Heterogeneity and biased T cell receptor alpha/beta repertoire of mucosal CD8+ cells from murine large intestine: implications for functional state.

Up to 90% of CD8+ intraepithelial lymphocytes (IEL) of the murine large intestine (LI) belong to the alpha/beta T cell lineage and consist of two subsets. One subset expresses both alpha and beta subunits of the CD8 coreceptor, and is uniformly Thy1+, CD5+, B220-, CD2+, CD28+. The CD8 alpha+beta+ LI-IEL exclude self-reacting V beta structures, and readily proliferate in vivo in response to T cell receptor-mediated stimuli. The CD8 alpha+beta- subset of TCR-alpha/beta+ LI-IEL is Thy1-/+, CD5-, B220+, CD2+/-, and CD28-. It contains cells with potentially self-reacting V beta s and is responsive in vivo to high doses of anti-TCR-alpha/beta monoclonal antibody (mAb), but not to bacterial superantigens. Both subsets are abundant in LI-IEL of old nude mice, and CD8 alpha+beta+ LI-IEL in nude mice undergo the same V beta deletions as in euthymic mice of the same background. Both subsets express the intestinal T cell-specific integrin alpha M290 beta 7, known to be a homing receptor for IEL. Unusually high proportions of CD69+ cells within both subsets indicate chronic activation. The proportions of CD69+ and alpha M290 beta 7+ cells within the CD8 alpha+beta+ subset increase with age, probably due to constant antigenic challenge. We propose that CD8 alpha+beta+ and CD8 alpha+beta- subsets of LI-IEL permanently reside in LI and represent a lineage different from spleen and lymph node CD8+ T cells. The CD8 alpha+beta+ undergoes negative selection, and is responsive to TCR-mediated stimuli. The CD8 alpha+beta- subset of LI-IEL is a subject of distinct selection mechanisms, and has low responsiveness to TCR-mediated stimuli.

Immunoregulation of murine myeloma in vitro. II. Suppression of MOPC-315 immunoglobulin secretion and synthesis by idiotype-specific suppressor T cells.

In previous studies, BALB/c mice immunized with trinitrophenyl-specific IgA protein (M315) produced by MOPC-315 developed idiotype (Id315)-specific T cells that suppressed M315 secretion in vivo. In the present in vitro studies, we show that inhibition of M315 secretion is mediated by a theta,Lyt-1-2+ cell that expresses a surface membrane receptor for Id315. The suppressor signal is a diffusable product that acts directly on M315-secreting myeloma cells. Inhibition of M315 secretion is T cell dose-dependent, Id315-specific, reversible, and occurs without any effect on MOPC-315 growth, viability, or surface membrane expression of M315. Inhibition of M315 secretion results from a selective inhibition of M315 synthesis in the myeloma cell. These studies provide new insight into the mechanisms of direct B cell regulation by idiotype-specific T cells.

CD4+ murine T cell clones that express high levels of immunoglobulin binding belong to the interleukin 4-producing T helper cell type 2 subset.

A panel of 20 murine CD4+ clones was examined for the presence of surface membrane receptors for IgA, IgM, IgD, IgE, and IgG. High level expression of multiple Fc receptors (FcRs) was found on all Th2 clones. FcR expression was low or undetected on the Th1 clones. The preferential expression of FcR on activated Th2 cells suggests potential mechanisms for immunoregulatory interactions with B cells.

Immunofluorescence studies of neurofilaments in the rat and human peripheral and central nervous system.

