_:b207643700 . _:b37533 . _:b37541 . _:b37527 . _:b37534 . . _:b37535 . . _:b37536 "NK T cells normally comprise a small percentage of the lymphocytes present in the spleen and lymph nodes (1\u20133); however, these cells are abundant in the liver (20, >>30<<). To quantitate peripheral NK T cells, lymphocytes from \u03B3c+, \u03B3c\u2212, or \u03B22m\u2212/\u2212 mice were isolated from the liver and spleen and the amount of V\u03B114\u2013J\u03B1281 mRNA was determined. NK T cells were clearly present in the liver and spleens of \u03B3c+" . _:b37528 . _:b37529 . . _:b37530 . _:b207643682 . _:b37531 . _:b37531 . _:b37532 . _:b37540 . _:b37505 . _:b37541 . _:b207643685 . . _:b207643672 . . _:b37536 . _:b37509 . _:b37537 . _:b37518 . _:b37512 "\u03B22m\u2212/\u2212 cells were used as control as it has been previously shown that NK T cells require the \u03B22m-associated CD1 molecules in order to be selected efficiently (4, 5, >>10<<, 11, 15). As shown in Fig. 1, C\u03B1 transcripts were found equally in all three cDNA preparations. The amount of V\u03B114\u2013J\u03B1281 mRNA was similar in both \u03B3c+ and \u03B3c\u2212 thymi (within threefold) and largely increased relative to \u03B22m\u2212/\u2212 thymi, which" . _:b37538 . _:b37519 . _:b37539 . _:b37512 . _:b207643689 . _:b207643686 . _:b207643669 . . _:b207643675 . _:b207643697 . _:b37513 "\u03B22m\u2212/\u2212 cells were used as control as it has been previously shown that NK T cells require the \u03B22m-associated CD1 molecules in order to be selected efficiently (4, 5, 10, >>11<<, 15). As shown in Fig. 1, C\u03B1 transcripts were found equally in all three cDNA preparations. The amount of V\u03B114\u2013J\u03B1281 mRNA was similar in both \u03B3c+ and \u03B3c\u2212 thymi (within threefold) and largely increased relative to \u03B22m\u2212/\u2212 thymi, which lack" . _:b37530 . _:b37532 "Exogenous IL-7 was able to restore the IL-4 response in vitro (>>29<<). NK thymocytes from \u03B3c\u2212 mice also manifest abnormal IL-4 production in vitro in the absence of IL-7R\u03B1 engagement; however, this can be restored with TSLP (Table 1)." . _:b37540 "Further studies using IL-2R\u03B2\u2013deficient mice (which can be considered as deficient in IL-2 and IL-15) (32) and IL-7R\u03B1-deficient mice (which inactivate IL-7 and TSLP) (>>33<<) should help to elucidate the \u03B3c-dependent interactions required for induction of NKR-P1 and Ly49 antigens on NK T cells and their export into the periphery." . _:b207643693 . _:b207643695 . _:b37537 . . _:b37514 . _:b37517 . _:b37502 "Reverse transcription and quantitative PCR amplification were carried out as previously described (>>23<<) using oligonucleotides specific for C\u03B1, V\u03B114, and J\u03B1281 (10)." . _:b37502 . . _:b37523 . _:b207643673 . _:b37535 . . _:b207643668 . _:b207643699 . _:b37525 . _:b37538 . _:b37499 "Liver lymphocytes were isolated using discontinuous Percoll gradients (>>20<<) with minor modifications." . _:b37501 "Total RNA was extracted with acid\u2013 guanidinium (>>22<<) and ethanol-precipitated with the addition of 5 \u03BCg of glycogen before resuspension in 20 \u03BCl of DEPC water." . _:b37527 "The unique ability of NK T cells to produce IL-4 after TCR triggering has been one main characteristic of this lymphoid subset (1, >>13<<, 14). When CD8\u2212 thymocytes from \u03B3c+ or \u03B3c\u2212 mice were cultured in vitro with soluble CD3 and antigen-presenting cells, IL-4 release could be detected in the supernatants from \u03B3c+ and \u03B3c\u2212 cells (Table 1), although IL-4 production from \u03B3c\u2212" . _:b37540 . . . _:b37516 "The TCR-\u03B2 repertoire of DN NK T cells is highly restricted, with \u223C50% of cells using V\u03B28 (4, >>7<<, 10). DN thymocytes from both \u03B3c+ and \u03B3c\u2212 mice contained a population of V\u03B28+ cells (\u223C5%), which was not detected in DN thymocytes from \u03B22m\u2212/\u2212 mice. Percentages