The elderly constitute the most rapidly growing subpopulation in the United States. This age group represents a significant burden on the healthcare system due, in part, to increases in morbidity and mortality associated with an increase in the incidence of intestinal infectious diseases. Our previous studies suggest that impaired homing of IgA immunoblasts from the Peyer's patches to the intestinal lamina propria contributes to the diminished intestinal immune response in the elderly. The present study employs flow cytometry and quantitative immunohistochemistry to assess age-related changes in the numbers of peripheral blood mononuclear cells expressing the homing integrin alpha4beta7 and vascular endothelial cells in the intestine expressing its specific receptor, the address in MAdCAM-1, in inbred Fischer 344 rats. The proportion of alpha4beta7-positive mononuclear cells in young rats is significantly greater than that measured in the blood of senescent animals. Although the density of intestinal lamina propria blood vessels with MAdCAM-1-positive endothelium was greater in young adult rats in comparison to old animals, this difference achieved only borderline statistical significance. This is the first study to examine the expression of these two critical lymphocyte homing molecules as a function of age.

Alternative delivery routes of the current Mycobacterium tuberculosis (Mtb) vaccine, intradermally (ID) delivered BCG, may provide better protection against tuberculosis, and be more easily administered. Here, we use rhesus macaques to compare the airway immunogenicity of BCG delivered via either ID or intragastric gavage vaccination. Ag-specific CD4 T cell responses in the blood were similar after BCG vaccination via gavage or ID injection. However, gavage BCG vaccination induced significantly lower T cell responses in the airways compared to intradermal BCG vaccination. Examining T cell responses in lymph node biopsies showed that ID vaccination induced T cell priming in skin-draining lymph nodes, while gavage vaccination induced priming in the gut-draining nodes, as expected. While both delivery routes induced highly functional Ag-specific CD4 T cells with a Th1* phenotype (CXCR3+CCR6+), gavage vaccination induced the co-expression of the gut-homing integrin α4β7 on Ag-specific Th1* cells, which was associated with reduced migration into the airways. Thus, in rhesus macaques, the airway immunogenicity of gavage BCG vaccination may be limited by the imprinting of gut-homing receptors on Ag-specific T cells primed in intestinal lymph nodes. IMPORTANCE Mycobacterium tuberculosis (Mtb) is a leading cause of global infectious disease mortality. The vaccine for Mtb, Bacillus Calmette-Guérin (BCG), was originally developed as an oral vaccine, but is now given intradermally. Recently, clinical studies have reevaluated oral BCG vaccination in humans and found that it induces significant T cell responses in the airways. Here, we use rhesus macaques to compare the airway immunogenicity of BCG delivered intradermally or via intragastric gavage. We find that gavage BCG vaccination induces Mtb-specific T cell responses in the airways, but to a lesser extent than intradermal vaccination. Furthermore, gavage BCG vaccination induces the gut-homing receptor a4ß7 on Mtb-specific CD4 T cells, which was associated with reduced migration into the airways. These data raise the possibility that strategies to limit the induction of gut-homing receptors on responding T cells may enhance the airway immunogenicity of oral vaccines.

The addition of sulfate to glycan structures can regulate their ability to serve as ligands for glycan-binding proteins. Although sulfate groups present on the monosaccharides glucosamine, uronate, N-acetylglucosamine and N-acetylgalactosamine are recognized by defined receptors that mediate important functions, the functional significance of galactose-6-O-sulfate (Gal6S) is not known. However, in vitro studies using synthetic glycans and sulfotransferase overexpression implicate Gal6S as a binding determinant for the lymphocyte homing receptor, L-selectin. Only two sulfotransferases have been shown to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase-1 (C6ST-1). In the present study, we use mice deficient in KSGal6ST and C6ST-1 to test whether Gal6S contributes to ligand recognition by L-selectin in vivo. First, we establish that KSGal6ST is selectively expressed in high endothelial venules (HEVs) in lymph nodes and Peyer's patches. We also determine by mass spectrometry that KSGal6ST generates Gal6S on several classes of O-glycans in peripheral lymph nodes. Furthermore, KSGal6ST, but not C6ST-1, is required for the generation of the Gal6S-containing glycan, 6,6'-disulfo-3'sLN (Siaα2→3[6S]Galβ1→4[6S]GlcNAc) or a closely related structure in lymph node HEVs. Nevertheless, L-selectin-dependent short-term homing of lymphocytes is normal in KSGal6ST-deficient mice, indicating that the Gal6S-containing structures we detected do not contribute to L-selectin ligand recognition in this setting. These results refine our understanding of the biological ligands for L-selectin and introduce a mouse model for investigating the functions of Gal6S in other contexts.

Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4(+) and CD8(+) T cells secreting INFgamma, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFgamma-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4(+) and CD8(+) T cell subsets, IFNgamma-secreting RV-specific CD8(+) but not CD4(+) T cells were detected in recently infected children. Using the same approach, both CD4(+) and CD8(+) RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFgamma-secreting CD4(+) T cells from adult volunteers preferentially express the intestinal homing receptor alpha4beta7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFgamma-secreting CD4(+) T cells preferentially express L-selectin but not alpha4beta7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFgamma is independent of the expression of these homing receptors.

CCR9 and α4β7 are the major trafficking receptors for lymphocyte migration to the gut, and their expression is induced during lymphocyte activation under the influence of retinoic acid (RA). We report here that BATF (basic leucine zipper transcription factor, ATF-like), an AP-1 protein family factor, is required for optimal expression of CCR9 and α4β7 by T helper cells. BATF-deficient (knockout [KO]) mice had reduced numbers of effector T and regulatory T cells in the intestine. The intestinal T cells in BATF KO mice expressed CCR9 and α4β7 at abnormally low levels compared with their wild-type (WT) counterparts, and BATF KO CD4(+) T cells failed to up-regulate the expression of CCR9 and α4β7 to WT levels in response to RA. Defective binding of RARα and histone acetylation at the regulatory regions of the CCR9 and Itg-α4 genes were observed in BATF KO T cells. As a result, BATF KO effector and FoxP3(+) T cells failed to populate the intestine, and neither population functioned normally in the induction and regulation of colitis. Our results establish BATF as a cellular factor required for normal expression of CCR9 and α4β7 and for the homeostasis and effector functions of T cell populations in the intestine.

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a "hub" gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract.

Using lymphocyte function-associated antigen (LFA)-1(-/-) mice, we have examined the role of LFA-1 and other integrins in the recirculation of lymphocytes. LFA-1 has a key role in migration to peripheral lymph nodes (pLNs), and influences migration into other LNs. Second, the alpha4 integrins, alpha4beta7 and alpha4beta1, have a hitherto unrecognized ability to compensate for the lack of LFA-1 in migration to pLNs. These findings are confirmed using normal mice and blocking LFA-1 and alpha4 monoclonal antibodies. Unexpectedly, vascular cell adhesion molecule (VCAM)-1, which is essential in inflammatory responses, serves as the ligand for the alpha4 integrins on pLN high endothelial venules. VCAM-1 also participates in trafficking into mesenteric LNs and Peyer's patch nodes where mucosal addressin cell adhesion molecule 1 (MAdCAM-1), the alpha4beta7-specific ligand, dominates. Both alpha4beta1, interacting with ligand VCAM-1, and also LFA-1 participate in substantial lymphocyte recirculation through bone marrow. These observations suggest that organ-specific adhesion receptor usage in mature lymphocyte recirculation is not as rigidly adhered to as previously considered, and that the same basic sets of adhesion receptors are used in both lymphocyte homing and inflammatory responses.

Whereas the role of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein (NLRP) 3 pathway in innate immunity has been extensively studied, little attention has been paid to its contribution to adaptive immunity. Studies in animal models and human subjects have shown the contribution of NLRP3 to the T cell compartment, and its role in B lymphocyte functions has been proposed. Here, we report that ablation of nlrp3 in mice led to altered B cell development in the bone marrow, and distorted expression of B cell subsets that play innate-like functions, that is, marginal zone B cells in the spleen and B-1a cells in the peritoneal cavity. Mechanistically, in the absence of NLRP3 expression, the transcription factor IRF4, previously found to interact with NLRP3 in the nucleus of lymphocytes, was up-regulated. NLRP3 ablation reduced the expression of the chemokine receptors CXCR4 and CCR7 in an IRF4-dependent manner, indicating that the presence of NLRP3 is critical for optimal expression of chemokine receptors on B cells. We conclude that activation of the NLRP3 inflammasome plays a role in B cell development, homing, and retention in lymphoid organs.

