Jens M. Chemnitz

CTLA-4 and PD-1 Receptors Inhibit T-Cell Activation by Distinct Mechanisms

ABSTRACT CTLA-4 and PD-1 are receptors that negatively regulate T-cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28-mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4-mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T-cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28-mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/AKT activation was dependent upon the immunoreceptor tyrosine-based switch motif located in its cytoplasmic tail, adding further importance to this domain in mediating PD-1 signal transduction. Lastly, PD-1 ligation is more effective in suppressing CD3/CD28-induced changes in the T-cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T-cell activation through distinct and potentially synergistic mechanisms.

SHP-1 and SHP-2 Associate with Immunoreceptor Tyrosine-Based Switch Motif of Programmed Death 1 upon Primary Human T Cell Stimulation, but Only Receptor Ligation Prevents T Cell Activation

To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.

Prostaglandin E2 Impairs CD4+ T Cell Activation by Inhibition of lck: Implications in Hodgkin's Lymphoma

Many tumors, including Hodgkins lymphoma, are associated with decreased cellular immunity and elevated levels of prostaglandin E(2) (PGE(2)), a known inhibitor of CD4+ T cell activation, suggested to be involved in immune deviation in cancer. To address the molecular mechanisms tumor-derived PGE(2) might have on primary human CD4+ T cells, we used a whole genome-based transcriptional approach and show that PGE(2) severely limited changes of gene expression induced by signaling through the T cell receptor and CD28. This data suggests an interference of PGE(2) at an early step of T cell receptor signaling: indeed, PGE(2) stimulation of T cells leads to inactivation of lck and reduced phosphorylation of ZAP70. Antiapoptotic genes escaped PGE(2)-induced inhibition resulting in partial protection from apoptosis in response to irradiation or Fas-mediated signaling. As a functional consequence, PGE(2)-treated CD4+ T cells are arrested in the cell cycle associated with up-regulation of the cyclin/cyclin-dependent kinase inhibitor p27(kip1). Most importantly, CD4+ T cells in Hodgkins lymphoma show similar regulation of genes that were altered in vitro by PGE(2) in T cells from healthy individuals. These data strongly suggest that PGE(2) is an important factor leading to CD4+ T cell impairment observed in Hodgkins lymphoma.

B and T Lymphocyte Attenuator-Mediated Signal Transduction Provides a Potent Inhibitory Signal to Primary Human CD4 T Cells That Can Be Initiated by Multiple Phosphotyrosine Motifs

The B and T lymphocyte attenuator (BTLA) is a recently identified member of the CD28 family of cell receptors. Initial reports demonstrated that mice deficient in BTLA expression were more susceptible to experimental autoimmune encephalomyelitis, indicating that BTLA was likely to function as a negative regulator of T cell activation. However, cross-linking of BTLA only resulted in a 2-fold reduction of IL-2 production, questioning the potency with which BTLA engagement blocks T cell activation. We established a model in which BTLA signaling could be studied in primary human CD4 T cells. We observed that cross-linking of a chimeric receptor consisting of the murine CD28 extracellular domain and human BTLA cytoplasmic tail potently inhibits IL-2 production and completely suppresses T cell expansion. Mutation of any BTLA tyrosine motifs had no effect on the ability of BTLA to block T cell activation. Only mutation of all four tyrosines rendered the BTLA cytoplasmic tail nonfunctional. We performed structure-function studies to determine which factors recruited to the BTLA cytoplasmic tail correlated with BTLA function. Using pervanadate as a means to phosphorylate the BTLA cytoplasmic tail, we observed both Src homology protein (SHP)-1 and SHP-2 recruitment. However, upon receptor engagement, we observed only SHP-1 recruitment, and mutations that abrogated SHP-1 recruitment did not impair BTLA function. These studies question whether SHP-1 or SHP-2 have any role in BTLA function and caution against the use of pervanadate as means to initiate signal transduction cascades in primary cells.

Anti-thymocyte globulins for post-transplant graft-versus-host disease prophylaxis—A systematic review and meta-analysis

BACKGROUND Despite advances made in allogeneic hematopoietic stem cell transplantation (alloSCT), graft versus host disease (GvHD) remains a major problem. The main strategy to combat GvHD is prophylaxis and ATG plays a major role in this arena. Conflicting reports on the effectiveness of ATG on GvHD prevention prompted us to address this question by means of a systematic review and meta-analysis. METHODS Six prospective randomized controlled trials (RCT) comparing the addition of ATG to standard immunosuppressive regimen as GvHD prophylaxis were analyzed. All ATG preparations were considered but homogeneity in type of preparation and dosage had to be observed within each trial. RESULTS Our meta-analysis reveals that the incidence of grade II-IV GvHD was significantly lower in patients receiving ATG. Addition of ATG had no impact on overall survival, relapse or non-relapse mortality. CONCLUSIONS Based on the current level of the data analyzed in this systematic review, we cannot conclude a general recommendation for the use of ATG for GvHD prophylaxis in alloSCT. In patients who are at high risk for severe GvHD it should be considered individually. However, due to the heterogeneity of the analyzable studies it seems likely that future studies might change the results of the pooled data of this meta-analysis. In order to improve the current level of data, further randomized studies in this topic are therefore urgently warranted.

