Leukocyte ß7 integrin targeted by Krüppel-like factors.

Constitutive expression of Krüppel-like factor 3 (KLF3, BKLF) increases marginal zone (MZ) B cell numbers, a phenotype shared with mice lacking KLF2. Ablation of KLF3, known to interact with serum response factor (SRF), or SRF itself, results in fewer MZ B cells. It is unknown how these functional equivalences result. In this study, it is shown that KLF3 acts as transcriptional repressor for the leukocyte-specific integrin ß7 (Itgb7, Ly69) by binding to the ß7 promoter, as revealed by chromatin immunoprecipitation. KLF2 overexpression antagonizes this repression and also binds the ß7 promoter, indicating that these factors may compete for target sequence(s). Whereas ß7 is identified as direct KLF target, its repression by KLF3 is not connected to the MZ B cell increase because ß7-deficient mice have a normal complement of these and the KLF3-driven increase still occurs when ß7 is deleted. Despite this, KLF3 overexpression abolishes lymphocyte homing to Peyer's patches, much like ß7 deficiency does. Furthermore, KLF3 expression alone overcomes the MZ B cell deficiency when SRF is absent. SRF is also dispensable for the KLF3-mediated repression of ß7. Thus, despite the shared phenotype of KLF3 and SRF-deficient mice, cooperation of these factors appears neither relevant for the formation of MZ B cells nor for the regulation of ß7. Finally, a potent negative regulatory feedback loop limiting KLF3 expression is shown in this study, mediated by KLF3 directly repressing its own gene promoter. In summary, KLFs use regulatory circuits to steer lymphocyte maturation and homing and directly control leukocyte integrin expression.

Intrinsic role of FoxO3a in the development of CD8+ T cell memory.

CD8(+) T cells undergo rapid expansion during infection with intracellular pathogens, which is followed by swift and massive culling of primed CD8(+) T cells. The mechanisms that govern the massive contraction and maintenance of primed CD8(+) T cells are not clear. We show in this study that the transcription factor, FoxO3a, does not influence Ag presentation and the consequent expansion of CD8(+) T cell response during Listeria monocytogenes infection, but plays a key role in the maintenance of memory CD8(+) T cells. The effector function of primed CD8(+) T cells as revealed by cytokine secretion and CD107a degranulation was not influenced by inactivation of FoxO3a. Interestingly, FoxO3a-deficient CD8(+) T cells displayed reduced expression of proapoptotic molecules BIM and PUMA during the various phases of response, and underwent reduced apoptosis in comparison with wild-type cells. A higher number of memory precursor effector cells and memory subsets was detectable in FoxO3a-deficient mice compared with wild-type mice. Furthermore, FoxO3a-deficient memory CD8(+) T cells upon transfer into normal or RAG1-deficient mice displayed enhanced survival. These results suggest that FoxO3a acts in a cell-intrinsic manner to regulate the survival of primed CD8(+) T cells.

Programming of marginal zone B-cell fate by basic Kruppel-like factor (BKLF/KLF3).

Splenic marginal zone (MZ) B cells are a lineage distinct from follicular and peritoneal B1 B cells. They are located next to the marginal sinus where blood is released. Here they pick up antigens and shuttle the load onto follicular dendritic cells inside the follicle. On activation, MZ B cells rapidly differentiate into plasmablasts secreting antibodies, thereby mediating humoral immune responses against blood-borne type 2 T-independent antigens. As Krüppel-like factors are implicated in cell differentiation/function in various tissues, we studied the function of basic Krüppel-like factor (BKLF/KLF3) in B cells. Whereas B-cell development in the bone marrow of KLF3-transgenic mice was unaffected, MZ B-cell numbers in spleen were increased considerably. As revealed in chimeric mice, this occurred cell autonomously, increasing both MZ and peritoneal B1 B-cell subsets. Comparing KLF3-transgenic and nontransgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-regulated/down-regulated on normal MZ B-cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor-receptor signaling, indicating that KLF3 may complement alternative nuclear factor-κB signaling. Thus, KLF3 is a driving force toward MZ B-cell maturation.

Impaired B cell development in the absence of Krüppel-like factor 3.

