RESUMO
CD4(+) Foxp3(+) T regulatory cells (Tregs ) are essential for maintaining immunological tolerance, which could be harnessed for novel cell-based therapies to prevent allograft rejection and control autoimmunity. However, the use of Tregs for therapy is hindered by the inability to generate sufficient cell numbers to inhibit desired immune response(s) and achieve stable engraftment of the donor-Treg cell inoculums. The present study was undertaken to investigate the in vivo requirements to promote engraftment of adoptively transferred Tregs and induce tolerance. We established that not only is peripheral space required, but competition from endogenous Tregs must be minimized for successful donor-Treg engraftment with IL-2 critical for driving their proliferation and survival. Moreover, these studies revealed a critical level of donor-Treg engraftment was required for tolerance induction to skin transplants. These mouse studies lay the foundation for development of novel Treg approaches for tolerance induction in the clinic involving not only organ or cellular transplantation, but also to re-establish self-tolerance in autoimmune settings.
Assuntos
Autoimunidade , Rejeição de Enxerto/imunologia , Subunidade alfa de Receptor de Interleucina-2/fisiologia , Linfócitos T Reguladores/imunologia , Doadores de Tecidos , Tolerância ao Transplante/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Feminino , Imunoterapia , Interleucina-2/imunologia , Interleucina-2/metabolismo , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dermatopatias/imunologia , Dermatopatias/metabolismo , Dermatopatias/terapia , Transplante de PeleRESUMO
Interleukin (IL)-2 and IL-15 are redundant in stimulating T-cell proliferation in vitro. Their precise role in vivo in governing T-cell expansion and T-cell homeostasis is less clear. Each may have distinct functions and regulate distinct aspects of T-cell activation. The functional receptors for IL-2 and IL-15 consist of a private alpha-chain, which defines the binding specificity for IL-2 or IL-15, and shared IL-2 receptor beta- and gamma-chains. The gamma-chain is also a critical signaling component of IL-4, IL-7 and IL-9 receptors. Thus, the gamma-chain is called the common gamma or gamma-c. As these receptor subunits can be expressed individually or in various combinations resulting in the formation of receptors with different affinities, distinct signaling capabilities or both, we hypothesized that differential expression of IL-2 and IL-15 receptor subunits on cycling T cells in vivo may direct activated T cells to respond to IL-2 or IL-15, thereby regulating the homeostasis of T-cell response in vivo. By observing in vivo T-cell divisions and expression of IL-2 and IL-15 receptor subunits, we demonstrate that IL-15 is a critical growth factor in initiating T cell divisions in vivo, whereas IL-2 limits continued T-cell expansion via downregulation of the gamma-c expression. Decreased gamma-c expression on cycling T cells reduced sustained Bcl-2 expression and rendered cells susceptible to apoptotic cell death. Our study provides data that IL-2 and IL-15 regulate distinct aspects of primary T-cell expansion in vivo.
Assuntos
Apoptose/fisiologia , Interleucina-15/fisiologia , Interleucina-2/fisiologia , Linfócitos T/citologia , Animais , Ativação Linfocitária/fisiologia , CamundongosRESUMO
Stimulation of a class II-restricted, antigen-specific T cell clone with interleukin 2 (IL-2) resulted in substantial increases in both cell surface IL-2 receptor (IL-2-R) and cytoplasmic IL-2-R messenger RNA (mRNA), whereas no increase was observed for cell-surface expression of Thy-1 and L3T4 antigens, and only a modest increase in Thy-1 mRNA was observed. These experiments demonstrate that, after initial acquisition of the IL-2-R, IL-2 as well as antigen is able to directly upregulate both the level of IL-2-R mRNA and cell surface IL-2-R molecules.
