FAS is highly expressed in the germinal center but is not required for regulation of the B-cell response to antigen.

In establishing the memory B-cell population and maintaining self-tolerance during an immune response, apoptosis mediates the removal of early, low-affinity antibody-forming cells, unselected germinal center (GC) cells, and, potentially, self-reactive B cells. To address the role of the apoptosis-signaling cell surface molecule FAS in the B-cell response to antigen, we have examined the T-cell-dependent B-cell response to the carrier-conjugated hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) in lpr mice in which the fas gene is mutated. High levels of FAS were expressed on normal GC B cells but the absence of FAS did not perturb the progressive decline in numbers of either GC B cells or extrafollicular antibody-forming cells. Furthermore, the rate of formation and eventual size of the NP-specific memory B-cell population in lpr mice were normal. The accumulation of cells with affinity-enhancing mutations and the appearance of high-affinity anti-NP IgG1 antibody in the serum were also normal in lpr mice. Thus, although high levels of FAS are expressed on GC B cells, FAS is not required for GC selection or for regulation of the major antigen-specific B-cell compartments. The results suggest that the size and composition of B-cell compartments in the humoral immune response are regulated by mechanisms that do not require FAS.

Fine specificity of the antibody response to myelin basic protein in the central nervous system in multiple sclerosis: the minimal B-cell epitope and a model of its features.

T cells, B cells, and antibody are found in the white matter of the central nervous system in multiple sclerosis. The epitope center for the antibody response to human myelin basic protein (MBP) fits precisely the minimal epitope Pro85-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro96 for that reported for HLA DR2b (DRB1*1501)-restricted T cells that recognize MBP [Wucherpfenning, K.W., Sette, A., Southwood, S., Oseroff, C., Matsui, M., Strominger, J. & Hafler, D. (1994) J. Exp. Med. 179, 279-290], and overlaps with the reported DR2a-restricted epitope for T cells reactive to MBP [Martin, R., Howell, M. D., Jaraquemada, D., Furlage, M., Richert, J., Brostoff, S., Long, E. O., McFarlin, D. E. & McFarland, H. F. (1991) J. Exp. Med. 173, 19-24]. We describe a molecular model of this epitope.

Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells.

The immunosuppressive drugs cyclosporin A and FK506 interfere with the inducible transcription of cytokine genes in T cells and in other immune cells, in part by preventing the activation of NF-AT (nuclear factor of activated T cells). We show that transcription factor NFAT1 in T cells is rapidly dephosphorylated on stimulation, that dephosphorylation occurs before translocation of NFAT1 into the cell nucleus, and that dephosphorylation increases the affinity of NFAT1 for its specific sites in DNA. Cyclosporin A prevents the dephosphorylation and the nuclear translocation of NFAT1 in T cells, B cells, macrophages, and mast cells, delineating at least one mechanism that contributes to the profound immunosuppressive effects of this compound.

Telomerase activity in normal and malignant hematopoietic cells.

Bone marrow and peripheral blood leukocytes from 19 leukemia patients were found to contain telomerase activity detectable by a PCR-based assay. Telomerase was also detectable in nonmalignant bone marrow and peripheral blood leukocytes from normal donors, including fractions enriched for granulocytes, T lymphocytes, and monocytes/B cells. Semiquantitative comparison revealed considerable overlap between telomerase activities in samples from normal subjects and leukemia patients, confounding evaluation of the role of telomerase in this disease. These data indicate that human telomerase is not restricted to immortal cells and suggest that the somatic expression of this enzyme may be more widespread than was previously inferred from the decline of human telomeres.

Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate.

The HS1 protein is one of the major substrates of non-receptor-type protein-tyrosine kinases and is phosphorylated immediately after crosslinking of the surface IgM on B cells. The mouse B-lymphoma cell line WEHI-231 is known to undergo apoptosis upon crosslinking of surface IgM by anti-IgM antibodies. Variants of WEHI-231 that were resistant to anti-IgM-induced apoptosis expressed dramatically reduced levels of HS1 protein. Expression of the human HS1 protein from an expression vector introduced into one of the variant cell lines restored the sensitivity of the cells to apoptosis induced by surface IgM crosslinking. These results suggest that HS1 protein plays a crucial role in the B-cell antigen receptor-mediated signal transduction pathway that leads to apoptosis.

Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VH1 gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2- B cells, most of which were in the G1/G0 phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-week-old mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.

Immortalization of human primary B lymphocytes in vitro with DNA.

