VH1-46 is the dominant immunoglobulin heavy chain gene segment in rotavirus-specific memory B cells expressing the intestinal homing receptor alpha4beta7

Memory B cells expressing the intestinal homing marker alpha4beta7 are important for protective immunity against human rotavirus (RV). It is not known whether the B cell repertoire of intestinal homing B cells differs from B cells of the systemic compartment. In this study, we analyzed the RV-specific VH and VL repertoire in human IgD- B cells expressing the intestinal homing marker alpha4beta7. The mean frequency of RV-specific B cells in the systemic compartment of healthy adult subjects was 0.6% (range, 0.2-1.2). The mean frequency of IgD- B cells that were both RV specific and alpha4beta7 was 0.04% (range, 0.01-0.1), and a mean of 10% (range, 1-32) of RV-specific peripheral blood human B cells exhibited an intestinal homing phenotype. We previously demonstrated that VH1-46 is the dominant Ab H chain gene segment in RV-specific systemic B cells from adults and infants. RV-specific systemic IgD- or intestinal homing IgD-/alpha4beta7+ B cells in the current study also used the gene segment VH1-46 at a high frequency, while randomly selected B cells with those phenotypes did not. These data show that VH1-46 is the immunodominant gene segment in human RV-specific effector B cells in both the systemic compartment and in intestinal homing lymphocytes. The mean replacement/silent mutation ratio of systemic compartment IgD- B cells was >2, consistent with a memory phenotype and antigenic selection. Interestingly, RV-specific intestinal homing IgD-/alpha4beta7+ B cells using the VH1-46 gene segment were not mutated, in contrast to systemic RV-specific IgD- B cells.     

Maturation and trafficking markers on rotavirus-specific B cells during acute infection and convalescence in children

We have previously studied B cells, from people and mice, that express rotavirus-specific surface immunoglobulin (RV-sIg) by flow cytometry with recombinant virus-like particles that contain green fluorescent protein. In the present study we characterized circulating B cells with RV-sIg in children with acute and convalescent infection. During acute infection, circulating RV-sIgD(-) B cells are predominantly large, CD38(high), CD27(high), CD138(+/-), CCR6(-), alpha4beta7(+), CCR9(+), CCR10(+), cutaneous lymphocyte antigen-negative (CLA(-)), L-selectin(int/-), and sIgM(+), sIgG(-), sIgA(+/-) lymphocytes. This phenotype likely corresponds to gut-targeted plasma cells and plasmablasts. During convalescence the phenotype switches to small and large lymphocytes, CD38(int/-), CD27(int/-), CCR6(+), alpha4beta7(+/-), CCR9(+/-) and CCR10(-), most likely representing RV-specific memory B cells with both gut and systemic trafficking profiles. Of note, during acute RV infection both total and RV-specific murine IgM and IgA antibody-secreting cells migrate efficiently to CCL28 (the CCR10 ligand) and to a lesser extent to CCL25 (the CCR9 ligand). Our results show that CCR10 and CCR9 can be expressed on IgM as well as IgA antibody-secreting cells in response to acute intestinal infection, likely helping target these cells to the gut. However, these intestinal infection-induced plasmablasts lack the CLA homing receptor for skin, consistent with mechanisms of differential CCR10 participation in skin T versus intestinal plasma cell homing. Interestingly, RV memory cells generally lack CCR9 and CCR10 and instead express CCR6, which may enable recruitment to diverse epithelial sites of inflammation.     

Immunodominance of the VH1-46 antibody gene segment in the primary repertoire of human rotavirus-specific B cells is reduced in the memory compartment through somatic mutation of nondominant clones

Detailed characterization of Ag-specific naive and memory B cell Ab repertoires elucidates the molecular basis for the generation of Ab diversity and the optimization of Ab structures that bind microbial Ags. In this study, we analyzed the immunophenotype and VH gene repertoire of rotavirus (RV) VP6-specific B cells in three circulating naive or memory B cell subsets (CD19+IgD+CD27-, CD19+IgD+CD27+, or CD19+IgD-CD27+) at the single-cell level. We aimed to investigate the influence of antigenic exposure on the molecular features of the two RV-specific memory B cell subsets. We found an increased frequency of CD19+IgD+CD27+ unclass-switched memory B cells and a low frequency of somatic mutations in CD19+IgD-CD27+ class-switched memory B cells in RV-specific memory B cells, suggesting a reduced frequency of isotype switching and somatic mutation in RV VP6-specific memory B cells compared with other memory B cells. Furthermore, we found that dominance of the VH1-46 gene segment was a prominent feature in the VH gene repertoire of RV VP6-specific naive B cells, but this dominance was reduced in memory B cells. Increased diversity in the VH gene repertoire of the two memory B cell groups derived from broader usage of VH gene segments, increased junctional diversity that was introduced by differential TdT activities, and somatic hypermutation.     

