B cell development requires the coordinated action of transcription factors and cytokines, in particular interleukin-7 (IL-7). We report that mice lacking the POZ (Poxvirus and zinc finger) domain of the transcription factor Miz-1 (Zbtb17(ΔPOZ/ΔPOZ)) almost entirely lacked follicular B cells, as shown by the fact that their progenitors failed to activate the Jak-Stat5 pathway and to upregulate the antiapoptotic gene Bcl2 upon IL-7 stimulation. We show that Miz-1 exerted a dual role in the interleukin-7 receptor (IL-7R) pathway by directly repressing the Janus kinase (Jak) inhibitor suppressor of cytokine signaling 1 (Socs1) and by activating Bcl2 expression. Zbtb17(ΔPOZ/ΔPOZ) (Miz-1-deficient) B cell progenitors had low expression of early B cell genes as transcription factor 3 (Tcf3) and early B cell factor 1 (Ebf1) and showed a propensity for apoptosis. Only the combined re-expression of Bcl2 and Ebf1 could reconstitute the ability of Miz-1-deficient precursors to develop into CD19(+) B cells.

IntroductionHematopoietic precursors differentiate into mature blood cell lineages through a series of well-coordinated steps. T cells are generated in the thymus, which is continuously replenished with lymphoid progenitors from the bone marrow via the bloodstream. 1 Early lymphoid progenitors (ELPs) enter the thymus and become early T lineage precursors (ETPs), defined as Lin Ϫ/low , CD117 high , and CD25 Ϫ . 2 The capacity of ELPs to migrate to the thymus has been attributed to their expression of CCR9. 3,4 In addition to CCR9 ϩ ELPs, other progenitors, such as CLPs, may home to the thymus and generate T cells. Recently, Ly6D has been used to identify the branch point of CLPs that gives rise to the first stages of B-cell development, B cell-biased lymphoid progenitor (BLP), and all-lymphoid progenitor (ALP), which contribute to the T-cell development. 5 The subsequent development of ETPs starts with CD4 Ϫ CD8 Ϫ double-negative 1 (DN1) cells. DN1s are subdivided into DN1a-e according to the expression of CD24 and CD117, DN1a/b corresponding to the ETP subset. 6 DN1s give rise to DN2a-b cells, which differentiate into DN3s, subdivided into DN3a-b based on their size and CD27 expression. 7 DN3a cells that have productively rearranged the T-cell receptor ␤-gene (TCR-␤) become activated by TCR-dependent signals (␤-selection), differentiate into DN3b, and become DN4 pre-T cells. The newly developed DN4s become CD4 ϩ CD8 ϩ double-positive (DP) cells and undergo positive/ negative selection before reaching the periphery as mature CD4 ϩ or CD8 ϩ T cells. 8 Pro-T-cell differentiation steps depend on the expression of Notch ligands, mainly ␦-like ligand 1 (DL1) and DL4 on thymic stroma, 9 and on cytokines, such as interleukin-7 (IL-7). 10 Notch signaling assures lineage commitment, survival, and development of ETPs into further DN subsets. 11 The IL-7/IL-7R pathway drives proliferation, survival, and progression of pro-T cells, 12 and also induces the rearrangement and transcription of the TCR-␥ locus. 13 The IL-7R signaling activates Janus kinase 1/3 (Jak1/3), which phosphorylate signal transducer and activator of transcription 5 (STAT5). Phosphorylated STAT5 then activates the transcription of IL-7-dependent target genes. 14 A key player in IL-7R cascade is the maintenance of cell survival by promoting a favorable balance of B-cell lymphoma-2 (Bcl-2) family members. 15 The expression of the antiapoptotic protein Bcl-2 is up-regulated after IL-7 stimulation. Some studies have shown that the up-regulation of Bcl-2 can be STAT5-dependent. [16][17][18] Other studies have shown that STAT5-mediated activation of AKT protein regulates the glucose metabolism of the cell and maintains prosurvival and growth functions. 19 Suppressor of cytokine signaling 1 (SOCS1) is known to inhibit phosphorylation of STAT proteins by directly binding to the Jak proteins and therefore inhibiting all further downstream signaling events to ensure a return to steady-state homeostasis after cytokine responses. 20 Miz-1 (Zbtb17) is a transcription f...

The human-specific p35 isoform of the invariant chain (Ii) includes an R-X-R endoplasmic reticulum (ER) retention motif that is inactivated upon HLA-DR binding. Although the masking is assumed to involve the cytoplasmic tails of class II molecules, the mechanism underlying this function remains to be investigated. Moreover, in light of the polymorphic nature of the class II cytosolic tails, little is known about the capacity of various isotypes or alleles to overcome the retention signal of Iip35. To gain further insights into these issues, we first addressed the proposed role of the HLA-DR cytoplasmic tails. As shown by flow cytometry, the presence of Iip35 in transfected HeLa cells prevented surface expression of HLA-DR molecules lacking their cytoplasmic tails (DRalphaTM/betaTM). These truncated class II molecules and Iip35 accumulated in the ER, and co-localized with calnexin, as determined by confocal microscopy. Sensitivity of DRalphaTM/betaTM to endoglycosidase H treatment confirmed that these molecules do not reach the trans-Golgi network when associated with Iip35. Further characterization revealed that the beta chain cytosolic tail is critical for efficient ER egress of class II/Iip35 complexes. Interestingly, our results clearly demonstrate for the first time that DP and DQ isotypes can also overcome the retention motif of Iip35 through a mechanism involving their very distinctive polymorphic beta chain cytoplasmic tails. Altogether, these results further dissect the masking of di-basic retention signals, and emphasize the interplay between class II molecules and Ii for the transport of the complex to the endocytic pathway.

