Expression Of Homing Molecules Research Articles

Parabiosis in Mice to Study Tissue Residency of Immune Cells.

Different populations of immune cells rely on their distinct migration patterns for immunosurveillance, immune regulation, tissue specific differentiation, and maturation. It is often important to clarify whether cells are recirculating or tissue resident, or whether tissue-specific cells are derived from blood-borne precursors or a tissue-resident population. Though migration or tissue residency of immune cells critically depends on the expression of different homing molecules (chemokine receptors, tissue retention molecules, etc.), characterization based solely on the expression of homing molecules may not faithfully reflect the migration patterns of immune cells. Therefore, a more reliable method to clarify migration patterns of immune cells is required. Parabiosis is a surgical connection of two mice resulting in a shared circulatory system, which allows reliable distinction of tissue-resident and circulating cells. Here, we describe a set of protocols for parabiosis, including technique details, pitfalls, and suggestions for optimization and troubleshooting. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of mice for parabiosis surgery Basic Protocol 2: Parabiosis surgery Basic Protocol 3: Recovery and use of mice after parabiosis surgery Basic Protocol 4: Reversal of parabiotic surgery Basic Protocol 5: Analysis of parabionts.

Single-Cell RNA Sequencing Reveals Tissue Compartment-Specific Plasticity of Mycosis Fungoides Tumor Cells.

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (TRM, CD69+CD27-NR4A1+RGS1+AHR+). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype (KLF2+TCF7+S1PR1+SELL+CCR7+), while retaining tissue-homing receptors (CLA, CCR10). The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory (IDO1) and Th2-associated mediators (CCL13, CCL17, CCL22). Given their expression of PVR, TNFRSF14 and CD80/CD86, they might be under direct control by TIGIT+CTLA4+CSF2+TNFSF14+ tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the TRM-like phenotype enables long-term skin residence of MF cells. Their switch to a TCM-like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF.

Benefit of Belatacept in Cord Blood-Derived Regulatory T Cell-Mediated Suppression of Alloimmune Response

The role of Regulatory T cells (Tregs) in tolerance induction post-transplantation is well-established, but Tregs adoptive transfer alone without combined immunosuppressants have failed so far in achieving clinical outcomes. Here we applied a set of well-designed criteria to test the influence of commonly used immunosuppressants (belatacept, tacrolimus, and mycophenolate) on cord blood-derived Tregs (CB-Tregs). Our study shows that while none of these immunosuppressants modulated the stability and expression of homing molecules by CB-Tregs, belatacept met all other selective criteria, shown by its ability to enhance CB-Tregs-mediated in vitro suppression of the allogeneic response without affecting their viability, proliferation, mitochondrial metabolism and expression of functional markers. In contrast, treatment with tacrolimus or mycophenolate led to reduced expression of functional molecule GITR in CB-Tregs, impaired their viability, proliferation and mitochondrial metabolism. These findings indicate that belatacept could be considered as a candidate in Tregs-based clinical immunomodulation regimens to induce transplant tolerance.

Characterization of gut-homing molecules in non-endstage livers of patients with primary sclerosing cholangitis and inflammatory bowel disease

