RESUMEN
Ever since the emergence of models for the processing and presentation of antigenic determinants by MHC class II molecules, the main view has been that proteins are unfolded, enzymatically cleaved into peptide lengths of about 12-25 amino acids and then loaded onto MHC class II molecules. There is, however, an alternative model stating that partially intact unfolding antigens are first bound by MHC class II molecules and then trimmed to fragments of a smaller size while remaining bound to the MHC class II molecule. In this analysis, we make the case that a considerable portion of the elutable peptide cargo belongs to this latter class.
Asunto(s)
Antígenos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Animales , Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos/metabolismo , Epítopos/química , Epítopos/inmunología , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunologíaRESUMEN
To determine the role that competition plays in a molecular mimic's capacity to induce autoimmunity, we studied the ability of naïve encephalitogenic T cells to expand in response to agonist altered peptide ligands (APLs), some capable of stimulating both self-directed and exclusively APL-specific T cells. Our results show that although the APLs capable of stimulating exclusively APL-specific T cells are able to expand encephalitogenic T cells in vitro, the encephalitogenic repertoire is effectively outcompeted in vivo when the APL is used as the priming immunogen. Competition as a mechanism was supported by: (i) the demonstration of a population of exclusively APL-specific T cells, (ii) an experiment in which an encephalitogenic T cell population was successfully outcompeted by adoptively transferred naïve T cells, and (iii) demonstrating that the elimination of competing T cells bestowed an APL with the ability to expand naïve encephalitogenic T cells in vivo. In total, these experiments support the existence of a reasonably broad T cell repertoire responsive to a molecular mimic (e.g., a microbial agent), of which the exclusively mimic-specific component tends to focus the immune response on the invading pathogen, whereas the rare cross-reactive, potentially autoreactive T cells are often preempted from becoming involved.
Asunto(s)
Autoinmunidad/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Proliferación Celular , Encefalomielitis Autoinmune Experimental/inmunología , Citometría de Flujo , Inmunización , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Oligopéptidos/inmunología , Bazo/citología , Bazo/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/citologíaRESUMEN
BACKGROUND: Non Obese Diabetic mice lacking B cells (NOD.Igµ(null) mice) do not develop diabetes despite their susceptible background. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age. METHODS: We have used the spectratyping technique to follow the T cell receptor (TCR) V beta repertoire of NOD.Igµ(null) mice following B cell reconstitution. This technique provides an unbiased approach to understand the kinetics of TCR expansion. We have also analyzed the TCR repertoire of reconstituted animals receiving cyclophosphamide treatment and following tissue transplants to identify common aggressive clonotypes. RESULTS: We found that B cell reconstitution of NOD.Igµ(null) mice induces a polyclonal TCR repertoire in the pancreas 10 weeks later, gradually diversifying to encompass most BV families. Interestingly, these clonotypic BV expansions are mainly confined to the pancreas and are absent from pancreatic lymph nodes or spleens. Cyclophosphamide-induced diabetes at 10 weeks post-B cell reconstitution reorganized the predominant TCR repertoires by removing potential regulatory clonotypes (BV1, BV8 and BV11) and increasing the frequency of others (BV4, BV5S2, BV9, BV16-20). These same clonotypes are more frequently present in neonatal pancreatic transplants under the kidney capsule of B-cell reconstituted diabetic NOD.Igµ(null) mice, suggesting their higher invasiveness. Phenotypic analysis of the pancreas-infiltrating lymphocytes during diabetes onset in B cell reconstituted animals show a predominance of CD19+ B cells with a B:T lymphocyte ratio of 4:1. In contrast, in other lymphoid organs (pancreatic lymph nodes and spleens) analyzed by FACS, the B:T ratio was 1:1. Lymphocytes infiltrating the pancreas secrete large amounts of IL-6 and are of Th1 phenotype after CD3-CD28 stimulation in vitro. CONCLUSIONS: Diabetes in NOD.Igµ(null) mice appears to be caused by a polyclonal repertoire of T cell accumulation in pancreas without much lymphoid organ involvement and is dependent on the help by B cells.
