Development of a coordinated allo T cell and auto B cell response against autosomal PTK2B after allogeneic hematopoietic stem cell transplantation

Abstract

+T-cell and auto-reactive antibody response against an autosomal antigen. ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o nThis antibody response was mapped to a small nonpolymorphic region of the protein. In conclusion, this is the first report demonstrating induction of a coordinated allo-T cell and auto-B cell response against an autosomal antigen after alloSCT. Methods Hematopoietic samplesPeripheral blood and bone marrow samples were obtained from patients and healthy individuals after approval by the Leiden University Medical Center Institutional Review Board and informed consent according to the Declaration of Helsinki. Antigen presentation assaysEBV-LCL were loaded with 1 mg/mL of synthetic peptides in IMDM with 2% FCS and incubated for 2 h at 37°C. Cells were washed twice and plated at 30,000 cells/well in a 96-well plate. Effector cells were added at 5000 T cells/well. After overnight coincubation, IFN-γ production was measured by ELISA (Sanquin, Burton upon Trent, UK) according to the manufacturer's instructions. Helper function assaysTo induce maturation of dendritic cells (DC), CD4 + T-cell clones and immature DC were seeded in a 1:1 ratio in a 24-well plate. After four days of co-incubation, cells were harvested, washed and stained with antibodies for FACS analysis. For induction of B-cell activation, CD4 + T-cell clones and isolated B cells were seeded in a 1:1 ratio and after two days of co-incubation, cells were harvested, washed and stained for FACS analysis. Western blot analysisWhole cell lysates of transfected HEK293T cells were obtained from OriGene (Rockville, USA). HeLa cells were retrovirally transduced with MP71 vector containing PTK2B or PI4K2B constructs; 20 mg of protein was loaded on each lane. SDS-Page was run on pre-cast NuPage® Novex 10% Bis-Tris Mini gels (Invitrogen) for 35 min at 30V under reducing conditions. Gels were blotted on PVDF membranes using XCell SureLock® Mini-Cell blotting system (Invitrogen) according to the manufacturer's instructions. Blots were blocked for 1 h at room temperature in phosphatebuffered saline with 0.05% Tween-20 and 5% BSA, and subsequently incubated with diluted (1:40) serum samples overnight at 4°C. Subsequently, the membrane was incubated with biotinylated anti-human IgG and streptavidin-QDots 625 (Invitrogen) for 1 h each and visualized under UV illumination. Suspension bead arrayThe suspension bead array was performed as previously described. 20,21 Purified proteins (20 mg) were coupled to carboxylated beads (Bio-Rad Laboratories B.V.) according to the manufacturers' instructions. Diluted serum samples (1:100 and 1:300) were pre-absorbed with 0.5% (w/v) polyvinylalcohol and 0.8% (w/v) polyvinylpyrrolidone (Sigma) (PVX) and 1% bovine serum albumin (BSA) for 1 h prior to incubation with the protein-coupled bead mix at room temperature on a shaker for 1 h. In specific blocking experiments, serum samples were pre-absorbed with purified recombinant proteins at 0.5 or 2.0 mg/mL in PVX and 1% BSA. Beads were washed and incubated for another...

Augmentation of Recipient Adaptive Alloimmunity by Donor Passenger Lymphocytes within the Transplant

“…Our findings suggest that inhibition of host NK cell alloresponses may enable GVH recognition by CD4 T cells within the subsequent donor infusion to provide promiscuous help for antibody production from residual host B cells that are concurrently engaging target antigen. This may explain reports documenting the development of humoral immunity against tumor antigen following establishment of mixed hematopoietic chimerism ( Bellucci et al., 2004 , Kremer et al., 2014 ). Similarly, the presence of donor CD4 T cells within donor lymphocyte infusions has been associated with loss of donor mixed hematopoietic chimerism ( Kim et al., 2004 , Hock et al., 2014 ), but rather than this being a bystander consequence of the general inflammatory milieu created by the GVH response ( Hock et al., 2014 ), our results suggest that the loss may instead be due to cognate recognition of MHC class II on the surface of recipient alloreactive B cells by donor CD4 T cells.…”

Allogeneic T cells: maestro in the co-ordination of the immune response after hematopoietic stem cell transplantation

“…[4][5][6][7] It is expected and predicted that the deeper the response the better the outcomes, where, today, outcome is considered in terms of overall survival, while tomorrow it is likely to be treatment-free survival. 8,9 Accordingly, the choice of treatment has traditionally been based on efficacy criteria including rate, time and depth of response. 8,9 This policy has sound clinical bases because CML is a cancer, and the ultimate objective is to provide a cure.…”

“…8,9 Accordingly, the choice of treatment has traditionally been based on efficacy criteria including rate, time and depth of response. 8,9 This policy has sound clinical bases because CML is a cancer, and the ultimate objective is to provide a cure. Consequently, outcome assessment in CML has, till now, been heavily disease oriented.…”

“…Currently, based on at least ten years of experience with imatinib and on the availability of other TKIs, less than 20% of patients are still at risk of dying of leukemia, less than 20% can achieve a treatment-free remission, and more than 60% are facing a situation of chronic, life-long treatment. 9 For many years, we have dedicated our efforts and resources to the evaluation of the response, achieving remarkable success in the standardization of the methods used to assess minimal residual disease (MRD), and widespread agreement on the evaluation of treatment Moving towards patient-centered decision-making in chronic myeloid leukemia: assessment of quality of life and symptom burden Michele Baccarani, 1 Fabio Efficace,…”

“…7,8 One of the identified antigens was derived from PTK2B, a protein belonging to the focal adhesion kinase family. As reported in this edition of the Journal, in their study, Kremer et al 9 chose to focus their attention on the response towards this specific antigen, as it has been documented that PTK2B can be an antibody target in certain transplanted patients treated with DLI for CML relapse. 10 However, it is still unclear whether the antibody response activated in these patients is of an allogeneic or an autologous nature and whether there is a specific T-cell response against PTK2B in this specific cohort of patients.…”