Cell migration relies on coordinated activity of chemotactic and guidance receptors. Here, we report a specific role for the RNA-binding protein ZFP36L1 in limiting the abundance of molecules involved in the homing of antibody-secreting cells (ASCs) to the bone marrow (BM). In the absence of ZFP36L1, ASCs build up in the spleen and the liver and show diminished accumulation in the BM. ZFP36L1 facilitates migration by directly regulating G protein-coupled receptor kinase 2 (GRK2) and the integrin chains α4 and β1 in splenic ASCs. Expression of CXCR4 and of the integrins α4 and β1 is differentially regulated on ASCs produced at the early and late stages of the immune response. Consequently, deletion of the Zfp36l1 gene has a stronger effect on BM accumulation of high-affinity ASCs formed late in the response. Thus, ZFP36L1 is an integral part of the regulatory network controlling gene expression during ASC homing.

Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. Using tetrameric complexes of human histocompatibility leukocyte antigen (HLA) class I to identify antigen-specific T cells ex vivo, we observed high frequencies of circulating MelanA-specific, A*0201-restricted cytotoxic T lymphocytes (A2-MelanA tetramer+ CTLs) in seven of nine HLA-A*0201-positive individuals with vitiligo. Isolated A2-MelanA tetramer+ CTLs were able to lyse A*0201-matched melanoma cells in vitro and their frequency ex vivo correlated with extent of disease. In contrast, no A2-MelanA tetramer+ CTL could be identified ex vivo in all four A*0201-negative vitiligo patients or five of six A*0201-positive asymptomatic controls. Finally, we observed that the A2-MelanA tetramer+ CTLs isolated from vitiligo patients expressed high levels of the skin homing receptor, cutaneous lymphocyte-associated antigen, which was absent from the CTLs seen in the single A*0201-positive normal control. These data are consistent with a role of skin-homing autoreactive melanocyte-specific CTLs in causing the destruction of melanocytes seen in autoimmune vitiligo. Lack of homing receptors on the surface of autoreactive CTLs could be a mechanism to control peripheral tolerance in vivo.

T cells expressing the cutaneous lymphocyte antigen (CLA) mediate pathogenic inflammation in atopic dermatitis (AD). The molecular alterations contributing to their dysregulation remain unclear. With the aim to elucidate putative altered pathways in AD we profiled DNA methylation levels and miRNA expression in sorted T cell populations (CD4+, CD4+CD45RA+ naïve, CD4+CLA+, and CD8+) from adult AD patients and healthy controls (HC). Skin homing CD4+CLA+ T cells from AD patients showed significant differences in DNA methylation in 40 genes compared to HC (p < 0.05). Reduced DNA methylation levels in the upstream region of the interleukin-13 gene (IL13) in CD4+CLA+ T cells from AD patients correlated with increased IL13 mRNA expression in these cells. Sixteen miRNAs showed differential expression in CD4+CLA+ T cells from AD patients targeting genes in 202 biological processes (p < 0.05). An integrated network analysis of miRNAs and CpG sites identified two communities of strongly interconnected regulatory elements with strong antagonistic behaviours that recapitulated the differences between AD patients and HC. Functional analysis of the genes linked to these communities revealed their association with key cytokine signaling pathways, MAP kinase signaling and protein ubiquitination. Our findings support that epigenetic mechanisms play a role in the pathogenesis of AD by affecting inflammatory signaling molecules in skin homing CD4+CLA+ T cells and uncover putative molecules participating in AD pathways.

Type 1 Diabetes Mellitus (T1DM) is characterized by an augmented pro-inflammatory immune state. This contributes to the increased risk for gestational complications observed in T1DM mothers. In normal pregnancies, critical immunological changes occur, including the massive recruitment of lymphocytes, particularly CD56bright NK cells, into early decidua basalis and a 2nd trimester shift towards Type 2 immunity. Decidual CD56bright NK cells arise at least partly from circulating progenitors expressing adhesion molecules SELL and ITGA4 and the chemokine receptors CXCR3 and CXCR4. In vitro studies show that T1DM reduces interactions between blood CD56+ NK cells and decidual endothelial cells by reducing SELL and ITGA4-based interactions. To address the mechanisms by which specific lymphocyte subsets may be recruited from the circulation during pregnancy and whether these mechanisms are altered in T1DM, flow cytometry was used to examine eight peripheral blood lymphocyte subsets (Type 1 (IL18R1+) and Type 2 (IL1RL1+) CD56bright NK, CD56dim NK, NKT and T cells) from control and T1DM women. Blood was collected serially over pregnancy and postpartum, and lymphocytes were compared for expression of homing receptors SELL, ITGA4, CXCR3, and CXCR4. The decline of Type 1/Type 2 immune cells in normal pregnancy was driven by an increase in Type 2 cells that did not occur in T1DM. CD56bright NK cells from control women had the highest expression of all four receptors with greatest expression in 2nd trimester. At this time, these receptors were expressed at very low levels by CD56bright NK cells from TIDM patients. Type 1/Type 2 NKT cell ratios were not influenced by either pregnancy or TIDM. Our results suggest that T1DM alters immunological balances during pregnancy with its greatest impact on CD56bright NK cells. This implicates CD56bright NK cells in diabetic pregnancy complications.

