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. 2017 Nov 10;8(1):1418.
doi: 10.1038/s41467-017-01631-z.

Allogeneic Stem Cell Transplantation in Fully MHC-matched Mauritian Cynomolgus Macaques Recapitulates Diverse Human Clinical Outcomes

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Free PMC article

Allogeneic Stem Cell Transplantation in Fully MHC-matched Mauritian Cynomolgus Macaques Recapitulates Diverse Human Clinical Outcomes

Benjamin J Burwitz et al. Nat Commun. .
Free PMC article

Abstract

Allogeneic hematopoietic stem cell transplantation (HSCT) is a critically important therapy for hematological malignancies, inborn errors of metabolism, and immunodeficiency disorders, yet complications such as graft-vs.-host disease (GvHD) limit survival. Development of anti-GvHD therapies that do not adversely affect susceptibility to infection or graft-vs.-tumor immunity are hampered by the lack of a physiologically relevant, preclinical model of allogeneic HSCT. Here we show a spectrum of diverse clinical HSCT outcomes including primary and secondary graft failure, lethal GvHD, and stable, disease-free full donor engraftment using reduced intensity conditioning and mobilized peripheral blood HSCT in unrelated, fully MHC-matched Mauritian-origin cynomolgus macaques. Anti-GvHD prophylaxis of tacrolimus, post-transplant cyclophosphamide, and CD28 blockade induces multi-lineage, full donor chimerism and recipient-specific tolerance while maintaining pathogen-specific immunity. These results establish a new preclinical allogeneic HSCT model for evaluation of GvHD prophylaxis and next-generation HSCT-mediated therapies for solid organ tolerance, cure of non-malignant hematological disease, and HIV reservoir clearance.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Primary and secondary engraftment failure in MHC-matched MCM post-allogeneic HSCT. a, b Longitudinal white blood cell (WBC), T cell, and platelet absolute counts in recipient macaques 32851 (a), 32846 (b), and 32849 (b). Immune conditioning regimens are indicated in top panels (Bu = busulfan, Flu = fludarabine, TBI = total body irradiation, CD3-IT = CD3-immunotoxin). CD8 depleting mAb was administered only to 32849, indicated by an asterisk (*). Colored circumflexes (^) indicate time points at which donor-derived cells were detected in whole blood by chimerism assays. Crosses (†) indicate time of necropsy. Normal WBC reference range = 5,600–184,000/μl; normal platelet reference range = 134,000–564,000/μl. Shaded gray boxes in ac indicate tacrolimus treatment period. Vertical dotted line in b indicates second transplant of 32846. c Longitudinal donor chimerism levels in 32846 and 32849 as measured by Illumina sequencing whole blood genomic DNA across SNPs differing between donor and recipient. Graphs display mean ± SEM frequencies of donor-derived cells as measured by sequencing two (32849) or three (32846) SNPs. d Mixed lymphocyte reactions assessing levels of CD4+ and CD8+ T cell alloreactivity in recipient macaque 32846 pre-HSCT and 28 days post-HSCT. Plots display proliferation of CFSE-labeled recipient T cells in response to irradiated donor cells, as measured by frequency of CFSE-lo cells. Plots are gated on live, CD3+ singlets, and either CD4+ or CD8+ cells. Recipient cells cultured alone (no stim) or with Staphylococcus enterotoxin B (SEB) served as negative and positive controls, respectively.
Fig. 2
Fig. 2
Lethal GvHD in fully MHC-matched MCM post-allogeneic HSCT. a Longitudinal white blood cell (WBC), T cell, and platelet absolute counts in recipient macaques 33450 and 33455. Immune conditioning regimens are indicated in top panels (Bu = busulfan, Flu = fludarabine, TBI = total body irradiation, CD3-IT = CD3-immunotoxin). *Fludarabine was administered only to 33450. Colored circumflexes (^) indicate timepoints at which donor-derived cells were detected in whole blood by chimerism assays. Crosses (†) indicate time of necropsy. Normal WBC reference range = 5,600–18,4000/μl; normal platelet reference range = 134,000–564,000/μl. Shaded gray boxes in a, b indicate tacrolimus treatment period. b Longitudinal donor chimerism levels as measured by Illumina sequencing whole blood genomic DNA across SNPs differing between donor and recipient. Graphs display mean ± SEM frequencies of donor-derived cells as measured by two SNPs. c Longitudinal clinical GvHD scoring. Scoring criteria described in Methods section. d Multifocally extensive exfoliative dermatitis of thoracic limb of recipient macaque 33450. e Blinded tissue histopathology GvHD scoring at time of necropsy. Scoring criteria described in Methods section. f Tissue donor chimerism levels at time of necropsy as measured by Illumina sequencing genomic DNA across SNPs differing between donor and recipient. Graphs display mean ± SEM frequencies of donor-derived cells as measured by sequencing two SNPs. To eliminate non-immune cell contamination, colon, jejunum, and liver cell preparations were sorted for CD45+ cells prior to genomic DNA extraction. g Frequencies of Ki67+ T cells in tissues at time of necropsy, as determined by intracellular Ki67 flow cytometric staining. h Mixed lymphocyte reactions assessing levels of CD4+ and CD8+ T cell alloreactivity in recipient macaques 33450 and 33455 post-HSCT (at time of necropsy). Plots display proliferation of CFSE-labeled recipient T cells in response to irradiated donor or pre-HSCT recipient cells, as measured by frequency of CFSE-lo cells. Plots are gated on live, CD3+ singlets, and either CD4+ or CD8+ cells. Recipient cells cultured alone (no stim) or with SEB served as negative and positive controls, respectively.
Fig. 3
Fig. 3
Stable, multi-lineage donor chimerism in MHC-matched MCM post-allogeneic HSCT. a Longitudinal white blood cell (WBC), T cell, and platelet absolute counts in recipient macaques 33454 and 34666. Immune conditioning regimens are indicated in top panels (Bu = busulfan, TBI = total body irradiation, CD3-IT = CD3-immunotoxin). Normal WBC reference range = 5,600–184,000/μl; normal platelet reference range = 134,000–564,000/μl. Shaded gray boxes in a, b indicate tacrolimus treatment period. b, c Donor chimerism levels as measured by Illumina sequencing genomic DNA across SNPs differing between donor and recipient. Specific populations were isolated by flow cytometric cell sorting of ACK-treated whole blood prior to genomic DNA extraction. Longitudinal whole blood (WB), granulocyte, and T cell donor chimerism levels shown in b. Timepoints of donor lymphocyte infusions (DLIs) into 33454 are indicated with arrows in b (bottom panel); DLI #1 = 1 × 107 CD3+ cells/kg, DLI #2 = 5 × 107 CD3+ cells/kg, DLI #3 = 5 × 107 CD3+ cells/kg. Comprehensive immune cell subset donor chimerism levels in whole blood (top) and lymph node (bottom) shown in c, measured 428 days (33454) or 193 days (34666) post-HSCT. N.D. = not determined due to insufficient cell numbers. d Mixed lymphocyte reactions assessing levels of CD4+ and CD8+ T cell alloreactivity in recipient macaques 33454 and 34666 post-HSCT (354 and 298 days post-HSCT, respectively). Plots display proliferation of CFSE-labeled recipient T cells in response to irradiated donor or pre-HSCT recipient cells, as measured by frequency of CFSE-lo cells. Plots are gated on live, CD3+ singlets, and either CD4+ or CD8+ cells. Recipient cells cultured alone (no stim) or with SEB served as negative and positive controls, respectively.

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