206 Prolonged Stimulation-Induced Pro-Apoptotic B Cells and Deficits in the Germinal Center Formation of Memory B Cells within One Year after Allogeneic HSCT

Track: Poster Abstracts
Wednesday, February 11, 2015, 6:45 PM-7:45 PM
Grand Hall CD (Manchester Grand Hyatt)
Angela Mensen, PhD , Institute for Medical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Youngseong Oh , Institute for Medical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Philipp Hemmati, MD , Hematology, Oncology and Tumor Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Christian Jehn, MD , Hematology, Oncology and Tumor Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Jörg Westermann, MD , Hematology, Oncology and Tumor Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Lam Vuong, PhD , Hematology, Oncology and Tumor Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Bernd Dörken, MD , Max Delbrück Center (MDC) for Molecular Medicine, Berlin, Germany
Carmen Scheibenbogen, MD , Institute for Medical Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Renate Arnold, MD , Hematology, Oncology and Tumor Immunology, Charité - Universitätsmedizin Berlin, Berlin, Germany
Il-Kang Na, MD , Experimental and Clinical Research Center (ECRC), Charité - Universitätsmedizin Berlin, Berlin, Germany
Presentation recording not available for download or distribution as requested by the presenting author.
B-cell immune dysfunction substantially contributes to the risk of severe infections after allogeneic hematopoietic stem cell transplantation (alloHSCT). B-cell numbers normalize within one year after transplantation, however many patients display a slow recovery of CD27+ memory B cells and week vaccination responses. Little is known about functional B-cell deficits associated with memory deficiency post-transplant.

In our study we quantitatively and phenotypically analysed B- and T-cell subsets in peripheral blood by flow cytometry at days 180 and 360 after alloHSCT in acute leukemic patients (n=36). In addition, apoptosis of B-cell subsets was investigated after stimulation with CpG and CD40L. To address the B-cell milieu cytokines and chemokines were measured with Luminex technology.

Half of patients at day 180 and all patients at day 360 displayed fully restored absolute B-cell numbers, although CD27+ memory B-cell subsets remained diminished (cells/µl±SEM: healthy control (HC) 26±4, alloHSCT 4±1; p≤0.001). All B-cell subsets were characterized by an activated/ pro-apoptotic phenotype with an increased CD86 and Fas but reduced Baff-R expression at day 180. Accordingly, an inflammatory milieu was present with increased serum levels of TNFa, IFNa2, G-CSF, IP-10, MCP-1, MIP-1b and Eotaxin in patients compared to HC.  While CD86 normalized at day 360 on most B-cell subsets, all subsets retained a strong pro-apoptotic phenotype with increased Fas and reduced Baff-R expression. Interestingly, stimulation of peripheral blood mononuclear cells with CpG and CD40L resulted in significantly increased percentages of apoptotic cells for the patients compared to HC (% of B cells±SEM: HC 11±2, alloHSCT 28±5 day180, 60±6 day 360; p≤0.001).

Regarding the lack of CD27+ memory B-cell recovery, we assumed a defective germinal center (GC) reaction. Supporting this suggestion we found (A) a reduced CXCR5 expression on naïve B cells, important for the entry into B-cell follicles (MFI±SEM: HC 5899±975, alloHSCT 3494±554; p≤0.01), (B) a low HLA-DR expression on naïve B cells, essential for antigen presentation (HC 20440±4742, alloHSCT 9150±2386; p≤0.01), (C) a normal number of CD27-IgD- double negative (DN) B cells, suggested to prematurely leave the GC reaction, (D) a remaining CD4+ T-cell deficiency (cells/µl±SEM: HC 972±77, alloHSCT 303±45; p≤0.001) and (E) a reduced percentage of circulating CXCR5+ follicular T helper cells, importantly involved in GC reactions (%±SEM: HC 6±2, alloHSCT 2±1).

We conclude that a sustained inflammatory milieu and possibly damage within lymphoid organs might contribute to a prolonged pro-apoptotic state of B-cell subsets and defects in the GC reaction long-term after allo-HSCT. Despite normal total B-cell numbers significant functional deficits exist more than one year after transplant, so that adoptive transfer of memory CD27+ B cells should be pursued.

Disclosures:
Nothing To Disclose