Summary of past research

Role of CD81 in B cell function and development

Tetraspanin CD81 is a component of the CD19/CD21 signaling complex in B cells, was originally discovered as a target of anti-proliferative antibody  in B cell lymphoma. However, the exact role of CD81 in B cell function is not known. Here we demonstrate that, upon BCR induction, B cells in its absence fluxed higher intercellular free calcium ion, increased phosphorylation of BCR-related proximal and distal substrates and increased proliferation. Similarly, polyclonal activation of CD81-deficient B cells with LPS also induced increased proliferation and antibody secretion. Consistent with foregoing observations, CD81-deficient mice also mount significantly higher immune response upon antigenic stimulation. Perisinusoidal B cells (IgM+IgD+) capable of mounting T-independent immune responses against blood-borne pathogens were also increased in CD81-deficient mice. These cells also displayed similarly increased calcium influx kinetics as splenic B cells and contributed to generation of antigen specific antibody forming cells (AFC). Taken together these results we propose that CD81 is involved in suppressing B cell activation.

Role of transcription factor Pbx1 proto-oncogene in B cell debelopment and function (Postdoctoral)

Role of the Pbx1 proto-oncogene in B cell development: Pbx1 is a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B cell leukemia.  It is a global regulator of embryonic development whose absence results in embryonic lethality. In leukemia, Pbx1 mutations are restricted to pre-B cells and result in in-frame fusions with the E2A gene, a critical regulator of B lineage development. Initially, I had determined the expression of Pbx1 proteins. Pbx1b but not the Pbx1a isoform was found in common lymphoid progenitors, early B cell progenitors and also in mature B cells. Involvement with malignant lymphopoiesis and expression during B cell development suggested that Pbx1 might also contribute to normal B lineage lymphopoiesis, however the lethality of Pbx1 null embryos at E15 prevents an analysis of their in situ lymphoid development. To circumvent this situation, I had created Pbx1 homozygous null embryonic stem (ES) cells by in vitro manipulation and injected into Rag1-/- blastocysts to generate somatic chimeras. Since Rag1 knockout mice lack mature B or T cells, their potential presence in chimeric mice must necessarily derive from the injected mutant or control ES cells. Analysis of Pbx1-/-Rag1-/- chimeras revealed that there were no mature B cells in the peripheral blood. Differential expression of Ly9.1 (Ly9.1+ donor ES cells versus Ly9.1- recipient C57Bl/6 blastocysts) was utilized to distinguish donor versus recipient B lineage cells in the chimeric mice. Despite significant chimerism, no B lineage cells were detected in the bone marrow or secondary lymphoid organs of Pbx1-/- chimeras. B cells differentiate from common lymphoid progenitors (CLP) which are distinguished by a Lin-CD127+c-KitintSca-1int surface phenotype. Absence of Ly9.1+ CLPs in  the bone marrow of the chimeric mice localized the defect at the earliest stage of B cell commitment. Re-expression of Pbx1 in Pbx1-/- ES cells using a lentiviral vector rescued B cell development starting at CLP stage in chimeric mice, thereby indicating that the requirement for Pbx1 in B cell development is cell intrinsic and establishing the early requirement of Pbx1 for B cell development. To address the role of Pbx1 at later stages of B cell development, I had inactivated Pbx1 at the pro-B cell stage in Pbx1-/floxedC19Cre mice.  Despite complete inactivation of Pbx1 from pro-B cells (CD19+) onward, B cell development was not affected. These studies demonstrate that Pbx1 is required for B cell commitment and subsequent development since this development: 1) fails completely in Pbx1-/-Rag1-/- blastocyst chimeras; 2) development of CLPs requires Pbx1; 3) is rescued by reintroduction of Pbx1; and, 4) proceeds normally despite Pbx1 inactivation from the pro-B (CD19+) stage onward. Thus, Pbx1 is required at a stage between hematopoietic stem cell development and B cell commitment.

To determine whether Pbx1 is also required for fetal liver lymphopoiesis, I had compared the lymphoid reconstitution potential of Pbx1-deficient fetal liver (FL) cells with that of normal littermates. By 12 weeks post-transplant, production of circulating mature B-lymphocytes in sub-lethally irradiated Rag2-/- mice reconstituted with Pbx1-deficient FL cells was significantly reduced as compared to wild-type littermate controls. In addition, de novo B-cell development were arrested prior to the pre-B (CD43- B220low) stage in the bone marrow of mice receiving Pbx1-deficient FL cells. Despite the markedly reduced numbers of circulating mature B-lymphocytes, chimeric mice reconstituted with Pbx1-/- FL cells mounted normal humoral response as monitored by production of anti-DNP (haptan-specific) IgG and IgM after immunization with DNP-conjugated keyhole limpet hemocyanin. These data demonstrate that Pbx1 is not essential for lymphocyte activation.  

