Membrane associations of p47 GTPases
Members of the p47 guanosine triphosphatase (GTPase) family are induced by IFN-γ and are responsible for immune control of intracellular pathogens in mice. The p47 GTPases are associated with structures such as the endoplasmic reticulum (ER), the Golgi membrane, and phagosomes containing bacteria, but specifics regarding these associations are unknown. Martens et al. (p. 2594 ) used immunofluorescence and cell fractionation to show that LRG-47, a p47 GTPase in mouse cells, was found in the cis-and medial-Golgi and ER following induction with IFN-γ; no LRG-47 was seen in untreated cells. Two other p47 GTPases, IGTP and IIGP1, localized only to the ER. LRG-47 quickly accumulated at the cell surface of IFN-γ-treated macrophages and fibroblasts where latex beads were being phagocytosed. Transfection of GTPase mutants into mouse cells showed that only the plasma membrane association of LRG-47 was nucleotide dependent and that the C-terminal domain of IIGP1 was essential for intracellular aggregate formation but not for membrane binding. Other mutants, analyzed by partitioning of transfected cell extracts, showed that the myristoylated N terminus of IIGP1 was sufficient for membrane association but that other membrane interactions were involved. Constructs containing G-domains or C-terminal domains, expressed with FLAG epitope tags, showed that the G-domains of both IIGP1 and LRG-47 were targeted to the plasma membrane. The C terminus of LRG-47, but not that of IIGP1, targeted the Golgi membranes; the sequence responsible was an amphipathic helix of 25 aa. The results show that p47 GTPases have a variety of mechanisms for associating with membranes of intracellular structures.
Peptide ligand promotes LFA-1-dependent adhesion of T cells to ICAM-1
Adhesion of LFA-1 to ICAM-1 on the surface of an APC results in activation of a T cell. Whereas both anti-CD3 Abs and PMA can promote the interaction, the effect of specific peptide-MHC (pMHC) tetramers has not been characterized. Mueller et al. (p. 2222 ) incubated purified T cells from OT-1 H2-Kb-restricted TCR transgenic mice with H2-Kb tetramers presenting the OVA peptide fragment (KbOVA). Adhesion of incubated cells to ICAM-1 was equivalent to that seen for OT-1 T cells stimulated with anti-CD3 or PMA and was blocked by Abs against LFA-1; pMHC containing altered OVA peptides or control peptides did not enhance adhesion. Inhibitors of src family tyrosine kinases and PI3K blocked T cell adhesion induced by KbOVA tetramers and by anti-CD3 mAb, but did not block adhesion induced by PMA. Small amounts (as low as 0.1 μg/ml) of KbOVA tetramers stimulated phosphorylation of both LAT-1 (linker for activation of T cells) and LFA-1-dependent T cell adhesion. The stimulated OT-1 T cells had increased blastogenesis and CD69 expression and proliferation. The data demonstrate that a high-affinity peptide ligand at low concentrations is sufficient to induce rapid LFA-1-dependent adhesion of T cells to ICAM-1, leading to immunological synapse formation, signal transduction, and full T cell activation.
STAT5 is required for fetal thymopoesis
Signaling by the IL-7R is required for proliferation, survival, and differentiation of thymic T cell precursors. Although the STAT5 kinase has been implicated in the IL-7R response, the mechanism of its action has not been determined. Kang et al. (p. 2307 ) found that thymic lymphopoiesis was approximately normal in 4- to 10-wk-old Stat5−/− mice (deficient in both Stat5a and Stat5b). In contrast, Stat5−/− fetal mice had a 5- to 10-fold reduction in the number of thymocytes due to increased cell death, and an enrichment in the earliest thymocyte precursor subsets compared with wild-type or Stat5 heterozygous controls. The greatest reduction was in the fetal γδ subset that gives rise to dendritic epidermal T cells, although there also was a reduction in fetal αβT cells. Stat5−/− mice at embryonic days 15–18 had nearly wild-type levels of Vγ3 gene rearrangements, but transcripts for the rearrangements were greatly diminished. Fetal Stat5−/− mice had reduced lymphoid tissue-inducing cells in their intestines, and adult Stat5−/− mice lacked Peyer’s patches. Total numbers of intraepithelial lymphocytes were reduced 2- to 3-fold in fetal Stat5−/− mice with the greatest reduction (10-fold) in the γδ subset compared with Stat heterozygotes. Young Stat5−/− mice also had reductions in splenic γδT cells. The findings support a critical role for STAT5 kinase in IL-7R-mediated peripheral γδT cell development in fetal, but not in adult, mice.
Diabetes induction by a promiscuous CD8+ T cell clone
CD8+ T cells cause destruction of pancreatic β cells in the early stages of type 1 diabetes (T1D) in the NOD mouse. Transgenic expression of the TCR from AI4, a CD8+ T cell clone isolated from the islets of a 5-wk-old female NOD mouse, accelerates NOD diabetes even in the absence of CD4+ T cells. Takaki et al. (p. 2530 ) found that AI4 cells lysed target cells in vitro that were pulsed with peptides eluted from H-2Db, but not from H-2Kd, molecules immunoaffinity-purified from a NOD-derived β cell line. However, transfer of splenocytes from a prediabetic NOD mouse expressing the AI4 TCR transgene induced diabetes only in congenic strains homozygously expressing both H-2Db and H-2Kd; mice heterozygous for either H-2 haplotype or homozygous for one did not develop the disease. The requirement for dual H-2 homozygosity was found in transfer experiments using bone marrow from prediabetic NOD/AI4 TCR donors and in in vitro killing of islet cells by AI4 T cells. AI4 lysed target cells pulsed with almost all mixtures of 9-aa target Ags bound to either H-2Db or H-2Kd, whereas another prediabetic CTL clone recognized one specific mimotope peptide bound to H-2Kd and no peptide bound to H-2Db. One H-2Db-binding nonapeptide elicited the best cytotoxic response from AI4 CTL. MHC tetramers containing that peptide stained splenocytes from A14 TCR transgenic NOD mice as well as in vitro IL-2 expanded CD8+ T cells from islets of nondiabetic NOD mice. The data show that AI4-like T cells represent a population of islet-infiltrating T cells in NOD mice that requires coexpression of both H-2Db and H-2Kd class I MHC molecules for efficient destruction of β cells.
