T11 target structure exerts effector function by activating immune cells in CNS against glioma where cytokine modulation provide favorable microenvironment

Glycoprotein T 11 target structure (T11TS), derived from sheep erythrocyte membrane, directly interacts with T cells to activate them to enter in the brain. When untreated, glioma exerts an immune-suppressive environment in its vicinity by secreting prostaglandin E2 (PGE2), IL-10, tumor growth factor beta, gangliosides etc. to dampen the immune attack. But exogenous administration of T11TS reverses the situation to pro-inflammatory immune active state by expressing enhanced IL-12 and tumor necrosis factor alpha (TNF-alpha) production and suppression of IL-4 and IL-10 levels. The T11TS activated lymphocytic accumulation along the capillary endothelium in brain and their penetration in the matrix was evident from histological sections. IL-6 with TNF-alpha facilitates leukocyte migration to glioma site to exert cytotoxic effector function. Brain infiltrated lymphocytes offer cytotoxic proximity to neoplastic glial cells, which lead them to apoptosis. In the Th1 dominated microenvironment microglial cells was found with enhanced phagocytic functions. Initially infiltrated lymphocytes with microglia showed increased production of TNF-alpha, interferon gamma (IFN-gamma) to facilitate their effector actions. Repeated dosing of T11TS shows glioma abrogation in rat model, but also a resurgence of anti-inflammatory cytokine environment found with increased IL-4, IL-10 and decreased IL-12, IL-6, TNF-alpha. This is a unique homeostatic regulation of total immune system after T11TS mediated carnage of glioma. The resultant balance of cytokines between interacting glioma cells, T cells and microglia in T11TS induced condition determines the success of its immunotherapeutic effect in glioma.     

Documentation of immune profile of microglia through cell surface marker study in glioma model primed by a novel cell surface glycopeptide T11TS/SLFA-3

STATEMENT OF THE PROBLEM: The sheep erythrocyte membrane glycoprotein T11TS/SLFA-3 can form a ligand-receptor complex with CD2 present on immunocyte and exert stimuli for activation and proliferation. Regression of brain tumor with the application of T11TS indicates the probable role of microglia, the chief immunomodulatory cell within the brain compartment. In the present study microglial activation and immunophenotypic modulation were assessed in T11TS treated brain tumor-bearing animal models. Rat glioma models induced by chemical carcinogen ENU were treated with three consecutive doses of T11TS. Microglial cells from brain were isolated and assessed through E-rosette formation, SEM and FACS for CD2, MHC class II, CD25, and CD4. The preliminary indication of presence of CD2 on microglia through E-rosette formation was confirmed by SEM and FACS. MHC class II and CD2 single and double positive subpopulations exist, and their expression is also modulated in different doses of T11TS. A general trend of highest receptor saturation and microglial activation, measured through the activation marker CD25 and CD4 expression, was observed in 2nd dose of T11TS administration, which was then dampened via a complex immune feedback mechanism in the 3rd dose.     

Sheep erythrocyte rosetting induces multiple alterations in T lymphocyte function: inhibition of T cell receptor activity and stimulation of T11/CD2

When T lymphocytes from human blood or lymphoid organs are prepared by the sheep red blood cell (SRBC) rosetting procedure, glycoproteins of the SRBC membrane interact intimately with the CD2 (T11) molecule on the T cell surface. We now show that rosette formation has measurable short- and long-term effects upon the T cells. First, for a period of 24-48 hr after rosetting, the signal transducing and activation functions of the T3/Ti T cell antigen receptor complex is paralyzed for anti-T3-induced calcium mobilization, with a concomitant decrease in proliferative response to mitogens or stimulatory anti-T3 antibodies. Calcium mobilization through the alternate pathway of T cell activation, the T11/CD2 SRBC receptor, was also inhibited by rosetting. Second, rosetting appears to confer a partial stimulatory signal through the T11/CD2 pathway. Thus, 72 hr or more after rosetting, there was increased expression of the T11(3) activation epitope, and rosetted T cells were stimulated to proliferate in the presence of anti-T11(3) antibodies alone. These results provide further details on the effects of SRBC-T cell interactions, with important methodological implications. Moreover, they suggest a hitherto unrecognized down-regulatory effect of engaging the CD2 molecule, and provide further evidence that the T cell receptor is functionally interconnected to the CD2 activation pathway.     

