Pre-B cells and other possible precursor lymphoid cell lines derived from patients with X-linked agammaglobulinemia

A group of unique Epstein-Barr virus-containing cell lines was derived from the bone marrow of three patients with X-linked agammaglobulinemia. Efforts to obtain cell lines from the peripheral blood of these patients were uniformly unsuccessful. Immunofluorescence analyses as well as biosynthetic studies with [(35)S]methionine indicated unusual patterns of Ig synthesis in many of these bone marrow derived lines. Seven of the lines were of particular interest in that two produced no Ig of any type; two others showed no Ig by fluorescence but small amounts by [(35)S]methionine labeling; one expressed only cytoplasmic μ chains without any evidence of light chain synthesis, and two produced primarily μ chains with only slight amounts of light chains. One of the lines without membrane or cytoplasmic Ig studied in detail grew like a typical lymphoid line and was carried in intermittent culture over a period of 2 yr without Ig expression. One line grew quite differently and resembled the round cell type described previously, which has been obtained from a variety of sources. The cell line with cytoplasmic μ chains and no light-chain expression had the characteristic properties of pre-B cells. Three normal type Ig-producing cell lines also were obtained from the patients. The accumulated evidence obtained in the present study indicates that these unusual cell lines represent normal precursor cells of the B-cell lineage; these grew out in these cases because of the virtual absence of mature B cells that ordinarily overgrow the culture system. However, the possibility that in certain instances they reflect abnormal Ig synthesis characteristic of the disease has not been ruled out.     

T cell receptor alpha-, beta-, and gamma-genes in T cell and pre-B cell acute lymphoblastic leukemia

We examined alpha-, beta-, and gamma-T cell receptor (TCR) gene activation within acute lymphoblastic leukemias (ALLs) that represent early stages of B and T cell development. We wished to determine if TCR rearrangement and expression was lineage restricted, showed any developmental hierarchy, or could identify new subsets of T cells. Rearrangement of gamma and beta TCR genes occurred early in development but in no set order, and most T-ALLs (22/26) were of sufficient maturity to have rearranged both genes. T-ALLs preferentially rearranged C gamma 2 versus the C gamma 1 complex; no preference within the beta locus was apparent. Once rearranged, the beta TCR continued to be expressed (11/13), whereas the gamma TCR was rarely expressed (3/14). The alpha TCR was expressed only in more mature T-ALLs (8/14) that usually displayed T3. The 3A-1 T cell associated antigen appeared earliest in development followed by T11 and T3. Within pre-B cell ALL a higher incidence of lineage spillover was noted for gamma TCR rearrangements (8/17) than for beta rearrangements (3/17). This also contrasts with the only occasional rearrangement of immunoglobulin (Ig) heavy chains (3/25) in T-ALL. However, in pre-B ALL the pattern of gamma TCR usage was distinct from that of T cells, with the C gamma 1 complex utilized more frequently. Almost all ALLs could be classified as pre-B or T cell in type by combining Ig and TCR genes with monoclonal antibodies recognizing surface antigens, although examples of lineage duality were noted. Unique subpopulations of cells were discovered including two genetically uncommitted ALLs that failed to rearrange either Ig or TCR loci. Moreover, two T lymphoblasts were identified that possessed the T3 molecule but failed to express alpha plus beta TCR genes. These T-ALLs may represent a fortuitous transformation of T cell subsets with alternative T3-Ti complexes.     

