Conference report: "Functional Glycomics in HIV Type 1 Vaccine Design" workshop report, Bethesda, Maryland, April 30-May 1, 2012.

A vital part of the renewed hope for a vaccine against the human immunodeficiency virus (HIV-1) is based on recent studies that have highlighted major sites of HIV-1 vulnerability that could be effectively targeted by a preventive vaccine. One of these potential vulnerabilities includes the dense cluster of carbohydrates surrounding HIV-1's envelope glycoproteins gp120 and gp41, typically referred to as the "glycan shield." Recent data from several laboratories have shown that glycans on the HIV-1 envelope form key epitopes for broadly neutralizing antibodies (bNAb). Moreover, HIV-1 envelope glycans play an important role in viral transmission, antigenicity, and immunogenicity. The recent availability of novel tools and technologies has now allowed investigators to leverage glycomic structure-function relationships in the design of candidate HIV-1 vaccines. Additionally, glycans modulate the immune response, playing an essential role in Fc receptor and complement activity. To promote cross-disciplinary collaboration and promote synergistic HIV-1- glycomics research, the National Institutes of Health (NIH) cosponsored and convened a 1.5-day workshop entitled "Functional Glycomics in HIV-1 Vaccine Design." The meeting focused on the role of glycan interactions with neutralizing antibodies, the influence of immunoglobulin G (IgG) Fc receptor glycosylation, newly available glycomics technologies, and how new information on the role of glycans could be applied in HIV-1 immunogen design strategies. This report summarizes the discussions of this workshop.

Bone marrow dendritic cell-mediated regulation of TLR and B cell receptor signaling in B cells.

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.

Memory T cells are enriched in lymph nodes of selectin-ligand-deficient mice.

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 (CD44(high)) T cells in LNs of FtDKO mice. We infected FtDKO mice with lymphocytic choriomeningitis virus to generate and track Ag-specific CD44(high)CD8 T cells in secondary lymphoid organs. Although frequencies were similar, total Ag-specific effector CD44(high)CD8 T cells were significantly reduced in LNs, but not blood, of FtDKO mice at day 8. In contrast, frequencies of Ag-specific memory CD44(high)CD8 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 CD44(high) 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.

Both systemic and mucosal LCMV immunization generate robust viral-specific IgG in mucosal secretions, but elicit poor LCMV-specific IgA.

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.

Naïve T cells re-distribute to the lungs of selectin ligand deficient mice.

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 (sLe(x)) 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 (sLe(x)) 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 (approximately 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.

The generation of influenza-specific humoral responses is impaired in ST6Gal I-deficient mice.

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 alpha-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 (microMT(-/-)) 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.

Systemic and mucosal infection program protective memory CD8 T cells in the vaginal mucosa.

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.

Lack of antigen-specific tissue remodeling in mice deficient in the macrophage galactose-type calcium-type lectin 1/CD301a.

Macrophage galactose-type C-type lectins (MGLs), which were recently named CD301, have 2 homologues in mice: MGL1 and MGL2. MGLs are expressed on macrophages and immature dendritic cells. The persistent presence of granulation tissue induced by a protein antigen was observed in wild-type mice but not in mice lacking an endogenous, macrophage-specific, galactose-type calcium-type lectin 1 (MGL1) in an air pouch model. The anti-MGL1 antibody suppressed the granulation tissue formation in wild-type mice. A large number of cells, present only in the pouch of MGL1-deficient mice, were not myeloid or lymphoid lineage cells and the number significantly declined after administration of interleukin 1 alpha (IL-1alpha) into the pouch of MGL1-deficient mice. Furthermore, granulation tissue was restored by this treatment and the cells obtained from the pouch of MGL1-deficient mice were incorporated into the granulation tissue when injected with IL-1alpha. Taken together, MGL1 expressed on a specific subpopulation of macrophages that secrete IL-1alpha was proposed to regulate specific cellular interactions crucial to granulation tissue formation.