Asunto(s)
Linfocitos B/inmunología , Diabetes Mellitus Experimental/inmunología , Cadenas mu de Inmunoglobulina/inmunología , Inmunofenotipificación/métodos , Islotes Pancreáticos/inmunología , Linfocitos T/inmunología , Animales , Animales Recién Nacidos , Linfocitos B/citología , Proliferación Celular , Células Clonales , Ciclofosfamida , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Rechazo de Injerto/complicaciones , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Memoria Inmunológica/inmunología , Islotes Pancreáticos/patología , Trasplante de Islotes Pancreáticos , Ratones , Ratones Endogámicos NOD , Fenotipo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Bazo/patología , Linfocitos T/citologíaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. Immunization of B10.PL mice with the Ac1-9 peptide, the immunodominant determinant of myelin basic protein (MBP), produced a single episode of EAE followed by recovery and resistance to reinduction of disease. Using the CDR3 length spectratyping technique, we characterized the clonal composition of the Ac1-9-specific T cell repertoire from induction through onset and resolution of disease. Two clonally restricted subsets within a heterogeneous self-reactive repertoire were found in mouse lymph nodes, spleen, and spinal cord soon after immunization, before any sign of EAE. These clonotypes, designated BV8S2/BJ2S7 and BV16/BJ2S5, were present in all mice examined and thus considered public. BV8S2/BJ2S7 was found in far greater excess; was exclusively Th1 polarized; disappeared from the spinal cord, spleen, and lymph nodes concomitantly with recovery; and transferred disease to naive recipients. In contrast, BV16/BJ2S5 and numerous private clonotypes were either Th1 or Th2 and persisted following recovery. These results are consistent with the hypothesis that the public clonotype BV8S2/BJ2S7 is a driver of disease and necessary for its propagation.
Asunto(s)
Regiones Determinantes de Complementariedad/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Epítopos de Linfocito T/inmunología , Esclerosis Múltiple/inmunología , Células TH1/inmunología , Células Th2/inmunología , Animales , Regiones Determinantes de Complementariedad/genética , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Epítopos de Linfocito T/genética , Ratones , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/genética , Esclerosis Múltiple/patología , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/inmunología , Proteína Básica de Mielina/toxicidad , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad , Recuperación de la Función/genética , Recuperación de la Función/inmunología , Células TH1/patología , Células Th2/patologíaRESUMEN
BACKGROUND: Tumor immune responses are first generated and metastases often begin in tumor sentinel lymph nodes (TSLN). Therefore, it is important to promote tumor immunity within this microenvironment. Mifepristone (RU486) treatment can interfere with cortisol signaling that can lead to suppression of tumor immunity. Here, we assessed whether treatment with RU486 in conjunction with an intratumor injection of Ad5IL-12 vector (a recombinant adenovirus expressing IL-12) could impact the TSLN microenvironment and prostate cancer progression. METHODS: The human PC3, LNCaP or murine TRAMP-C1 prostate cancer cell lines were used to generate subcutaneous tumors in NOD.scid and C57BL/6 mice, respectively. Adjuvant effects of RU486 were looked for in combination therapy with intratumor injections (IT) of Ad5IL-12 vector in comparison to PBS, DL70-3 vector, DL70-3 + RU486, RU486 and Ad5IL-12 vector treatment controls. Changes in tumor growth, cell cytotoxic activity and populations of CD4+/FoxP3+ T regulatory cells (Treg) in the TSLN were evaluated. RESULTS: Treatment of human PC3 prostate xenograft or TRAMP-C1 tumors with combination Ad5IL-12 vector and RU486 produced significantly better therapeutic efficacy in comparison to controls. In addition, we found that combination therapy increased the capacity of TSLN lymphocytes to produce Granzyme B in response to tumor cell targets. Finally, combination therapy tended towards decreases of CD4+/FoxP3+ T regulatory cell populations to be found in the TSLN. CONCLUSION: Inclusion of RU486 may serve as a useful adjuvant when combined with proinflammatory tumor killing agents by enhancement of the immune response and alteration of the TSLN microenvironment.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos , Interleucina-12/administración & dosificación , Metástasis Linfática , Mifepristona/uso terapéutico , Neoplasias de la Próstata/terapia , Animales , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Interleucina-12/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patologíaRESUMEN
