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T J Kipps

Publications and source records attributed to T J Kipps.

At least 73 records · Page 4Linked to original sources
PubMed1995

Analysis of immunoglobulin VH gene repertoire by an anchored PCR-ELISA.

We developed a novel technique to analyze the relative concentration of the expressed immunoglobulin (Ig) VH genes using an enzyme-linked immunosorbent assay (ELISA). Expressed Ig cDNA are amplified via anchored PCR and then subjected to a "nested PCR" reaction that attaches biotin to the 5' end of the antisense strand. This allows us to tether the antisense strand of PCR products onto avidin-coated ELISA plates. Digoxigenin-labeled oligonucleotides specific for the leader sequence sense strand of each major Ig VH gene subgroup are used to probe the plate-tethered, alkaline-denatured, and single-stranded antisense cDNA. Bound probes then are detected with alkaline-phosphatase-conjugated anti-digoxigenin antibodies. Using this method, we assessed the distribution of Ig VH genes used by IgM-expressing blood B cells of normal adults. We found the predominant subgroup is VH3, representing approximately half (range 41-59%) of the expressed IgM repertoire. The next largest subgroups used are VH4 (19-23%), VH1 (15-17%), and VH5 (7-11%). The VH2, VH6, and VH7 subgroups each constitute less than 3% of the expressed IgM repertoire. These results agree with those obtained using traditional and more laborious methods that analyze the distribution of Ig clones in cDNA libraries. In addition, we find that this method compares favorably in sensitivity and specificity to more conventional techniques for assessing the clonality of blood or tissue B-cell populations. This rapid and nonradioactive method should have utility for assessing the Ig repertoires expressed by normal, autoimmune, or neoplastic B-cell populations.

Adult↗
PubMed1995

Expression of CD27 and its ligand, CD70, on chronic lymphocytic leukemia B cells.

Crosslinking the CD27 antigen on T cells provides a costimulatory signal that, in concert with T-cell receptor crosslinking, can induce T-cell proliferation and cellular immune activation. We find that chronic lymphocytic leukemia (CLL) B cells from most patients coexpress both membrane-bound and soluble CD27, along with its newly identified ligand, CD70. The expression of soluble CD27 may preclude leukemic B cells from stimulating T cells via CD70, thereby potentially impairing their ability to function as effective antigen-presenting cells. We find that leukemic B-cell expression of soluble and membrane-bound CD27 can be downmodulated through a CD40-dependent signal. This signal also induces enhanced expression of CD70 on both normal and leukemic B cells. We find that tumor necrosis factor (TNF)-alpha, or the Th1 cytokine interferon (IFN)-gamma, also can induce downmodulation of CD27, whereas Th2-associated cytokines interleukin-4 (IL-4) or IL-10 can enhance leukemic B-cell expression of this accessory molecule. The modulation of CD27 induced by these conditions is accompanied by reciprocal changes in the expression levels of CD70, suggesting that these accessory molecules may be engaged in reciprocal receptor-ligand downmodulation. Consistent with this, we observe that co-culture of CLL B cells with transfected murine plasmacytoma cells that express human CD70 affects downmodulation of CD27 and enhanced expression of CD70 on leukemic B cells, but does not affect expression of CD27 mRNA. However, we find that CD40-crosslinking, in addition to reducing the level of CD27 protein, also reduces leukemic B-cell expression of CD27 mRNA. This argues that the changes in the expression levels of CD27 following CD40-signaling are not simply due to induced increases in the expression levels of CD70. Finally, we demonstrate that reciprocal changes in expression of CD27 and CD70 may contribute to the enhanced antigen-presenting capacity of CLL B cells after CD40-dependent leukemic B-cell activation. These findings expand the understanding of the regulation of costimulatory molecules important in antigen presentation and also have implications for the immunobiology of and therapy for CLL.

Antigens, CD↗
PubMed1995

Tumor necrosis factor-alpha facilitates induction of CD80 (B7-1) and CD54 on human B cells by activated T cells: complex regulation by IL-4, IL-10, and CD40L.