Role of the Pbx1 proto-oncogene in T cell development

To determine whether Pbx1 also contribute to T cell development I had utilized above mentioned Rag1-deficient blastocyst complementation assays using Pbx1 homozygous knockout embryonic stem (ES) cells. Although, Pbx1 null ES cells produced circulating CD4 and CD8 single positive T cells, in contrast to the typical naive T cell phenotype (CD62LhighCD44lowCD45RBhigh) in control mice they exclusively displayed a characteristic memory T cell phenotype (CD62LlowCD44highCD45RBlow). Analysis of the thymi of the chimeric mice for T cell developmental precursors showed the complete absence of CD4/CD8 double-positive intermediates. Rather, T cells in the thymus, and all secondary lymphoid organs, were single positive CD4 or CD8 cells that exclusively displayed a similar memory T cell phenotype typical of T cells derived from bone marrow resident committed T cell progenitors (CTP). The striking absence of classical CD4/CD8 double-positive cells in the thymus indicates that Pbx1 deficiency interrupts the intrathymic T cell developmental pathway. Furthermore, the phenotypic resemblance of Pbx1 null T cells with T cells derived from bone marrow resident CTP strongly suggests that T cell development in the absence of Pbx1 may occur via an extrathymic pathway.

Identification of transcriptional targets for proto-oncogene Pbx1

Pbx1 nullizygus embryos display several abnormalities which include abnormal skeletal, hematopoietic, kidney, spleen and heart development before intra uterine death at E15/16. The molecular mechanism underlying the contribution of this protein to development is not clear. It is also not known what are the downstream transcriptional targets for this protein. To address these issues I had utilized Pbx1 nullizygus mouse embryonic fibroblasts (MEFs) and cDNA microarray technology to identify the downstream targets. It was observed that the mRNA for decorin, a member of a family of small proteoglycanes called Small Leucin-Rich Proteoglycans (SLRP) was upregulated in Pbx1 null MEFs. It is the most prominent member of this family, containing Leucin-Rich Repeat (LRR) – a structural motif used in diverse molecular recognition processes like cell adhesion, signal transduction, DNA repair and RNA processing. mRNA for other members of this family including fibromodulin and lumicane were also consistently upregulated in Pbx1 null MEFs. This observation was further validated by northern blot and real-time PCR analysis. Decorin regulates collagen fibrillogenesis - a process essential in development, tissue repair and metastasis, it binds to TGF-beta and sequesters this growth stimulatory factor and it binds to EGF receptor and increases intracellular p21 leading to phosphorylation of MAP Kinase. Decorin inhibits in vitro growth of ovarian cancer cell lines by up-regulation of p21 protein. Lack of decorin accelerates lymphoma tumorigenesis in a mouse model predisposed to cancer (p53). Overall decorin is a growth suppressive molecule. Growth suppression and premature differentiation is the prominent cellular phenomena in the Pbx1 nullizygus embryos. Aberrant expression of decorin and other SLRP family members may mediate the Pbx1 null embryonic phenotypes.

In vitro differentiation of Pbx1 nullizygus embryonic stem cell (ES)

I have  developed an in vitro differentiation strategies for differention of ES cells to hematopoietic stem cells and subsequently to lymphoid cells. In this effort I am utilizing recently published OP9/ES cell coculture system, hanging drop embryoid body differentiation and combination of both to generate B and T lymphocyte in vitro. 

Understanding the role of neurogenic inflammation in the etiology of psoriasis using mouse-human chimeric model

During this period I was studying the etiology and pathophysiology of psoriasis. In this study a human skin xenotransplantation model of psoriasis in SCID mouse had been developed. In brief, Skin from normal individuals and from the psoriatic patients was transplanted to the SCID mice. The autologous lymphocytes from the patient blood were injected to these mice to reconstruct the patient’s immune system in these mice.  Using this mouse: human chimeric animal model the role of neurogenic inflammation in initiation of psoriasis was studied.

Mechanism of control of human trophoblast invasion and differentiation using in vitro differentiation model (Doctoral research)

During my graduate program, I had been studying collagenase-IV and plasminogen activator secretion by cultured human trophoblast cells and the mechanism of control of trophoblast proliferation. During this period, primary trophoblast cell culture model was developed. In this model, trophoblast cells isolated from human term placenta were purified and cultured. When maintained under culture condition, these cells differentiate from mononucleated cytotrophoblast to multinucleated syncitiotrophoblast by membrane fusion. This fusion process was studied by light and electron microscopy. Once differentiated, trophoblasts become endocrine cells and start secreting an array of hormones, growth factors and cytokines. Differentiated syncitiotrophoblast cells secrete two major hormones, human chorionic gonadotropin (hCG) and progesterone. The secretion profile of these two hormones was monitored by quantitative measurement in spent media using specific radioimmunoassay, and correlated with trophoblastic differentiation. The novelty of this model is that it is an in vitro reproduction of the in vivo phenomena. The secretion profiles of collagenase and plasminogen activator, the two markers for cellular invasiveness, were monitored by assay of their activity and zymography using spent media at different time points of culture. It was observed that trophoblast cells secreted these enzymes before differentiation. At this stage, under in vivo condition trophoblast cells are invasive and proliferative. In addition, it was also demonstrated that collagenase-IV is necessary for trophoblast differentiation. Finally, the role of different growth factors and cytokines on trophoblast proliferation were studied using MTT assay, BrdU incorporation combined with flowcytometry and Ki67 immunolabelling. 

In addition, I had also been studying the role of nitric oxide on trophoblast proliferation and differentiation. NADPH-Diaphorase cytochemical staining technique revealed the presence NOS related activity in trophoblast cells. Immunohistochemical study further confirmed the presence of  only neuronal type of nitric oxide but not endothelial and inducible form  in cytotrophoblast, syncytiotrophoblast and JEG-3 cells - a trophoblast derived choriocarcinoma cell line. nNOS was demonstrated in the cytoplasm using confocal laser scanning microscopy. In addition, it was demonstrated that NO induced trophoblast proliferation and reduced differentiation.