Dendritic cells (DCs) play an essential role in regulation of immune responses. In the periphery, Ag presentation by DCs is critical for adaptive responses; for this reason, DCs are often targets of adjuvants that enhance vaccine responses. Activated mature DCs enhance B cell activation and differentiation by providing cytokines like BAFF and a proliferation-inducing ligand. However, the role of immature DCs in B cell tolerance is not well studied. Recently, mouse immature bone marrow-derived DCs (iBMDCs) have been shown to suppress anti-IgM–induced B cell activation. In this study, we tested the ability of mouse DCs to modulate B cell functions during TLR activation. We found that iBMDCs potently suppressed proliferation and differentiation of various B cell subsets on TLR stimulation. However, iBMDCs did not affect CD40-mediated B cell activation. Optimal suppression of B cell activation by iBMDCs required cell contact via the CD22 receptor on B cells. The B cell suppression was a property of iBMDCs or DCs resident in the bone marrow (BM), but not mature BM-derived DCs or DCs resident in the spleen. Presence of iBMDCs also enhanced the Ag-induced apoptotic response of BM B cells, suggesting that the suppressive effects of iBMDCs may have a role in B cell tolerance.

Abstract The first step in leukocyte trafficking into tissues and peripheral organs is mediated by selectin binding. Mice deficient in α1,3 fucosyltransferases-VII and -IV (FtDKO) are unable to synthesize sLex, which is required for E-, P-, and L-selectin binding. We recently reported that due to an inability to enter peripheral lymph nodes, FtDKO mice show increased numbers of T cells in peripheral blood and increased T cell accumulation in the lung. We now report that FtDKO mice show substantial increases in granulocyte populations in peripheral blood, with an ~20-fold increase in Ly6G+Mac-1+ neutrophil populations in the lung parenchyma. To determine whether increased neutrophil localization in the lung influences disease processes, we infected FtDKO mice with influenza. As previously reported, FtDKO mice show impaired neutrophil trafficking to the peritoneal cavity following inflammation due to loss of selectin-mediated binding. However, FtDKO mice show rapid and enhanced neutrophil trafficking to broncho-alveolar regions of the lung following influenza infection. These data suggest that instead of reducing inflammatory cell populations, loss of selectin-mediated binding may paradoxically enhance accumulation of neutrophils in the lung parenchyma, which may in turn facilitate the influx of these cells into broncho-alveolar regions of the lung following inflammation or injury.

ST6Gal I is a glycosyltransferase highly expressed by lymphocytes and cancer cells that catalyzes the addition of α2,6 sialic acid to galactose (Siaα2-6Gal) on N-linked glycoproteins. We discovered that ST6Gal I shows dramatic changes in gene expression during memory CD8 T cell differentiation that corresponds with lectin binding, but the significance of this regulated carbohydrate modification remained a mystery. To identify the significance of these regulated glyco-phenotypic changes, we infected ST6Gal I-/- mice and found that viral-specific cell expansion was impaired. Moreover, defective viral-specific CD8 T cell expansion was due to early intrinsic defects in proliferation of terminal effector cells, whereas memory precursor development was enhanced. We provide direct evidence that loss of ST6Gal I expression by CD8 T cells resulted in delayed surface expression of IL-2Rα in vivo due to impaired IL-2/IL-2R signaling. These results suggest that CD8 T cells regulate ST6Gal I expression to promote proliferation induced by cytokine-dependent signaling.

Immunoglobulins in secretions play a critical role in protection at mucosal surfaces. We examined the generation of viral-specific IgG and IgA in plasma and mucosal secretions of mice following systemic or mucosal immunization with lymphocytic choriomeningitis virus (LCMV), a widely used experimental model of viral infection. While there are early differences in humoral responses depending on the route of viral entry, we show that both routes generate comparably robust viral-specific IgG in plasma, vaginal, lung, and nasal secretions of immune mice. In contrast, LCMV elicited poor viral-specific IgA responses. Mice that were infected IN showed elevated viral-specific IgA in nasal and lung washes compared to IP-infected mice; however, LCMV-specific IgG overwhelmingly contributed to the humoral response in all mucosal secretions examined. Thus similarly to HIV-1, and several other mucosally-encountered microbial infections, these data suggest that LCMV infection fails to induce vigorous viral-specific IgA responses.

