Islet β-Cell-Specific T Cells Can Use Different Homing Mechanisms to Infiltrate and Destroy Pancreatic Islets
2007; Elsevier BV; Volume: 170; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.060142
ISSN1525-2191
AutoresArno Hänninen, Rita Nurmela, Mikael Maksimow, Jarkko Heino, Sirpa Jalkanen, Christian Kurts,
Tópico(s)Diabetes and associated disorders
ResumoOrgan infiltration by T cells depends on the adhesion molecules expressed in these sites and on homing receptors expressed by the T cells. Here, we have studied which form of priming can enable T cells to home to pancreatic islets. To this end, we have used transgenic mice expressing the model autoantigen ovalbumin in pancreatic islets and transgenic ovalbumin-specific CD4 and CD8 T cells. We demonstrate that these T cells were imprinted with homing receptor patterns characteristic for the site of priming, such as α4β7 integrin for mucosal antigen delivery or functionally active α4β1 integrin for islet autoantigens. The adhesion molecules corresponding to these receptors were found to be constitutively expressed in islets, enabling T cells bearing these receptors to infiltrate the islets and to cause diabetes. Disease was prevented only by blockade of the endothelial adhesion molecule, ligand of homing receptors with which the T cells were imprinted. Thus, different priming locations induced different homing mechanisms, allowing T cells to target the islets. This may contribute to the susceptibility of islets to T-cell-mediated attack. Furthermore, it may pertain to the design of adhesion-modulating therapies alone or in combination with external autoantigen administration. Organ infiltration by T cells depends on the adhesion molecules expressed in these sites and on homing receptors expressed by the T cells. Here, we have studied which form of priming can enable T cells to home to pancreatic islets. To this end, we have used transgenic mice expressing the model autoantigen ovalbumin in pancreatic islets and transgenic ovalbumin-specific CD4 and CD8 T cells. We demonstrate that these T cells were imprinted with homing receptor patterns characteristic for the site of priming, such as α4β7 integrin for mucosal antigen delivery or functionally active α4β1 integrin for islet autoantigens. The adhesion molecules corresponding to these receptors were found to be constitutively expressed in islets, enabling T cells bearing these receptors to infiltrate the islets and to cause diabetes. Disease was prevented only by blockade of the endothelial adhesion molecule, ligand of homing receptors with which the T cells were imprinted. Thus, different priming locations induced different homing mechanisms, allowing T cells to target the islets. This may contribute to the susceptibility of islets to T-cell-mediated attack. Furthermore, it may pertain to the design of adhesion-modulating therapies alone or in combination with external autoantigen administration. Circulating T cells use homing receptors to interact with adhesion molecules on vascular endothelium and to egress from vasculature. These homing receptors are acquired during priming in secondary lymphatics1Campbell DJ Butcher EC Rapid acquisition of tissue-specific homing phenotypes by CD4(+) T cells activated in cutaneous or mucosal lymphoid tissues.J Exp Med. 2002; 195: 135-141Crossref PubMed Scopus (431) Google Scholar, 2Mora JR Bono MR Manjunath N Weninger W Cavanagh LL Rosemblatt M Von Andrian UH Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells.Nature. 2003; 424: 88-93Crossref PubMed Scopus (903) Google Scholar, 3Johansson-Lindbom B Svensson M Wurbel MA Malissen B Marquez G Agace W Selective generation of gut tropic T cells in gut-associated lymphoid tissue (GALT): requirement for GALT dendritic cells and adjuvant.J Exp Med. 2003; 198: 963-969Crossref PubMed Scopus (392) Google Scholar to help guide T cells preferentially into the tissue where the priming antigen was derived and where a causative pathogen is most likely to reside.4Butcher EC Picker LJ Lymphocyte homing and homeostasis.Science. 