Expression of immune accessory molecules, such as CD80 (B7-1), on antigen-presenting cells governs whether such cells can activate antigen-specific T cells. As such, the factors that regulate the expression of these accessory molecules may determine whether presentation of antigen leads to immune activation or anergy. We previously reported that anti-CD3-activated T cells (Ta) can induce expression of CD80 and CD54 (ICAM-1) on human B cells through a contact-dependent signal delivered to the CD40 molecule via the CD40 ligand. Here, we demonstrate that another molecule in the CD40-ligand family, namely tumor necrosis factor-alpha (TNF-alpha), also plays a role in the Ta-mediated induction of CD80 or CD54 on human B cells. Neutralizing mAbs specific for TNF-alpha can inhibit B cell expression of CD80 or CD54 that is induced when B cells are cultured with Ta cells or in Ta-cell conditioned media. Moreover, soluble, recombinant TNF-alpha or TNF-beta can induce significant increases in B cell expression of CD80 and CD54. The phenotypic changes effected by TNF-alpha can be recapitulated by crosslinking CD120b (p75 TNF-receptor), but not CD120a (p55 TNF-receptor), with mAbs presented on Fc gamma RII (CD32)-expressing L cells. IL-4 augments the expression of CD80 induced by crosslinking either CD40 or CD120b. However, although IL-10 augments CD40-induced expression of CD80, this cytokine inhibits the expression of CD80 that is induced by crosslinking CD120b. Further regulation of TNF-mediated CD80 expression may occur at the level of CD120b expression itself. We find that stimulation with exogenous IL-4 or CD40-cross-linking induces B cell expression of CD120b, but not CD120a. This study identifies an ancillary, TNF-mediated pathway, whereby activated T cells can induce B cells to express enhanced levels of the important costimulatory molecules, CD80 and CD54.

Antigen-Presenting Cells↗
PubMed1995

Origin of high-grade lymphomas in Richter syndrome.

Patients with B cell chronic lymphocytic leukemia (CLL) occasionally develop high-grade B cell lymphomas that are associated with constitutional symptoms, rapidly progressive lymphadenopathy, and swift clinical deterioration. Now known as Richter syndrome, this symptom complex develops in approximately 5% of all patients with CLL. Structural and molecular analysis of the immunoglobulin (Ig) genes have allowed investigators to define the clonal relationship between the leukemia and lymphoma cells of a given patient. In most cases the aggressive lymphoma evolves from the original leukemia cell clone. However, in some cases the lymphoma apparently represents a second malignancy. Differentiation between these two types of lymphoma may have clinical significance. Further investigation is required to allow for identification of CLL patients who are at risk for developing Richter syndrome and to understand factors involved in its etiopathogenesis.

Clone Cells↗
PubMed1994

Structure-function studies on a polyreactive (natural) autoantibody. Polyreactivity is dependent on somatically generated sequences in the third complementarity-determining region of the antibody heavy chain.

SMI is a previously characterized IgM kappa polyreactive (natural) autoantibody. The variable regions of the heavy and light chains of SMI are respectively encoded by a nonmutated VH1 gene, designated 51p1, and a conserved nonmutated V kappa gene, designated Humkv325. These V genes seem to be over-represented in the autoimmune and fetal B cell repertoires, and to be frequently expressed in malignant B cells during certain lymphoid proliferations such as chronic lymphocytic leukemia. Polyreactive natural autoantibodies are thought to rely mainly on the use of such V genes in germ-line configuration. However, this model underestimates the contribution of the somatically generated heavy chain third complementarity-determining region (HCDR3) to autoantibody specificity. We used oligonucleotide site-directed mutagenesis to permute the sequence of the SMI-HCDR3 to generate a family of mutant proteins, each of which differed from the original SMI-IgM kappa by one amino acid residue. This allowed us to examine the relative contribution of selected amino acid residues in this region to the binding affinity of SMI against a panel of self-Ags. We found that a single amino acid substitution within the HCDR3 could dramatically alter the specificity of this autoantibody. Some substitutions abrogated the reactivity with all the tested Ags, whereas others changed the affinity or spectrum of reactivity for certain self-Ags. These results demonstrate that the autoantibody-binding activity of these conserved autoantibody-associated germ-line V genes is dependent upon heavy chain junctional sequences that are generated somatically during Ig gene rearrangement.

