Thomas M. Yankee

Visualization of Syk-Antigen Receptor Interactions Using Green Fluorescent Protein: Differential Roles for Syk and Lyn in the Regulation of Receptor Capping and Internalization

The cross-linking of the B cell Ag receptor (BCR) is coupled to the stimulation of multiple intracellular signal transduction cascades via receptor-associated, protein tyrosine kinases of both the Src and Syk families. To monitor changes in the subcellular distribution of Syk in B cells responding to BCR cross-linking, we expressed in Syk-deficient DT40 B cells a fusion protein consisting of Syk coupled to green fluorescent protein. Treatment of these cells with anti-IgM Abs leads to the recruitment of the kinase from cytoplasmic and nuclear compartments to the site of the cross-linked receptor at the plasma membrane. The Syk-receptor complexes aggregate into membrane patches that redistribute to form a cap at one pole of the cell. Syk is not demonstrably associated with the internalized receptor. Catalytically active Syk promotes and stabilizes the formation of tightly capped BCR complexes at the plasma membrane. Lyn is not required for the recruitment of Syk to the cross-linked receptor, but is required for the internalization of the clustered BCR complexes. In the absence of Lyn, receptor-Syk complexes at the plasma membrane are long lived, and the receptor-mediated activation of the NF-AT transcription factor is enhanced. Thus, Lyn appears to function to negatively regulate aspects of BCR-dependent signaling by stimulating receptor internalization and down-regulation.

The small 11kDa nonstructural protein of human parvovirus B19 plays a key role in inducing apoptosis during B19 virus infection of primary erythroid progenitor cells

Human parvovirus B19 (B19V) infection shows a strong erythroid tropism and drastically destroys erythroid progenitor cells, thus leading to most of the disease outcomes associated with B19V infection. In this study, we systematically examined the 3 B19V nonstructural proteins, 7.5 kDa, 11 kDa, and NS1, for their function in inducing apoptosis in transfection of primary ex vivo-expanded erythroid progenitor cells, in comparison with apoptosis induced during B19V infection. Our results show that 11 kDa is a more significant inducer of apoptosis than NS1, whereas 7.5 kDa does not induce apoptosis. Furthermore, we determined that caspase-10, an initiator caspase in death receptor signaling, is the most active caspase in apoptotic erythroid progenitors induced by 11 kDa and NS1 as well as during B19V infection. More importantly, cytoplasm-localized 11 kDa is expressed at least 100 times more than nucleus-localized NS1 at the protein level in primary erythroid progenitor cells infected with B19V; and inhibition of 11 kDa expression using antisense oligos targeting specifically to the 11 kDa-encoding mRNAs reduces apoptosis significantly during B19V infection of erythroid progenitor cells. Taken together, these results demonstrate that the 11 kDa protein contributes to erythroid progenitor cell death during B19V infection.

Maternal PD-1 regulates accumulation of fetal antigen-specific CD8+ T cells in pregnancy

The failure to reject the semi-allogeneic fetus suggests that maternal T lymphocytes are regulated by potent mechanisms in pregnancy. The T cell immunoinhibitory receptor, Programmed Death-1 (PD-1), and its ligand, B7-H1, maintain peripheral tolerance by inhibiting activation of self-reactive lymphocytes. Here, we investigated the role of the PD-1/B7-H1 pathway in maternal tolerance of the fetus. Antigen-specific maternal T cells both proliferate and upregulate PD-1 in vivo at mid-gestation in response to paternally inherited fetal antigen. In addition, when these cells carry a null deletion of PD-1, they accumulate excessively in the uterus-draining lymph nodes (P<0.001) without a concomitant increase in proliferation. In vitro assays showed that apoptosis of antigen-specific CD8(+) PD-1(-/-) cells was reduced following peptide stimulation, suggesting that the accumulation of these cells in maternal lymph nodes is due to decreased cell death. However, the absence of neither maternal PD-1 nor B7-H1 had detectable effects on gestation length, litter size, or pup weight at birth in either syngeneic or allogeneic pregnancies. These results suggest that PD-1 plays a previously unrecognized role in maternal-fetal tolerance by inducing apoptosis of paternal antigen-specific T cells during pregnancy, thereby controlling their abundance.