Abstract ST6Gal I, a glycosyltransferase highly expressed by B cells, catalyzes the addition of alpha-2, 6 sialic acid to galactose (Siaα2-6Gal), a modification found on N-linked glycoproteins. Aberrant glycosylation has been associated with the pathogenesis of IgA nephropathy (IgAN), a disease characterized by immune complex deposits in the glomerular mesangium, which can lead to end-stage renal failure in 20-40% of diagnosed patients. Previously, we have shown that ST6Gal I-/- mice infected with influenza A/HKx31 demonstrated profound impairment in the generation of viral-specific humoral responses, although memory IgG responses were mostly normal. We now show that influenza infected ST6Gal I-/- mice had higher levels of influenza-specific IgA and total IgA in sera at memory compared to WT mice. Additionally, we observed increased deposition of IgA in the glomeruli of kidneys of ST6Gal I-/- mice, although no differences were noted in urinalysis. These data suggest that loss of expression of an alpha-2,6 sialyltransferase, together with influenza viral infection, can contribute to increased deposition of IgA in mouse kidney glomeruli, and may contribute to the pathogenesis of one of the most common forms of glomerulonephritis in humans.

Background Selectin mediated tethering represents one of the earliest steps in T cell extravasation into lymph nodes via high endothelial venules and is dependent on the biosynthesis of sialyl Lewis X (sLex) ligands by several glycosyltransferases, including two fucosyltransferases, fucosyltransferase-IV and –VII. Selectin mediated binding also plays a key role in T cell entry to inflamed organs. Methodology/Principal Findings To understand how loss of selectin ligands (sLex) influences T cell migration to the lung, we examined fucosyltransferase-IV and –VII double knockout (FtDKO) mice. We discovered that FtDKO mice showed significant increases (∼5-fold) in numbers of naïve T cells in non-inflamed lung parenchyma with no evidence of induced bronchus-associated lymphoid tissue. In contrast, activated T cells were reduced in inflamed lungs of FtDKO mice following viral infection, consistent with the established role of selectin mediated T cell extravasation into inflamed lung. Adoptive transfer of T cells into FtDKO mice revealed impaired T cell entry to lymph nodes, but selective accumulation in non-lymphoid organs. Moreover, inhibition of T cell entry to the lymph nodes by blockade of L-selectin, or treatment of T cells with pertussis toxin to inhibit chemokine dependent G-coupled receptor signaling, also resulted in increased T cells in non-lymphoid organs. Conversely, inhibition of T cell egress from lymph nodes using FTY720 agonism of S1P1 impaired T cell migration into non-lymphoid organs. Conclusions/Significance Taken together, our results suggest that impaired T cell entry into lymph nodes via high endothelial venules due to genetic deficiency of selectin ligands results in the selective re-distribution and accumulation of T cells in non-lymphoid organs, and correlates with their increased frequency in the blood. Re-distribution of T cells into organs could potentially play a role in the initiation of T cell mediated organ diseases.

Fucosyltransferase-IV and -VII double knockout (FtDKO) mice reveal profound impairment in T cell trafficking to lymph nodes (LNs) due to an inability to synthesize selectin ligands. We observed an increase in the proportion of memory/effector (CD44high) T cells in LNs of FtDKO mice. We infected FtDKO mice with lymphocytic choriomeningitis virus to generate and track Ag-specific CD44highCD8 T cells in secondary lymphoid organs. Although frequencies were similar, total Ag-specific effector CD44highCD8 T cells were significantly reduced in LNs, but not blood, of FtDKO mice at day 8. In contrast, frequencies of Ag-specific memory CD44highCD8 T cells were up to 8-fold higher in LNs of FtDKO mice at day 60. Because wild-type mice treated with anti-CD62L treatment also showed increased frequencies of CD44high T cells in LNs, we hypothesized that memory T cells were preferentially retained in, or preferentially migrated to, FtDKO LNs. We analyzed T cell entry and egress in LNs using adoptive transfer of bone fide naive or memory T cells. Memory T cells were not retained longer in LNs compared with naive T cells; however, T cell exit slowed significantly as T cell numbers declined. Memory T cells were profoundly impaired in entering LNs of FtDKO mice; however, memory T cells exhibited greater homeostatic proliferation in FtDKO mice. These results suggest that memory T cells are enriched in LNs with T cell deficits by several mechanisms, including longer T cell retention and increased homeostatic proliferation.

Posttranslational modification of proteins, such as glycosylation, can impact cell signaling and function. ST6Gal I, a glycosyltransferase expressed by B cells, catalyzes the addition of alpha-2,6 sialic acid to galactose, a modification found on N-linked glycoproteins such as CD22, a negative regulator of B cell activation. We show that SNA lectin, which binds a-2,6 sialic acid linked to galactose, shows high binding on plasma blasts and germinal center B cells following viral infection, suggesting ST6Gal I expression remains high on activated B cells in vivo. To understand the relevance of this modification on the antiviral B cell immune response, we infected ST6Gal I-/- mice with influenza A/HKx31. We demonstrate that the loss of ST6Gal I expression results in similar influenza infectivity in the lung, but significantly reduced early influenza-specific IgM and IgG levels in the serum, as well as significantly reduced numbers of early viral-specific Ab-secreting cells. At later memory time points, ST6Gal I-/- mice show comparable numbers of IgG influenza-specific memory B cells and long-lived plasma cells, with similarly high antiviral IgG titers, with the exception of IgG2c. Finally, we adoptively transfer purified B cells from wild-type or ST6Gal I-/- mice into B cell-deficient (mu MT-/-) mice. Recipient mice that received ST6Gal I-/- B cells demonstrated reduced influenza-specific IgM levels, but similar levels of influenza-specific IgG, compared with mice that received wild-type B cells. These data suggest that a B cell intrinsic defect partially contributes to the impaired antiviral humoral response. The Journal of Immunology, 2009, 182: 4721-4727.

