Microchimerism Detection by HLA-Specific Real-Time Polymerase Chain Reaction Analysis in Recipients of Allogeneic Epstein Barr Virus Specific Cytotoxic T Lymphocytes.

Adoptive immunotherapy with allogeneic EBV specific cytotoxic T lymphocytes (CTL) has been used as a treatment strategy for EBV induced lymphoproliferative disease in stem cell transplant and organ transplant recipients, and is currently being investigated for patients with EBV positive Hodgkin’s disease. Currently available molecular methods for detecting chimerism, such as single tandem repeat (STR) analysis, have a sensitivity range that does not permit the detection of allogeneic effector cells when doses of 10⁶ to 10⁷ cells/kg are infused. We have demonstrated that the infusion of EBV CTL from allogeneic donors can be associated with clinical responses in patients with therapy refractory Hodgkin’s disease, however donor cells could not be detected by STR analysis. We employed HLA-specific real-time polymerase chain reaction analysis (Q-PCR) to test for donor microchimerism in two groups of subjects with relapsed, therapy refractory, EBV positive Hodgkin’s disease. Non-shared, donor-specific HLA sequences (HLA B, DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1) were targeted to detect the DNA equivalent of 1 donor cell in a background of 500,000 host cells. The first cohort consisted of three subjects, each receiving three allogeneic EBV CTL infusions consisting of 5 x 10⁶ cells/kg/dose, with no chemotherapy prior to the infusions. A second cohort received a single infusion of 1.5 x 10⁷ EBV CTL/kg, which was preceded by fludarabine 30 mg/m²/day for three days (in order to attempt to achieve donor CTL chimerism). Informative HLA-specific Q-PCR assays were available for one subject not receiving chemotherapy and for the three subjects receiving fludarabine prior to a single CTL infusion. Results are expressed as genome equivalent number of donor cells per million host cells (gEq/mil). The subject who did not receive fludarabine had no detectable donor DNA prior to the CTL infusion and had 105 gEq/mil 2 days following the first infusion, 230 gEq/mil one day following the second infusion, and only 12 gEq/mil one day after the third infusion. This data suggests that the subject had been sensitized following the first CTL infusion and possibly rejected subsequent infusions. All three subjects who received fludarabine prior to a single EBV CTL infusion had increases in donor DNA post-infusion. One of these subjects had 13,061 gEq/mil one day post-infusion, which decreased to slowly to 124 gEq/mil by 21 days post-infusion, and was at 193 gEq at 3.5 months post-infusion. The other two CTL recipients who received immunosuppressive chemotherapy prior to CTL had increases in donor DNA post-infusion but not of the same maximum amplitude (5026 and 740 gEq/mil at one week post-infusion for each of these subjects). Since subsequent CTL infusions were not given to the second cohort, it is unclear whether the decrease in donor cells over time in this group represented cell rejection or the natural kinetics of allogeneic CTL infused in this setting. These studies demonstrate that quantification of donor cell microchimerism is feasible using Q-PCR for mismatched HLA alleles for subjects receiving allogeneic CTL.