RESUMEN
Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LATG135D) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LATG135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LATG135D T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LATG135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Linfocitos T , Ratones , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Receptores de Antígenos de Linfocitos T/metabolismo , Activación de Linfocitos , Fosforilación , Fosfoproteínas/genéticaRESUMEN
The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the in vitro analyses of CD8 T cell responses to this epitope make use of an altered peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.
Asunto(s)
Antígenos Virales/inmunología , Glicoproteínas/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Proteínas Virales/inmunología , Traslado Adoptivo , Animales , Antígenos Virales/química , Antígenos Virales/genética , Antígenos CD8/metabolismo , Citocinas/metabolismo , Epítopos/química , Epítopos/inmunología , Glicoproteínas/química , Glicoproteínas/genética , Antígenos H-2/inmunología , Activación de Linfocitos/inmunología , Coriomeningitis Linfocítica/virología , Ratones , Ratones Transgénicos , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Unión Proteica , Transducción de Señal , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
In response to infection, naïve CD4+ T cells differentiate into two subpopulations: T follicular helper (TFH) cells, which support B cell antibody production, and non-TFH cells, which enhance innate immune cell functions. Interleukin-2 (IL-2), the major cytokine produced by naïve T cells, plays an important role in the developmental divergence of these populations. However, the relationship between IL-2 production and fate determination remains unclear. Using reporter mice, we found that differential production of IL-2 by naïve CD4+ T cells defined precursors fated for different immune functions. IL-2 producers, which were fated to become TFH cells, delivered IL-2 to nonproducers destined to become non-TFH cells. Because IL-2 production was limited to cells receiving the strongest T cell receptor (TCR) signals, a direct link between TCR-signal strength, IL-2 production, and T cell fate determination has been established.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Expresión Génica , Interleucina-2/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Cromatina/metabolismo , Genes Reporteros , Activación de Linfocitos/genética , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Antígenos de Linfocitos T/genética , Factores de Transcripción/metabolismoRESUMEN
TCR affinity for peptide MHC dictates the functional efficiency of T cells and their propensity to differentiate into effectors and form memory. However, in the context of chronic infections, it is unclear what the overall profile of TCR affinity for Ag is and if it differs from acute infections. Using the comprehensive affinity analysis provided by the two-dimensional micropipette adhesion frequency assay and the common indirect affinity evaluation methods of MHC class II tetramer and functional avidity, we tracked IAb GP61-80-specific cells in the mouse model of acute (Armstrong) and chronic (clone 13) lymphocytic choriomeningitis virus infection. In each response, we show CD4 T cell population affinity peaks at the effector phase and declines with memory. Of interest, the range and average relative two-dimensional affinity was equivalent between acute and chronic infection, indicating chronic Ag exposure did not skew TCR affinity. In contrast, functional and tetramer avidity measurements revealed divergent results and lacked a consistent correlation with TCR affinity. Our findings highlight that the immune system maintains a diverse range in TCR affinity even under the pressures of chronic Ag stimulation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Glicoproteínas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Afinidad de Anticuerpos/inmunología , Ligando CD27/metabolismo , Femenino , Memoria Inmunológica/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
A robust primary immune response has been correlated with the precursor number of antigen-specific T cells, as identified using peptide MHCII tetramers. However, these tetramers identify only the highest-affinity T cells. Here we show the entire CD4+ T-cell repertoire, inclusive of low-affinity T cells missed by tetramers, using a T-cell receptor (TCR) signalling reporter and micropipette assay to quantify naive precursors and expanded populations. In vivo limiting dilution assays reveal hundreds more precursor T cells than previously thought, with higher-affinity tetramer-positive T cells, comprising only 5-30% of the total antigen-specific naive repertoire. Lower-affinity T cells maintain their predominance as the primary immune response progresses, with no enhancement of survival of T cells with high-affinity TCRs. These findings demonstrate that affinity for antigen does not control CD4+ T-cell entry into the primary immune response, as a diverse range in affinity is maintained from precursor through peak of T-cell expansion.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Precursoras de Linfocitos T/inmunología , Adyuvantes Inmunológicos , Traslado Adoptivo , Animales , Adyuvante de Freund , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Noqueados , Mycobacterium tuberculosis/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Toxina del Pertussis/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Bazo/citología , Regulación hacia ArribaRESUMEN
T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below â¼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.
