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Abstract In clinical situations, peripheral blood accessible CD3+CD4+CXCR5+ T-follicular helper (TFH) cells may have to serve as a surrogate indicator for dysregulated germinal center responses in tissues. To determine the heterogeneity of TFH cells in peripheral blood versus tonsils, CD3+CD4+CD45RA–CXCR5+ cells of both origins were sorted. Transcriptomes, TCR repertoires and cell-surface protein expression were analysed by single-cell RNA sequencing, flow cytometry and immunohistochemistry. Reassuringly, all blood-circulating CD3+CD4+CXCR5+ T-cell subpopulations also appear in tonsils, there with some supplementary TFH characteristics, while peripheral blood-derived TFH cells display markers of proliferation and migration. Three further subsets of TFH cells, however, with bona fide T-follicular gene expression patterns, are exclusively found in tonsils. One additional, distinct and oligoclonal CD4+CXCR5+ subpopulation presents pronounced cytotoxic properties. Those ‘killer TFH (TFK ) cells’ can be discovered in peripheral blood as well as among tonsillar cells but are located predominantly outside of germinal centers. They appear terminally differentiated and can be distinguished from all other TFH subsets by expression of NKG7 (TIA-1), granzymes, perforin, CCL5, CCR5, EOMES, CRTAM and CX3CR1. All in all, this study provides data for detailed CD4+CXCR5+ T-cell assessment of clinically available blood samples and extrapolation possibilities to their tonsil counterparts.

Abstract The adoptive transfer of regulatory T cells is a promising strategy to prevent graft-versus-host disease after allogeneic bone marrow transplantation. Here, we use a major histocompatibility complex-mismatched mouse model to follow the fate of in vitro expanded donor regulatory T cells upon migration to target organs. Employing comprehensive gene expression and repertoire profiling, we show that they retain their suppressive function and plasticity after transfer. Upon entering non-lymphoid tissues, donor regulatory T cells acquire organ-specific gene expression profiles resembling tissue-resident cells and activate hallmark suppressive and cytotoxic pathways, most evidently in the colon, when co-transplanted with graft-versus-host disease-inducing conventional T cells. Dominant T cell receptor clonotypes overlap between organs and across recipients and their relative abundance correlates with protection efficacy. Thus, this study reveals donor regulatory T cell selection and adaptation mechanisms in target organs and highlights protective features of Treg to guide the development of improved graft-versus-host disease prevention strategies.

Summary: Background: Induction of donor-specific tolerance is a promising approach to achieve long-term graft patency in transplantation with little to no maintenance immunosuppression. Changes to the recipient’s T cell receptor (TCR) repertoire are understood to play a pivotal role in the establishment of a robust state of tolerance in chimerism-based transplantation protocols. Methods: We investigated changes to the TCR repertoires of patients participating in an ongoing prospective, controlled, phase I/IIa trial designed to evaluate the safety and efficacy of combination cell therapy in living donor kidney transplantation. Using high-throughput sequencing, we characterized the repertoires of six kidney recipients who also received bone marrow from the same donor (CKBMT), together with an infusion of polyclonal autologous Treg cells instead of myelosuppression. Findings: Patients undergoing combination cell therapy exhibited partial clonal deletion of donor-reactive CD4+ T cells at one, three, and six months post-transplant, compared to control patients receiving the same immunosuppression regimen but no cell therapy (p = 0.024). The clonality, R20 and turnover rates of the CD4+ and CD8+ TCR repertoires were comparable in both groups, showing our protocol caused no excessive repertoire shift or loss of diversity. Treg clonality was lower in the case group than in control (p = 0.033), suggesting combination cell therapy helps to preserve Treg diversity. Interpretation: Overall, our data indicate that combining Treg cell therapy with CKBMT dampens the alloimmune response to transplanted kidneys in humans in the absence of myelosuppression. Funding: This study was funded by the Vienna Science and Technology Fund (WWTF).

