Frederick J. Schnell

Junction adhesion molecule is a receptor for reovirus.

Virus attachment to cells plays an essential role in viral tropism and disease. Reovirus serotypes 1 and 3 differ in the capacity to target distinct cell types in the murine nervous system and in the efficiency to induce apoptosis. The binding of viral attachment protein sigma1 to unidentified receptors controls these phenotypes. We used expression cloning to identify junction adhesion molecule (JAM), an integral tight junction protein, as a reovirus receptor. JAM binds directly to sigma1 and permits reovirus infection of nonpermissive cells. Ligation of JAM is required for reovirus-induced activation of NF-kappaB and apoptosis. Thus, reovirus interaction with cell-surface receptors is a critical determinant of both cell-type specific tropism and virus-induced intracellular signaling events that culminate in cell death.

Signal Regulatory Protein (SIRPα), a Cellular Ligand for CD47, Regulates Neutrophil Transmigration

Recent studies have demonstrated that CD47 plays an important role in regulating human neutrophil (PMN) chemotaxis. Two ligands for CD47, thrombospondin and SIRPα, have been described. However, it is not known if SIRP-CD47 interactions play a role in regulating PMN migration. In this study, we show that SIRPα1 directly binds to the immunoglobulin variable domain loop of purified human CD47 and that such SIRP-CD47 interactions regulate PMN transmigration. Specifically, PMN migration across both human epithelial monolayers and collagen-coated filters was partially inhibited by anti-SIRP monoclonal antibodies. Similar kinetics of inhibition were observed for PMN transmigration in the presence of soluble, recombinant CD47 consisting of the SIRP-binding loop. In contrast, anti-CD47 monoclonal antibodies inhibited PMN transmigration by markedly different kinetics. Results of signal transduction experiments suggested differential regulation of PMN migration by SIRPversus CD47 by phosphatidylinositol 3-kinase and tyrosine kinases, respectively. Immunoprecipitation followed by Western blotting after SDS-PAGE under nonreducing conditions suggested that several SIRP protein species may be present in PMN. Stimulation of PMN with fMLP resulted in increased surface expression of these SIRP proteins, consistent with the existence of intracellular pools. Taken together, these results demonstrate that PMN migration is regulated by CD47 through SIRPα-dependent and SIRPα-independent mechanisms.

Reduced Expression of Junctional Adhesion Molecule and Platelet/Endothelial Cell Adhesion Molecule-1 (CD31) at Human Vascular Endothelial Junctions by Cytokines Tumor Necrosis Factor-α Plus Interferon-γ Does Not Reduce Leukocyte Transmigration Under Flow

The combination of tumor necrosis factor (TNF)-alpha plus interferon (IFN)-gamma has been shown previously to promote redistribution of platelet/endothelial cell adhesion molecule-1 (PECAM-1) (CD31), junctional adhesion molecule (JAM), and VE-cadherin away from lateral junctions of human umbilical vein endothelial cell monolayers. In parallel, neutrophil transmigration was significantly reduced. Because PECAM-1 and JAM have been implicated in leukocyte transmigration, the observed redistribution by cytokine activation was presumed to represent the mechanism causing decreased transmigration under static conditions. The current results confirm that culture of human umbilical vein endothelial cells with TNF-alpha plus IFN-gamma caused a decrease in surface-expressed and junctional-localized JAM and PECAM-1, but did not cause decreased leukocyte transmigration in an in vitro flow assay. Furthermore, blocking monoclonal antibody to PECAM-1 still significantly reduced monocyte transmigration, demonstrating that it retains a functional role even though its levels were reduced and redistributed away from junctions, whereas a panel of monoclonal antibodies to JAM failed to reduce leukocyte transmigration. Given the alterations in junction protein location, permeability function was assessed. IFN-gamma alone or TNF-alpha plus IFN-gamma significantly increased permeability, but TNF-alpha alone did not, suggesting lack of correlation between transmigration and loss of permeability. In conclusion, cytokine activation induced loss and redistribution of PECAM-1 and JAM away from lateral junctions, but per se does not negatively regulate either neutrophil or monocyte transmigration under flow.

Murine pregnancy leads to reduced proliferation of maternal thymocytes and decreased thymic emigration.

During mammalian pregnancy the maternal thymus undergoes significant involution, and then recovers in size after birth. The mechanism behind this involution is not known, but it has been suggested that elevated levels of hormones during pregnancy induce the involution. We have recently shown that injection of 17β‐oestradiol into mice causes loss of early thymocyte precursors and inhibits proliferation of developing thymocytes. This suggests that elevated oestrogen in pregnancy may contribute to thymic involution. We have investigated this idea by examining the fate of thymocytes during mouse pregnancy in much greater detail than has been previously reported. Looking over a broad time–course, we find that pregnancy does not affect thymocyte precursor populations in the bone marrow, but induces a profound loss of early thymic progenitors in the thymus as early as day 12·5 of pregnancy. This loss is accompanied by decreased thymocyte proliferation, which returns to normal 2–4 days postpartum. No enhancement of apoptosis is detectable at any stage of pregnancy. We also find that there is a reduction in recent thymic emigrants after oestrogen treatment and at day 17·5 of pregnancy, suggesting that thymic involution during pregnancy influences the peripheral T‐cell repertoire. The similarities between oestrogen‐mediated involution and pregnancy‐mediated involution suggest that oestrogen is a significant contributor to loss of thymocyte cellularity during pregnancy, and probably functions primarily by reducing thymocyte proliferation.

