As antibodies to tumor necrosis factor (TNF) suppress immune responses in Crohn’s disease by binding to membrane-bound TNF (mTNF), we created a fluorescent antibody for molecular mTNF imaging in this disease. Topical antibody administration in 25 patients with Crohn’s disease led to detection of intestinal mTNF+ immune cells during confocal laser endomicroscopy. Patients with high numbers of mTNF+ cells showed significantly higher short-term response rates (92%) at week 12 upon subsequent anti-TNF therapy as compared to patients with low amounts of mTNF+ cells (15%). This clinical response in the former patients was sustained over a follow-up period of 1 year and was associated with mucosal healing observed in follow-up endoscopy. These data indicate that molecular imaging with fluorescent antibodies has the potential to predict therapeutic responses to biological treatment and can be used for personalized medicine in Crohn’s disease and autoimmune or inflammatory disorders.

BackgroundA major complication after allogeneic hematopoietic stem cell transplantation (aSCT) is the reactivation of herpesviruses such as cytomegalovirus (CMV) and Epstein–Barr virus (EBV). Both viruses cause significant mortality and compromise quality of life after aSCT. Preventive transfer of virus-specific T cells can suppress reactivation by re-establishing functional antiviral immune responses in immunocompromised hosts.MethodsWe have developed a good manufacturing practice protocol to generate CMV/EBV-peptide-stimulated T cells from leukapheresis products of G-CSF mobilized and non-mobilized donors. Our procedure selectively expands virus-specific CD8+ und CD4+ T cells over 9 days using a generic pool of 34 CMV and EBV peptides that represent well-defined dominant T-cell epitopes with various HLA restrictions. For HLA class I, this set of peptides covers at least 80% of the European population.ResultsCMV/EBV-specific T cells were successfully expanded from leukapheresis material of both G-CSF mobilized and non-mobilized donors. The protocol allows administration shortly after stem cell transplantation (d30+), storage over liquid nitrogen for iterated applications, and protection of the stem cell donor by avoiding a second leukapheresis.ConclusionOur protocol allows for rapid and cost-efficient production of T cells for early transfusion after aSCT as a preventive approach. It is currently evaluated in a phase I/IIa clinical trial.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1498-3) contains supplementary material, which is available to authorized users.

BackgroundWe have recently shown that memory B cells from murine CMV immune donor animals adoptively transferred into immunodeficient mice were highly effective in protecting from a viral infection indicating a therapeutic potential of virus specific memory B cells. These preclinical data provided evidence that a cell-based strategy supporting the humoral immune response might be effective in a clinical setting of immunodeficiency after allogeneic hematopoietic stem cell transplantation. As adoptive transfer of B cells has not been used before in a clinical setting it was necessary to establish a technology for the generation of good manufacturing practice (GMP)-grade B cell products.MethodsStarting from the leukapheresis product of healthy blood donors, B cells were purified by two different separation strategies using GMP-grade microbeads and the CliniMACS system. A one-step protocol was used for positive enrichment of B lymphocytes with anti-CD19 microbeads. In a two-step enrichment protocol, first T lymphocytes were depleted by anti-CD3 microbeads and the remaining fraction was positively selected by anti-CD19 microbeads.ResultsThe purity and recovery after enrichment of B lymphocytes from the leukapheresis material in both separations strategies was not statistically different. However, contamination of the B-cell product with T cells was significantly lower after the two-step protocol (0.16%, range 0.01–0.43% after two-step separation and 0.55%, range 0.28–0.85% after one-step separation, p < 0.05). Therefore, a combined CD3 depletion and CD19 enrichment was used for the production of GMP-conform B-cell products from the leukapheresis material of 17 healthy stem cell donors. The absolute B-cell numbers obtained in the final product was 4.70 ± 3.64 × 108 with a purity of 95.98 ± 3.31% B lymphocytes and a recovery of 18.9 ± 10.6%. Importantly, the contamination with CD3+ T cells was extremely low in the final B- cell products (0.10 ± 0.20%). Purified B cells exhibited normal antibody production after in vitro stimulation and showed excellent viability after cryopreservation.ConclusionsA GMP-grade B-cell product can be obtained with high purity and very low T-cell contamination using the two-step enrichment protocol based on CliniMACS® technology.