IntroductionThe co-occurrence of inflammatory bowel disease (IBD) in up to 80% of patients with primary sclerosing cholangitis (PSC) suggests a relation between the gut and the liver in patients with both PSC and IBD. One hypothesis suggests that aberrantly expressed homing molecules in the liver drive infiltration of gut-homing memory T-cells that are originally primed in intestinal environment. One of the main findings supporting this hypothesis is the expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in PSC livers. Expression of homing molecules in early PSC remains unclear. The aim of this study was to investigate expression patterns of homing chemokines and adhesion molecules in PSC-IBD colons and livers, and to study whether changes are already present in early stages of PSC. MethodsNeedle biopsies from livers of 20 PSC patients with short-term PSC (PSC-IBDST) as well as explant liver biopsies of 8 patients with long-term PSC (PSC-IBDLT) were collected (median disease duration 0 and 22 years, respectively). Only patients with concomitant IBD were included (89% ulcerative colitis and 11% Crohn’s disease). Expression and distribution of MAdCAM-1, VAP-1, integrin β7, CCL25, CCL28, CXCL12, αE (CD103) and E-cadherin were assessed in both liver and colon tissue. Liver tissue collected from obstructive cholangitis in resection specimens for Klatskin tumors or resection specimens from hepatic metastasis, liver tissue of patients with hepatitis C virus (HCV) and of patients with primary biliary cholangitis (PBC) served as controls. ResultsMAdCAM-1 expression in livers of PSC-IBDLT patients was increased compared to controls. The proportion of CD3+ T-cells expressing integrin β7 did not differ between PSC-IBDST and control groups, but was higher in liver tissue of PSC-IBDLT patients. There was no difference in αE+ T-cells between PSC-IBDLT and control groups. The chemokine CCL28 was highly expressed in biliary epithelial cells. This intense staining pattern was more pronounced in PSC-IBDST, but overall did not significantly differ from controls. ConclusionsWe confirm that aberrant gut lymphocyte homing to the liver exists in PSC, linking gut and liver disease pathology in PSC-IBD. Our data suggests that this phenomenon increases over time in later stages of the disease, worsening ongoing inflammation.

B cell subset alteration and the expression of tissue homing molecules in dengue infected patients

BackgroundB cells play an essential role during dengue viral infection. While a major expansion of antibody secreting cells (ASCs) was observed, the importance of these increased frequencies of ASCs remains unclear. The alteration of B cell subsets may result from the expression of tissue specific homing molecules leading to their mobilization and distribution to different target organs during acute dengue viral infection.MethodsIn this study, whole blood samples were obtained from thirty pediatric dengue-infected patients and ten healthy children and then stained with fluorochrome-conjugated monoclonal antibodies against CD3, CD14, CD19, CD20, CD21, CD27, CD38, CD45, CD138 and homing molecules of interest before analyzed by polychromatic flow cytometry. B cell subsets were characterized throughout acute infection period.ResultsData shows that there were no detectable differences in frequencies of resting, activated and tissue memory cells, whereas the frequency of ASCs was significantly increased and associated with the lower frequency of naïve cells. These results were found from patients with both dengue fever and dengue hemorrhagic fever, suggesting that such change or alteration of B cells was not associated with disease severity. Moreover, several homing molecules (e.g., CXCR3 and CCR2) were found in ASCs, indicating that ASCs may distribute to inflamed tissues and various organs.ConclusionsFindings from this study provide insight into B cell subset distribution. Furthermore, organ mobilization according to homing molecule expression on different B cell subsets during the course of dengue viral infection also suggests they are distributed to inflamed tissues and various organs.

IL-2 Enhances Gut Homing Potential of Human Naive Regulatory T Cells Early in Life.

Recent evidence suggests early environmental factors are important for gut immune tolerance. Although the role of regulatory T (Treg) cells for gut immune homeostasis is well established, the development and tissue homing characteristics of Treg cells in children have not been studied in detail. In this article, we studied the development and homing characteristics of human peripheral blood Treg cell subsets and potential mechanisms inducing homing molecule expression in healthy children. We found contrasting patterns of circulating Treg cell gut and skin tropism, with abundant β7 integrin+ Treg cells at birth and increasing cutaneous lymphocyte Ag (CLA+) Treg cells later in life. β7 integrin+ Treg cells were predominantly naive, suggesting acquisition of Treg cell gut tropism early in development. In vitro, IL-7 enhanced gut homing but reduced skin homing molecule expression in conventional T cells, whereas IL-2 induced a similar effect only in Treg cells. This effect was more pronounced in cord compared with adult blood. Our results suggest that early in life, naive Treg cells may be driven for gut tropism by their increased sensitivity to IL-2-induced β7 integrin upregulation, implicating a potential role of IL-2 in gut immune tolerance during this critical period of development.

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in the CD34+CD133+CXCR4low fraction.

The feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, the expression of CD34, CD133, CD90, CD45RA, CD26 and CD9 was determined on sorted CD34+ cells according to CXCR4 (“neg”, “low” “bright”) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in the steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained only in the CD133+CXCR4low cells. The failure of the CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood cells, as well as to those issued from the bone marrow. These data represent the first phenotypic characterization of steady-state blood cells exhibiting short- and long-term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation.