Protective intestinal anti-rotavirus B cell immunity is dependent on alpha 4 beta 7 integrin expression but does not require IgA antibody production

Rotavirus (RV) is the main cause of severe gastroenteritis in young children; protection has been correlated with intestinal Ab responses. Using a mouse model of RV infection and beta(7)-deficient (beta(7)(-/-)) mice, which do not express alpha(4)beta(7) integrin, we demonstrated the importance of alpha(4)beta(7) integrin in B cell-mediated anti-RV immunity. beta(7)(-/-) mice acutely infected with murine RV resolved infection and developed normal serum IgG Abs but had diminished intestinal IgA responses. alpha(4)beta(7)(-/-) immune B cells did not resolve RV infection when adoptively transferred into RV-infected Rag-2-deficient mice. Fewer RV-specific B cells were found in the intestine of Rag-2-deficient mice transferred with beta(7)(-/-) B cells compared with wild type. The absence of alpha(4)beta(7) expression and/or a lower frequency of IgA-producing cells among transferred beta(7)(-/-) B cells could have accounted for the inability of these cells to resolve RV infection following passive transfer. To distinguish between these possibilities, we studied the importance of IgA production in RV infection using IgA-deficient (IgA(-/-)) mice. IgA(-/-) mice depleted of CD8(+) T cells were able to clear primary RV infection. Similarly, adoptive transfer of immune IgA(-/-) B cells into chronically infected Rag-2-deficient mice resolved RV infection. We further demonstrated in both wild-type and IgA(-/-) mice that, following oral RV infection, protective B cells reside in the alpha(4)beta(7)(high) population. Our findings suggest that alpha(4)beta(7) integrin expression is necessary for B cell-mediated immunity to RV independent of the presence of IgA.     

Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.     

Redundant role of chemokines CCL25/TECK and CCL28/MEC in IgA+ plasmablast recruitment to the intestinal lamina propria after rotavirus infection

Rotaviruses (RV) are the most important cause of severe childhood diarrheal disease. In suckling mice, infection with RV results in an increase in total and virus-specific IgA(+) plasmablasts in the small intestinal lamina propria (LP) soon after infection, providing a unique opportunity to study the mechanism of IgA(+) cell recruitment into the small intestine. In this study, we show that the increase in total and RV-specific IgA(+) plasmablasts in the LP after RV infection can be blocked by the combined administration of Abs against chemokines CCL25 and CCL28, but not by the administration of either Ab alone. RV infection in CCR9 knockout mice still induced a significant accumulation of IgA(+) plasmablasts in the LP, which was blocked by the addition of anti-CCL28 Ab, confirming the synergistic role of CCL25 and CCL28. The absence of IgA(+) plasmablast accumulation in LP following combined anti-chemokine treatment was not due to changes in proliferation or apoptosis in these cells. We also found that coadministration of anti-CCL25 and anti-CCL28 Abs with the addition of anti-alpha(4) Ab did not further inhibit IgA(+) cell accumulation in the LP and that the CCL25 receptor, CCR9, was coexpressed with the intestinal homing receptor alpha(4)beta(7) on IgA(+) plasmablasts. Finally, we showed that RV infection was associated with an increase in both CCL25 and CCL28 in the small intestine. Hence, our findings indicate that alpha(4)beta(7) along with either CCR9 or CCR10 are sufficient for mediating the intestinal migration of IgA(+) plasmablasts during RV infection.     

B2 but not B1 cells can contribute to CD4+ T-cell-mediated clearance of rotavirus in SCID mice

Studies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4+ T cells, and CD8+ T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected alphabeta T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of B1 cells suggested that B1 cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding. We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and leads to the production of high levels of RV-specific intestinal IgA. In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA. Cotransfer of mixtures of purified B1 cells and B1-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (B1-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA. To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4+ T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4+ T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4+ T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4+ T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.     