Rat lymphoid cell lines with human T cell leukemia virus production. I. Biological and serological characterization

Cocultivation of spleen cells, lymph node cells, and thymocytes of female Wistar-King-Aptekman rats with short-term cultured male adult T cell leukemia (ATL) cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) resulted in the establishment of rat lymphoid cell lines, TARS-1, TARL-2, and TART-1. Cytogenetic analysis of the three cell lines showed a female rat karyotype with 42 chromosomes. The surface phenotypes of TARS-1 and TART-1 were those of rat T cells. TARL-2 was only positive for rat Ia and leukocyte common antigens. The cell lines continuously produced a type C retrovirus, human T cell leukemia virus (HTLV) and expressed ATL-associated antigens. TARS-1 and TART-1, but not TARL-2 were transplantable into newborn syngeneic rats and nude mice. These results strongly indicate that HTLV not only immortalizes, but also transforms rat T cells in vitro. Adult rats immunized with either TARS-1 or TARL-2 produced antibodies specific for HTLV. The biochemical analysis of the antigens that reacted with rat sera revealed that they are the two HTLV-specific polypeptides, p24 and p28.     

Cytotoxic T cells specific for antigens expressed on surface immunoglobulin-positive cells

C.B-20 mice were immunized with splenocytes or B leukemia cells (BCL1) from Ig H chain allotype congenic strains. Spleen cells from these immunized mice were rechallenged in vitro to generate H-2-restricted cytotoxic T cells that were specific for target antigens controlled by genes linked to the Ig H chain locus. The anti-Ig H cytotoxic T cells detected an antigen(s) expressed only on surface Ig+ cells. Thus, T cell lymphoblasts, eight BALB/c myeloma cell lines, and a T cell lymphoma were not lysed by the effector cells. In contrast, B cell lymphoblasts and the surface Ig+ BCL1 cells were sensitive to lysis. A surface Ig- hybridoma (which secretes the IgM from the BCL1 cells) generated by fusing BCL1 cells to X63 myeloma cells was not killed by the effector cells. These data indicate that cytotoxic T cells specific for antigenic determinants on either surface IgM+ or IgD+ or on a molecule that is coordinately expressed on IgM+ or IgD+ cells can be generated and that such cells might play a role in regulating the growth of normal B cells or surface Ig+ tumor cells in vivo.     

B cell growth factors and B cell differentiation factor from human T hybridomas. Two distinct kinds of B cell growth factor and their synergism in B cell proliferation

Human T hybridomas secreting B cell growth factors (BCGF) and B cell differentiation factor (BCDF) have been established. Hybrid clones 77- A, 94-C, and 98-F secreted BCGF that induced proliferation of anti-IgM- stimulated normal B cells. The culture supernatant from 77-A cells could also maintain continuous proliferation of colony-forming B cells, but the factor from 94-C could not. The addition of the supernatant from 94-C cells to that from 77-A cells, however, synergistically augmented the proliferation of colony-forming B cells, demonstrating the existence of two distinct kinds of BCGF and the synergism between them. These supernatants, however, showed no interleukin 2 (IL-2) or BCDF activity. A hybrid clone, 90-E, secreted BCDF. The culture supernatant induced Ig production in Cowan I-stimulated normal B cells or in a transformed B cell line, CESS. However, the supernatant had no BCGF or IL-2 activity. Anti-Ig-stimulated B cells, but not IL-2- dependent T cells, absorbed BCGF activity and CESS cells absorbed BCDF activity but not BCGF activity in the culture supernatants from T hybridomas. Taken collectively, the results demonstrated that IL-2, BCGF, and BCDF were different molecules and acceptors specific for the each molecule are present on the each target cell.     

Rat lymphoid cell lines producing human T cell leukemia virus. II. Constitutive expression of rat interleukin 2 receptor

Three rat lymphoid cell lines (TARS-1, TARL-2, and TART-1) (12) transformed by human T cell leukemia/lymphoma virus I (HTLV-I) had rearrangement of the beta chain gene of the T cell antigen receptor, and had integrated proviral DNA from HTLV-I in their genomes. As is the case with adult T cell leukemia (ATL)-derived human T cell lines transformed by HTLV-I, these rat cell lines unequivocally expressed interleukin 2 (IL-2) receptor, as determined by radiolabeled IL-2 binding. By Scatchard plot analysis, one of the cell lines, TART-1, proved to have high affinity receptors (Ka = 1.3 X 10(11)/M and 8.8 X 10(9)/M). Rat IL-2 receptor, not human IL-2 receptor, was expressed on HTLV+ rat cell lines, as demonstrated by the fact that they expressed antigens reactive with monoclonal antibodies (ART-18) against rat IL-2 receptor, but not with anti-Tac antibodies. The collective evidence indicates that the endogenous IL-2 receptor gene is activated in human and rat lymphoid cell lines with HTLV-I production. The mechanism of abnormal IL-2 receptor expression in HTLV infection is discussed.     

