T cell activation is driven by the TCR and complemented by costimulation. We have studied the dynamics of ligand-engagement of the costimulatory receptor CD2 in T cell/APC couples. Thousands of ligand-engaged CD2 molecules were included in a large T cell invagination at the center of the cellular interface within 1 min of cell couple formation. The structure and regulation of this invagination shared numerous features with phagocytosis and macropinocytosis. Three observations further characterize the invagination and the inclusion of CD2: 1) numerous ligand-engaged receptors were enriched in and internalized through the T cell invagination, none as prominently as CD2; 2) dissolution of the T cell invagination and CD2 engagement were required for effective proximal T cell signaling; and 3) the T cell invagination was uniquely sensitive to the affinity of the TCR for peptide-MHC. Based on this characterization, we speculate that the T cell invagination, aided by CD2 enrichment, internalizes parts of the TCR signaling machinery to reset T cell signaling upon agonist-mediated, stable APC contact.

Virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) can be used as a scaffold to facilitate the delivery of antigens to induce cell-mediated immune responses. In this study, we investigated the immune response to lymphocytic choriomeningitis virus-derived peptide antigen (gp33) delivered by RHDV VLP. The gp33 peptides were incorporated into the VLP in 2 different forms, either recombinantly expressed inside the VLP (VLP-gp33r) or chemically coupled to the surface of the VLP (VLP-gp33c). We showed that VLP-gp33r induced a greater level of cytotoxicity than VLP-gp33c against gp33-coated target cells in vivo. Both VLP, when delivered as prophylactic vaccines, inhibited the growth of Lewis' lung carcinoma tumors expressing gp33 (LL-LCMV) in mice to a similar degree. Studies to investigate the mechanism induced by these VLP showed that 2 CD11c DC subsets, CD8α and CD8α, acquired VLP in vivo and in vitro, and VLP-gp33r were cross-presented by both these subsets to prime CD8 T cells through a TAP-independent, endosomal recycling pathway. Depletion of Langerin DC in vivo before and after vaccination with VLP-gp33r, lead to reduced cytotoxicity implicating these cells in the induction of cytotoxic effector cells. These results suggest that recombinant VLP expressing tumor peptides targeted to Langerin DC may have clinical application. Finally we found that VLP-gp33r were more effective antitumor vaccines than VLP-gp33c when delivered therapeutically. The findings of this study suggest the potential of VLP as a platform for delivery of tumor-associate antigen and elicit protective immunity against tumors.

Upon interaction with resting B lymphocytes, IL-2-propagated NK cells can initiate the process of Ig constant region switch recombination (CSR) by inducing germ line transcripts for gamma2a (Igamma2a) as well as increased levels of mRNA for activation-induced cytidine deaminase enzyme. Whereas both these processes are necessary for CSR, they are not sufficient because the cells do not proceed to the expression of mature mRNA for gamma2a (VDJCgamma2a). In addition, NK cells can also upregulate mRNA for the T-box transcription factor (T-bet) in B cells without being able to induce further differentiation. Using transgenic B cells with B cell receptor specificity for nitrophenol (NP), we have now shown that NP-Ficoll-stimulated B cells can be induced by NK cells to express IgG2a as well as IgG1 presumably due to the completion of the process of switch recombination. The inductive ability of NK cells does not require IFN-gamma but does require signals transmitted via CD48 by direct cell contact. In addition, NP-Ficoll on its own can induce proliferation of antigen-specific B cells as well as germ line transcripts of gamma1; however, expression of VDJCgamma1 mRNA also requires NK cell interaction with B lymphocytes. Therefore, in the presence of antigen, NK cells can provide a necessary signal that substitutes for cytokines in the induction of IgG2a as well as IgG1 expression. This in vitro analysis provides a mechanistic basis for understanding the documented NK cell effects on T-independent B cell responses in vivo.

Ag presentation to CD4 T cells can be mediated by a number of cell types depending on the anatomical site in which Ag is first encountered. For blood borne Ags, cells localized in situ in the spleen should be major players. There is now much evidence that B cell Ag presentation may be particularly important in the priming of memory T cells. The majority of NK cells are also localized the spleen. Inasmuch as we have previously shown that NK cells can modulate various aspects of B cell differentiation, we entertained the possibility that NK cells can also influence Ag presentation by B cells. By specific depletion of NK cells before immunization, we show herein that NK cells play an important role in modulating the ability of B cells to process and present Ag to T cells. These effects are particularly important in the generation of memory T cells. The findings are further substantiated by in vitro experiments showing that the enhancement does not require IFN-γ but is mediated by direct cell-cell interaction. These results show, for the first time, that the rapid activation of a component of the innate response can even exert effects on the Ag-specific memory response.

B6.Sle1b mice, which contain the Sle1b gene interval derived from lupus prone NZM2410 mice on a C57BL/6 background, present with gender-biased, highly penetrant anti-nuclear antibody (ANA) production. To obtain some insight into the possible induction mechanism of autoantibodies in these mice we compared antigen specific T dependent (TD) and T independent (TI-II) responses between B6.Sle1b and B6 mice before the development of high ANA titers. Our results show that B6.Sle1b mice mount enhanced responses to a TI-II antigen. Additionally, the memory T cell response generated by a TD antigen was also increased. This enhancement correlates with the greater ability of B cells from B6.Sle1b mice to present antigen to T cells. The SLAM Associated Protein (SAP) is critical for signaling of many of the molecules encoded by the SLAM/CD2 gene cluster, candidates for mediating the Sle1b phenotype; therefore, we also investigated the effect of sap deletion in these strains on the TD and TI-II responses as well as on ANA production. The results of these studies of responses to non-self antigens provide further insight for the mechanism by which responses to selfantigens might be initiated in the context of specific genetic alterations.