Tumor-necrosis factor impairs CD4+ T cell-mediated immunological control in chronic viral infection

Persistent viral infections are characterized by the simultaneous presence of chronic inflammation and T cell dysfunction. In prototypic models of chronicity—infection with human immunodeficiency virus (HIV) or lymphocytic choriomeningitis virus (LCMV)—we used transcriptome-based modeling to reveal that CD4+ T cells were co-exposed not only to multiple inhibitory signals but also to tumor-necrosis factor (TNF). Blockade of TNF during chronic infection with LCMV abrogated the inhibitory gene-expression signature in CD4+ T cells, including reduced expression of the inhibitory receptor PD-1, and reconstituted virus-specific immunity, which led to control of infection. Preventing signaling via the TNF receptor selectively in T cells sufficed to induce these effects. Targeted immunological interventions to disrupt the TNF-mediated link between chronic inflammation and T cell dysfunction might therefore lead to therapies to overcome persistent viral infection.

OCTET-CY: a phase II study to investigate the efficacy of post-transplant cyclophosphamide as sole graft-versus-host prophylaxis after allogeneic peripheral blood stem cell transplantation.

Post‐transplant cyclophosphamide is increasingly used as graft‐versus‐host disease (GvHD) prophylaxis in the setting of bone marrow transplantation. No data have been published on the use of single‐agent GvHD prophylaxis with post‐transplant cyclophosphamide in the setting of peripheral blood stem cell transplantation (PBSCT).

Activated primary human B cells efficiently induce early CD40L and CD107a expression in CD4+ T cells.

To the editor: In the February issue of Blood , Chakrabarty and colleagues present data suggesting that early activation of CD4+ T cells, measured by CD40L expression, is significantly induced by CD14hi monocytes, myeloid dendritic cells and monocytic cell lines in vitro. In contrast, B cells (

Pretransplant Comorbidities Maintain Their Impact on Allogeneic Stem Cell Transplantation Outcome 5 Years Posttransplant: A Retrospective Study in a Single German Institution

The introduction of reduced-intensity conditioning regimens has allowed elderly patients with preexisting comorbidities access to the potentially curative allogeneic stem cell transplantation. Patients comorbidities at the time of treatment consideration play a significant role in transplant outcome in terms of both overall survival (OS) and nonrelapse mortality (NRM). The hematopoietic stem cell transplantation comorbidity index (HCT-CI) quantifies these patient specific risks and has established itself as a major tool in the pretransplant assessment of patients. Many single center and multicenter studies have assessed the HCT-CI score and reported conflicting outcomes. The present study aimed to evaluate the HCT-CI in a single large European transplant centre. 245 patients were retrospectively analyzed and the predictive value of the score was assessed with respect to OS and NRM. We confirm that the HCT-CI predicts outcome for both OS and NRM. Moreover, we identified age of the patient as an independent prognostic parameter for OS. Incorporation of age in the HCT-CI would improve its ability to prognosticate and allow the transplant physician to assess the patient specific risks appropriately at the time of counseling for transplant.

Cytomorphologic signs of severe pernicious anemia obscured in a patient with heterozygous hemoglobin Stanleyville II.

Here, we report a rare coincidence of heterozygous hemoglobinopathy (Hb) Stanleyville II and severe pernicious anemia due to autoimmune gastritis. Hb Stanleyville II is characterized by a single base exchange (AAC→AAA) resulting in a substitution Asn→Lys at position 78 of hemoglobin α2‐chain. Under normal conditions this hemoglobinopathy does not cause any symptoms even if present as homozygous variant. However, in our case diagnosis of pernicious anemia was hampered by the absence of typical erythrocytic macrocytosis and hyperchromasia. In addition, interpretation of bone marrow smears was difficult as characteristic findings for pernicious anemia were little pronounced. All known reasons for the absence of typical cytomorphologic signs in pernicious anemia as underlying iron deficiency and thalassemia could be excluded.