Regulation of B cell differentiation : from actin dynamics to cancer evolution

Primary immunodeficiency (PID) disorders specify a heterogeneous group of diseases characterized by poor or absent function in one or more components of the immune system. Most often they arise because of congenital monogenetic mutations. Wiskott-Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are two types of PIDs occurring as a consequence of loss-of-function and gain-of- function mutation in the Wiskott-Aldrich Syndrome protein (WASp), respectively. WASp is a regulator of the actin cytoskeleton, which is required for many hematopoietic and immune cell functions, including blood circulation, effective migration, and immune synapse formation. Besides compromised immune response giving autoimmunity, PID patients are predisposed to develop malignancies (e.g. lymphoma) of poor prognosis.

In Paper I, we studied B cell affinity maturation in the germinal center (GC) of XLN patients and murine models. XLN patients had normal levels of naïve and plasma blasts B cells, but decreased B cell proliferation at the expense of IgA switched B cells as well as memory B cells. Immunized XLN mice presented smaller GCs but had increased plasma blasts and plasma cells output. In vitro XLN murine B cells resulted in reduced Ig class switching and faulty cell division. Overall, XLN patients and animal models showed decreased B cell proliferation, reduced IgA switching, and increased genomic instability.

In Paper II, we investigated the nuclear function of WASp in WAS and XLN mouse models during differentiation from naïve to activated B cells. We performed nuclear-cytosol fractionation and WASp immunoprecipitation to reveal the presence of WASp in the nucleus of B cells besides its known localization in the cytoplasm. Chromatin immunoprecipitation (ChIP) of RNA polymerase II (RNA pol II) revealed an intrinsic role of WASp at transcription start sites (TSS), considering the decrease of RNA pol II coverage in WASp-KO activated B cells. Flow cytometry analysis of purified nuclei highlighted the different chromatin states of naïve (repressed, resting state) and activated (open, actively transcribing) B cells.

Differences in the nucleus of WT, XLN and WASp-KO naïve and activated B cells were reflected by differentially expressed gene analysis identified by bulk RNA sequencing. In summary, our work suggests a coordinated activity of WASp and RNA pol II in murine naïve and activated B cells.

In Paper III, we utilized a single cell-based approach coupled with VDJ sequencing to understand the mechanisms of lymphomagenesis during childhood. We collected aged-matched reactive lymph nodes, pediatric Burkitt Lymphoma (pBL), pediatric T cell lymphoblastic lymphoma (pTLBL) and pediatric Hodgkin lymphoma (pHL). We identified in pBL and pTLBL lymphoma cells by the hyperexpanded VDJ clone as well as infer copy number variation. pBL shared similarity with GC B cells, plasma cells and memory B cells, while pTLBL occupied a unique niche. pHL cells were characterized by expression of the CD30 marker both in single cell data and by special transcriptomics. HL cells were surrounded by exhaustion T cells supporting their growth. Our data revealed shared and unique transcriptomic profiles in BL, TLBL and HL patients and pediatric reactive lymph nodes.

In Paper IV, we tested the small molecule CK666, an Arp2/3 inhibitor, in dendritic cells (DCs) to mimic WASp-KO DCs that have increased DC cross-presentation capacity. CK666-treated DCs maintained a neutral phagosome pH and increased T cell proliferation as a readout of an efficient cross-presentation when primed with ovalbumin (OVA). The combination of CK666-treated DCs with activated T cells increased the survival of mice with the aggressive B16-melanoma tumor. Mice survival was longer when we combined CK666-treated DCs and activated T cells with a-PD-1 checkpoint inhibitor therapy. Our results highlight the efficacy of a drug that impair the actin cytoskeleton to facilitate tumor killing.

List of scientific papers

I. He M, Saeed MB, Record J, Keszei M, Gonçalves Pinho L, Vasconcelos-Fontes L, D'Aulerio R, Vieira R, Oliveira MMS, Geyer C, Bohaumilitzky L, Thiemann M, Deordieva E, Buedts L, Matias Lopes JP, Pershin D, Hammarström L, Xia Y, Zhao X, Cunningham-Rundles C, Thrasher AJ, Burns SO, Cotta-de-Almeida V, Liu C, Shcherbina A, Vandenberghe P, Westerberg LS. Overactive WASp in X-linked neutropenia leads to aberrant B- cell division and accelerated plasma cell generation. Journal of Allergy and Clinical Immunology (2022) https://doi.org/10.1016/j.jaci.2021.07.033

II. D'Aulerio R, Machado Matos G, Gonçalves Pinho L, Doukoumopoulos E, Calixto Vieira R, He M, Xue L, Gautier J, Westerberg LS. The actin regulator WASp associates with chromatin and regulates RNA polymerase II transcription in B cells. [Manuscript]

III. D'Aulerio R*, Yong T*, Oertlin C, Record J, Elliot A, Kwiecinska A, Baecklund F*, Westerberg LS *. Clonal evolution analysis of pediatric lymphoma reveals a diversified transcriptome and targets for precision medicine. [Manuscript] *equal contribution

IV. Oliveira MMS, D'Aulerio R, Yong T, He M, Baptista MAP, Nylén S, Westerberg LS. Increased cross-presentation by dendritic cells and enhanced anti-tumor therapy using the Arp2/3 inhibitor CK666. British Journal of Cancer (2023) https://doi.org/10.1038/s41416-022-02135-4