Changes in the Immune Functions during ALL Induction Therapy - Prospective Immune Monitoring in 23 Children.

Introduction: Inadequate lymphocyte functions are an important clinical problem complicating chemotherapy by severe bacterial and fungal infections. During an ongoing immune response, cytokines produced by T-cells are critical to the effectiveness of that response. To understand what happens with the immune system in the beginning of therapy, we investigated lymphocyte subpopulations and functioning (cytokines, immunoglobulins).

Patients: 23 children (median age 5y;range 2m-14y;15 female, 8 male) with B precursor ALL were treated according to the ALL-BFM 2000 protocol. Blood samples were drawn at diagnosis and on days 8, 15, 33 and 64 and before beginning of protocol M, protocol 2 and during maintenance therapy.

Methods: We analyzed lymphocyte subpopulations using flow cytometry. Intracellular cytokines (IFNg, IL2, TNFa, IL4, IL5, and IL10) were determined by FACS after in vitro stimulation with PMA, ionomycin and brefeldin for 24h. Additionally, we measured various cytokines in unstimulated sera by ELISA. Furthermore, we studied TRECs and Spectratype values and immunoglobulins.

Results: As to lymphocyte subpopulations, B-cells decreased rapidly from a median value of 301+120/mm_ before chemotherapy to 85+138/mm_ during chemotherapy (“day 33”) and did not reappear until the end of therapy.

T cells decreased at the beginning of chemotherapy, but there was a partial recovery proportional to the intensity of chemotherapy. Also we found a shift towards CD8+ cells. NK cells did not show significant changes.

As to cytokines in CD3+ T-cells, we detected an increase of IFNg (diagnosis:11+9%, day 33: 22+7%). We could detect a correlation between gd-TCR and IFNg production (FACS) as well as IFNg values (ELISA) and high memory cell count. Also CD45RA+cells correlate with CD4IL2+cells. Finally TGFb in unstimulated sera correlated strongly (p<0,01) with CD19+cells. TGFb was also produced by blasts. We detected difference in cytokine profile between conditioning with dexamethasone vs. prednisone, especially the IFNg secretion was much higher in the prednisone branch (p<0,01). TREC values were higher in the dexamethason branch, but this might be due to the age, because younger children had significantly higher TREC values. Analyses of TCR repertoire show a slightly higher complexity in the prednisone branch. Until d15 the repertoire complexities were reduced and increase slowly continuingly during therapy. We could detect Vb gene families with high complexity during the whole therapy (for example BV22, BV23) and with low complexity (BV13b, BV6a). The complexity correlated positively with the number of naïve T-cells and with the age (p<0,01).

Summary: We detected a shift towards Th1-cytokines during ALL-induction-therapy. B cells were erased during chemotherapy. This might be of clinical importance of the possible necessity of prophylactic immunoglobulins to prevent serious infection because of the lack of B-cells.