The chromodomain helicase DNA-binding proteins (CHDs) are known to affect transcription through their ability to remodel chromatin and modulate histone deacetylation. In an effort to understand the functional role of the CHD2 in mammals, we have generated a Chd2 mutant mouse model. Remarkably, the Chd2 protein appears to play a critical role in the development, hematopoiesis and tumor suppression. The Chd2 heterozygous mutant mice exhibit increased extramedullary hematopoiesis and susceptibility to lymphomas. At the cellular level, Chd2 mutants are defective in hematopoietic stem cell differentiation, accumulate higher levels of the chromatin-associated DNA damage response mediator, γH2AX, and exhibit an aberrant DNA damage response after X-ray irradiation. Our data suggest a direct role for the chromatin remodeling protein in DNA damage signaling and genome stability maintenance.

Selectin mediated tethering represents one of the earliest steps in T cell extravasation into lymph nodes via HEVs. Fucosyltransferases (FucT) -IV and -VII are required for production of the sialyl Lewis X moiety, which is essential for selectin mediated T cell entry into LNs. We examined T cell trafficking to lymphoid and non-lymphoid compartments such as the lung using FucT -IV and -VII double knockout (FucT DKO) mice. Examination of uninfected FucT DKO mice revealed significantly increased numbers of T cells localized in lungs, with no evidence of induced bronchial associated lymphoid tissue (iBALT). Following LCMV viral infection, we noted similar numbers of antigen specific CD8 T cells, but reduced numbers of total activated CD8 T cells in the lungs compared to wildtype mice. Finally, we transferred bona fide naïve or memory CD8 T cells into recipient mice and show increased trafficking of T cells into lungs of uninfected FucT DKO mice. We conclude from these studies that while selectin mediated entry may play an important role in T cell trafficking to the inflamed lung, in the absence of selectin ligand expression, T cells preferentially migrate to and are retained in the lung under non-pathological conditions. In situations where normal trafficking of T cells to LNs are compromised, tissues such as the lung may become sites for T cell accumulation, and this may play a role in the initiation of T cell mediated lung diseases. This work was supported by NIH grants AI057719, GM62116 (CFG), and University of Tennessee start up funds

Post‐translational modification of proteins, such as glycosylation can affect the localization of cell surface glycoproteins on the cell membrane, impacting cell signaling and function. ST6Gal‐I is a glycosyltransferase expressed by T and B cells that catalyzes the addition of alpha2,6 sialic acid to galactose, a modification typically found on N‐linked glycoproteins. In this study, we show that in contrast to activated T cells, ST6Gal‐I expression remains high on plasma blasts and germinal center B cells following viral infection. To determine the in vivo role of the loss of this enzyme during viral infection, we infected ST6Gal‐I null mice with influenza A/HK x31. We demonstrate that loss of ST6Gal‐I expression results in similar infectivity in the lung at day 3, but significantly reduced influenza‐specific IgM and IgG levels in the serum, as well as significantly reduced antibody‐forming cells (AFCs) in the MedLN and spleen in the acute phase of influenza infection. Following the acute phase of the response, we measured similar overall levels of influenza‐specific antibody in the serum and similar numbers of influenza specific memory B cells. These studies suggest that loss of ST6Gal‐I expression significantly impairs the acute viral specific B cell immune response and that changes in surface carbohydrate modification may affect B cell differentiation in vivo.