1996; 272: 60-66Crossref PubMed Scopus (2514) Google Scholar Furthermore, preferential T-cell homing may avert activated effector lymphocytes from other tissues where they might cause unwanted immune reactions. Important determinants of tissue-selective lymphocyte homing identified thus far include homing receptors α4β7 integrin, P-selectin ligand PSGL-1, and L-selectin5Butcher EC Williams M Youngman K Rott L Briskin M Lymphocyte trafficking and regional immunity.Adv Immunol. 1999; 72: 209-253Crossref PubMed Google Scholar and chemokine receptors CCR9, CCR4, and CCR10.6Rot A von Andrian UH Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells.Annu Rev Immunol. 2004; 22: 891-928Crossref PubMed Scopus (1021) Google Scholar In addition to these, numerous other adhesion molecules contribute to lymphocyte homing from blood into tissues. Several adhesion molecules, most convincingly very late antigen-4 (VLA-4, integrin α4), have been implicated in the pathogenesis of diabetes in the nonobese diabetic (NOD) mouse.7Baron JL Reich EP Visintin I Janeway Jr, CA The pathogenesis of adoptive murine autoimmune diabetes requires an interaction between alpha 4-integrins and vascular cell adhesion molecule-1.J Clin Invest. 1994; 93: 1700-1708Crossref PubMed Scopus (110) Google Scholar, 8Burkly LC Jakubowski A Hattori M Protection against adoptive transfer of autoimmune diabetes mediated through very late antigen-4 integrin.Diabetes. 1994; 43: 529-534Crossref PubMed Scopus (53) Google Scholar, 9Yang XD Michie SA Tisch R Karin N Steinman L McDevitt HO A predominant role of integrin alpha 4 in the spontaneous development of autoimmune diabetes in nonobese diabetic mice.Proc Natl Acad Sci USA. 1994; 91: 12604-12608Crossref PubMed Scopus (89) Google Scholar, 10Yang XD Michie SA Mebius RE Tisch R Weissman I McDevitt HO The role of cell adhesion molecules in the development of IDDM: implications for pathogenesis and therapy.Diabetes. 1996; 45: 705-710Crossref PubMed Scopus (58) Google Scholar VLA-4 consists of two integrin chains, α4 and β1, linked together to form a heterodimer (α4β1). Integrin α4 also forms a heterodimer with the β7 chain, termed α4β7 (lymphocyte Peyer's patch adhesion molecule, LPAM-1; mucosal homing receptor), which binds to mucosal addressin cell adhesion molecule-1 (MAdCAM-1),11Berlin C Berg EL Briskin MJ Andrew DP Kilshaw PJ Holzmann B Weissman IL Hamann A Butcher EC Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1.Cell. 1993; 74: 185-195Abstract Full Text PDF PubMed Scopus (1278) Google Scholar a ligand distinct from that of α4β1 (VCAM-1, vascular cell adhesion molecule-1). In addition, α4β7 and its ligand MAdCAM-1 have been implicated in the development of diabetes in the NOD mouse.12Yang XD Sytwu HK McDevitt HO Michie SA Involvement of beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the development of diabetes in obese diabetic mice.Diabetes. 1997; 46: 1542-1547Crossref PubMed Google Scholar, 13Hänninen A Taylor C Streeter PR Stark LS Sarte JM Shizuru JA Simell O Michie SA Vascular addressins are induced on islet vessels during insulitis in nonobese diabetic mice and are involved in lymphoid cell binding to islet endothelium.J Clin Invest. 1993; 92: 2509-2515Crossref PubMed Scopus (201) Google Scholar, 14Hänninen A Jaakkola I Jalkanen S Mucosal addressin is required for the development of diabetes in nonobese diabetic mice.J Immunol. 1998; 160: 6018-6025PubMed Google Scholar The principal site of activation of diabetogenic T cells is the pancreatic lymph node (PaLN).15Gagnerault MC Luan JJ Lotton C Lepault F Pancreatic lymph nodes are required for priming of beta cell reactive T cells in NOD mice.J Exp Med. 2002; 196: 369-377Crossref PubMed Scopus (229) Google Scholar However, the expression of the two heterodimers, α4β1 and α4β7, or that of other homing determinants has not been studied on T cells primed in this lymph node. It is also unclear to what extent various homing receptors contribute to homing of T cells into islets. To study this, we used a transgenic mouse model expressing ovalbumin (OVA) as a model autoantigen in islet β-cells and allowing activation of naïve, OVA-reactive, and thus β-cell-reactive T cells in the PaLN.16Kurts C Heath WR Carbone FR Allison J Miller JF Kosaka H Constitutive class I-restricted exogenous presentation of self antigens in vivo.J Exp Med. 1996; 184: 923-930Crossref PubMed Scopus (525) Google Scholar Furthermore, we used transgenic mice expressing OVA in islet β-cells but unable to present