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Adoptive transfer of alloantigen-specific immunomodulatory cells generated ex vivo with anti-CD80/CD86 mAbs (2D10.4/IT2.2) holds promise for operational tolerance after transplantation. However, good manufacturing practice is required to allow widespread clinical application. Belatacept, a clinically approved cytotoxic T-lymphocyte antigen 4-immunoglobulin that also binds CD80/CD86, could be an alternative agent for 2D10.4/IT2.2. With the goal of generating an optimal cell treatment with clinically approved reagents, we evaluated the donor-specific immunomodulatory effects of belatacept- and 2D10.4/IT2.2-generated immunomodulatory cells. Immunomodulatory cells were generated by coculturing responder human peripheral blood mononuclear cells (PBMCs) (50 x 10(6) cells) with irradiated donor PBMCs (20 x 10(6) cells) from eight human leukocyte antigen-mismatched responder-donor pairs in the presence of either 2D10.4/IT2.2 (3 mu g/10(6) cells) or belatacept (40 mu g/10(6) cells). After 14 days of coculture, the frequencies of CD4(+) T cells, CD8(+) T cells, and natural killer cells as well as interferon gamma (IFN-gamma) production in the 2D10.4/IT2.2- and belatacept-treated groups were lower than those in the control group. The percentage of CD19(+) B cells was higher in the 2D10.4/IT2.2- and belatacept-treated groups than in the control group. The frequency of CD4(+)CD25(+)CD127(low)FOXP3(+) T cells increased from 4.1 +/- 1.0% (preculture) to 7.1 +/- 2.6% and 7.3 +/- 2.6% (day 14) in the 2D10.4/IT2.2- and belatacept-treated groups, respectively (p<0.05). Concurrently, delta-2 FOXP3 mRNA expression increased significantly. Compared with cells derived from the no-antibody treated control group, cells generated from both the 2D10.4/IT2.2- and belatacept-treated groups produced lower IFN-gamma and higher interleukin-10 levels in response to donor-antigens, as detected by enzyme-linked immunospot. Most importantly, 2D10.4/IT2.2- and belatacept-generated cells effectively

QC 20161028

Allogeneic hematopoietic stem cell transplantation is associated with several complications and risk factors, for example, graft versus host disease (GVHD), viral infections, relapse, and graft rejection. While high levels of CD3+ cells in grafts can contribute to GVHD, they also promote the graft versus leukemia (GVL) effect. Infusions of extra lymphocytes from the original stem cell donor can be used as a treatment after transplantation for relapse or poor immune reconstitution but also they increase the risk for GVHD. In peripheral blood, 95% of T-cells express the alpha beta T-cell receptor and the remaining T-cells express the gamma delta. T-cell receptor. As alpha beta T-cells are the primary mediators of GVHD, depleting them from the graft should reduce this risk. In this pilot study, five patients transplanted with HLA-matched related and unrelated donors were treated with alpha beta T-cell depleted stem cell boosts. The majority of gamma delta T-cells in the grafts expressed V delta 2 and/or V gamma 9. Most patients receiving alpha beta-depleted stem cell boosts increased their levels of white blood cells, platelets, and/or granulocytes 30 days after infusion. No signs of GVHD or other side effects were detected. A larger pool of patients with longer follow-up time is needed to confirm the data in this study., QC 20170419

The molecular basis underlying T-cell recognition of MHC molecules presenting altered peptide ligands is still not well-established. A hierarchy of T-cell activation by MHC class I-restricted altered peptide ligands has been defined using the T-cell receptor P14 specific for H-2D b in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H-2D b in complex with the alanine-substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H-2D b/gp33 was strictly enthalpy driven, recognition of the weak agonist H-2D b/Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H-2D b/gp33 compared with H-2D b/Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex., QC 20121213

QC 20191001