T-bet deficiency attenuates cardiac remodelling in rats.

Previous studies have suggested the involvement of CD4 + T lymphocytes in cardiac remodelling. T-bet can direct Th1 lineage commitment. This study aimed to investigate the functional significance of T-bet in cardiac remodelling induced by pressure overload using T-bet global knockout rats. Increased T-bet levels were observed in rodent and human hypertrophied hearts. T-bet deficiency resulted in a less severe hypertrophic phenotype in rats. CD4 + T-lymphocyte reconstitution in T-bet-/- rats resulted in aggravated cardiac remodelling. T-cell homing molecule expression and cytokine secretion were altered in T-bet-deficient rat hearts. Administration of exogenous interferon-γ (IFN-γ) offset T-bet deficiency-mediated cardioprotection. Cardiomyocytes cultured in T-bet-/- CD4 + T-cell-conditioned media showed a reduced hypertrophic response after hypertrophic stimuli, which was abolished by an IFN-γ-neutralizing antibody. Taken together, our findings show that T-bet deficiency attenuates pressure overload-induced cardiac remodelling in rats. Specifically, targeting T-bet in T cells may be of great importance for the treatment of pathological cardiac remodelling and heart failure.

Single and combined effect of retinoic acid and rapamycin modulate the generation, activity and homing potential of induced human regulatory T cells.

Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) has been successfully utilized to treat graft versus host disease and represents a promising strategy for the treatment of autoimmune diseases and transplant rejection. The aim of this study was to evaluate the effects of all-trans retinoic acid (atRA) and rapamycin (RAPA) on the number, phenotype, homing markers expression, DNA methylation, and function of induced human Treg cells in short-term cultures. Naive T cells were polyclonally stimulated and cultured for five days in the presence of different combinations of IL-2, TGF-β1, atRA and RAPA. The resulting cells were characterized by the expression of FOXP3, activation, surface and homing markers. Methylation of the Conserved Non-coding Sequence 2 was also evaluated. Functional comparison of the different culture conditions was performed by suppression assays in vitro. Culturing naive human T cells with IL-2/TGFβ1 resulted in the generation of 54.2% of Treg cells (CD4+CD25+FOXP3+) whereas the addition of 100 nM atRA increased the yield of Treg cells to 66% (p = 0.0088). The addition of RAPA did not increase the number of Treg cells in any of these settings. Treg cells generated in the presence of atRA had an increased expression of the β7 integrin to nearly 100% of the generated Treg cells, while RAPA treated cells showed enhanced expression of CXCR4. The differential expression of homing molecules highlights the possibility of inducing Treg cells with differential organ-specific homing properties. Neither atRA nor RAPA had an effect on the highly methylated CNS2 sites, supporting reports that their contribution to the lineage stability of Treg cells is not mediated by methylation changes in this locus. Treg cells generated in the presence of RAPA show the most potent suppression effect on the proliferation of effector cells.

Gut-Homing Conventional Plasmablasts and CD27− Plasmablasts Elicited after a Short Time of Exposure to an Oral Live-Attenuated Shigella Vaccine Candidate in Humans

Currently, there is no licensed Shigella vaccine; however, various promising live-attenuated vaccine candidates have emerged, including CVD1208S (ΔguaBA, Δset, Δsen S. flexneri 2a), which was shown to be safe and immunogenic in Phase 1 clinical trials. Here, we report the immune responses elicited in an outpatient Phase 2 clinical trial in which subjects were vaccinated with CVD 1208S. Oral immunization with CVD 1208S elicited high anti-S. flexneri 2a LPS and IpaB antibody responses as well as an acute plasmablast (PB) infiltration in peripheral blood 7 days after immunization. PB sorted based on their expression of homing molecules confirmed that cells expressing integrin α4β7 alone or in combination with CD62L were responsible for antibody production (as measured by ELISpot). Furthermore, using high-color flow-cytometry, on day 7 after immunization, we observed the appearance of conventional PB (CPB, CD19dim CD20− CD27+high CD38+high CD3−), as well as a PB population that did not express CD27 (CD27− PB; pre-plasmablasts). The pattern of individual or simultaneous expression of homing markers (integrin α4β7, CD62L, CXCR3, and CXCR4) suggested that CPB cells homed preferentially to the inflamed gut mucosa. In contrast, ~50% CD27− PB cells appear to home to yet to be identified peripheral lymphoid organs or were in a transition state preceding integrin α4β7 upregulation. In sum, these observations demonstrate that strong immune responses, including distinct PB subsets with the potential to home to the gut and other secondary lymphoid organs, can be elicited after a short time of exposure to a shigella oral vaccine.