Following Leishmania major infection, the early LACK (Leishmania homolog of receptors for activated C kinase)-induced IL-4 response appears to determine disease susceptibility in BALB/c mice. Therefore, we sought to manipulate the pathogenic T cell responses to the immunodominant epitope with the use of altered peptide ligands (APLs). Conservative and non-conservative substitutions for each amino acid of the LACK 161-175 peptide determinant were tested for their stimulatory capacity in four different LACK-reactive T cell systems. From these results, we propose a likely LACK 163-171/I-A(d) core peptide register and show that APLs with changes at putative T cell receptor (TCR) contacts provide the greatest potential for immune deviation. In particular, the TCR-contact H164V APL expanded Th1 cells upon in vitro recall of naïve splenocytes from LACK-specific BV4 T cell receptor transgenic mice and stimulated IFN-gamma secretion from a Th2-committed LACK-reactive T cell line. We also observed that non-conservative substitutions flanking the core determinant had strong agonistic effects for proliferation and Th1/Th2 modulation. However, upon immunization, the H164V APL considerably downregulated proliferation and cytokine responses to the wild type LACK 161-175 peptide, while immunization with the weak agonist, MHC contact APL S171K, increased the IFN-gamma/IL-4 ratio to the wild type peptide. In these instances, a hyporesponsive T cell response to the wild-type peptide was achieved by immunizing with an APL possessing non-conservative substitutions at TCR contact sites, while immune deviation was accomplished using a weak-agonist APL that retained the core determinant. Thus, certain LACK-APLs are able to induce T cell responses with a protective phenotype in an infectious disease such as leishmaniasis.
Asunto(s)
Antígenos de Protozoos/inmunología , Epítopos Inmunodominantes/inmunología , Leishmania major/inmunología , Péptidos/inmunología , Proteínas Protozoarias/inmunología , Células Th2/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos de Protozoos/química , Línea Celular , Proliferación Celular , Reactividad Cruzada/inmunología , Citocinas , Inmunización , Interferón gamma/inmunología , Interleucina-4/inmunología , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Datos de Secuencia Molecular , Péptidos/química , Proteínas Protozoarias/química , Vacunas Antiprotozoos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Células Th2/citologíaRESUMEN
Tumor cells can evolve to evade immune responses by down-modulating surface MHC class I expression and become refractory to T cell-directed immunotherapy. We employed a strategy to bypass this escape mechanism using a recombinant adenovirus vector expressing interleukin-12 (Ad5IL-12) to target natural killer (NK) cell-mediated killing of human prostate tumors in NOD.scid mice. Fluorescence-activated cell sorting analysis revealed that LNCaP tumor cells bear negligible levels of MHC class I molecules; yet, they express MICA/B molecules, ligands for the NKG2D receptors found on NK cells. Transduction of LNCaP cells with the Ad5IL-12 vector prevented tumor formation in NOD.scid mice, indicating that NK cells alone can conduct tumor immunosurveillance and mediate protection. Intratumor injection of the Ad5IL-12 vector to established LNCaP tumors in NOD.scid mice resulted in a significant delay of tumor growth mediated by NK cell killing activity. The dependency of NK cells in this protective response was shown by the complete loss of Ad5IL-12 therapeutic efficacy on LNCaP tumors established in NOD.Cg-Rag1(tm1Mom)Prf1(tm1Sdz) congenic mice, which are devoid of NK cell activity. More pronounced attenuation of tumor growth and enhanced NK killing activity was observed when pharmacologic adrenalectomy with mitotane was done in combination with Ad5IL-12 vector treatment. The Ad5IL-12 vector treatment also induced killing of MICA/B-negative MHC class I-positive PC3 tumors formed in NOD.scid mice. Together, these results indicate that a targeted NK cell response could provide a generic approach for cancer immunotherapy, and that enhancing the NK cell response via control of cortisol levels may provide an additional therapeutic avenue in cancer.
