Marcus Svensson

Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing

Gut-associated lymphoid tissue (GALT) dendritic cells (DCs) display a unique ability to generate CCR9+ α 4 β 7 + gut-tropic CD8+ effector T cells. We demonstrate efficient induction of CCR9 and α 4 β 7 on CD8+ T cells in mesenteric lymph nodes (MLNs) after oral but not intraperitoneal (i.p.) antigen administration indicating differential targeting of DCs via the oral route. In vitro, lamina propria (LP)–derived DCs were more potent than MLN or Peyers patch DCs in their ability to generate CCR9+ α 4 β 7 + CD8+ T cells. The integrin α chain CD103 (α E) was expressed on almost all LP DCs, a subset of MLN DCs, but on few splenic DCs. CD103+ MLN DCs were reduced in number in CCR7−/− mice and, although CD8+ T cells proliferated in the MLNs of CCR7−/− mice after i.p. but not oral antigen administration, they failed to express CCR9 and had reduced levels of α 4 β 7. Strikingly, although CD103+ and CD103− MLN DCs were equally potent at inducing CD8+ T cell proliferation and IFN-γ production, only CD103+ DCs were capable of generating gut-tropic CD8+ effector T cells in vitro. Collectively, these results demonstrate a unique function for LP-derived CD103+ MLN DCs in the generation of gut-tropic effector T cells.

Selective Generation of Gut Tropic T Cells in Gut-associated Lymphoid Tissue (GALT) Requirement for GALT Dendritic Cells and Adjuvant

In the current study, we address the underlying mechanism for the selective generation of gut-homing T cells in the gut-associated lymphoid tissues (GALT). We demonstrate that DCs in the GALT are unique in their capacity to establish T cell gut tropism but in vivo only confer this property to T cells in the presence of DC maturational stimuli, including toll-like receptor-dependent and -independent adjuvants. Thus, DCs from mesenteric LNs (MLNs), but not from spleen, supported expression of the chemokine receptor CCR9 and integrin α4β7 by activated CD8+ T cells. While DCs were also required for an efficient down-regulation of CD62L, this function was not restricted to MLN DCs. In an adoptive CD8+ T cell transfer model, antigen-specific T cells entering the small intestinal epithelium were homogeneously CCR9+α4β7 +CD62Llow, and this phenotype was only generated in GALT and in the presence of adjuvant. Consistent with the CCR9+ phenotype of the gut-homing T cells, CCR9 was found to play a critical role in the localization of T cells to the small intestinal epithelium. Together, these results demonstrate that GALT DCs and T cell expression of CCR9 play critical and integrated roles during T cell homing to the gut.

Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes

β1 integrins are ubiquitously expressed receptors that mediate cell–cell and cell–extracellular matrix interactions. To analyze the function of β1 integrin in skin we generated mice with a keratinocyte‐restricted deletion of the β1 integrin gene using the cre–loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of α6β4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin‐5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal–epidermal junction. In contrast, the integrity of the basement membrane surrounding the β1‐deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of β1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiaton and proliferation, and in the formation and/or maintenance of hemidesmosomes.

CCL25 mediates the localization of recently activated CD8alphabeta(+) lymphocytes to the small-intestinal mucosa.

The recruitment of antigen-specific T lymphocytes to the intestinal mucosa is central to the development of an effective mucosal immune response, yet the mechanism by which this process occurs remains to be fully defined. Here we show that the CC chemokine receptor 9 (CCR9) is selectively and functionally expressed on murine alpha(E)beta(7)(+) naive CD8alphabeta(+) lymphocytes and a subset of recently activated CD69(+) CD8alphabeta(+) lymphocytes. Using a T cell receptor transgenic transfer model, we demonstrate that CCR9 expression is functionally maintained on CD8alphabeta(+) lymphocytes following activation in mesenteric lymph nodes but rapidly downregulated on CD8alphabeta(+) lymphocytes activated in peripheral lymph nodes. These recently activated CCR9(+) CD8alphabeta(+) lymphocytes selectively localized to the small-intestinal mucosa, and in vivo neutralization of the CCR9 ligand, CCL25, reduced the ability of these cells to populate the small-intestinal epithelium. Together these results demonstrate an important role for chemokines in the localization of T lymphocytes to the small-intestinal mucosa and suggest that targeting CCL25 and/or CCR9 may provide a means to selectively modulate small-intestinal immune responses.