Surrogate or conventional light chains are required for membrane immunoglobulin mu to activate the precursor B cell transition [published erratum appears in J Exp Med 1997 Jan 6;185(1):183]

To examine the role of light chains in early B cell development we combined RAG-1 and lambda 5 mutations to produce mice that expressed neither conventional nor surrogate light chains (RAG-1-/-, lambda 5-/- ). Unique heavy and light chain genes were then introduced into the double and single mutant backgrounds. Membrane immunoglobulin (Ig)mu (mIg mu) associated with Ig alpha-Ig beta but was unable to activate the pre-B cell transition in RAG-1-/-lambda 5-/- mice. Either lambda 5 or kappa light chains were sufficient to complement this deficiency. Therefore light chains are absolutely required for a functional Ig signaling module in early B cell development. Our data provide direct evidence for the existence of two pathways for induction of early B cell development: one which is activated through surrogate light chains and mIg mu, and an alternative pathway which uses conventional light chains and mIg mu.     

Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow cytometry and were clonal by Southern blotting. Surprisingly, two of the mu/lambda-expressing cell lines contained both kappa alleles in germline configuration, and synthesis/expression of conventional lambda L chains was directly proven by immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in one of them. Thus, human fetal bone marrow B lineage cells harbor the capacity to make functional lambda L chain gene rearrangements without rearranging or deleting either kappa allele. A third unusual cell line, designated 30.30, was observed to coexpress cell surface kappa and lambda L chains associated with mu H chains. The 30.30 cell line had a diploid karyotype, a single H chain rearrangement, both kappa alleles rearranged, and a single lambda rearrangement. Immunoprecipitation/SDS-PAGE confirmed that 30.30 cells synthesized and expressed kappa and lambda L chains. Multiparameter flow cytometry was used to demonstrate the existence of kappa+/lambda+ cells in fetal bone marrow and fetal spleen at frequencies of 2-3% of the total surface Ig+ B cell population. The flow cytometry data was confirmed by two-color immunofluorescence microscopy. The existence of normal human B cells expressing cell surface kappa and lambda refutes the widely accepted concept that expression of a single L chain isotype is immutable. The kappa+/lambda+ cells may represent transients undergoing L chain isotype switching.     

The mouse vitronectin receptor is a T cell activation antigen

In this report, we demonstrate that the T cell activation antigen, recognized by monoclonal antibody H9.2B8, is the murine homologue of the vitronectin receptor (VNR) and, thereby, we provide initial evidence that VNR is expressed on lymphoid cells. VNR is expressed on a variety of T cell lines, tumors, and Con A-activated splenocytes, but not resting T cells, and is capable of binding to the extracellular matrix proteins fibronectin, fibrinogen, and vitronectin, via the tripeptide sequence RGD. There was no evidence of novel beta chains pairing with the VNR alpha chain, as has been demonstrated in some human cells. In view of recent studies demonstrating that this molecule functions as an accessory molecule in T cell activation, the VNR may play an important role in mouse T cell functions.     