Chimeric antigen receptor (CAR-) T cell therapy causes serious side effects including cytokine release syndrome (CRS). CRS-related coagulopathy is associated with hypofibrinogenemia that is thus far considered the result of disseminated intravascular coagulation (DIC) and liver dysfunction. We investigated incidence and risk factors for hypofibrinogenemia in 41 consecutive adult patients with hematologic malignancies (median age 69 years, range 38-83 years) receiving CAR-T cell therapy between 01/2020 and 05/2023 at the University Medical Center Regensburg. CRS occurred in 93% of patients and was accompanied by hypofibrinogenemia already from CRS grade 1. Yet, DIC and liver dysfunction mainly occurred in severe CRS (≥ grade 3). After an initial increase during CRS, fibrinogen levels dropped after administration of tocilizumab in a dose dependent manner (r = -0.44, p = 0.004). In contrast, patients who did not receive tocilizumab had increased fibrinogen levels. Logistic regression analysis identified tocilizumab as an independent risk factor for hypofibrinogenemia (odds ratio = 486, p < 0.001). We thus hypothesize that fibrinogen synthesis in CRS is upregulated in an interleukin-6-dependent acute phase reaction compensating for CRS-induced consumption of coagulation factors. Tocilizumab inhibits fibrinogen upregulation resulting in prolonged hypofibrinogenemia. These observations provide novel insights into the pathophysiology of hypofibrinogenemia following CAR-T cell therapy and emphasize the need for close fibrinogen monitoring after tocilizumab treatment of CRS.

IntroductionChronic graft-versus-host disease (cGvHD) is a serious late complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT).MethodsThis multicenter analysis determined the cumulative incidence (CI) of cGvHD and late acute GvHD (laGvHD) and its impact on transplantation-related mortality (TRM), relapse (R), and overall survival (OS) in 317 patients [296 adults, 21 pediatrics (

Abstract Background Total body irradiation (TBI)-based-conditioning before allogeneic hematopoietic stem cell transplantation (allo-HSCT) is standard of care in patients with acute myeloid leukemia (AML) but can cause long-term morbidity. Data on the impact of chronic Graft-versus-host disease (cGvHD) on cognitive function (CF) and quality of life (QoL) of long-term transplant survivors are sparse. Methods We analyzed patient-reported outcomes focusing on progression-free AML patients and 1st allo-HSCT applying a standardized TBI-technique with an average dose rate of 4 cGy/min to the total body and lung shielding in case of doses > 8 Gy. Instruments included the Functional Assessment of Cancer Therapy-Bone marrow transplant (FACT-BMT, version 4), the FACT-Cognition Function (FACT-Cog, version 3) and the Patient Health Questionaire-4 (PHQ-4). We put focus on the impact of cGvHD and compared the results to normative data derived from the general population. Results Out of 41 eligible patients contacted, 32 (78.0%) patients with a medium follow-up of 154 months (Interquartile range 113, 191 months) participated in the study. Eleven patients (34.4%) had active cGvHD, 11 (34.4%) resolved cGvHD and 10 (31.3%) never had cGvHD. Patients with active cGvHD had poorer FACT-BMT, FACT-Cog and higher PHQ-4 scores compared to patients with resolved cGvHD or who never had cGvHD. Outcomes were similar in patients with resolved cGvHD and those who never had cGvHD. Patients with active cGvHD had similar FACT-Cog, but lower FACT-BMT in comparison to normative data. However, the overall patient sample had similar FACT-BMT and FACT-Cog in comparison to normative data. Conclusion Our data indicate that CF of long-term survivors upon TBI-based allo-HSCT is not impaired, even in the presence of active cGvHD. However, active cGvHD has a negative impact on QoL. Trial registration The local Ethics Board of the University of Regensburg approved this study (Number 20-1810_1-101).

