Larry W. Arnold

Identification of a Precursor to Phosphatidyl Choline-Specific B-1 Cells Suggesting That B-1 Cells Differentiate from Splenic Conventional B Cells In Vivo: Cyclosporin A Blocks Differentiation to B-1

The origin of B-1 cells is controversial. The initial paradigm posited that B-1 and B-2 cells derive from separate lineages. More recently it has been argued that B-1 cells derive from conventional B cells as a result of T-independent Ag activation. To understand B-1 cell differentiation, we have generated Ig transgenic (Tg) mice using the H and L chain genes (VH12 and Vκ4) of anti-phosphatidyl choline (anti-PtC) B cells. In normal mice anti-PtC B cells segregate to B-1. Segregation is intact in VH12 (6-1) and VH12/Vκ4 (double) Tg mice that develop large numbers of PtC-specific B cells. However, if B-1 cell differentiation is blocked, anti-PtC B cells in these Tg mice are B-2-like in phenotype, suggesting the existence of an Ag-driven differentiative pathway from B-2 to B-1. In this study, we show that double Tg mice have a population of anti-PtC B cells that have the phenotypic characteristics of both B-2 and B-1 cells and that have the potential to differentiate to B-1 (B-1a and B-1b). Cyclosporin A blocks this differentiation and induces a more B-2-like phenotype in these cells. These findings indicate that these cells are intermediate between B-2 and B-1, further evidence of a B-2 to B-1 differentiative pathway.

A VH12 Transgenic Mouse Exhibits Defects in Pre-B Cell Development and Is Unable to Make IgM+ B Cells

VH12 B cells undergo stringent selection at multiple checkpoints to favor development of B-1 cells that bind phosphatidylcholine. Selection begins with the VH third complementarity-determining region (CDR3) at the pre-B cell stage, in which most VH12 pre-B cells are selectively eliminated, enriching for those with VHCDR3s of 10 aa and a fourth position Gly (designated 10/G4). To understand this selection, we compared B cell differentiation in mice of two VH12 transgenic lines, one with the favored 10/G4 VHCDR3 and one with a non-10/G4 VHCDR3 of 8 aa and no Gly (8/G0). Both H chains drive B cell differentiation to the small pre-BII cell stage, and induce allelic exclusion and L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are deficient in cell division and unable to differentiate to B cells. We suggest that this is due to poor 8/G0 pre-B cell receptor expression and to an inability to form an 8/G0 B cell receptor. Our findings also suggest that VH12 H chains have evolved such that association with surrogate and conventional L chains is most efficient with a 10/G4 CDR3. Thus, selection for phosphatidylcholine-binding B-1 cells is most likely the underlying evolutionary basis for the loss of non-10/G4 pre-B cells.

Autoantibodies to phosphatidylcholine. The murine antibromelain RBC response.

The observation that murine B-cell populations can contain relatively large numbers of cells that produce IgM with the ability to lyse bromelain-treated mouse erythrocytes (BrMRBC), but not normal untreated MRBC, was made nearly 20 years ago. The major observations regarding the antigen specificity, the cells that produce this IgM, and the immunoglobulin V genes that encode them are summarized in this report. The epitope on BrMRBC that is recognized has been identified as the head group of phosphatidylcholine (PtC); B cells whose IgM has this specificity can be easily identified by their ability to bind fluorescent synthetic liposomes whose membrane contains PtC. The cells producing IgM specific for PtC all derive from the Ly-1 B-cell subset, and they use primarily two VH/VL gene pairs to encode the anti-PtC antibodies. The VH genes used describe two new VH gene families, VH11 and VH12. The genes encoding anti-PtC are unmutated and have characteristics and restricted VDJ constructions. The cells with this specificity, within individual mice, are polyclonal. These criteria are consistent with a primary antigen-driven clonal selection mechanism as the basis for the development of this immune specificity.

Practical issues in high-speed cell sorting.

Modern flow cytometric cell sorters are all capable of so‐called “high‐speed sorting.” However, there is confusion about exactly how fast a “high‐speed” cell sorter can sort cells. There are many considerations in achieving the fastest sorting speed, as well as the highest quality sort results—cell recovery, purity, and functionality. This requires the same considerations required for “slow‐speed sorting”; however, a more precise implementation is required for high‐speed sorting. The modern cell sorters enable high‐speed sorting because of advances in high‐speed electronics and data processing. We discuss the practical considerations of high‐speed sorting in terms of the theory and practical aspects of the mechanical and software components of sorting, statistics of sorting, cell preparation and viability, instrument setup, sort strategies, and biosafety. Curr. Protoc. Cytom. 51:1.24.1‐1.24.30.

