Reconstitution of IL-17-Producing T Helper Cells After Allogeneic Hematopoietic Cell Transplantation

Abstract 4167

Acute graft-versus-host disease (GvHD) following allogeneic hematopoietic cell transplantation (HCT) has been classically assumed to be mediated by T helper cells type 1 (Th1), characterized by the production of interferon-γ (IFN-γ). Recently, Interleukin 17A (IL-17)-producing CD4⁺ T helper type 17 (Th17) cells have also drawn attention as possible effector cells of acute GvHD in murine models. Their role following allogeneic HCT in humans is unknown. We hypothesized that IFN-γ/IL-17-production and quantity of T helper cells might depend on the time-point after HCT, immune responses to allo-antigens (GvHD) or pathogens (e.g. bacterial infection, CMV reactivation) and the presence of T cell depleting antibodies (e.g. ATG). To explore this hypothesiswe initiated a prospective study to investigate the reconstitution of Th1, Th1/17 and Th17 cells in patients after HCT.

80 consecutive patients with various hematologic disorders undergoing allogeneic human leukocyte antigen (HLA)-matched HCT at our center between 12/2009 and 9/2010 were included into the study. Blood samples were collected once in the 1^st month, 2^nd month and 3^rd month after HCT. To quantify IL-17- and IFN-γ-producing T helper cells, we used surface staining for CD3 and CD4 followed by intracellular cytokine staining for IFN-γ and IL-17 in PBMCs. T helper cells producing both IFN-γ and IL-17 (IFN-γ⁺IL-17⁺) were termed Th1/17 cells, T helper cells producing only either cytokine alone are indicated as Th17 cells (IFN-γ⁻IL-17⁺) or Th1 cells (IFN-γ⁺IL-17⁻). For each time period patient cohorts were defined according to the subsequent criteria: (i) bacterial infection (C-reactive protein >50 mg/L, positive blood culture and/or fever in the absence of viral or fungal infection), (ii) CMV reactivation (positive CMV-specific PCR), (iii) acute GvHD (according to the Seattle criteria) and (iv) ATG in the conditioning regimen (dose of 20mg/kg on day -3, -2 and -1 before HCT). As a reference group we chose time-matched and age-matched patients that did not meet any of the criteria under investigation. Student′s t test (two sided, unpaired) was used for statistical evaluation.

In all patients with no relevant complication (absence of bacterial infection, CMV reactivation, acute GvHD) and no ATG in the conditioning regimen Th1, Th1/17 and Th17 cells were detectable within the first month after HCT. However, these T helper cell subsets did not reconstitute to levels of healthy controls within the first 3 month after HCT. In contrast to Th1 cells, no further expansion of Th1/17 and Th17 cells was observed following the 1^st month after HCT. ATG during conditioning significantly reduced the frequency of Th17 cells at all time-points analyzed (median decrease: 1^st month, 71.5%, P=0.0049; 2^nd month, 82.5%, P=0.0002; late engraftment, 71.4%, P=0.0011). Th1/17 cells were also suppressed in patients with ATG, although this reduction was less prominent and reached no significance following the 2^nd month after HCT (median decrease: 1^st month, 76.19%, P=0.012; 2^nd month, 70.11%, P=0.054; late engraftment, 50.7%, P=0.69). Finally, Th1 cells were not significantly reduced in patients receiving ATG compared to time-matches controls (median decrease: 1^st month: 89.18%, P=0.34; 2^nd month: 62.7%, P=0.21; late engraftment: 19.9%, P=0.8), indicating that the suppressive effect of ATG is less pronounced on Th1 and TH1/17 cells, compared to Th17 cells. Acute GvHD°I was not associated with significant changes in the size of the Th1, TH1/17 or Th17 cell subsets. In patients with GvHD°II-IV a tendency towards increased counts of Th1, TH1/17 and TH17 cells in the peripheral blood was observed. However, these changes were not statistically different compared to time-matched controls. CMV reactivation triggered the expansion of all T helper subsets and Th1 cells showed the strongest increase (median increase: Th1, 449.1%, P=0.00075; Th17, 74.9%, P=0.00069; Th1/17, 97.1%, P=0.00012). In contrast, no significant changes were found in the T helper cell compartment of patients with bacterial infection compared to time matched controls.

In conclusion, quantitative reconstitution of Th1, Th1/17 and Th17 cells is impaired within the first 3 months after HCT, especially when ATG is administered during conditioning. CMV reactivation, but not bacterial infection, triggered the absolute expansion of these T cell subsets.