T(h) cells have long been divided into two subsets, T(h)1 and T(h)2; however, recently, T(h)17 and inducible regulatory T (iTreg) cells were identified as new T(h) cell subsets. Although T(h)1- and T(h)2-polarizing cytokines have been shown to suppress T(h)17 and iTreg development, transcriptional regulation of T(h)17 and iTreg differentiation by cytokines remains to be clarified. In this study, we found that expression of the growth factor independent 1 (Gfi1) gene, which has been implicated in T(h)2 development, was repressed in T(h)17 and iTreg cells compared with T(h)1 and T(h)2 lineages. Gfi1 expression was enhanced by the IFN-gamma/STAT1 and IL-4/STAT6 pathways, whereas it was repressed by the transforming growth factor-beta1 stimulation at the promoter level. Over-expression of Gfi1 strongly reduced IL-17A transcription in the EL4 T cell line, as well as in primary T cells. This was due to the blockade of recruitment of retinoid-related orphan receptor gammat to the IL-17A promoter. In contrast, IL-17A expression was significantly enhanced in Gfi1-deficient T cells under T(h)17-promoting differentiation conditions as compared with wild-type T cells. In contrast, the impacts of Gfi1 in iTregs were not as strong as in T(h)17 cells. Taken together, these data strongly suggest that Gfi1 is a negative regulator of T(h)17 differentiation, which represents a novel mechanism for the regulation of T(h)17 development by cytokines.

The αβ T cell receptor (TCR) repertoire on mature T cells is selected in the thymus, but the basis for thymic selection of MHC-restricted TCRs from a randomly generated pre-selection repertoire is not known. Here we perform comparative repertoire sequence analyses of pre-selection and post-selection TCR from multiple MHC-sufficient and MHC-deficient mouse strains, and find that MHC-restricted and MHC-independent TCRs are primarily distinguished by features in their non-germline CDR3 regions, with many pre-selection CDR3 sequences not compatible with MHC-binding. Thymic selection of MHC-independent TCR is largely unconstrained, but the selection of MHC-specific TCR is restricted by both CDR3 length and specific amino acid usage. MHC-restriction disfavors TCR with CDR3 longer than 13 amino acids, limits positively charged and hydrophobic amino acids in CDR3β, and clonally deletes TCRs with cysteines in their CDR3 peptide-binding regions. Together, these MHC-imposed structural constraints form the basis to shape VDJ recombination sequences into MHC-restricted repertoires.

The transcriptional repressor Gfi1 is a nuclear zinc-finger protein that is expressed in T cell precursors in the thymus, but is down-regulated in mature, resting T cells. Gfi1 expression rises transiently to levels seen in thymocytes upon antigenic activation. We show here that lack of Gfi1 causes delayed cell cycle entry and apoptosis after antigenic stimulation in both mature CD4 + and CD8 + T cells ex vivo. DNA micro-array analysis demonstrated that this correlated with an up-regulation of the death receptor CD95, the proapoptotic factors Bad and Apaf1 and the cell cycle inhibitor p21, and a downregulation of Bcl-2 expression in Gfi1 -/-T cells. Surprisingly, while Gfi1-deficient CD4 + T cells showed the same defective behavior in vivo, Gfi1-deficient CD8 + T cells showed no aberration in vivo and were fully able to mount an anti-viral immune response. This indicates that Gfi1 exerts different functions in CD4 + and CD8 + T cells very likely by maintaining different genetic programs in both cell types, and appears to be essential for the CD4 helper T cell immune response but dispensable for the function of cytotoxic CD8 + T cells.

Twenty years ago, Dr. François A. Auger, the founder of the Laboratory of Experimental Organogenesis (LOEX), introduced the self-assembly technique. This innovative technique relies on the ability of dermal fibroblasts to produce and assemble their own extracellular matrix, differing from all other tissue-engineering techniques that use preformed synthetic scaffolds. Nevertheless, the use of the self-assembly technique was limited for a long time due to its main drawbacks: time and cost. Recent scientific breakthroughs have addressed these limitations. New protocol modifications that aim at increasing the rate of extracellular matrix formation have been proposed to reduce the production costs and laboratory handling time of engineered tissues. Moreover, the introduction of vascularization strategies in vitro permits the formation of capillary-like networks within reconstructed tissues. These optimization strategies enable the large-scale production of inexpensive native-like substitutes using the self-assembly technique. These substitutes can be used to reconstruct three-dimensional models free of exogenous materials for clinical and fundamental applications.

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