Epstein-Barr virus (EBV) is a human DNA tumor virus that efficiently immortalizes human primary B lymphocytes in vitro. Although viral genes that are expressed in latently infected B lymphocytes have been shown to function in cellular growth control, their detailed genetic analysis has been cumbersome for two reasons. The viral genome is too large to permit genetic engineering and human primary B lymphocytes, the only targets for infection by EBV in vitro, are both intractable in culture and recalcitrant to DNA transfection. To overcome these obstacles, we have assembled all the essential genes of EBV on a single recombinant vector molecule in Escherichia coli. We show here that this mini-EBV plasmid can yield immortalized B cells upon transfer of its naked DNA into human primary B lymphocytes. Established cell lines carry recombinant vector DNA and cannot support virus production. Because this DNA can be easily manipulated in E. coli, mutant mini-EBVs as well as foreign genes can now be introduced and studied successfully in recipient B lymphocytes from any human donors. These mini-EBVs therefore are potentially useful for human gene therapy.

Blockade of T- and B-lymphocyte development by antibody to the gamma c subunit of the receptors for interleukins 2, 4, and 7.

Cytokines are important regulators of hematopoesis. Mutations in gamma c, which is a subunit shared by the receptors for interleukin (IL) 2, IL-4, and IL-7, have been causally associated with human X chromosome-linked severe combined immunodeficiency disease. This finding indicates a mandatory role for cytokine receptor signaling at one or more stages of lymphocyte development. To evaluate the cellular level at which gamma c is critical for lymphopoiesis, the effect of monoclonal antibodies to gamma c on the capacity of syngeneic bone marrow cells to reconstitute the hematopoietic compartment of lethally irradiated recipient mice was examined. We show that monoclonal antibody to gamma c blocked lymphocyte development at or before the appearance of pro-B cells and prior to or at the seeding of the thymus by precursor cells while erythromyeloid cell development was normal. These results suggest that one level of lymphocyte development that requires gamma c is a point in hematopoietic cell differentiation near the divergence of lymphopoiesis and erythromyelopoesis.

Overexpressed Ly-6A.2 mediates cell-cell adhesion by binding a ligand expressed on lymphoid cells.

The Ly-6 locus encodes several cell surface proteins whose functions are unknown. Although it is hypothesized that these proteins may be receptors, there is no direct evidence that they bind a ligand. Herein we present evidence that Ly-6A.2, a Ly-6 protein expressed on T lymphocytes, binds a ligand expressed on normal thymocytes and splenic B and T cells. We find that transgenic thymocytes that overexpress Ly-6A.2 spontaneously aggregate in culture. This homotypic adhesion requires the overexpression of Ly-6A.2 because it is not observed in cultures of nontransgenic thymocytes. The aggregation of Ly-6A.2 transgenic thymocytes is inhibited by phosphatidylinositol-specific phospholipase C (which removes Ly-6A.2 and other glycosylphosphatidylinositol-anchored proteins from the membrane). Some anti-Ly-6A.2 monoclonal antibodies, including nonactivating ones and Fab' fragments, inhibit this aggregation. In contrast, other anti-Ly-6A.2 monoclonal antibodies increase the aggregation of transgenic but not nontransgenic thymocytes. To further examine whether Ly-6A.2 mediates adhesion (versus inducing another adhesion pathway) reaggregation assays were performed with paraformaldehyde-fixed Tg+ thymocytes. Paraformaldehyde-fixed Tg+ thymocytes reaggregate in culture and this aggregation is also blocked by phosphatidyl-inositol-specific phospholipase C and anti-Ly-6A.2 monoclonal antibodies. These results indicate that the homotypic adhesion of cultured Ly-6A.2 transgenic thymocytes is directly mediated by Ly-6A.2 and, more importantly, strongly suggests that Ly-6A.2 binds a ligand that is expressed on thymocytes. Tg+ thymocytes also bind to nontransgenic thymocytes, B cells, and T cells, indicating that normal cells naturally express the Ly-6A.2 ligand.

B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes.

Treatment of small resting B cells with soluble F(ab')2 fragments of anti-IgM, an analogue of T-independent type 2 antigens, induced activation characterized by proliferation and the expression of surface CD5. In contrast, B cells induced to proliferate in response to thymus-dependent inductive signals provided by either fixed activated T-helper 2 cells or soluble CD40 ligand-CD8 (CD40L) recombinant protein displayed elevated levels of CD23 (Fc epsilon II receptor) and no surface CD5. Treatment with anti-IgM and CD40L induced higher levels of proliferation and generated a single population of B cells coexpressing minimal amounts of CD5 and only a slight elevation of CD23. Anti-IgM- but not CD40L-mediated activation was highly sensitive to inhibition by cyclosporin A and FK520. Sp-cAMPS, an analogue of cAMP, augmented CD40L and suppressed surface IgM-mediated activation. Taken together these results are interpreted to mean that there is a single population of small resting B cells that can respond to either T-independent type 2 (surface IgM)- or T-dependent (CD40)-mediated activation. In response to different intracellular signals these cells are induced to enter alternative differentiation pathways.

