A renewable source of donor cells for repetitive monitoring of T- and B-cell alloreactivity.
A major impediment to repetitive monitoring of alloreactivity or tolerance is the limited supply of donor cells available for assays of host-versus-graft T- and B-cell reactivity. In this paper, we describe the use of CD40L stimulated CD19(+) B cells as targets or stimulators in flow cytometric crossmatching (FXM), mixed lymphocyte reactivity and IFN-gamma ELISPOT assays. Stimulated B cells (sBc) express high levels of MHC class I and II, as well as the costimulatory molecules CD80 and CD86. They can be polyclonally expanded and frozen for later use. We describe the use of sBc in ELISPOT, mixed lymphocyte cultures and FXM. CD4(+) T cells exposed to sBc express a similar cytokine profile as those stimulated with unfractionated PBMC. We further analyzed T- and B-cell responses in 14 patients on the renal transplant waiting list, finding that those with an elevated panel reactive antibody (PRA) (>60%) had higher alloreactive T-cell precursor frequencies as measured by CDFSE MLR and IFN-gamma ELISPOT. We conclude that sBc are a renewable source of donor-specific target/stimulator cells for use in repetitive and coordinate assays of B- and T-cell alloreactivity.
Zand
Martin S
MS
Nephrology Unit, Department of Medicine, University of Rochester Medical Center, Rochester, NY, USA. Martin_Zand@URMC.rochester.edu
Bose
Anirban
A
Vo
Thuong
T
Coppage
Myra
M
Pellegrin
Tina
T
Arend
Lois
L
Lee
F Eun-Hyung
FE
Bozorgzadeh
Adel
A
Leong
Nufatt
N
D000906:Antibodies / Q000737:chemistry; D015703:Antigens, CD / Q000096:biosynthesis; D018941:Antigens, CD19 / Q000096:biosynthesis; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018122:B7-1 Antigen / Q000096:biosynthesis; D017252:CD3 Complex / Q000096:biosynthesis; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D023201:CD40 Ligand / Q000378:metabolism; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D017690:Cell Transplantation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods; D005838:Genotype; D006801:Humans; D007108:Immune Tolerance; D007074:Immunoglobulin G / Q000737:chemistry; D007371:Interferon-gamma / Q000378:metabolism; D016030:Kidney Transplantation; D000075362:Leukocyte Immunoglobulin-like Receptor B1; D008198:Lymph Nodes / Q000473:pathology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism; D010641:Phenotype; D011961:Receptors, Fc / Q000737:chemistry; D011971:Receptors, Immunologic / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D013997:Time Factors; D014019:Tissue Donors
D000906:Antibodies; D015703:Antigens, CD; D018941:Antigens, CD19; D018122:B7-1 Antigen; D017252:CD3 Complex; D007074:Immunoglobulin G; C105299:LILRB1 protein, human; D000075362:Leukocyte Immunoglobulin-like Receptor B1; D011961:Receptors, Fc; D011971:Receptors, Immunologic; D023201:CD40 Ligand; D007371:Interferon-gamma