E. Dijke

Pediatric Thymic Tissue as a Novel Source for the Expansion of Stable Regulatory T Cells for Cellular Therapy.: Abstract# 2273

2274 Suppression of CD8+ Memory T Cell Proliferation and Expansion of T Regulatory Cells By Bone Marrow Derived Autologous Mesenchymal Stem Cells in Nonhuman Primates. T. Oura, K. Hotta, B. Cosimi, G. Benichou, T. Kawai. Transplant Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA. Background: We have previously reported successful induction of tolerance of MHC mismatched renal allografts in nonhuman primates (NHPs) via a mixed hematopoietic chimerism approach. In these studies, we found that the effective suppression of CD8+ memory T cells (TMEM) without inhibition/deletion of CD4+ regulatory T cells (Tregs) is critically important. To date, however, no clinically available modality that can effectively inhibit CD8+ TMEM without jeopardizing Treg function has been identifi ed. In this current NHPs study, we evaluated the immunological properties of bone marrow (BM) derived mesenchymal stem cells (MSCs). Methods: MSCs were characterized as CD73+CD90+CD105+CD34-CD45-. In vitro: Allogeneic CFSE-MLRs were performed with MSCs co-cultured at 1:1 and 1:10 (responder: MSC) ratios. CD4+CD25T cells were co-cultured with or without MSCs at 1:10 (responder: MSC) ratio and cytokines (IL-2: 200 U/ml, TGF-b: 1 ng/ml). In vivo: Four doses of 1x106 autologous MSCs / kg were administered intravenously over one week without conditioning (n=3). Before and after MSC infusion, T cell phenotypes including effector memory T cell (TEM), central memory T cell (TCM), and naïve T cell (TNaive) were monitored. Results: In vitro: MSCs suppressed allogeneic MLR in a dose dependent manner (n = 4) (Fig. A). Similar effects were observed after polyclonal T cell stimulation with beads coated with antibodies against CD2/CD3/CD28 (n = 4). In contrast, Tregs were signifi cantly expanded by MSCs after allo-stimulation (n = 3) (Fig. B). In vivo: Absolute CD8+ TEM counts were markedly decreased, while the frequencies of CD8+ TNaive increased after treatment with MSCs (Fig. C). Finally, a reduction of total CD4+ TEM numbers without a decrease in Treg counts was also observed. Conclusion: Autologous MSCs inhibit the expansion of allospecifi c TMEM (especially CD8+ TEM), while preserving/expanding Tregs and therefore represent a promising adjunctive therapy for induction of chimerism and tolerance in primates. Abstract# 2275 Interferon-gamma and Interleukin-17 Modifi ed Mesenchymal Stem Cells (MSC) Directly or Indirectly Modulate T Cell Responses By Expressing Inhibitory Factors, Downregulating T Cell Activation and Inducing Regulatory T Cells. K. Sivanathan,1,2 C. Hope,1,2 D. RojasCanales,1,2 R. Carroll,1,2 S. Gronthos,3 P. Coates.1,2 1Centre for Clinical and Experimental Transplantation (CCET), Royal Adelaide Hospital, Adelaide, Australia; 2School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, Australia; 3Mesenchymal Stem Cell Group, Department of Hematology, SA Pathology, Adelaide, Australia. 2275 Interferon-gamma and Interleukin-17 Modifi ed Mesenchymal Stem Cells (MSC) Directly or Indirectly Modulate T Cell Responses By Expressing Inhibitory Factors, Downregulating T Cell Activation and Inducing Regulatory T Cells. K. Sivanathan,1,2 C. Hope,1,2 D. RojasCanales,1,2 R. Carroll,1,2 S. Gronthos,3 P. Coates.1,2 1Centre for Clinical and Experimental Transplantation (CCET), Royal Adelaide Hospital, Adelaide, Australia; 2School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, Australia; 3Mesenchymal Stem Cell Group, Department of Hematology, SA Pathology, Adelaide, Australia. MSC have immunosuppressive properties benefi cial for transplantation cellular therapy. We modified human bone marrow-derived MSC with inflammatory cytokines as a strategy to enhance MSC immunosuppression on T cells. MSC were preconditioned with infl ammatory cytokines IFN-γ, TNF-α, IL-1β, IL-2, IL-12 and IL-17 for 5d and were co-cultured with T cells activated by the mitogen phytohemagglutinin (PHA). Modifi ed MSC displayed greater immunosuppression on T cell proliferation compared to unmodified-MSC (UT:MSC). IL-6 gene expression significantly increased in TNF-α and IL-1β-modified MSC following 5d of preconditioning. MSCγ and TNF-α-modifi ed MSC upregulated cyclooxygenase gene expression relative to UT:MSC while only IFN-γ induced indoleamine 2,3-dioxygenase expression in MSC. TGF-β1 gene expression was unaffected following cytokine modifi cation of MSC and IL-10 was undetectable. Mechanistically, only IFN-γ modifi ed MSC (MSCγ) increased the T cell negative co-stimulatory molecule B7-H1. Neutralization of B7-H1 failed to restore T cell proliferative responses suggesting a partial but non-exclusive role of B7-H1 in MSCγ immunosuppression. Furthermore, UT:MSC, MSCγ and IL-17 preconditioned MSC (MSC17) downregulated the T cell activation marker CD25 on CD4+ and CD8+ T cells to a similar extent but does not affect CD69 on T cells. Additionally, an increase in proportion of CD4+CD25hiCD127loFoxp3+ regulatory T cells (Tregs) was observed in PHA-activated T cells co-cultured with MSCγ (1.8 to 4.2-fold) and MSC17 (2.3 to 3-fold) relative to UT:MSC; an effect dependent on the MSC donor and responder T cells. In subsequent experiments, MSCγ and MSC17 that were co-cultured with PHAactivated CD4+CD25T cells showed greater induction of Tregs (iTregs) compared to UT:MSC, with MSC17 showing highest iTregs up to 8.2-fold increase relative to UT:MSC. In 2 of 3 MSC donors, prostaglandin-2 protein levels were highest in MSC17 suggesting a possible involvement of prostaglandin-2 in MSC17-mediated Treg induction. In conclusion, proinfl ammatory cytokine preconditioning enhances immunosuppression in MSC. Abstract# 2276 Myeloid Progenitors Can Induce Tolerance in the Context of Autologous HSC Transplantation. J. Domen,1 Y. Li,1 L. Sun,1 P. Simpson,2 K. Gandy.3 1Cardiac Surgery, Children’s Mercy Hospital and Clinics, Kansas City, MO; 2Pediatrics, Div Quantitative Health Sciences, Medical College of Wisconsin, Milwaukee, WI; 3Biomedical and Health Informatics, University of Missouri-Kansas City, Kansas City, MO. 2276 Myeloid Progenitors Can Induce Tolerance in the Context of Autologous HSC Transplantation. J. Domen,1 Y. Li,1 L. Sun,1 P. Simpson,2 K. Gandy.3 1Cardiac Surgery, Children’s Mercy Hospital and Clinics, Kansas City, MO; 2Pediatrics, Div Quantitative Health Sciences, Medical College of Wisconsin, Milwaukee, WI; 3Biomedical and Health Informatics, University of Missouri-Kansas City, Kansas City, MO. i. Purpose. We have published that tolerance for skin graft transplantation can be consistently induced with Myeloid Progenitor Cells (MP) without long-term highlevel donor-derived engraftment. Importantly, MP are in clinical trials to test their ability to lower infections in bone marrow transplantation, making them clinically available. MP can be given either two months prior to or at the time of organ transplantation for tolerance induction. Using MP from B10;B6-Rag2-/-Il2rg-/mice (devoid of T, B or NK cells) we have established that donor-matched lymphoid cells are not required. Here we test whether tolerance is preserved when MP are combined

