Lusijah S. Rott

Lymphocyte Trafficking and Regional Immunity

Publisher Summary This chapter discusses the lymphocyte trafficking and regional immunity. The chapter discusses the present understanding of tissue-selective lymphocyte trafficking, focusing in particular on the gut and its associated lymphoid tissues. The sophisticated cellular, developmental, and molecular mechanisms involved are discussed in the chapter with emphasis on the importance of these mechanisms for the understanding and manipulation of regional immune responses in the gastrointestinal tract. Lymphocytes are migratory cells, trafficking from their sites of origin in the bone marrow and thymus and homing to and recirculating through specialized lymphoid and extra lymphoid tissues in the periphery. Like all leukocytes, lymphocytes develop with characteristic trafficking properties. A central tenet of current thinking in the field is that this tissue-specific targeting of antigen-reactive populations must increase the efficiency of immune surveillance by circulating memory cells as well as enhancing local immune responses. From the perspective of regional physiology and pathology, selective trafficking may also provide a mechanism for segregating the specialized immune response modalities characteristic of intestinal versus systemic immune responses.

Rules of chemokine receptor association with T cell polarization in vivo

Current concepts of chemokine receptor (CKR) association with Th1 and Th2 cell polarization and effector function have largely ignored the diverse nature of effector and memory T cells in vivo. Here, we systematically investigated the association of 11 CKRs, singly or in combination, with CD4 T cell polarization. We show that Th1, Th2, Th0, and nonpolarized T cells in blood and tissue can express any of the CKRs studied but that each CKR defines a characteristic pool of polarized and nonpolarized CD4 T cells. Certain combinations of CKRs define populations that are markedly enriched in major subsets of Th1 versus Th2 cells. For example, although Th0, Th1, and Th2 cells are each found among blood CD4 T cells coordinately expressing CXCR3 and CCR4, Th1 but not Th2 cells can be CXCR3(+)CCR4(-), and Th2 but only rare Th1 cells are CCR4(+)CXCR3(-). Contrary to recent reports, although CCR7(-) cells contain a higher frequency of polarized CD4 T cells, most Th1 and Th2 effector cells are CCR7(+) and thus may be capable of lymphoid organ homing. Interestingly, Th1-associated CKRs show little or no preference for Th1 cells except when they are coexpressed with CXCR3. We conclude that the combinatorial expression of CKRs, which allow tissue- and subset-dependent targeting of effector cells during chemotactic navigation, defines physiologically significant subsets of polarized and nonpolarized T cells.

Expression of mucosal homing receptor alpha4beta7 by circulating CD4+ cells with memory for intestinal rotavirus.

The integrin alpha4beta7 mediates lymphocyte binding to mucosal addressin cell adhesion molecule-1, and its expression defines lymphocytes capable of trafficking through the intestines and the intestinal lymphoid tissues. We examined the ability of discrete alpha4beta7(hi) and alpha4beta7- subsets of circulating memory phenotype (CD45RA-) CD4+ T cells to proliferate in response to rotavirus, a ubiquitous intestinal pathogen. alpha4beta7(hi) memory (CD45RA-) CD4+ T cells displayed much greater reactivity to rotavirus than alpha4beta7- memory or naive (CD45RA+) CD4+ T cells. In contrast, alpha4beta7- memory cells were the predominant population responsive to mumps antigen after intramuscular vaccination. Our results are consistent with the conclusion that natural rotavirus infection, an enteric pathogen, results in a specific circulating memory CD4+ response that is largely limited to the gut-homing alpha4beta7+ subpopulation. This phenotype is not shared with memory cells elicited by intramuscular immunization (shown here) or by skin contact allergens. The results support the hypothesis that gut trafficking memory CD4+ T cells comprise cellular memory for intestinal antigens and suggest that regulated expression of alpha4beta7 helps target and segregate intestinal versus systemic immune response.

Correlation of Tissue Distribution, Developmental Phenotype, and Intestinal Homing Receptor Expression of Antigen-Specific B Cells During the Murine Anti-Rotavirus Immune Response

The intestinal homing receptor, α4β7, helps target lymphocytes to Peyer’s patches (PP) and intestinal lamina propria (ILP). We have previously shown that protective immunity to rotavirus (RV), an intestinal pathogen, resides in memory B cells expressing α4β7. In this study, using a novel FACS assay, we have directly studied the phenotype of B cells that express surface RV-specific Ig during the in vivo RV immune response. During primary infection, RV-specific B cells first appear as large IgD−B220lowα4β7− and α4β7+ cells (presumptive extrafollicular, Ab-secreting B cells), and then as large and small IgD−B220highα4β7− cells (presumptive germinal center B cells). The appearance of B cells with the phenotype of large IgD−B220lowα4β7+ cells in PP and most notably in mesenteric lymph nodes coincides with the emergence of RV-specific Ab-secreting cells (ASC) in the ILP. Thus, these B lymphocytes are good candidates for the migratory population giving rise to the RV-specific ASC in the ILP. RV-specific long-term memory B cells preferentially accumulate in PP and express α4β7. Nine months after infection most RV-specific IgA ASC are found in PP and ILP and at lower frequency in bone marrow and spleen. This study is the first to follow changes in tissue-specific homing receptor expression during Ag-specific B cell development in response to a natural host, tissue-specific pathogen. These results show that α4β7 is tightly regulated during the Ag-specific B cell response to RV and is expressed concurrently with the specific migration of memory and effector B cells to intestinal tissues.