Documentation of immune profile of microglia through cell surface marker study in glioma model primed by a novel cell surface glycopeptide T11TS/SLFA-3

Statement of the Problem: The sheep erythrocyte membrane glycoprotein T11TS/SLFA-3 can form a ligand-receptor complex with CD2 present on immunocyte and exert stimuli for activation and proliferation. Regression of brain tumor with the application of T11TS indicates the probable role of microglia, the chief immunomodulatory cell within the brain compartment. In the present study microglial activation and immunophenotypic modulation were assessed in T11TS treated brain tumor-bearing animal models. Rat glioma models induced by chemical carcinogen ENU were treated with three consecutive doses of T11TS. Microglial cells from brain were isolated and assessed through E-rosette formation, SEM and FACS for CD2, MHC class II, CD25, and CD4. The preliminary indication of presence of CD2 on microglia through E-rosette formation was confirmed by SEM and FACS. MHC class II and CD2 single and double positive subpopulations exist, and their expression is also modulated in different doses of T11TS. A general trend of highest receptor saturation and microglial activation, measured through the activation marker CD25 and CD4 expression, was observed in 2nd dose of T11TS administration, which was then dampened via a complex immune feedback mechanism in the 3rd dose. Published in 2004.     

Immunoregulatory function of CD3+, CD4-, and CD8- T cells. Gamma delta T cell receptor-positive T cells from nude mice abrogate oral tolerance

Previous work has shown that abrogation of oral tolerance is mediated by T cells which are found in the CD3+, L3T4- (CD4-), and Lyt-2- (CD8-) subset (termed double-negative; DN) in mice. Inasmuch as it is known that athymic, nude (nu/nu) mice possess Thy 1+, CD4-, and CD8- T cells which also exhibit a functionally rearranged TCR gamma-chain, we investigated whether this subset of nude T cells contained functional immunoregulatory cells. In this report, we examined the phenotype and distribution of CD3+ T cells in the spleen and in the mesenteric and peripheral lymph nodes of BALB/c nu/nu mice in comparison with normal mice (+/+). In the spleens of nude mice, the predominant CD3+ T cell subpopulation was DN. Further, in mesenteric and peripheral lymph nodes, approximately one-third and one-half of the CD3+ T cells were double negative, respectively. In contrast, CD3+, DN T cells represent a small subpopulation in normal (+/+) mice. We next showed that functional regulatory T cells which possess the ability to abrogate oral tolerance were induced in nu/nu mice by Ag priming. BALB/c nude mice were immunized with SRBC, and the splenic CD3+, Vicia villosa-adherent cells were obtained by panning. Adoptive transfer of CD3+, V. villosa-adherent T cells to orally tolerant BALB/c mice restored responsiveness to SRBC, whereas V. villosa nonadherent cells were without effect. In other experiments, CD3+ T cells from the spleens of SRBC-primed mice were further enriched for the CD5+, DN phenotype and adoptive transfer of this subset completely abrogated oral tolerance to SRBC. To characterize the nature of the TCR expressed on these CD3+, DN T cells, we developed a rabbit antibody to a synthetic peptide (residues 209-218: Tyr-Ala-Asn-Ser-Phe-Asn-Asn-Glu-Lys-Leu) which was synthesized from a deduced sequence of the murine delta-gene. Immunoprecipitation of a cell membrane fraction from CD3+, DN T cells with anti-delta TCR antibody isolated a 45-kDa band. Furthermore, immunoprecipitation of these cells with anti-CD3 (145-2C11) revealed bands at 45 and 35 kDa (corresponding to delta- and gamma-chains, respectively). Taken together, these results are the first to show that gamma delta-TCR bearing CD3+, CD4-, and CD8- T cells are functional and reverse oral tolerance when adoptively transferred.     