Interleukin 2 inhibits in vitro growth of human T cell lines carrying retrovirus

Four human T cell lines, TL-Mor, TL-Su, TL-TerI, and TL-OmI, carrying human T cell leukemia virus (HTLV), were established previously. TL-Mor, TL-Su, and TL-TerI were derived from interleukin 2 (IL-2)-dependent parental cell lines cloned from peripheral blood leukocytes (PBL) of three healthy HTLV carriers, while TL-OmI was directly established from PBL of a patient with adult T cell leukemia. These four TL cell lines grow autonomously without IL-2. When they were cultured in the presence of IL-2, their growth was inhibited after a few days. This growth inhibition depended on the dose of IL-2, and the effective dose significantly promoted growth of their parental IL-2-dependent cell lines. The growth inhibition is demonstrated to be due to specific binding of IL-2 to receptors on the TL cells.     

Monoclonal antibody against human T cell leukemia virus p19 defines a human thymic epithelial antigen acquired during ontogeny

Using monoclonal antibody 12/1-2 against a 19,000-dalton human T cell leukemia virus (HTLV) protein (anti-p19), previously demonstrated to be reactive with HTLV-infected human cells, but not in numerous other uninfected cells, we found a reactive antigen to be expressed on the neuroendocrine component of human thymic epithelial cells but not on any other normal epithelial or neuroendocrine human tissues. Moreover, this reactive antigen is acquired on neuroendocrine thymic epithelium during thymic ontogeny--first appearing on fetal thymic epithelial cells between 8 and 15 wk gestation. While only a portion of thymic epithelial cells in the subcapsular cortical region of 15- and 24-wk fetal thymuses contained anti-p19+ epithelial cells, the entire subcapsular cortical region of newborn thymus epithelium was anti-p19+. By age 3 yr, normal subjects' entire subcapsular cortical and medullary thymic epithelium was anti-p19+. Using antibody against HTLV core protein, p24, and c-DNA probes for HTLV DNA, neither HTLV-specific p24 protein nor proviral DNA could be demonstrated in anti-p19+ thymic epithelial tissue. However, thymic epithelial extracts, disrupted HTLV extracts, as well as purified HTLV p19 antigen all inhibited the binding of anti-p19 antibody to thymic epithelium. Thus, anti-p19 may recognize a determinant on an HTLV-encoded 19,000-dalton structural protein that is shared by human thymic epithelium. Alternatively, anti- p19 defines a host encoded protein that is selectively expressed by normal thymic epithelium, and is induced to be expressed in HTLV- infected malignant T cells.     

Immunoglobulin mu-chain gene rearrangement in a patient with T cell acute lymphoblastic leukemia

A 6-yr-old girl with T cell acute lymphoblastic leukemia (ALL) is described. She had a mediastinal mass and her leukemic cells expressed T cell-associated antigens (Leu 1+, OKT3+, OKT9+, and OKT10+). When we examined genomic DNA from the leukemic cells, we detected Ig mu-chain gene rearrangement with germ-line configuration of light chain genes. As reported recently, detecting Ig gene rearrangement has become an important procedure for further classifying B cell precursor cells. This case, however, suggests that there is also heterogeneity among patients with T cell ALL, not only at the level of cell surface phenotypes, but also at the level of the Ig gene. These findings have major implications when we consider both the ontogenesis of these leukemic cells and the normal differentiation of human lymphocytes.     

Generation of an HLA-restricted cytotoxic T cell line reactive against cultured tumor cells from a patient infected with human T cell leukemia/lymphoma virus

Lymphocytes from a patient who had an unusually long survival after therapy for a human T cell leukemia/lymphoma virus (HTLV)-associated T cell lymphoma were stimulated in vitro with an autologous tumor cell line, and the generation of cytotoxic T lymphocytes (CTL) was studied. CTL generated were directed against autologous (HTLV-associated tumor cells. These propagated CTL were OKT3+, OKT4-, and OKT8+. The cytotoxic activity required target tumor cells that were infected with HTLV and also expressed histocompatibility antigens in common with the patient, suggesting a major histocompatibility complex-restricted associative recognition of target antigens expressed on the tumor cell membrane.     