Intestinal immunoglobulin A (IgA) is strongly involved in microbiota homeostasis. Since microbiota disruption is a major risk factor of acute graft-versus-host disease (GvHD), we addressed the kinetics of intestinal IgA-positive (IgA+) plasma cells by immunohistology in a series of 430 intestinal biopsies obtained at a median of 1,5 months after allogeneic stem cell transplantation (allo-SCT) from 115 patients (pts) at our center. IgA+ plasma cells were located in the subepithelial lamina propria and suppressed in the presence of histological aGvHD (GvHD Lerner stage 0: 131+/-8 IgA+ plasma cells/mm2; stage 1-2: 108+/-8 IgA+ plasma cells/mm2; stage 3-4: 89+/-16 IgA+ plasma cells/mm2; P=0.004). Overall, pts with IgA+ plasma cells below median had an increased treatment related mortality (P=0.04). Time courses suggested a gradual recovery of IgA+ plasma cells after day 100 in the absence but not in the presence of GvHD. Vice versa IgA+ plasma cells above median early after allo-SCT were predictive of relapse and relapse-related mortality (RRM): pts with low IgA+ cells had a 15% RRM at 2 and at 5 years, while pts with high IgA+ cells had a 31% RRM at 2 years and more than 46% at 5 years; multivariate analysis indicated high IgA+ plasma cells in biopsies (hazard ratio =2.7; 95% confidence interval: 1.04-7.00) as independent predictors of RRM, whereas Lerner stage and disease stage themselves did not affect RRM. In contrast, IgA serum levels at the time of biopsy were not predictive for RRM. In summary, our data indicate that IgA+ cells are highly sensitive indicators of alloreaction early after allo-SCT showing association with TRM but also allowing prediction of relapse independently from the presence of overt GvHD.

Abstract Burkitt leukemia (BL) represents a highly aggressive lymphoma characterized by proliferation rates of around 100%, and a frequent spread into the central nervous system. If standard frontline chemotherapy fails, the prognosis is usually dismal, and reports on successful effective salvage therapy strategies for patients with relapsed/refractory BL are scant. Here, we report on a 40‐year‐old female patient who suffered an early relapse of BL three months after the completion of frontline chemoimmunotherapy. Strikingly, after only one cycle of R‐DHAP chemotherapy, the patient showed CR of BL enabling swift transition to a consolidating allogeneic stem cell transplantation. A 40‐year‐old previously healthy woman presented to the hospital with fatigue and incessant epistaxis, and a diagnosis of BL was made upon histological examination of a bone marrow biopsy. Treatment was initiated according to the GMALL 2002 B‐NHL/ALL protocol, which could induce complete molecular remission. Nevertheless, three months after chemotherapy, the patient exhibited BL relapse in the bone marrow, and on Fluorodeoxyglucose (FDG)–PET‐imaging. The relapse therapy was started with R‐DHAP, and after only one cycle, the patient once again entered complete remission (CR) paving the way for allogeneic stem cell transplantation. Unfortunately, the patient again relapsed five months after transplantation prompting salvage therapy with R‐DHAC and the execution of the second stem cell transplantation. However, one month after the second transplantation the patient presented with chemorefractory meningeosis leukemia resulting in the initiation of palliative care treatment. In summary, we report on rapid CR of relapsed BL after a single cycle of rituximab‐DHAP. Given a paucity of clinical trials on the treatment of patients with r/r BL, we intend to highlight the potential efficacy of rituximab‐DHAP as salvage therapy in those patients.

Reactivation of human cytomegalovirus (HCMV) from latency is a frequent complication following hematopoietic stem cell transplantation (HSCT). The development of acute graft-versus-host disease (GVHD) is a significant risk factor for HCMV disease. Using a murine GVHD model in animals latently infected with murine CMV (MCMV), we studied preventive and therapeutic interventions in this high-risk scenario of HSCT. Mice latently infected with MCMV experienced reactivated MCMV and developed disseminated MCMV infection concomitant with the manifestations of GVHD. Dissemination was accompanied by accelerated mortality. We demonstrate that MCMV reactivation and dissemination was modulated by MCMV-specific antibodies, thus demonstrating in vivo protective activity of antiviral antibodies. However, the efficacy of serum therapy required repetitive doses of high-titer immune serum secondary to the shortened serum half-life of IgG in animals with GVHD. In a complementary approach, treatment of GVHD by adoptive transfer of donor-derived Tregs facilitated production of MCMV-specific antibodies from newly developing donor-derived B cells. Together, our findings strongly suggest that antibodies play a major role in controlling recurrent MCMV infection that follows GVHD, and they argue for reassessing the potential of antibody treatments as well as therapeutic strategies that enhance de novo antibody development against HCMV.

