All-Trans Retinoic Acid (ATRA) Targets IRF4 Deficient, NOTCH2-Activated B Cells from Chronic Gvhd Patients
Authors: Poe Jonathan C. , Jia Wei , Su Hsuan , Li Zhiguo , Hakim Frances T. , Pavletic Steven Z. , Rose Jeremy J. , Rizzieri David A , Yang Yiping , Chen Benny J. , Anand Sarah , Ritz Jerome , Siebel Christian W. , Chao Nelson J. , Maillard Ivan , Sarantopoulos Stefanie 
Published: October 2019

Abstract
Allogeneic hematopoietic stem cell transplantation (HCT)-related immune pathology severely limits patient (Pt) survival, largely due to infections. Chronic graft versus host disease (cGVHD) Pts are particularly immune incompetent with impaired memory B cell responses. Paradoxically, cGVHD Pts are hypogammaglobulinemic and yet are variably immune tolerant with potentially pathogenic alloantibodies frequently produced. B cells with marginal zone (MZ)-like properties are expanded in cGVHD Pts with high BAFF/B-cell ratios (rev. Sarantopoulos, Blood 2015), similar to findings in BAFF-overexpressing mice. We previously found that the increased BCR responsiveness of cGVHD B cells depended on NOTCH2 signaling, since a specific monoclonal antibody (Genentech) blocked hyperactivation (ASH 2015 oral abstract: Blood. 2015. 126:145). Our current objective is to define the molecular defects underlying this aberrant NOTCH2-BCR axis.
Increased NOTCH2 expression/activity is found in mice genetically deficient in the transcription factor IRF4 (J. Exp. Med. 2013. 210:2887-2902). Likewise, IRF4 levels are subnormal in unstimulated B cells from active cGVHD Pts relative to healthy donors (Blood. 2014. 123: 2108-15). Thus, we hypothesized that a B cell-intrinsic IRF4 deficiency contributes to the NOTCH2-BCR axis in cGVHD. We studied multi-center (Duke Univ., NCI-NIH, Dana Farber Cancer Inst.) de-identified clinical samples from 38 Pts with or without NIH diagnostic criteria of cGVHD who were >12 months post-HCT and not receiving high dose steroids. We employed our in vitro culture system utilizing NOTCH ligand-expressing feeder cells and measured proliferative responses (Ki-67% by flow cytometry) to surrogate Ag. First, adding to our previous findings, B cells from active cGVHD Pts (n=8) proliferated robustly to minimal BCR ligation when NOTCH was activated compared to B cells from Pts without cGVHD (n=7) (38.1% vs 15.3%, P =0.006). To test our new hypothesis, IRF4 levels were determined by quantitative PCR analysis in B cells isolated from HCT Pts with active cGVHD and with no cGVHD stimulated ex vivo for 24 hours (h) with a limiting amount of surrogate Ag (anti-IgM). BCR-engaged B cells from active cGVHD Pts (Figure 1A, open bars) maintained significantly lower IRF4 levels compared to B cells from Pts with no cGVHD (Figure 1A, filled bars; n=4 in each group, P =0.01 in a two-sided t-test).
Importantly, this IRF4 deficiency may be correctable therapeutically in cGVHD Pts, since the ex vivo treatment of human B cells with all-trans retinoic acid (ATRA) increases IRF4 expression (J. Immunol. 2015. 195:2601-11), and ATRA has demonstrated efficacy in a mouse model of cGVHD (Blood. 2012. 119:285-95). When ATRA (100 nM) was added to cultures of surrogate Ag-stimulated B cells (Figure 1A, right), IRF4 levels increased significantly in both active cGVHD B cells (n = 4, P <0.05 in a two-sided t-test) and no cGVHD B cells (n = 4, P < 0.01 in a two-sided t-test). Nevertheless, even with ATRA treatment the level of IRF4 remained significantly less in B cells from active cGVHD Pts compared to no cGVHD Pts (P <0.01 in a two-sided t-test). To determine whether IRF4 expression could also be enhanced when the NOTCH2-BCR axis is engaged, as could occur in vivo in Pts, we subjected active cGVHD B cells to NOTCH ligand-expressing feeder cells. When assessed by intracellular staining and flow cytometry, IRF4 protein increased following 24h of ATRA treatment relative to vehicle alone, either in the presence or absence of surrogate Ag (Figure 1B and data not shown, one of 6 representative Pts assessed displayed). Strikingly, ATRA treatment completely abolished NOTCH2-BCR hyper-responsiveness of active cGVHD B cells, as evaluated by Ki-67 staining after 72h using flow cytometry (Figure 1C, n=6 in each group, P <0.01 in a two-sided t-test).
In summary, we have revealed skewing of a key B cell maturation pathway in active cGVHD. Our data suggest that an intrinsic defect in IRF4 expression dictates heightened NOTCH2-BCR responsiveness. This defect may be corrected by retinoids or similar agents that promote IRF4 expression. Thus, we have identified a candidate molecular target for blocking aberrant B cell activation and potentially inducing functional B cell development.
This work was supported by a Translational Research Program grant from the Leukemia & Lymphoma Society and a National Institutes of Health grant, NIH (NHLBI) R01 HL 129061-01.
Figure 1. Figure 1.

Disclosures
Ritz: Kiadis: Membership on an entity's Board of Directors or advisory committees. Siebel:Genentech Inc.: Employment, Equity Ownership.