Immunity to Trichinella spiralis infection in vitamin A-deficient mice.

Vitamin A-deficient (A-) mice make strikingly poor IgG responses when they are immunized with purified protein antigens. Previously, we showed that A- T cells overproduce interferon gamma (IFN-gamma), which then could inhibit interleukin 4 (IL-4)-stimulated B cell IgG responses. To determine if the altered IFN-gamma regulation pattern and its immunological consequences would extend to a natural infection, we studied mice infected with the parasitic helminth Trichinella spiralis. The course of the infection was similar in A- and A-sufficient (A+) mice. These mice did not differ with respect to newborn larvae/female/hour produced in the intestine, or muscle larvae burden 5 wk postinfection. They also did not differ in the intestinal worm expulsion rate until day 15, when A- mice still harbored parasites, whereas A+ mice had cleared intestinal worms. Vitamin A deficiency reduced both the frequency of B lymphocytes secreting IgG1 antibodies to parasite antigens, and the bone marrow eosinophilia associated with helminth infection. The cytokine secretion patterns in infected mice were consistent with these observations and with previous studies. Mesenteric lymph node cells from infected A- mice secreted significantly more IFN-gamma, and significantly less IL-2, IL-4, and IL-5 than infected A+ controls. A- splenocytes secreted significantly more IFN-gamma, and equivalent amounts of IL-2, IL-4, and IL-5 compared with A+ controls. Interestingly, CD4-CD8- cells secreted the majority of the IL-4 produced in the spleen. The IL-2, IL-4, and IL-5 steady-state transcript levels correlated with secreted protein levels, but IFN-gamma transcripts did not. Although they secreted more protein, A- cells contained fewer IFN-gamma transcripts than A+ cells. These results suggest two vitamin A-mediated regulation steps in IFN-gamma gene expression: positive regulation of IFN-gamma transcript levels, and negative regulation posttranscriptionally. The essentially unaltered outcome of T. spiralis infection in vitamin A-deficient mice probably reflects a balance between cellular and humoral responses. The IFN-gamma overproduction might have a positive effect on the gut inflammatory response, but the decrease eosinophilia, cytokine production in mesenteric lymph node, and IgG1-secreting cell frequency might have a negative effect on T. spiralis immunity.

Characterization of transport of newly assembled, T cell-stimulatory MHC class II-peptide complexes from MHC class II compartments to the cell surface.

In human B cells MHC class II molecules acquire antigenic peptides in lysosome-related compartments called the MHC class II compartments (MIIC). How assembled complexes, capable of activating T cells, then reach the cell surface has not been fully resolved. We have used selective ablation of early and recycling endosomes to determine whether newly peptide-loaded class II molecules require functional recycling endosomes to exit to the cell surface. Cellular compartments accessed by transferrin-horseradish peroxidase conjugates were functionally inactivated by cross-linking with diaminobenzidine and hydrogen peroxide. Cells with ablated endosomal compartments were unable to recycle transferrin to the cell surface and could not deliver exogenous Ag for processing and presentation to T cells. In contrast, cells that had taken up Ag and assembled intracellular class II-peptide complexes before endosome ablation were still able to deliver class II-peptide complexes to the cell surface and stimulate T cell proliferation. This delivery was abolished in the presence of brefeldin A. These data show that the parts of the endocytic apparatus necessary for Ag delivery to the MIIC are not required for functional class II-peptide complexes to reach the cell surface. Moreover, the brefeldin A sensitivity of this final step in the class II molecule biosynthetic pathway suggests a vesicular intermediate for transport between the MIIC and the plasma membrane.

Functional early endosomes are required for maturation of major histocompatibility complex class II molecules in human B lymphoblastoid cells.

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place.

Evidence for differential induction of helper T cell subsets during Trichinella spiralis infection.

The H-2-compatible mouse strains, AKR and B10.BR, exhibit disparate responses to infection with the parasitic nematode Trichinella spiralis. The resistant AKR mice expel intestinal adult worms faster than susceptible B10.BR mice. We tested antibody and lymphokine responses in these strains. With respect to antibody responses, the B10.BR mice had 3- to 10-fold more serum IgE and T. spiralis-specific IgG1 and IgA than AKR mice. The B10.BR mice also had greater numbers of IgG and IgA plaque-forming cells than AKR mice. In contrast, AKR mice produced T. spiralis-specific IgG2a, whereas the B10.BR mice did not. The antibody response kinetics of these strains were similar. We also analyzed lymphokine secretion after restimulating lymphocytes in vitro with T. spiralis Ag. The AKR mesenteric lymph node cells produced more IFN-gamma and less IL-4 than the B10.BR mesenteric lymph node cells. The B10.BR splenocytes produced more IL-4 than the AKR splenocytes, although splenocyte IFN-gamma production was not different. The kinetics of IL-4 production also differed between the two strains. In summary, resistant AKR mice produced more IFN-gamma and T. spiralis-specific IgG2a than susceptible B10.BR mice, which produced more IL-4, IgE, and T. spiralis-specific IgG1. Our results are consistent with differential activation of Th cell subsets in T. spiralis-infected AKR and B10.BR mice.

Influence of resistant and susceptible genotype, IL-1, and lymphoid organ on Trichinella spiralis-induced cytokine secretion.