Epicutaneous Immunotherapy-Induced Regulatory T Cells Could Migrate To More Various Sites Of Allergen Exposure Compared To Sublingual Or Subcutaneous Immunotherapy In Mice Sensitized To Peanut

In mice, epicutaneous immunotherapy (EPIT) increases Foxp3+ regulatory T cells (Tregs) (Dioszeghy, J Immunol 2011). Tregs could display distinct efficacy based on their expression of homing molecules. The aim of this study was to evaluate homing receptors expression by Tregs induced during EPIT compared to sublingual (SLIT) or subcutaneous (SCIT) immunotherapy.

Distinct Immunomodulatory and Migratory Mechanisms Underpin the Therapeutic Potential of Human Mesenchymal Stem Cells in Autoimmune Demyelination

Mesenchymal stem cells (MSCs) are efficacious in a variety of intractable diseases. While bone marrow (BM)-derived MSCs (BM-MSCs) have been widely investigated, MSCs from other tissue sources have also been shown to be effective in several autoimmune and inflammatory disorders. In the present study, we simultaneously assessed the therapeutic efficacy of human BM-MSCs, as well as MSCs isolated from adipose tissue (Ad-MSCs) and umbilical cord Wharton's jelly (UC-MSCs), in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Prior to in vivo experiments, we characterized the phenotype and function of all three MSC types. We show that BM-MSCs were more efficient at suppressing the in vitro proliferation of mitogen or antigen-stimulated T-cell responses compared to Ad-MSCs and UC-MSCs. Notably BM-MSCs induced the differential expression of cytokines from normal and stimulated T-cells. Paradoxically, intravenous transplantation of BM-MSCs into C57Bl/6 mice with chronic progressive EAE had a negligible effect on the disease course, even when multiple MSC injections were administered over a number of time points. In contrast, Ad-MSCs had the most significant impact on clinical and pathological disease outcomes in chronic progressive and relapsing-remitting EAE models. In vivo tracking studies revealed that Ad-MSCs were able to migrate to the central nervous system (CNS), a property that most likely correlated with their broader expression of homing molecules, while BM-MSCs were not detected in this anatomic region. Collectively, this comparative investigation demonstrates that transplanted Ad-MSCs play a significant role in tissue repair processes by virtue of their ability to suppress inflammation coupled with their enhanced ability to home to the injured CNS. Given the access and relatively ease for harvesting adipose tissue, these data further implicate Ad-MSCs as a cell therapeutic that may be used to treat MS patients.

Life history of cognate antigen exposure indicates skewing of mucosal lymphocyte populations during aging (P3229)

Abstract The impact of aging on mucosal immune responses, particularly IgA production, is controversial. Many previous studies haven’t separated effects of the aged microenvironment (i.e. aged T cells, homing molecule expression, antigen exposure) from intrinsic B cell defects. We hypothesized that life-history of cognate antigen exposure influences function and distribution of mucosal B cell populations during aging. These studies used immunoglobulin transgenic mice, whose cognate antigen nitrophenyl is not naturally encountered. Mice were periodically immunized orally from birth with T cell-dependent or -independent antigens. At various ages, intestines were analyzed for relative abundance, size, and cellular composition of Peyer’s Patches and isolated lymphoid follicles. Results revealed significantly more isolated lymphoid follicles in the distal region of aged mice when compared to young. Comparing immunized and control mice, flow cytometry data reveals a significant decrease in percentages of transgenic B cells in both tissues, and in animals receiving T cell independent antigen there were changes in CD4 and CD8 T cell distribution in aged mice. There was also a significant decrease of transgenic B cells in aged mice immunized with a T cell-dependent antigen for both Peyer’s Patches and isolated lymphoid follicles. Hence, preliminary data suggests significant role of cognate antigen exposure and further elucidates the mechanisms of age-related immune dysfunction.