of V\u03B28+ cells amongst mature (HSAlo) DN thymocytes were also comparable" . . . _:b37522 "The coabsence of Ly49 family molecules on \u03B3c\u2212 NK thymocytes is consistent with the NK-associated markers being encoded by their genetically linked loci or the NK gene complex (>>27<<), the regulation of which appears to ensure the simultaneous expression of negative (Ly49) and positive (NKR-P1) signaling molecules on NK cells and NK T cells." . _:b207643672 . _:b207643673 . _:b207643674 . _:b207643675 . _:b207643676 . _:b207643677 . . _:b207643678 . _:b207643679 . _:b207643668 . _:b37498 . _:b207643669 . _:b207643670 . _:b207643670 . _:b207643671 . _:b37507 _:b37536 . _:b37507 _:b37537 . _:b37507 _:b37538 . _:b37507 _:b37539 . "PMC0" . _:b37507 _:b37540 . _:b207643669 "3"^^ . _:b37507 _:b37541 . _:b37517 "The TCR-\u03B2 repertoire of DN NK T cells is highly restricted, with \u223C50% of cells using V\u03B28 (4, 7, >>10<<). DN thymocytes from both \u03B3c+ and \u03B3c\u2212 mice contained a population of V\u03B28+ cells (\u223C5%), which was not detected in DN thymocytes from \u03B22m\u2212/\u2212 mice. Percentages of V\u03B28+ cells amongst mature (HSAlo) DN thymocytes were also comparable between" . . _:b207643668 "3"^^ . . _:b37526 "The unique ability of NK T cells to produce IL-4 after TCR triggering has been one main characteristic of this lymphoid subset (>>1<<, 13, 14). When CD8\u2212 thymocytes from \u03B3c+ or \u03B3c\u2212 mice were cultured in vitro with soluble CD3 and antigen-presenting cells, IL-4 release could be detected in the supernatants from \u03B3c+ and \u03B3c\u2212 cells (Table 1), although IL-4 production from" . _:b207643671 "3"^^ . _:b37521 . _:b207643676 . _:b207643670 "3"^^ . _:b37538 "Moreover, IL-7\u2013deficient mice display normal percentage of thymic and splenic NK T cells with a normal phenotype (>>29<<), and we have not detected a decrease in V\u03B114\u2013J\u03B1281 transcripts in the thymus, spleen and liver of IL-2\u2212/\u2212, IL-4\u2212/\u2212, or IL-7\u2212/\u2212 mice compared with wildtype controls (Lantz, O., and J.P. DiSanto, unpublished data)." . _:b37507 "results and discussion" . . _:b37520 . _:b37523 "a similar degree in the thymus of \u03B3c\u2212 mice as in \u03B3c+ mice, and in the absence of NK-associated markers, the major determinant of NK T cell selection remains the invariant V\u03B114\u2013J\u03B1281 TCR-\u03B1 chain paired with the restricted TCR-\u03B2 chains (>>10<<, 16). Although we cannot rule out a lower avidity reaction in the absence of NKR-P1 or Ly49, we would suggest that the expression of NK-associated markers are probably the result of additional maturation events after selection, rather" . _:b207643680 . _:b207643694 . . _:b37510 "\u03B22m\u2212/\u2212 cells were used as control as it has been previously shown that NK T cells require the \u03B22m-associated CD1 molecules in order to be selected efficiently (>>4<<, 5, 10, 11, 15)." . _:b37507 _:b37512 . _:b207643696 . _:b37507 _:b37513 . _:b207643697 . _:b37507 _:b37514 . _:b207643698 . _:b37534 . _:b37507 _:b37515 . _:b207643699 . _:b37507 _:b37516 . _:b207643700 . _:b37507 _:b37517 . _:b207643701 . _:b37507 _:b37518 . _:b37507 _:b37519 . _:b207643688 . . _:b207643689 . _:b207643690 . _:b37507 . _:b207643691 . _:b37507 _:b37508 . _:b207643677 "2"^^ . _:b207643692 . _:b37507 _:b37509 . _:b207643696 . _:b207643693 . _:b37507 _:b37510 . _:b207643694 . _:b37507 _:b37511 . _:b207643695 . _:b37507 _:b37528 . _:b207643676 "2"^^ . _:b207643680 . _:b37507 _:b37529 . . _:b37507 _:b37530 . _:b207643681 . _:b207643682 . . _:b37507 _:b37531 . _:b207643683 . _:b37507 _:b37532 . _:b207643679 "2"^^ . _:b207643684 . _:b37507 _:b37533 . _:b207643685 . _:b37507 _:b37534 . _:b207643686 . _:b37507 _:b37535 . _:b207643687 . . _:b37507 _:b37520 . _:b207643678 "2"^^ . _:b37507 _:b37521 . _:b37507 _:b37522 . _:b37507 _:b37523 . _:b37507 _:b37524 . _:b207643673 "3"^^ . _:b37507 _:b37525 . _:b37507 _:b37526 . _:b37507 _:b37527 . _:b207643672 "3"^^ . _:b207643675 "2"^^ . _:b37519 "The ability of NK thymocytes to develop in the absence of \u03B3c clearly distinguishes this lymphoid subset from classical NK cells, which have an absolute requirement for \u03B3c-dependent interactions in their development (>>19<<)." . _:b207643674 "2"^^ . _:b207643683 . _:b207643685 "2"^^ . . . _:b207643674 . _:b207643684 "2"^^ . . _:b207643687 "2"^^ . _:b207643686 "2"^^ . _:b37531 "These results are in accord with the recent observations in IL-7 knockout mice, in which NK thymocytes develop but demonstrate a functional defect in IL-4 production after CD3 stimulation (>>29<<). Exogenous IL-7 was able to restore the IL-4 response in vitro (29). NK thymocytes from \u03B3c\u2212 mice also manifest abnormal IL-4 production in vitro in the absence of IL-7R\u03B1 engagement; however, this can be restored with TSLP (Table 1)." . _:b37514 "\u03B22m\u2212/\u2212 cells were used as control as it has been previously shown that NK T cells require the \u03B22m-associated CD1 molecules in order to be selected efficiently (4, 5, 10, 11, >>15<<). As shown in Fig. 1, C\u03B1 transcripts were found equally in all three cDNA preparations. The amount of V\u03B114\u2013J\u03B1281 mRNA was similar in both \u03B3c+ and \u03B3c\u2212 thymi (within threefold) and largely increased relative to \u03B22m\u2212/\u2212 thymi, which lack NK T" . _:b37534 "NK T cells normally comprise a small percentage of the lymphocytes present in the spleen and lymph nodes (>>1<<\u20133); however, these cells are abundant in the liver (20, 30)." . _:b207643681 "2"^^ . _:b37520 "NK T thymocytes express a unique constellation of cell surface markers, including intermediate density TCR-\u03B1\u03B2 (TCR-\u03B1\u03B2int) and coexpression of NK-associated antigens, including members of the NKR-P1 and Ly49 families (>>1<<\u20133). Next, we investigated whether NK T thymocytes from \u03B3c\u2212 mice maintained this particular phenotype using mice on the C57BL/6 background." . _:b207643680 "2"^^ . _:b207643683 "2"^^ . _:b207643682 "2"^^ . _:b37508 . _:b207643693 "2"^^ . _:b37499 . _:b207643692 "2"^^ . . . _:b207643684 . _:b207643669 . . _:b207643695 "2"^^ . _:b207643668 . _:b207643694 "2"^^ . _:b207643671 . _:b207643689 "2"^^ . _:b207643670 . _:b207643688 "2"^^ . . _:b37504 . _:b207643691 "2"^^ . _:b207643690 "2"^^ . _:b207643698 . _:b37511 . _:b207643701 "2"^^ . _:b207643687 . _:b37528 "The unique ability of NK T cells to produce IL-4 after TCR triggering has been one main characteristic of this lymphoid subset (1, 13, >>14<<). When CD8\u2212 thymocytes from \u03B3c+ or \u03B3c\u2212 mice were cultured in vitro with soluble CD3 and antigen-presenting cells, IL-4 release could be detected in the supernatants from \u03B3c+ and \u03B3c\u2212 cells (Table 1), although IL-4 production from \u03B3c\u2212 cells" . _:b207643700 "2"^^ . _:b207643677 . _:b37497 "materials and methods" . . _:b37533 . _:b207643676 . . _:b37500 . _:b37524 "similar degree in the thymus of \u03B3c\u2212 mice as in \u03B3c+ mice, and in the absence of NK-associated markers, the major determinant of NK T cell selection remains the invariant V\u03B114\u2013J\u03B1281 TCR-\u03B1 chain paired with the restricted TCR-\u03B2 chains (10, >>16<<). Although we cannot rule out a lower avidity reaction in the absence of NKR-P1 or Ly49, we would suggest that the expression of NK-associated markers are probably the result of additional maturation events after selection, rather than" . _:b37508 "A semiquantitative PCR approach (>>23<<) was used to enumerate NK T cells by exploiting the fact that these cells exhibit a restricted TCR-\u03B1 chain repertoire, using the V\u03B114 segment joined to J\u03B1281 (10)." . _:b207643679 . _:b207643697 "2"^^ . _:b37515 . _:b207643678 . _:b207643696 "2"^^ . _:b207643673 . _:b37503 "Reverse transcription and quantitative