Analysis of a T cell receptor gene as a target of the somatic hypermutation mechanism

In an effort to identify cis-acting elements required for targeting of the somatic hypermutation process in mice, we examined whether a T cell receptor (TCR) transgene under the control of the immunoglobulin (Ig) heavy (H) chain intron enhancer would be mutated in antigen-stimulated B cells. Hybridomas were established from splenic B cells of mice carrying two copies of the TCR transgene after hyperimmunization with phosphorylcholine keyhole limpet hemocyanin. Northern analysis revealed that all of the transgene-containing hybridomas expressed the TCR mRNA. Multiple somatic point mutations were found in seven of eight endogenous Ig VH genes examined. In contrast, 29 of 32 TCR genes examined contained no mutations. One potential mutation was seen in each of the three other TCR genes. Our data indicate that although the TCR transgene is expressed in B cells, it is not efficiently targeted by the mutator mechanism. Furthermore, the presence of an Ig H chain enhancer is itself not sufficient for targeting of the somatic hypermutation mechanism.     

Autologous lymphoma vaccines induce human T cell responses against multiple, unique epitopes

The clonotypic surface Ig receptor expressed by malignant B cells, idiotype, is a tumor-specific antigen and an attractive target for active immunotherapy. While Ab's specific for tumor idiotype have been well described in patients with B cell malignancies, the precise antigenic epitopes in human idiotype recognized by autologous T cells remain largely unknown. We report here that T cell lines generated from lymphoma patients actively immunized with idiotype protein specifically recognized multiple, unique immunodominant epitopes in autologous tumor idiotype. Synthetic peptides corresponding to hypervariable, but not framework, regions of Ig heavy chain specifically stimulated CD4(+) and CD8(+) T cells to proliferate and secrete proinflammatory cytokines in an MHC-associated manner. Detailed analysis revealed a minimal determinant of an immunodominant epitope, comprising critical residues at the amino terminus that may be a product of somatic hypermutation. Association of idiotype-specific T cell responses with previously documented molecular remissions in idiotype-vaccinated patients suggests that the newly identified T cell epitopes may be clinically relevant. Such antigenic epitopes may serve as candidates for novel peptide-vaccine strategies, and as tools to selectively expand tumor antigen-specific T cells for adoptive immunotherapy and for monitoring T cell immunity in vaccinated patients.     

Human T cell hybridomas secreting factors for IgA-specific help, polyclonal B cell activation, and B cell proliferation

Human T-T hybridomas were established by fusion of concanavalin A- activated OKT-4+ T cells with hypoxanthine guanine phosphoribosyl transferase-deficient as well as nondeficient T cell lines. Four hybrids were selected for further study. Supernatant from hybrid clone J1.3 specifically enhanced IgA production and secretion by isolated human B cells, with increases in IgA plaque-forming cells approaching those seen with addition of autologous T cells and pokeweed mitogen. A monoclonal lymphocytic leukemia with membrane IgA also differentiated to IgA plasma cells by this supernatant. Evidence suggests that this hybrid supernatant acts on post-switch IgA-committed B cells. The other hybrids were not isotype specific; hybrid J2S1 enhanced polyclonal Ig secretion and hybrids K1 and K8 induced B cell proliferation without induction of Ig secretion.     

Specificity and function of a human autologous reactive T cell

Normal human peripheral blood contains a population of T cells (autologous reactive cells [ARC]) capable of proliferating in response to signals from autologous B cells and monocytes. Selective suicide of proliferating ARC with 5-bromo-2-deoxyuridine and light demonstrated that this ARC was responsive to signals coded for by genes more closely linked to the HLA-DR, than to the HLA-A, or HLA-B, loci. Density-gradient fractionation of T cells indicated that populations enriched in ARC reactivity were also enriched for helper influences required for Ig synthesis by autologous B cells. In contrast, populations negatively selected for proliferating ARC were deficient in helper activity. These studies indicate that the ARC is responsive, at least in part, to products of genes closely linked to the HLA-DR locus and can function as a helper cell.