Abnormal development of the thymus in "nude" mice.

Nude mice bearing grafts of normal thymus reject skin grafts and have low, but higher than usual, lymphocyte counts. Nude bone marrow can successfully repopulate the thymus and thymus-derived areas of lethelly irradiated recipient mice. Attempts to reconstitute nude mice with normal fetal liver failed. The so-called thymic rudiment of nude mice when grafted to normal mice did not develop thymocytes. These experiments show that nude mice suffer from a defect of the epithelial portion of the thymus rather than of the precursors of thymocytes.

Influence of Igh-linked gene products on the generation of T helper cells in the response to sheep erythrocytes.

The role of Igh-linked loci in the generation and expression of T cell help for antibody responses to sheep erythrocytes (SRBC) was investigated. The production of IgM, IgG3, IgG1, IgG2b, and IgG2a antibody to SRBC was shown to be T cell dependent. The Igh-congenic mouse pair CBA/Tufts (Ighj) and CBA.Ighb gave equivalent responses to SRBC. CBA.nude mice (Ighj) supplemented with peripheral T cells of either Ighj or Ighb genotype produced equivalent, high responses. Therefore, T cell-B cell mismatching for the Igh haplotype is not in itself a bar to the generation or expression of help. In contrast, T cells primed in an environment that lacks Ighj-linked products are inefficient helpers for Ighj B cells. These results suggest that antigen-primed B cells or their products prime a set of T cells that can help B cells that bear matching, Igh-linked gene products.

Strain variation in the frequency of Abelson murine leukemia virus-transformed fetal liver pre-B cells bearing complete immunoglobulin heavy chain rearrangements.

Fetal liver Abelson pre-B cell lines obtained from CBA/Tufts.xid and (CBA/Tufts.xid x CBA/Tufts)F1 mice have complete VDJH rearrangements on at least one allele. Such high frequencies of VDJH rearrangements have previously been observed in adult derived but not fetal liver derived Abelson pre-B cell lines. Genetic analyses suggest that CBA/Tufts.xid carries an autosomal dominant gene(s) that determines the predominance of VDJH rearrangements among transformants. This autosomal gene(s) might affect the intrinsic development of the early B cell lineage in the fetus or the fetal microenvironment, expanding pre-B cells of the "more mature" VDJH phenotype.

Lack of mature B cells in nude mice with X-linked immune deficiency.

Mice were bred that simultaneously expressed the mutations nude and x-linked immune deficiency (xid). These doubly deficient animals had less than 10% of normal serum immunoglobulin levels. Their spleen cells did not respond to thymus-independent antigens in vitro nor did they respond to lipopolysaccharide. There was a virtual absence of cells with surface mu, kappa, or lambda 1, as detected by fluorescence. Sections of lymphoid organs revealed an absence of primary B cell follicles. Taken together, these results indicate a lack of mature B cells in nude xid mice. The possibility is considered that mature B cells belong to two subpopulations representing two lineages, one controlled by alleles at the xid locus and the other by alleles at the nude locus.

Mouse spleen dendritic cells present soluble antigens to antigen-specific T cell hybridomas.

In the presence of the soluble polypeptide antigens ovalbumin and keyhole limpet hemocyanin, purified mouse spleen dendritic cells induce the secretion of IL-2 by antigen-specific T cell hybridomas. This response is H-2 restricted and can be specifically inhibited by monoclonal anti-I-A. These data indicate that dendritic cells can present soluble antigen to H-2-compatible T cells.

Bruton's tyrosine kinase links the B cell receptor to nuclear factor kappaB activation.

The recognition of antigen by membrane immunoglobulin M (mIgM) results in a complex series of signaling events in the cytoplasm leading to gene activation. Bruton's tyrosine kinase (BTK), a member of the Tec family of tyrosine kinases, is essential for the full repertoire of IgM signals to be transduced. We examined the ability of BTK to regulate the nuclear factor (NF)-kappaB/Rel family of transcription factors, as the activation of these factors is required for a B cell response to mIgM. We found greatly diminished IgM- but not CD40-mediated NF-kappaB/Rel nuclear translocation and DNA binding in B cells from X-linked immunodeficient (xid) mice that harbor an R28C mutation in btk, a mutation that produces a functionally inactive kinase. The defect was due, in part, to a failure to fully degrade the inhibitory protein of NF-kappaB, IkappaBalpha. Using a BTK-deficient variant of DT40 chicken B cells, we found that expression of wild-type or gain-of-function mutant BTK, but not the R28C mutant, reconstituted NF-kappaB activity. Thus, BTK is essential for activation of NF-kappaB via the B cell receptor.

