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The proximal promoter of lck directs gene expression exclusively in T cells. To investigate the developmental regulation of the lck proximal promoter activity and its relationship to T cell lineage commitment, a green fluorescence protein (GFP) transgenic (Tg) mouse in which the GFP expression is under the control of the proximal promoter of lck was created. In the adult GFP-Tg mice, >90% of CD4+CD8+ and CD4+CD8– thymocytes, and the majority of CD4–CD8+ and CD4–CD8– [double-negative (DN)] thymocytes were highly positive for GFP. Slightly lower but substantial levels of expression of GFP was also observed in mature splenic T cells. No GFP+ cells was detected in non-T lineage subsets, including mature and immature B cells, CD5+ B cells, and NK cells, indicating a preserved tissue specificity of the promoter. The earliest GFP+ cells detected were found in the CD44+CD25– DN thymocyte subpopulation. The developmental potential of GFP– and GFP+ cells in the CD44+CD25– DN fraction was examined using in vitro culture systems. The generation of substantial numbers of αβ and γδ T cells as well as NK cells was demonstrated from both GFP– and GFP+ cells. However, no development of B cells or dendritic cells was detected from GFP+ CD44+CD25– DN thymocytes. These results suggest that the progenitors expressing lck proximal promoter activity in the CD44+CD25– DN thymocyte subset have lost most of the progenitor potential for the B and dendritic cell lineage. Thus, progression of T cell lineage restriction in the earliest thymic population can be visualized by lck proximal promoter activity, suggesting a potential role of Lck in the T cell lineage commitment. [ABSTRACT FROM PUBLISHER]

Murine NKT cells can recognize α-galactosylceramide (α-GalCer) in the context of a class Ib CD1d molecule. Here we show that α-GalCer can selectively activate freshly isolated human Vα24+Vβ11+ cells, functionally defining the human NKT cells. The naive human NKT cell repertoire consisted of cells expressing an invariant Vα24JαQ chain and a diverse array of β chains derived from a single Vβ11 gene segment. Stimulation with α-GalCer expanded a polyclonal subset of the human NKT cell repertoire carrying a novel complementarity-determining region (CDR) 3β consensus motif that may directly interact with the sugar moiety of α-GalCer. Our data suggest that certain redundancy is allowed for CDR3β of NKT antigen receptor to interact with the ligand and provide a first clue to understand the novel protein–carbohydrate interaction mechanisms. [ABSTRACT FROM PUBLISHER]

We isolated and sequenced the rearranged genomic variable (V) and joining (J) gene segments of T cell receptor α-chain gene from two independent keyhole limpet hemocyanin (KLH)-specific suppressor T cell (T) hybridomas (BW5147 x C57BL/6 KLH-T. These nucieotide sequences were compared with those of germline DNA from kidney and also with cDNA of α-chain (VJ28l) previously isolated from T hybridoma (348-281) with KLH/H-2 T activity. The entire V, and J sequences of all three T hybridomas were exactly identical and were encoded by the germline V, and J gene segments without any mutations, except for 2-nucieotide deletions from both the 3' end of V and the 5' end of J gene segments, respectively, and a 1-nucleotide (guanine) insertion in the junctional (N) region which was not encoded by the germline gene. Six additional KLH-T hybridomas, further analyzed, also possessed the same α-chain, indicating the preferential usage of the particular α-chain in these hybridomas. As chromosome analysis demonstrated a different pattern in each clone, these hybridomas appear to be independent. More surprisingly, 0.5–1.5% of the total functional T cell α-chain mRNA in the thymus and spleen of unprimed C57BL/6 mice was found to be of this particular α-chain. These results suggest that the repertoire of KLH-T is strictly limited. [ABSTRACT FROM PUBLISHER]