Asunto(s)
Adenoviridae , Genes MHC Clase I , Terapia Genética/métodos , Hidrocortisona/metabolismo , Interleucina-12/uso terapéutico , Células Asesinas Naturales/fisiología , Neoplasias de la Próstata/terapia , Animales , Antineoplásicos Hormonales/uso terapéutico , Terapia Combinada , Vectores Genéticos , Humanos , Inmunidad Celular , Inmunoterapia/métodos , Interleucina-12/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mitotano/uso terapéutico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Degeneracy in the immune system not only refers to the recognition by its specific receptors, but also at the level of functional degeneracy of reactivity by its lymphocytes. Features of the immune system such as dominance and competition occur at each of these two levels. Another characteristic of the immune system as a fuzzy system, is its continuum of states, where difficult choices lead to the emergence of unpredictable phenotypes.
Asunto(s)
Anticuerpos/inmunología , Antígenos/inmunología , Sistema Inmunológico , Animales , Anticuerpos/fisiología , Antígenos/fisiología , Humanos , Receptores de Antígenos/inmunología , Receptores de Antígenos/fisiologíaRESUMEN
Glutamic acid decarboxylase (GAD65) is one of the autoantigens that initiates pathogenic T cell responses against insulin-secreting pancreatic beta cells in Type 1 diabetes (T1D). Previously it was shown that spontaneously arising pathogenic T cell responses in the NOD mouse model are confined to GAD530-543 (p530). However, regulatory T cell subpopulations, which can prevent diabetes, can also be generated, for example, by immunization with GAD524-538 (p524) or GAD524-543. Interestingly, two functionally distinct subpopulations of T cells which recognize overlapping determinants of GAD524-543, p524 and p530, utilize distinct TCR Vbeta families, Vbeta4 for pathogenic, and Vbeta12 for regulatory T cells. We characterized T cell receptors (TCRs) from each subpopulation of T cells and visualized p524-specific TCR/p524/I-A(g7) and p530-specific TCR/p530/I-A(g7) complexes via molecular modeling to help us understand, at a molecular level, the in vivo expansion of p524- or p530-specific T cells in the NOD model of T1D. The absolute restriction in Vbeta usage but not Valpha usage and conserved CDR3beta lengths for both T cell subpopulations demonstrates that the beta chains are main contributors in shaping both p524/I-A(g7) and p530/I-A(g7) restricted TCRs. However, only Vbeta4+ T cells but not Vbeta12+ T cells contain a common motif (DWG) in CDR3beta and may involve all of CDR1beta, CDR2beta, and CDR3beta in the recognition of the C-terminus of p530. These observations imply that the spontaneously arising p530-restricted TCRs may be selected under stringent structural frameworks to bind p530/I-A(g7) with high affinity. Thus, the pathogenic p530-specific T cells may arise from a small pool of autoreactive T cells upon breaking tolerance.