Differential homing commitments of antigen-specific T cells after oral or parenteral immunization in humans

Animal experiments show that lymphocytes activated in the intestine circulate through mesenteric lymph nodes, lymphatics, and blood, returning to the gut. Homing into intestinal lamina propria is mediated by lymphocyte surface homing receptors, mainly the alpha4beta7-integrin. We studied in humans whether intestinal T cells entering the blood upon antigenic activation would exhibit homing commitments to the gut. Volunteers were immunized with keyhole limpet hemocyanin (KLH) first orally and then parenterally or only parenterally, and the expression of alpha4beta7 on T cells specific for KLH or tetanus toxoid was studied. Circulating T cells were depleted of alpha4beta7+ cells by immunomagnetic selection. This depletion removed a significant proportion of the KLH-specific cells (mean decrease in proliferative response of 71%) in the orally immunized volunteers. No difference in the KLH-induced proliferation was found between the total and the alpha4beta7-depleted populations in volunteers parenterally immunized with KLH, regardless of whether a preceding mucosal priming had taken place or not. In both immunization groups, the depletion of alpha4beta7+ cells had no influence on the proliferative response to tetanus toxoid. We conclude that, in contrast to T cells activated by parenteral immunization, gut-derived T cells have preferential homing commitments to the gut. This commitment was no longer observed after a subsequent parenteral Ag administration. Besides showing that the site of Ag encounter determines the expression of homing receptors, the present study is the first to provide evidence for a circulation of newly activated Ag-specific intestinal T cells back to the gut in humans.     

Critical role of the fifth domain of E-cadherin for heterophilic adhesion with alpha E beta 7, but not for homophilic adhesion

The integrin alpha(E)beta(7) is expressed on intestinal intraepithelial T lymphocytes and CD8(+) T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between alpha(E)beta(7)(+) T and epithelial cells is mediated by the adhesive interaction of alpha(E)beta(7) and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and alpha(E)beta(7), we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When alpha(E)beta(7)(+) K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of alpha(E)beta(7)(+) K562 cells. In contrast, the binding of alpha(E)beta(7)(+) K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with alpha(E)beta(7)(+) K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with alpha(E)beta(7), but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to alpha(E)beta(7) and may be a potential target for treatments altering the adhesion of alpha(E)beta(7)(+) T cells to epithelial cells in inflammatory epithelial diseases.     

The microenvironment of human Peyer's patches inhibits the increase in CD38 expression associated with the germinal center reaction

Analysis of B cells in the human tonsils identified CD38 expression as a hallmark of germinal center (GC) B cells. However, the signals responsible for the in vivo induction of CD38 have not been determined. The primary site for generation of memory and plasma cells in the gastrointestinal tract is the GCs of Peyer's patches (PP). PP and intestinal mucosa, but not tonsils or oral mucosa, express mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The ligand for MAdCAM-1, integrin alpha(4)beta(7), is expressed on naive B cells and memory B cells that traffic to the gastrointestinal tract. In this study we determine that, unlike tonsil, human PP GC B cells do not express significant levels of CD38. PP B cells can be induced to express CD38 upon culture with CD40 ligand, anti-B cell receptor, and IFN-gamma. However, coculture of tonsil naive B cells with an Ab directed against integrin beta(7) inhibits IFN-gamma-induced CD38 hyperexpression. The absence of CD38 on PP GCs suggests that there are tissue-specific pathways of B cell development that differ between tonsil and PP. The differential expression pattern of MAdCAM-1, together with the observation that ligation of beta(7) can inhibit the induction of CD38 expression, suggests that ligation of alpha(4)beta(7) in vivo may contribute to a PP-specific GC phenotype.     

Cutting edge: dichotomy of homing receptor dependence by mucosal effector B cells: alpha(E) versus L-selectin

The common mucosal immune system may be compartmentalized because lymphocyte homing to the upper respiratory tract appears to be mediated by L-selectin interactions rather than alpha(4)beta(7) interactions, as is the case for gut-associated lymphoreticular tissue. To assess the role of L-selectin in effector B cell immunity, L-selectin-deficient mice were intranasally immunized with cholera toxin (CT), and mucosal immune responses were compared with C57BL/6 mice. The absence of L-selectin correlated with a reduction in CT-specific secretory-IgA responses in nasal passages and reproductive tract, but not intestinal lamina propria. Cell sorting experiments showed that an L-selectin-dependent subset was responsible for CT-specific responses in nasal passages and reproductive tract, whereas an alpha(E)beta(7)(+) B cell subset was responsible for L-selectin-independent intestinal immunity. This study provides evidence for compartmentalization of the common mucosal immune system into "intestinal" vs "nonintestinal" effector sites.     