The ability of CD40L, but not lipopolysaccharide, to initiate immunoglobulin switching to immunoglobulin G1 is explained by differential induction of NF-kappaB/Rel proteins.

Antibodies of the immunoglobulin G1 class are induced in mice by T-cell-dependent antigens but not by lipopolysaccharide (LPS). CD40 engagement contributes to this preferential isotype production by activating NF-kappaB/Rel to induce germ line gamma1 transcripts, which are essential for class switch recombination. Although LPS also activates NF-kappaB, it poorly induces germ line gamma1 transcripts. Western blot analyses show that CD40 ligand (CD40L) induces all NF-kappaB/Rel proteins, whereas LPS activates predominantly p50 and c-Rel. Electrophoretic mobility shift assays show that in CD40L-treated cells, p50-RelA and p50-RelB dimers are the major NF-kappaB complexes binding to the germ line gamma1 promoter, whereas in LPS-treated cells, p50-c-Rel and p50-p50 dimers are the major binding complexes. Transfection of expression plasmids for NF-kappaB/Rel fusion proteins (forced dimers) indicates that p50-RelA and p50-RelB dimers activate the germ line gamma1 promoter and that p50-c-Rel and p50-p50 dimers inhibit this activation by competitively binding to the promoter without activating the promoter. Therefore, germ line gamma1 transcription depends on the composition of NF-kappaB/Rel proteins.

Regulation of nuclear localization and transcriptional activity of TFII-I by Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is required for normal B-cell development, as defects in Btk lead to X-linked immunodeficiency (xid) in mice and X-linked agammaglobulinemia (XLA) in humans. Here we demonstrate a functional interaction between the multifunctional transcription factor TFII-I and Btk. Ectopic expression of wild-type Btk enhances TFII-I-mediated transcriptional activation and its tyrosine phosphorylation in transient-transfection assays. Mutation of Btk in either the PH domain (R28C, as in the murine xid mutation) or the kinase domain (K430E) compromises its ability to enhance both the tyrosine phosphorylation and the transcriptional activity of TFII-I. TFII-I associates constitutively in vivo with wild-type Btk and kinase-inactive Btk but not xid Btk. However, membrane immunoglobulin M cross-linking in B cells leads to dissociation of TFII-I from Btk. We further show that while TFII-I is found in both the nucleus and cytoplasm of wild-type and xid primary resting B cells, nuclear TFII-I is greater in xid B cells. Most strikingly, receptor cross-linking of wild-type (but not xid) B cells results in increased nuclear import of TFII-I. Taken together, these data suggest that although the PH domain of Btk is primarily responsible for its physical interaction with TFII-I, an intact kinase domain of Btk is required to enhance transcriptional activity of TFII-I in the nucleus. Thus, mutations impairing the physical and/or functional association between TFII-I and Btk may result in diminished TFII-I-dependent transcription and contribute to defective B-cell development and/or function.

ABL oncogenes directly stimulate two distinct target cells in bone marrow from 5-fluorouracil-treated mice.

Mice reconstituted with BCR/ABL-infected 5-fluorouracil-treated bone marrow are considered a model system for human chronic myelogenous leukemia, a malignancy that arises in hematopoietic stem cells. These animals develop multiple types of hematopoietic tumors, which could arise either from undifferentiated cells that mature during tumor development or from progenitors committed to different lineages. To examine the BCR/ABL-sensitive target cells present in the marrow of mice treated with 5-fluorouracil, we used a single-step in vitro assay. These experiments revealed that both the P210 and P185 BCR/ABL proteins and the related v-abl protein induce lymphoid and myeloid colonies, colony types that mimic two of the prominent types of tumors found in the reconstitution model. The lymphoid colonies were similar to lymphoid colonies found following infection of normal bone marrow with respect to differentiation state and tumorigenicity. The cells in the myeloid colonies were differentiated and non-tumorigenic. Fluorescence-activated cell sorting revealed that most of the lymphoid and myeloid colonies arose from distinct precursors and that the lymphoid colonies arose from B-lineage-committed cells. These data suggest that most of the lymphomas observed in the reconstitution model arise from committed progenitors that are distinct from those involved in the myeloid disease.