In this registry-based study, we compared outcomes of allogeneic hematopoietic cell transplantation (allo-HCT) in adult patients with acute lymphoblastic leukemia (ALL) transplanted in first complete remission (CR-1), following conditioning with total body irradiation (TBI) at a standard 12-Gray or at a lower 8-Gray total dose. Patients received fludarabine (flu) as the sole chemotherapy complementing TBI. Eight-Gray TBI/flu was used in 494 patients and 12-Gray TBI/flu in 145 patients. Eighty-eight (23.1%) and 36 (29%) of the patients had Ph-negative B-ALL, 222 (58.3%) and 53 (42.7%) had Ph-positive B-ALL, 71 (18.6%) and 35 (28.2%) T-ALL, respectively (P = 0.008). Patients treated with 8-Gray were older than ones received 12-Gray (median 55.7 versus 40.3 years, P < 0.0001) and were more frequently administered in vivo T-cell depletion (71% versus 40%, P

The vitamin D receptor (VDR) is critical in regulating intestinal homeostasis and emerging evidence demonstrates that VDR deficiency is a critical factor in inflammatory bowel disease pathology. However, no clinical data exist regarding the intestinal expression of VDR in patients after allogeneic haematopoietic stem cell transplantation (HSCT). Analyzing intestinal biopsies from 90 patients undergoing HSCT with mortality follow-up, we demonstrated that patients with severe acute gastrointestinal graft versus host disease (GI-GvHD) showed significant downregulation of VDR gene expression compared to mild or no acute GI-GvHD patients (p = 0.007). Reduced VDR expression was already detectable at acute GI-GvHD onset compared to GvHD-free patients (p = 0.01). These results were confirmed by immunohistochemistry (IHC) where patients with severe acute GI-GvHD showed fewer VDR+ cells (p = 0.03) and a reduced VDR staining score (p = 0.02) as compared to mild or no acute GI-GvHD patients. Accordingly, low VDR gene expression was associated with a higher cumulative incidence of treatment-related mortality (TRM) (p = 1.6x10-6) but not with relapse-related mortality (RRM). A multivariate Cox regression analysis identified low VDR as an independent risk factor for TRM (p = 0.001, hazard ratio 4.14, 95% CI 1.78-9.63). Furthermore, VDR gene expression significantly correlated with anti-microbial peptides (AMPs) gene expression (DEFA5: r = 0.637, p = 7x10-5, DEFA6: r 0 0.546, p = 0.001). In conclusion, our findings suggest an essential role of the VDR in the pathogenesis of gut GvHD and the prognosis of patients undergoing HSCT.

The role of IL-22 in adult patients undergoing allogeneic stem cell transplantation (SCT) is of major interest since animal studies showed a protective and regenerative effect of IL-22 in graft versus host disease (GvHD). However, no clinical data exist on the tissue expression. Here we demonstrate that patients not suffering from transplant-related mortality (TRM) show significantly upregulated IL22 expression during histological and clinical GI-GvHD (p = 0.048 and p = 0.022, respectively). In contrast, in GvHD patients suffering from TRM, IL22 was significantly lower (p = 0.007). Accordingly, lower IL22 was associated with a higher probability of TRM in survival analysis (p = 0.005). In a multivariable competing risk Cox regression analysis, low IL22 was identified as an independent risk factor for TRM (p = 0.007, hazard ratio 2.72, 95% CI 1.32 to 5.61). The expression of IL22 seemed to be microbiota dependent as broad-spectrum antibiotics significantly diminished IL22 expression (p = 0.019). Furthermore, IL22 expression significantly correlated with G-protein coupled receptor (GPR)43 (r = 0.263, p = 0.015) and GPR41 expression (r = 0.284, p = 0.009). In conclusion, our findings reveal an essential role of IL22 for the prognosis of patients undergoing allogeneic SCT.