Variable effects of transgenic c-Maf on autoimmune diabetes.

Autoimmune diabetes is associated with T helper 1 polarization, but protection from disease can be provided by the application of T helper 2 (Th2) cytokines. To test whether genetic manipulation of T-cells can provide protective Th2 responses, we developed transgenic mice in which T-cells express the interleukin-4-specific transcription factor c-Maf. When crossed with a transgenic model that combines a class II restricted T-cell receptor specific for influenza hemagglutinin with islet beta-cell expression of hemagglutinin, the c-Maf transgene provided significant protection from spontaneous autoimmunity but not from adoptively transferred diabetes. In a second transgenic model in which islet cells express the lymphocytic choriomeningitis virus nucleoprotein, the virus infection triggers autoimmune diabetes within a few weeks involving both CD4 and CD8 T-cells; here too transgenic c-Maf provided significant protection. Surprisingly, when the c-Maf transgene was backcrossed with the NOD model of spontaneous disease, no protection was evident. Thus, transgenic c-Maf can strongly influence autoimmune disease development in some models, but additional factors, such as background genetic differences, can influence the potency of its effect.

T-cell production of an inducible interleukin-10 transgene provides limited protection from autoimmune diabetes.

In a number of animal models of spontaneous autoimmune diabetes, pathogenesis has been highly correlated with autoreactive T-cell production of the type 1 cytokine interferon-gamma (IFN-gamma), while protection from disease was associated with type 2 cytokines such as interleukin (IL)-4. Curiously, in some models, diabetes is associated with unexpected cytokine patterns; for example, diabetes can develop in NOD mice lacking a functional IFN-gamma gene. In another situation, acceleration of diabetes occurs in transgenic mice with constitutive beta-cell expression of the type 2 cytokine IL-10. IL-10 has generally been associated with immunosuppression, including the modulation of class II expression on antigen-presenting cells and the generation of regulatory CD4 T-cells. Because it is possible that unregulated expression of any cytokine might lead to unphysiological effects in vivo, we tested the notion that an inducible T-cell-specific IL-10 transgene might yet mediate a more physiological protection from autoimmune diabetes. Our results show that indeed, regulated T-cell production of IL-10 does not accelerate diabetes and instead can provide significant protection from disease. These results help rectify the apparent discrepancies between the effect of IL-10 on various models of autoimmune diabetes.

RelB regulation of chemokine expression modulates local inflammation.

The resolution of acute inflammation is incompletely understood but presumably requires the elimination of both inflammatory cells and production of inflammatory cytokines. In the case of recruited bone-marrow-derived inflammatory cells such as granulocytes and macrophages, their short life span helps eliminate these cells and the cytokines they produce. By contrast, resident permanent cells such as fibroblasts require other mechanisms to stop the production of chemokines generated in response to inflammatory triggers such as lipopolysaccharide. Here we demonstrate that RelB is an important regulator of chemokine expression in fibroblasts, thereby playing a key role in the resolution of acute inflammation. Activation of normal fibroblasts by lipopolysaccharide induced a transient production of chemokines, closely followed by induction of RelB expression. However, stimulated RelB-/- fibroblasts exhibited dramatic persistent induction of seven chemokines (RANTES, MIP-1 alpha, MIP-1 beta, MIP-2, IP-10, JE/MCP-1, and KC/CINC). The persistent overexpression of chemokines correlated with increased NF- kappa B binding as well as with increased p50, p65/RelA, and I kappa B alpha expression. Transfection of RelB cDNA into RelB-deficient fibroblasts reversed the lipopolysaccharide-induced chemokine overexpression. In vivo, activated RelB-/- fibroblasts dramatically increased recruitment of granulocytes into tissues. In view of the apparent role of RelB in the resolution of acute inflammation in tissues and previous work showing a requirement for RelB in the initiation of immune responses through the differentiation of antigen-presenting cells, RelB may be an important factor regulating the transition from innate to adaptive immunity.

Construction and characterization of human CD7-specific single-chain Fv immunotoxins.

