Malay Mandal

β-Catenin/Tcf Determines the Outcome of Thymic Selection in Response to αβTCR Signaling

Journal of Immunology, Sep 15, 2009

Thymic maturation of T cells depends on the intracellular interpretation of αβTCR signals by proc... more Thymic maturation of T cells depends on the intracellular interpretation of αβTCR signals by processes that are poorly understood. In this study, we report that α-catenin/Tcf signaling was activated in double-positive thymocytes in response to TCR engagement and impacted thymocyte selection. TCR engagement combined with activation of β-catenin signaled thymocyte deletion, whereas Tcf-1 deficiency rescued from negative selection. Survival/apoptotis mediators including Bim, Bcl-2, and Bcl-x L were alternatively influenced by stabilization of β-catenin or ablation of Tcf-1, and Bim-mediated β-catenin induced thymocyte deletion. TCR activation in double-positive cells with stabilized β-catenin triggered signaling associated with negative selection, including sustained overactivation of Lat and Jnk and a transient activation of Erk. These observations are consistent with β-catenin/Tcf signaling acting as a switch that determines the outcome of thymic selection downstream the αβTCR cascade. Productive rearrangement of the TCRα gene at the CD4 + CD8 + double-positive (DP) 6 stage of thymocyte development leads to the surface assembly of a mature αβTCR. This initiates a cascade of processes, ultimately leading to development of the various T cell lineages and shaping of the T cell repertoire. The outcome of thymocyte selection depends on the quality of αβTCR interactions with self-peptide-MHC complexes. Most DP thymocytes fail to

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A shared Runx1-bound Zbtb16 enhancer directs innate and innate-like lymphoid lineage development

Nature Communications, Oct 16, 2017

Zbtb16-encoded PLZF is a signature transcription factor (TF) that directs the acquisition of T-he... more Zbtb16-encoded PLZF is a signature transcription factor (TF) that directs the acquisition of T-helper effector programs during the development of multiple innate lymphocyte lineages, including natural killer T cell, innate lymphoid cell, mucosal-associated invariant T cell and γδ lineages. PLZF is also essential in osteoblast and spermatogonial development. How Zbtb16 itself is regulated in different lineages is incompletely understood. Here, by systematic CRISPR/Cas9-assisted deletions of chromatin accessible regions within the Zbtb16 locus in mouse, we identify a critical enhancer controlling PLZF expression exclusively in innate lymphoid lineages. Multiple sites within this enhancer express canonical motifs for the TF Runx1, which is essential for the development of these lineages. Notably, some regulatory sites control the kinetic rather than the overall level of PLZF expression. Thus, our comprehensive, unbiased analysis of regulatory elements in vivo reveals critical mechanisms of Zbtb16 regulation shared between innate and innate-like lymphoid lineages.

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Ddx3xregulates B-cell development and light chain recombination in mice

bioRxiv (Cold Spring Harbor Laboratory), Oct 25, 2018

Ddx3x encodes a DEAD box RNA helicase implicated in antiviral immunity and tumorigenesis. We find... more Ddx3x encodes a DEAD box RNA helicase implicated in antiviral immunity and tumorigenesis. We find that hematopoietic Ddx3x deficiency in Vav1-Cre mice (Ddx3x) results in altered leukocyte composition of secondary lymphoid tissues, including a marked reduction in mature B cells. This paucity of peripheral B cells is associated with deficits in B-cell development in the bone marrow, including reduced frequencies of small pre-B cells. Bone marrow chimera experiments reveal a B-cell intrinsic effect of Ddx3x deletion. Mechanistically, Ddx3x small pre-B cells exhibit lower expression of Brwd1, a histone reader that restricts recombination at the immunoglobulin kappa (Igk) locus. In fact, the B-cell deficits in Ddx3x mice resemble those of Brwd1 mutant mice, and both strains of mice exhibit defective Igk rearrangement in small pre-B cells. The contribution of Ddx3x to Brwd1 expression and light chain rearrangement constitutes the first evidence of a role for an RNA helicase in promoting B-cell development.

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Identification, purification and characterization of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle plasma membrane

Molecular and Cellular Biochemistry, 2004

Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatin... more Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatinase: MMP-2; E.C. 3.4.24.24) as revealed by immunoblot studies of its smooth muscle plasma membrane suspension with polyclonal MMP-2 antibody. In this report, we described the purification and partial characterization of MMP-2 in the plasma membrane fraction of the smooth muscle. MMP-2 has been purified from plasma membrane fraction

It Takes Three Receptors to Raise a B Cell

Trends in Immunology, Jul 1, 2020

As the unique source of diverse immunoglobulin repertoires, B lymphocytes are an indispensable pa... more As the unique source of diverse immunoglobulin repertoires, B lymphocytes are an indispensable part of humoral immunity. B cell progenitors progress through sequential and mutually exclusive states of proliferation and recombination, coordinated by cytokines and chemokines. Mutations affecting the crucial pre-B cell checkpoint result in immunodeficiency, autoimmunity, and leukemia. This checkpoint was previously modeled by the signaling of two opposing receptors, IL-7R and the pre-BCR. We provide an update to this model in which three receptors, IL-7R, pre-BCR, and CXCR4, work in concert to coordinate both the proper positioning of B cell progenitors in the bone marrow (BM) microenvironment and their progression through the pre-B checkpoint. Furthermore, signaling initiated by all three receptors directly instructs cell fate and developmental progression.