The relative importance of cell-mediated inflammatory responses and antibody-mediated responses in controlling parasitic helminth infection is debated. To study the relationship between these responses and resistance or susceptibility to primary Trichinella spiralis infection, we infected resistant AKR mice and susceptible B10.BR mice and analyzed the lymphokines IL-2, IFN-gamma, and IL-5 produced by their T cells as a function of time and lymphoid organ. IL-2-secretors occurred maximally between days 3 and 6 postinfection, whereas IL-5-secretors peaked between days 6 and 9. Previously, we found that IFN-gamma producers peaked before day 6, whereas IL-4 producers peaked between days 6 and 9. Most cytokine secretors were CD4+. The simultaneous development of IL-2- and IFN-gamma-secreting cells, and IL-4- and IL-5-secreting cells, suggests that the infection may be stimulating T cells to differentiate into cells capable of secreting specific cytokine sets, analogous to the postulated Th1 and Th2 subsets. In the spleen and mesenteric lymph nodes, cells from B10.BR mice secreted more IL-5 than cells from AKR mice, as we found previously for IL-4. For both strains, mesenteric lymph node cells produced more IL-5 than splenocytes. The AKR mesenteric lymph node cells produced more IL-2 than the B10.BR cells, but the reverse occurred in splenocytes. The AKR peripheral lymph node cells also secreted more IFN-gamma than the B10.BR cells, but the strains were equivalent for peritoneal exudate cell IFN-gamma production. Thus, the lymphoid organ microenvironment plays an important role in regulating cytokine-secreting cell development in this system. We also tested the possible regulatory role of IL-1. Exogenous rIL-1 alpha increased IFN-gamma secretion early but not late in mesenteric lymph node cells from both strains; this reflected an increased IFN-gamma-secreting cell frequency, not a change in IFN-gamma mRNA transcript level. Exogenous rIL-1 alpha did not consistently affect IL-2, IL-4, or IL-5 secretion. These data suggest that IL-1 alpha availability in vivo may regulate IFN-gamma-secreting cell development. In sum, early activation of IFN-gamma-secreting T cells in lymph nodes, with little subsequent activation of IL-4- and IL-5-secreting cells, distinguished the resistant from susceptible strain responses to T. spiralis infection, and IL-1 alpha and lymphoid organ environment influence IFN-gamma-secreting cell activation.

Clathrin-coated lattices and buds on MHC class II compartments do not selectively recruit mature MHC-II.

Newly synthesized major histocompatibility complex class II molecules (MHC-II) are transported to MHC-II-containing endosomal and lysosomal compartments (MIICs) for the degradation of associated invariant chain and peptide loading. Subsequently MHC-II is transported to the plasma membrane, in part through direct fusion of MIICs with the plasma membrane. In search of potential alternative pathway(s) we studied the 3-dimensional structure of MIICs and the subcellular distribution of MHC-II by immuno electronmicroscopy on whole-mount preparations and cryosections of Mel JuSo cells. Intracellular MHC-II and invariant chain mainly localized to lamp-1 positive compartments suggesting that the majority of MHC-II exits the endocytic tract at lysosomes. Clathrin-coated lattices and buds were found to be associated with these organelles, but MHC-II was not found to be enriched in the clathrin-coated domains. Moreover, leupeptin, a drug that interferes with Ii-processing and delays delivery of newly synthesized MHC-II to the plasma membrane, was not found to decrease the relative amount of MHC-II in clathrin-coated areas. Together these data indicate clathrin-mediated exit site(s) from lysosomes but suggest that they do not selectively recruit mature MHC-II, consistent with the notion that transport to the plasma membrane occurs independently of the cytoplasmic domains of the MHC-II (&agr;) and (beta) chains.

A role for acidic residues in di-leucine motif-based targeting to the endocytic pathway.

Recent reports have suggested that major histocompatibility complex class II molecules load peptide through a specialized compartment of the endocytic pathway and are targeted to this pathway by association with invariant chain (Iip31). Therefore we used a site-directed mutagenesis approach to determine whether Iip31 possesses novel protein targeting signals. Our results indicate that two di-leucine-like pairs mediate Iip31 targeting and that an acidic amino acid residue four or five residues N-terminal to each Iip31 di-leucine-like pair is required for endocytic targeting. Results from additional testing with hybrid Iip31 molecules indicate that the acidic residues N-terminal to di-leucine pairs are critical for accumulation of these molecules in large endocytic vesicles and in some cases provide a structure favorable for internalization. The acidic residues N-terminal to di-leucine pairs are important in some sequence contexts in providing a structure favorable for internalization, whereas in other contexts an acidic residue is critical for targeting to, and formation of, large endocytic vesicles. Although our results do not support the idea that Iip31 possesses unique protein targeting motifs, they do suggest that di-leucine motifs may be recognized as part of a larger secondary structure. In addition, our data imply that the targeting motif requirements for internalization may differ from the requirements for further transport in the endocytic pathway.

Nonclassical behavior of the mouse CD1 class I-like molecule.

The mouse CD1 (mCD1) molecule is a class I-like molecule that is encoded outside of the MHC. We show here that mCD1 shares several properties with Ag-presenting class I molecules, including a requirement for beta2-microglobulin for stable cell-surface expression in T lymphocyte transfectants and thymocytes. mCD1 is also capable of binding to mouse CD8alphabeta heterodimers participating in the activation of CD8+ T cells in a manner similar to classical class I molecules. However, mCD1 surface expression is not decreased at high temperatures in cells that lack the transporter associated with Ag processing (TAP), including both RMA-S and Drosophila melanogaster cells. The data indicate that mCD1 does not require TAP to be expressed in a stable fashion at the cell surface. We speculate that the ability of mCD1 to reach the cell surface in transporter-deficient cells may reflect its ability to present a distinct set of ligands. The properties of mCD1 described here can account, in part, for the selection of the diverse populations of T cells that are known to be mCD1 reactive.