Contrasuppressor cells that break oral tolerance are antigen-specific T cells distinct from T helper (L3T4+), T suppressor (Lyt-2+), and B cells

Continuous gastric intubation of mice with the T cell-dependent antigen sheep erythrocytes (SRBC) leads to a state of systemic unresponsiveness to parenteral SRBC challenge, a state termed oral tolerance. The systemic unresponsiveness of mice rendered orally tolerant to SRBC, however, is converted to humoral immune responsiveness by adoptive transfer of effector T contrasuppressor (Tcs) cells. In this study, the authors have isolated and characterized the Tcs cell subset, from the spleens of orally immunized mice, which abrogates oral tolerance. This Tcs cell is a novel cell type, which can be separated from functional T suppressor (Lyt-2+) and T helper (L3T4+) cells, and the effector Tcs cell exhibits a Lyt-1+, 2-, L3T4- phenotype. Furthermore, contrasuppression is not mediated by B cells, including those of the Lyt-1+ phenotype. Adoptive transfer of splenic Lyt-1+, 2-, L3T4- T cells from C3H/HeJ mice given oral SRBC for 21 to 28 days and splenic Lyt-1+, 2-, L3T4- T cells of C3H/HeN mice orally immunized for a shorter interval abrogated oral tolerance. Furthermore, separation of Lyt-1+ T cells into L3T4+ and L3T4- subsets by flow cytometry resulted in Lyt-1+, L3T4+ T cells with helper but not contrasuppressor function, whereas the Lyt-1+, L3T4- T cell fraction abrogated oral tolerance even though it was without helper activity. This Tcs cell subset was also effective when added to cultures of tolerized spleen cells derived from SRBC-fed mice. The effector Tcs cells are antigen-specific, because Tcs cells from SRBC-immunized mice reverse tolerance to SRBC but not to horse erythrocytes (HRBC), and Tcs cells from HRBC-immunized mice reverse tolerance to HRBC but not to SRBC. When splenic T3 (CD3)-positive T cells (Lyt-1+, 2-, and L3T4-) were separated into Vicia villosa-adherent and nonadherent subpopulations, active contrasuppression was associated with the T3-positive and Vicia villosa-adherent T cell fraction. Thus, a distinct Lyt-1+, 2-, L3T4- T cell subset that contains a T3-T cell receptor complex, which can regulate oral tolerance, is present in spleens of orally immunized mice.     

Skin immune responses to peptide and protein antigen are TLR4 independent

Little is known about the innate immune mechanisms regulating adaptive immune responses elicited through the skin. Tissue injury is postulated to liberate Toll like receptor 4 (TLR4) ligands. In this study, we determined whether TLR4 signaling modulates the response to epidermal injury induced by tape stripping (TS) and whether it alters humoral and cellular immune responses generated through epicutaneous immunization with peptide+cholera toxin (CT). The combined use of cholera toxin and TS with antigen promoted optimal antigen-specific CD4(+) and CD8(+) T cell proliferation in Balb/c and C57BL/6 mice, respectively. TLR4 mutant mice had similar T cell responses to wild type mice. Further, OVA-protein specific IgG, IgG(1), IgG(2a), and IgE titers were similar in wild type and TLR4 mutant mice. Thus, TLR4 signaling was not required for the generation of epicutaneous T cell or antibody mediated immune responses and did not alter the quality of the immune responses elicited.     

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.     

Suppressor T-cell activity in MRL/Mp-lpr/lpr mice: differential effects on primary and memory antibody responses of MRL/Mp-lpr/lpr and MRL/Mp-+/+ spleen cells to thymus dependent and thymus independent antigens in vitro