Asunto(s)
Glutamato Descarboxilasa/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Secuencias de Aminoácidos , Animales , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Ratones , Ratones Endogámicos NOD , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/químicaRESUMEN
The available T cell repertoire directed against self is appreciable owing to the escape of many clones from negative selection, largely because many determinants on self proteins are cryptic and not presented adequately. In addition, the degeneracy of T cell receptor specificity permits each lymphocyte a broad recognitive potential. Within the available self-reactive repertoire are T cells with high affinity, and these can compete favorably with other T cells with the same specificity. We have studied a "driver clone" and its two specific regulators in the B10.PL model of experimental autoimmune encephalomyelitis and found that each of these repertoires is highly limited. There is a single major clonal family comprising the aggressive driver population, which is public and of high affinity, and just one other minor public clonotype. The receptors of this Vbeta8.2/Jbeta2.7 driver are presented to a CD4 regulator and a CD8 suppressor, each of limited clonality, the latter killing the driver clone by apoptosis, completing a feedback control loop. This tightly regulated group of three cell types furnishes an excellent example of the immune homunculus.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Animales , Humanos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Here, we demonstrate that the Wistar Kyoto (WKY/NHsd) rat, which bears the same RT1(l) haplotype as the experimental autoimmune encephalomyelitis (EAE)-susceptible Lewis rat strain, is highly resistant to myelin basic protein (MBP)-induced EAE. No differences between Lewis and WKY strains were found in T cell proliferative specificity or the use of Vbeta8.2 T cell receptors in response to MBP. A Th2 cytokine bias correlated with WKY's EAE resistance. MBP challenge of WKY-into-Lewis adoptive transfer recipients produced a novel biepisodic EAE. The WKY strain should be useful in studies of many tissue-specific autoimmune diseases to which the Lewis rat is susceptible.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Proteína Básica de Mielina/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/citología , División Celular/inmunología , Citocinas/análisis , Inmunidad Innata/inmunología , Inmunización , Masculino , Proteína Básica de Mielina/farmacología , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WKY , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Especificidad de la EspecieRESUMEN
BACKGROUND: Nasal instillation is an effective method for inducing antigen-specific immune tolerance. However, it is not clear how a tolerization scheme established in one mouse strain will perform when used in a mouse of a different haplotype. OBJECTIVES: To compare the antigen-specific recall responses in four mouse strains--BALB/c, C57BL/6, NOD, and B10.PL--that were pretreated nasally with 50 micrograms of hen egg-white lysozyme prior to parenteral immunization with homologous antigen. METHODS: Mice were nasally treated with a prototype antigen, HEL, and then immunized with the same antigen emulsified in complete Freund's adjuvant. Spleens and lymph nodes were assayed for T cell proliferation measured by tritiated thymidine incorporation. Cytokine production was measured using ELISPOT assay. Serum antibody response to HEL was measured using an enzyme-linked immunosorbent assay. RESULTS: Proliferative recall responses to HEL in B10.PL, C57BL/6, and BALB/c were greatly reduced compared to control mice, but non-obese diabetic mice were resistant to the tolerization regime. Despite their susceptibility to nasally induced suppression, the mechanisms responsible for tolerance induction differed in BALB/c and C57BL/6 mice. CONCLUSIONS: Our findings demonstrate that while mucosal contacts with specific antigen consistently affect the outcome of subsequent exposure to the same antigen, the observed response will vary non-predictably, depending on the genetic background of the animal.