it to T cells in the PaLN.17Miller JF Kurts C Allison J Kosaka H Carbone F Heath WR Induction of peripheral CD8+ T-cell tolerance by cross-presentation of self antigens.Immunol Rev. 1998; 165: 267-277Crossref PubMed Scopus (70) Google Scholar In these mice, we modeled the situation of T-cell activation in response to antigens that enter the body by the gastrointestinal or the subcutaneous route, giving rise to islet-reactive T cells via, eg, antigenic mimicry. Our findings imply more than one receptor-ligand pair in T-cell homing into islets and that the receptor-ligand pair that mediates homing depends on the site of activation of effector T cells. OT-I, OT-II, RIP-mOVA, and RIP-OVAlo mice,17Miller JF Kurts C Allison J Kosaka H Carbone F Heath WR Induction of peripheral CD8+ T-cell tolerance by cross-presentation of self antigens.Immunol Rev. 1998; 165: 267-277Crossref PubMed Scopus (70) Google Scholar backcrossed >12 times to C57/BL 6, were generated and provided by Dr. W.R. Heath and Dr. F.R. Carbone, Melbourne, Australia, and were bred and maintained in the animal facilities of Turku and Bonn Universities. All experiments were approved by the Institutional Board of Animal Experiments. Lymph node and spleen cells of OT-II and OT-I mice were isolated using standard techniques. Their number was determined by staining for TCR Vα2 and Vβ5 chains and CD8 or CD4, respectively. OT-I cells were also detected by staining for CD8 and the H2-Kb-SIINFEKL-specific tetramer (Beckman Coulter, Fullerton, CA). Diabetes was induced in RIP-mOVA and RIP-OVAlo mice by adoptive transfer of a mixture of 0.3 × 106 OT-II and 0.2 × 106 OT-I cells or with 2.0 × 106Rot A von Andrian UH Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells.Annu Rev Immunol. 2004; 22: 891-928Crossref PubMed Scopus (1021) Google Scholar OT-I cells, as indicated in figure legends. OVA [lipopolysaccharide (LPS) (grade V; Sigma, St. Louis, MO) concentration ≥1.2 U/ml as measured by the LAL assay (BioWhittaker, Walkersville, MD), which corresponds to ∼100 ng/ml LPS from Escherichia coli18Watanabe J Miyazaki Y Zimmerman GA Albertine KH McIntyre TM Endotoxin contamination of ovalbumin suppresses murine immunologic responses and development of airway hyper-reactivity.J Biol Chem. 2003; 278: 42361-42368Crossref PubMed Scopus (96) Google Scholar] was applied either intragastrically via a plastic feeding gauge (3 mg in 300 μl of phosphate-buffered saline at day 0 and 2) or subcutaneously (200 μg in incomplete Freund's adjuvant at day 0) in the base of the tail. Blood glucose values were measured on day 10 after cell transfer using a MediSense glucometer, and mice with a reading more than 14.3 mmol/L were considered diabetic. Blood glucose levels were measured again on day 14 and similar results were obtained (not shown). Lymph node and spleen lymphocytes from OT-I and OT-II mice were labeled with carboxyfluorescein succinimidyl ester (Molecular Probes, Eugene, OR). OT-I and OT-II cells (4 × 106) were injected intravenously into RIP-mOVA or RIP-OVAlo mice. After adoptive cell transfer, RIP-OVAlo mice recipients were either fed OVA once or immunized subcutaneously with OVA. Cells were stained with Alexa 647-conjugated anti-CD4 and PerCP-Cy5.5-conjugated anti-CD8 and with phycoerythrin (PE)-conjugated anti-integrin α4 monoclonal antibody (mAb) (R1-2), anti-α4β7 heterodimer (DATK-32), or with biotinylated anti-integrin β1 chain mAb (Ha2/5), or anti-CD62L mAb (MEL-14), or with isotype-matched control antibodies, all from Becton, Dickinson and Company, San Jose, CA. In case of biotinylated mAbs, phycoerythrin-conjugated streptavidin (SA-PE; Becton, Dickinson and Company) was used as the second-step reagent. For detection of P-selectin ligand, cells were stained using chimeric P-selectin-human IgG fusion protein (Becton, Dickinson and Company) in a buffer with divalent cations, followed by biotinylated anti-human IgG and SA-PE. Flow cytometry was performed on an LSR-II flow cytometer, and data were analyzed with WinMDI2.8 software. Pancreata were isolated from unmanipulated C57BL/6 mice to study constitutive expression of adhesion molecules. To study their expression during insulitis and to study homing receptor expression of infiltrating CD4 and CD8 T cells, pancreata were isolated