Robust B cell immunity but impaired T cell proliferation in the absence of CD134 (OX40).

CD134 (OX40) is a member of the TNF receptor family that is expressed on activated T lymphocytes. T cells from mice that lack expression of CD134 made strong responses to a range of challenges, but they showed impaired proliferation in response to direct stimulation through the TCR with monoclonal anti-CD3epsilon Ab. CD134-deficient mice controlled infection with Leishmania major, Nippostrongylus brasiliensis, and Theiler's murine encephalomyelitis virus, and they made overtly normal Ab responses to a variety of antigens. Thus, CD134 is not essential for many T cell responses in vivo, nor is it required for the provision of help to B cells. Nonetheless, a subtle role in the regulation of T cell reactivity is suggested by the effect of CD134 deficiency on in vitro T cell responses.

OX40 is differentially expressed on activated rat and mouse T cells and is the sole receptor for the OX40 ligand.

OX40, a member of the tumor necrosis factor (TNF) receptor/nerve growth factor (NGF) receptor superfamily was first identified as a marker of activated rat CD4+ cells with the MRC OX40 monoclonal antibody (mAb). A ligand for OX40 (called OX40 ligand or OX40L) has recently been identified and has sequence similarity to TNF. Mouse OX40L-immunoglobulin fusion protein (OX40L-Ig) binds to activated mouse CD4+ and CD8+ cells (Baum, P. R. et al., EMBO J. 1994. 13: 3992) suggesting that OX40 could have a differential pattern of expression on mouse and rat T cells. This, however, did not rule out the presence of an alternative receptor on CD8+ cells that also binds the OX40L. We have compared the binding of the MRC OX40 mAb with that of OX40L-Ig to activated rat lymph node cells and show that both recognize the same protein, namely OX40 which is expressed on CD4+ and CD4+ CD8 alpha+ cells, but not on CD4-CD8+ cells. We have raised a new mAb (MRC OX86) using recombinant mouse OX40 protein and show by two-color flow cytometry that mouse OX40 is expressed on CD4 and CD8 single-positive cells. In addition, the new MRC OX86 mAb, unlike the MRC OX40 mAb, did not block binding of the OX40L. We conclude that OX40 is differentially expressed on activated mouse and rat T cells and is the sole receptor for the OX40L.

Use of isolated immature-stage B cells to understand negative selection and tolerance induction at the molecular level.

Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.

Gene structure and chromosomal localization of the mouse homologue of rat OX40 protein.

The OX40 protein is expressed only on activated rat CD4+ T blasts and is a member of a superfamily of cell surface molecules which includes CD40, CD30, CD95 (Fas), CD27, 4-1BB antigens and the receptors for tumor necrosis factor (TNF) and nerve growth factor (NGF). The proteins of this group are related to each other by having three to six repeats of a cysteine-rich sequence in their extracellular domains. Members of this family of receptors have also been shown to bind to ligands which are structurally related to TNF. The mouse homologue of the rat OX40 protein was cloned at the cDNA and genomic levels. The gene structure shows that there are several intron/exon borders shared between OX40 and CD27, CD40, TNF receptor type I, CD95 and 4-1BB genes. This group of genes is less closely related structurally to the gene structure of the NGF receptor. The gene encoding murine OX40 has been placed on mouse chromosome 4, in an area which contains the genes for TNF receptor type II and 4-1BB, and is syntenic with a region of human chromosome 1 which contains human TNF receptor type II, OX40, and CD30 genes.

Progressive changes in CD45RB phenotype and lymphokine production by murine CD4+ T cells after alloantigen exposure.

Changes in CD45R expression correlate with changes in phenotype in mouse, rat and human T cells. It has been shown in mouse that CD45RB high T cells produce mostly interleukin-2 (IL-2) while CD45RB low T cells produce more IL-4 than IL-2 after mitogen stimulation in vitro. CD45RB expression also decreases when T cells are stimulated. In this study we compared responses of CD45RB high and low CD4+ T cells to alloantigens. Although a majority of unstimulated murine T cells from unimmunized mice are CD45RB high, we were able to isolate and purify sufficient numbers of T cells to study their response to alloantigens. When separated cells were stimulated in vitro with alloantigen the CD45RB high T cells became heterogeneous for their expression of CD45RB, indicating that high cells decrease their expression of CD45RB epitopes. Surprisingly, only CD45RB high T cells from unimmunized mice were alloreactive, as measured by proliferation and lymphokine secretion. In contrast, both CD45RB high and low populations from mice primed with allogeneic spleen were responsive to alloantigens. The 'primary' response of T cells from alloantigen-primed mice to third party stimulators is 10-fold greater in the CD45RB high population than in the CD45RB low, as would be predicted by our results in the primary mixed lymphocyte/leucocyte reaction (MLR). Furthermore, the response of CD45RB low T cells from unprimed mice and the response to third party alloantigen from primed mice was reconstituted by the addition of exogenous IL-2. The response of CD45RB low cells from primed mice was specific, as the third party alloresponsive cells were again primarily contained within the CD45RB high population. In the CD45RB high population in the secondary MLR we observed an increase in the production of IL-4 relative to IL-2.

