Modeling Chronic Graft-Versus-Host-Disease: A New MHC-Matched Model of Late-Onset Scleroderma After Low-Dose Conditioning and Hematopoietic Cell Transplantation That Affects Only Male Recipients of Female Grafts.
Authors: Mueller Antonia MS , Miklos David , Shizuru Judith 
Published: October 2019

Abstract
Abstract 3560
Poster Board III-497
Chronic graft-vs-host disease (GVHD) is the most common late complication of allogeneic hematopoietic cell transplantation (HCT), is associated with high morbidity, and presents a major problem even after reduced intensity conditioning. Many aspects of the syndrome, such as sclerodermatous skin changes resemble autoimmune diseases. Unlike acute GVHD, which is a donor T cell (TC) driven inflammatory event, the pathophysiology of chronic GVHD remains poorly elucidated. One reason for this limited understanding is the lack of mouse models that recapitulate the late-onset scenario of collagenous sclerosis observed in humans. Here, we present a MHC-matched model in which mice develop profound sclerotic skin changes beyond day (d) 100 post-HCT. Importantly, the syndrome occurs only in males receiving female grafts, a donor/recipient gender combination which in humans is associated with increased risk of developing chronic GVHD. FACS-purified hematopoietic stem cells (HSC: cKIT+Thy1.1loSca1+Lin-) alone or supplemented with TC from female AKR/J donors were infused into sublethally (4 Gy) irradiated BALB.K mice (both H2k). Recipients underwent a transient phase of mild acute GVHD (skin reddening, weight loss), but recovered fully. While none of 24 female hosts displayed any signs of chronic GVHD over an observation period of more than 200d, 17 of 27 males (63%) developed massive skin thickening with severe bruising and excoriations. The progressively indurated lesions appeared between d100-200, primarily on the trunk, were tightly fixed to the underlying fascia, and subsequently resulted in alopecia. Histologically, hyperkeratosis and massive thickening of trichrome-positive collagen bundles in dermis and subcutis predominated the picture. Late-onset scleroderma was not associated with significant weight loss or rapid death. In this non-myeloablative setting all mice with chronic GVHD were mixed chimeras in their myeloid, T and B cell lineages. Mice that had rejected their grafts remained healthy. The composition of mononuclear cells (MNC) in bone marrow (BM), spleen (SP), lymph nodes (LN), thymus, liver, and skin of affected animals was FACS analyzed and compared with disease-free female recipients and normal controls. Chronic GVHD afflicted mice had B lymphopenia (30-50% decrease) in BM, SP and LN. Both donor and host TC were increased in the livers (each 7-17%), but normal in the other organs. NK cells were markedly reduced in liver (1-2.5%) and SP (<1%), compared with unmanipulated BALB.K mice which have ∼5% NK cells in liver and SP. Single cell suspensions of MNC infiltrating the skin were made by enzymatic digestion, enriched using a Ficoll gradient, and analyzed by FACS, Wright Giemsa staining, and PCR for chimerism assessment of sorted cell subsets. Regarding their morphology on cytospin slides the majority of skin MNC resembled macrophages with a central small round nucleus and a low nuclear / cytoplasmic ratio. By FACS 35-55% of cells were Mac1high (vs 8% in females and controls), 20-35% Mac1lo (vs 15-20%). Mac1high cells comprised granulocytes (30% Gr1+), mature NK cells (∼30% DX5+CD122+), and macrophages (Gr1-DX5-). Skin of affected mice (but not females and controls) also contained 15-25% CD4 TC, but only few CD8 TC (1-5%), and no B cells. Neither in the skin nor in any other organ did PMA-stimulated lymphocytes express increased amounts of IFNγ or IL-17. All cellular subsets were mixed chimeric. In summary: here, we present a novel MHC-matched model of non-myeloablative HCT that resembles human chronic GVHD in many key aspects, including late onset, development of scleroderma-like fibrosis, preponderance of male recipients given female grafts, and no clear association to graft type. Similarities with autoimmune scleroderma are striking with regard to increased thickness of the skin with hyperkeratosis, broad sclerotic collagen bundles in dermis and subcutis, predominance of CD4 over CD8 TC, presence of macrophages, and mixed microchimerism in the skin. We continue to pursue the identity of the cells responsible for this syndrome and the targets of immune reactivity.

Disclosures:
Miklos: Novartis: Research Funding.