Interaction of rotavirus with human myeloid dendritic cells

We have previously shown that very few rotavirus (RV)-specific T cells that secrete gamma interferon circulate in recently infected and seropositive adults and children. Here, we have studied the interaction of RV with myeloid immature (IDC) and mature dendritic cells (MDC) in vitro. RV did not induce cell death of IDC or MDC and induced maturation of between 12 and 48% of IDC. Nonetheless, RV did not inhibit the maturation of IDC or change the expression of maturation markers on MDC. After treatment with RV, few IDC expressed the nonstructural viral protein NSP4. In contrast, a discrete productive viral infection was shown in MDC of a subset of volunteers, and between 3 and 46% of these cells expressed NSP4. RV-treated IDC secreted interleukin 6 (IL-6) (but not IL-1beta, IL-8, IL-10, IL-12, tumor necrosis factor alpha, or transforming growth factor beta), and MDC released IL-6 and small amounts of IL-10 and IL-12p70. The patterns of cytokines secreted by T cells stimulated by staphylococcal enterotoxin B presented by MDC infected with RV or uninfected were comparable. The frequencies and patterns of cytokines secreted by memory RV-specific T cells evidenced after stimulation of peripheral blood mononuclear cells (PBMC) with RV were similar to those evidenced after stimulation of PBMC with RV-infected MDC. Finally, IDC treated with RV strongly stimulated naive allogeneic CD4+ T cells to secrete Th1 cytokines. Thus, although RV does not seem to be a strong maturing stimulus for DC, it promotes their capacity to prime Th1 cells.     

TLR2, TLR4 and the MyD88 Signaling Are Crucial For the In Vivo Generation and the Longevity of Long-Lived Antibody-Secreting Cells

This study was undertaken to gain better insights into the role of TLRs and MyD88 in the development and differentiation of memory B cells, especially of ASC, during the Th2 polarized memory response induced by Natterins. Our in vivo findings demonstrated that the anaphylactic IgG1 production is dependent on TLR2 and MyD88 signaling, and that TLR4 acts as adjuvant accelerating the synthesis of high affinity-IgE. Also, TLR4 (MyD88-independent) modulated the migration of innate-like B cells (B1a and B2) out of the peritoneal cavity, and the emigration from the spleen of B1b and B2 cells. TLR4 (MyD88-independent) modulated the emigration from the spleen of Bmem as well as ASC B220^pos. TLR2 triggered to the egress from the peritoneum of Bmem (MyD88-dependent) and ASC B220^pos (MyD88-independent). We showed that TLR4 regulates the degree of expansion of Bmem in the peritoneum (MyD88-dependent) and in BM (MyD88-independent) as well as of ASC B220^neg in the spleen (MyD88-independent). TLR2 regulated the intensity of the expansion of Bmem (MyD88-independent) and ASC B220^pos (MyD88-dependent) in BM. Finally, TLR4 signals sustained the longevity of ASC B220^pos (MyD88-independent) and ASC B220^neg into the peritoneum (MyD88-dependent) and TLR2 MyD88-dependent signaling supported the persistence of B2 cells in BM, Bmem in the spleen and ASC B220^neg in peritoneum and BM. Terminally differentiated ASC B220^neg required the cooperation of both signals through TLR2/TLR4 via MyD88 for longevity in peritoneum, whereas Bmem required only TLR2/MyD88 to stay in spleen, and ASC B220^pos rested in peritoneum dependent on TLR4 signaling. Our data sustain that earlier events on memory B cells differentiation induced in secondary immune response against Natterins, after secondary lymph organs influx and egress, may be the key to determining peripheral localization of innate-like B cells and memory B cells as ASC B220^pos and ASC B220^neg.     