CD43 modulates severity and onset of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis characterized by infiltration of activated CD4(+) T lymphocytes into tissues of the CNS. This study investigated the role of CD43 in the induction and progression of EAE. Results demonstrate that CD43-deficient mice have reduced and delayed clinical and histological disease severity relative to CD43(+/+) mice. This reduction was characterized by decreased CD4(+) T cell infiltration of the CNS of CD43(-/-) mice but similar numbers of Ag-specific T cells in the periphery, suggesting a defect in T cell trafficking to the CNS. The absence of CD43 also affected cytokine production, as myelin oligodendrocyte glycoprotein (MOG) 35-55-specific CD43(-/-) CD4(+) T cells exhibited reduced IFN-gamma and increased IL-4 production. CD43(-/-) CD4(+) MOG-primed T cells exhibited reduced encephalitogenicity relative to CD43(+/+) cells upon adoptive transfer into naive recipients. These results suggest a role for CD43 in the differentiation and migration of MOG(35-55)-specific T cells in EAE, and identify it as a potential target for therapeutic intervention.

TCR signal transduction in antigen-specific memory CD8 T cells.

Memory T cells are more responsive to Ag than naive cells. To determine whether memory T cells also have more efficient TCR signaling, we compared naive, effector, and memory CD8 T cells of the same antigenic specificity. Surprisingly, initial CD3 signaling events are indistinguishable. However, memory T cells have more extensive lipid rafts with higher phosphoprotein content before TCR engagement. Upon activation in vivo, they more efficiently induce phosphorylation of-LAT (linker for activation of T cells), ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38. Thus, memory CD8 T cells do not increase their TCR sensitivity, but are better poised to augment downstream signals. We propose that this regulatory mechanism might increase signal transduction in memory T cells, while limiting TCR cross-reactivity and autoimmunity.

Primary and secondary immunocompetence in mixed allogeneic chimeras.

Targeted disruption of T cell costimulatory pathways, particularly CD28 and CD40, has allowed for the development of minimally myeloablative strategies for the induction of mixed allogeneic chimerism and donor-specific tolerance across full MHC barriers. In this study we analyze in depth the ability of mixed allogeneic chimeras in two strain combinations to mount effective host-restricted and donor-restricted antiviral CD4 and CD8 responses, as well as the impact of development of mixed chimerism on the maintenance of pre-existing memory populations. While antiviral CD8 responses in mixed chimeras following acute viral infection with lymphocytic choriomeningitis virus Armstrong or vaccinia virus are largely host-restricted, donor-restricted CD8 responses as well as host- and donor-restricted CD4 responses are also readily detected, and virus is promptly cleared. We further demonstrate that selection of donor-restricted T cells in mixed chimeras is principally mediated by bone marrow-derived cells in the thymus. Conversely, we find that mixed chimeras exhibit a deficit in their ability to deal with a chronic lymphocytic choriomeningitis virus clone 13 infection. Encouragingly, pre-existing memory populations are largely unaffected by the development of high level mixed chimerism and maintain the ability to control viral rechallenge. Our results suggest that while pre-existing T cell memory and primary immunocompetence to acute infection are preserved in mixed allogeneic chimeras, MHC class I and/or class II tissue matching may be required to fully preserve immunocompetence in dealing with chronic viral infections.

Cutting edge: persistent viral infection prevents tolerance induction and escapes immune control following CD28/CD40 blockade-based regimen.

A continuing concern with CD28 and/or CD40 blockade-based strategies to induce tolerance and mixed chimerism is their potential to disrupt protective immunity to preexisting infections. In this report, we find that preexisting persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13 prevents the induction of tolerance, mixed chimerism, and donor-reactive T cell deletion. Mice continue to be refractory to tolerance induction even after viremia has been resolved and virus is present only at very low levels in peripheral tissues. Conversely, we find that the full tolerance regimen, or costimulation blockade alone, specifically inhibits already ongoing antiviral immune responses, leading to an inability to control viremia. These findings suggest that ongoing T cell responses continue to depend on costimulatory interactions in the setting of a chronic infection and provide insight into potential risks following costimulation blockade posed by chronic or latent viral infections such as hepatitis C, EBV, and CMV.

Dynamic regulation of T cell immunity by CD43.