Animals↗
PubMed1994

Transforming growth factor beta as endogenous growth inhibitor of chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) B cells are hyporesponsive or refractory to mitogens and growth factors in vitro. This study examined whether transforming growth factor beta (TGF-beta), a potent inhibitor of lymphocyte proliferation may play a role in the growth regulation of CLL B cells. CLL B cells from all donors treated expressed detectable TGF-beta 1 mRNA. In vitro release of TGF-beta by unstimulated cultures, or cultures stimulated by antibody to cell surface immunoglobulin (anti-mu) plus phorbol 12-myristate 13-acetate (PMA) was higher in CLL than in normal B cells. High levels of TGF-beta activity were also detected in plasma samples of CLL patients. The role of TGF-beta in growth regulation of CLL B cells was tested in assays using different B cell activators. Purified neoplastic B cells from most CLL patients proliferated in response to anti-mu, or the combination of anti-mu plus PMA. Levels of CLL B cell proliferation were lower than observed in normal B cells. Some CLL were refractory to these stimuli. Antibody to CD40 induced proliferation of CLL B cells from all donors tested when presented on Fc gamma RII (CDw32)-expressing L cells. Neutralizing antibodies to TGF-beta increased CLL B cell proliferation in the absence or presence of additional stimuli. These effects were dose dependent and specific. Exogenous TGF-beta completely inhibited CLL B cell proliferation induced by anti-mu, PMA, and anti-TGF-beta. CLL B cell proliferation induced by anti-CD40 was reduced by exogenous TGF-beta. However, even at high doses, TGF-beta did not completely inhibit the anti-CD40 effect. In summary, TGF-beta is overexpressed in CLL. CLL B cells are sensitive to TGF-beta and this cytokine functions as an autocrine growth inhibitor accounting at least in part for reduced proliferative responses of these leukemic cells and for the slow progression of the malignant process in vivo.

Aged↗
PubMed1994

Elevated expression of CD80 (B7/BB1) and other accessory molecules on synovial fluid mononuclear cell subsets in rheumatoid arthritis.

OBJECTIVE: To assess the expression of important accessory molecules such as CD80 (B7/BB1) and CD54 (intercellular adhesion molecule 1) on potential antigen-presenting cells (APC) in the synovial fluid (SF) of patients with rheumatoid arthritis (RA). METHODS: The level of expression of various accessory molecules on T cells, monocytes, and CD5+ or CD5- B cells from RA SF was compared, by multiparameter flow cytometric analysis, with the expression on peripheral blood (PB) mononuclear cells from the same patients and from normal controls. The functional significance of increased expression of these accessory molecules was assessed by the ability of the different cell populations to stimulate T cells in the mixed lymphocyte reaction. RESULTS: Monocytes, T cells, and B cells isolated from the SF of inflamed joints of patients with RA expressed significantly higher levels of CD80 and CD54 than those from the PB of RA patients or normal controls. CD11b and CD11c expression also were increased on SF B cells, while SF monocytes exhibited enhanced levels of CD40. Further, we found that the elevated CD80 and CD54 levels in RA SF cells may enhance the capacity of these cells to act as stimulatory APC. CONCLUSION: This is the first demonstration that specific cell subsets constitutively express increased levels of CD80 at a site of autoimmune pathology, which could potentially contribute to the initiation or maintenance of autoimmune disease.

Antigen-Presenting Cells↗
PubMed1994

Anti-B cell autoantibodies encoded by VH 4-21 genes in human fetal spleen do not require in vivo somatic selection.

We isolated immunoglobulin (Ig) VH4 genes that were rearranged in the genomic DNA of 160 day human fetal spleen. Productively rearranged VH 4-21 genes were cloned into pRTM1, a human IgM expression vector. This allowed us to generate IgM kappa-expressing transfectomas by co-transfecting each of these constructs with pSVG-V kappa 3, an Ig kappa light-chain expression vector that has a variable region encoded Humkv325, a conserved V kappa gene that is frequently expressed early B cell ontogeny. We find that all transfectomas expressing IgM kappa encoded by VH 4-21 make IgM autoantibodies reactive with i, a linear poly-N-acetyllactosamine determinant present on neonatal red blood cells and a B cell-restricted isoform of the CD45 surface molecule. In contrast, a transfectoma expressing pSVG-V kappa 3 and pRTM1 containing a rearranged VH4-59 (V71-4) gene isolated from a chronic lymphocytic leukemia B cell population, designated WIL, produced IgM kappa antibodies that had no detectable anti-i binding activity. However, transfectomas expressing VH 4-21 fused onto the Ig heavy-chain third complementarity determining region (CDR3) of WIL are found to make anti-B cell autoantibodies with anti-i activity. These studies indicate that VH 4-21 genes rearranged in human fetal B cell ontogeny can encode anti-B cell autoantibodies with a binding specificity that does not require in vivo somatic selection.