We have established an anti-sense RNA system which is capable of regulating expression of the class II (ia) molecule coded for by the major histocompatibility complex in cultured mouse cells. Various areas of the I-A β chain gene were subcioned in an anti-sense orientation to the 3′ of the dihydrofolate reductase (DHFR) cDNA under the control of the human metallothionein IIa gene promoter. These anti-sense DNA constructs were transfected into M12.4 cells, a BALB/c B lymphoma cell line which expresses both I-A and I-E molecules on the cell surface. I-A expression of selected clones transfected with anti-sense DNA encompassing the 5′ untranslated region (UT) (100 or 310 bp) including the translation start site or the poly(A) addition signalling sequence in the 3′ UT (250 bp) of the I-A β chain gene were specifically reduced to <5% of the control M12.4 cell surface I-A expression. These clones had normal levels of I-E expression. However, transfection of the anti-sense DNA to the β1 domain (510 bp) including the splicing donor and acceptor sequences did not affect the expression of I-A molecules. The same antisense DNA constructs (100 bp of the 5′ UT or 250 bp of the 3′ UT) without the DHFR cDNA (710 bp) did not down-regulate the expression of I-A molecules, indicating that either the physical length of the anti-sense RNA or specific DHFR cDNA sequences are also important. Moreover, we found that the ratio of the amounts of the exogenous anti-sense and the endogenous sense RNA In the transfectants was correlated with the extent of the reduction of I-A expression. Antigen presenting cell activities of the transfectants were also depressed in relation to the reduction of I-A molecules on the transfectants. [ABSTRACT FROM PUBLISHER]

An obituary for professor and "International Immunology" founder Tomio Tada is presented.

Memory CD4(+) T cells promote protective humoral immunity; however, how memory T cells acquire this activity remains unclear. This study demonstrates that CD4(+) T cells develop into antigen-specific memory T cells that can promote the terminal differentiation of memory B cells far more effectively than their naive T-cell counterparts. Memory T cell development requires the transcription factor B-cell lymphoma 6 (Bcl6), which is known to direct T-follicular helper (Tfh) cell differentiation. However, unlike Tfh cells, memory T cell development did not require germinal center B cells. Curiously, memory T cells that develop in the absence of cognate B cells cannot promote memory B-cell recall responses and this defect was accompanied by down-regulation of genes associated with homeostasis and activation and up-regulation of genes inhibitory for T-cell responses. Although memory T cells display phenotypic and genetic signatures distinct from Tfh cells, both had in common the expression of a group of genes associated with metabolic pathways. This gene expression profile was not shared to any great extent with naive T cells and was not influenced by the absence of cognate B cells during memory T cell development. These results suggest that memory T cell development is programmed by stepwise expression of gatekeeper genes through serial interactions with different types of antigen-presenting cells, first licensing the memory lineage pathway and subsequently facilitating the functional development of memory T cells. Finally, we identified Gdpd3 as a candidate genetic marker for memory T cells., (© The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Activation of NK cells is triggered by multiple receptors. We demonstrate here that SLP-76 is required for CD16- and NKG2D-mediated NK cell cytotoxicity, while MIST negatively regulates these responses in an SLP-76-dependent manner. Exceptionally, MIST acts as a positive regulator of cytotoxicity against YAC-1 cells, although SLP-76 plays a more key role. SLP-76 acts as a dominant positive regulator for both NKG2D-mediated and YAC-1 cell-triggered IFN-gamma production. Although NKG2D-mediated IFN-gamma production depends on phospholipase C (PLC) gamma 2, YAC-1 cell-triggered IFN-gamma production is PLC gamma 2- and Syk/ZAP-70 independent and nuclear factor-kappa B mediated. SLP-76 is required for this process in the presence of MIST but is dispensable in the absence of MIST. Thus, YAC-1 cell-triggered NKG2D-independent IFN-gamma production appears to be regulated by SLP-76-dependent and -independent pathways, in which the latter is negatively regulated by MIST. Taken together, these results suggest that SLP-76 and MIST distinctly but interactively regulate NK cell cytotoxicity and IFN-gamma production.

Sarcoidosis is a systemic disorder associated with granuloma characterized by an abnormal T(h)1-type cytokine production and accumulation of T(h)1 CD4 T cells in the granuloma lesions, suggesting an importance of T(h)1 responses in sarcoidosis. However, the pathogenesis of sarcoidosis remains to be solved. Here, we investigated the nature of V(alpha)24 NKT cells with immunoregulatory functions in sarcoidosis. Patients with non-remitting sarcoidosis displayed a decrease in the number of V(alpha)24 NKT cells in peripheral blood, but an accumulation of these cells in granulomatous lesions. When stimulated with the specific glycolipid ligand, alpha-galactosylceramide, peripheral blood V(alpha)24 NKT cells from patients with non-remitting disease produced significantly less IFN-gamma than those from healthy volunteers, but normal levels of IL-4. The reduced IFN-gamma production was observed only in V(alpha)24 NKT cells and not conventional CD4 T cells, but was normal in patients with remitting disease, suggesting that non-remitting sarcoidosis involves an insufficient IFN-gamma production of V(alpha)24 NKT cells which is well correlated with disease activity. Thus, these results suggest that V(alpha)24 NKT cells play a crucial role in the disease status of sarcoidosis.