Islet Lymphocyte Subsets in Male and Female NOD mice are Qualitatively Similar but Quantitatively Distinct

Islet-infiltrating lymphocytes of individual male and female non-obese diabetic (NOD) mice were examined with the purpose of determining the differences that lead to a predominance of diabetes in female versus males NOD mice. When normalized for the amount of islet lymphocytes recovered, the infiltrating lymphocytes of female NOD mice were indistinguishable from those of male NOD mice. The only observed difference was that islet inflammation progressed at an increased rate in female compared to male NOD mice. There was no difference in the composition of islet infiltrates in male and female NOD mice. Unexpectedly, the ratio of CD4+:CD8+ T cells was tightly controlled in the islets throughout diabetogenesis. The frequency of IL-4+ CD4+ T cells started high but quickly fell to 3% of the population that was maintained with increasing inflammation. A significant portion of the CD8+ T cells were islet-specific glucose-6-phosphatase catalytic subunit-related protein specific in both male and female NOD mice and this population was antigen experienced and increased at high levels of islet inflammation. Surprisingly, a large pool of antigen inexperienced naïve T cells was detected in the islets. We conclude the underlying immunological processes in both male and female NOD mice are similar while the rates differ and the presence of naïve T cell in the islets may contribute to epitope spreading.

Disrupted B-lymphocyte development and survival in interleukin-2-deficient mice.

Interleukin‐2‐deficient (IL‐2−/−) mice develop a spontaneous, progressive, CD4+ T‐cell‐mediated colitis with an age‐related decrease in the number of B lymphocytes. The aim of this study was to determine the mechanisms of B‐cell loss in IL‐2−/− mice. Serum immunoglobulin G1 (IgG1) levels in 8‐week‐old IL‐2−/− mice were above normal but then decreased dramatically with advancing age. Between 8 and 11 weeks of age, the number of B‐cell progenitors (B220+ IgM−) in the bone marrow of IL‐2−/− mice was less than half of those in IL‐2+/+ littermates. By 22 weeks of age, very few progenitor cells remained in the bone marrow of most mice, and spleens were almost devoid of B cells. Likewise, B1 cells were not present in the peritoneal cavity of aged IL‐2−/− mice. Flow cytometry analysis of B‐cell differentiation in the bone marrow suggested a progressive loss of B cells from the most mature to the least mature stages, which was not dependent on IL‐2 receptor‐α (IL‐2Rα) expression. B cells transferred from normal animals had similar survival rates in IL‐2−/− and wild‐type mice. We conclude that conventional B cells in older IL‐2−/− mice are lost by attrition owing to a derangement in B‐cell development. Because B1 cells are less dependent on the bone marrow, a separate mechanism for their loss is suggested.

Isotype Switching in CD5 B Cells

Antibodies are diverse both with respect to antigen specificity and with respect to the biological consequences that follow antigen binding. The former results from the structure of the assembled V, D, and J heavy chain and V and J light chain genes, and the latter depends upon which particular heavy chain constant region gene is expressed. Isotype switching is the process by which mature IgM-expressing B cells, having selected a particular V,-D-J, and V,-J,-C, combination for immunological relevance, rearrange the ZgH locus, C region DNA sequences to express those selected V-region and light-chain combinations on IgG, IgE, or IgA constant-region genes. This process allows the enormous diversity of antigen-combining sites generated by V-D-J recombination, heavy-light chain associations, and somatic hypermutation to be expressed on immunoglobulin (Ig) molecules with a variety of functional capabilities. In this report we will summarize our work with the murine CD5+ B-cell lymphoma CHlZLX, which switches spontaneously in vitro from the expression of IgM to any one of several IgG subclasses or IgA. We began with the observation that variants of CH12,LX, expressing isotypes other than IgM, could be isolated in vitro by fluorescence-activated cell sorting. I We than asked three related questions about this process: (1) Can we develop a method by which the spontaneous isotype-switch frequency from IgM to each of the available isotypes (encoded by C, genes located 3’ of C p ) can be quantified? (2) Do clones of CH12.LX expressing IgG3, IgGl, or IgG2b retain the ability to undergo further isotype switching, and if so, to which isotypes can they switch? and (3) Can we use this method of enumerating switch frequency to determine the effect of T cell-derived interleukins, cytokines, mitogens, antigen, or other factors on (a) isotype-switch frequency and (b) the relative frequency of switching to each of the available isotypes? There have been several studies with conventional splenic B cells that have been interpreted as showing that interleukin-4 (IL-4)2*3 and transforming growth factor-@ (TGF-@r regulate isotype switching by directing IgM-expressing B cells to switch to particular downstream isotypes. Such conclusions are difficult to support when working with a highly heterogeneous population of B cells in a variety of stages of differentiation, because the soluble factor@) may, in fact, be selecting a small subpopulation of B cells precommitted to switching to a particular isotype. We hoped that by working with a clone of in vitro-adapted B cells with defined antigen specificity, we could establish an in vitro model of switching that would enable us to address these and other questions about switch regulation.