Assuntos
Interleucina-2/imunologia , Receptores Imunológicos/biossíntese , Linfócitos T/imunologia , Animais , Membrana Celular/imunologia , Células Clonais/imunologia , Citoplasma/metabolismo , Ativação Linfocitária , Camundongos , Peptídeos/imunologia , Polímeros , RNA Mensageiro/metabolismo , Receptores de Interleucina-2 , Linfócitos T/metabolismoRESUMO
Mice that lack a functional gamma c subunit of the receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 display profound defects in lymphoid development. The IL-7/IL-7R system represents a critical interaction for conventional T and B cell development. In this report, the role of IL-7R alpha in the development of lymphoid lineages other than conventional T and B cells was examined. We demonstrate that gamma delta + T cells were absent in IL-7R alpha-deficient mice, whereas the development and function of natural killer cells were normal. Thus, IL-7R alpha function is required for the development of gamma delta + T cells but not natural killer cells.
Assuntos
Antígenos CD/fisiologia , Células Matadoras Naturais/citologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Interleucina/fisiologia , Subpopulações de Linfócitos T/citologia , Animais , Diferenciação Celular , Citometria de Fluxo , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-7 , Baço/citologia , Subpopulações de Linfócitos T/imunologia , Timo/citologiaRESUMO
This study has characterized the primary T cell subpopulations that secrete IL-2 in response to recognition of either class I or class II MHC encoded determinants. The addition to culture of anti-IL-2-R mAb inhibited the consumption of IL-2 by activated lymphocytes during the response period, permitting a much more accurate assessment of the amount of IL-2 produced in the response cultures. Using this response system, we found that primary T cell populations contain two IL-2-secreting T cell subsets that express reciprocal phenotypes and different MHC recognition specificities: an L3T4+, Lyt-2- T cell subset responsive to both class I and class II MHC alloantigens, and an L3T4-Lyt-2+ T cell subset responsive only to class I MHC alloantigens. The L3T4+ T cell subset expressed a broad functional response repertoire in that L3T4+ T cells were triggered to secrete IL-2 upon recognition of unmodified self-Ia determinants, allogeneic Ia determinants, and class I alloantigens presented by self-Ia determinants. The activation of L3T4+ IL-2-secreting T cells, even those responsive to class I MHC alloantigens, could be blocked completely by anti-Ia mAbs, confirming that the L3T4+ T cell subset was in fact class II restricted. In contrast, the Lvt-2+ T cell subset expressed a narrow functional response repertoire in that they were triggered to secrete IL-2 only in response to allogeneic class I MHC determinants, and were not triggered to secrete IL-2 even in response to TNP-modified self-MHC determinants. The specificity of Lyt-2+ IL-2-secreting T cells for class I MHC allodeterminants was confirmed by the observations that: (a) their activation could be blocked completely by anti-class I mAbs, (b) they could be triggered by Ia- cell lines which expressed class I MHC alloantigens and possessed accessory function, and (c) they responded to class I MHC alloantigens but failed to respond to class II MHC alloantigens, even in the presence of exogenously added second signals that circumvented the requirement for alloantigen-bearing accessory cells. Finally, the frequency of primary Lyt-2+ T cells that secreted IL-2 in response to class I (Kbm1) MHC alloantigens was shown to be only minimally lower than that of L3T4+ T cells that secreted IL-2 in response to class II (I-Abm12) MHC alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Celular , Interleucina-2/biossíntese , Complexo Principal de Histocompatibilidade , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo , Antígenos de Diferenciação de Linfócitos T , Antígenos de Superfície/análise , Células Cultivadas , Ativação Linfocitária , Camundongos , Receptores Imunológicos/imunologia , Receptores de Interleucina-2 , Linfócitos T/classificação , Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Trinitrobenzenos/imunologiaRESUMO
We have identified a single rat monoclonal antibody, G7, that is a potent inducer of interleukin (IL-2) production from all functioning T cell hybridomas as well as from normal T cells. G7 is also mitogenic for normal T cells and is a very effective inducer of IL-2 receptor expression. On fluorescence-activated cell sorter analysis, G7 recognized a pan-T cell antigen. Immunoprecipitation studies demonstrated that G7 recognized a cell surface molecule of 28-32 kD that appeared to be identical to Thy-1 in coprecipitation studies. In addition, G7 precipitated a protein of 50 kD. The possible relationship of the putative molecular complex identified by G7 on murine cells to the molecular complex identified on human T cells with anti-T3 reagents is discussed. In addition, G7 should prove to be a very useful reagent for studying the early events of lymphocyte activation as well as an inducer of lymphokine-rich supernatants.