from RIP-mOVA and RIP-OVAlo mice 10 days after adoptive transfer of OT-I and OT-II cells (and antigen exposure of RIP-OVAlo mice). Cryosections of normal pancreata were stained for VCAM-1 using the rat IgG1 mAb 6C7 (gift from Dr. D. Vestweber, Max-Planck Institute of Molecular Biomedicine, Münster, Germany), for MAdCAM-1 using MECA-367 (rat IgG2a), and for P-selectin using polyclonal rabbit anti-human P-selectin (Becton, Dickinson and Company). As controls, we used anti-human CD44 (hermes-1 prepared in our laboratory) for VCAM-1 and MAdCAM-1 staining or normal rabbit serum for P-selectin staining. Staining was detected by fluorescein isothiocyanate-conjugated goat anti-rat IgG (heavy and light chains; Southern Biotechnology, Birmingham, AL). Pancreatic islets were visualized by staining for insulin using rabbit anti-mouse insulin (Santa Cruz Biotechnology, Santa Cruz, CA), followed by Alexa 546-conjugated anti-rabbit IgG (Molecular Probes). Cryosections of prediabetic pancreata were stained for VCAM-1, MAdCAM-1, and P-selectin as described above and for infiltrating T cells using PE-conjugated anti-CD4 and anti-CD8. To detect homing receptor expression on infiltrating T cells, PE-conjugated anti-integrin α4 or α4β7 heterodimer or biotinylated anti-CD62L were used, followed by a mixture of fluorescein isothiocyanate-conjugated anti-CD4 and CD8 mAbs. Biotinylated anti-CD62L was revealed by SA-PE. The following antibodies were raised from hybridomas, precipitated with ammonium sulfate, purified using protein G columns (Pharmacia Amersham, Piscataway, NJ) and filtered sterile: anti-VCAM-1, anti-MAdCAM-1, anti-CD62L (MEL-14, rat IgG2a), anti-P-selectin (RB40.34, rat IgG1) and anti-PSGL-1 (4RA10, rat IgG1), the last two obtained as gifts from Drs. D. Vestweber and M.K. Wild (see above). Isotype controls included antibodies 2D10 (anti-human VAP-1, rat IgG1 produced in our laboratory) and hermes-1 (rat IgG2a). Antibody treatment was started 2 days after adoptive transfer of OT cells, and in RIP-OVAlo mice, simultaneous exposure to antigen occurred (oral OVA or subcutaneous OVA with adjuvant). Mice received intraperitoneal injections of 200 μg of mAb (purified as indicated below) or isotype control every other day until 14 days after cell transfer. Anti-P-selectin antibody was used at 100 μg per injection, a dose previously shown to block macrophage accumulation after vascular injury.19Phillips JW Barringhaus KG Sanders JM Hesselbacher SE Czarnik AC Manka D Vestweber D Ley K Sarembock IJ Single injection of P-selectin or P-selectin glycoprotein ligand-1 monoclonal antibody blocks neointima formation after arterial injury in apolipoprotein E-deficient mice.Circulation. 2003; 107: 2244-2249Crossref PubMed Scopus (107) Google Scholar Blood glucose values were measured at 10 and 14 days after transfer. To analyze insulitis, nonparallel sections of paraffin-embedded pancreata were stained with hematoxylin and eosin (H&E). Each pancreas was assigned an insulitis score by analyzing the level of lymphocytic infiltration in each of 50 to 100 islets (0, no insulitis; 1, peri-insulitis or scant intra-islet insulitis; 2, insulitis covering 50% of islet area) per pancreas. Islets were counted in a blinded manner by the same reader. Statistical significance for the differences in incidence of diabetes between treated and control mice in each experiment was analyzed using χ2 test. Differences in insulitis scores were calculated using two-tailed, unpaired Student's t-test with Welch's correction. To study expression of homing receptors by autoreactive T cells, a 1:1 mixture of carboxyfluorescein succinimidyl ester-labeled OVA-specific CD4 (OT-II) and CD8 (OT-I) T cells was transferred into transgenic recipient mice expressing this model antigen in pancreatic islets. As recipients, we used either RIP-mOVA or RIP-OVAlo mice, in which OVA is expressed on islet β-cells and presented in pancreatic (and kidney)-draining lymph nodes sufficient to drive T-cell activation (RIP-mOVA) or expressed on islet β-cells in amounts too low to result in such activation but sufficient to permit CTL-mediated lysis of islet β-cells (RIP-OVAlo).17Miller JF Kurts C Allison J Kosaka H Carbone F Heath WR Induction of peripheral CD8+ T-cell tolerance by cross-presentation of