RESUMEN
The generation of thymic NK1.1(+)alpha beta T (NKT) cells involves positive selection of cells enriched for V(alpha)14/V(beta)8 TCR by CD1d MHC class I molecules. However, it has not been determined whether positive selection is preceded by pre-TCR-dependent beta selection. Here we studied NKT cell development in CD3 signaling-deficient mice (CD3 zeta/eta(-/-) and/or p56(lck-/-)) and TCR alpha-deficient mice. In contrast to wild-type mice, NK1.1(+) thymocytes in CD3 signaling-deficient mice are approximately 10-fold reduced in number, do not exhibit V(alpha)14-J(alpha)281 rearrangements and fail to express alpha beta TCR at the cell surface. However, they exhibit TCR beta VDJ rearrangements and pre-T alpha mRNA, suggesting that they contain pre-NKT cells. Strikingly, pre-NKT cells of CD3 zeta/Lck double-deficient mice fail to express TCR beta mRNA and protein. Whereas in wild-type NKT cells TCR beta VDJ junctions are selected for productive V(beta)8 and against productive V(beta)5 rearrangements, V(beta)8 and V(beta)5 rearrangements are non-selected in pre-NKT cells of CD3 signaling-deficient mice. Thus, pre-NKT cell development in CD3 signaling-deficient mice is blocked after rearrangement of TCR beta VDJ genes but before expression of TCR beta proteins. Most NKT cells of TCR alpha-deficient mice exhibit cell surface gamma delta TCR. In contrast to pre-NKT cells of CD3 signaling-deficient mice, approximately 25% of NKT cells of TCR alpha-deficient mice exhibit intracellular TCR beta polypeptide chains. Moreover, both V(beta)8 and V(beta)5 families are selected for in-frame VDJ joints in the TCR beta(+) NKT cell subset of TCR alpha-deficient mice. The data suggest that CD3 signals regulate initial TCR beta VDJ gene expression prior to beta selection in developing pre-NKT cells.
Asunto(s)
Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Células Asesinas Naturales/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Citometría de Flujo , Receptores de Hialuranos/metabolismo , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Precursores del ARN/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/biosíntesis , Complejo Receptor-CD3 del Antígeno de Linfocito T/deficiencia , Receptores de Antígenos de Linfocitos T alfa-beta/deficienciaAsunto(s)
Complejo CD3/metabolismo , Células Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T/inmunología , Animales , Ciclo Celular , Diferenciación Celular , Regulación de la Expresión Génica , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Células Asesinas Naturales/citología , Ratones , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/citologíaRESUMEN
Immunotherapeutic strategies in advanced stages of solid tumors have generally met with little success. Various mechanisms have been discussed permitting the escape of tumor cells from an effective antitumoral immune response. Solid tumors are known to develop regions with acidic interstitial pH. In a recent study performed in the human system, we were able to demonstrate that non-MHC-restricted cytotoxicity is inhibited by an acidic microenvironment. To get more insight into the mechanisms leading to this reduced cytotoxic activity, we have now investigated the influence of an acidic extracellular pH (pH(e)) on the killing process in detail. Unstimulated PBMC and LAK cells were used as effector cells. Both populations are able to kill tumor cells in a MHC-independent manner via perforin/granzymes or TNFalpha, whereas only IL-2-activated cells can use the killing pathway via Fas/FasL. We studied the influence of a declining pH(e) on the different killing pathways against TNFalpha-sensitive and -resistant, as well as Fas-positive and -negative, target cells. Experiments in the absence of extracellular Ca(2+) were used to discriminate the Ca(2+)-dependent perforin-mediated killing. Here we show that the release of perforin/granzyme-containing granules, the secretion of TNFalpha, and also the cytotoxic action of Fas/FasL interaction or of membrane-bound TNFalpha were considerably inhibited by declining pH(e). Furthermore, the secretion of the activating cytokine IFNgamma, as well as the release of the down-regulating cytokines IL-10 and TGF-beta(1), was strictly influenced by surrounding pH. As a pH(e) of 5.8 resulted in a nearly complete loss of cytotoxic effector cell functions without affecting their viability, we investigated the influence of pH(e) on basic cellular functions, e.g. , mitochondrial activity and regulation of intracellular pH. We found an increasing inhibition of both functions with declining pH(e). Therefore, an acidic pH(e) obviously impairs fundamental cellular regulation, which finally prevents the killing process. In summary, our data show a strict pH(e) dependence of various killer cell functions. Thus, an acidic microenvironment within solid tumors may contribute to the observed immunosuppression in vivo, compromising antitumoral defense and immunotherapy in general, respectively.