Localization of antisera to neurofilament antigens derived from rat peripheral nerve was carried out in tissues of rat and human peripheral and central nervous systems by indirect immunofluorescence. Unfixed and chloroform-methanol-fixed frozen sections of tissues were incubated in purified IgG of the experimental rabbit antisera and subsequently exposed to goat anti-rabbit IgG conjugated with fluorescein isothiocyanate. Control studies were conducted on identical tissue preparations incubated in the same concentrations of nonspecific rabbit IgG or in experimental rabbit IgG absorbed with extracts of rat peripheral nerve containing neurofilament antigen. Extensive immunofluorescence was observed in rat and human peripheral and central nervous systems. The distribution and configuration of immunofluorescence corresponded to neurofilament-rich structural components of these tissues. Prominent immunofluorescence was also noted in neuronal cell bodies of spinal sensory ganglia, especially in perikarya of the large neuronal type. Immunofluorescence of the central nervous system was located predominantly in myelinated axons of the white matter in cerebrum, cerebellum, brain stem, and spinal cord. Less intense immunofluorescence was also seen in neuronal perikarya and in short thin linear processes of grey matter.

Suppressor T cell action inhibits the expression of an excluded immunoglobulin gene.

Cells of the murine plasmacytoid line MOPC-315 synthesize two distinct immunoglobulin light chains: a normal lambda II protein, which is incorporated into secretory and surface-bound immunoglobulin, and a truncated, nonfunctional lambda I protein found only in the cytoplasm. Idiotype-specific suppressor T lymphocytes selectively inhibit the expression of both lambda II- and lambda I-specific messenger RNA by MOPC-315 cells. This finding demonstrates that phenotypically excluded light chain genes can be subject to immunoregulatory control and suggests that the expression of divergent lambda isotypes may be coordinately regulated in immunoglobulin-secreting cells.

Expansion of Fc receptor-bearing T lymphocytes in patients with immunoglobulin G and immunoglobulin A myeloma.

Lymphocytes obtained from the blood of normal individuals and six patients with newly diagnosed multiple myeloma were separated into T and non-T cell populations by rosette-formation with sheep erythrocytes, and were then assayed for the presence of surface membrane Fc receptors. When compared with normal individuals, four patients with IgG myeloma had a three- to fourfold increase in T cells with IgG receptors (T gamma cells) and two patients with IgA myeloma had a two- to threefold increase in T cells with IgA receptors (T alpha cells). Patients with IgG or IgA myeloma had normal numbers of non-T lymphocytes with surface receptors for IgG and IgA, respectively. The finding that human myeloma is accompanied by elevated numbers of T cells with Fc receptors for the heavy chain class of the myeloma protein: (1) may account for the apparent "monoclonal" lymphocyte population in patients with myeloma; (b) extends to humans similar observations made in mice with secretory plasmacytomas; and (c) is of interest because T cells with Fc receptors are immunoregulatory lymphocytes.

Immunoglobulin-specific suppressor T cells.

This article has considered evidence that supports the occurrence and functional importance of suppressor T cells that are directed to B cell targets. Cells with these features have been demonstrated in experimental animals and in humans. The designation "suppressor" comes from the serologic phenotype of these cells as well as from their functional property of noncytotoxic inhibition of B cell function. Distinct suppressor T cells with these properties have been identified that effect antigen-, idiotype-, isotype-, and allotype-specific suppression of B cell function. While such cells had been suspected from earlier studies of normal immune responses, the development of monoclonal B cell models using tumor cells has provided a means to readily detect these suppressor T cells and to investigate the mechanisms by which they mediate their effects. Tumor models have proved to be powerful tools in the effort to identify and analyze the elements that underlie the complexity of immune responses. Combined with the insights provided by molecular genetic approaches and flow cytometry, functional and responsive lymphoid tumor cells are being used with increasing frequency to address basic immunoregulatory issues. An important family of suppressor T cells with B cell targets are those that express surface Fc receptors, elaborate immunoglobulin-binding factors, and appear to participate in the regulation of immunoglobulin heavy chain class expression. In addition to their importance in the regulation of heavy chain class expression during normal immune responses, alterations in FcR+ T cells in a number of disease states may provide clues that will lead to a better understanding of disorders of immune regulation.

A murine model for central nervous system leukemia and its possible relevance to human leukemia.