Effects of Immunonutrition for Cystectomy on Immune Response and Infection Rates: A Pilot Randomized Controlled Clinical Trial

UNLABELLED After radical cystectomy (RC), patients are at risk for complications including infections. The expansion of myeloid-derived suppressor cells (MDSCs) after surgery may contribute to the lower resistance to infection. Immune response and postoperative complications were compared in men consuming either specialized immunonutrition (SIM; n=14) or an oral nutrition supplement (ONS; n=15) before and after RC. MDSC count (Lin- CD11b+ CD33+) was significantly different between the groups over time (p=0.005) and significantly lower in SIM 2 d after RC (p<0.001). MDSC count expansion from surgery to 2 d after RC showed a weak association with an increase in infection rate 90 d after surgery (p=0.061). Neutrophil:lymphocyte ratio was significantly lower in SIM compared with ONS 3h after the first incision (p=0.039). Participants receiving SIM had a 33% reduction in postoperative complication rate (95% confidence interval [CI], 1-64; p=0.060) and a 39% reduction in infection rate (95% CI, 8-70; p=0.027) during late-phase recovery. The small sample size limits the study findings. PATIENT SUMMARY Results show that the immune response to surgery and late infection rates differ between radical cystectomy patients receiving specialized immunonutrition versus oral nutrition supplement in the perioperative period. TRIAL REGISTRATION ClinicalTrials.gov NCT01868087.

Depletion and recovery of lymphoid subsets following morphine administration

BACKGROUND AND PURPOSE Opioid use and abuse has been linked to significant immunosuppression, which has been attributed, in part, to drug‐induced depletion of lymphocytes. We sought to define the mechanisms by which lymphocyte populations are depleted and recover following morphine treatment in mice.

The Gads (GrpL) adaptor protein regulates T cell homeostasis

Little is known about the role of the Gads (GrpL) adaptor protein in mature T cell populations. In this study we show that the effects of Gads deficiency on murine CD4+ and CD8+ T cells are markedly different. Gads−/− CD4+ T cells were markedly deficient in the spleen and had an activated phenotype and a rapid turnover rate. When transferred into a wild-type host, Gads−/− CD4+ T cells continued to proliferate at a higher rate than wild-type CD4+ T cells, demonstrating a defect in homeostatic proliferation. Gads−/− CD8+ T cells had a memory-like phenotype, produced IFN-γ in response to ex vivo stimulation, and underwent normal homeostatic proliferation in wild-type hosts. Gads−/− T cells had defective TCR-mediated calcium responses, but had normal activation of ERK. Gads−/− CD4+ T cells, but not CD8+ T cells, had a severe block of TCR-mediated proliferation and a high rate of spontaneous cell death and were highly susceptible to CD95-induced apoptosis. This suggests that the rapid turnover of Gads−/− CD4+ T cells is due to a defect in cell survival. The intracellular signaling pathways that regulate homeostasis in CD4+ and CD8+ T cells are clearly different, and the Gads adaptor protein is critical for homeostasis of CD4+ T cells.

CD95/Fas induces cleavage of the GrpL/Gads adaptor and desensitization of antigen receptor signaling

The balance between cell survival and cell death is critical for normal lymphoid development. This balance is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95/Fas. In some cells, ligating the B cell antigen receptor can protect the cell from apoptosis induced by CD95. Here we report that ligation of CD95 inhibits antigen receptor-mediated signaling. Pretreating CD40-stimulated tonsillar B cells with anti-CD95 abolished B cell antigen receptor-mediated calcium mobilization. Furthermore, CD95 ligation led to the caspase-dependent inhibition of antigen receptor-induced calcium mobilization and to the activation of mitogen-activated protein kinase pathways in B and T cell lines. A target of CD95-mediated caspase 3-like activity early in the apoptotic process is the adaptor protein GrpL/Gads. GrpL constitutively interacts with SLP-76 via its C-terminal SH3 domain to regulate transcription factors such as NF-AT. Cleavage of GrpL removes the C-terminal SH3 domain so that it is no longer capable of recruiting SLP-76 to the membrane. Transfection of a truncated form of GrpL into Jurkat T cells blocked T cell antigen receptor-induced activation of NF-AT. These results suggest that CD95 signaling can desensitize antigen receptors, in part via cleavage of the GrpL adaptor.