Assuntos
Anticorpos Monoclonais/fisiologia , Soro Antilinfocitário/farmacologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Interleucina-2/biossíntese , Interfase , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Camundongos Nus , Ratos , Ratos Endogâmicos Lew , Receptores Imunológicos/imunologia , Receptores de Interleucina-2 , Linfócitos T/citologia , Antígenos Thy-1RESUMO
This study characterizes the T helper (Th) cells that initiate primary cytotoxic T lymphocyte (CTL) responses against allogeneic and trinitrophenyl (TNP)-modified self class I major histocompatibility (MHC) determinants. We show that two distinct Th cell subsets participate in allospecific CTL responses: (a) an L3T4+,Lyt-2- class II-restricted Th cell population, and (b) an L3T4-,Lyt-2+ class I-restricted Th cell population. Both of these T cell subpopulations were shown to function in allospecific CTL responses as helper cells by their ability to show synergy with allospecific CTL precursors. Thus, primary class I allospecific CTL responses represent an immune response involving not only L3T4+ Th cells, but Lyt-2+ Th cells as well. One of the necessary functions performed by both L3T4+ and Lyt-2+ Th cell populations in allospecific CTL responses was found to be the secretion of interleukin 2. Finally, despite the many similarities between anti-allo- and anti-TNP-CTL responses, anti-TNP-CTL responses were found to be mediated by only L3T4+ Th cells, not by Lyt-2+ Th cells. Consequently, Lyt-2+ Th cells appear to be a helper cell population that is primarily involved in MHC-specific immune responses.
Assuntos
Antígenos de Histocompatibilidade/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Antígenos Ly/imunologia , Citotoxicidade Imunológica , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-2/biossíntese , Isoantígenos/imunologia , Camundongos , Quimera por Radiação , Trinitrobenzenos/imunologiaRESUMO
The Ly-6 locus controls the expression and/or encodes for alloantigenic specificities found primarily on subpopulations of murine T and B lymphocytes. We have recently identified and characterized a new rat mAb, D7, that recognizes a nonpolymorphic Ly-6 specificity. After crosslinking by anti-Ig reagents or by Fc receptor-bearing accessory cells, mAb D7 could induce IL-2 production from T cell hybridomas, and in the presence of PMA could trigger a vigorous proliferative response in resting peripheral T cells. The addition of mAb D7 to cultures of antigen- and alloantigen-, but not mitogen-stimulated T cells resulted in a marked augmentation of the proliferative response. A number of other well-characterized mAbs to Ly-6 locus products could also stimulate a T cell proliferative response after crosslinking by anti-Ig and in the presence of PMA. These results strongly suggest that Ly-6 molecules may play a critical role in the T cell activation cascade, either as receptors for an unidentified soluble or cell-associated ligand or as transducing molecules that modulate signals initiated by antigen stimulation of the T3-Ti complex.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Ly/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/fisiologia , Cálcio/metabolismo , Hibridomas/metabolismo , Interleucina-2/metabolismo , Camundongos , Mitógenos/farmacologia , Receptores Fc/fisiologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
B cells cultured with anti-IgM, BSF-p1, and B15-TRF will differentiate into high rate IgM-synthesizing cells in the presence of supernatants from EL-4 cells that have been induced with phorbol myristate acetate. These supernatants contain two molecular species (EL-TRFs) that have differentiative activity. One co-migrates with interleukin 2 (IL-2) and its activity is blocked by antibody to the IL-2 receptor. Furthermore, molecularly cloned IL-2, at concentrations of 100 U/ml or more, expresses such EL-TRF activity. The EL-TRF activity of cloned IL-2 can also be inhibited by antibody to the IL-2 receptor. The other material with EL-TRF activity has a molecular weight of approximately 32,000. This material lacks IL-2 activity. Antibody to the IL-2 receptor does not impair its function. B cells stimulated with anti-IgM and BSF-p1, with or without B15-TRF, express determinants that react with two monoclonal antibodies which recognize distinct epitopes on the T cell IL-2 receptor. These determinants are present at much lower density (approximately 100-fold) on stimulated B cells that on HT-2 cells, an IL-2-dependent T cell line. Very small amounts of [3H]IL-2 (less than 1,000 molecules per cell) bind to activated B cells. These results indicate that IL-2 binds to a receptor on appropriately prepared B cells and causes them to differentiate into high rate IgM-synthesizing cells. The physiologic significance of the B cell differentiative activity of IL-2 remains to be investigated.