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 β7-deficient (β7−/−) mice, which do not express α4β7 integrin, we demonstrated the importance of α4β7 integrin in B cell-mediated anti-RV immunity. β7−/− mice acutely infected with murine RV resolved infection and developed normal serum IgG Abs but had diminished intestinal IgA responses. α4β7−/− 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 β7−/− B cells compared with wild type. The absence of α4β7 expression and/or a lower frequency of IgA-producing cells among transferred β7−/− 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 α4β7high population. Our findings suggest that α4β7 integrin expression is necessary for B cell-mediated immunity to RV independent of the presence of IgA.

Rotavirus NSP1 Protein Inhibits Interferon-Mediated STAT1 Activation

ABSTRACT Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the NSP1 gene and was observed in a simian SA11 monoreassortant that encoded porcine OSU NSP1 but not in wild-type SA11 or a reassortant encoding simian RRV NSP1.

In vivo distribution and characterization of two novel mononuclear phagocyte differentiation antigens in mice

Flow cytometry, immunohistology, and SDS‐ PAGE Western blot analysis were used to characterize two novel anti‐mouse mononuclear phagocyte differentiation antigens defined by monoclonal antibodies. One antibody, Monts‐4, recognized an 80‐100 kd cell‐surface protein expressed on resident macrophages in the peritoneum and stained macrophages in the splenic white pulp and marginal zone, liver, lymph node paracortical regions, Peyers patches, and cortex of the thymus. Monts‐4 did not stain blood monocytes or monocyte‐derived inflammatory macrophages in the peritoneal cavity. Macrophages that were Monts‐4 positive became Monts‐4 negative within 24‐72 h after culturing in vitro. Monoclonal antibody SK39 recognized a 180 kd cell‐surface molecule expressed on inflamed peritoneal monocytes/macro‐ phages and stained macrophages in the splenic red pulp, cortex of the thymus, subcapsule and medullary regions of lymph nodes, and in sites of acute and chronic inflammation. No differences were seen in the expression of these antigens in the immunodeficient SCID versus normal BALB/c mouse. A comparison of the distribution and molecular weights of the Monts‐4 and SK39 antigens with other described mononuclear phagocyte‐specific antigens, including F4/80 and those defined by the Ml/70, ERTR9, MOMA1, MOMA2, and SER4 monoclonal antibodies, shows these to be novel mononuclear phagocyte‐specific differentiation antigens.

Plasmablast frequency and trafficking receptor expression are altered in pediatric ulcerative colitis

Background: The incidence of pediatric ulcerative colitis (UC), a chronic autoinflammatory disease of the colon, is on the rise. Although an increased infiltration of B cells from the peripheral blood into the colon occurs in UC, B‐cell trafficking is understudied. We hypothesized that the frequency of circulating plasmablasts (PBs) and their trafficking receptor (TR) expression may be indicative of the location and degree of pathology in pediatric UC. Methods: We conducted multicolor flow cytometry analyses of circulating IgA+/− PBs and IgA+ memory B cells (MBCs) in pediatric UC patients with remission, mild, moderate, and severe state of disease (n = 12), and healthy pediatric (n = 2) and adult donors (n = 11). Results: Compared to healthy donors the average frequency of PBs among total peripheral blood lymphocytes is increased 30‐fold during severe UC activity, and positively correlates with Pediatric Ulcerative Colitis Activity Index score, C‐reactive protein level, and erythrocyte sedimentation rate. A greater percent of PBs in severe patients express the gut‐homing receptors &agr;4&bgr;7 and CCR10, and the inflammatory homing molecule P‐selectin ligand (P‐sel lig). The percent of IgA+ MBCs expressing &agr;4&bgr;7, however, is reduced. Furthermore, expression of the small intestine TR CCR9 is decreased on &agr;4&bgr;7high PBs, and on &agr;4&bgr;7high/CCR10high PBs and MBCs in these patients, consistent with preferential cell targeting to the colon. Conclusions: Peripheral blood PBs with a colon‐homing phenotype (&agr;4&bgr;7/CCR10/P‐sel lig) are elevated in children with severe UC. Screening this B‐cell subset may provide a complementary approach in monitoring disease activity or therapeutic efficacy in pediatric UC. (Inflamm Bowel Dis 2012;)