Jörg Kirberg

Kit Regulates HSC Engraftment across the Human-Mouse Species Barrier

In-depth analysis of the cellular and molecular mechanisms regulating human HSC function will require a surrogate host that supports robust maintenance of transplanted human HSCs in vivo, but the currently available options are problematic. Previously we showed that mutations in the Kit receptor enhance engraftment of transplanted HSCs in the mouse. To generate an improved model for human HSC transplantation and analysis, we developed immune-deficient mouse strains containing Kit mutations. We found that mutation of the Kit receptor enables robust, uniform, sustained, and serially transplantable engraftment of human HSCs in adult mice without a requirement for irradiation conditioning. Using this model, we also showed that differential KIT expression identifies two functionally distinct subpopulations of human HSCs. Thus, we have found that the capacity of this Kit mutation to open up stem cell niches across species barriers has significant potential and broad applicability in human HSC research.

Impaired B cell development in the absence of Krüppel-like factor 3

Krüppel-like factor 3 (Klf3) is a member of the Klf family of transcription factors. Klfs are widely expressed and have diverse roles in development and differentiation. In this study, we examine the function of Klf3 in B cell development by studying B lymphopoiesis in a Klf3 knockout mouse model. We show that B cell differentiation is significantly impaired in the bone marrow, spleen, and peritoneal cavity of Klf3 null mice and confirm that the defects are cell autonomous. In the bone marrow, there is a reduction in immature B cells, whereas recirculating mature cells are noticeably increased. Immunohistology of the spleen reveals a poorly structured marginal zone (MZ) that may in part be caused by deregulation of adhesion molecules on MZ B cells. In the peritoneal cavity, there are significant defects in B1 B cell development. We also report that the loss of Klf3 in MZ B cells is associated with reduced BCR signaling strength and an impaired ability to respond to LPS stimulation. Finally, we show increased expression of a number of Klf genes in Klf3 null B cells, suggesting that a Klf regulatory network may exist in B cells.

Peripheral T Lymphocytes Recirculating Back into the Thymus Can Mediate Thymocyte Positive Selection

The thymus continuously produces T lymphocytes that contribute to the maintenance of the peripheral T cell pool. Since peripheral recirculating T cells represent a very minor population among total thymocytes in normal animals, the relationship between the thymus and secondary lymphoid organs is generally considered unidirectional. Recently, several reports have described the presence of recirculating T cells in the thymus, raising issues regarding their possible function. In this article, we show that the niche for recirculating T cells in the thymus, i.e., their absolute number, is the same in lymphopenic and normal mice. Using a novel combination of TCR-transgenic mice in which the ligand necessary for positive selection of host T cells is only expressed by transferred donor T cells, we show that mature T cells recirculating back to the thymus can mediate positive selection.

Programming of marginal zone B cell fate by basic krüppel-like factor (BKLF/KLF3)

Splenic marginal zone (MZ) B cells are a lineage distinct from follicular and peritoneal B1 B cells. They are located next to the marginal sinus where blood is released. Here they pick up antigens and shuttle the load onto follicular dendritic cells inside the follicle. On activation, MZ B cells rapidly differentiate into plasmablasts secreting antibodies, thereby mediating humoral immune responses against blood-borne type 2 T-independent antigens. As Krüppel-like factors are implicated in cell differentiation/function in various tissues, we studied the function of basic Krüppel-like factor (BKLF/KLF3) in B cells. Whereas B-cell development in the bone marrow of KLF3-transgenic mice was unaffected, MZ B-cell numbers in spleen were increased considerably. As revealed in chimeric mice, this occurred cell autonomously, increasing both MZ and peritoneal B1 B-cell subsets. Comparing KLF3-transgenic and nontransgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-regulated/down-regulated on normal MZ B-cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor-receptor signaling, indicating that KLF3 may complement alternative nuclear factor-κB signaling. Thus, KLF3 is a driving force toward MZ B-cell maturation.