Étude de la migration des populations de lymphocytes B du sang de patients infectés par le virus d’immunodéficience humaine (VIH)

La dérégulation du compartiment de cellules B est une conséquence importante de l’infection par le virus de l’immunodéficience humaine (VIH-1). On observe notamment une diminution des nombres de lymphocytes B sanguins ainsi qu’une variation des fréquences relatives des différentes populations de lymphocytes B chez les individus infectés par rapport aux contrôles sains. Notre laboratoire a précédemment démontré l’implication des cellules dendritiques dans la dérégulation des lymphocytes B via la roduction excessive de BLyS/BAFF, un stimulateur des cellules B. De plus, lors l’études menées chez la souris transgénique présentant une maladie semblable au SIDA, et chez la souris BLyS/BAFF transgénique, l’infection au VIH-1 fut associée à une expansion de la zone marginale (MZ) de la rate. De façon intéressante, nous observons chez les contrôleurs élites une diminution de la population B ‘mature’ de la MZ. Il s’agit du seul changement important chez les contrôleurs élites et reflète possiblement un recrutement de ces cellules vers la périphérie ainsi qu’une implication dans des mécanismes de contrôle de l’infection. Pour tenter d’expliquer et de mieux comprendre ces variations dans les fréquences des populations B, nous avons analysé les axes chimiotactiques CXCL13-CXCR5, CXCL12-CXCR4/CXCR7, CCL20-CCR6 et CCL25-CCR9. L’étude longitudinale de cohortes de patients avec différents types de progression clinique ou de contrôle de l’infection démontre une modulation des niveaux plasmatiques de la majorité des chimiokines analysées chez les progresseurs rapides et classiques. Au contraire, les contrôleurs élites conservent des niveaux normaux de chimiokines, démontrant leur capacité à maintenir l’homéostasie. La migration des populations de cellules B semble être modulée selon la progression ou le contrôle de l’infection. Les contrôleurs élites présentent une diminution de la population B ‘mature’ de la MZ et une augmentation de la fréquence d’expression du récepteur CXCR7 associé à la MZ chez la souris, suggérant un rôle important des cellules de la MZ dans le contrôle de l’infection au VIH-1. De façon générale, les résultats dans cette étude viennent enrichir nos connaissances du compartiment de cellules B dans le contexte de l’infection au VIH-1 et pourront contribuer à élaborer des stratégies préventives et thérapeutiques contre ce virus.

Mechanisms of B and T lymphocyte accumulation in tumor-draining lymph nodes

Thesis (Master's)--University of Washington, 2016-06

Regulatory T-cells infiltrate periodontal disease tissues

CD4(+) CD25(+) regulatory T ( Tr) cells are critical in regulating the immune response and thereby play an important role in the defense against infection and control of autoimmune diseases. Our previous studies demonstrated the involvement of autoimmune responses in periodontitis. The aim of this study was to identify CD4(+) CD25(+) Tr cells in periodontitis tissues and compare them with those in gingivitis tissues. Immunohistological analysis of CD4, CD25, and CTLA- 4 and the gene expression analysis of FOXP3, TGF- beta 1, and IL-10 on gingival biopsies revealed the presence of CD4(+) CD25(+) Tr cells in all tissues. In periodontitis, the percentage of CD4(+) CD25(+) Tr cells increased with increasing proportions of B-cells relative to T- cells. FOXP3, a characteristic marker for CD4(+) CD25(+) Tr cells, TGF- beta 1 and IL-10 were expressed more highly in periodontitis compared with gingivitis. These findings suggest that CD4(+) CD25(+) Tr cells and possibly other regulatory T- cell populations do exist and may play regulatory roles in periodontal diseases.

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (he) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG(4) Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of he genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant he and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

Decreased efficiency of trypsinization of cells following photodynamic therapy:evaluation of a role for tissue transglutaminase

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cellular damage are to be fully understood. The relationship between sensitizer, fluence rate and the removal of cells by trypsinization was studied using the RIF-1 cell line. Following treatment of RIF-1 cells with pyridinium zinc (II) phthalocyanine (PPC), or polyhaematoporphyrin at 10 mW cm−2 (3 J cm−2), there was a significant number of cells that were not removed by trypsin incubation compared to controls. Decreasing the fluence rate from 10 to 2.5 mW cm−2 resulted in a two-fold increase in the number of cells attached to the substratum when PPC used as sensitizer; however, with 5,10,15,20 meso-tetra(hydroxyphenyl) chlorin (m-THPC) there was no resistance to trypsinization following treatment at either fluence rate. The results indicate that resistance of cells to trypsinization following PDT is likely to be both sensitizer and fluence rate dependent. Increased activity of the enzyme tissue-transglutaminase (tTGase) was observed following PPC-PDT, but not following m-THPC-PDT. Similar results were obtained using HT29 human colonic carcinoma and ECV304 human umbilical vein endothelial cell lines. Hamster fibrosarcoma cell (Met B) clones transfected with human tTGase also exhibited resistance to trypsinization following PPC-mediated photosensitization; however, a similar degree of resistance was observed in PDT-treated control Met B cells suggesting that tTGase activity alone was not involved in this process.