self antigens.Immunol Rev. 1998; 165: 267-277Crossref PubMed Scopus (70) Google Scholar This allowed study of homing receptor expression by T cells activated by autoantigen expressed in islet β-cells (RIP-mOVA) or introduction of autoantigen in other locations (RIP-OVAlo), for example subcutaneously or orally, and study of islet-directed CTL effectors resulting from such priming. Priming via these routes resulted in antigen presentation to OT cells, as shown for OT-I cells (Figure 1A) in the corresponding lymph nodes (subcutaneous or mesenteric). Homing receptor expression in consecutive generations of dividing cells was evaluated separately for OT-II and OT-I cells 4 days (96 hours) after adoptive co-transfer. Only few OT-II cells were driven into cell cycle by OVA expressed as nonlymphoid autoantigen in RIP-mOVA mice (Figure 1B) as previously reported.20Behrens GM Li M Davey GM Allison J Flavell RA Carbone FR Heath WR Helper requirements for generation of effector CTL to islet beta cell antigens.J Immunol. 2004; 172: 5420-5426PubMed Google Scholar On these few dividing OT-II cells, integrin α4 and β1 chains (which form the α4β1 heterodimer VLA-4) were slightly up-regulated, whereas significant up-regulation occurred on OT-I cells (Figure 1C). Expression of the α4β7 heterodimer remained low on both OT-II and OT-I cells responding to pancreatic autoantigen. L-selectin expression declined slightly but remained on an intermediate level, whereas P-selectin ligand activity decreased on both OT-II and OT-I cells (Figure 1, B and C). To study expression of homing receptors on autoreactive T cells activated by orally consumed autoantigen, OVA was fed to RIP-OVAlo mice expressing low-dose OVA in pancreatic islets. In mesenteric lymph nodes of these mice, integrin α4 and β1 chains and α4β7 heterodimer were up-regulated both on dividing OT-II and OT-I cells (Figure 1, B and C). To exclude that up-regulation of α4β7 heterodimer was attributable to LPS contaminations in the OVA preparations used, we injected 100 μg of LPS intraperitoneally into RIP-mOVA mice. This did not induce expression of α4β7 heterodimer or otherwise change expression of homing receptors by OT-I cells proliferating in PaLNs (data not shown), indicating that factors other than LPS were responsible for the differences in homing receptor expression. In addition, the antigen dose did not affect the pattern of homing receptor expression, as revealed by titration of orally administered antigen to the smallest possible dose that induced proliferation of OT-II or OT-I cells. Only in response to autoantigen administered subcutaneously, OT-I cells clearly up-regulated P-selectin ligand activity after several rounds of proliferation, although this was true for some, but not the majority, of OT-II cells. L-selectin expression remained high in the majority of both OT-II and OT-I cells, but in contrast to cells being activated elsewhere, OT-II and OT-I cells activated by subcutaneous autoantigen down-regulated integrin α4 chain (Figure 1, B and C). Neither the dose of subcutaneous antigen nor its combination with LPS or incomplete Freund's adjuvant affected this expression pattern (data not shown). For homing to pancreatic islets, the acquired differences of homing receptor expression should persist until these T cells reach target tissue. We therefore studied expression of integrin α4, α4β7 heterodimer, and of L-selectin in islet-infiltrating T cells by immunohistochemistry. Islet infiltrating T cells (Figure 2) expressed the type of homing receptors that was induced during the respective way of priming (Figure 1) with a few exceptions. These exceptions were the expression of integrin α4 on infiltrating T cells also when OT-II and OT-I cells were activated in peripheral lymph nodes, expression of L-selectin on scattered T cells after T-cell activation in PaLN, and relative scarcity of L-selectin-expressing cells after T-cell activation in response to oral antigen. Importantly, T cells expressing α4β7 heterodimer were detected only if OT-II and OT-I cells were activated in response to oral antigen, and L-selectin was expressed most frequently and at highest intensity on islet-infiltrating T cells when OT-II and OT-I cells were activated in peripheral lymph nodes (Figure 2). These