To develop novel therapeutic agents for treatment of human T cell malignancies, we constructed two single-chain Fv (sFv) immunotoxins specific for the T cell-associated Ag CD7. The sFv fragments were derived from the murine hybridomas 3A1e and 3A1f and were expressed as soluble proteins in Escherichia coli. Surface plasmon resonance analyses demonstrated that the purified 3A1e and 3A1f sFv fragments specifically bound CD7 with high affinity, 8.1 and 1.8 nM, respectively. The difference in affinity is chiefly due to a slower dissociation rate for the 3A1f sFv fragment. Despite this difference, both monovalent sFv fragments were comparably internalized by CD7+ human T leukemic cells within 30 min. These data support findings of previous studies suggesting that CD7 internalization does not require cross-linking. The sFv immunotoxins were assembled by linking ricin toxin A chain to the C termini of the sFv fragments via disulfide bonds. Both sFv immunotoxins were comparably potent in their ability to inhibit protein synthesis in vitro in CD7+ Jurkat cells (50% inhibiting concentration = 15 pM). Further preclinical studies on the use of the 3A1e and 3A1f sFv immunotoxins to treat human T cell diseases therefore appear warranted.

High-level secretion of two antibody single chain Fv fragments by Pichia pastoris.

The diagnostic and therapeutic applications of antibody single-chain Fv (sFv) fragments often require large amounts of protein that can be problematic and expensive to obtain. Here we report the secretion of two sFv fragments by the yeast Pichia pastoris at levels up to 250 mg/l. Soluble sFv fragments were purified from culture supernatants in one step by affinity or metal-chelating chromatography, and were indistinguishable from their bacterially expressed counterparts in terms of affinity. Secretion of functional sFv fragments by Pichia pastoris provides a low cost, high yield alternative to current sFv expression systems.

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.

Association of DNA content and proliferative activity with clinical outcome in patients with diffuse mixed cell and large cell non-Hodgkin's lymphoma.

Formalin-fixed and paraffin-embedded lymph node biopsy specimens from 52 untreated patients with newly diagnosed diffuse large cell (n = 48) or mixed cell (n = 4) non-Hodgkin's lymphoma (NHL) were analyzed for DNA content and proliferative activity (PA) by flow cytometry. The results obtained by flow cytometry were compared with the results of cytogenetic studies performed on 28 of the specimens. The median age of the patients was 65 years (range, 15-84 years) and the male to female ratio was 3 to 2. All patients were uniformly staged and uniformly treated with cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine, and prednisone. The flow cytometric results were compared statistically by univariate analysis with the rate and duration of complete remission and survival. Tumors with low PA (greater than or equal to 80% of cells in G0/G1 phase) were found in 65% of the patients; 74% of those with low PA versus only 44% of those with high PA achieved an initial complete remission (P less than 0.02). DNA aneuploidy was detected in tumors of 56% of the patients and was associated with a significantly longer duration of complete remission (P less than 0.01). Both low PA and aneuploidy independently predicted longer survival. The predicted 2-year actuarial survival for patients with tumors with low PA was 68% versus 10% for those with high PA (P less than 0.01). Similarly, the 2-year survival of patients with aneuploid tumors was 60% versus 36% for those with diploid tumors (P less than 0.01). The combination of PA and DNA content categorized the patients into four groups with decreasing 2-year survivals: low PA/aneuploid (n = 20), 77%; low PA/diploid (n = 14), 57%; high PA/aneuploid (n = 9), 32%; high PA/diploid (n = 9), 0%. The flow cytometric results correlated well with those of the cytogenetic studies. We conclude that low PA and DNA aneuploidy, both separately and in combination, predict a favorable clinical outcome for patients with diffuse mixed cell and large cell NHL.

Reactive T cells in the immune repertoire: self-restricted and allo-restricted helper T-cell clones to Epstein-Barr virus.

Helper T-cell clones were generated by stimulation with autologous or allogeneic lymphoblastoid B cells (B-LCL) transformed by the Epstein-Barr virus (EBV). Some of these T-cell clones were allo-reactive and others were specific to EBV-transformed B-LCL. Helper T-cell clones specific to EBV-transformed B-LCL were restricted either by class-I or by class-II HLA molecules of self. T-cell clones restricted by class-I HLA molecules were stained by OKT3 and OKT8 monoclonal antibodies (MAbs), whereas class-II-restricted clones stained with OKT3 and OKT4. Not all helper T-cell clones specific to EBV-transformed B-LCL were restricted to self: one clone restricted by allo-HLA antigen was established. This finding suggests that in humans, as in mice, some T cells in the T-cell repertoire can be allo-restricted. This allo restriction may represent cross-reactivity of T cells, whereby "self + X" equals "allo + Y." Activation of these cross-reacting T cells restricted by allogeneic HLA molecules during infectious mononucleosis will give a T-cell response which may appear unrestricted by self HLA molecules. This mechanism helps to explain, at least in part, the HLA unrestricted cytotoxicity to B-LCL observed in infectious mononucleosis.