Expression and functional importance of collagen-binding integrins,alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1)beta(1) parallels that of viral-specific effector CD8(+) T cells (defined by tetramer and IFN-gamma staining). In an adoptive transfer model, mAb-mediated blockade of these integrins on activated effector and memory T cells inhibited Ag-specific delayed-type hypersensitivity responses; similar decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect of alpha(1)beta(1) blockade on the delayed-type hypersensitivity response could be bypassed by direct injection of Ag-specific T cells to inflammatory sites, demonstrating for the first time in vivo that collagen-binding integrins are involved in leukocyte migration into tissues.     

Circulating human antibody-secreting cells during vaccinations and respiratory viral infections are characterized by high specificity and lack of bystander effect

Surges of serum Abs after immunization and infection are highly specific for the offending Ag, and recent studies demonstrate that vaccines induce transient increases in circulating Ab-secreting cells (ASCs). These ASCs are highly enriched but not universally specific for the immunizing Ag, suggesting that a fraction of these ASCs could arise from polyclonal bystander stimulation of preexisting memory cells to unrelated Ags. This model is proposed to explain maintenance of long-lived serological memory in the absence of Ag exposure. To test this model, we measure the ability of respiratory syncytial virus and influenza virus infection or immunizations to influenza virus, tetanus toxoid, hepatitis B Ag, and human papillomavirus to stimulate bystander memory cells specific for other major environmental Ags that represent a large fraction of the preexisting memory B compartment. Bystander or nonspecific ASC responses to respiratory syncytial virus and tetanus could not be detected above the background levels in healthy adults, despite the presence of circulating memory B cells specific for the corresponding Ags. Nonspecific ASC responses in the healthy subjects and cord blood samples were similar. In contrast, both vaccination and infection induce massive expansion of circulating Ag-specific ASCs without significant increases in the frequencies of ASCs against unrelated Ags. Hence, nonspecific stimulation of memory B cells is unlikely to contribute to the mechanisms of long-term serological memory against major human pathogens. Additionally, high specificity of circulating ASCs after antigenic challenge highlights the diagnostic value of interrogating ASCs as an ideal single-time-point diagnostic immune surrogate for serology during acute infection.     

Systemic dissemination and persistence of Th2 and type 2 cells in response to infection with a strictly enteric nematode parasite

Oral infection with the nematode parasite Heligmosomoides polygyrus H. polygyrus is entirely restricted to the small intestine. Although the evoked Th2 response has been extensively studied in secondary lymphoid organs, little is known about the systemic dissemination of Th2 cells or type 2 associated eosinophils and basophils. In this study we use bicistronic 4get IL-4 reporter mice to directly visualize the type 2 response to H. polygyrus infection. We observed that CD4(+)/GFP(+) Th2 cells spread systemically and found that these cells accumulated in nonlymphoid "hot spots" in the liver, the lung airways, and the peritoneal cavity. Interestingly, the total number of Th2 cells in the peritoneal cavity was comparable to those found in the draining mesenteric lymph node or the spleen. Peritoneal Th2 cells were distinguished by an exceptionally low apoptotic potential and high expression of the intestinal homing receptor alpha(4)beta(7) integrin. CD4(+)/GFP(+) Th2 cells from these peripheral sites were fully functional as indicated by rapid IL-4 production upon polyclonal or Ag-specific restimulation. Th2 cells persisted in the intestinal tissue and the peritoneal cavity of drug-cured mice for weeks. The presence of peripheral memory Th2 cells in the intestine might be crucial for immunity to recall infections. These findings have important implications for the design of vaccination strategies because it may be necessary to establish and maintain memory CD4(+) T cells at the potential future site of infection.     

Cutting edge: instructive role of peripheral tissue cells in the imprinting of T cell homing receptor patterns

Tissue-specific homing of effector and memory T cells to skin and small intestine requires the imprinting of specific combinations of adhesion molecules and chemokine receptors by dendritic cells in the draining lymph nodes. In this study, we demonstrate that CD8(+) T cells activated by Ag-pulsed bone marrow-derived dendritic cells were induced to express the small intestine homing receptors alpha(4)beta(7) integrin and chemokine receptor CCR9 in coculture with small intestinal epithelial cells. In contrast, in coculture with dermal fibroblasts the skin-homing receptor E-selectin ligand was induced. Interestingly, the imprinting of gut homing receptors on anti-CD3/anti-CD28 stimulated T cells was induced by soluble factors produced by small intestinal epithelial cells. Retinoic acid was identified as a crucial factor. These findings show that peripheral tissue cells directly produce homing receptor imprinting factors and suggest that dendritic cells can acquire their imprinting potential already in the peripheral tissue of origin.     