Treatment of a transplantable leukemia in AKR mice with both amphotericin B and 1,3-Bis(2-chloroethyl)-1-nitrosourea cured a significant percentage of animals with advanced disease. Some long-term survivors developed paralysis, and they invariably demonstrated central nervous system (CNS) leukemia. Some of these animals had a systemic relapse of their leukemia, and the CNS appeared to act as a focus for systemic dissemination. The occurrence patterns and histopathologic features of the CNS leukemia in the long-term survivors were strikingly similar to those observed in humans with acute lymphoblastic leukemia.

In vivo growth advantage of the MOPC-315 mouse myeloma over its immunoselected variants.

Myeloma cells of the "wild type" that produce complete immunoglobulin molecules and those of the more usual variant type that display only one kind of chain [either light (L) or heavy (H)] were cocultivated ip and sc in syngeneic BALB/c mice. With each of six deliberately selected variants, a progressive increase in the proportion of wild-type cells was observed; the rate of change suggested that these variants had an approximately 10% slower growth rate than that of the wild-type tumor. In contrast, a variant that arose spontaneously overgrew the wild-type cells. The results may account for a) the stable capacity of most wild-type tumors to produce complete immunoglobulin molecules (L- plus H-chains) over many years, even though they frequently generate variant cells that produce only L- or only H-chains; and b) the occasional spontaneous change of myeloma cell populations from predominantly wild-type to variant cells.

Regulatory roles for FcgammaRIII (CD16) and FcgammaRII (CD32) in the development of T- and B-lineage lymphoid cells.

IgG Fc receptors (FcgammaR) occur on all hematopoietic lineages and participate in a diversity of functions that reflect the combined effects of the molecular heterogeneity of FcgammaR and the inherent specialization of the FcgammaR+ cells. An extensive literature describes the functions of FcgammaR on mature myeloid and lymphoid cells in humans and mice but little has been published about FcgammaR on lineage progenitor cells. Several studies suggest that FcgammaR can influence leukocyte development and that FcgammaRII (CD32) and FcgammaRIII (CD16) can regulate murine T- and B-lineage development at stages before the expression of clonal antigen receptors. The nominal ligand of FcgammaR is IgG and the physiologically relevant ligand is the IgG-antigen complex, but it is also known that alternative, non-Ig ligands exist for Fc receptors. A role for FcgammaR in the regulation of leukocyte development has potential relevance for clinical situations in which the levels of nominal and/or alternative ligands of FcgammaR are elevated, or the production of soluble forms of FcgammaR is increased.

Anti-idiotypic regulation of IgA expression in myeloma cells.

MOPC-315 (alpha lambda 2) is a BALB/c plasmacytoma that produces an anti-TNP antibody (M315). In addition to being secreted M315 is also expressed on the surface membranes of MOPC-315 cells. In the present studies an in vitro-adapted line of MOPC-315 was used to study the effect of affinity-purified, isologous anti-idiotypic antibodies (a-Id315) and idiotype-specific T cells on M315 expression. We observed that both monoclonal and polyclonal a-Id315 mediated a reversible clearance of surface membrane M315 but did not influence M315 secretion or MOPC-315 growth even when the myeloma cells were cultured in the continuous presence of a-Id315 for three weeks. Clearance of surface M315 was rapid, reversible, and a-Id315 dose-dependent. M315:a-Id315 complexes were shed from MOPC-315 cells in the form of microscopic membranous vesicles and a-Id315 was consumed in the process. Protein synthesis was required for re-expression of surface M315 only if a presynthesized internal pool of M315 had previously been depleted. In contrast, idiotype-specific T cells mediated specific inhibition of M315 secretion without influencing surface M315 expression. Although the anti-idiotypic antibodies and the anti-idiotypic T-cells are both directed to the surface membrane immunoglobulin on the cloned B cell, the anti-idiotypic antibodies regulate surface membrane expression of immunoglobulin while the anti-idiotypic T-cells regulate secretion of immunoglobulin. These observations support the view that in idiotype regulation, surface membrane immunoglobulin molecules function as a focusing device for regulatory effectors which actually determine the quality of the effect achieved.