findings indicate that the homing receptor expression profile acquired during activation was at least partly maintained during recirculation and islet homing of activated T cells. To evaluate the ability of T cells expressing various homing receptors to interact with pancreatic vasculature, we determined expression of the endothelial ligands VCAM-1, MAdCAM-1, and P-selectin on pancreatic vasculature in normal C57BL/6 mice and in RIP-mOVA and RIP-OVAlo mice during insulitis. Intriguingly, constitutive low-level expression of all these ligands was detected even in pancreas tissue from nontransgenic C57BL/6 control mice (Figure 3A). This expression was not located directly on islet vasculature but on several small- and middle-sized vessels close to islets. During insulitis, all of these molecules were expressed on islet vessels irrespective of the type of activation of OT-II and OT-I cells (Figure 3B). Thus, pancreatic and islet vessels expressed the adhesion molecules required to permit entry of autoreactive T cells activated in all locations tested. We next tested the efficacy by which each form of priming induced diabetes and mononuclear cell accumulation into islets in the experimental settings described above. A combination of 0.3 × 106 CD4 and 0.2 × 106 cells CD8 T cells was used because both cell types contribute to the pathogenesis of diabetes.21Bach JF Immunotherapy of insulin-dependent diabetes mellitus.Curr Opin Immunol. 2001; 13: 601-605Crossref PubMed Scopus (26) Google Scholar These cell numbers created a better window for intervention and were closer to T-cell numbers in a normal repertoire than those previously used in this system.22Kurts C Carbone FR Barnden M Blanas E Allison J Heath WR Miller JF CD4+ T cell help impairs CD8+ T cell deletion induced by cross-presentation of self-antigens and favors autoimmunity.J Exp Med. 1997; 186: 2057-2062Crossref PubMed Scopus (271) Google Scholar In one experiment, mice were sacrificed at day 6 for pancreatic histology and, in another, followed until day 17 for the occurrence of hyperglycemia. Subcutaneous priming was most effective, followed by endogenous priming (RIP-mOVA), both of which induced diabetes in the majority of mice. Oral priming was least effective and induced milder insulitis than subcutaneous or endogenous priming (Figure 4). In conclusion, all priming routes tested were able to induce immune-mediated diabetes, albeit at different extents. To test whether the adhesion molecules expressed in the islets were relevant for infiltration by autoreactive T cells activated in the three situations tested, we treated RIP-mOVA mice with function-blocking anti-VCAM-1 or isotype control mAb from 2 days after adoptive transfer of OT cells. To test that antibody treatment did not affect expansion of effector cells in response to priming, we determined in separate experiments the numbers of OT-I cells in lymph nodes and the spleen 10 days after adoptive transfer using the H2-Kb-SIINFEKL-tetramer and flow cytometry and found that numbers of OT-I cells were not affected by anti-VCAM-1 treatment (not shown). Borderline hyperglycemia (diabetes) developed in only 1 of 13 recipients treated with anti-VCAM-1, whereas 7 of 14 recipients treated with control antibody developed diabetes (P = 0.012; Figure 5A). Anti-MAdCAM-1 was not able to inhibit development of diabetes in RIP-mOVA mice, as 6 of 14 recipients became diabetic compared with 5 of 13 control recipients. The mean level of insulitis in pancreata of mice that were normoglycemic at the end of the experiment was significantly lower in the group of mice that had received anti-VCAM-1 compared with those receiving control antibody. These findings imply a functional role of VCAM in islet homing of diabetogenic T cells when these were activated in the pancreatic LN. To determine whether up-regulation of α4β7 expression was functionally relevant in homing of GALT-activated T cells into the pancreas and whether these cells were able to use also α4β1 for their homing into pancreas, we treated RIP-OVAlo mice with anti-VCAM-1 or anti-MAdCAM-1 antibody starting 2 days after adoptive transfer of OT cells and oral OVA administration. Blockade of VCAM-1 was without an effect, as 5 of 11 anti-VCAM-1-treated mice (and 5 of 11 controls) became