TGN1412 Induces Lymphopenia and Human Cytokine Release in a Humanized Mouse Model

Therapeutic monoclonal antibodies (mAbs) such as the superagonistic, CD28-specific antibody TGN1412, or OKT3, an anti-CD3 mAb, can cause severe adverse events including cytokine release syndrome. A predictive model for mAb-mediated adverse effects, for which no previous knowledge on severe adverse events to be expected or on molecular mechanisms underlying is prerequisite, is not available yet. We used a humanized mouse model of human peripheral blood mononuclear cell-reconstituted NOD-RAG1-/-Aβ-/-HLADQ(tg+ or tg-)IL-2Rγc-/- mice to evaluate its predictive value for preclinical testing of mAbs. 2–6 hours after TGN1412 treatment, mice showed a loss of human CD45+ cells from the peripheral blood and loss of only human T cells after OKT3 injection, reminiscent of effects observed in mAb-treated humans. Moreover, upon OKT3 injection we detected selective CD3 downmodulation on T cells, a typical effect of OKT3. Importantly, we detected release of human cytokines in humanized mice upon both OKT3 and TGN1412 application. Finally, humanized mice showed severe signs of illness, a rapid drop of body temperature, and succumbed to antibody application 2–6 hours after administration. Hence, the humanized mouse model used here reproduces several effects and adverse events induced in humans upon application of the therapeutic mAbs OKT3 and TGN1412.

KLF2- A Negative Regulator of Pre-B Cell Clonal Expansion and B Cell Activation

Maturation as well as antigen-dependent activation of B cells is accompanied by alternating phases of proliferation and quiescence. We and others have previously shown that Krüppel-like factor 2 (KLF2), a regulator of T cell quiescence and migration, is upregulated in small resting precursor (pre)-B cells after assembly of the immature pre-B cell receptor (pre-BCR) and is downregulated upon antigen-induced proliferation of mature B cells. These findings suggest that KLF2, besides its function in maintaining follicular B cell identity, peripheral B cell homeostasis and homing of antigen-specific plasma cells to the bone marrow, also controls clonal expansion phases in the B cell lineage. Here, we demonstrate that enforced expression of KLF2 in primary pre-B cells results in a severe block of pre-BCR-induced proliferation, upregulation of the cell cycle inhibitors p21 and p27 and downregulation of c-myc. Furthermore, retroviral KLF2 transduction of primary B cells impairs LPS-induced activation, favors apoptosis and results in reduced abundance of factors, such as AID, IRF4 and BLIMP1, that control the antigen-dependent phase of B cell activation and plasma cell differentiation. Hence, we conclude that KLF2 is not only a key player in terminating pre-B cell clonal expansion but also a potent suppressor of B cell activation.

Innate, antigen‐independent role for T cells in the activation of the immune system by Propionibacterium acnes

Propionibacterium acnes is a human commensal but also an opportunistic pathogen. In mice, P. acnes exerts strong immunomodulatory activities, including formation of intrahepatic granulomas and induction of LPS hypersensitivity. These activities are dependent on P. acnes recognition via TLR9 and subsequent IL‐12‐mediated IFN‐γ production. We show that P. acnes elicits IL‐12p40 and p35 mRNA expression in macrophages, and IFN‐γ mRNA in liver CD4+ T cells and NK cells. After priming with P. acnes, CD4+ T cells serve as the major IFN‐γ mRNA source. In the absence of CD4+ T cells, CD8+ T cells (regardless of antigenic specificity) or NK cells can produce sufficient IFN‐γ to induce the P. acnes‐driven immune effects. Moreover, in the absence of αβT cells, γδT cells also enable the development of strongly enhanced TNF‐α and IFN‐γ responses to LPS and intrahepatic granuloma formation. Thus, under microbial pressure, different T‐cell types, independent of their antigen specificity, exert NK‐cell‐like functions, which contribute decisively to the activation of the innate immune system.

An evolutionary stability perspective on oncogenesis control in mature T-cell populations.