Enhancer mediated suppression of epsilon heavy-chain gene expression in a murine IgE-producing hybridoma.

In vitro co-culture of IgE-secreting hybridoma cells (B53) with spleen cells harvested from mice with established B53 tumours results in a specific, T cell-dependent suppression of epsilon-chain expression in the B53 cells. The role of immunoglobulin enhancers in the suppression of IgE synthesis in B53 cells was examined by transfecting B53 cells with CAT expression vectors containing the immunoglobulin heavy- or kappa light-chain intron enhancers or a Rous sarcoma virus (RSV) LTR. When epsilon-chain expression of transfected cells was suppressed in vitro. CAT expression was also suppressed in cells transfected with vectors containing the immunoglobulin heavy-chain gene enhancer, but not in cells transfected with vectors containing the kappa enhancer or RSV LTR. Thus, the T cell-dependent suppression of IgE synthesis in B53 cells correlates with a specific inactivation of the immunoglobulin heavy chain enhancer, strongly suggesting that T cell-mediated suppression of Ig synthesis can normally occur through specific repression of Ig enhancer function. This represents a new regulatory pathway involved in the control of IgE synthesis and is the first indication that the enhancer mediated expression of Ig genes in B cells can be modulated through T cell-dependent processes.

Increased T epsilon cells in BALB/c mice with an IgE-secreting hybridoma.

Since it has been proposed that T cells with receptors for the Fc portion of IgE (T epsilon) in rats, mice and humans are IgE-specific regulatory cells, it was investigated whether mice with an IgE-secreting hybridoma might be a source of large numbers of T epsilon cells. BALB/c mice with ascitic tumors of the IgE hybridoma B53 (epsilon, kappa, anti-DNP) developed high serum concns of monoclonal IgE which was followed by the appearance of large numbers of Lyt1-2+ L3T4- T epsilon cells. In contrast, mice bearing a non-IgE producing variant of B53 failed to develop T epsilon cells. Mice infused with cell-free ascites fluid (containing high levels of monoclonal IgE) from the IgE-secreting B53 hybridoma developed high serum IgE levels and also developed high numbers of T epsilon cells. Mice infused with cell-free ascites obtained from the non-IgE-secreting variant ENP-1 (containing very low amounts of IgE) did not develop either high serum IgE levels or T epsilon cells. These findings suggest that high serum IgE concns induce large numbers of T cells that express phenotypic markers of suppressor cells and have surface IgE-Fc receptors. These studies extend to IgE the principle that hybridomas and plasmacytomas induce large numbers of immunoregulatory T cells that express Fc receptors specific for the heavy chain class of the secreted monoclonal immunoglobulin. Since T epsilon cells are normally present only in small numbers, their marked increase in mice with IgE-secreting hybridomas identifies a ready source of large numbers of T epsilon cells that can be used to investigate their regulatory properties.

The Fc epsilon RII/CD23 gene is actively transcribed during all stages of murine B-lymphocyte development.

It is generally accepted that the expression of Fc epsilon RII/CD23 on the surface of the B-lineage cells is restricted to the stage of the resting, mature (sIgM+/sIgD+) B-lymphocyte. However, it is unknown whether activation of the Fc epsilon RII/CD23 gene is also restricted to the stage of the mature B-lymphocyte. To address this question we investigated a panel of B-lineage cell lines for the presence of transcripts encoding Fc epsilon RII/CD23. We detected transcripts in 16 of 26 B-lineage cell lines representing the entire spectrum of B-cell development. In most cases (13 of 16) active transcription of the murine Fc epsilon RII/CD23 gene was not coupled with the expression of cell surface Fc epsilon RII/CD23 expression did not hold for all murine B-cell lines. One post-switch B-cell line (sIgM-/sIgG+) expressed Fc epsilon RII/CD23 on the cell surface and another could be induced with IL-4 and LPS to express surface Fc epsilon RII/CD23. Transcription of the murine CD23 gene in the absence of cell surface expression of Fc epsilon RII/CD23 does not appear to simply be an aberrant feature of transformed B-cells since we found transcripts, but not surface expression, in some normal splenic and peritoneal B-lymphocytes. Our findings suggest that the potential for expression of Fc epsilon RII/CD23 may occur over a much broader development window of the B-lineage than previously suspected. Transcription of the Fc epsilon RII/CD23 gene, in the absence of detectable cell surface protein expression in B-lineage cell lines, and in sort-purified B-lymphocyte subpopulations, implies that in addition to regulatory mechanisms already known, murine CD23 is also regulated through post-transcriptional mechanisms that have not yet been characterized.