Complementary signaling through flt3 and interleukin-7 receptor alpha is indispensable for fetal and adult B cell genesis

Extensive studies of mice deficient in one or several cytokine receptors have failed to support an indispensable role of cytokines in development of multiple blood cell lineages. Whereas B1 B cells and Igs are sustained at normal levels throughout life of mice deficient in IL-7, IL-7Rα, common cytokine receptor gamma chain, or flt3 ligand (FL), we report here that adult mice double deficient in IL-7Rα and FL completely lack visible LNs, conventional IgM+ B cells, IgA+ plasma cells, and B1 cells, and consequently produce no Igs. All stages of committed B cell progenitors are undetectable in FL−/− × IL-7Rα−/− BM that also lacks expression of the B cell commitment factor Pax5 and its direct target genes. Furthermore, in contrast to IL-7Rα−/− mice, FL−/− × IL-7Rα−/− mice also lack mature B cells and detectable committed B cell progenitors during fetal development. Thus, signaling through the cytokine tyrosine kinase receptor flt3 and IL-7Rα are indispensable for fetal and adult B cell development.

CCL25/CCR9 promotes the induction and function of CD103 on intestinal intraepithelial lymphocytes.

The integrin CD103 and the chemokine receptor CCR9 are co‐expressed on small intestinal CD8+ intraepithelial lymphocytes (IEL), naïve murine CD8+ T cells and by a small population of effector/memory CD8+ T cells, indicating a potential role for CCR9 in regulating CD103 expression and function. Here, we demonstrate that CD103, in contrast to CCR9, is down‐regulated on CD8+ T cells following their activation in mesenteric lymph nodes and that effector CD8+ T cells upon initial entry into the small intestinal epithelium are CCR9+CD103–. CD103 was rapidly induced on wild‐type CD8+ T cells subsequent to their entry into the small intestinal epithelium, however, CCR9–/– CD8+ T cells exhibited a significant delay in CD103 induction at this site. In addition, the CCR9 ligand, CCL25, that is constitutively expressed in the small intestinal epithelium, induced transient, dose‐dependent and pertussis toxin‐sensitive CD103‐mediated adhesion of CD8+ small intestinal IEL to a murine E‐cadherin human Fc (mEFc) fusion protein. Together, these results demonstrate a role for CCR9/CCL25 in promoting the induction and function of CD103 on CD8+ IEL and suggest that this chemokine receptor/chemokine pair may function to regulate lymphocyte‐epithelial interactions in the small intestinal mucosa.

β1 Integrin Is Not Essential for Hematopoiesis but Is Necessary for the T Cell-Dependent IgM Antibody Response

Several experimental evidences suggested that beta1 integrin-mediated adhesion of hematopoietic stem cells (HSC) is important for their function in the bone marrow (BM). Using induced deletion of the beta1 integrin gene restricted to the hematopoietic system, we show that beta1 integrin is not essential for HSC retention in the BM, hematopoiesis, and trafficking of lymphocytes. However, immunization with a T cell-dependent antigen resulted in virtually no IgM production and an increased secretion of IgG in mutant mice, while the response to a T cell-independent type 2 antigen showed decreases in both IgM and IgG. These data suggest that beta1 integrins are necessary for the primary IgM antibody response.