During a viral response, Ag-specific effector T cells show dramatically increased binding by the mAb 1B11 and the lectin peanut agglutinin (PNA). We investigated the contribution of CD43 expression to 1B11 and PNA binding as well as its role in generation and maintenance of a CD8 T cell response. Analysis of CD43(-/-) mice revealed no increased 1B11 binding and reduced PNA binding on virus-specific CD8 T cells from -/- mice compared with +/+ mice. Furthermore, we examined the role of CD43 in the kinetics of an immune response. We show that CD43 expression modestly effects generation of a primary virus-specific CD8 T cell response in vivo but plays a more significant role in trafficking of CD8 T cells to tissues such as the brain. More interestingly, CD43 plays a role in the contraction of the immune response, with CD43(-/-) mice showing increased numbers of Ag-specific CD8 T cells following initial expansion. Following the peak of expansion, Ag-specific CD8 T cells from -/- mice show similar proliferation but demonstrate increased Bcl-2 levels and decreased apoptosis of Ag-specific effector CD8 T cells in vitro. Consistent with a delay in the down-modulation of the immune response, following chronic viral infection CD43(-/-) mice show increased morbidity. These data suggest a dynamic role of CD43 during an immune response: a positive regulatory role in costimulation and trafficking of T cells to the CNS and a negative regulatory role in the down-modulation of an immune response.

Generation of mice deficient for macrophage galactose- and N-acetylgalactosamine-specific lectin: limited role in lymphoid and erythroid homeostasis and evidence for multiple lectins.

Macrophage receptors function in pattern recognition for the induction of innate immunity, in cellular communication to mediate the regulation of adaptive immune responses, and in the clearance of some glycosylated cells or glycoproteins from the circulation. They also function in homeostasis by initiating the engulfment of apoptotic cells. Evidence has suggested that macrophage receptors function to recognize cells that are destined for programmed cell death but not yet overtly apoptotic. We have examined the function of a macrophage receptor specific for unsialylated glycoproteins, known as the mouse macrophage galactose- and N-acetylgalactosamine-specific lectin (mMGL) (Ii et al., J. Biol. Chem. 265:11295-11298, 1990; Sato et al., J. Biochem. [Tokyo] 111:331-336, 1992; Yamamoto et al., Biochemistry 33:8159-8166, 1994). With targeted disruption, we tested whether mMGL is necessary for macrophage function, controlled thymic development, the loss of activated CD8 T cells, and the turnover of red blood cells. Evidence indicates that mMGL may play a nonessential role in several of these macrophage functions. Experiments are presented that indicate the existence of another galactose- and N-acetylgalactosamine-recognizing lectin distinct from mMGL. This may explain the absence of a strong phenotype in mMGL-deficient mice.

Molecular cloning and characterization of a novel mouse macrophage C-type lectin, mMGL2, which has a distinct carbohydrate specificity from mMGL1.

A novel mouse macrophage galactose-type C-type lectin 2 (mMGL2) was identified by BLAST analysis of expressed sequence tags. The sequence of mMGL2 is highly homologous to the mMGL, which should now be called mMGL1. The open reading frame of mMGL2 contains a sequence corresponding to a type II transmembrane protein with 332 amino acids having a single extracellular C-type lectin domain. The 3'-untranslated region included long terminal repeats of mouse early transposon. The Mgl2 gene was cloned from a 129/SvJ mouse genomic library and sequenced. The gene spans 7,136 base pairs and consists of 10 exons, which is similar to the genomic organization of mMGL1. The reverse transcriptase-PCR analysis indicates that mMGL2 is expressed in cell lines and normal mouse tissues in a macrophage-restricted manner, also very similar to that of mMGL1. The mMGL2 mRNA was also detected in mMGL1-positive cells, which were sorted from thioglycollate-induced peritoneal cells with a mMGL1-specific monoclonal antibody, LOM-8.7. The soluble recombinant proteins of mMGL2 exhibited carbohydrate specificity for alpha- and beta-GalNAc-conjugated soluble polyacrylamides, whereas mMGL1 preferentially bound Lewis X-conjugated soluble polyacrylamides in solid phase assays. These two lectins may function cooperatively as recognition and endocytic molecules on macrophages and related cells.