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Modelos Animales de Enfermedad , Tolerancia Inmunológica/inmunología , Inmunización/métodos , Ratones Endogámicos BALB C/inmunología , Ratones Endogámicos C57BL/inmunología , Ratones Endogámicos NOD/inmunología , Muramidasa/inmunología , Muramidasa/uso terapéutico , Animales , Enfermedades Autoinmunes/inducido químicamente , Evaluación Preclínica de Medicamentos , Epítopos/genética , Epítopos/inmunología , Haplotipos/genética , Haplotipos/inmunología , Tolerancia Inmunológica/genética , Inmunidad Mucosa/inmunología , Instilación de Medicamentos , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C/genética , Ratones Endogámicos C57BL/genética , Ratones Endogámicos NOD/genética , Mucosa Nasal , Linfocitos T/inmunologíaAsunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Inmunoensayo/métodos , Vaina de Mielina/inmunología , Análisis por Matrices de Proteínas/métodos , Vacunas de ADN/inmunología , Vacunas de ADN/uso terapéutico , Animales , Tolerancia a Medicamentos , Encefalomielitis Autoinmune Experimental/diagnóstico , Ratones , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Mapeo de Interacción de Proteínas/métodos , Resultado del TratamientoRESUMEN
OBJECTIVE: Immunotherapy using peptides from the ß-cell antigen GAD65 can preserve glucose homeostasis in diabetes-prone NOD mice; however, the precise mechanisms that arrest islet-reactive T cells remain unresolved. Our previous work revealed that a dominant GAD65 epitope contained two overlapping I-A(g7)-restricted determinants, 524-538 and 530-543, with the former associated with amelioration of hyperglycemia. Here, we sought to discover whether p524-538-specific T cells could directly regulate islet-reactive T cells. RESEARCH DESIGN AND METHODS: Prediabetic NOD mice were used to determine the relationship between peptide p524-538-induced interleukin (IL)-13 and regulation of islet autoimmunity. Pancreatic lymph node (PLN) cells from mice at distinct stages of islet inflammation, peri-insulitis versus invasive insulitis, were harvested to establish the expression pattern of IL-13 receptor α1 (IL-13Rα1) on islet-associated T cells. RESULTS: Peptide p524-538 preferentially induced IL-13-producing T cells that antagonized the release of γ-interferon by spontaneously arising GAD65 autoimmunity, and recombinant human IL-13 inhibited proliferation of islet-reactive clonotypic T cells. A subset of CD4(+) T cells in NOD and NOD.BDC2.5 T cell receptor transgenic mice expressed functional IL-13Rα1, which induced phosphorylation of signal transducer and activator of transcription 6 in the presence of cognate cytokine. Notably, the number of IL-13Rα1(+) T cells was heightened in the PLN of young NOD mice when compared with older female counterparts with advanced insulitis. Immunization with p524-538 preserved IL-13Rα1 expression on PLN T cells. CONCLUSIONS: IL-13 may be important for regulating autoimmunity in the early stages of insulitis, and the loss of IL-13Rα1 on islet-reactive T cells may be a biomarker for fading regional immune regulation and progression to overt diabetes.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Animales , Autoinmunidad/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Femenino , Citometría de Flujo , Glutamato Descarboxilasa/farmacología , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Interferón gamma/metabolismo , Interleucina-13/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Fragmentos de Péptidos/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT6/metabolismoRESUMEN
Alterations in presenting self determinants to T cells may depend upon the availability of sites on the molecule adjacent to known determinants to different processing enzymes, or, at the level of amino acid sequence or conformation of a single determinant. We have studied three possible ways that could modulate the processing and presentation of T cell determinants of a diabetes autoantigen, glutamic acid decarboxylase (GAD) 65, which could contribute to induction of GAD65-specific regulatory versus pathogenic T cells in type 1 diabetes (T1D): 1) enhanced presentation of subdominant/cryptic determinants to T cells that have not been well-tolerized, which may activate T cells of high affinity and high aggressiveness; 2) trimming or truncating flanking residues which may otherwise provide needed binding energy to determinants that activate regulatory cells, thus releasing autoaggressive T cells from suppression; 3) biochemical or chemical modifications of self antigens in an inflammatory environment or within activated antigen presenting cells (APC) which may convert a previously regulatory antigen or determinant into a disease-causing one that activates autoreactive T cells at a higher affinity.