Krüppel-like factor 3 (Klf3) is a member of the Klf family of transcription factors. Klfs are widely expressed and have diverse roles in development and differentiation. In this study, we examine the function of Klf3 in B cell development by studying B lymphopoiesis in a Klf3 knockout mouse model. We show that B cell differentiation is significantly impaired in the bone marrow, spleen, and peritoneal cavity of Klf3 null mice and confirm that the defects are cell autonomous. In the bone marrow, there is a reduction in immature B cells, whereas recirculating mature cells are noticeably increased. Immunohistology of the spleen reveals a poorly structured marginal zone (MZ) that may in part be caused by deregulation of adhesion molecules on MZ B cells. In the peritoneal cavity, there are significant defects in B1 B cell development. We also report that the loss of Klf3 in MZ B cells is associated with reduced BCR signaling strength and an impaired ability to respond to LPS stimulation. Finally, we show increased expression of a number of Klf genes in Klf3 null B cells, suggesting that a Klf regulatory network may exist in B cells.

Peripheral T lymphocytes recirculating back into the thymus can mediate thymocyte positive selection.

The thymus continuously produces T lymphocytes that contribute to the maintenance of the peripheral T cell pool. Since peripheral recirculating T cells represent a very minor population among total thymocytes in normal animals, the relationship between the thymus and secondary lymphoid organs is generally considered unidirectional. Recently, several reports have described the presence of recirculating T cells in the thymus, raising issues regarding their possible function. In this article, we show that the niche for recirculating T cells in the thymus, i.e., their absolute number, is the same in lymphopenic and normal mice. Using a novel combination of TCR-transgenic mice in which the ligand necessary for positive selection of host T cells is only expressed by transferred donor T cells, we show that mature T cells recirculating back to the thymus can mediate positive selection.

Prevention of Functional Decline by Reframing the Role of Nursing Homes?

Institutionalization is generally a consequence of functional decline driven by physical limitations, cognitive impairments, and/or loss of social supports. At this stage, intervention to reverse functional losses is often too late. To be more effective, geriatric medicine must evolve to intervene at an earlier stage of the disability process. Could nursing homes (NHs) transform from settings in which many residents dwell to settings in which the NH residents and those living in neighboring communities benefit from staff expertise to enhance quality of life and maintain or slow functional decline? A task force of clinical researchers met in Toulouse on December 2, 2015, to address some of these challenges: how to prevent or slow functional decline and disabilities for NH residents and how NHs may promote the prevention of functional decline in community-dwelling frail elderly. The present article reports the main results of the Task Force discussions to generate a new paradigm.

The role of nicotinic acetylcholine receptors in lymphocyte development.

The sizes of lymphocyte populations in lymphoid organs of nicotinic acetylcholine receptor knockout and chimera (knockout/wild-type) mice were studied by flow cytometry. The absence of beta2 subunit decreased, while nicotine treatment increased B lymphocyte numbers in the bone marrow. In chimera mice, either beta2 or alpha7 subunits influenced lymphocyte populations in primary lymphoid organs, while in the spleen, only alpha7 receptors were critical. More annexin V-positive B cells were found in the bone marrow of knockout than wild-type animals. We conclude that nicotinic receptors are involved in regulating lymphocyte development and control the B lymphocyte survival.

Structure, chromosomal localization and expression of the mouse regulator of G-protein signaling10 gene (mRGS10).

Regulator of G-protein signaling (RGS) proteins negatively regulate signaling pathways involving seven transmembrane receptors and heterotrimeric G proteins. The purpose of this study was to determine the chromosomal localization, structure and expression profile of the gene coding for mouse regulator of G-protein signaling10 (mRGS10). Fluorescence in situ hybridization analysis indicated that mRGS10 maps to band F3-F4 of the mouse chromosome 7. Sequence analysis revealed that the RGS10 gene encompasses six exons spanning more than 40 kb of genomic DNA. The RGS domain is encoded by exons 3-6; alternative splicing of the first exons allows the generation of two isoforms in the mouse system which differ in their N-terminal portion. Thus, mRGS10 encodes two intracellular proteins of 167 and 181 amino-acids which are highly homologous to the human and rat polypeptides. The deduced amino-acid sequences of mouse RGS10 show 92% sequence identity to their orthologues from human. The mRGS10 gene is expressed predominantly in brain and testis but it is also found in heart, lung, bone marrow, lymph node and spleen. Differential display between mature B lymphocytes and marginal zone B cells, as well as reverse transcription-polymerase chain reaction and Northern blot, showed that mRGS10 is differentially transcribed during B-cell differentiation. Finally, mRGS10 protein was detected in plasma cells of secondary lymphoid organs by immunofluorescence.

Peripheral T cells in the thymus: have they just lost their way or do they do something?

