Tocilizumab administration in cytokine release syndrome is associated with hypofibrinogenemia after chimeric antigen receptor T-cell therapy for hematologic malignancies.

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.

Distinct Mechanisms of IgM Antibody-Mediated Acquired von Willebrand Syndrome and Successful Treatment with Recombinant von Willebrand Factor in One Patient.

Acquired von Willebrand Syndrome (AVWS) is a rare coagulation disorder which can be associated with IgM paraproteinaemia. Recently, recombinant von Willebrand factor (rVWF) has become available for the treatment of bleedings in patients with inherited von Willebrand disease, but experience in patients with AVWS is limited. We report on 2 patients with AVWS with underlying IgM paraproteinaemia with distinct underlying pathomechanisms. In 1 patient, the paraprotein built unspecific complexes with von Willebrand factor (VWF). In the other patient, we were able to detect an IgM antibody against VWF. Bleeding in this patient was successfully treated with rVWF. To our knowledge, this is the first report about the successful use of rVWF in a patient with AVWS with the detection of a VWF-specific antibody.

Reconstitution and phenotype of Tregs in CMV reactivating patients following allogeneic hematopoietic stem cell transplantation.

In experimental and clinical settings Tregs prevent graft-versus-host disease (GvHD) by inhibiting the proliferation and function of conventional T cells (Tconv). The suppressive potency of Tregs might also lead to the inhibition of protective antiviral T cell responses. As the control of CMV reactivation is important to improve the clinical outcome in allogeneic HSCT, we analyzed the Treg reconstitution in CMV reactivating patients with and without GvHD (n=47) in the first 6 months following transplantation. Most importantly, CMV reactivation does not correlate with the numerical reconstitution of CD4(+)CD25(high)CD127(-) Tregs. During CMV reactivation the proportion of Tregs within the CD4(+) T cell population decreased significantly independent of GvHD manifestation. A comprehensive FACS analysis was performed in order to characterize the phenotype of Tregs and Tconv cells in greater detail for activation, co-stimulation, proliferation, suppressive function and migratory capability. Interestingly, Tregs of patients with CMV reactivation showed a significantly higher CXCR3 expression. CD4(+) Tconv cells expressed significantly higher protein levels of the proliferation marker Ki67 correlating with a numerical increase of CD4(+) T cells. Our results indicate that Tregs are not inhibiting pathogen clearance by Tconv following HSCT, which is of high relevance for future Treg cell-based clinical trials in allogeneic HSCT.

Isolation strategies of regulatory T cells for clinical trials: phenotype, function, stability, and expansion capacity.

Recent clinical results demonstrate the highly effective potency of regulatory T cells (Tregs) to control graft-versus-host disease (GvHD). In this presented study, we directly compared different Treg subpopulations in order to define the most promising Treg target cell population for cellular intervention studies with respect to their phenotype, functional properties, stability, and expansion capacity. Different Treg cell populations have been isolated from healthy donors and characterized by fluorescence activated cell sorting (FACS) analysis for their phenotypic marker and purity, functional properties by suppression assay, stability by Treg-specific demethylated region (TSDR) of the Foxp3 promoter and their in vitro expansion capacity. The direct comparison of the respective Treg target cell populations identified CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs as the most promising Treg population for fresh cell infusions in clinical trials with respect to cell yield, phenotype, function, and stability. The CD4(+)CD25(hi) Tregs qualified as the best candidate for in vitro expansion combining a highly stable phenotype with strong suppressive potential and attractive cell yield after repetitive stimulation. The suppressive capacity of freshly isolated CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs is comparable to freshly isolated CD4(+)CD25(hi), but inferior to CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs. In vitro expansion of CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs resulted in cell populations with less suppressive potency compared with CD4(+)CD25(hi) expanded Tregs correlating well with a higher TSDR demethylation level. In conclusion, future clinical trials should favor CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs for direct Treg cell transfer, whereas CD4(+)CD25(hi) Tregs qualify as best candidate for in vitro expansion.