Tyrosine kinase 2 (TYK2) belongs to the Janus kinase (JAK) family of tyrosine kinases, which transmit signals from activated cytokine receptors. GWAS have consistently implicated TYK2 in psoriasis susceptibility. We performed an in-depth association analysis of TYK2 using GWAS and resequencing data. Strong genetic association of three nonsynonymous variants in the exonic regions of the TYK2 gene (rs34536443, rs12720356, and rs2304256) were found. rs12720356 encoding I684S is predicted to be deleterious based on its location in the pseudokinase domain. We analyzed PBMCs from 29 individuals representing the haplotypes containing each of the significantly associated signals. STAT4 phosphorylation was evaluated by phospho-flow cytometry after CD3/CD28 activation of cells followed by IL-12 stimulation. Individuals carrying the protective I684S variant manifested significantly reduced p-STAT4 levels in CD4 + CD25 + CD45RO+ (mean Stimulation Index (S.I.) 48.08, n = 10) and CD8 + CD25 + CD45RO + cells (S.I. 55.71, n = 10), compared to controls homozygous for the ancestral haplotype (S.I. 68.19, n = 10 (p = 0.002) and 76.76 n = 10 (p = 0.0008) respectively). Reduced p-STAT4 levels were also observed in skin-homing, cutaneous lymphocyte associated antigen (CLA)-positive CD4 and CD8 cells from I684S carriers. No significant changes in p-STAT4 for the psoriasis-associated variant rs34536443 was found. These data establish the functional significance of the TYK2 I684S variant in psoriasis susceptibility.

The majority of T lymphocytes that infiltrate psoriatic lesions express cutaneous lymphocyte antigen (CLA), a skin homing receptor involved in the influx of memory T cells to cutaneous sites. We investigated CLA expression on normal human peripheral blood mononuclear cells (PBMCs) and evaluated its association with IL-12 receptors, chemokine receptor, CXCR3, and IL-2Ralpha. PBMCs were stimulated in vitro with or without polyclonal activators (mitogen, or superantigens, or anti-CD3+anti-CD28) in the presence or absence of exogenous rhIL-12. The percentage of CLA+ T lymphocytes increased significantly after superantigen stimulation compared to anti-CD3+anti-CD28 or mitogen activation. The majority of activation induced CLA+ T lymphocytes co-expressed IL-12Rbeta1, IL-12Rbeta2, CXCR3, and CD25 in the presence of rhIL-12. Our results indicate that CLA expression on activated T lymphocytes is IL-12 and activation dependent and correlates with the expression of IL-12 receptors, IL-2Ralpha, and CXCR3. Monitoring the levels of Th1 differentiation markers such as CXCR3 and IL-12Rbeta2 along with activation marker, CD25 on skin homing CLA+ T lymphocytes may provide insight into the mechanism of action of immunotherapies directed against Th1 type skin inflammatory diseases.

TECK (thymus-expressed chemokine), a recently described CC chemokine expressed in thymus and small intestine, was found to mediate chemotaxis of human G protein-coupled receptor GPR-9-6/L1.2 transfectants. This activity was blocked by anti-GPR-9-6 monoclonal antibody (mAb) 3C3. GPR-9-6 is expressed on a subset of memory alpha4beta7(high) intestinal trafficking CD4 and CD8 lymphocytes. In addition, all intestinal lamina propria and intraepithelial lymphocytes express GPR-9-6. In contrast, GPR-9-6 is not displayed on cutaneous lymphocyte antigen-positive (CLA(+)) memory CD4 and CD8 lymphocytes, which traffic to skin inflammatory sites, or on other systemic alpha4beta7(-)CLA(-) memory CD4/CD8 lymphocytes. The majority of thymocytes also express GPR-9-6, but natural killer cells, monocytes, eosinophils, basophils, and neutrophils are GPR-9-6 negative. Transcripts of GPR-9-6 and TECK are present in both small intestine and thymus. Importantly, the expression profile of GPR-9-6 correlates with migration to TECK of blood T lymphocytes and thymocytes. As migration of these cells is blocked by anti-GPR-9-6 mAb 3C3, we conclude that GPR-9-6 is the principal chemokine receptor for TECK. In agreement with the nomenclature rules for chemokine receptors, we propose the designation CCR-9 for GPR-9-6. The selective expression of TECK and GPR-9-6 in thymus and small intestine implies a dual role for GPR-9-6/CCR-9, both in T cell development and the mucosal immune response.