Agglutinins↗
PubMed1993

Autoantigen inhibits apoptosis of a human B cell leukemia that produces pathogenic rheumatoid factor.

We studied a variant CD5- B cell chronic lymphocytic leukemia (CLL) cell population that produces pathologic IgM kappa rheumatoid factor autoantibodies. In contrast to common CD5+ B cell CLL, this variant leukemia cell population displays intraclonal diversity in its expressed Ig V genes, similar to that noted for follicular B cell non-Hodgkin's lymphomas. Also, in contrast to common B cell CLL, these leukemia cells rapidly undergo cell death hours after being placed in tissue culture. We find that addition of Ag (aggregated human IgG) enhances significantly the survival of these cells in vitro. Leukemia cell survival also could be enhanced by exogenous IFN-gamma or anti-CD40 presented on Fc gamma RII (CDw32)-expressing L cells, but not by exogenous IL-4, IL-6, or monomeric human IgG. We find that Ag acts directly on the leukemia B cells to inhibit apoptosis. This effect could be mimicked by cross-linking the leukemia cells' surface IgM receptors with immobilized murine mAb specific for human Ig mu-chains, but not by immobilized mAb of irrelevant specificity. In contrast to most follicular NHL, this leukemia B cell population does not have evidence of bcl-2 gene rearrangement. Also, in contrast to non-Hodgkin's lymphomas and most B cell CLL, these cells do not express detectable amounts of bcl-2. Finally, although capable of inhibiting apoptosis, surface Ig receptor cross-linking does not induce expression of bcl-2 in these variant leukemia cells. We hypothesize that the lack of bcl-2 expression may render these leukemia cells particularly dependent upon the survival signal(s) derived from surface Ig receptor cross-linking. This state may represent an early stage in leukemia/lymphomagenesis, possibly accounting for the intraclonal diversity observed in the Ig V genes expressed by certain CD5- B cell leukemias and lymphomas.

Antigens, CD↗
PubMed1993

Common clonal origin of chronic lymphocytic leukemia and high-grade lymphoma of Richter's syndrome.

Patients with B-cell chronic lymphocytic leukemia (CLL) infrequently may develop high-grade B-cell lymphoma, or Richter's syndrome lymphoma (RS lymphoma). Such lymphomas differ from the original leukemia in both histology and clinical behavior. Studies seeking to define the clonal relationship between the cells of the two malignancies in any one patient have yielded conflicting reports. We examined the clonal relationship between the early and late neoplastic cells of a patient who underwent Richter's transformation. In contrast to the original leukemia cells, the secondary high-grade lymphoma was CD5-. However, both the leukemia cells and the evolved RS lymphoma expressed surface IgM lambda reactive with Lc1, a murine monoclonal antibody specific for a supratypic cross-reactive idiotype encoded by a subset of human Ig variable region genes of the VH4 subgroup. Nucleic acid sequence analyses of the heavy and light chain variable region genes expressed by both leukemia and lymphoma cells show that the CD5- B-cell lymphoma constitutes a clonal expansion of mutant cells derived from the original CD5+ B-cell leukemia. Moreover, certain sets of somatic mutations distinguish the Ig variable region genes used by RS lymphoma from those expressed by the CLL B cells. This is the first study to establish the clonal relationship between CLL and RS lymphoma through primary structural analyses of the expressed Ig genes.

Amino Acid Sequence↗
PubMed1993

Immunoglobulin VH3 gene products: natural ligands for HIV gp120.

