Michael P. Brown

Stat5a and Stat5b Proteins Have Essential and Nonessential, or Redundant, Roles in Cytokine Responses

A variety of cytokines mediate the activation of Janus protein tyrosine kinases (Jaks). The Jaks then phosphorylate cellular substrates, including members of the signal transducers and activators of transcription (Stat) family of transcription factors. Among the Stats, the two highly related proteins, Stat5a and Stat5b, are activated by a variety of cytokines. To assess the role of the Stat5 proteins, mutant mice were derived that have the genes deleted individually or together. The phenotypes of the mice demonstrate an essential, and often redundant, role for the two Stat5 proteins in a spectrum of physiological responses associated with growth hormone and prolactin. Conversely, the responses to a variety of cytokines that activate the Stat5 proteins, including erythropoietin, are largely unaffected.

Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice.

Inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome) is characterized by failure of immunoglobulin isotype switching and severe defects of cell-mediated immunity. To test the potential for gene transfer therapy to correct this disorder, we transduced murine bone marrow or thymic cells with a retroviral vector containing the cDNA for the murine CD40 ligand (CD40L) and injected them into CD40L–/– mice. Even low-level, constitutive expression of the transgene stimulated humoral and cellular immune functions in these mice. With extended follow-up, however, 12 of 19 treated mice developed T-lymphoproliferative disorders, ranging from polyclonal increases of lymphoblasts to overt monoclonal T-Lymphoblastic lymphomalymphomas that involved multiple organs. Our findings show that constitutive (rather than tightly regulated), low-level expression of CD40L can produce abnormal proliferative responses in developing T lymphocytes, apparently through aberrant interaction between CD40L+ and TCRαβ+CD40+ thymocytes. Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades.

CD40 Ligand (CD154) Enhances the Th1 and Antibody Responses to Respiratory Syncytial Virus in the BALB/c Mouse

CD40 ligand (CD40L) is a cell surface costimulatory molecule expressed mainly by activated T cells. CD40L is critically important for T-B cell and T cell-dendritic cell interactions. CD40L expression promotes Th1 cytokine responses to protein Ags and is responsible for Ig isotype switching in B cells. Respiratory syncytial virus (RSV) is an important pathogen of young children and the elderly, which causes bronchiolitis and pneumonia. Studies of mice infected with RSV suggest that a Th2 cytokine response may be responsible for enhanced pulmonary disease. To investigate the effect CD40L has on RSV immunity, mice were infected simultaneously with RSV and either an empty control adenovirus vector or one expressing CD40L or were coimmunized with plasmid DNA vectors expressing CD40L and RSV F and/or G proteins and subsequently challenged with RSV. The kinetics of the intracellular and secreted cytokine responses, the cytotoxic T lymphocyte precursor frequency, NO levels in lung lavage, rates of virus clearance, and anti-RSV Ab titers were determined. These studies show that coincident expression of CD40L enhances the Th1 (IL-2 and IFN-γ) cytokine responses, increases the expression of TNF-α and NO, accelerates virus clearance, and increases the anti-F and anti-G Ab responses. These data suggest that CD40L may have the adjuvant properties needed to optimize the safety and efficacy of RSV vaccines.

POTENT ANTITUMOR EFFECTS OF CD154 TRANSDUCED TUMOR CELLS IN EXPERIMENTAL BLADDER CANCER

PURPOSEnCurrent intravesical immunotherapy for bladder cancer with bacillus Calmette-Guerin instillations is standard treatment for patients with high risk superficial tumors but relapses are common. We evaluated the tumor vaccine concept in murine bladder cancer by comparing tumor cell transduction with genes coding for the immunostimulatory molecules CD154, interleukin (IL)-12 and CD80 to design a novel vaccination strategy.nnnMATERIALS AND METHODSnAdenoviral vectors were used to transduce murine bladder cancer MB-49 cells with genes coding for CD154, IL-12 and CD80. Parental or transduced MB-49 cells were injected subcutaneously into syngeneic mice. The effects of transgene expression on tumorigenicity and the generation of protective immunological memory against challenge with parental tumor were studied.nnnRESULTSnAll 76 animals injected with parental MB-49 cells had tumors within 8 to 12 days. Tumor cell expression of CD154 combined with IL-12 completely inhibited tumor outgrowth with all 21 mice tumor-free and CD154 transduction alone was almost as effective with 33 of 35 tumor-free. IL-12 production by tumor cells delayed tumor outgrowth and 4 of 10 mice remained tumor-free. Over expression of CD80 had no effect on tumorigenicity. CD154 expressing tumors were rapidly infiltrated with large numbers of CD4+ and CD8+ T cells. Mice vaccinated 4 times with adenoviral CD154 transduced MB-49 cells were completely protected against challenge with parental tumor. Co-injection of CD154 modified cells with parental MB-49 cells retarded tumor growth.nnnCONCLUSIONSnOur experimental results suggest that the potent antitumor effects of CD154 gene transduction should be considered for immunostimulatory gene therapy for bladder cancer.

Detection of TNF α and Fas ligand mRNA within synovial mononuclear cells by fluorescence in-cell labeling PCR (FICL-PCR)

T cells that infiltrate the synovial lesions of rheumatoid arthritis may play a key role in its pathogenesis. To learn more about their functional nature, we determined the frequency of synovial T cells that harbored the TNF alpha and Fas ligand transcript by a technique, called Fluorescence In-Cell Labeling Polymerase Chain Reaction (FICL-PCR). The mRNA of interest was detected in fixed cells by the incorporation during PCR of a fluorescein-12-dUTP label following an initial reverse transcription PCR step. Using this technique the CD3 transcript was detected in the T leukemic cell line, MOLT-4, with calculated sensitivity and specificity values of 91% and 100%, respectively. The percentage mean (+/-S.D.) of TNF alpha mRNA positive cells and Fas ligand mRNA positive cells in peripheral blood mononuclear cells from 12 rheumatoid arthritis patients were 5.1+/-2.3% and 4.8+/-3.1%, respectively. The percentage mean (+/-S.D.) of TNF alpha mRNA positive cells and Fas ligand mRNA positive cells among synovial mononuclear cells from six rheumatoid arthritis patients was 16.8+/-8.3% and 10.8+/-1.8%, respectively. This result indicates that the cytotoxic T cells expressing TNF alpha accumulate in rheumatoid arthritic lesions where they may play a pathogenic role.