High levels of CD44 expression distinguish virgin from antigen-primed B cells.

The in vitro polyclonal stimulation of B cells through their surface immunoglobulin (Ig) induces substantial increases in CD44 protein levels within 24 hours, whereas other stimuli (e.g., lipopolysaccharide, phorbol 12,13 dibutyrate, and interleukin 4) fail to significantly upregulate CD44. The marked increase in CD44 protein expression on anti-Ig-treated B lymphocytes correlates with an increase in CD44-specific mRNA. Cell sorting experiments with B cells isolated from trinitrophenyl-keyhole limpet hemocyanin-immunized mice demonstrate that both short-term antigen-specific, IgG-secreting cells and long-term antigen-primed B cells are exclusively CD44high. We speculate that the rapid and sustained increase in CD44 expression mediated by surface Ig stimulation may alter the homing properties of antigen-primed B cells.

Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation.

Leukocytes express a family of plasma membrane proteins called CD45 or the leukocyte common antigen. Isoforms of various molecular masses, 180-240 kDa, are produced by alternative splicing and usage of three exons, named A, B, and C, that encode the N-terminal portion of the external domain. By using monoclonal antibodies that precipitate B exon-dependent and B exon-independent isoforms we find that both murine CD4+ and murine CD8+ T cells selectively down-regulate the B exon-dependent forms of CD45 during an immune response. This change was monitored by using fluorescence-activated cell sorter (FACS) analysis and immunoprecipitation from surface radioiodinated and metabolically labeled cells. The loss of the 190-kDa B exon-dependent isoform during T-cell activation is accompanied by an increased production of a 180-kDa form, which does not contain the B exon-encoded sequence. This accounts for our observation that the overall expression of CD45, as assessed by FACS analysis, does not change.

Epitopes on CD45R [T200] molecules define differentiation antigens on murine B and T lymphocytes.

In the course of isolating hybridomas from rats that had been immunized with anti-Ig activated murine B lymphoblasts, we have identified three new mAb reactive with the T200 or leukocyte common antigen family (CD45). One, MB4B4, reacts with T200 on all leukocytes, and immunoprecipitates all the molecules that are recognized by an existing mAb to this family. Two others, MB23G2 and MB15C11, react with a subset of T200 molecules that has a unique tissue and cellular distribution. By FACS analysis and by immunocytochemical labeling of tissue sections, MB23G2/15C11 bind to most, if not all, small B and T lymphocytes, but react weakly with macrophages and dendritic cells. Expression of the MB23G2/15C11 T200 epitope exhibits four distinct features on lymphocytes: 1) activation of CD4+ cells in the mixed leukocyte reaction results in a decreased expression of MB23G2/15C11, on a subset of 30-60% of the T-cell blasts; 2) of 16 T-helper clones examined, clones of the TH2 phenotype express moderate to high levels of MB23G2/15C11 (2.5-19-fold increase in median fluorescence over control) while the TH1 clones express low to moderate levels (1.2-6.4-fold increase in median fluorescence over control) of the antigen recognized by these mAb; 3) The MB23G2/15C11 mAb do not react with germinal center cells in tissue sections of spleen, lymph node, and Peyer's patches; 4) MB23G2/15C11 reacts primarily with a subset of large thymocytes localized to the medulla. Therefore, MB23G2/15C11 define a subset-restricted form of T200 (CD45R) on murine lymphocytes. The tissue distribution of this T200 associated epitope is distinct from previously defined CD45 antigens and suggests a role during several pathways of lymphocyte development.

T-independent and T-dependent steps in the murine B cell response to antiimmunoglobulin.

Sepharose-anti-Ig and purified populations of small, high-density B cells have been used to study the formation and function of B lymphoblasts. Sepharose-anti-Ig converts small, Ia-poor B cells with a high-buoyant density to large, Ia-rich, B blasts with a low-buoyant density. We find that this response proceeds efficiently in the absence of IL-4 (BSF-1) as well as most T cells, macrophages, and dendritic cells. Further development of the blasts requires an additional stimulus, such as LPS or the conditioned medium of stimulated EL-4 thymoma cells. Within 6 h, blasts begin to enter S phase and within 24 h most divide. At later times (48-72 h) most of the blasts are actively secreting IgM. Recombinant IL-1, -2, -3, and -4 have little or no effect on the B blasts, and a neutralizing mAb to IL-4 does not block the response to EL-4 Sn. We conclude that Sepharose-anti-Ig induces B cell blastogenesis in a T-independent fashion and that these blasts represent a highly enriched population of cells that respond to distinct, T cell-derived lymphokines.