We have previously shown that suppressor-T-cell (TS) activity in the spleens of autoimmune MRL/Mp-lpr/lpr (MRL/l) mice is increased after 2 months of age. The TS suppress the in vitro primary IgM response to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC) of B and T cells from young congenic MRL/Mp-+/+ (MRL/n) mice which lack the lymphoproliferation (lpr) gene. The TS are nylon wool nonadherent, Thy 1.2 positive, and radiation sensitive. The studies presented here were done to further characterize the TS and to attempt to determine the mechanism of action of these cells. We found that increased TS activity was also present in the proliferating lymph nodes of old MRL/l mice but not in lymph nodes of young MRL/l or MRL/n mice. The splenic TS equally suppressed the primary IgM SRBC response of both young MRL/l and MRL/n B and T cells, indicating that MRL/l SRBC-specific B and T cells are not resistant to suppression. The IgM response of MRL/n B and T cells to the T-independent (TI) antigen trinitrophenyl conjugated to Brucella abortus (TNP-BA) was not suppressed by the TS, although the IgM response to TNP was suppressed when TNP was coupled to the TD carrier SRBC. The results of kinetics studies of TS expression showed that when the TS were added on Day 0 of culture the SRBC response was suppressed as early as Day 2 of culture; however, when the TS were added on Days 1, 2, or 3 of culture, the suppression was reduced. The TS suppressed the in vitro memory IgG response of spleen cells from MRL/n mice which had been primed with SRBC; the memory IgG responses of spleen cells from MRL/l mice were variably suppressed. Taken together, these results suggest that the TS suppress TH function in early events of antibody production and that some activated B or T cells may be resistant to the effects of the TS. Increased TS activity was not present in the spleens of aged New Zealand Black X NZ White (NZB/W) F1 mice. Possible reasons for the presence of increased TS activity in MRL/l mice and its relation to autoimmune disease is discussed.     

Regulatory function for murine intraepithelial lymphocytes. Two subsets of CD3+, T cell receptor-1+ intraepithelial lymphocyte T cells abrogate oral tolerance

The murine intraepithelial lymphocyte (IEL) population is enriched in T cells that express the gamma delta-TCR, however, the biologic function served by these T cells remains obscure. IEL are considered to be major effector cells in mucosal immunity, and we have investigated whether IEL subsets could reverse orally induced systemic unresponsiveness (oral tolerance; OT) and support secondary type responses when adoptively transferred to mice orally tolerized with SRBC. When purified CD3+ IEL from mice orally primed with SRBC were transferred to adoptive hosts and challenged with SRBC, splenic IgM, IgG1, IgG2b, and IgA anti-SRBC plaque-forming cell responses were observed. However, CD3+ IEL from HRBC orally primed mice did not abrogate SRBC induced OT. Further, HRBC-primed CD3+, IEL converted HRBC-specific OT but not SRBC-specific OT. CD3+ IEL could be separated into four subsets based on expression of CD4 and CD8. CD3+, CD4-, 8+ T cells were the major subset (74.5%), with smaller numbers of CD4- and CD8- (double negatives, DN) (7.8%), CD4+, 8- (7.6%) and CD4+, CD8+ (double positives) (10.1%) T cells. Interestingly, both the CD3+, CD8+, and the CD3+, DN IEL subsets abrogated OT, resulting in significant IgM, IgG1, IgG2b, and IgA anti-SRBC plaque-forming cell responses when adoptively transferred to mice with OT. However, neither CD3+, CD4+, CD8-, nor double positive T cells affected OT when studied in this system. The CD3+, CD8+ IEL subset could be further separated into Thy-1+ (16.6%) and Thy-1- (83.4%) cells; adoptive transfer of Thy-1- cells abrogated oral tolerance whereas the Thy-1+ subset was without effect. When the expression of TCR on IEL with this biologic function was determined by use of monoclonal anti-alpha beta TCR (H57.597), TCR2-, CD3+ IEL possessed immunoregulatory function whereas the alpha beta-TCR+ (TCR2+) fraction did not abrogate OT. Immunoprecipitation of membrane fractions obtained from purified CD3+, CD4-, CD8+, Thy-1- IEL with polyclonal anti-delta peptide (Tyr-Ala-Asn-Ser-Phe-Asn-Asn-Glu-Lys-Leu) antibody revealed bands of 45 and 35 kDa, corresponding to the delta- and gamma-chains, respectively. These results suggest that gamma delta-TCR+ IEL possess a regulatory function, namely the restoration of immune responses in a state of oral tolerance. Further, both CD3+, CD4-, CD8+, Thy-1-, and CD3+, DN IEL T cells exhibit this effector contrasuppressor function.     

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.     