Here we present a mathematical model for the dynamics of oncogenesis control in mature T-cell populations within the blood and lymphatic system. T-cell homeostasis is maintained by clonal competition for trophic niches (survival signals stimulated through interactions with self-antigens bound to major histocompatibility molecules), where a clone is defined as the set of T cells carrying the same antigen specific T-cell receptor (TCR). We analytically derive fitness functions of healthy and leukemic clone variants, respectively, that capture the dependency of the stability of the healthy T-cell pool against leukemic invaders on clonal diversity and kinetic parameters. Similar to the stability of ecosystems with high biodiversity, leukemic mutants are suppressed within polyclonal T-cell populations, i.e., in the presence of a huge number of different TCRs. To the contrary, for a low clonal diversity the leukemic clone variants are able to invade the healthy T-cell pool. The model, therefore, describes the experimentally observed phenomenon that preleukemic clone variants prevail in quasi-monoclonal experimental settings (in mice), whereas in polyclonal settings the healthy TCR variants are able to suppress the outgrowth of tumours. Between the two extremal situations of mono- and polyclonality there exists a range of coexistence of healthy and oncogenic clone variants with moderate fitness (stability) each. A variation of cell cycle times considerably changes the dynamics within this coexistence region. Faster proliferating variants increase their chance to dominate. Finally, a simplified niche variation scheme illustrates a possible mechanism to increase clonal T-cell diversity given a small niche diversity.

Leukocyte β7 Integrin Targeted by Krüppel-like Factors

Constitutive expression of Krüppel-like factor 3 (KLF3, BKLF) increases marginal zone (MZ) B cell numbers, a phenotype shared with mice lacking KLF2. Ablation of KLF3, known to interact with serum response factor (SRF), or SRF itself, results in fewer MZ B cells. It is unknown how these functional equivalences result. In this study, it is shown that KLF3 acts as transcriptional repressor for the leukocyte-specific integrin β7 (Itgb7, Ly69) by binding to the β7 promoter, as revealed by chromatin immunoprecipitation. KLF2 overexpression antagonizes this repression and also binds the β7 promoter, indicating that these factors may compete for target sequence(s). Whereas β7 is identified as direct KLF target, its repression by KLF3 is not connected to the MZ B cell increase because β7-deficient mice have a normal complement of these and the KLF3-driven increase still occurs when β7 is deleted. Despite this, KLF3 overexpression abolishes lymphocyte homing to Peyer’s patches, much like β7 deficiency does. Furthermore, KLF3 expression alone overcomes the MZ B cell deficiency when SRF is absent. SRF is also dispensable for the KLF3-mediated repression of β7. Thus, despite the shared phenotype of KLF3 and SRF-deficient mice, cooperation of these factors appears neither relevant for the formation of MZ B cells nor for the regulation of β7. Finally, a potent negative regulatory feedback loop limiting KLF3 expression is shown in this study, mediated by KLF3 directly repressing its own gene promoter. In summary, KLFs use regulatory circuits to steer lymphocyte maturation and homing and directly control leukocyte integrin expression.

Concepts in mature T-cell lymphomas – highlights from an international joint symposium on T-cell immunology and oncology*

Abstract Growing attention in mature T-cell lymphomas/leukemias (MTCL) is committed to more accurate and meaningful classifications, improved pathogenetic concepts and expanded therapeutic options. This requires considerations of the immunologic concepts of T-cell homeostasis and the specifics of T-cell receptor (TCR) affinities and signaling. Scientists from various disciplines established the CONTROL-T research unit and in an international conference on MTCL they brought together experts from T-cell immunity, oncology, immunotherapy and systems biology. We report here meeting highlights on the covered topics of diagnostic pitfalls, implications by the new WHO classification, insights from discovered genomic lesions as well as TCR-centric concepts of cellular dynamics in host defense, auto-immunity and tumorigenic clonal escape, including predictions to be derived from in vivo imaging and mathematical modeling. Presentations on novel treatment approaches were supplemented by strategies of optimizing T-cell immunotherapies. Work packages, that in joint efforts would advance the field of MTCL more efficiently, are identified.