diabetic (Figure 5B). However, MAdCAM-1 blockade was effective, as only 2 of 24 anti-MAdCAM-1-treated mice (including one borderline hyperglycemia) (and 8 of 24 control) became diabetic (P = 0.033). Anti-MAdCAM-1 treatment also significantly reduced the accumulation of T cells in islets, implying a functional role of MAdCAM-1 in islet homing of diabetogenic T cells when these were activated in the gut. Anti-MAdCAM-1 treatment did not inhibit expansion of OT-I cells, tested as described above (not shown). Integrin β1 was up-regulated on OT cells activated in all locations tested (Figure 1), and α4 after priming in PaLN and GALT. Therefore, it was surprising that blocking of the α4β1 ligand VCAM-1 only affected diabetogenesis and insulitis after activation of OT cells in the PaLN. We speculated that expression of α4β1 integrin in its high-affinity state occurring in VLA-423Hynes RO Integrins: bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6889) Google Scholar might be restricted to T cells activated in the PaLN. To test this hypothesis, we used an antibody reacting only with an activation-associated conformational β1 epitope.24Lenter M Uhlig H Hamann A Jeno P Imhof B Vestweber D A monoclonal antibody against an activation epitope on mouse integrin chain beta 1 blocks adhesion of lymphocytes to the endothelial integrin alpha 6 beta 1.Proc Natl Acad Sci USA. 1993; 90: 9051-9055Crossref PubMed Scopus (232) Google Scholar Indeed, activation in response to islet-derived antigen in the PaLN led to appearance of the activated form of β1 on T cells, and after six rounds of cell division most T cells had β1 in its active conformation (Figure 5C). This change in conformation was not observed when T cells were activated by external antigen in other lymphoid tissues. After subcutaneous injection of OVA, most OT-II cells lost expression of integrin α4 and α4β7 heterodimer, and only a few OT-I cells up-regulated integrin α4. However, both expressed L-selectin, and all OT-I cells up-regulated P-selectin ligand (Figure 1, B and C). Blockade of VCAM-1 did not inhibit diabetogenesis by these effector cells (Figure 6) nor did blockade of either selectin alone or P-selectin ligand and L-selectin in combination. Although we induced diabetes by using both CD4 and CD8 T cells, it was important to examine islets for potential differences in infiltration by these T-cell subsets after different forms of priming and after therapies intervening in adhesion molecule function. Although inhibition of VCAM-1 function diminished insulitis (and partially prevented diabetes) in RIP-mOVA mice and inhibition of MAdCAM-1 function had a similar effect after oral priming in RIP-OVAlo mice, in both cases infiltration of islets by CD4 and CD8 T cells was similarly affected; ie, the remaining infiltrates contained both subsets. Both CD4 and CD8 T cells were also seen in islets of mice primed by subcutaneous OVA, thus excluding major differences in CD4 versus CD8 T-cell homing into islets after different forms of priming (Figure 7). Recent studies have demonstrated that T cells acquire the capacity to home to and infiltrate organs such as the gut mucosa,1Campbell DJ Butcher EC Rapid acquisition of tissue-specific homing phenotypes by CD4(+) T cells activated in cutaneous or mucosal lymphoid tissues.J Exp Med. 2002; 195: 135-141Crossref PubMed Scopus (431) Google Scholar, 2Mora JR Bono MR Manjunath N Weninger W Cavanagh LL Rosemblatt M Von Andrian UH Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells.Nature. 2003; 424: 88-93Crossref PubMed Scopus (903) Google Scholar, 3Johansson-Lindbom B Svensson M Wurbel MA Malissen B Marquez G Agace W Selective generation of gut tropic T cells in gut-associated lymphoid tissue (GALT): requirement for GALT dendritic cells and adjuvant.J Exp Med. 2003; 198: 963-969Crossref PubMed Scopus (392) Google Scholar, 25Iwata M Hirakiyama A Eshima Y Kagechika H Kato C Song SY Retinoic acid imprints gut-homing specificity on T cells.Immunity. 2004; 21: 527-538Abstract Full Text Full Text PDF PubMed Scopus (1218) Google Scholar the brain,26Calzascia T Masson F Di Berardino-Besson W Contassot E Wilmotte R Aurrand-Lions M Ruegg C Dietrich PY Walker PR Homing phenotypes of tumor-
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