A Mathematical Model to Elucidate Brain Tumor Abrogation by Immunotherapy with T11 Target Structure

T11 Target structure (T11TS), a membrane glycoprotein isolated from sheep erythrocytes, reverses the immune suppressed state of brain tumor induced animals by boosting the functional status of the immune cells. This study aims at aiding in the design of more efficacious brain tumor therapies with T11 target structure. We propose a mathematical model for brain tumor (glioma) and the immune system interactions, which aims in designing efficacious brain tumor therapy. The model encompasses considerations of the interactive dynamics of glioma cells, macrophages, cytotoxic T-lymphocytes (CD8+ T-cells), TGF-β, IFN-γ and the T11TS. The system undergoes sensitivity analysis, that determines which state variables are sensitive to the given parameters and the parameters are estimated from the published data. Computer simulations were used for model verification and validation, which highlight the importance of T11 target structure in brain tumor therapy.     

Age-associated decline in effective immune synapse formation of CD4(+) T cells is reversed by vitamin E supplementation

Aging is associated with reduced IL-2 production and T cell proliferation. Vitamin E supplementation, in aged animals and humans, increases cell division and IL-2 production by naive T cells. The immune synapse forms at the site of contact between a T cell and an APC and participates in T cell activation. We evaluated whether vitamin E affects the redistribution of signaling proteins to the immune synapse. Purified CD4(+) T cells, from the spleens of young and old mice, were treated with vitamin E before stimulation with a surrogate APC expressing anti-CD3. Using confocal fluorescent microscopy, we observed that CD4(+) T cells from old mice were significantly less likely to recruit signaling proteins to the immune synapse than cells from young mice. Vitamin E increased the percentage of old CD4(+) T cells capable of forming an effective immune synapse. Similar results were found following in vivo supplementation with vitamin E. When compared with memory cells, naive T cells from aged mice were more defective in immune synapse formation and were more responsive to vitamin E supplementation. These data show, for the first time, that vitamin E significantly improves age-related early T cell signaling events in naive CD4(+) T cells.     

CD4+ CD25+ regulatory T cell repertoire formation in response to varying expression of a neo-self-antigen

We have examined the development of self-peptide-specific CD4+ CD25+ regulatory T cells in lineages of transgenic mice that express the influenza virus PR8 hemagglutinin (HA) under the control of several different promoters (HA transgenic mice). By mating these lineages with TS1-transgenic mice expressing a TCR that recognizes the major I-E(d)-restricted determinant from HA (site 1 (S1)), we show that S1-specific T cells undergo selection to become CD4+ CD25+ regulatory T cells in each of the lineages, although in varying numbers. In some lineages, S1-specific CD4+ CD25+ regulatory T cells are highly abundant; indeed, TS1xHA-transgenic mice can contain as many S1-specific CD4+ T cells as are present in TS1 mice, which do not express the neo-self HA. In another lineage, however, S1-specific thymocytes are subjected to more extensive deletion and far fewer S1-specific CD4+ CD25+ regulatory T cells accumulate in the periphery. We show that radioresistant stromal cells can direct both deletion and CD4+ CD25+ regulatory T cell selection of S1-specific thymocytes. Interestingly, even though their numbers can vary, the S1-specific CD4+ CD25+ regulatory T cells in all cases coexist with clonally related CD4+ CD25- T cells that lack regulatory function. These findings show that the formation of the CD4+ CD25+ regulatory T cell repertoire is sensitive to variations in the expression of self-peptides.     

Glycan analysis of therapeutic glycoproteins

Therapeutic monoclonal antibodies (mAbs) are glycoproteins produced by living cell systems. The glycan moieties attached to the proteins can directly affect protein stability, bioactivity, and immunogenicity. Therefore, glycan variants of a glycoprotein product must be adequately analyzed and controlled to ensure product quality. However, the inherent complexity of protein glycosylation poses a daunting analytical challenge. This review provides an update of recent advances in glycan analysis, including the potential utility of lectin-based microarray for high throughput glycan profiling. Emphasis is placed on comparison of the major types of analytics for use in determining unique glycan features such as glycosylation site, glycan structure, and content.     

Rap1-GTP is a negative regulator of Th cell function and promotes the generation of CD4+CD103+ regulatory T cells in vivo

The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.     

Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus

Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.     