The chemokine stromal cell-derived factor-1 alpha modulates alpha 4 beta 7 integrin-mediated lymphocyte adhesion to mucosal addressin cell adhesion molecule-1 and fibronectin

The interaction between the integrin alpha(4)beta(7) and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that alpha(4)beta(7)-mediated adhesion of CD4(+) T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1alpha in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4(+) T cells were targets of SDF-1alpha-triggered increased adhesion. In addition, SDF-1alpha augmented alpha(4)beta(7)-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4(+) and RPMI 8866 cells to SDF-1alpha, enhanced alpha(4)beta(7)-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Galpha(i)-dependent as well as Galpha(i)-independent signaling. Accordingly, we show that SDF-1alpha induced a rapid and transient association between its receptor CXCR4 and Galpha(i), whereas association of pertussis toxin-insensitive Galpha(13) with CXCR4 was slower and of a lesser extent. SDF-1alpha also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of alpha(4)beta(7)-mediated lymphocyte adhesion in response to SDF-1alpha, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1alpha of lymphocyte adhesion mediated by alpha(4)beta(7) could contribute to lymphocyte homing to secondary lymphoid tissues.     

Sphingosine 1-phosphate regulates the egress of IgA plasmablasts from Peyer's patches for intestinal IgA responses

It is well established that Peyer's patches (PPs) are sites for the differentiation of IgA plasma cell precursors, but molecular and cellular mechanisms in their trafficking remain to be elucidated. In this study, we show that alterations in type 1 sphingosine 1-phosphate (S1P) receptor expression during B cell differentiation in the PPs control the emigration of IgA plasma cell precursors. Type 1 S1P receptor expression decreased during the differentiation of IgM(+)B220(+) B cells to IgA(+)B220(+) B cells, but recovered on IgA(+)B220(-) plasmablasts for their emigration from the PPs. Thus, IgA(+)B220(-) plasmablasts migrated in response to S1P in vitro. Additionally, IgA(+) plasmablasts selectively accumulated in lymphatic regions of PPs when S1P-mediated signaling was disrupted by FTY720 treatment. This accumulation of IgA(+) plasmablasts in the PPs led to their reduction in the intestinal lamina propria and simultaneous impairment of Ag-specific intestinal IgA production against orally administered Ag. These findings suggest that S1P regulates the retention and emigration of PP B cells and plays key roles in the induction of intestinal IgA production.     

Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine.

We have revealed that 100-200 clusters, filled with closely packed lymphocytes, can be found throughout the length of the antimesenteric wall of the mouse small intestine. They are composed of a large B cell area, including a germinal center, and epithelia overlying the clusters contain M cells. A large fraction of B cells displays B220+ CD19+ CD23+ IgM(low)IgD(high)CD5(-)Mac-1(-) phenotype, and the composition of IgA+ B cells is smaller but substantial. To our knowledge, these clusters are the first identification of isolated lymphoid follicles (ILF) in mouse small intestine. ILF can be first detected at 7 (BALB/c mice) and 25 (C57BL/6 mice) days after birth, and lymphoid clusters equivalent in terms of cellular mass to ILF are present in germfree, athymic nude, RAG-2(-/-), TCR-beta(-/-), and Ig mu-chain mutant (mu(-/-)) mice, although c-kit+ cells outnumber B220+ cells in germfree and athymic nude mice, and most lymphoid residents are c-kit+ B220(-) in RAG-2(-/-), TCR-beta(-/-), and mu(-/-) mice. ILF develop normally in the progeny of transplacentally manipulated Peyer's patch (PP)-deficient mice, and decreased numbers of conspicuously atrophied ILF are present in IL-7Ralpha(-/-) PP(null) mice. Neither ILF nor PP are detectable in lymphotoxin alpha(-/-) and aly/aly mice that retain well-developed cryptopatches (CP) and thymus-independent subsets of intraepithelial T cells, whereas ILF, PP, CP, and thymus-independent subsets of intraepithelial T cells disappear from common cytokine receptor gamma-chain mutant mice. These findings indicate that ILF, PP, and CP constitute three distinct organized gut-associated lymphoid tissues that reside in the lamina propria of the mouse small intestine.