Analysis of changes in recipient and donor hemopoietic cell origin is extremely useful to monitor the effect of stem cell transplantation (SCT) and sequential adoptive immunotherapy by donor lymphocyte infusions (DLI). We developed a sensitive and accurate method to quantify the percentage of recipient and donor cells by real-time PCR using single nucleotide polymorphisms (SNPs) as markers. Allele-specific PCR of seven SNPs resulted in specific markers for donor or recipient in 97% of HLA-identical sibling pairs. Both, recipient-and donor-derived hemopoietic cells can be simultaneously analyzed in 67% sibling pairs. We expect this can be increased to approximately 99% by developing three additional SNP-PCR. Serial dilution of SNP-positive DNA into either SNP-negative DNA or water revealed a detection limit of 0.1-0.01% depending on the amount of input DNA and start C t of the used SNP-PCR. Application of our real-time SNP-PCR method for a CML patient treated by allogeneic SCT and DLI demonstrated its feasibility to follow donor T-cell chimerism and early detection of residual and recurrent autologous hemopoiesis in response to treatment. This detailed monitoring of the genetic origin of hemopoietic cells, in particular immune effector cells and target cells after SCT and DLI, may substantially contribute to understanding of the mechanisms that play a role in the success of treatment.

The quantitative method is based on a real-time PCR (PerkinElmer Applied Biosystems, ABI Prism 7700), with allele-specific primers for DNA sequences containing single nucleotide polymorphisms (SNPs) and target DNA-specific probes. 1,2 The nonextendible hybridization probes are labeled with a reporter fluorescent dye at the 5 0 end and a quencher at the 3 0 end, which results in a fluorescence signal after cleavage by 5 0 -3 0 nuclease activity of Taq polymerase. A charge-coupled device camera attached to ABI Prism 7700 detects target-specific signals at a threshold of 10 standard deviations above the baseline fluorescence. Normalized signals minus baseline signals of the reporter dyes (DR n ) are plotted against PCR cycle numbers, which results in a logarithmic amplification function. The cycle number of DNA amplification that generates the first specific fluorescence signal (DR n ) above the threshold is called cycle threshold (C t ). Therefore, C t represents directly the relative amount of target DNA in the analyzed samples.Allele-specific amplification revealed that each biallelic SNP acts as a specific marker in 24-50% of 80 sibling pairs. The discriminative capacity of each SNP marker depends on the frequency of the SNPs in the human population. Calculation of the percentage donor and recipient cells is based on the relation of specific amplification signals from both recipient and donor DNA to specific amplification signals obtained by calibration curves for both recipient and donor markers. The amount of input DNA is simultaneously calibrated by amplifying a DNA fragment of the albumin gene. Protocol DNA preparationGenomic DNA is isolated from PBMC, purified hemopoietic cell populations, and EBV-transformed B cells with a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) following the protocol of the kit. Samples 450 000 cells are eluted with 200 ml and DNA concentrations are determined by spectrophotometry. Samples r50 000 cells are eluted with 2 Â 25 ml. DNA and the amount of input DNA is only determined by realtime PCR of the albumin gene.DNA from four EBV-transformed B cell lines that contain all 14 SNP alleles are used as calibrators. Aliquots (20 ml) of DNA dilutions, containing 100, 30, 10, 3, 1, 0.3, 0.1 and 0.03 ng/ml, are stored at À801C. In total, 5 ml of these dilutions are amplified by allele-specific real-time SNP-PCR and by the albumin genespecific real-time PCR to construct calibration curves. 3

T lymphocytes used for adoptive immunotherapy are often cultured before transfer to generate sufficient amounts of effector cells with desired specificity. Modification of lymphocytes induced by in vitro activation and expansion may influence their potential effector capacity by altering the survival and trafficking patterns after transfer. In this report, the authors show that the culture period of T cells after ConA/IL-2 stimulation strongly influences the retention and tissue distribution of these cells after infusion into syngeneic C57BL/6 mice. Infused labeled cells that have been cultured for 3 days remained in the peripheral blood and organs in at least a ten-fold higher number than cells cultured for 8 days. In addition, cells cultured for 3 days preferentially migrate to lungs and liver shortly after infusion and subsequently to lymph nodes and spleen. Cells cultured for 8 days preferentially migrate to liver and can be hardly detected in lymph nodes. In contrast, labeled cells cultured for 3 days are predominantly present in lymph nodes starting from day 8 until day 28. We showed that accurate monitoring of transferred cells is feasible, which may contribute to understanding response to adoptive immunotherapy.

Introduction of the HSV-Tk suicide gene into allogeneic T cells offers the possibility to control developing host-reactive cells within the context of allogeneic bone marrow transplantation (BMT). Sensitive quantitative detection methods are a prerequisite to monitor genetically modified T cells in peripheral blood and tissues to study their involvement in graft-versus-host disease (GVHD)-induced lesions as well as their disappearance or persistence after ganciclovir (GCV)-induced suicide. We monitored the alloreactivity of HSV-Tk-transduced T cells after BMT by studying their in vivo distribution and quantity in peripheral blood and in tissues in a WAG/Rij into Brown Norway fully mismatched rat allogeneic BMT model. Genetically modified T cells were quantified in blood and tissues by fluorescence-activated cell sorting, immunohistochemical analysis, and real-time quantitative polymerase chain reaction (PCR) analysis. A significant increase in the number of allogeneic HSV-Tk(+) T cells was found in particular in spleen and lymph nodes and large numbers were found in tongue, skin, and intestines. In blood, an increase in HSV-Tk(+) T cells closely preceded clinical symptoms of GVHD. Real-time quantitative PCR proved to be a fast and accurate tool by which to quantify transduced T cells both in blood and tissues. This enables the study of the in vivo alloreactivity of retrovirus-transduced cells and the response of HSV-Tk-expressing T cells to GCV-induced suicide therapy. Furthermore, we showed the potential use to study specific cause-effect relationships in a broad range of animal and clinical studies involving genetically engineered cells.