5867 Changes in chromatin structure brought about by various factors are known to play a critical role in gene regulation. Epigenetic regulation and alterations in chromatin structure allow various nuclear factors to access certain regions of DNA to carry out DNA replication, repair, recombination, and transcription. The chromatin modifiers are classified broadly into two classes: histone-modifying enzymes and ATP-dependent chromatin-remodeling factors. Chromodomain helicase DNA binding proteins (CHD) are a group of highly conserved proteins sharing sequence motifs and functional domains and are classified under the group of ATP-dependent chromatin-remodeling factors. CHD2 is one of the poorly characterized CHD proteins. The human gene encoding CHD2 is mapped to chromosome15q26.2. This region has been associated with rare genetic disorders that lead to growth retardation, cardiac defects and early post natal lethality. In an effort to understand the functional role of the chromodomain helicase DNA binding protein 2 (Chd2) in mammals, we have generated a Chd2 mutant mouse model. Our data show that Chd2 is an essential protein in mammals and regulates hematopoietic stem cell differentiation along the erythroid lineage. The Chd2 protein also appears to play a critical role in tumor suppression as the Chd2 heterozygous mutant mice are highly susceptible to lymphomas. In addition, Chd2 mutant cells exhibit cell cycle defects, accumulate higher levels of the chromatin associated DNA damage response mediator, γH2AX, and exhibit an aberrant DNA damage response after X-ray irradiation. In essence our data indicate that the chromatin remodeling protein, Chd2, functions in tumor suppression by affecting DNA damage responses.

Glycosylation can affect the localization and interactions of cell surface glycoproteins, impacting cell signaling and function. ST6Gal‐I is a glycosyltransferase expressed by T and B cells that catalyzes the addition of a2,6 sialic acid to galactose on N‐linked glycoproteins such as CD45. We show that changes in expression of ST6Gal‐I during memory CD8 T cell differentiation results in changes in cell surface sialylation as demonstrated by SNA lectin binding. Moreover, lectin binding revealed heterogeneity within the memory CD8 T cell population isolated from tissues, identifying SNAhigh and SNAlow memory CD8 T cells. To further understand the role of this modification in T cell function in vivo, we examined ST6Gal‐I null mice following LCMV infection. Our analysis revealed lower numbers of Ag‐specific CD8 T cells at day 8 post‐infection compared to WT mice. In addition, adoptive transfer of P14×ST6Gal‐I+/− or P14×ST6Gal‐I−/− cells into WT recipient mice show reduced numbers of transgenic null CD8 T cells on day 8, suggesting the defect is T cell intrinsic. Finally we demonstrate using in vivo CFSE labelling of purified P14×ST6Gal‐I+/− or P14×ST6Gal‐I−/− CD8 T cells into WT recipient mice that null transgenic CD8 T cells show substantially reduced proliferation. These studies suggest that differential expression of surface carbohydrates can significantly impair CD8 T cell activation and proliferation in vivo.

Abstract In this study we compared the efficacy of two different routes of viral infection on the generation, maintenance, and localization of antigen specific CD8T cells in lymphoid, non-lymphoid, and mucosal tissues during peak effector and memory phases of an immune response. We used an adoptive transfer system of naïve transgenic CD8 T cells, followed by intra-peritoneal (i.p) or intra-nasal (i.n) LCMV infection. Surprisingly, on day 8 the peak of the primary response, i.p. infection resulted in higher numbers of antigen-specific CD8T cells in the vaginal mucosa and ILN only, in addition to 2–3x more antigen specific CD8 T cells that co-expressed both IFNγ and TNFα, when compared to the i.n. route of infection. During memory, equivalent numbers of antigen specific CD8T cells were detected at all sites, irrespective of the route of infection. Following intravaginal re-challenge, both i.p. and i.n generated memory CD8 T cells showed robust secondary responses in the vaginal tract, however the i.n. memory CD8 T cell response was consistently greater. These data suggest that both systemic and mucosal routes of infection can generate a similar quantity of memory CD8 T cells in mucosal sites. However, the initial route of infection appears to influence the programming of the memory CD8T cells generated, and these early differences in priming may impact the quality of protective secondary responses.

Whether mucosal immunization is required for optimal protective CD8 T cell memory at mucosal surfaces is controversial. In this study, using an adoptive transfer system, we compare the efficacy of two routes of acute lymphocytic choriomeningitis viral infection on the generation, maintenance, and localization of Ag-specific CD8 T cells in tissues, including the vaginal mucosa. Surprisingly, at day 8, i.p. infection results in higher numbers of Ag-specific CD8 T cells in the vaginal mucosa and iliac lymph node, as well as 2-3x more Ag-specific CD8 T cells that coexpress both IFN-gamma and TNF-alpha in comparison to the intranasal route of infection. Expression of the integrin/activation marker CD103 (alphaEbeta7) is low on vaginal mucosal Ag-specific CD8 T cells in comparison to gut mucosal intraepithelial lymphocytes. At memory, no differences are evident in the number, cytokine production, or protective function of Ag-specific CD8 T cells in the vaginal mucosa comparing the two routes of infection. However, differences persist in the cytokine profile of genital tract vs peripheral Ag-specific CD8 T cells. So although the initial route of infection, as well as tissue microenvironment, appear to influence both the magnitude and quality of the effector CD8 T cell response, both systemic and mucosal infection are equally effective in the differentiation of protective memory CD8 T cell responses against vaginal pathogenic challenge.