Immature single-positive CD8+ thymocytes represent the transition from Notch-dependent to Notch-independent T-cell development

Early in T-cell development, cells proceed through stages that are critically dependent on signaling through the Notch receptor. As cells mature, thymocytes transition from being Notch dependent to being Notch independent, but the stage of development during which this transition occurs is unknown. We used an in vitro differentiation system in which thymocytes can be cultured in the presence or absence of a Notch ligand to identify the stage of development in which thymocytes transition from being Notch responsive to Notch non-responsive. We identified the immature single-positive (ISP) CD8(+) stage of T-cell development as being this transition point. ISP thymocytes were responsive to Notch, but ISP cells responded to Notch ligation in a manner that was distinct from the response by double-negative (DN) thymocytes. Fewer ISP thymocytes proliferated and more ISP cells died in culture than DN thymocytes. Further, fewer double-positive (DP) thymocytes generated by culturing ISP thymocytes were in the S, G2 or M phase of the cell cycle as compared with DP thymocytes derived from DN thymocytes. These data indicate that the DP population created varied depending on the input population. In summary, the data presented here indicate that ISP thymocytes responded to Notch differently than DN thymocytes and ISP thymocytes represent the transition stage from Notch-dependent survival and proliferation to Notch-independent survival and proliferation.

Gads−/− Mice Reveal Functionally Distinct Subsets of TCRβ+ CD4−CD8− Double-Negative Thymocytes

TCRβ expression in CD4−CD8− double-negative (DN) thymocytes induces signaling pathways that promote survival and proliferation, as well as differentiation into CD4+CD8+ double-positive thymocytes. The signaling pathways that regulate survival, proliferation, and differentiation remain unclear. We used Gads-deficient mice to investigate the signaling pathways that regulate these cell fates. During this investigation, we focused on TCRβ+ DN thymocytes and found that there are at least three functionally distinct subsets of TCRβ+ DN thymocytes: TCRβ+ DN3E, TCRβ+ DN3L, and TCRβ+ DN4. Survival and proliferation of TCRβ+ DN3E were independent of Gads, but survival and proliferation of TCRβ+ DN3L cells were Gads dependent. Likewise, expression of Bcl-2 in TCRβ+ DN3E cells was Gads independent, but Gads was necessary for Bcl-2 expression in TCRβ+ DN3L cells. Bcl-2 expression was not dependent on Gads in TCRβ+ DN4 cells, but proliferation of TCRβ+ DN4 cells was Gads dependent. Gads was not required for the differentiation of DN thymocytes into DP thymocytes. In fact, Gads−/− DN3E cells differentiated into DP thymocytes more readily than wild-type cells. We conclude that signaling pathways required to initiate TCRβ-induced survival and proliferation are distinct from the pathways that maintain survival and proliferation. Furthermore, signaling pathways that promote survival and proliferation may slow differentiation.

The effect of omeprazole on the development of experimental autoimmune encephalomyelitis in C57BL/6J and SJL/J mice

BackgroundGastric disturbances such as dyspepsia are routinely encountered by multiple sclerosis (MS) patients, and these conditions are often treated with gastric acid suppressors such as proton pump inhibitors, histamine H2 receptor antagonists, or antacids. The proton pump inhibitor omeprazole can alter the gut flora and immune responses, both of which can influence the course of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The objective of the current study was to examine the effect of omeprazole treatment on the development of EAE. Bacterial microbiome analysis of mouse fecal pellets was determined in C57BL/6J EAE mice chronically treated with omeprazole, and spleen immune cell content, clinical scores, weight, rotarod latency, and histopathology were used as outcome measures in C57BL/6J and SJL/J mice with EAE.ResultsOmeprazole treatment resulted in decreases in Akkermansia muciniphila and Coprococcus sp. and an increase in unidentified bacteria in the family S24-7 (order Bacteroidales) in C57BL/6J mice with EAE. Omeprazole did not alter spleen immune cell content compared to vehicle in EAE mice, but differences independent of treatment were observed in subsets of T cells between early and advanced disease in C57BL/6J mice as well as between the two strains of mice at an advanced disease stage. Omeprazole caused no difference in clinical scores in either strain, but significantly lowered weight gain compared to vehicle in the C57BL/6J mice with EAE. Omeprazole also did not alter rotarod behavior or hindbrain inflammatory cell infiltration compared to vehicle in both strains of mice with EAE. Rotarod latency did reveal a negative correlation with clinical scores during active disease in both mouse strains, but not during clinical remission in SJL/J mice, suggesting that rotarod can detect disability not reflected in the clinical scores.ConclusionsDespite alterations in the gut microbiota and weight gain in the C57BL/6J EAE model, omeprazole had no effect on multiple measures of disease activity in C57BL/6J and SJL/J mice with EAE, supporting the notion that omeprazole does not substantially influence disease activity in MS patients.