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Patients after allogeneic stem cell transplantation (alloSCT) show a long lasting immune deficiency involving both T- and B-lymphocytes. Antibody responses to vaccination, as a measure of functional B-cell immunity, are insufficient in the first months after alloSCT and usually recover within 1-2 years. To improve B-cell immune reconstitution after alloSCT, we developed a novel concept of adoptive transfer of memory B-cells from the original stem cell donor (Klenovsek et al., Blood 110:3472-79, 2007). To this end, we first established the technology for the manufactoring of a GMP-qualified B-cell product and initiated a first-in-man phase I/IIa clinical trial in patients after alloSCT to evaluate safety and tolerability of adoptively transferred donor B-cells. B-cells were manufactured under GMP conditions from 16 unstimulated leukapheresis products derived from the original stem cell donor. The separation protocol consists of two subsequent separation steps on the CliniMACS® System (Miltenyi Biotec) including depletion of CD3+ T-cells and subsequent positive separation of CD19+ B-cells by magnetic beads. In all of the 16 cell products high purities of CD20+ B-cells (average 96%, range 85-99%) with only low frequencies of contaminating T-cells (average 0.10 %, range 0.01-0.82%) were achieved. After manufacturing, B-cell products were cryopreserved for single administration to 4 groups of patients with escalating doses. To prevent GvH reactions the absolute number of CD3+ T lymphocytes in the B-cell product had to be below 4 x 104 CD3+ cells/kg BW. The clinical trial was designed as an open label dose escalating study using the traditional 3+3 design with 4 dose levels of 0.5-1-2-4x106 B-cells/kg BW. Patients were enrolled on day +140 ± 20 after alloSCT after tapering of immunosuppression. Patients with acute GVHD grade III/IV, chronic GVHD with intermediate to high risk, EBV reactivation (>10,000 EBV copies/ml) or after therapy with B-cell antibodies (i.e. rituximab) were not eligible. Up to now, 13 patients after alloSCT received donor B-cells in 4 different B-cell dosages (average day +147, range from day +130 to d +160). Three patients received 0.5x106 B-cells per kg BW, 3 patients 1.0x106 B-cells/kg BW, 5 patients 2.0x106 B-cells per kg BW, and 2 patients 4.0x106 B-cells/kg BW, respectively. The median number of infused T cells was 0.06x104 T-cells per kg BW (range 0.01-0.13 x104/kg BW). Regarding primary safety data, the B-cell transfer was well tolerated without any acute adverse reactions. Importantly, neither significant EBV-viremia nor acute or chronic GvHD reactions were observed during the observation period of 4 months after the transfer of B-cells. In addition to the analysis of the primary safety endpoints of the trial, the activity of infused donor memory B-cells in vivo were tested. A characteristic memory B-cell response is defined as a mobilization of plasmablasts (PBs) 7 days after booster vaccination into the peripheral blood (Odendahl et al., Blood 105: 1614-21, 2005). Preliminary results obtained from patients that received the donor derived B-cells demonstrated significant mobilization of PBs in some of the patients after re-vaccination with a pentavalent vaccine (Pentavac®), suggestive for a memory B-cell response. A detailed analysis of the vaccination data from the study patients in comparison to a control group of transplanted patients without B-cell transfer after routine re-vaccination will be presented. Furthermore, the analysis of immunoglobulin VH-sequences by next-generation-sequencing in memory B-cell populations of two individual patients after B-cell transfer revealed that at least 50% of all memory B-cell clones identified in the recipient are unambiguously contained within the memory B-cell population of the infused B-cell product. In summary, our data show that the manufacturing of a B-cell product from the original stem cell donor under GMP conditions without significant T-cell contamination is feasible. The transfer of donor-derived B-cells into patients after alloSCT is safe with regard to EBV reactivation and induction of acute or chronic GvHD. Future clinical trials will evaluate whether the adoptive transfer of memory B-cells from the donor can significantly reduce the frequency of infections after alloSCT. (Supported by the Deutsche Forschungsgemeinschaft through SFB 643 "Strategies of cellular immune intervention") Disclosures No relevant conflicts of interest to declare.