ICOSL+ plasmacytoid dendritic cells as inducer of graft-versus-host disease, responsive to a dual ICOS/CD28 antagonist

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). CD146 and CCR5 are proteins that mark activated T helper 17 (Th17) cells. The Th17 cell phenotype is promoted by the interaction of the receptor ICOS on T cells with ICOS ligand (ICOSL) on dendritic cells (DCs). We performed multiparametric flow cytometry in a cohort of 156 HCT recipients and conducted experiments with aGVHD murine models to understand the role of ICOSL+ DCs. We observed an increased frequency of ICOSL+ plasmacytoid DCs, correlating with CD146+CCR5+ T cell frequencies, in the 64 HCT recipients with gastrointestinal aGVHD. In murine models, donor bone marrow cells from ICOSL-deficient mice compared to those from wild-type mice reduced aGVHD-related mortality. Reduced aGVHD resulted from lower intestinal infiltration of pDCs and pathogenic Th17 cells. We transplanted activated human ICOSL+ pDCs along with human peripheral blood mononuclear cells into immunocompromised mice and observed infiltration of intestinal CD146+CCR5+ T cells. We found that prophylactic administration of a dual human ICOS/CD28 antagonist (ALPN-101) prevented aGVHD in this model better than did the clinically approved belatacept (CTLA-4-Fc), which binds CD80 (B7-1) and CD86 (B7-2) and interferes with the CD28 T cell costimulatory pathway. When started at onset of aGVHD signs, ALPN-101 treatment alleviated symptoms of ongoing aGVHD and improved survival while preserving antitumoral cytotoxicity. Our data identified ICOSL+-pDCs as an aGVHD biomarker and suggest that coinhibition of the ICOSL/ICOS and B7/CD28 axes with one biologic drug may represent a therapeutic opportunity to prevent or treat aGVHD

AMG 330-induced cytotoxicity in the presence of healthy donor T-cells.

<p>Forty-one primary AML specimens were incubated with increasing concentrations of AMG 330 and T-cells from a single healthy donor at various effector:target (E:T) cell ratios as indicated. After 48 hours, cell counts were determined and cytotoxicity was assessed with DAPI staining to quantify drug-specific cytotoxicity. Results are shown as mean±SEM.</p

AMG 330-induced cytotoxicity in the presence of healthy donor T-cells, stratified by disease stage and risk.

<p>AMG 330-induced cytotoxicity at 48 hours, stratified by <b>(A, B)</b> disease stage (newly diagnosed AML [n = 21] and relapsed/refractory AML [n = 20]), and <b>(C, D)</b> cytogenetic/molecular disease risk (favorable-risk [n = 5]; intermediate-risk [n = 26]); and adverse-risk [n = 10]) in the presence of T-cells from a single healthy donor at an E:T cell ratio of 1:3 and 1:1, as indicated.</p

AKT Signaling as a Novel Factor Associated with <em>In Vitro</em> Resistance of Human AML to Gemtuzumab Ozogamicin

<div><p>Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ₁ derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ₁ resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance <em>in vitro</em> in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ₁ with particularly pronounced effects in otherwise GO or free calicheamicin-γ₁ -resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ₁. Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance <em>in vitro</em> may provide a novel approach to improve the <em>in vivo</em> efficacy of GO/calicheamicin-γ₁ and, by extrapolation, other DNA damage-based therapeutics.</p> </div