Asunto(s)
ADN/administración & dosificación , Activación de Linfocitos , Mecanotransducción Celular , Nanopartículas del Metal/administración & dosificación , Receptores de Antígenos de Linfocitos T/fisiología , Fenómenos Biomecánicos , Antígenos CD8/fisiología , Calcio/metabolismo , Oro , Humanos , Molécula 1 de Adhesión Intercelular/fisiología , Antígeno-1 Asociado a Función de Linfocito/fisiologíaRESUMEN
Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC, the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming, low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo, as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice, but not in high affinity-primed mice. Mechanistically, low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge, low affinity-primed CD45RB(hi) cells became CD45RB(lo), demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus, these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency, low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Rechazo de Injerto/inmunología , Antígenos Comunes de Leucocito/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Trasplante de Piel , Animales , Diferenciación Celular , Células Cultivadas , Reacciones Cruzadas , Rechazo de Injerto/prevención & control , Memoria Inmunológica , Antígenos Comunes de Leucocito/genética , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Mutación , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Linfocitos T CD8-positivos/citología , Proliferación Celular , Supervivencia Celular , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad/metabolismo , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Monitorización Inmunológica , Transducción de SeñalRESUMEN
Antigen receptor diversity underpins adaptive immunity by providing the ground for clonal selection of lymphocytes with the appropriate antigen reactivity. Current models attribute T cell clonal selection during the immune response to T-cell receptor (TCR) affinity for either foreign or self peptides. Here, we report that clonal selection of CD4(+) T cells is also extrinsically regulated by B cells. In response to viral infection, the antigen-specific TCR repertoire is progressively diversified by staggered clonotypic expansion, according to functional avidity, which correlates with self-reactivity. Clonal expansion of lower-avidity T-cell clonotypes depends on availability of MHC II-expressing B cells, in turn influenced by B-cell activation. B cells clonotypically diversify the CD4(+) T-cell response also to vaccination or tumour challenge, revealing a common effect.
Asunto(s)
Linfocitos B/fisiología , Células Clonales/fisiología , Linfocitos T Colaboradores-Inductores/fisiología , Animales , Células de la Médula Ósea , Linfocitos T CD4-Positivos/fisiología , Células Clonales/inmunología , Regulación de la Expresión Génica/fisiología , Ratones , Ratones EndogámicosRESUMEN
Signal regulatory protein α (SIRPα), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRPα binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of innate immune defense against pathogens. However, little is known about molecular details of the structural interaction of Sp-D with SIRPs. In the present work, we examined the molecular basis of Sp-D binding to SIRPα using domain-deleted mutant proteins. We report that Sp-D binds to the membrane-proximal Ig domain (D3) of SIRPα in a calcium- and carbohydrate-dependent manner. Mutation of predicted N-glycosylation sites on SIRPα indicates that Sp-D binding is dependent on interactions with specific N-glycosylated residues on the membrane-proximal D3 domain of SIRPα. Given the remarkable sequence similarity of SIRPα to SIRPß and the lack of known ligands for the latter, we examined Sp-D binding to SIRPß. Here, we report specific binding of Sp-D to the membrane-proximal D3 domain of SIRPß. Further studies confirmed that Sp-D binds to SIRPα expressed on human neutrophils and differentiated neutrophil-like cells. Because the other known ligand of SIRPα, CD47, binds to the membrane-distal domain D1, these findings indicate that multiple, distinct, functional ligand binding sites are present on SIRPα that may afford differential regulation of receptor function.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Antígeno CD47/metabolismo , Calcio/metabolismo , Neutrófilos/metabolismo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Receptores Inmunológicos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación/genética , Antígeno CD47/genética , Células CHO , Cricetinae , Cricetulus , Glicosilación , Células HEK293 , Células HL-60 , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Proteína D Asociada a Surfactante Pulmonar/genética , Receptores Inmunológicos/genética , Eliminación de SecuenciaRESUMEN
Interaction of SIRPα with its ligand, CD47, regulates leukocyte functions, including transmigration, phagocytosis, oxidative burst, and cytokine secretion. Recent progress has provided significant insights into the structural details of the distal IgV domain (D1) of SIRPα. However, the structural roles of proximal IgC domains (D2 and D3) have been largely unstudied. The high degree of conservation of D2 and D3 among members of the SIRP family as well as the propensity of known IgC domains to assemble in cis has led others to hypothesize that SIRPα forms higher order structures on the cell surface. Here we report that SIRPα forms noncovalently linked cis homodimers. Treatment of SIRPα-expressing cells with a membrane-impermeable cross-linker resulted in the formation of SDS-stable SIRPα dimers and oligomers. Biochemical analyses of soluble recombinant extracellular regions of SIRPα, including domain truncation mutants, revealed that each of the three extracellular immunoglobulin loops of SIRPα formed dimers in solution. Co-immunoprecipitation experiments using cells transfected with different affinity-tagged SIRPα molecules revealed that SIRPα forms cis dimers. Interestingly, in cells treated with tunicamycin, SIRPα dimerization but not CD47 binding was inhibited, suggesting that a SIRPα dimer is probably bivalent. Last, we demonstrate robust dimerization of SIRPa in adherent, stimulated human neutrophils. Collectively, these data are consistent with SIRPα being expressed on the cell surface as a functional cis-linked dimer.