Activation of B-cells by sIgM cross-linking induces accumulation of CD5 mRNA.

The surface membrane molecule CD5 is expressed on mature T cells and on the B-1a subpopulation of B cells. These CD5 positive B cells express an antibody repertoire with a relatively high frequency of self-reactivity. There is uncertainty about the origins of CD5 B cells and the reasons for this are reviewed. Recent reports which relate to the lineage/selection debate are discussed. For instance, an increase in the frequency of CD5 B cells is a feature of several genetically determined polysystem autoimmune syndromes. In the case of motheaten (me, mev) the pathogenesis of this increase in CD5 B cells is not yet understood, even though the mutation has been mapped to the Hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Another mutation which affects B cell development, X-linked immunodeficiency (xid), encodes a point mutation in a B cell cytoplasmic tyrosine kinase. Expression of xid in otherwise normal mice causes a lack of CD5 B cells and a shift in the antibody repertoire. Interestingly, expression of both xid and motheaten results in an amelioration of autoantibody production. Evidence is presented that in B cells regulation of expression of CD5 can occur at the level of mRNA and that cross-linking of sIgM can induce the accumulation of CD5 mRNA. The overall concept advanced is that cells expressing natural autoantibodies are triggered via sIgM ligation to become CD5 B cells.

Effect of immunosuppressive therapy for renal allografts on the number of circulating sheep red blood cells rosetting cells.

Patients receiving renal allografts from relatively incompatible donors were randomly assigned to one of two immunosuppressive regimens: azathioprine and prednisone with or without a 2-week course of horse antihuman thymocyte globulin (ATG). The number of circulating lymphocytes fell to about one-third of pretreatment values in both groups of patients. In patients given ATG, the proportion of sheep red blood cell-rosetting lymphocytes (SRBC-R) fell promptly to less than 10% of pretreatment values. In contrast, the percentage of SRBC-R remained at about 70% in patients receiving only prednisone and azathioprine. The addition of ATG reduced the number of SRBC-R/mm3 to one-tenth to one-thirtieth the number seen in non-ATG-treated patients. Plasma of patients undergoing therapy did not inhibit rosetting in vitro. It is proposed that the monitoring of circulating rosetting cells may be a useful clinical guide to the degree of T cell immunosuppression. It is also suggested that the regimen of azathioprine, prednisone, and ATG results in a more effective suppression of circulating T cells than that produced by azathioprine and prednisone alone.

Surface markers, heavy chain sequences and B cell lineages.

A unifying theory of B cell development and lineage commitment is presented. There are two firmly established B lineages: cells which normally arise only from fetal sources and lack N insertions in their rearranged heavy chains; and N-containing cells which arise from adult bone marrow precursors (and perhaps from late fetal sources). Commitment to the expression of CD5 and the capacity for long-life (or self-renewal) are induced as a consequence of sIg cross-linking, typically by a repeating epitope, thymus independent type two antigen. Alternatively, activation resulting from cognate interaction with a helper T cell does not induce CD5 but results in lower expression of J11d. In this case activation occurs in the absence of sIg cross-linking. It is further proposed that differences in the Ig repertoire make it highly likely that fetal/neonatal, but not adult derived B cells will be induced to express CD5. The model offers a plausible explanation for the correlation of CD5 expression and natural autoantibody production by neonatal B cells. Possible sources of pathogenic autoantibody are discussed in the context of this model.

Loss of CD23 is a consequence of B-cell activation. Implications for the analysis of B-cell lineages.

When splenic CD5- B cells are stimulated with antiimmunoglobulin they become CD5+ and have a prolonged in vitro life. Further treatment with IL-6 induces a loss of surface CD23 and IgD; that is, they resemble freshly isolated peritoneal CD5+ cells. These data suggest that the CD5 phenotype is induced after sIg-mediated B-cell activation. Additional support for this view arises from the observation that the loss of CD23 and IgD can be induced by another activation inducer, LPS, although in this case CD5 is not expressed. Thus, activation by anti-Ig plus IL-6 or by LPS induces CD23 loss. Consistent with the hypothesis that the loss of CD23 is a consequence of activation, we now report that the surface expression of CD23 varies inversely with the amount of total cellular RNA. We also find both CD23 positive and negative B cells among freshly isolated splenic CD5- B cells. In young mice a proportion of small splenic CD5+ B cells are CD23+, providing additional evidence that CD23 is present on all B cells prior to activation. A comparison of the features of CD5+ B cells and the antibody responses to thymus-dependent and thymus-independent antigens leads us to hypothesize that the CD5 phenotype arises as a consequence of thymus-independent type 2 (TI-2) stimulation. The relationship of CD5 expression to B-cell lineage (fetal vs. adult bone marrow) is discussed.

