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The Use of Ex Vivo Generated Regulatory T-Cell Preparations in a Canine Lung Allograft Model

Abrams, Kraig V. BA1; Hwang, Billanna MPH, DHSc2; Nash, Richard A. MD1,3; Georges, George E. MD1,4; Lamm, Wayne BS5; Storer, Barry PhD1; Madtes, David K. MD1,4,5; Glenny, Robert MD4,5; Mulligan, Michael S. MD2,4

doi: 10.1097/TP.0000000000001868
Letter

1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA.

2 Department of Surgery, University of Washington School of Medicine, Seattle, WA.

3 Colorado Blood Cancer Institute, Denver, CO.

4 Department of Medicine, University of Washington School of Medicine, Seattle, WA.

5 Division of Pulmonary and Critical Care Medicine, University of Washington School of Medicine, Seattle, WA.

Received 6 January 2017. Revision received 5 June 2017.

Accepted 7 June 2017.

The authors are grateful for the research funding from the National Institutes of Health, Bethesda, MD grants HL077420-01A2, DK42716 from NIH, Bethesda, MD.

V.K.A. drafted and revised the article, conducted the studies, analyzed and interpreted the data. B.H. conducted the studies, analyzed and interpreted the data, and revised the article. R.A.N. codesigned the experiment, supervised the studies, interpreted the data, and revised the article. G.E.G. codesigned the experiment. W.L. performed the pulmonary function tests and interpreted the data. B.S. provided statistical support. D.K.M. participated in the lung transplants, revised the article and interpreted the data. R.G. revised the article and interpreted the data. M.S.M. performed the lung transplants and revised the article.

Correspondence: Michael S. Mulligan, MD, Cardiothoracic Surgery, University of Washington 1959 NE Pacific St, Seattle, WA 98195. (msmmd@uw.edu).

Allograft survival is dependent on allorecognition due to peptide display differences by a major histocompatibility complex (MHC) distinct from self (major), or presentation of a novel peptide by identical MHC (minor). In the dog leukocyte antigen (DLA)-identical setting without immune suppression, robust lung allograft rejection based on minor antigen differences occurs within 2 weeks in normal recipients. We previously observed an increase in peripheral regulation in DLA-identical mixed hematopoietic chimeras (MC) as compared with normal dogs and saw a further regulation increase in MC with functioning solid organ transplants from their hematopoietic donors.1 Because tolerance induced thru regulation of minor antigens via MC could replace immune suppression, but would require a toxic conditioning regimen, we investigated the administration of ex vivo generated regulatory T-cell preparations (Treg preps) as a safer route to tolerance.

In vitro generated, MHC-mismatched alloantigen-primed peripheral blood mononuclear cells contain highly suppressive CD25+ cells expressing 10-fold more FOXP3 than CD25+ cells in blood.2 We hypothesized that “off-the-shelf”3 recipient-derived major alloantigen-primed CD25-selected cells could regulate incoming minor antigen presentation differences in a DLA-identical lung transplantation model and would extend the duration of allografts compared with that of a historic control group.1 Dogs in both groups received orthotopic lung allografts4 without any immune suppression and underwent pulmonary function tests (PFT) to determine allograft duration. Tested controls had early function (median, 5 days; range, 2-7; n = 4) and were electively, serially sacrificed (median, 19 days; range, 6-33 days; n = 7) with allografts from day 8 onward found engorged at necropsy.1 The experimental group (n = 7) underwent the same surgery with only the addition of an intraoperative, intravenous Treg prep infusion immediately after allograft reperfusion. PFT were performed a week posttransplant and monthly thereafter; all evaluable recipients had early allograft function (median, 8 days; range, 5-8; n = 6), whereas 3 had extended duration with functional allografts on days 29, 48, and 183.

Treg preps were variable in composition with a higher percentage of CD8+CD25+ cells found in preps with allograft rejection times as observed in the control group, whereas the highest CD4+CD25+ percentages were seen in the 2 preps with the longest allograft duration. There was no correlation between allograft duration and cell number infused; the longest duration was with the least number of cells, total, and per kg (Table 1). The in vitro suppressive capacity of a Treg prep correlated with the in vivo lung allograft duration (R2 = 0.81; P = 0.01; n = 6). Taken together, this suggests that quality of a Treg preparation is paramount to quantity.5 Improvements could employ CD8-depletion before infusion.

TABLE 1

TABLE 1

Because alloantigen-primed Treg preps can suppress a DLA-mismatched MLR, a DLA-mismatched primed Treg prep of recipient origin might prolong DLA-mismatched allograft function if educated against the incoming organ donor's antigens. This technique would require donor access for over a week and would be clinically difficult. However, if the few cells presenting minor antigen differences in an MHC-matched allograft can be regulated in an organ that contains many passenger leukocytes (lung), then perhaps all cells bearing major antigen differences in organs with few passenger leukocytes (kidney, heart) can be regulated using “off-the-shelf” Treg preps.

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REFERENCES

1. Nash RA, Yunusov M, Abrams VK, et al. Immunomodulatory effects of mixed hematopoietic chimerism: immune tolerance in canine model of lung transplantation. Am J Transplant. 2009;9:1037–1047.
2. Abrams VK, Hwang B, Lesnikova M, et al. A novel monoclonal antibody specific for canine CD25 (P4A10): selection and evaluation of canine Tregs. Vet Immunol Immunopathol. 2010;135:257–265.
3. Steiner D, Brunicki N, Blazar BR, et al. Tolerance induction by third-party “off-the-shelf” CD4 + CD25+ Treg cells. Exp Hematol. 2006;34:66–71.
4. Farivar AS, Yunusov MY, Chen P, et al. Optimizing a canine survival model of orthotopic lung transplantation. Transplant Proc. 2006;38:1638–1640.
5. Koenen HJ, Joosten I. Antigen-specific regulatory T-cell subsets in transplantation tolerance regulatory T-cell subset quality reduces the need for quantity. Hum Immunol. 2006;67:665–675.
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