Asunto(s)
Glutamato Descarboxilasa/inmunología , Linfocitos T Reguladores/inmunología , Animales , Animales Modificados Genéticamente , Técnicas de Inactivación de Genes , HumanosRESUMEN
OBJECTIVE: Efforts to map non-major histocompatibility complex (MHC) genes causing type 1 diabetes in NOD mice identified Slc11a1, formerly Nramp1, as the leading candidate gene in the Idd5.2 region. Slc11a1 is a membrane transporter of bivalent cations that is expressed in late endosomes and lysosomes of macrophages and dendritic cells (DCs). Because DCs are antigen-presenting cells (APCs) known to be critically involved in the immunopathogenic events leading to type 1 diabetes, we hypothesized that Slc11a1 alters the processing or presentation of islet-derived antigens to T-cells. RESEARCH DESIGN AND METHODS: NOD mice having wild-type (WT) or mutant Slc11a1 molecules and 129 mice having WT or null Slc11a1 alleles were examined for parameters associated with antigen presentation. RESULTS: We found that Slc11a1 enhanced the presentation of a diabetes-related T-cell determinant of GAD65, and its function contributed to the activation of a pathogenic T-cell clone, BDC2.5. An enhanced generation of interferon (IFN)-gamma-producing T-cells was also associated with functional Slc11a1. The alteration of immune responsiveness by Slc11a1 genotype did not correlate with altered MHC class II expression in DCs; however, functional Slc11a1 was associated with accelerated phagocytosis and phagosomal acidification in DCs. CONCLUSIONS: The association of variants encoding Slc11a1 with type 1 diabetes may reflect its function in processing and presentation of islet self-antigens in DCs. Thus, non-MHC genes could affect the MHC-restricted T-cell response through altered antigen processing and presentation.
Asunto(s)
Presentación de Antígeno/inmunología , Autoinmunidad/inmunología , Proteínas de Transporte de Catión/fisiología , Islotes Pancreáticos/inmunología , Linfocitos T/inmunología , Animales , Antígeno B7-1/metabolismo , Western Blotting , Proteínas de Transporte de Catión/genética , Proliferación Celular , Células Cultivadas , Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/fisiopatología , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Fagocitosis/fisiología , Linfocitos T/citologíaRESUMEN
Competitive capture is a process by which different determinants of an unfolding antigen compete for binding to the same MHC class II molecule. The "winning" determinant is then dominantly displayed. For self antigens, T cells with specificity for dominantly displayed determinants will be subject to strong tolerance induction. With this in mind we set out to characterize the determinant hierarchy of the junctional region of the Golli-MBP complex. Within this region the MBP 1-9 determinant is known to be a strong inducer of experimental autoimmune encephalomyelitis. We found that the Golli-MBP junctional region contains a triad of three overlapping determinants: LDVM1-5, MBP 1-9, and MBP 7-20. We demonstrate that these three determinants are unique and compete for binding to I-A(u) and that a determinant hierarchy exists with MBP 7-20 being the most dominantly displayed determinant. Because of the prevention of MBP1-9 access to MHC-II, the residual T cell repertoire to this determinant remains complete, thereby permitting its highest affinity members to drive the response, and to convert MBP1-9 into a dominant determinant, despite its poor MHC binding capacity.
Asunto(s)
Presentación de Antígeno , Encefalomielitis Autoinmune Experimental/inmunología , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Linfocitos T/inmunología , Animales , Autoantígenos/inmunología , Unión Competitiva/inmunología , Modelos Animales de Enfermedad , Epítopos de Linfocito T/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Hibridomas , Activación de Linfocitos , Ratones , Esclerosis Múltiple/inmunología , Proteína Básica de Mielina , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/inmunología , Autotolerancia/inmunología , Linfocitos T/metabolismo , Factores de Transcripción/química , Factores de Transcripción/inmunologíaRESUMEN
