Cytokine Patterns and Lymphocyte Subsets in Patients with Chronic GVHD after Allogeneic Stem Cell Transplantation with Reduced Intensity Conditioning.

Background .Chronic GVHD (cGVHD) is the most common and severe late complication after allogeneic stem cell transplantation (SCT), because of increasing use of matched unrelated donors, peripheral blood stem cells and reduced intensity conditioning (RIC). Impaired immune reconstitution and unbalanced Th1-type or Th2-type immune response with cytokines dysregulation seem to play a key role in the pathogenesis of chronic GVHD (cGVHD).

Aims. In order to study the immunologic mechanisms involved in cGVHD, we prospectively evaluated the Th1 (TNF-alpha, IFN-gamma) and Th2 (IL-4, IL-6, IL-10) cytokine patterns and lymphocyte subsets in the setting of allogeneic SCT-RIC.

Patients and methods. We assessed by ELISA the serum levels of TNF-alpha, IFN-gamma, IL-4, IL-6, IL-10 and soluble tumor necrosis factor receptors I and II (sTNF-R) in 8 healthy donors and in 19 patients undergoing allogeneic SCT-RIC (13 patients with related and 6 patients with unrelated donor). Serum levels were assessed before transplantation and monthly from second to twelfth month after SCT. Total lymphocytes and their subsets with different co-stimulatory molecules (CD4, CD8, CD19, CD28/3,CD25/4, CD134/4, CD152/3, CD16/56, CD4/45ro, CD4/45ra, CD8/45ro, CD8/45ra) were evaluated by flow cytometry in peripheral blood (PB) monthly after SCT. Cyclosporine A was used as GVHD prophylaxis in all patients from third month until its tapering or until the beginning of the therapy against extensive cGVHD. Wilcoxon test was used for statistical analysis of the data.

Results. Twelve out of 19 patients developed cGVHD at a median time of 7 months (range, 6–10); cGVHD was extensive in 8 patients at a median time of 8 months (range, 6–12). The levels of cytokines did not differ significantly in patients pre-SCT and healthy donors. Patients with cGVHD differed from those without cGVHD because of:

1. significantly higher levels of TNF-alpha from third to sixth month after SCT (3^rd, p=0.003; 4^th p=0.001; 5^th, p=0.0005; 6^th, p=0.01);

2. significantly higher levels of sTNF-R II at 6^th (p=0.01);

3. significantly higher levels of IL-10 (p= 0.004) at 4th month;

4. significantly lower number of NK cells in PB from third to sixth month after SCT (3^rd, p=0.004; 4^th p=0.009; 5^th, p=0.0007; 6^th, p=0.0003);

5. significantly lower number of CD 152/3 cells in PB from third to sixth month after SCT (3^rd, p=0.009; 4^th p=0.0004; 5^th, p=0.0004; 6th, p=0.007);

6. significantly lower number of CD 4/25 cells in PB at 4^th (p=0.0004) and 5^th (p=0.01)

Discussion. Our sequential study showed:

1. increased levels of Th1 (TNF-alpha) and Th2 (IL-10) cytokines with different kinetics after SCT and before the onset of cGVHD;

2. a decrease of NK and T cells with regulatory molecules such as CD152 after SCT and before the onset of cGVHD.

These results suggest an overall prevalence of a TNF-alpha oriented response in patients with cGVHD. Defects of immunoregulatory cells could be related to these fluctuating and unbalanced cytokine patterns. However, further studies with more patients are required to support these preliminary results.