Humans with Wiskott-Aldrich syndrome display a progressive immunological disorder associated with compromised Wiskott-Aldrich Syndrome Interacting Protein (WIP) function. Mice deficient in WIP recapitulate such an immunodeficiency that has been attributed to T cell dysfunction; however, any contribution of B cells is as yet undefined. Here we have shown that WIP deficiency resulted in defects in B cell homing, chemotaxis, survival, and differentiation, ultimately leading to diminished germinal center formation and antibody production. Furthermore, in the absence of WIP, several receptors, namely the BCR, BAFFR, CXCR4, CXCR5, CD40, and TLR4, were impaired in promoting CD19 co-receptor activation and subsequent PI3 kinase (PI3K) signaling. The underlying mechanism was due to a distortion in the actin and tetraspanin networks that lead to altered CD19 cell surface dynamics. In conclusion, our findings suggest that, by regulating the cortical actin cytoskeleton, WIP influences the function of CD19 as a general hub for PI3K signaling.

IL-7 is essential for the development and homeostasis of T and B lymphocytes and is critical for neonatal lymph node organogenesis because Il7-/- mice lack normal lymph nodes. Whether IL-7 is a continued requirement for normal lymph node structure and function is unknown. To address this, we ablated IL-7 function in normal adult hosts. Either inducible Il7 gene deletion or IL-7R blockade in adults resulted in a rapid loss of lymph node cellularity and a corresponding defect in lymphocyte entry into lymph nodes. Although stromal and dendritic cell components of lymph nodes were present in normal numbers and representation, innate lymphoid cell (ILC) subpopulations were substantially decreased after IL-7 ablation. Testing lymphocyte homing in bone marrow chimeras reconstituted with Rorc-/- bone marrow confirmed that ILC3s in lymph nodes are required for normal lymphocyte homing. Collectively, our data suggest that maintenance of intact lymph nodes relies on IL-7-dependent maintenance of ILC3 cells.

The antigenic heterogeneity of the reticular framework of the white pulp and marginal zone is well documented in the human adult spleen. Immunostaining of α-smooth muscle actin characterizes the heterogeneity of the reticular framework of the white pulp and marginal zone. In the human spleen, the blood cells flow in an open circulation. T and B lymphocytes flow out from the arterial terminal, and migrate in the reticular framework. Homing of lymphocytes to lymphoid tissues is regulated by selective interactions between cell surface homing receptors and tissue vascular addressins at sites of lymphocyte recruitment from the blood. In the present study, mucosal addressin cell adhesion molecule-1 was selectively expressed on α-smooth muscle actin-positive reticular framework. The reticular framework may function in lymphocyte homing and segregation into the periarteriolar lymphoid sheath, lymph follicle and marginal zone.

Chemokine receptors direct T lymphocytes to the site of an infection by following coordinated chemokine gradients, which allow their recruitment to specific tissues. Although identification of receptors needed for homing to some mucosal sites, such as skin and gut, have been elucidated, the receptors that direct lymphocytes to the genital mucosa remain relatively uncharacterized. In this study we identify that the chemokine receptors CXCR3 (chemokine (C-X-C motif) receptor 3) and CCR5 (chemokine (C-C motif) receptor 5) are pivotal for T-lymphocyte access to the genital tract during Chlamydia trachomatis infection. Chlamydia-specific CD4(+) transgenic T cells that lack CXCR3 or CCR5 do not accumulate in the genital mucosa following infection. Loss of either CXCR3 or CCR5 impairs the protective capacity of Chlamydia-specific T cells, whereas T cells lacking both receptors are completely nonprotective. These results show that CXCR3 and CCR5 are the predominant chemokine receptors that act cooperatively to promote homing to the genital mucosa during Chlamydia infection.

T cell subsets are considered central orchestrators of inflammation and homeostasis in the intestine and are established targets for the treatment of inflammatory bowel disease. While approaches aimed at the neutralization of T cell effector cytokines have provided significant benefits for pediatric and adult patients, more recent strategies aimed at inhibiting the infiltration of pathogenic T cell subsets have also emerged. In this review, we describe current knowledge surrounding the function of T cell subsets in pediatric inflammatory bowel disease and outline approaches aimed at targeting T cell trafficking to the intestine which may represent a new treatment option for pediatric inflammatory bowel disease.