A major complication of hemophilia A therapy is the development of alloantibodies (inhibitors) that neutralize intravenously administered coagulation factor VIII (FVIII). Immune tolerance induction therapy (ITI) by repetitive FVIII injection can eradicate inhibitors, and thereby reduce morbidity and treatment costs. However, ITI success is difficult to predict and the underlying immunological mechanisms are unknown. Here, we demonstrated that immune tolerance against FVIII under nonhemophilic conditions was maintained by programmed death (PD) ligand 1–expressing (PD-L1–expressing) regulatory T cells (Tregs) that ligated PD-1 on FVIII-specific B cells, causing them to undergo apoptosis. FVIII-deficient mice injected with FVIII lacked such Tregs and developed inhibitors. Using an ITI mouse model, we found that repetitive FVIII injection induced FVIII-specific PD-L1⁺ Tregs and reengaged removal of inhibitor-forming B cells. We also demonstrated the existence of FVIII-specific Tregs in humans and showed that such Tregs upregulated PD-L1 in patients with hemophilia after successful ITI. Simultaneously, FVIII-specific B cells upregulated PD-1 and became killable by Tregs. In summary, we showed that PD-1–mediated B cell tolerance against FVIII operated in healthy individuals and in patients with hemophilia A without inhibitors, and that ITI reengaged this mechanism. These findings may impact monitoring of ITI success and treatment of patients with hemophilia A.

Since the COVID-19 pandemic, mRNA-based vaccines have gained prominence and opened up new opportunities for vaccine development. Rapid and long-lasting immune responses to antigens are crucial for the success of vaccines. Chemokines, cytokines, and chemokine receptors play an essential role in this process; thus, in this study, we aim to investigate their regulation and the resulting immune cell profile during mRNA-based SARS-CoV2 vaccination. SARS-CoV-2 neutralizing antibody titers were examined by ELISA. Chemokine abundance before and after 2^nd dose of the mRNA-1273 vaccine on time points d-1, d+1/2, d+3/4, and d+7 was examined in serum samples using bead-based immunoassays. In addition, flow cytometric analysis of blood samples was performed to characterize chemokine receptor expression on several immune cell populations associated with vaccination success. Our results demonstrated that mRNA-1273 vaccination increased serum levels of type 1 (IFNγ), type 2 (IL-4), other pro-inflammatory cytokines (IL-1α, IL-1β) and chemokines (CXCL9, CXCL10, CXCL11, CCL2, CCL4) between day 1 and day 4 post vaccination. Vaccination altered chemokine receptor expression on various cell types. Despite high interindividual differences, enhanced expression was observed for CXCR5 on activated CD4 T cells. Furthermore, CCR5 expression was increased on RBD⁺ B cells after vaccination, while CCR1, CCR2, CCR4, CCR5, and CXCR5 were elevated on different monocyte subsets. A notable finding was the positive correlation between elevated CXCR4 expression on RBD⁺ B cells and a high SARS-CoV-2 neutralizing antibody (NAb) titer ratio. We here demonstrate a dynamic, cell-specific chemokine regulation early after mRNA booster vaccination. This chemokine modulation likely facilitates an efficient induction of innate immune cell and adaptive T cell responses.

Acute-Graft- versus -Host disease (aGvHD) is a life-threatening complication after allogeneic stem-cell-transplantation. It is mediated by alloreactive T cells whose trafficking to aGvHD target organs is orchestrated by chemokines. We here asked whether CCL17 and its corresponding receptor CCR4 are involved in aGvHD development and severity. We applied an experimental mouse model of aGvHD in CCR4/CCL17 knockout mice and analyzed gut biopsies of GvHD patients. We show that the absence of CCR4 in transplanted T cells induced significantly less severe aGvHD. This was accompanied by reduced expression of Gata3. Mechanistically, only CD4 ⁺ , but not CD8 ⁺ CCR4 ^-/- T cells protected from aGvHD. We next identified dendritic cells in the small intestine to produce CCL17, which selectively recruited CD4 ⁺ T cells. IL-4 production by intestinal CD4 ⁺ T cells promoted proliferation of CD8 ⁺ T cells. In line, we detected an upregulation of CCL17 and Gata3 in human aGvHD samples. Our results indicate that local CCL17 production in aGvHD target organs recruits T cells, reinforcing local tissue damage and immune cell recruitment. We identified the JAK1/2-inhibitor ruxolitinib to dampen CCL17-expression, thereby reducing GvHD severity. We here dissect a to date unknown role of the CCL17-CCR4 axis in aGvHD, which might help to develop novel therapeutic strategies.

Recurrent genetic and chromosomal aberrations drive multiple myeloma (MM) pathogenesis. Among these, the t(4;14) translocation leads to overexpression of fibroblast growth factor receptor 3 (FGFR3) and is associated with poor prognosis. However, therapeutic approaches directly targeting FGFR3-driven myeloma progression remain limited. Here, we investigated the single-agent activity of ponatinib, a multikinase inhibitor, in MM. KMS18 and U266 myeloma cell lines were treated with ponatinib, and apoptosis induction, as well as VEGF and IL-6 secretion, was assessed. RNA sequencing of MM cells revealed pathway alterations induced by ponatinib treatment, which were subsequently validated by Western blot analysis. In vivo, mice inoculated with 5T33 myeloma cells received ponatinib, and survival was monitored. Notably, ponatinib exerted potent single-agent antimyeloma activity in an FGFR3-dependent manner by inducing apoptosis and suppressing VEGF and IL-6 secretion through inhibition of JAK/STAT, PI3K/AKT, and MAPK signaling. In vivo administration prolonged survival in myeloma-bearing mice. Collectively, our findings demonstrate the therapeutic efficacy of ponatinib in FGFR3-expressing MM beyond selective FGFR3 inhibition, suggesting that concurrent suppression of multiple signaling pathways is a critical mechanism of action. These results highlight the therapeutic potential of combined FGFR3-targeted strategies in multiple myeloma and provide a rationale for further clinical investigation.