To investigate mechanisms of stem cell graft rejection we studied the allo‐stimulatory potential of G‐CSF mobilized peripheral blood progenitor cells (PBPC). CD34+ cells were purified (> 95%) in a two‐step procedure using immunoaffinity columns for CD34 selection and T‐depletion. The capacity of CD34+ cells to stimulate allogeneic T‐cell responses was compared with other cells from the same individual. CD34+ cells induced potent proliferative responses at stimulator:responder ratios of 1:20, but were approximately 50‐fold less efficient compared to dendritic cells. Furthermore, CD34+ cells primed responses from partially matched allogeneic T cells in bulk cultures. Dual‐colour flow cytometry showed that the co‐stimulatory molecules B7.1, CD40 and ICAM‐1 were absent on resting CD34‐positive progenitor cells, but were induced during incubation with allogeneic lymphocytes due to a cytokine‐mediated effect. Up‐regulation of accessory molecules on CD34+ cells was reproduced by incubation with interferon‐γ or GM‐CSF which enhanced the allo‐stimulatory activity of CD34+ cells. Blocking studies with inhibitory antibodies suggested co‐stimulatory functions for B7.2, ICAM‐3, CD40 and LFA‐3. CD34+ cells were more efficient in inducing allogeneic T‐cell responses when compared to the unprocessed leukapheresis products. The reduced allo‐stimulatory ability of G‐CSF mobilized PBPC could be explained by the presence of CD3+4+ and CD3+8+ lymphocytes with suppressor activity. We conclude that current methods of stem cell selection for transplantation do not avoid allo‐sensitization of the recipient and that further graft manipulation with add‐back of lymphocytes or selection of subsets of CD34+ cells with reduced allo‐stimulatory ability may reduce graft rejection.
Pl A1 and Pl A2 are alternative platelet-specific alloantigens in the Pl A1 system. Sensitization to Pl A1 underlies most cases of neonatal alloimmune thrombocytopenia (NAIT) and posttransfusion purpura (PTP) in white populations. A rapid and simple method for large-scale platelet phenotyping is desirable for identifying expectant mothers at risk of allosensitization and for identifying Pl A1 -negative donors when transfusions are indicated for treatment of NAIT or PTP. We investigated the effectiveness of a solid-phase microplate immunoassay for this purpose. Platelet-rich donor plasmas were tested using the Capture-P ® kit (Immucor, Norcross, GA). Platelet monolayers in microtiter wells were incubated with anti-Pl A1 , washed, and exposed to red blood cells (RBCs) precoated with anti-human IgG. Adherence of RBCs in a diffuse pattern across the well surface indicated the attachment of anti-Pl A1 to Pl A1 -positive platelets whereas sedimentation of unattached RBCs into a central pellet indicated the platelets were Pl A1 -negative. Of 520 donors, 15 (2.88%) tested Pl A1 -negative, which correlates well with the reported Pl A2,2 frequency in whites of 2.25 percent. Results were confirmed by DNA genotyping and/or immunoblotting. This screening technique permits phenotyping donors for Pl A1 alloantigen with minimal specialized equipment. Confirmatory testing for Pl A2 alloantigen can be reserved for donors that test negative for Pl A1 .
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