Infection with human immunodeficiency virus-type 1 (HIV-1) depletes T cells expressing CD4 and B cells expressing immunoglobulin (Ig) VH3 gene products. A subpopulation of normal B cells from non-HIV-infected individuals was shown to bind to HIV gp120 by means of membrane Ig; most of these B cells expressed VH3 family Ig. Serum VH3 IgM from uninfected individuals also avidly bound gp120. Finally, gp120 selectively induced Ig secretion by VH3 B cells, indicating that the binding of gp120 functionally activated these cells. These results indicate that naturally occurring VH3 Ig is a second ligand for gp120 and a candidate superantigen for VH3 B cells.

Adolescent↗
PubMed1993

Induction of antibodies to a kappa V region by gene immunization.

Direct gene transfer into muscle can lead to sustained gene expression at the injected sites. Here we tested the ability of in vivo gene transfection to immunize mice against an isolated human IgV region. The Humkv325 germline kappa L chain V gene encodes the kappa L chain in V regions of several human IgM autoantibodies and is used frequently in chronic lymphocytic leukemia. This gene was inserted into a mammalian expression vector, pREP7, to produce pREVk3. Mice injected i.m. with pREVk3 produced antibodies against the V region of Glo, a human monoclonal IgM paraprotein whose kappa L chain is encoded by Humkv325. Co-injection of an expression vector encoding IL-2 enhanced anti-Glo antibody production fivefold and induced a localized delayed hypersensitivity reaction. Antibody production also was induced when vectors encoding Humkv325 and IL-2 were injected s.c. These experiments demonstrate that gene immunization vectors can stimulate immune responses to antibody V region determinants.

Animals↗
PubMed1993

Systemic immunological effects of cytokine genes injected into skeletal muscle.

Somatic gene therapy is an interesting approach for the delivery of cytokines for prolonged periods. The present experiments show that direct injections into mouse skeletal muscle of cDNA expression vectors encoding interleukin 2 (IL-2), IL-4, or type beta 1 transforming growth factor (TGF-beta 1) induce biological effects characteristic of these cytokines in vivo. Mice injected intramuscularly with a vector encoding IL-2 had enhanced humoral and cellular immune responses to an exogenous antigen, transferrin, that was delivered at a separate site. These IL-2 effects were abolished by coadministration of a vector directing synthesis of TGF-beta 1. The TGF-beta 1 vector by itself depressed the anti-transferrin antibody response and caused an 8-fold increase in plasma TGF-beta 1 activity. The TGF-beta 1 plasmid injection did not cause muscle infiltration with monocytes or neutrophils and there was no evidence for fibrotic changes. Muscle injection with a cDNA encoding IL-4 selectively increased IgG1 levels but did not alter the cellular immune response to transferrin. In lupus-prone mice (MRL/lpr/lpr), injection with IL-2 expression vectors increased and TGF-beta 1 vectors decreased auto-antibodies to chromatin. These results demonstrate that intramuscular injection of cytokine genes, in the absence of infectious viral vectors, can regulate humoral and cellular immune responses in vivo.

Animals↗
PubMed1993

Lack of extensive mutations in the VH5 genes used in common B cell chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (CLL) is a malignancy of the CD5+ B cells. Prior studies indicated that CLL B cells generally express immunoglobulin (Ig) VH and VL genes with little or no somatic mutations. However, a recent report indicated that VH251, one of three VH genes belonging to the VH5 subgroup (e.g., VH251, VH32, and VH15), not only is frequently rearranged in this disease, but also has extensive and selective mutations when expressed by CLL B cells. The extent and nature of these mutations contrasts markedly from the low level of mutations noted in VH5 genes used by normal B cells or other Ig V genes found expressed in CLL. To determine whether this difference reflects a unique property of VH251 or a previously unrecognized subgroup of CLL, we examined for VH5 Ig gene rearrangements in leukemia cells from 68 patients that satisfied clinical and diagnostic criteria for CD5+ B cell CLL. Southern blot hybridization studies with probes for VH251 and the JH locus revealed that only 7 (10%) of the 68 monoclonal CLL cell populations had undergone Ig gene rearrangement involving VH5 genes. Two (3%) were found to have functionally rearranged VH5 genes that shared > or = 98% sequence homology with 5-2R1, a VH251 gene isolated from a pre-B cell acute lymphocytic leukemia. The other five CLL (7%) had functionally rearranged VH5 genes that each shared > or = 99% nucleic acid sequence homology with a germline VH32 isolated from human sperm DNA. These data indicate that VH251 or VH32 also may be expressed by CD5+ CLL B cells with little or no somatic mutation. These findings contrast with a recently published study on VH5 gene expression in B CLL and contest the hypothesis that extensive somatic mutation is a common property of the VH5 genes used in this disease. Further work to define the clinical and/or phenotypic characteristics of patients with leukemia cells that express mutated versus nonmutated Ig V genes may reveal subsets of CLL that possibly differ in their cytogenesis, etiopathogenesis, and/or clinical behavior.