Assuntos
Imunoglobulina M/biossíntese , Interleucina-2/fisiologia , Animais , Linfócitos B/metabolismo , Diferenciação Celular/efeitos dos fármacos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Receptores Imunológicos/metabolismo , Receptores de Interleucina-2RESUMO
Antigen-activated T lymphocytes produce within 24 h of stimulation a factor that is indistinguishable biochemically and functionally from the B cell co-stimulating growth factor, BCGF-I, originally identified in induced EL4 supernatants: Supernatants from antigen-stimulated T cell lines are not directly mitogenic for resting B cells, but synergize in an H-2-unrestricted manner with anti-Ig activated B cells to produce polyclonal proliferation but not antibody-forming-cell development; biochemical studies reveal the B cell co-stimulating factor present in antigen-stimulated T cell line supernatants is identical by phenyl Sepharose chromatography and isoelectric focusing (IEF) to EL4 supernatant BCGF-I. We thus conclude that normal T cells produce BCGF-I in response to antigenic stimulation. Analysis of the mechanism of BCGF-I production by antigen-stimulated T cells showed that optimum amounts of BCGF-I were obtained as quickly as 24 h post-stimulation, and that the factor producing cells in the T cell line investigated bore the Lyt-1+2- phenotype. As few as 10(4) T cells produced sufficient BCGF-I to support the proliferation of 5 X 10(4) purified anti-Ig activated B cells. Finally, the activation of normal T cell lines to produce BCGF-I required either antigen presented in the context of syngeneic antigen-presenting cells (APC) or interleukin 2 (IL-2).
Assuntos
Substâncias de Crescimento/metabolismo , Interleucina-2/fisiologia , Linfócitos T/metabolismo , Animais , Linhagem Celular , Cromatografia , Interleucina-4 , Focalização Isoelétrica , Camundongos , Modelos TeóricosRESUMO
TCR-mediated activation of T cell hybridomas induces programmed cell death by a Fas-dependent pathway. We now show that costimulation of 2B4 cells, in the absence or presence of transgenic Bcl-2, with anti-CD3 epsilon and forskolin, an activator of cAMP signaling, resulted in antagonism of Fas-dependent activation-induced cell death that was always accompanied by selective downregulation of the nuclear levels of NF-kappa B p65-p50 (RelA-p50) transcription factor. Forskolin not only inhibited activation-induced cell death and NF-kappa B activation, but also suppressed expression of Fas and Fas ligand (Fas-L). Furthermore, NF-kappa B p65 antisense oligonucleotide down-regulated nuclear levels of NF-kappa B, inhibited cell surface expression of Fas-L and apoptosis of 2B4. Collectively, these finding demonstrate a potential role of NF-kappa B in the regulation of activation-induced apoptosis in T lymphocytes.