Immunoglobulin (Fc) receptors on murine T- and B-lymphocytes: investigations using tumor models.

Lymphoid tumors are productive experimental models for the study of lymphocyte immunoglobulin receptors. Investigations with Fc receptor expressing lymphoid tumor cells have generated much useful information about: (a) the developmental expression of the different classes of Fc receptors on lymphoid cells of the T- and B-lineages; (b) the biochemical steps involved in the regulation of Fc receptor expression on lymphoid cells; (c) the structures of lymphoid cell Fc receptors and their genes; (d) the signals that induce alterations in the expression of Fc receptors on lymphoid cells; and (e) the molecular specificity of the binding of immunoglobulin to lymphoid cells Fc receptors. In addition, tumors that secrete immunoglobulins are providing useful models for analysis of the mechanisms by which B-cells influence Fc receptor expression and function on T-cells. An interesting, bi-directional immunoregulatory circuit involving Fc epsilon R+ host T-cells and IgE-secreting hybridoma cells has been identified that could prove useful in the analysis of the regulation of epsilon heavy chain expression. The studies discussed in this article and elsewhere in this volume serve to emphasize that, in addition to being clonal sources of key molecules such as Fc receptors and their messenger RNAs, lymphoid tumor cells that express Fc receptors are powerful and unique experimental models for investigating the developmental biology, regulation and function of lymphocyte Fc receptors.

Characterization of novel FcepsilonRII/CD23 isoforms lacking the transmembrane (TM) segment in human cell lines.

Human FcepsilonRII/CD23 is an approximately 45 kDa type II transmembrane glycoprotein belonging to the C-type animal-lectin family, and has two isoforms (a and b) that only differ in their intracytoplasmic tails. We previously found that in several human and mouse cell lines there were two additional CD23 transcripts (a' and b') lacking the exon 3 that encodes the entire transmembrane segment and a part of cytoplasmic tails. In this study, we analyzed the putative CD23a' and CD23b' products at protein levels and characterized with rabbit polyclonal antibodies against novel amino-acid sequences of the putative CD23a' and CD23b' molecules (anti-CD23a' Ab, anti-CD23b' Ab). Western blots in COS cells transfected with CD23a' or CD23b' cDNA as well as in vitro translation assays showed that the a' and b' CD23 transcripts were translated to about 40 kDa molecules. These 40 kDa molecules were also recognized by a polyclonal antibody against 25 kDa soluble fragment of human CD23. We also found that human cells having mRNAs for CD23a' and CD23b' expressed protein products recognized specifically by anti-CD23a' or anti-CD23b' Ab, respectively. In addition, the CD23a' and CD23b' molecules in transfected COS cells were resistant to Endo H(f) and PNGase F, although these truncated forms as well as the membrane-associated forms had an asparagine residue responsible for the N-linked glycosylation. Taken together, our results show that the a' and b' CD23 transcripts are expressed and translated in human lymphoid cells and that their translated products are retained in the cytoplasm where they might play an unique regulatory role in the expression of the full-length CD23 on the cell surface.