In young adult mice, the thymus produces about a million newly formed T cells every day that colonize peripheral lymphoid tissues. Mostly regarded as a primary lymphoid organ only, the relationship between the thymus and peripheral lymphoid organs is considered unidirectional. However, this perception has been challenged by reports showing that peripheral lymphocytes, mostly T cells, can migrate back into the thymus. The presence of recirculating T cells in the thymus is rather incongruous and raises the question: is the presence of 'peripheral' T cells in the thymus superfluous or do these cells fulfill some relevant physiologic functions? There is now evidence that cells of the hematopoietic lineage, including T cells, can play an active role during thymocyte selection, a role generally considered the exclusive property of thymic epithelial cells and dendritic cells. Although, on a per cell basis, peripheral T cells in the thymus may be less efficient than thymus epithelial cells or dendritic cells at thymocyte positive and negative selection, they may nevertheless contribute to selection by influencing the selectable TCR repertoire and post-selection T cell functionality. Here, peripheral lymphocytes re-entering the thymus may be envisioned as Trojan horses as these cells may introduce antigens necessary for both positive and negative selection of T cells.

T cell receptor contact to restricting MHC molecules is a prerequisite for peripheral interclonal T cell competition.

T cell survival and homeostatic proliferation in the periphery requires T cell receptor (TCR) binding to restricting major histocompatability complex (MHC)-encoded molecules, as well as the availability of certain lymphokines. However, the exact mechanisms by which these signals interrelate and contribute to homeostasis are not understood. By performing T cell transfers into TCR transgenic hosts we detected a hierarchical order of homeostatic proliferation for T cells differing in MHC restriction, such that OT1 cells (K(b) restricted) proliferated in P14 (D(b)-restricted TCR) recipients, but not vice versa. Using K(b) mutant mice, we demonstrated that proliferation of OT1 cells in P14 recipients, as well as the ability of host OT1 cells to hinder the proliferation of donor P14 cells, were dependent on OT1-TCR binding to K(b) molecules. However, interclonal T cell competition was not mediated simply by competition for physical access to the MHC-bearing cell. This was shown in parabiotic pairs of OT1 and K(b) mutant mice in which P14 cells failed to proliferate, even though the OT1 cells could not interact with half of the APCs in the system. Thus, we conclude that the interaction between the TCR and restricting MHC molecule influences the ability to compete for trophic resources not bound to the stimulating APC. This mechanism allows a local competitiveness that extends beyond a T cell's specificity.

S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage.

During the postnatal development, astrocytic cells in the neocortex progressively lose their neural stem cell (NSC) potential, whereas this peculiar attribute is preserved in the adult subventricular zone (SVZ). To understand this fundamental difference, many reports suggest that adult subventricular GFAP-expressing cells might be maintained in immature developmental stage. Here, we show that S100B, a marker of glial cells, is absent from GFAP-expressing cells of the SVZ and that its onset of expression characterizes a terminal maturation stage of cortical astrocytic cells. Nevertheless, when cultured in vitro, SVZ astrocytic cells developed as S100B expressing cells, as do cortical astrocytic cells, suggesting that SVZ microenvironment represses S100B expression. Using transgenic s100b-EGFP cells, we then demonstrated that S100B expression coincides with the loss of neurosphere forming abilities of GFAP expressing cells. By doing grafting experiments with cells derived from beta-actin-GFP mice, we next found that S100B expression in astrocytic cells is repressed in the SVZ, but not in the striatal parenchyma. Furthermore, we showed that treatment with epidermal growth factor represses S100B expression in GFAP-expressing cells in vitro as well as in vivo. Altogether, our results indicate that the S100B expression defines a late developmental stage after which GFAP-expressing cells lose their NSC potential and suggest that S100B expression is repressed by adult SVZ microenvironment.

Peripheral T cell lymphopenia and concomitant enrichment in naturally arising regulatory T cells: the case of the pre-Talpha gene-deleted mouse.

In pre-Talpha (pTalpha) gene-deleted mice, the positively selectable CD4+ CD8+ double-positive thymocyte pool is only 1% that in wild-type mice. Consequently, their peripheral T cell compartment is severely lymphopenic with a concomitant increase in proportion of CD25+ FoxP3+ regulatory T cells. Using mixed bone marrow chimeras, where thymic output was 1% normal, the pTalpha(-/-) peripheral T cell phenotype could be reproduced with normal cells. In the pTalpha(-/-) thymus and peripheral lymphoid organs, FoxP3+ CD4+ cells were enriched. Parabiosis experiments showed that many pTalpha(-/-) CD4+ single-positive thymocytes represented recirculating peripheral T cells. Therefore, the enrichment of FoxP3+ CD4+ single-positive thymocytes was not solely due to increased thymic production. Thus, the pTalpha(-/-) mouse serves as a model system with which to study the consequences of chronic decreased thymic T cell production on the physiology of the peripheral T cell compartment.