Vaccinating against mouse mammary tumors
All BALB/c mice transgenic for the HER-2/neu oncogene (NeuT mice) develop mammary carcinomas. Lollini and colleagues previously demonstrated survival of 90–100% of NeuT mice treated with a combination of IL-12 and MHC allogeneic tumor cells expressing p185neu. In the paper on p. 2288 from the same laboratory (Nanni et al.), they report that no vaccinated NeuT mice crossed with IFN-γ−/− BALB/c mice (NeuT-IFN-γ−/−) survived. In comparison, vaccinated NeuT mice crossed with mice deficient in B cell immunity (NeuT-μMT) had increased survival times, compared with unvaccinated NeuT controls, but mortality eventually reached 100%. Tumors that developed in vaccinated NeuT-IFN-γ−/− and NeuT-μMT mice were histologically similar to those of untreated NeuT mice. Two-thirds of NeuT-μMT mice produced high-titer Abs against the vaccine. Those responder mice had prolonged survival and few hyperplastic nodules in their mammary glands at 16 wk of age; their in vitro stimulated splenocytes released IFN-γ at levels comparable to those of stimulated splenocytes from NeuT mice. Vaccinated NeuT-IFN-γ−/− and NeuT-μMT mice produced IgG1, but only the NeuT-μMT animals produced IgG2a and IgG2b. The IgG2a:IgG2b ratio was reversed from that seen in vaccinated NeuT mice. The data demonstrate the requirement for IFN-γ and a robust anti-p185neu Ab response in preventing tumor development in NeuT mice vaccinated with IL-12 and allogeneic tumor cells.
Inducing alloantigen-specific T cell tolerance
One approach to preventing transplant rejection is to induce T cell tolerance by engaging CTLA-4 at the same time interactions between TCR and MHC alloantigens occur. Vasu et al. (p. 2866 ) used a bispecific Ab (BiAb) that bound both a thyroid Ag expressed by M12 (H2d) cells and the CTLA-4 expressed by attacking host T cells. Spleen cells from H-2k mice, injected with BiAb-coated M12 cells, had reduced ex vivo T cell proliferation, diminished IL-2 and IFN-γ responses, and increased production of IL-4, IL-10, and TGF-β1 when exposed to M12 cells compared with spleen cells from control mice. Ex vivo stimulation of the H-2k cells with M12 cells did not break the tolerance. Spleen and lymph node cells from BiAb tolerant mice, challenged ex vivo with M12 cells, had increased numbers of Treg (CD4+CD25+T) cells with increased expression of CTLA-4 and CD62L and decreased expression of CD69 compared with controls. Hyporesponsiveness of the T cells could be reversed in vitro by a high concentration of IL-2 with M12 cells, by anti-TGF-β1 Ab with or without anti-IL-10 Ab, or by anti-CTLA-4 Ab; reversal required continuous exposure to the reagents. The proliferative response of immune CD4+CD25− T cells to M12 cells was suppressed by CD4+CD25+ T cells from tolerant mice; direct cell contact was required for maximum suppression. M12 tolerant CD4+CD25+ T cells adoptively transferred the suppression in vivo. The experiments demonstrate effective generation of Treg cells by engagement of both CTLA-4 and an alloantigen.
Regulating peripheral B cell development
Only one of the three receptors to which B cell-activating factor (BAFF) binds is essential for the generation and maintenance of the mature B cell pool. Yet the precise role of that receptor, BAFF-R, in B cell development has not been defined. Sasaki et al. (p. 2245 ) used gene targeting to develop Baff-r−/− mice that had a dramatic reduction in recirculating mature B cells in bone marrow, spleen, lymph nodes, and peritoneal cavity compared with wild-type and heterozygous controls. Analyses of surface markers on splenocytes by flow cytometry and immunostaining indicated that the mutant mice had a normal number of transitional 1 (T1) B cells. However, the sizes of the T2 and T3 populations (CD23+) were reduced compared with those of control mice, and marginal zone (MZ) cells (CD21+) were absent. Serum levels of all Ig classes except IgA were decreased in Baff-r−/− mice. The IgM and IgG3 responses of the Baff-r−/− mice to a T1 Ag were lower than controls; the IgG1 response to a TD Ag was significantly reduced, but the IgM response was nearly the same as that of control animals. B cells continuously formed germinal centers in mesenteric lymph nodes in the mutant mice. Overexpression of the anti-apoptotic protein Bcl-2 through genetic manipulation increased the sizes of mature B cell compartments to equal those in controls. The authors conclude that BAFF-R is essential for the survival of peripheral B2 cells from the T2 stage although the humoral response is partially independent of BAFF-R.
Summaries written by Dorothy L. Buchhagen, Ph.D.