Assuntos
Apoptose/fisiologia , Genes bcl-2/fisiologia , Ativação Linfocitária/fisiologia , Glicoproteínas de Membrana/metabolismo , NF-kappa B/fisiologia , Linfócitos T/fisiologia , Receptor fas/fisiologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Complexo CD3/imunologia , Colforsina/farmacologia , Proteína Ligante Fas , Hibridomas , Ativação Linfocitária/genética , Linfócitos T/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Five cytokines, IL-2, IL-4, IL-7, IL-9, and IL-15, form one group that is characterized by utilizing the common gamma chain (gamma c) as a receptor subunit. Examination of the phenotype of various cytokine or cytokine receptor "knockout" mice demonstrates that these cytokines are critical for normal lymphocyte development and subsequent functional activity of the peripheral immune compartment. This review summarizes the structural and functional properties of each of these five cytokines and their receptors, including the known redundant pathways for each cytokine or receptor. The contribution of these cytokines and receptors will then be considered in detail with respect to regulation of T lymphocyte development and homeostasis of the peripheral T cell compartment.
Assuntos
Homeostase , Interleucinas/imunologia , Receptores de Interleucina/imunologia , Linfócitos T/imunologia , Animais , Ligação Genética , Humanos , Interleucinas/química , Interleucinas/classificação , Camundongos , Camundongos Mutantes , Receptores de Interleucina/química , Receptores de Interleucina/classificação , Imunodeficiência Combinada Severa/imunologia , Cromossomo XRESUMO
Mice lacking a functional gamma c subunit of cytokine receptors exhibit profound defects in the development of multiple lymphoid lineages. To investigate the role of gamma c-dependent cytokines in T cell development, the phenotype of developing T cells was compared in interleukin (IL)-7Ralpha-deficient mice and anti-gamma c mAb-treated chimeric mice reconstituted with adult bone marrow cells or subsets of pro-T cells. These studies indicate that gamma c contributes to T cell development at multiple stages of pro-T cell maturation and that IL-7/IL-7R is the primary cytokine for thymic-dependent T cell development. However, our data also implicate other gamma c-dependent cytokines during thymic T cell development. By contrast, substantial intestinal intraepithelial lymphocytes (IEL) development was observed in the intestinal intraepithelium in both types of mice. Analysis of IL-7Ralpha-deficient mice indicates that the IL-7/IL-7R system is critical only for the development of TCR gammadelta+ IEL. However, the inhibitory activity of the anti-deltac mAb in the chimeric mouse model suggests that additional gamma cutilizing cytokines regulate the development of the remaining subsets of IEL.
Assuntos
Anticorpos Monoclonais/farmacologia , Citocinas/fisiologia , Cadeias gama de Imunoglobulina/imunologia , Receptores de Citocinas/antagonistas & inibidores , Linfócitos T/citologia , Animais , Quimera , Células Epiteliais/citologia , Feminino , Interleucina-7/fisiologia , Intestinos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina/análise , Receptores de Interleucina/deficiência , Receptores de Interleucina/metabolismo , Receptores de Interleucina-17 , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Linfócitos T/efeitos dos fármacos , Timo/citologiaRESUMO
Although the mouse IL-2 receptor (IL-2R) beta and gamma c subunits have been identified by molecular cloning, the biochemical identity of these subunits has not yet been established. In the present study, the mouse IL-2R was biochemically characterized from cell lines expressing normal and aberrant IL-2R. Using novel monoclonal antibodies specific for the beta or gamma c subunits, we established that the M(r) of the beta chain is 90,000-100,000 and that of the gamma c subunit is 75,000-80,000. Analysis of transfected EL4 cells that expressed alpha, gamma c, and truncated beta subunits or mutant EL4 cells, which selectively lacked cell surface gamma c, revealed that no other material migrated to a position on SDS-PAGE characteristic of IL-2/IL-2R beta and IL-2/IL-2R gamma c cross-linked complexes, respectively. Thus, the beta and gamma c subunits appear to be the sole IL-2R constituents of these IL-2 cross-linked complexes. The IL-2/IL-2R gamma c, but not the IL-2/IL-2R beta, complex exhibited enhanced mobility after SDS-polyacrylamide gel electrophoresis under nonreducing conditions, suggesting a more compact structure for gamma c as a result of intrachain disulfide bonds. The primary posttranslational modification of the mouse beta and gamma c subunits is N-linked glycosylation. These biochemical studies reconcile past uncertainties concerning the subunit composition of the mouse IL-2R and are consistent with a model of the IL-2R containing only three subunits.