Immune Correlates of Resistance to Trichinella spiralis Reinfection in Mice

The immune correlate of host resistance induced by reinfection of Trichinella spiralis remains unclear. In this study, we investigated immune correlates between the resistance and serum IgG antibody level, CD23⁺ IgM⁺ B cells, and eosinophil responses induced by T. spiralis reinfection. Mice were primarily infected with 10 or 100 T. spiralis larvae (10 TS, 100 TS), respectively, and after 4 weeks, they were challenge infected with 100 T. spiralis larvae (10–100 TS, 100-100 TS). Upon challenge infections, 10–100 TS mice induced significantly higher levels of T. spiralis-specific total IgG antibody responses in sera and antibody secreting cell responses in spleens compared to 100-100 TS mice, resulting in significantly reduced worm burdens in 10–100 TS mice (60% and 70% reductions for adult and larvae, respectively). Higher levels of eosinophils were found in mice primarily infected with 10 TS compared to those of 100 TS at week 8 upon challenge. CD23⁺ IgM⁺ B cells were found to be increased significantly in mice primarily infected with 10 TS. These results indicate that primary infection of 10 larvae of T. spiralis, rather than 100 larvae, induces significant resistance against reinfection which closely correlated with T. spiralis-specific IgG, eosinophil, and CD23⁺ IgM⁺ B cell responses.     

Immune interactions with CD4+ T cells promote the development of functional osteoclasts from murine CD11c+ dendritic cells

Dendritic cells (DC) are innate immune effectors and are critically involved in regulating T cell immunity. Osteoclasts (OC) are bone-resorbing cells derived from the monocyte/macrophage lineage in response to receptor activator of NF-kappaB ligand (RANKL). DC and T cells form aggregates in the inflammatory infiltrates at active disease sites in human and in experimental rheumatoid arthritis and periodontitis. We investigated whether DC interactions with T cells in the bone environment can support the development of functional OC. In the present study, we demonstrate that upon proper activation by microbial or protein Ags (namely Actinobacillus actinomycetemcomitans, bovine insulin, and outer membrane protein-1) and during immune interactions with CD4+ T cells in vitro, murine BM-derived and splenic CD11c+ DC (CD11b- F4/80- Ly-6C- CD31-) develop into TRAP+ CT-R+ cathepsin-k+ functional OC in a RANKL/RANK-dependent manner. Rescue and blocking experiments using CD11c+ DC derived from Csf-1(-/-) op/op mice show that M-CSF is required "before" developing such osteoclastogenic potential upstream of RANKL/RANK signaling, suggesting that immature CD11c+ DC can indeed act like OC precursors. In addition, these CD11c+ DC-derived OC are capable of inducing bone loss after adoptive transfer in vivo. These data suggest a direct contribution of DC during immune interactions with CD4+ T cells to inflammation-induced osteoclastogenesis. Therefore, our findings not only provide further evidence for DC plasticity, but also extend the current paradigm of osteoimmunology.     

Glycosylation, galectins and cellular signaling

Glycosylation is a common posttranslational modification of proteins and lipids of the secretory pathway that generates binding sites for galactose-specific lectins or galectins. Branching of Asn-linked (N-)glycans by the N-acetylglucosaminyltransferases (Mgat genes) increases affinity for galectins. Both tissue-specific expression of the enzymes and the metabolic supply of sugar-nucleotides to the ER and Golgi regulate glycan distribution while protein sequences specify NXS/T site multiplicity, providing metabolic and genetic contributions to galectin-glycoprotein interactions. Galectins cross-link glycoproteins forming dynamic microdomains or lattices that regulate various mediators of cell adhesion, migration, proliferation, survival and differentiation. There are a similar number of galactose-specific galectins in C. elegans and humans, but expression of higher-affinity branched N-glycans are a more recent feature of vertebrate evolution. Galectins might be considered a reading code for repetition of the minimal units of binding [Gal(NAc)β1-3/4GlcNAc] and NXS/T site multiplicity in proteins. The rapidly evolving and structurally complex Golgi modifications to surface receptors are interpreted through affinity for the lattice, which regulates receptor levels as a function of the cellular environment, and thereby the probability of various cell fates. Many important questions remain concerning the regulation of the galectins, the glycan ligands and lattice interaction with other membrane domains and endocytic pathways.