/imunologia , Lectinas Tipo C/análise , Lectinas Tipo C/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos Ly/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Lectinas Tipo C/metabolismo , Ligantes , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Ratos , Receptores Semelhantes a Lectina de Células NK
15.
J Immunol ; 174(5): 2702-11, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15728478

RESUMO

The Ly49 family of lectin-like receptors in rodents includes both stimulatory and inhibitory members. Although NK alloreactivity in mice is regulated primarily by inhibitory Ly49 receptors, in rats activating Ly49 receptors are equally important. Previous studies have suggested that activating rat Ly49 receptors are triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, RT1-CE/N/M, while inhibitory Ly49 receptors bind to widely expressed classical class Ia molecules encoded from the RT1-A region. To further investigate rat Ly49-mediated regulation of NK alloreactivity, we report in this study the identification and characterization of two novel paired Ly49 receptors that we have termed Ly49 inhibitory receptor 5 (Ly49i5) and Ly49 stimulatory receptor 5 (Ly49s5). Using a new mAb (mAb Fly5), we showed that Ly49i5 is an inhibitory receptor that recognizes ligands encoded within the class Ib region of the u and l haplotypes, while the structurally related Ly49s5 is an activating receptor that recognizes class Ib ligands of the u haplotype. Ly49s5 is functionally expressed in the high NK-alloresponder PVG strain, but not in the low alloresponder BN strain, in which it is a pseudogene. Ly49s5 is hence not responsible for the striking anti-u NK alloresponse previously described in BN rats (haplotype n), which results from repeated alloimmunizations with u haplotype cells. The present studies support the notion of a complex regulation of rat NK alloreactivity by activating and inhibitory Ly49 members, which may be highly homologous in the extracellular region and bind similar class Ib-encoded target ligands.


Assuntos
Antígenos Ly/isolamento & purificação , Antígenos Ly/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Ativação Linfocitária/imunologia , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/biossíntese , Antígenos Ly/química , Antígenos Ly/genética , Antígenos Ly/imunologia , Clonagem Molecular/métodos , Feminino , Haplótipos , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Imunofenotipagem , Células Matadoras Naturais/química , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Lectinas Tipo C , Ligantes , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Oligopeptídeos , Peptídeos/genética , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores Semelhantes a Lectina de Células NK , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
16.
Eur J Immunol ; 34(12): 3389-400, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15517613

RESUMO

Two populations of CD8(+) IEL generally express restricted, but apparently random and non-overlapping TCR repertoires. Previous studies in mice suggested that this could be explained by a dual origin of CD8(+) IEL, i.e. that CD8alphabeta(+) IEL derive from a few peripheral CD8(+) T cell lymphoblasts stimulated by microbial antigens in gut-associated lymphoid tissue, whereas CD8alphaalpha(+) IEL descend from an inefficient intestinal maturation pathway. We show here that the gut mucosa, instead, becomes seeded with surprisingly broad and generally non-overlapping CD8 IEL repertoires and that oligoclonality is induced locally after microbial colonization. In germ-free (GF) rats, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed surprisingly diverse TCR Vbeta repertoires, although beta-chain diversity tended to be somewhat restricted in the CD8alphaalpha(+) subset. CDR3 length displays in individual Vbeta-Cbeta and Vbeta-Jbeta combinations generally revealed polyclonal distributions over 6-11 different lengths, similar to CD8(+) lymph node T cells, and CDR3beta sequencing provided further documentation of repertoire diversity. By contrast, in ex-GF rats colonized with normal commensal microflora, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed oligoclonal CDR3 length distributions for most of the Vbeta genes analyzed. Our data suggest that microbial colonization induces apparently random clonal expansions of CD8alphabeta(+) and CD8alphaalpha(+) IEL locally in the gut.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Animais , Divisão Celular/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Intestino Delgado/citologia , Intestino Delgado/microbiologia , Ratos , Receptores de Antígenos de Linfócitos T/imunologia
17.
J Immunol ; 172(10): 6144-51, 2004 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15128801

RESUMO

The SH2D2A gene, encoding the T cell-specific adapter protein (TSAd), is rapidly induced in activated T cells. In this study we investigate the regulation of the SH2D2A gene in Jurkat T cells and in primary T cells. Reporter gene assays demonstrated that the proximal 1-kb SH2D2A promoter was constitutively active in Jurkat TAg T cells and, to a lesser extent, in K562 myeloid cells, Reh B cells, and 293T fibroblast cells. The minimal SH2D2A promoter was located between position -236 and -93 bp from the first coding ATG, and transcriptional activity in primary T cells depended on a cAMP response element (CRE) centered around position -117. Nuclear extracts from Jurkat TAg cells and activated primary T cells contained binding activity to this CRE, as observed in an EMSA. Consistent with this observation, we found that a cAMP analog was a very potent inducer of SH2D2A mRNA expression in primary T cells as measured by real-time RT-PCR. Furthermore, activation of SH2D2A expression by CD3 stimulation required cAMP-dependent protein kinase activity. Thus, transcriptional regulation of the SH2D2A gene in activated T cells is critically dependent on a CRE in the proximal promoter region.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , AMP Cíclico/análogos & derivados , Regiões Promotoras Genéticas , Ativação Transcricional , Proteínas de Transporte/biossíntese , Proteínas de Transporte/fisiologia , Linhagem Celular , Separação Celular , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica/imunologia , Humanos , Células Jurkat , Células K562 , Ativação Linfocitária , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , RNA Mensageiro/biossíntese , Complexo Receptor-CD3 de Antígeno de Linfócitos T/fisiologia , Elementos de Resposta/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Tionucleotídeos/farmacologia , Ativação Transcricional/imunologia
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