Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture.

Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S bioassay with regards to specificity, sensitivity, detection limit, and reproducibility. We have found the optimal assay conditions to be 1 x 10 (4) CT.h4S cells/well deprived of IL-4 for 24 h and preincubated for 7 h followed by 18 h of incubation with tritiated methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. With the use of 1x10(6) responder cells/well in HLA-mismatched MLC, we found limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S bioassay is a reliable and specific method for quantification of IL-4 accumulation in cultures of human MNC. The difference in optimal timing for IL-2 (day 3) and IL-4 (>/=day 12) detection and evidence of very low IL-4 producing HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay questionable.

Alloreactivity and the predictive value of anti-recipient specific interleukin 2 producing helper T lymphocyte precursor frequencies for alloreactivity after bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) is a treatment modality with the potential of curing otherwise lethal diseases. The predominant indications for BMT are haematological malignancies. In BMT alloreactivity plays a pivotal role for the outcome. Graft-versus-host disease (GvHD) and graft-versus-leukaemia (GvL) are correlated manifestations of alloreactivity. Severe GvHD is one of the main causes of morbidity and mortality post-BMT. In the absence of GvL the risk of relapse is high. The main effector cells are T lymphocytes. Donor leukocyte infusion (DLI) for treatment of leukaemic relapse after BMT can induce durable remissions. DLI causes GvHD in the majority of the responding patients. However, a GvL effect may be present without evidence of GvHD and vise versa. The importance of alloreactivity for the treatment outcome prompted the interest for a predictive test of alloreactivity. Interleukin 2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies determined by limiting dilution analysis (LDA) in the graft-versus-host direction were explored. The HTLp assay was optimized and the sources of error minimized to ensure sensitive and reproducible results. The IL-2 dependent cell line, CTLL-2 was optimized to detect 0.6 pg IL-2. UV-B irradiation of the cells was demonstrated to effectively terminate proliferation of the responder cells and thus allow IL-2 to be detected in the whole culture volume. The design of the assay was explored by Monte Carlo simulations resulting in a design yielding frequencies with a coefficient of variation of 20% in the range of 1:20,000-1:1,000,000. The influence of autoreactivity of the donor and recipient cells was minimized as well as the risk of the stimulator cells producing IL-2. The HTLp frequencies correlated with the degree of human leukocyte antigen (HLA) disparity and the assays were able to detect minor histocompatibility antigen mismatches. The HTLp frequencies of 28 HLA-identical sibling BMT pairs and 20 HLA-matched unrelated and partially HLA-matched related BMT pairs were determined. HTLp frequencies from the HLA-identical sibling BMT pairs had a median of 1:557,362 (range 1:9.511 to < 1:2,500,000). The HTLp frequencies from the HLA-matched unrelated and partially HLA-matched related BMT pairs had a median of 1:88,110 (range 1:4.139-1:736,123). Analysis of the HLA-identical sibling BMT pairs in a high and a low HTLp frequency group above and below 1:500,000 showed a trend towards a higher risk of acute GvHD > or = grade II and a significantly higher risk of chronic GvHD in the high HTLp frequency group. This group had a significantly lower risk of relapse as well as a significantly better overall survival and leukaemia free survival. The HLA-matched unrelated and partially HLA-matched related BMT pairs were split evenly in a high and a low HTLp frequency group above and below 1:90,000. There was a significantly higher risk of acute GvHD > or = grade II and a trend towards a higher treatment related mortality (TRM) in the high HTLp frequency group. There were no differences in chronic GvHD, risk of relapse, overall survival and leukaemia free survival. Analyzing all 48 patients the risk of acute GvHD > or = grade II and TRM was significantly higher with HTLp frequencies > 1:100,000 and there was a trend towards a higher risk of relapse with low HTLp frequencies < 1:400,000. Patients in the intermediate HTLp frequency group 1:100,000-1:400,000 had a trend towards improved survival. The HTLp frequency seems to detect clinically significant differences in alloreactivity, that can be useful in donor selection, graft-engineering, T cell add-back and the pharmacological immunosuppression used after BMT.