Adult↗
PubMed1993

Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal.

Cognate interactions between antigen-presenting B and T cells play crucial roles in immunologic responses. T cells that have been activated via the crosslinking of CD3 are able to induce B cell proliferation and immunoglobulin secretion in a major histocompatibility complex-unrestricted and contact-dependent manner. We find that such activated human CD4+ T cells, but not control Ig-treated T cells, may induce normal or leukemic B cells to express B7/BB1 and significantly higher levels of CD54 intercellular adhesion molecule 1 via a process that also requires direct cell-cell contact. To discern what cell surface molecule(s) may be responsible for signalling B cells to express B7/BB1, we added various monoclonal antibodies (mAbs) specific for T or B cell accessory molecules or control mAbs to cocultures of alpha-CD3-activated T cells and resting B cells. We find that only alpha-CD40 mAbs can significantly inhibit the increased expression of B7/BB1, suggesting that the ligand for CD40 expressed on activated T cells may be an important inducer of B7/BB1 expression. Subsequent experiments in fact demonstrate that alpha-CD40 mAbs, but not control mAbs, induce changes in B cell phenotype similar to those induced by activated T cells when the mAbs are presented on Fc gamma RII (CDw32)-expressing L cells. These phenotypic changes have significant effects on B cell function. Whereas chronic lymphocytic leukemia (CLL) B cells normally are very poor stimulators in allogeneic mixed lymphocyte reactions (MLRs), CLL-B cells preactivated via CD40 crosslinking are significantly better presenters of alloantigen, affecting up to 30-fold-greater stimulation of T cell proliferation than that induced by control treated or nontreated CLL-B cells. Similarly, the MLR of T cells stimulated by allogeneic nonleukemic B cells can be enhanced significantly if the stimulator B cells are preactivated via CD40 crosslinking. The enhanced MLR generated by such preactivated B cells may be inhibited by blocking B7/BB1-CD28 interaction with CTLA4Ig. These studies demonstrate a novel, CD40-dependent pathway for inducing B cell expression of B7/BB1 and enhancing B cell antigen-presenting cell activity that can be initiated via cell-cell contact with alpha-CD3-stimulated CD4+ T cells.

Animals↗
PubMed1993

Relationship of deoxycytidine kinase and cytoplasmic 5'-nucleotidase to the chemotherapeutic efficacy of 2-chlorodeoxyadenosine.

The agent 2-chlorodeoxyadenosine (2-CdA) has chemotherapeutic activity in hairy cell leukemia (HCL) and in refractory chronic lymphocytic leukemia (CLL). The cytotoxic activity of 2-CdA requires the intracellular accumulation of 2-CdA nucleotides. Deoxycytidine kinase (dCK) and cytoplasmic 5'-nucleotidase (5'-NT) are the principal enzymes that phosphorylate 2-CdA and dephosphorylate 2-CdA 5'-monophosphate, respectively. The net accumulation of 2-CdA nucleotides may therefore depend on both dCK and 5'-NT. The purpose of the present experiments was to determine if there is a relationship between pretreatment levels of dCK and 5'-NT in HCL and in CLL cells, and the clinical outcome of 2-CdA treatment. As measured by a direct immunoassay for dCK in 25 CLL patients, and by a 5'-NT activity assay in 23 patients, mean dCK levels were significantly higher in 2-CdA responders than in nonresponders (P < .01), whereas mean 5'-NT levels were significantly lower in 2-CdA responders than in nonresponders (P < .05). Mean dCK levels were higher in six HCL 2-CdA responders than in one nonresponder, whereas mean 5'-NT levels were lower in the 2-CdA responders than in the nonresponder. These results suggest that both dCK and 5'-NT are determinants of 2-CdA responsiveness.

5'-Nucleotidase↗