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BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.
Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations
(2021)
Transcription factors play a crucial role in regulating biological processes such as cell
growth, differentiation, organ development and cellular signaling. Within this group, proteins
equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding
transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a
variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary
glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which
serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown
to be critically important for efficient regulation of the BCL11B target genes and could therefore
represent a promising target for novel drug therapies. Here, we report the structural basis for
BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure
prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy
transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of
the single ZF domain and directly involved in forming the dimer interface. These findings not only
provide deep insight into how BCL11B acquires its active structure but also represent an important
step towards rational design or selection of potential inhibitors.
Generation of Inducible BCL11B Knockout in TAL1/LMO1 Transgenic Mouse T Cell Leukemia/Lymphoma Model
(2022)
The B-cell CLL/lymphoma 11B gene (BCL11B) plays a crucial role in T-cell development, but its role in T-cell malignancies is still unclear. To study its role in the development of T-cell neoplasms, we generated an inducible BCL11B knockout in a murine T cell leukemia/lymphoma model. Mice, bearing human oncogenes TAL BHLH Transcription Factor 1 (TAL1; SCL) or LIM Domain Only 1 (LMO1), responsible for T-cell acute lymphoblastic leukemia (T-ALL) development, were crossed with BCL11B floxed and with CRE-ER/lox mice. The mice with a single oncogene BCL11Bflox/floxCREtg/tgTAL1tg or BCL11Bflox/floxCREtg/tgLMO1tg were healthy, bred normally, and were used to maintain the mice in culture. When crossed with each other, >90% of the double transgenic mice BCL11Bflox/floxCREtg/tgTAL1tgLMO1tg, within 3 to 6 months after birth, spontaneously developed T-cell leukemia/lymphoma. Upon administration of synthetic estrogen (tamoxifen), which binds to the estrogen receptor and activates the Cre recombinase, the BCL11B gene was knocked out by excision of its fourth exon from the genome. The mouse model of inducible BCL11B knockout we generated can be used to study the role of this gene in cancer development and the potential therapeutic effect of BCL11B inhibition in T-cell leukemia and lymphoma.
Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.
Molekularbiologische Untersuchungen haben eine Bedeutung für die Diagnostik, die Therapieüberwachung, die Prognoseeinschätzung und das pathogenetische Verständnis hämatologischer Malignome. Für die Festlegung unkontrollierten Wachstums ist die Klonalitätsanalyse wichtig. Neben den spezifischen Translokationen können hierzu auch klonale Immunglobulin oder T-Zellrezeptor-Genumlagerungen genutzt werden. Der T-Zellrezeptor delta-Genort eignet sich besonders zur Untersuchung von atypischen klonalen Rearrangements, da er im Vergleich zu den anderen T-Zellrezeptoren nur über ein begrenztes Repertoire an Rekombinationselementen verfügt. Interessanterweise lassen sich circa 20% der Umlagerungen im TCR delta-Genort nicht durch Umlagerungen mit bekannten Gensegmenten erklären lassen. Das Ziel dieser Arbeit war es, Gensegmente aufzudecken, die zu derartigen atypischen Banden führen. In Vorarbeiten der Arbeitsgruppe wurde eine atypische Rekombination gefunden, an welcher das Gensegment D3 des T-Zellrezeptor delta-Gens und die drei ersten Exone des BCL11B Gens beteiligt sind. Beide Gene (TCR delta als auch BCL11B) sind auf Chromosom 14 lokalisiert. In der vorliegenden Arbeit konnte gezeigt werden, dass neben der Inversion auf Chromosom 14 (inv(14)(q11;q32.31)) weitere Bruchstücke der Chromosomen drei, elf und zwanzig in der Bruchpunktregion (t(14;20;3;11)(q11;q11;p21;p12)) lokalisiert waren. Dies ist ein Hinweis darauf, dass die Chromatinorganisation bei dieser Leukämie neben der Inversion 14 wesentlich stärker gestört ist. Weiterhin führt diese Translokation zu einem Fusionsgen und in der betreffenden Leukämie zu einem Fusionstranskript. Die Bedeutung dieses Ereignisses für die maligne Transformation war nicht Inhalt dieser Arbeit und wurde nicht weiter untersucht. Bei weiteren atypischen Umlagerungen konnten bekannte Gene mit teilweise neuen Bruchpunkten identifiziert werden.