Differential homing mechanisms regulate regionalized effector CD8αβ+ T cell accumulation within the small intestine

The CC chemokine receptor (CCR)9 is expressed on the majority of small intestinal, but few colonic, T cells, whereas its ligand CCL25 is constitutively expressed by small intestinal epithelial cells. As such, CCR9/CCL25 have been proposed to play a central role in regulating small intestinal but not colonic immune responses and thus to organize regionalized immunity within the intestinal mucosa. Here, we demonstrate that CCL25 is expressed at reduced levels by epithelial cells in the distal compared with proximal small intestine, which correlated with less efficient CCR9-dependent effector CD8αβ+ T cell entry into the ileal epithelium. In vitro-generated α4β7+ effector CD8αβ+ T cell entry into the lamina propria was less dependent on CCR9 than entry into the epithelium along the entire length of the small intestine and in particular in the ileum. CCR9-independent α4β7+ effector CD8αβ+ T cell entry was pertussis toxin-sensitive, suggesting a role for additional GαI-linked G protein-coupled receptors. Finally, in vivo-primed effector CD8αβ+ T cells displayed regionalized differences in their entry to the small intestinal epithelium with enhanced CCR9-independent entry to the ileum. These results highlight a hitherto underappreciated compartmentalization of immune responses within the small intestine and have direct implications for targeting strategies aimed at regulating T cell localization to the small intestinal mucosa.

Functional Characterization of the CCL25 Promoter in Small Intestinal Epithelial Cells Suggests a Regulatory Role for Caudal-Related Homeobox (Cdx) Transcription Factors

The chemokine CCL25 is selectively and constitutively expressed in the small intestinal epithelium and plays an important role in mediating lymphocyte recruitment to this site. In this study, we demonstrate that CCL25 expression in murine small intestinal epithelial cells is independent of signaling through the lymphotoxin β receptor and is not enhanced by inflammatory stimuli, pathways involved in driving the expression of most other chemokines. We define a transcriptional start site in the CCL25 gene and a region −141 to −5 proximal of exon 1 that is required for minimal promoter activity in the small intestinal epithelial cell lines, MODE-K and mICc12. These cell lines expressed far less CCL25 mRNA than freshly isolated small intestinal epithelial cells indicating that they are missing important factors driving CCL25 expression. The CCL25 promoter contained putative binding sites for the intestinal epithelial-associated Caudal-related homeobox (Cdx) transcription factors Cdx-1 and Cdx-2, and small intestinal epithelial cells but not MODE-K and mICc12 cells expressed Cdx-1 and Cdx-2. EMSA analysis demonstrated that Cdx proteins were present in nuclear extracts from freshly isolated small intestinal epithelial cells but not in MODE-K or mICcl2 cells, and bound to putative Cdx sites within the CCL25 promoter. Finally, cotransfection of MODE-K cells with Cdx transcription factors significantly increased CCL25 promoter activity as well as endogenous CCL25 mRNA levels. Together these results demonstrate a unique pattern of regulation for CCL25 and suggest a role for Cdx proteins in regulating CCL25 transcription.

Involvement of CCL25 (TECK) in the generation of the murine small-intestinal CD8alpha alpha+CD3+ intraepithelial lymphocyte compartment.

The CC chemokine CCL25 (TECK) is selectively expressed in the thymus and small intestine, indicating a potential role in T lymphocyte development. In the present study we examined the role of CCL25 in the generation of the small‐intestinal CD8α α+CD3+ intraepithelial lymphocyte (IEL) compartment. CCL25 mRNA expression in the murine small intestine increased at three weeks of age and corresponded with the appearance of CD8α α+CD3+ lymphocytes in the small‐intestinal epithelium. Administration of monoclonal neutralizing anti‐CCL25 antibody to two‐week‐old mice led to a ∼50% reduction in the total number of CD8α α+TCRγ δ+ and CD8α α+TCRα β+ IEL at four weeks of age. Freshly isolated murine CD8α α+CD3+ IEL migrated in response to CCL25 and expressed the CCL25 receptor, CCR9. Analysis of CCR9 expression on putative IEL precursor populations demonstrated the presence of both CCR9– and CCR9+ cells and indicated that up‐regulation of this receptor occurred during IEL precursor differentiation. Finally, data from wild‐type and RAG–/– mice suggested that the reduction in CD8α α+CD3+ IEL in anti‐CCL25 antibody treated mice resulted primarily from defective maintenance and/or development of IEL precursors rather than a direct effect on mature CD8α α+CD3+ IEL.