Assuntos
Receptores de Interleucina-2/química , Amidoidrolases/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Células Cultivadas , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Glicosilação , Camundongos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Receptores de Interleucina-2/imunologia , Receptores de Interleucina-2/isolamento & purificação , Receptores de Interleucina-2/metabolismoRESUMO
The Ly-6 locus encodes a group of cell surface molecules found predominantly on lymphoid cells in the mouse. These proteins share several structural and functional characteristics with Thy-1, a molecule expressed in both lymphoid and neuronal tissue. Utilizing anti-Ly-6A/E monoclonal antibodies, the present results demonstrate in situ expression of these molecules in brain tissue. The findings also indicated that these molecules are not expressed during embryonic or neonatal stages of development. Moreover, although Ly-6b haplotype mice exhibited staining primary associated with vascular elements throughout the brain, Ly-6a mice exhibited staining predominantly associated with white matter limited to the hippocampal and midbrain regions. Although cultured glial and neuronal cells expressed marginally detectable levels of Ly-6A/E, the majority of GFAP+ cells in these cultures expressed high levels of Ly-6A/E following incubation with cytokines including rIFN-gamma. In addition, northern blot analysis of RNA from enriched astrocytic cultures corroborated the induction of Ly-6A/E expression. These findings have therefore established that Ly-6 is amongst those groups of genes expressed in both brain and lymphoid tissues.
Assuntos
Envelhecimento/metabolismo , Antígenos Ly/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica , Medula Espinal/metabolismo , Animais , Anticorpos Monoclonais , Antígenos de Superfície/metabolismo , Astrócitos/citologia , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Células Cultivadas , Imuno-Histoquímica , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimentoRESUMO
Serologic and structural comparisons of the rabbit IgA-g allotypes revealed that 1) the IgA-g allotypes have multiple allotypic determinant sites, 2) the g74, g76 and g77 allotypic specificities have several allotypic determinants in common whereas g75 molecules do not appear to have allotypic determinants in common with g74, g76 and g77 molecules, 3) the partial amino acid sequence of alpha chain from g75 and g76 Fc2alpha fragments differ by at least one amino acid residue, and 4) the g74 alpha-chains may have the "extra" intradomain disulfide bond in the Calpha2 domain whereas the g75 and g76 alpha chains lack this disulfide bond. Thus, multiple mutational events must have occurred during the evolution of g74, g75 and g77 genes.
Assuntos
Imunoglobulina A/classificação , Alótipos de Imunoglobulina/classificação , Sequência de Aminoácidos , Animais , Reações Cruzadas , Dissulfetos , Epitopos , Imunoglobulina A/imunologia , Fragmentos Fab das Imunoglobulinas , Fragmentos Fc das Imunoglobulinas , CoelhosRESUMO
As an initial approach to macrophage (M phi) antigen handling, guinea pig M phi pulsed with the radiolabeled terpolymer L-glutamic acid, L-lysine, L-tyrosine (GLT) were modified with the cell surface-specific probe 2,4,6-trinitrobenzenesulfonic acid either immediately after pulsing (fresh) or 24 h after pulsing (aged); nonidet P-40 extracts were prepared from these cells and analysis of these extracts with anti-trinitrophenyl antisera was performed in a quantitative radioimmunoassay. Such experiments indicated that fresh GLT-pulsed M phi contained a pool of intracellular GLT as well as a distinct pool of cell surface GLT. In contrast, aged GLT-pulsed M phi lacked intracellular GLT but expressed GLT on the cell surface. In addition, a pool of cell surface GLT was also detected on Ia-M phi and on M phi from guinea pigs which were nonresponders to GLT. Thus, this study provides a direct demonstration of the presence of exogenous antigen on the M phi cell surface. However, the cell surface expression of GLT does not appear to be an exclusive property expressed by functional Ia+ antigen-presenting M phi.