Effects of increasing IL-7 availability on lymphocytes during and after lymphopenia-induced proliferation.

IL-7 is critically involved in regulating peripheral T cell homeostasis. To investigate the role of IL-7 on lymphopenia-induced proliferation of polyclonal lymphocytes, we have transferred CFSE-labeled cells into a novel T-lymphopenic, IL-7-transgenic mouse line. Results obtained indicate that T and B cells do not respond in the same way to IL-7-homeostatic signals. Overexpression of IL-7 enhances proliferation of both CD4(+) and CD8(+) T cells but with distinctly temporal effects. Expansion of naturally arising CD4(+)-regulatory T cells was like that of conventional CD4(+) T cells. IL-7 had no effect on B cell proliferation. By immunohistology, transferred T cells homed to T cell areas of spleen lymphoid follicles. Increasing IL-7 availability enhanced T cell recovery by promoting cell proliferation and reducing apoptosis during early stages of lymphopenia-induced proliferation. Taken together, these results provide new insights into the pleiotropic effects of IL-7 on lymphopenia-induced T cell proliferation.

Differential expression of regulator of G-protein signalling transcripts and in vivo migration of CD4+ naïve and regulatory T cells.

The immune response of T lymphocytes to pathogens is initiated in draining secondary lymphoid organs, and activated cells then migrate to the site of infection. Thus, control of naive and regulatory CD4+ T-cell migration is crucial; however, it is poorly understood in physiological and pathological conditions. We found that CD4+ subpopulations displayed characteristic regulator of G-protein signalling (RGS) gene expression profiles. Regulatory T cells express higher levels of RGS1, RGS9 and RGS16 than naive cells. These genes are up-regulated upon cell activation and their level of expression correlates with in vivo cell migration. Using parabiosis, we showed that regulatory T lymphocytes migrate less than naive T cells and that migrant naive T cells express even lower RGS levels than their static counterparts. Our results show an inverse correlation between the capacity to migrate and the levels of RGS1, RGS9 and RGS16 for both naive and regulatory T cells. Taken together, these results suggest a role for RGS molecules in chemokine-induced lymphocyte migration and demonstrate the peculiarity of regulatory T cells in terms of phenotype and migration ability, providing new insights into their function.

B lymphocyte life span, rate of division and differentiation are regulated by total cell number.

I analyzed how B cell survival, the rate of B cell division and the rate of B cell activation may be affected in mice with different numbers of peripheral B cells. For this purpose, I created bone marrow (BM) chimeras with different BM B cell production; I analyzed peripheral B cell dynamics in these animals and studied the fate of carboxyfluorescein succinimidyl ester-labeled lymph node B cells transferred into these mice. I found that in lymphopenic mice, B cell life span is shortened and the rates of terminal B cell differentiation and cell death increased. As a consequence, I did not observe accumulation of B cells in mice with reduced but constant B-cell-poiesis. These results suggest that peripheral B cell survival relies not only on the number of competitors, as previously shown, but also on the rate of throughput of cells in a particular compartment.

B-cell homeostasis, competition, resources, and positive selection by self-antigens.

In adult mice, the number of B lymphocytes remains constant under homeostatic control, in spite of the fact that B cells are produced continuously in numbers that largely exceed the number required to replenish the peripheral pools. It follows that each newly formed lymphocyte can only persist if another lymphocyte dies. In an immune system where the total number of cells is limited, cell survival is no longer a passive phenomenon but rather a continuous active process where each lymphocyte must compete with other lymphocytes to survive. Consequently, the number and the life expectancy of a B-cell clone vary according to the presence or absence of competitor populations. This process of lymphocyte competition is likely controlled by a common need for resources that are in limited supply. The number of peripheral B-cells varies according to the availability of B-cell receptor (BCR) ligands. Indeed, it is possible to modify steady-state B-cell numbers by antigen manipulation. Moreover, conventional self-reactive B cells can undergo positive selection. We showed that the fate of a self-reactive B cell is determined by the quantity of self-antigens, the number of antigen-specific receptors engaged, and its overall antigen-binding avidity rather than the affinity of individual BCRs.