PCR amplification were carried out as previously described (23) using oligonucleotides specific for C\u03B1, V\u03B114, and J\u03B1281 (>>10<<). It should be stressed that because there is no allelic exclusion for the \u03B1 chain locus of the TCR (24), only enrichment for a certain VJ combination in a peculiar sample can be detected by PCR analysis using PCR primers specific for V\u03B1" . _:b207643699 "2"^^ . _:b207643672 . _:b207643679 . _:b207643698 "2"^^ . . _:b37503 . _:b207643675 . _:b37535 "NK T cells normally comprise a small percentage of the lymphocytes present in the spleen and lymph nodes (1\u20133); however, these cells are abundant in the liver (>>20<<, 30). To quantitate peripheral NK T cells, lymphocytes from \u03B3c+, \u03B3c\u2212, or \u03B22m\u2212/\u2212 mice were isolated from the liver and spleen and the amount of V\u03B114\u2013J\u03B1281 mRNA was determined. NK T cells were clearly present in the liver and spleens of \u03B3c+" . _:b207643674 . . _:b37524 . _:b37530 "Therefore, TSLP can effectively substitute for IL-7 in stimulating NK thymocytes in vitro (>>28<<). These results are in accord with the recent observations in IL-7 knockout mice, in which NK thymocytes develop but demonstrate a functional defect in IL-4 production after CD3 stimulation (29). Exogenous IL-7 was able to restore the" . . _:b37515 "The TCR-\u03B2 repertoire of DN NK T cells is highly restricted, with \u223C50% of cells using V\u03B28 (>>4<<, 7, 10). DN thymocytes from both \u03B3c+ and \u03B3c\u2212 mice contained a population of V\u03B28+ cells (\u223C5%), which was not detected in DN thymocytes from \u03B22m\u2212/\u2212 mice. Percentages of V\u03B28+ cells amongst mature (HSAlo) DN thymocytes were also comparable" . _:b207643685 . _:b37504 "It should be stressed that because there is no allelic exclusion for the \u03B1 chain locus of the TCR (>>24<<), only enrichment for a certain VJ combination in a peculiar sample can be detected by PCR analysis using PCR primers specific for V\u03B1 and J\u03B1 segments." . _:b37541 "Complementary results from Arase et al. (>>34<<) showed that NK1.1+ TCR-\u03B1\u03B2 T cells, as well as mainstream \u03B1\u03B2 T cells, are absent in CD3-\u03B6-deficient mice, whereas NK cells were present." . _:b207643684 . _:b207643687 . _:b207643686 . . _:b207643681 . _:b207643671 . . _:b207643690 . _:b207643680 . _:b207643677 . _:b207643683 . . _:b207643682 . _:b37539 . _:b207643693 . _:b37510 . _:b207643692 . _:b207643695 . _:b37520 . _:b207643681 . _:b207643694 . _:b207643689 . _:b207643688 . _:b37500 "Cells were stained using combinations of directly conjugated mAbs: FITC\u2013anti-TCR-\u03B1\u03B2 (clone H57), biotin\u2013anti-heat-stable antigen (HSA, clone J11d), and biotin\u2013 anti-V\u03B28 (clone F23.1); FITC\u2013anti-IL-7R\u03B1 chain (>>21<<) (purified and locally conjugated by standard methods), PE\u2013anti-HSA, biotin\u2013anti-TCR-\u03B1\u03B2, FITC\u2013anti-IL-2R\u03B2, FITC\u2013anti-Ly49C, and PE\u2013anti-NK1.1 (all from PharMingen, San Diego, CA); PE\u2013antiCD4, FITC\u2013anti-CD8, and Tricolor\u2013streptavidin" . . _:b207643691 . _:b37521 "Concerning the potential function of NKR-P1 molecules as coreceptors for the recognition of CD1 during selection of NK T cells (>>2<<), our results show that NKR-P1 expression is not strictly required for positive selection of NK T cells on CD1, although we cannot rule out that additional interactions are afforded to the selection process by NKR-P1 molecules." . _:b207643690 . _:b207643701 . _:b37525 "Indeed, the invariant \u03B1 chain appears selected at the protein level rather than being produced through a genetic program that selectively recombines V\u03B114 and J\u03B1281 (>>10<<)." . _:b37526 . _:b207643700 . _:b37529 . _:b37501 . . . _:b37537 "Although IL-2\u2013deficient mice have reduced numbers of NK cells (>>31<<), NK thymocytes are present in IL-2\u2212/\u2212 mice