Pregnancy programs epigenetic and transcriptional exhaustion in memory CD8+ T cells

Research Square (Research Square), Apr 5, 2023

Alloreactive memory T cells, unlike naive T cells, fail to be restrained by transplantation toler... more Alloreactive memory T cells, unlike naive T cells, fail to be restrained by transplantation tolerance protocols or regulatory T cells, and therefore represent a critical barrier to long-term graft acceptance. Using female mice sensitized by rejection of fully-mismatched paternal skin allografts, we show that subsequent semi-allogeneic pregnancy successfully reprograms memory fetus/graft-specific CD8 + T cells (TFGS) towards hypofunction in a manner that is mechanistically distinct from naive TFGS. Post-partum memory TFGS were durably hypofunctional, exhibiting enhanced susceptibility to transplantation tolerance induction. Furthermore, multi-omics studies revealed that pregnancy induced extensive phenotypic and transcriptional modifications in memory TFGS reminiscent of T cell exhaustion. Strikingly, at loci transcriptionally modified in both naive and memory TFGS during pregnancy, chromatin remodeling was observed exclusively in memory and not naive TFGS. These data reveal a novel link between T cell memory and hypofunction via exhaustion circuits and pregnancy-mediated epigenetic imprinting. This conceptual advance has immediate clinical relevance to pregnancy and transplantation tolerance.

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A divide and conquer approach (DACA) to predict high fidelity structure of large multidomain protein BRWD1

bioRxiv (Cold Spring Harbor Laboratory), Jul 11, 2023

Therapeutic importance in inhibiting Bromodomain and WD Repeat Domain containing BRWD1 against nu... more Therapeutic importance in inhibiting Bromodomain and WD Repeat Domain containing BRWD1 against numerous human pathophysiological processes including cancers prompts prediction of a workable structure of this large protein. Here, a novel divide and conquer strategy was adopted to utilize smaller overlapping sequence-fragments of BRWD1 to further utilize their predicted structures as derived templates for prediction of complete BRWD1 structure in absence of its desired homologues in the template database. The novelty of this methodology stemmed from the requirement of templates of high sequence similarity in any comparative model based predictors whereas, the own fragments of the same target protein, BRWD1 could successfully fulfill this criteria. Additionally, the outputs of different high performing predictors including AlphaFold and RoseTTAFold were systematically integrated under the premise of Inductive Reasoning. The resultant structures were validated using existing validation parameters. Finally, a new validation paradigm was adopted to screen the best structure from the result presenting in-silico studies of known interactions of BRWD1 with various small molecules like, BD inhibitors, modified histone tails, DNA motifs and interacting proteins. The algorithm proposed in this work also paved the way for prediction of authentic structures of large size proteins.

format_quoteUtilized AlphaFold for BRWD1's structural prediction while addressing limitations of existing predictive models for large proteins.format_quote

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BRWD1 establishes epigenetic states for germinal center initiation, maintenance, and function

B-1a Cells Acquire Their Unique Characteristics by Bypassing the Pre-BCR Selection Stage

Social Science Research Network, 2019

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the perit... more B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/ STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

format_quoteTransgenic studies demonstrate that receptor swapping can convert B-2 cells to B-1a, indicating flexible developmental paths.format_quote

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B-1a Cells Acquire Their Unique Characteristics by Bypassing the Pre-BCR Selection Stage

Social Science Research Network, 2019

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the perit... more B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/ STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

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B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage

format_quoteLower IL-7R/STAT5 signaling promotes alternative pathways favoring B-1a cell development with characteristic self-reactive receptors.format_quote

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BRWD1 orchestrates small pre-B cell chromatin topology by converting static to dynamic cohesin

Nature Immunology

STAT3 signaling in B cells controls germinal center zone organization and recycling

Cell Reports, May 1, 2023

Ikaros and Aiolos Inhibit Pre-B-Cell Proliferation by Directly Suppressing c-Myc Expression