Assuntos
Antígenos de Superfície/análise , Macrófagos/análise , Linfócitos T/imunologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Sobrevivência Celular , Relação Dose-Resposta Imunológica , Genes MHC da Classe II , Cobaias , Hemocianinas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/metabolismo , Octoxinol , Peptídeos/imunologia , Polietilenoglicóis/farmacologia , Polímeros , Radioimunoensaio , Linfócitos T/análise , Linfócitos T/metabolismo , Trinitrobenzenos/imunologiaRESUMO
The IL 2 receptor isolated from the IL 2-dependent CTL-L cell line was subjected to biochemical analysis. Pulse-chase and tunicamycin studies, as well as digestion with the endoglycosidases, Endo-F and Endo-H, of 35S-methionine-labeled IL 2 receptors suggested a single protein precursor of 32,000 (p32) daltons. The p32 precursor was rapidly processed by addition of high-mannose-containing core N-linked sugars to intracytoplasmic precursor intermediates of 38,000 (p38) and 40,000 (p40) daltons, which undergo further processing to yield a mature surface receptor with heterogeneous apparent m.w. of 52,000 to 65,000 (p58). Two-dimensional gel studies indicated that p58 exhibited broad charge heterogeneity between pH 4.6 and 6.3. Endo-F digestions of p58 shifted the isoelectric focus point to a more basic 5.5 to 7.4. This considerable charge heterogeneity is consistent with the possibility that other posttranslational modifications to the mouse IL 2 receptor occur besides addition of complex N-linked glycans. Immunoprecipitations of the IL 2 receptor from surface iodinated cells also revealed an additional band at 110,000 (p110) daltons. IEF vs SDS-PAGE two-dimensional gel studies demonstrated that p110 also had an isoelectric focus point identical to p58. Western blot studies with an anti-IL 2 receptor monoclonal antibody (7D4) demonstrated that p38, p40, p58, and p110 each expressed the epitope recognized by this antibody. Thus, it is likely that p110 is not a unique molecule that coprecipitates with the IL 2 receptor. Western blot analysis of mitogen-stimulated T and B lymphocytes also revealed bands similar to p58 and p110, although these bands had an average apparent m.w. 3000 to 6000 less than those seen for CTL-L cells.
Assuntos
Interleucina-2/metabolismo , Receptores Imunológicos/isolamento & purificação , Animais , Anticorpos Monoclonais , Reações Antígeno-Anticorpo , Colódio , Eletroforese em Gel de Poliacrilamida , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Peso Molecular , Papel , Testes de Precipitina , Receptores Imunológicos/biossíntese , Receptores Imunológicos/imunologia , Receptores de Interleucina-2 , Dodecilsulfato de Sódio , Linfócitos T Citotóxicos/metabolismoRESUMO
The proteins cross-linked to the IL-2R p55 subunit were biochemically compared in distinct cell populations that varied in their capacity to express high affinity IL-2R. We directly cross-linked p75 to p55 in the absence of IL-2 for the cell populations that bear only high affinity IL-2R. Furthermore, although no endogenous IL-2 production was detected, p75 was readily cross-linked to p55 for EL4J-3.4, a p55 transfectant of EL4 that bears high affinity IL-2R. These results strongly suggest that high affinity IL-2R exist as a preformed heterodimer of p55 and p75 which do not require IL-2 for their association. Furthermore, cross-linking of three other proteins of apparent Mr of 100,000, 135,000, and 180,000 to p55 was also seen, raising the possibility of a more complex subunit composition for the IL-2R.