Randomized, Double-Blind, Phase III Trial of Enzastaurin Versus Placebo in Patients Achieving Remission After First-Line Therapy for High-Risk Diffuse Large B-Cell Lymphoma

PURPOSE To compare disease-free survival (DFS) after maintenance therapy with the selective protein kinase C β (PKCβ) inhibitor, enzastaurin, versus placebo in patients with diffuse large B-cell lymphoma (DLBCL) in complete remission and with a high risk of relapse after first-line therapy. PATIENTS AND METHODS This multicenter, phase III, randomized, double-blind, placebo-controlled trial enrolled patients who were at high risk of recurrence after rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Patients (N = 758) with stage II bulky or stage III to IV DLBCL, three or more International Prognostic Index risk factors at diagnosis, and a complete response or unconfirmed complete response after 6 to 8 cycles of R-CHOP were assigned 2:1 to receive oral enzastaurin 500 mg daily or placebo for 3 years or until disease progression or unacceptable toxicity. Primary end point was DFS 3 years after the last patient entered treatment. Correlative analyses of biomarkers, including cell of origin by immunohistochemistry and PKCβ expression, with efficacy outcomes were exploratory objectives. RESULTS After a median follow-up of 48 months, DFS hazard ratio for enzastaurin versus placebo was 0.92 (95% CI, 0.689 to 1.216; two-sided log-rank P = .541; 4-year DFS, 70% v 71%, respectively). Independent of treatment, no significant associations were observed between PKCβ protein expression or cell of origin and DFS or overall survival. CONCLUSION Enzastaurin did not significantly improve DFS in patients with high-risk DLBCL after achieving complete response to R-CHOP. Achievement of a complete response may have abrogated the prognostic significance of cell of origin by immunohistochemistry.

Control of Recent Thymic Emigrant Survival by Positive Selection Signals and Early Growth Response Gene 1

Early growth response gene 1 (Egr1) is a transcriptional regulator whose expression can be induced by multiple signals including the TCR. Egr1 has been shown to promote positive selection, but an investigation of its role in T cell homeostasis has not been reported. The possibility that similar signals control both positive selection and peripheral T cell homeostasis led us to investigate the role of Egr1 in the maintenance of peripheral T cells. We have found that on TCR transgenic backgrounds, Egr1-deficient mice have a reduction in their number of naive T cells. Although Egr1-deficient animals have a low percentage of mature thymocytes due to inefficient positive selection, the absolute number of mature thymocytes is only slightly reduced due to increased thymus size in Egr1-deficient mice. Despite possessing near normal numbers of mature thymocytes, we find that Egr1-deficient mice have poor accumulation of recent thymic emigrants (RTE) in the periphery. The poor accumulation of RTE in Egr1-deficient mice appears to originate from decreased survival of mature thymocytes and RTE, which we have observed both in vitro and in vivo. These findings suggest that an Egr1-mediated signal during positive selection promotes not only the production of single positive thymocytes, but also the survival of selected thymocytes until they can become established in the periphery.

Early Growth Response Gene 1 Provides Negative Feedback to Inhibit Entry of Progenitor Cells into the Thymus

The size of the thymus can be greatly influenced by changes in the small number of early progenitors in the thymus. However, it is not known whether thymic cellularity feeds back to regulate the recruitment, survival, and expansion of progenitors. The transcription factor early growth response gene 1 (Egr1) has been implicated in controlling proliferation and survival in many cell types. We have previously shown that mice deficient in Egr1 have increased thymic cellularity. We now show that Egr1 regulates a negative feedback signal that controls the entry of cells into the thymus. Egr1-deficient mice have higher percentages of early T lineage progenitors in the thymus, yet Egr1-deficient mice have normal numbers of myelolymphoid progenitors in the bone marrow, and Egr1-deficient thymocytes show normal rates of apoptosis and proliferation at all stages of development. Evidence from mixed bone marrow chimeras shows that the ability of Egr1 to control progenitor recruitment is mediated by bone marrow-derived cells, but is not cell autonomous. Furthermore, Egr1-deficient thymuses have increased P-selectin expression. The data suggest that Egr1 mediates a feedback mechanism whereby the number of resident double negative thymocytes controls the entry of new progenitors into the thymus by regulating P-selectin expression on thymic endothelial cells.

CD8+ T cell responses to a viral escape mutant epitope: active suppression via altered SHP-1 activity.

One mechanism viruses use to subvert immune surveillance is through mutation of MHC contact residues of antigenic epitopes that weaken T cell recognition to the point that the immune system is ignorant of the infection. However, in contrast to ignorance, results presented herein demonstrate that intracellular signaling does occur upon stimulation with a lymphocytic choriomeningitis virus-derived escape mutant as demonstrated by the sustained activation of Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1). In addition to the increased SHP-1 activity, we found that the mutated epitope failed to induce oxidation of SHP-1, further enhancing enzymatic activity. Sustained activation of SHP-1 in a reduced form correlated with ERK and early growth response gene 1 activation and failure of T cells to commit to the effector lineage. Thus, instead of immune ignorance, these studies demonstrate the activation of a negative signaling pathway that actively suppresses T cell responses and limits recognition of viral escape mutants.

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.