CD3 expression, modulation and signalling in T-cell subpopulations from MRLMp-lprlpr mice

The expanded T-cell population of MRL/Mp-lpr2lpr mice is abnormal from a variety of standpoints. We have already shown that T-cell receptor expression and modulation are aberrant in the predominant CD4- CD8 (DN) T cell population. To investigate these abnormalities further, we examined CD3 expression and modulation in subpopulations of +/+ and lpr T cells and measured mitogen-induced Ca++ mobilization in DN lpr T cells. We found that expression and modulation of CD3 in CD4hi and CD8hi lpr single positive (SP) T cells are similar to that in +/+ T cells. We have, however, identified additional lpr cell subsets that are CD4lo or CD8lo. Their expression and modulation of CD3 are intermediate, between that of SP and DN lpr T cells. These subpopulations may thus represent a transitional stage between the SP and DN populations. The rapid modulation of CD3 in the DN population does not appear to be merely related to the lack of expression of CD4 or CD8, and may in fact cause (rather than result from) low CD3 expression. In addition, we observed impairment of CA++ mobilization in DN lpr T cells in response to concanavalin A or anti-CD3 antibody. These findings further define the abnormalities of T cells from lpr mice.

The CH Series of Murine B Cell Lymphomas: Identification of Cross-reactive Idiotypes and Restricted Antigen Specificities

Mice of the double congenic strain, B10.H-2aH-4bp/Wts (2a4b), display a high incidence of lymphoma following intense adoptive immunization with sheep erythrocytes (SRBC). The tumors which arise comprise a heterogenous group, including the UNC series of T cell lymphomas (Corley 1983), the CH series of B cell lymphomas (Lanier 1982), and a set of lymphomas which are not readily characterized as belonging to either the B cell or T cell lineage (unpublished data). Presently available data are insufficient to define the etiology of this disease, but we previously had found that heterologous anti-idiotype sera raised against the surface IgM of CHI and of CH2 each reacted with one other of the first 11 B cell lymphomas described (CH9 and CH5, respectively) (Arnold 1982). This suggested that a limited subset of the total normal idiotype repertoire might be expressed by CH lymphomas. We also had found that the surface immunoglobulin of CH12 was specifically reactive with an epitope present on SRBC, suggesting that adoptive immunization had led to the expansion of a subset of B cells with neoplastic potential (Arnold 1983).

CD5' B Cell Lymphomas of Mice

Spontaneous murine lymphomas commonly arise from CD5 B cells. They appear late in life and the frequency of their occurrence can be increased by transferring syngeneic lymphoid cells from old animals to young. Such transfer experiments show that although the lymphomas are not detected until late in life, the commitment to neoplasia is made much earlier and is followed by an indolent phase before the neoplastic phenotype becomes manifest. The repertoire of immunoglobulin (Ig) V genes expressed is not random, but certain VH genes appear repeatedly and, at least in some cases, are specifically associated with particular VK genes. This unbalanced repertoire is similar to that expressed by normal CD5 B cells of young adult mice, implying that the process of lymphomagenesis operates after the normal CD5 B cell repertoire has been established. Development of new CD5 B cells from pre-B cells only occurs before and shortly after birth; in the adult, the population is self sustaining and is not replenished from Ig negative precursors. The newly generated population of CD5 B cells present at birth more nearly approximates a random selection from the germ line genes. Skewing of the repertoire occurs during the first few weeks of post-natal life and results from antigen driven clonal selection by self antigens, predominantly type 2 thymus independent antigens. From 5-20% of normal adult CD5 B cells (and lymphomas) produce antibody reactive with phosphatidyl choline. Almost exclusively, these cells express VH11 or VH12 family genes in combination with specific VK genes and displaying severe constraints as to size and sequence of VDJ regions. These VH genes have not been seen expressed by conventional B cells. Neither do conventional B cells produce antibody of this specificity. In vivo, murine CD5 B cell lymphomas produce IgM; in vitro they can respond to differentiative signals and undergo isotype switching, with a marked predilection for IgA. Somatic mutation of assembled Ig genes has not been seen in these tumors and idiotype-loss variants have not been seen to result from anti-idiotype therapy.