and have normal expression of the NKR-P1 and IL-2R\u03B2." . . _:b37498 "Mice deficient for the common cytokine receptor \u03B3 chain, \u03B3c (initially identified as the IL-2 receptor \u03B3c, reference >>19<<), were maintained in our conventional animal facility and were of a mixed background (129/Ola/BALB/c or 129/Ola/BL/6)." . . _:b207643691 . _:b37511 "\u03B22m\u2212/\u2212 cells were used as control as it has been previously shown that NK T cells require the \u03B22m-associated CD1 molecules in order to be selected efficiently (4, >>5<<, 10, 11, 15)." . . _:b207643697 . . _:b207643696 . _:b207643699 . _:b207643678 . _:b37513 . _:b37529 "We have recently identified a novel cytokine, TSLP, which shares many functional similarities to IL-7 (>>25<<), and uses the IL-7R\u03B1 chain, but not the \u03B3c chain for signaling, which can maintain \u03B3c+ and \u03B3c\u2212 thymocytes in vitro (Park et al., unpublished data)." . _:b207643698 . . . _:b37497 . . _:b37534 . . _:b37518 "these results suggest that NK T cells are found at the same relative frequency in \u03B3c\u2212 thymi as in \u03B3c+ thymi, although their absolute numbers are reduced by 20-fold in parallel with the overall decrease in thymopoiesis seen in \u03B3c\u2212 mice (>>19<<). These results suggest that generation of NK T cells after interactions with CD1 molecules can proceed in the absence of \u03B3c." . _:b37536 . _:b37500 . _:b37501 . . _:b207643692 . _:b37502 . . _:b37534 . _:b37503 . _:b37497 . _:b37498 . _:b37533 "This concept is supported by recent observations using V\u03B114\u2013J\u03B1281 transgenic mice, which demonstrate an increased frequency of IL-4\u2013producing NK T cells resulting in increased basal levels of serum IgG1 and IgE (>>16<<). Our results are consistent with the idea that positive selection of the V\u03B114\u2013J\u03B1281-bearing TCRs confers the IL-4\u2013producing phenotype. Furthermore, we demonstrate that this unique ability of NK T cells to secrete IL-4 is not dependent on" . _:b37499 . _:b37539 "Further studies using IL-2R\u03B2\u2013deficient mice (which can be considered as deficient in IL-2 and IL-15) (>>32<<) and IL-7R\u03B1-deficient mice (which inactivate IL-7 and TSLP) (33) should help to elucidate the \u03B3c-dependent interactions required for induction of NKR-P1 and Ly49 antigens on NK T cells and their export into the periphery." . _:b207643701 . _:b207643688 . _:b37508 . . _:b37509 . _:b37506 "and subsequent in vitro culture of splenocyte suspensions for IL-4 production were performed exactly as described (>>13<<). To evaluate cytokine production following stimulation in vitro, CD8\u2212 thymocytes were purified after a one-step killing with anti-CD8 mAb (TiB-211; American Type Culture Collection, Rockville, MD) plus low-toxic M rabbit complement" . . _:b37510 . _:b37511 . . . . _:b37504 . _:b37520 . _:b37509 "A semiquantitative PCR approach (23) was used to enumerate NK T cells by exploiting the fact that these cells exhibit a restricted TCR-\u03B1 chain repertoire, using the V\u03B114 segment joined to J\u03B1281 (>>10<<). We quantitated the amounts of V\u03B114\u2013J\u03B1281 TCR-\u03B1 chain in CD8\u2212 (CD4\u2212CD8\u2212 [DN] and CD4+ single-positive [SP]) thymocytes from \u03B3c+, \u03B3c\u2212, and \u03B22m\u2212/\u2212 mice." . _:b37505 . _:b37506 . _:b37516 . _:b37506 . _:b37507 . _:b37516 . _:b37517 . . _:b37522 . _:b37518 . _:b37528 . . _:b37505 "Indeed, polyclonal sequencing of such V\u03B114\u2013J\u03B1281 PCR products demonstrated their polymorphism (reference >>10<<; data not shown)." . _:b37519 . _:b37512 . . . _:b37513 . . _:b37514 . . _:b37515 . _:b37524 . . _:b37525 . _:b37497 _:b37498 . _:b37497 _:b37499 . _:b37497 _:b37500 . _:b37526 . _:b37497 _:b37501 . _:b37497 _:b37502 . _:b37497 _:b37503 . _:b37497 _:b37504 . _:b37527 . _:b37497 _:b37505 . _:b37497 _:b37506 . . _:b37520 . . . _:b37521 . . _:b37522 . . _:b37523 . _:b37532 .