Molecular and Cellular Biology, Sep 1, 2010

Pre-B-cell expansion is driven by signals from the interleukin-7 receptor and the pre-B-cell rece... more Pre-B-cell expansion is driven by signals from the interleukin-7 receptor and the pre-B-cell receptor and is dependent on cyclin D3 and c-Myc. We have shown previously that interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to suppress pre-B-cell proliferation. However, the molecular mechanisms through which Ikaros and Aiolos exert their growth inhibitory effect remain to be determined. Here, we provide evidence that Aiolos and Ikaros bind to the c-Myc promoter in vivo and directly suppress c-Myc expression in pre-B cells. We further show that downregulation of c-Myc is critical for the growth-inhibitory effect of Ikaros and Aiolos. Ikaros and Aiolos also induce expression of p27 and downregulate cyclin D3 in pre-B cells, and the growth-inhibitory effect of Ikaros and Aiolos is compromised in the absence of p27. A time course analysis further reveals that downregulation of c-Myc by Ikaros and Aiolos precedes p27 induction and cyclin D3 downregulation. Moreover, downregulation of c-Myc by Ikaros and Aiolos is necessary for the induction of p27 and downregulation of cyclin D3. Collectively, our studies identify a pre-B-cell receptor signaling induced inhibitory network, orchestrated by Ikaros and Aiolos, which functions to terminate pre-B-cell expansion.

format_quoteIkaros and Aiolos bind directly to c-Myc promoter, indicating their role in repressing c-Myc in pre-B cells.format_quote

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B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage

Nature Communications, Oct 18, 2019

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the perit... more B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/ STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

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RAG Represents a Widespread Threat to the Lymphocyte Genome

Cell, Aug 1, 2015

Highlights d The RAG endonuclease binds to thousands of sites in the lymphocyte genome d Off-targ... more Highlights d The RAG endonuclease binds to thousands of sites in the lymphocyte genome d Off-target RAG activity is rare outside of the antigen receptor genes d Substrates for RAG cleavage are preferentially depleted at sites of RAG binding d RSSs inserted in the genome create a recombination and translocation hot spot

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Regulation of T-cell progenitor survival and cell-cycle entry by the pre-T-cell receptor

Immunological Reviews, Feb 1, 2006

Pre-T-cell receptor (pre-TCR) functions and the study of early thymocyte development continue to ... more Pre-T-cell receptor (pre-TCR) functions and the study of early thymocyte development continue to fascinate immunologists more than 10 years after the first description and cloning of the receptor. Although multiple reports have addressed several aspects of pre-TCR signaling and function, its ability to regulate diverse functions, including proliferation, survival, and allelic exclusion of the TCR-beta locus, remains an open question. What fascinates us is its central role in the fine balance between physiological differentiation and thymocyte transformation that leads to T-cell leukemia and lymphomas. In this review, we integrate pre-TCR signaling pathways and study their effects on the regulation of T-cell progenitor cell-cycle entry and cell survival. We also connect aberrant pre-TCR signaling to deregulated proliferation and apoptotic balances and thymocyte transformation.

Targeting the NF-κB signaling pathway in Notch1-induced T-cell leukemia

Nature Medicine, Dec 17, 2006

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Igβ ubiquitination activates PI3K signals required for endosomal sorting

Journal of Experimental Medicine, Nov 15, 2017

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Regulation of lymphocyte progenitor survival by the proapoptotic activities of Bim and Bid

Proceedings of the National Academy of Sciences of the United States of America, Dec 30, 2008

On their entry into the thymus, developing lymphocyte progenitors depend on signaling from the pr... more On their entry into the thymus, developing lymphocyte progenitors depend on signaling from the pre-T cell receptor (pre-TCR), which orchestrates differentiation, cell proliferation, and survival. The exact mechanism of pre-TCR-mediated suppression of T cell death remains unclear and controversial. Here, we identify Bim and Bid, 2 members of the BH3-only group of the BCL2 family, as important regulators of pre-T cell death. Both factors are highly expressed in proapoptotic thymocytes and their expression is suppressed on signaling through the pre-TCR. Their expression is directly regulated by the transcription factors FoxO3a and p53. Bid expression and p53 activity are related to the ongoing rearrangement of the TCR loci and induced DNA damage responses. Bim expression and FoxO3a nuclear translocation are directly controlled by the pre-TCR by means of its downstream kinase Akt/PKB. Interestingly, deletion of either gene on a pre-TCR ؊/؊ background rescues survival, but fails to induce further progenitor differentiation uncoupling the 2 processes. BCL2 family ͉ cell death ͉ pre-TCR signaling ͉ T cell development Results Bim and Bid Are Overexpressed in Proapoptotic, Pre-TCR Deficient T Cell Progenitors. We have initially compared BCL2-family gene expression in pre-TCR nonexpressing, proapoptotic thymocytes, and cells that receive pre-TCR signals, suppress apoptosis, and differentiate. Thus, we have purified ''small'' (based on FSC axis), pre-TCR-nonexpressing ''DN3'' (CD25 ϩ 44-c-kit Ϫ) and compared them with ''large,'' pre-TCR-expressing ''DN4''

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