Immunodominance in self-Ag-reactive pathogenic CD4(+) T cells has been well established in several experimental models. Although it is clear that regulatory lymphocytes (Treg) play a crucial role in the control of autoreactive cells, it is still not clear whether immunodominant CD4(+) Treg clones are also involved in control of autoreactivity. We have shown that TCR-peptide-reactive CD4(+) and CD8(+) Treg play an important role in the spontaneous recovery and resistance from reinduction of experimental autoimmune encephalomyelitis in B10.PL mice. We report, by sequencing of the TCR alpha- and beta-chain associated with CD4(+) Treg, that the TCR repertoire is limited and the majority of CD4(+) Treg use the TCR Vbeta14 and Valpha4 gene segments. Interestingly, sequencing and spectratyping data of cloned and polyclonal Treg populations revealed that a dominant public CD4(+) Treg clonotype expressing Vbeta14-Jbeta1.2 with a CDR3 length of 7 aa exists in the naive peripheral repertoire and is expanded during the course of recovery from experimental autoimmune encephalomyelitis. Furthermore, a higher frequency of CD4(+) Treg clones in the naive repertoire correlates with less severity and more rapid spontaneous recovery from disease in parental B10.PL or PL/J and (B10.PL x PL/J)F(1) mice. These findings suggest that unlike the Ag-nonspecific, diverse TCR repertoire among the CD25(+)CD4(+) Treg population, TCR-peptide-reactive CD4(+) Treg involved in negative feedback regulation of autoimmunity use a highly limited TCR V-gene repertoire. Thus, a selective set of immunodominant Treg as well as pathogenic T cell clones can be targeted for potential intervention in autoimmune disease conditions.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Epítopos Inmunodominantes/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Reguladores/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/inmunología , Modelos Animales de Enfermedad , Femenino , Inmunización , Epítopos Inmunodominantes/química , Ratones , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta/químicaRESUMEN
A diabetes-associated peptide in the glutamic acid decarboxylase 65 (GAD65) molecule, p524-543, activates two distinct populations of T cells, which apparently play opposite roles in the development of diabetes in NOD mice. By comparing the fine specificity of these two T cell repertoires using a nested set of truncated peptides that cover the p524-543 region, we found, surprisingly, that all clones recognized the same core within this peptide, p530-539. The core itself was non-immunogenic, but the residues flanking this shared sequence played the crucial role in selecting T cells to activate. A peptide missing N-terminal flanking residues at position 528 and 529 was stimulatory in NOD but not in MHC-matched, NOD-resistant (NOR) mice, suggesting that a protective response in the resistant mice may require T cell recognition of one or more of the N-terminal flanking residues. T cell repertoire studies demonstrated selective clonal expansions within the BV4 TCR family that dominates the p524-543 response in NOD but not in NOR mice. These data suggest that processing or trimming events affecting T cell recognition of very few flanking residues of diabetes-associated determinants might be involved in the protective response in NOR mice.
Asunto(s)
Antígenos/inmunología , ADN Intergénico/genética , Diabetes Mellitus/enzimología , Diabetes Mellitus/inmunología , Glutamato Descarboxilasa/metabolismo , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Animales , Diabetes Mellitus/genética , Glutamato Descarboxilasa/química , Glutamato Descarboxilasa/genética , Inmunización , Activación de Linfocitos/efectos de los fármacos , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/efectos de los fármacosRESUMEN
We examined the TCR repertoire used by naive SJL mice in their in vitro spontaneous response to proteolipid protein (PLP) 139-151 by Vbeta-Jbeta spectratyping and compared it to that used after immunization with the peptide. T cells from immunized mice use the public rearrangement Vbeta10-Jbeta1.1, but naive mice do not; in contrast, TCR CDR3-beta rearrangements of Vbeta18-Jbeta1.2 and Vbeta19-Jbeta1.2 consistently are associated with the spontaneous response. T cells involved in spontaneous and induced responses can each recognize PLP(139-151) presented in vivo, but its s.c. administration has different consequences for the two repertoires. Four days after immunization, T cells associated with spontaneous responsiveness appear in the draining lymph nodes but disappear by day 10 and never appear elsewhere. Simultaneously, Vbeta10-Jbeta1.1 T cells are likewise activated in the lymph nodes by day 4 and spread to the spleen by day 10. Eight- to 10-wk-old naive mice use a narrower repertoire of TCRs than do immunized age-matched mice. Induced Vbeta10-Jbeta1.1 T cells home to the CNS during experimental autoimmune encephalomyelitis, whereas we failed to detect Vbeta18-Jbeta1.2 and Vbeta19-Jbeta1.2 TCR rearrangements in the CNS. Thus, we observe that administration of PLP(139-151) primes a T cell repertoire distinct from the one responsible for spontaneous responsiveness. This "immunized" repertoire substitutes for the naive one and becomes dominant at the time of disease onset.