Randomized clinical trial of antithymocyte globulin in cadaver renal allograft recipients: importance of T cell monitoring.

Despite incontrovertable evidence demonstrating the unique immunosuppressive capabilities of antihymocyte globulin (ATG) in animals, its value in clinical transplantation has remained inconclusive. A multicenter, prospective study was undertaken in an attempt to determine this value. Cadaver renal allograft recipients were randomized into two treatment groups: prednisone and azathioprine only, or prednisone, azathioprine, and ATG. Four to 36 month follow-up of our first 50 recipients, supported by results in over 200 patients from other centers, permits the following observations. (1) Allograft survival can be improved by nearly 20 percent in recipients treated with active ATG preparations. (2) Presently available assays performed in vitro and on subhuman primate allograft survival, though capable of excluding inactive preparations, cannot quantitate precisely the immunosuppressive capacity of active batches of ATG. (3) Monitoring circulating T cell levels in patients receiving ATG appears to offer the best means of defining immunosuppressive potency and the variation in response to the agent among identically treated individuals. It is concluded that ATG therapy can be beneficial in renal transplantation; but monitoring of recipients' T cell level is desirable during therapy to determine the dosage required. Furthermore, the optimal immunosuppressive protocol can be approximated by modifying the regimen according to individual variations in response, thus limiting the infectious complications of excessive immunosuppression.

Pleiotropic effects of the nude mutation.

Mice homozygous for the autosomal mutation nude suffer from thymic dysgenesis, hairlessness and abnormal development of gonads and salivary glands. Their immunologic and reproductive defects can be overcome by grafts of whole thymus or thymic epithelium. Thymic replacement does not prevent or reverse the lack of hair growth. Skin from nude mice grafted to histocompatible or immune-suppressed phenotypically normal mice does not grow hair. Conversely, normal skin, successfully grafted to nude mice, maintains hair growth. Thus, the hairlessness of the nude mouse is not a secondary result of thymic dysgenesis; rather, it appears that thymus and skin have a single common defect.

CD4-, CD8- gamma/delta cells from normal mice respond to a syngeneic B cell lymphoma and can induce its differentiation.

Lymph nodes and spleens from normal unimmunized mice contain small numbers of CD3+, CD4-, CD8- (double negative, DN) T cells. Of these, approximately one-third express the marker Ly-5(B220) in a form previously seen only on normal B cells and a population of DN T cells found in mice genetically prone to develop autoimmunity. DN T cells proliferate when co-cultured with a syngeneic surface Ig+ lymphoma, CH12. After one cycle of stimulation with CH12 almost all of the responding CD3+ DN cells express Ly-5(B220), suggesting that it is an activation marker for some DN T cells. The CH12 responding population also contains cells with two other phenotypes, Thy-1+, CD4-, CD8-, Ly-5(B220)+, sIgM-, CD3- and Thy-1+, CD4+, CD8-, Ly-5(B220)-, sIgM-, CD3+. The Ly-5(B220)+, CD3- population is no longer found after repeated stimulation. While the relationship between these three populations is unknown, DN T cells can proliferate in the absence of CD4+ or CD8+ cells and therefore their proliferation is not dependent on the presence of other T cells or lymphokines produced by CD4+ or CD8+ T cells. Anti-CD3 immunoprecipitation of CH12-responding cells reveals at least seven different receptor proteins of which five can also be precipitated with an anti-(C gamma 1/C gamma 2) monoclonal antibody. Thus at least three different TCR gamma-delta heterodimers are expressed by CH12-responding T cells. The Thy-1+, CD4-, CD8-, Ly-5(B220)+ cells can provide help to CH12 cells for Ig secretion even in the absence of the nominal antigen for the B lymphoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)