Intrarectal Immunization and IgA Antibody-Secreting Cell Homing to the Small Intestine

According to the current paradigm, lymphocyte homing to the small intestine requires the expression of two tissue-specific homing receptors, the integrin α4β7 and the CCL25 receptor CCR9. In this study, we investigated the organ distribution and the homing molecule expression of IgA Ab-secreting cells (ASCs) induced by intrarectal immunization with a particulate Ag, in comparison with other mucosal immunization routes. Intrarectal immunization induces gut-homing IgA ASCs that localize not only in the colon but also in the small intestine, although they are not responsive to CCL25, unlike IgA ASCs induced by oral immunization. The mucosal epithelial chemokine CCL28, known to attract all IgA ASCs, does not compensate for the lack of CCL25 responsiveness, because the number of Ag-specific cells is not decreased in the gut of CCR10-deficient mice immunized by the intrarectal route. However, Ag-specific IgA ASCs induced by intrarectal immunization express the integrin α4β7, and their number is considerably decreased in the gut of β7-deficient mice immunized by the intrarectal route, indicating that α4β7 enables these cells to migrate into the small intestine, even without CCL25 responsiveness. In contrast, IgA ASCs induced by intranasal immunization express low α4β7 levels and are usually excluded from the gut. Paradoxically, after intranasal immunization, Ag-specific IgA ASCs are significantly increased in the small intestine of β7-deficient mice, demonstrating that lymphocyte homing is a competitive process and that integrin α4β7 determines not only the intestinal tropism of IgA ASCs elicited in GALTs but also the intestinal exclusion of lymphocytes primed in other inductive sites.

A Critical Role for the Retinoic Acid Signaling Pathway in the Pathophysiology of Gastrointestinal Graft-Versus-Host Disease

Abstract 341Damage to the gastrointestinal tract during graft-versus-host disease (GVHD) is one of the major causes of morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. However, the molecular mechanism(s) responsible for colonic GVHD induction remain obscure. All-trans retinoic acid (RA) is an active metabolite of vitamin A and has recently emerged as a critical regulator of gut immunity. We hypothesized that administration of RA might be a strategy to reduce inflammatory responses during GVHD, particularly within the colon microenvironment. In a B6→Balb/c MHC-mismatched murine allogeneic bone marrow transplantation (BMT) model, we found unexpectedly that exposure of recipient mice to exogenous RA resulted in significantly accelerated GVHD-associated mortality compared with DMSO-treated animals (p<0.0001). Histological examination of GVHD target organs 5 weeks after transplantation revealed a significant increase in pathologic damage in the colons of RA-treated mice compared with DMSO-treated animals (9.7 ± 0.8 versus 3.3 ± 0.9, p<0.001), whereas there was no significant difference in pathology scores in either the lung or liver. RA treatment resulted in significantly increased expression of the gut-homing molecules, CCR9 and α4β7, on donor T cells in mesenteric lymph nodes early after BMT. Notably, expression of other chemokine receptors that have been implicated in GVHD pathogenesis, such as CCR5, CXCR3, and CCR10, was not affected by RA treatment, demonstrating a selective role of RA in imprinting gut-homing donor T cells. Examination of GVHD target organs three weeks after transplantation revealed a significant increase (eight-fold) in the absolute numbers of both CD4+IFN-γ+ (TH1) and CD4+IL-17+ (TH17) cells in the colon of RA-treated mice versus DMSO-treated animals, whereas there were no statistical differences in TH1 or TH17 cells in the spleen, liver and lung. There was also a significant increase in the absolute number of regulatory T cells (Tregs) in the colon of RA-treated mice. However, the increase in Tregs was noted to be more modest (three-fold) than was observed for TH1 and TH17 cells, suggesting a pro-inflammatory role of RA. To further confirm a role for RA in imprinting gut-homing donor T cells during GVHD, we generated vitamin A-deficient mice and employed them as recipients. Depletion of RA in recipient mice by vitamin A deprivation resulted in a dramatic reduction of gut homing molecule expression on donor T cells after allogeneic BMT. Retinoic acid receptor-α (RAR-α) signaling appears to be critical in mediating the effects of RA on alloreactive T cells. In mixed lymphocyte culture, when B6 T cells were stimulated with Balb/c dendritic cells, the presence of an RAR-α agonist was able to significantly enhance the expression of gut-homing molecules on T cells. In contrast, the addition of a selective RAR-α antagonist abolished the effects of RA on T cells. We then measured endogenous RA levels in the colons of mice undergoing either syngeneic or allogeneic BMT by mass spectrometry. These studies revealed that RA was significantly increased in the colon tissue of allogeneic transplant recipients compared with syngeneic transplant recipients early after BMT (day 7). In addition, Real-Time q-PCR analysis revealed a mean 3.5-fold increase in RAR-α expression in purified donor splenic CD4+ T cells 4 days post transplantation when compared to purified naïve CD4+ T cells obtained from original donor animals. Finally, we examined the ability of RAR-α-deficient T cells to cause GVHD. We found that recipients of RAR-α-deficient marrow grafts had significantly decreased GVHD-associated mortality when compared with the recipients of wild type T cells (93% versus 36% at day 90, p=0.002). Histological examination of GVHD target organs on days 10–12 after transplantation revealed a significant reduction in pathologic damage in the colon of mice that received RAR-α-deficient T cells compared with control mice (7.3 ± 2.5 versus 0.8 ± 1.0, p<0.01). In summary, we have identified RA as a novel and critical molecule in the induction of gastrointestinal GVHD. These findings provide evidence that blockade of the RA signaling pathway may represent a novel strategy to mitigate the severity of colonic GVHD. Disclosures:No relevant conflicts of interest to declare.

Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination

Mesenchymal stem/stromal cells (MSCs) can be isolated from most adult tissues and hold considerable promise for tissue regenerative therapies. Some of the potential advantages that MSCs have over other adult stem cell types include: (1) their relative ease of isolation, culture and expansion; (2) their immunomodulatory properties; (3) they can provide trophic support to injured tissues; (4) they can be transduced by retroviral vectors at a high efficiency; (5) they have an ability to home to sites of inflammation and injury. Collectively these characteristics suggest that MSCs are attractive vehicles for cell and gene therapy applications. In the current study, we investigated whether transplantation of human adipose-derived MSCs (Ad-MSCs) engineered to overexpress the anti-inflammatory cytokine interleukin (IL)-4 was efficacious in experimental autoimmune encephalomyelitis (EAE). Ad-MSCs transduced with a bicistronic lentiviral vector encoding mouse IL-4 and enhanced green fluorescent protein (Ad-IL4-MSCs) stably expressed, relatively high levels of both transgenes. Importantly the phenotypic and functional attributes of Ad-IL4-MSCs, such as the expression of homing molecules and differentiation capacity, was not altered by the transduction process. Notably, the early administration of Ad-IL4-MSCs in mice with EAE at the time of T-cell priming attenuated clinical disease. This protective effect was associated with a reduction in peripheral MOG-specific T-cell responses and a shift from a pro- to an anti-inflammatory cytokine response. These data suggest that the delivery of Ad-MSCs genetically engineered to express anti-inflammatory cytokines may provide a rational approach to promote immunomodulation and tissue protection in a number of inflammatory and degenerative diseases including multiple sclerosis.

Epithelial Expression of the Cytosolic Retinoid Chaperone Cellular Retinol Binding Protein II Is Essential for in Vivo Imprinting of Local Gut Dendritic Cells by Lumenal Retinoids

