Saliva of many vector arthropods contains factors that inhibit haemostatic responses in their vertebrate hosts. Less is known about the effect of vector saliva on host immune responses. We investigated the effect of Aedes aegypti salivary gland extracts on antigen-stimulated responses of transgenic OVA-TCR DO11 mouse splenocytes in vitro. T-cell proliferation was inhibited in a dose-dependent manner, with greater than 50% inhibition at 0.3 salivary gland pair (SGP) equivalents/mL. LPS-stimulated B-cell proliferation was also inhibited. Secretion of the Th1 cytokines IL-2 and IFN-gamma was reduced by 50% or more with 0.45-0.6 SGP/mL, as was secretion of the pro-inflammatory cytokines GM-CSF and TNF-alpha, and the Th2 cytokine IL-5. The Th2 cytokines IL-4 and IL-10 were similarly reduced with 0.6-2 SGP/mL. Inhibition of lymphocyte function involved modulation of viable T-cells at low salivary gland extract (SGE) concentrations, and decreased viability at higher concentrations. Dendritic cells were not killed by salivary gland extracts at concentrations as high as 25 salivary gland pairs/mL, but secretion of IL-12 was inhibited by 87% following exposure to 0.6 SGP/mL. Activity is present in saliva and extracts of female but not male salivary glands, and it is depleted from salivary glands of blood-fed mosquitoes. The activity is denatured by boiling and by digestion with the protease papain, indicating a protein; gel filtration HPLC indicates a mass of about 387 kDa. These results suggest that A. aegypti saliva exerts a marked immunomodulatory influence on the environment at the bite site.

T cells recognizing self-peptides that mediate autoimmune disease and those that are responsible for efficacious immunity against pathogens may differ in affinity for antigen due to central and peripheral tolerance mechanisms. Here we utilize prototypical self-reactive (myelin) and viral-specific (LCMV) T cells from T cell receptor (TCR) transgenic mice (2D2 and SMARTA, respectively) to explore affinity differences. The T cells responsive to virus possessed >10,000 fold higher 2D affinity as compared to the self-reactive T cells. Despite their dramatically lower affinity for their cognate ligand, 2D2 T cells respond with complete, albeit delayed, activation (proliferation and cytokine production). SMARTA activation occurs rapidly, achieving peak phosphorylation of p38 (1 minute), Erk (30 minutes), and Jun (3 hours) as well as CD69 and CD25 upregulation (3 and 6 hours, respectively), with a corresponding early initiation of proliferation. 2D2 stimulation with MOG results in altered signaling – no phospho-Erk or phospho-p38 accumulation, significantly delayed activation kinetics of Jun (12 hours), and delayed but sustained SHP-1 activity – as well as delayed CD69 and CD25 expression (12–24 hours), and slow initiation of proliferation. This delay was not intrinsic to the 2D2 T cells, as a more potent antigen with >100-fold increased 2D affinity restored rapid response kinetics in line with those identified for the viral antigen. Taken together, these data demonstrate that time can offset low TCR affinity to attain full activation and suggest a mechanism by which low affinity T cells participate in autoimmune disease.

Our lab has demonstrated that encephalitogenic T cells can be effectively anergized by treatment with MHC variant peptides, which are analogues of immunogenic peptides containing an amino acid substitution at an MHC anchor residue. The MHC variant peptide of myelin oligodendrocyte glycoprotein (MOG)35–55 proves an effective treatment as it does not induce symptoms of experimental autoimmune encephalomyelitis and fails to recruit macrophages or MOG35–55-specific T cells to the CNS. In this study, we sought to characterize the signaling pathways required for the induction of anergy by building upon the observations identifying the tyrosine phosphatase SHP-1 as a critical regulator of T cell responsiveness. Motheaten viable heterozygous mice, which contain a mutation in the SHP-1 gene resulting in a reduction in functional SHP-1, were challenged with MOG35–55 or the MOG35–55 MHC variant 45D. These mice display symptoms of experimental autoimmune encephalomyelitis upon immunization with MHC variant peptide and have significant CNS infiltration of tetramer-positive CD4+ cells and macrophages, unlike B6 mice challenged with the variant peptide. The effects of SHP-1 are directly on the T cell as Motheaten viable heterozygous mice autoreactive T cells are not anergized in vitro. Lastly, we demonstrate no distinguishable difference in the initial interaction between the TCR and agonist or MHC variant. Rather, an unstable interaction between peptide and MHC attenuates the T cell response, seen in a decreased half-life relative to MOG35–55. These results identify SHP-1 as a mediator of T cell anergy induced by destabilized peptide:MHC complexes.

A 171,000 M(r )polypeptide of Podisus maculiventris (Say) (Heteroptera: Pentatomidae) that constituted 16% of the protein in eggs also constituted up to 25% of the protein in hemolymph of fed females. It was identified as the major or sole apoprotein of vitellogenin. Eggs contained major polypeptides of 171, 106, and 51 kDa. The hemolymph polypeptide was identified with a polypeptide (vitellin) in egg extracts by comparing molecular weights, specificity of occurrence in fed females, and immunological reactivities. Females, starved for 5 days after eclosion to assure complete previtellogenic development, produced vitellogenin within a day after feeding on larval Galleria mellonella, and within 4 days after feeding on an artificial diet. Appearance of vitellogenin preceded ovarian growth by 2-3 days. Two monoclonal antibodies raised against egg proteins of P. maculiventris were selected for their strong reaction against egg extract and female hemolymph and null reaction against male hemolymph. Only one 170-kDa band in egg and hemolymph reacted with the antibodies on denaturing Western blots. These monoclonal antibodies are being used to develop an enzyme-linked immunosorbent assay (ELISA) to quantitate reproductive response of females to diets of differing quality.

Previous studies have found that a 95% reduction in TCR expression does not adversely affect response to foreign Ags, indicating that T cells have an excess of TCR for Ag recognition. Because self-reactive T cells may have low affinity for peptide:MHC, we investigated whether myelin-reactive T cells require these excess TCR for optimal response. To test this concept, mAb were used to effectively reduce the TCR of Vα3.2 and Vβ11 TCR transgenic mice (referred to as 2D2). After masking the TCR with either continuous or prepulsed anti-Vα3.2 Ab, 2D2 cells were immediately stimulated with myelin oligodendrocyte glycoprotein (MOG)35–55. These cells have a dramatic Ab dose-dependent reduction in proliferation, with a small reduction in TCR expression leading to a 50% reduction in proliferation in vitro. Additionally, 2D2 cells, treated with anti-Vα3.2 Ab and peptide for 7 days, were re-stimulated with MOG and continue to have a dose-dependent reduction in proliferation. TCR quantitation identified the same amount of TCR on the Ab/peptide treatment compared with the peptide-only control. These results point out that the combination of reduced TCR and peptide challenge leads to a phenotypic change resulting in T cell anergy. Importantly, adoptive transfer of these anergic T cells upon autoimmune disease induction had a marked reduction in disease severity compared with untreated MOG-specific CD4+ T cells, which had significant autoimmune disease manifested by optic neuritis and death. Thus, reduction of TCR expression may provide a potential therapy for self-reactive T cells involved in autoimmune diseases through the induction of anergy.