Increased Expression of the Inhibitory B-Cell Molecule CD22 on CD27+IgM+ B Cells Containing ABO-Antibody-Secreting Cells: A Role in Susceptibility to B-Cell Tolerance after ABO-Incompatible Infant Heart Transplantation?: 2329

Purpose: Immune immaturity allows ABO-incompatible heart transplantation (ABOi HTx) to be performed safely in infants and generally results in B-cell tolerance to donor ABO antigens. ABO antibodies are thought to arise in a T-independent (TI) manner; TI B-cell activation has been shown to be inhibited by the interaction of the inhibitory B-cell co-receptor CD22 with sialic acids on cells and tissues, leading to B-cell tolerance. It is unknown whether CD22 plays a role in regulating B-cell responses to ABO antigens in the transplant setting. Due to the generally reduced immune response to TI-antigens in early childhood, we hypothesized an enhanced role for CD22 during infancy compared to later in life. In this study we examined CD22 expression on various B-cell subsets. In addition, we performed functional assays to determine CD22 expression after B-cell stimulation and to assess the presence of ABO antigen-specific IgM antibody-secreting cells (ASC). Methods: We analyzed human splenocytes isolated from organ donors (n=41; ages 4 days 74 years). Flow cytometric analysis was performed to quantify the expression levels of CD22, CD27, CD38, IgM and IgG on the surface of CD19+ B cells. CD27+IgM+ B cells and CD27-IgM+ B cells were isolated from human splenocytes (n=5) by automated magnetic activated cell sorting, labelled with Cell Proliferation Dye eFluor450 and stimulated with CpG plus IL-2, IL-10 and IL-15. After one week, CD22 expression was examined by flow cytometry and the frequency of ABO antigen-specific ASC detected by ELISPOT. Results: Significant differences were observed when comparing the median fluorescence intensity (MFI) of CD22 amongst various B-cell subsets (p< 0.0001; Kruskal Wallis test). Post testing using the Dunn‘s multiple comparison test revealed that CD27+IgM+ B cells had higher expression (p< 0.001) than other B-cell subsets. Furthermore, the MFI of CD22 on the CD27+IgM+ B cells was inversely correlated with age (p=0.001), with infant samples having the highest level of CD22, and expression decreasing with increasing age. After culture, downregulated CD22 expression was observed in proliferating CD27+IgM+ B cells compared to non-proliferating cells. ELISPOT analysis showed that the vast majority of ABO antigen-specific ASC were derived from the CD27+IgM+ B-cell population. Conclusion: Based on previous studies showing the inhibitory role of CD22 on B-cell activation, our findings suggest that the increased expression of CD22 on the CD27+IgM+ B-cell subset, which includes precursors of ABO antigen-specific ASC, may cause these infant B cells to be more susceptible to down-regulation of B-cell signaling leading to subsequent inactivation. CD22 may therefore play an inhibitory role in immune responses to ABO antigens in the ABOi HTx setting. Studies are underway to determine the role of CD22 in B-cell signalling and regulation during infancy. 2374