Alternative transcripts of the human CD23/Fc epsilon RII. A possible novel mechanism of generating a soluble isoform in the type-II cell surface receptor.

Human CD23/Fc epsilon RII is a 45 kDa type-II membrane glycoprotein having two isoforms (a and b) that only differ in the structures of their intracytoplasmic tails. CD23/Fc epsilon RII has been demonstrated to have multiple roles in the immune system such as regulation of lymphocyte growth and differentiation and IgE-mediated immune responses. Here, we found that the human B-cell line RPMI8866, in addition to a and b transcripts, contained shorter transcripts (a' and b') that lack the entire third exon. These alternative transcripts were also detected in peripheral blood lymphocytes as well as other hematopoietic cell lines with CD23/Fc epsilon RII. Because exon 3 encodes all of the transmembrane segment and the anchoring region of the cytoplasmic tail, it is suggested that a' and b' transcripts encode secretory forms of CD23/Fc epsilon RII or they may function as regulatory transcripts involved in the control of CD23/Fc epsilon RII expression.

Expression of IgA and IgM Fc receptors on murine T lymphocytes.

Fc receptors are induced on T cells following activation via the TCR. T cells that express Fc receptors transiently have the ability to use two different cognate systems: the TCR and immunoglobulins bound to the Fc receptors. The studies discussed in this article are focused on the Fc alpha and Fc mu receptors that can be induced on certain subsets of murine T lymphocytes. The article emphasizes the role of the T cell receptor for antigen in the expression of Fc alpha and Fc mu receptors on murine T cells and reviews experimental observations that suggest significant molecular heterogeneity of these Fc receptors. The finding that regulation of expression of Fc alpha receptors and Fc mu receptors on T lymphocytes is linked to cellular activation via the CD3/TCR complex implies that these Fc receptors might mediate important functions in the biology and pathology of T cells.

Plasmacytoma spleen colonization: a sensitive, quantitative in vivo assay for idiotype-specific immune suppression of MOPC-315.

A spleen colony-forming assay for the measurement of idiotype-specific transplantation resistance to MOPC-315 is described. The assay is highly quantitative, sensitive, reproducible, less time consuming and distinctly superior to conventional in vivo assays which measure tumor incidence, tumor size, and/or host survival time after tumor challenge. The assay directly measures those clonogenic cells of the MOPC-315 myeloma which have a sufficient proliferative capacity to form macroscopic splenic foci within 14 days after intravenous challenge.

Developmental regulation of Fc epsilon RII/CD23 expression in B-lineage cells: evidence for transcriptional and post-transcriptional levels of control.

The present studies show that the expression of cell surface Fc epsilon RII/CD23, detected with the monoclonal anti-Fc epsilon RII/CD23 antibody B3B4 or by the binding of IgE, is not restricted to the stage of resting mature virgin B lymphocytes. Murine CD23 was detected as a cell surface protein on two sIgG+ B-cell lines. Moreover, we detected full-length transcripts for Fc epsilon RII/CD23 in several members of a panel murine B lymphoid lineage cell lines representative of all stages of murine B lymphocyte development. Our findings suggest that regulation of CD23 translation may differ between B-cell lines at various stages of differentiation. The detection of mRNA transcripts for Fc epsilon RII/CD23 was not restricted to transformed cell lines. Fc epsilon RII/CD23 transcripts were amplified by RT-PCR from peritoneal and splenic B lymphocyte subpopulations that were sorted by flow cytometry into populations that did not express surface Fc epsilon RII/CD23. Our findings suggest that Fc epsilon RII/CD23 transcription and translation are not necessarily restricted to the narrow developmental window of murine B lymphocyte differentiation as previously thought. Our findings imply that Fc epsilon RII/CD23 may be expressed at the protein level at various stages of murine B lymphocyte differentiation. Investigations into the expression patterns and potential mechanisms of regulation of Fc epsilon RII/CD23 could provide insight into the basis for the wide range of immunological functions that have been proposed for Fc epsilon RII/CD23.