Dendritic cells (DCs) use all-trans retinoic acid (ATRA) to promote characteristic intestinal responses, including Foxp3(+) Treg conversion, lymphocyte gut homing molecule expression, and IgA production. How this ability to generate ATRA is conferred to DCs in vivo remains largely unstudied. Here, we observed that among DCs, retinaldehyde dehydrogenase (ALDH1), which catalyzes the conversion of retinal to ATRA, was preferentially expressed by small intestine CD103(+) lamina propria (LP) DCs. Retinoids induced LP CD103(+) DCs to generate ATRA via ALDH1 activity. Either biliary or dietary retinoids were required to confer ALDH activity to LP DCs in vivo. Cellular retinol-binding protein II (CRBPII), a cytosolic retinoid chaperone that directs enterocyte retinol and retinal metabolism but is redundant to maintain serum retinol, was required to confer ALDH activity to CD103(+) LP DCs. CRBPII expression was restricted to small intestine epithelial cells, and ALDH activity in CRBPII(-/-) DCs was restored by transfer to a wild-type recipient. CD103(+) LP DCs from CRBPII(-/-) mice had a decreased capacity to promote IgA production. Moreover, CD103(+) DCs preferentially associated with the small intestine epithelium and LP CD103(+) DC ALDH activity, and the ability to promote IgA production was reduced in mice with impaired DC-epithelia associations. These findings demonstrate in vivo roles for the expression of epithelial CRBPII and lumenal retinoids to imprint local gut DCs with an intestinal phenotype.

Dendritic cell homing and immune cell function in crohn's anal fistulae

IntroductionThe aetiology of perianal fistulating Crohn's disease remains obscure but genetic, microbiological and immunological factors play a role. Dendritic cells (DC) are antigen presenting cells which sample mucosal-associated bacteria and...

The α4β1 Integrin in Localization of Mycobacterium tuberculosis–Specific T Helper Type 1 Cells to the Human Lung

Rapid mobilization of antigen-specific T helper (Th) type 1-like CD4(+) T cells to the lung appears to be critically important for control of the respiratory pathogen Mycobacterium tuberculosis (M. tb), and for protection against pulmonary tuberculosis, the most contagious form of the disease. Accordingly, the preferential circulation of memory lymphocytes back to the tissues in which they first encountered antigen (i.e., "homing") may underlie the limited efficacy of current intradermal vaccination with the M. bovis strain bacillus Calmette-Guerrin. We previously developed a method of bronchoscopic antigen challenge with purified protein derivative of M. tb (PPD) to model local recall responses of healthy PPD-positive individuals who were infected via respiratory exposure to M. tb. Bronchoscopic challenge with PPD results in recruitment of additional antigen-specific Th1-like cells into challenged lung segments of healthy M. tb-infected individuals but not those of PPD-negative control subjects. In this study, we assessed the role of homing molecule expression in localization of M. tb-specific recall responses to the lung. Compared with peripheral blood, baseline bronchoalveolar lavage is significantly enriched for CD4(+) T cells expressing the α4β1 integrin homing molecule. This skewing is continued after PPD-induced recruitment of CD4(+) T cells, and is even more pronounced for recruited CD4(+) cells that display PPD-specific production of IFN-γ, of which over 83% express α4β1. Expression of the α4β1 integrin, therefore, appears likely to optimize localization of M. tb-specific Th1-like recall responses to the human lung.

Modulation of human plasmacytoid DC function by IFN-λ1 (IL-29)

The type III family of IFNs displays immunomodulatory and antiviral activity. Each member (IFN-lambda1, -2, and -3) signals through the same heterodimeric receptor complex, which consists of the binding and signaling subunit (IL-28Ralpha) plus the IL-10Rbeta chain. Although the receptor has a wide tissue distribution, the direct effects of IFN-lambda on various immune cell subsets have not been fully characterized. We have identified high levels of IL-28Ralpha mRNA in pDC from peripheral blood and hypothesized that IFN-lambda plays an important role in pDC maturation and development. We show that stimulation of pDC with HSV or Imiquimod causes an increase in IL-28Ralpha mRNA. In these cells, IFN-lambda1 alters expression of the costimulatory molecules CD80 and ICOS-L and synergizes with IFN-alpha to up-regulate CD83. In addition, IFN-lambda1 has a variable effect on the homing molecule expression of pDC and mDC. IFN-lambda1-treated pDC display a marked difference in their ability to stimulate production of the signature cytokines IL-13, IFN-gamma, and IL-10 in a MLR. This work characterizes the variable effects of IFN-lambda on DC surface molecule expression and identifies a role in pDC activation and immunostimulatory potential.