Dirk Baumjohann

Embryonic hematopoiesis is thought to occur exclusively in the extraembryonic yolk sac before hematopoietic stem cells (HSCs) arise in the aorta–gonad–mesonephros region. Here we identify a previously unrecognized hematopoietic site at the maternal–fetal interface in mice, termed MIXER (Maternally Interfaced eXtraEmbRyonic) tissue, which develops contemporaneously with the yolk sac and exhibits robust hematopoietic activity. MIXER contains abundant hematopoietic precursors, including classical CSF1R ⁺ erythro-myeloid progenitors and a unique embryonic CD4 ⁺ CSF1R ⁻ Erythro-Myeloid PRogenitOR (eEMPROR) population. Lineage-tracing experiments demonstrate that eEMPROR cells contribute to select macrophage subpopulations during fetal development, primarily in the perivascular space. Notably, early human placental villi, before HSC emergence, also harbor CD4 ⁺ nucleated erythrocytes, indicating a conserved hematopoietic role for the maternal–fetal interface. These findings establish MIXER as an anatomically distinct niche for embryonic hematopoiesis and expand the developmental map of blood cell emergence in mammals.

In recent years, demand for human‐based in vitro models has grown substantially. In this regard, human secondary lymphoid organ (SLO)‐derived ex vivo immune organoids are gaining recognition, as they provide a platform for studying highly differentiated, tissue‐specific immune cell subsets found in SLOs, such as lymph nodes, tonsils, or spleen. These systems support the study of adaptive immune responses mediated by T and B cells, e.g., due to the presence of T follicular helper cells and germinal center B cells within these immune organoids. Here, we describe procedures for the isolation of human tonsil‐ or adenoid‐derived immune cells, their cryopreservation and thawing, the establishment of immune organoids in vitro, and a detailed flow cytometry protocol for longitudinal phenotypic profiling of the immune organoids. In summary, these protocols serve as a foundation for downstream functional assays and complementary analyses in a human‐first approach. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Cell isolation from human tonsils and adenoids Basic Protocol 2 : Thawing of cryopreserved tonsil and adenoid cells and establishment of immune organoids Basic Protocol 3 : Flow cytometry for longitudinal phenotypical profiling of human tonsil/adenoid‐derived immune organoids.

T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells play critical roles in regulating the activity of the germinal center (GC), which is essential for the generation of high-affinity antibodies. In the GC, Tfh cells help B cells to proliferate and to differentiate into memory B cells and long-lived plasma cells. In contrast, Tfr cells, a specialized subset of regulatory T cells (Tregs), modulate the humoral immune response by suppressing excessive or autoreactive B-cell activity. Here, we established an in vitro differentiation protocol for mouse CD4⁺ T cells that yielded CXCR5⁺FoxP3⁺ Tfr cells that exhibited a Bcl6 ^hi PD-1 ^hi CD25 ^lo GITR ^int phenotype and were distinct from Treg and Tfh cells. Functionally, in vitro-generated Tfr cells potently suppressed Tfh cell-driven B-cell class switching to IgG1 and downregulated the expression of B-cell costimulatory ligands. While in vitro-generated Bcl6 -deficient Tfh cells were impaired in providing help to B cells for efficient class switching to IgG1, in vitro-generated Bcl6 -deficient Tfr cells failed to inhibit Tfh cell-driven B-cell class switching to IgG1. Mechanistically, we showed that Tfr cells emerged from FoxP3 ⁺ precursors in low-IL-2 environments through a TGF-β- and c-Maf-dependent pathway, allowing for reprogramming and reinforcement of the follicular regulatory cell program in CD4 ⁺ T cells in vitro.

Cellular polarization plays a crucial role in regulating immunological processes and is often associated with reorientation of the centrosome. During immune synapse formation, centrosome repositioning in lymphocytes assists in T cell activation. While a single centrosome, consisting of two centrioles, is present in T cells, antigen-presenting cells such as dendritic cells amplify centrioles during maturation and immune activation. How centriole amplification in antigen-presenting cells affects immune synapse formation and T cell activation is unclear. In this study, we combine experimental data with mathematical and computational modelling to provide evidence that extra centrioles in dendritic cells form over-active microtubule organizing centers, which cluster during dendritic cell-T cell interactions and, unlike in T cells, localize close to the cell center. Perturbing either centrosome integrity or centriole numbers and configuration in dendritic cells results in impaired T cell activation. Collectively, our results highlight a crucial role for centriole amplification and optimal centrosome positioning in antigen-presenting cells for controlling T cell responses.

T follicular helper (T_FH) cells are the primary CD4⁺ T helper cell subset providing help to B cells for efficient antibody responses in vaccination, allergy, autoimmunity, and infectious diseases. Despite their critical involvement in immunity, T_FH cells’ specific role in filarial infections remains unclear. Using the rodent filarial model Litomosoides sigmodontis, we examined T_FH and germinal center (GC) B cell responses in lung‐draining mediastinal lymph nodes (medLNs) over a 110‐day infection period in naive and infected wildtype (WT) BALB/c mice, as well as eosinophil‐deficient dblGATA mice, using flow cytometry and ELISA. We observed robust and prolonged T_FH and GC B cell responses in medLNs of infected BALB/c mice, along with enduring IgG1 antibody responses next to a persistent systemic humoral immune response. We further provide evidence of dysregulated T_FH/T follicular regulatory (T_FR) cell ratios in medLNs. Finally, elevated T_FH cell frequencies in medLNs of dblGATA mice reaffirm the significant role of eosinophils during chronic infection. In conclusion, our findings provide novel insights into population changes of T_FH and GC B cells during filarial infection.

The centrosome is a microtubule orchestrator, nucleating and anchoring microtubules that grow radially and exert forces on cargos. At the same time, mechanical stresses from the microenvironment and cellular shape changes compress and bend microtubules. Yet, centrosomes are membraneless organelles, raising the question of how centrosomes withstand mechanical forces. Here, we discover that centrosomes can deform and even fracture. We reveal that centrosomes experience deformations during navigational pathfinding within motile cells. Coherence of the centrosome is maintained by Dyrk3 and cNAP1, preventing fracturing by forces. While cells can compensate for the depletion of centriolar-based centrosomes, the fracturing of centrosomes impedes cellular function by generating coexisting microtubule organizing centers that compete during path navigation and thereby cause cellular entanglement in the microenvironment. Our findings show that cells actively maintain the integrity of the centrosome to withstand mechanical forces. These results suggest that centrosome stability preservation is fundamental, given that almost all cells in multicellular organisms experience forces.

Studying the human immune system in vivo is challenging and often not possible. Therefore, most human immunology studies have been predominantly confined to peripheral blood analyses, which by themselves have inherent limitations, as many immune reactions take place within tissues. For example, potent antibody responses that contribute to fighting infections and provide protection following vaccination require cellular interactions between B cells and T cells in specialized micro-anatomical structures called germinal centers, which are found in secondary lymphoid organs such as spleen, lymph nodes, and tonsils. Thus, there is a clear demand for novel enhanced experimental systems that faithfully recapitulate the intricate dynamics of the human immune system as much as possible. In this review, we discuss recent advances in versatile human tonsil/adenoid tissue-based ex vivo immune organoid cultures as well as related cancer and autoimmunity-focused experimental setups. These systems have been implemented as translational immunology platforms for in-depth analyses of human B and T cell-mediated immune responses, thereby facilitating mechanistic studies as well as drug and vaccine testing in a human-first approach.

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8⁺ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.