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Gut 2004;53:339-345. doi: 10.1136/gut.2003.020107 Background and aims: Interleukin-12 (IL-12), a p35/p40 heterodimer, plays a pivotal role in the immune response in Crohn's disease (CD). Since IL-12 p40 dimers act as [...]

Recent insight into the balance of self-tolerance and auto-aggression has raised interest in using human regulatory T (Treg) cells for adoptive immunotherapy of unlimited autoimmune diseases including type-1 diabetes, rhematoid arthritis and multiple sclerosis. The therapeutic use of Treg cells, however, is so far hampered by the inefficiency of current protocols in making them accessible for genetic manipulations. We report here that TCR/CD3 stimulation that is accompanied by extensive CD28 costimulation makes human Treg cells susceptible to retroviral gene transfer ex vivo while preserving their properties in vitro and in vivo. To show the power of genetic manipulation of human Treg cells, we engineered ‘designer Treg cells’ by retroviral expression of a chimeric immunoreceptor with defined specificity, which activates Treg cells in a ligand-dependent manner to proliferate, to secrete high amounts of interleukin-10 and to repress an ongoing cytolytic T-cell response in vivo. The procedure in genetically modifying human Treg cells ex vivo will open a panel of applications for their use in the adoptive therapy of deregulated immune responses. [ABSTRACT FROM AUTHOR]

Human T lymphocytes can be redirected with a new defined specificity by expression of a chimeric T-cell receptor (immunoreceptor) for the use in adoptive immunotherapy of cancer. Whereas standard procedures use retroviral gene transduction to constitutively express immunoreceptors in T cells, we here explored for the first time mRNA electroporation to achieve transient immunoreceptor expression, and thereby minimizing the risk of persistence of potential autoaggression. CD4+ and CD8+ T cells were efficiently transfected with immunoreceptors specific for ErbB2 and CEA. The immunoreceptor expression was transient with half-maximal expression at day 2 and no detectable immunoreceptor expression at day 9 after electroporation. Immunoreceptor-transfected T cells were specifically activated upon coincubation with ErbB2+ and CEA+ tumor cells, respectively, resulting in secretion of interferon-γ (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNFα). Furthermore, immunoreceptor-transfected CD8+ T cells specifically lysed ErbB2+ and CEA+ tumor cells, respectively. The RNA-transfected T cells retained their cytotoxic function after 2 days of activation and exhibited cytolytic activities like retrovirally transduced T cells. RNA electroporation of T cells thereby provides a versatile tool for transient immunoreceptor expression, which may be of advantage in avoiding the persistence of unintended autoaggression.Gene Therapy (2009) 16, 596–604; doi:10.1038/gt.2008.189; published online 22 January 2009 [ABSTRACT FROM AUTHOR]

Background and aims: The prognosis of metastatic colorectal cancer is still poor, raising the need for alternative therapeutic approaches, particularly by manipulating the antitumour immune response. Advanced tumour stages, however, are frequently accompanied by functional T cell defects which may be critical for a T cell based anticancer immunotherapy. The aim of this study was to address whether T cells from colorectal cancer patients with advanced tumour stages can be specifically antigen activated against their autologous tumour cells. Methods: T cells were isolated from colorectal cancer patients and retrovirally transduced to express a recombinant immunoreceptor that has an extracellular binding domain for carcinoembryonic antigen (CEA) and an intracellular CD3ζ signalling domain with and without CD28 costimulation for T cell activation. Results: Peripheral blood T cells from colorectal cancer patients were successfully engineered to express the anti-CEA immunoreceptor on the cell surface. On coincubation with autologous CEA+ tumour cells, T cells with anti-CEA immunoreceptor are specifically activated to secrete interferon γ (IFN-γ) and to lyse autologous tumour cells whereas T cells without immunoreceptor are not. T cells equipped with combined CD3ζ-CD28 signalling receptor are more efficiently activated to secrete IFN-γ compared with T cells with CD3ζ signalling receptor. Induction of interleukin 2 secretion on targeting towards autologous tumour cells requires triggering of T cells by the CD3ζ-CD28 costimulatory receptor. Conclusions: T cells from advanced colorectal cancer patients can be tumour specifically activated with high efficiency by engraftment with a combined CD3ζ-CD28 immunoreceptor to break tolerance against autologous tumour cells. [ABSTRACT FROM AUTHOR]

Background and aims: Interleukin-12 (IL-12), a p35/p40 heterodimer, plays a pivotal rote in the immune response in Crohn's disease (CD). Since IL-12 p40 dimers act as IL-12 antagonists, we assayed p40 dimer proteins to modulate chronic intestinal inflammation. Methods: We generated a fusion protein consisting of the IL-12(p40) subunit fused to the constant region of IgG2b. IL-12(p40)-IgG2b was tested in a murine 2,4,6,-trinitrobenzene sulphonic acid (TNBS) colitis model and in lamina propria mononuclear cells (LPMNC) from patients with CD in vitro. Results: Dimeric IL-12(p40)-IgG2b fusion protein bound specifically to the IL-12 receptor. In concentrations <10-7 M, it acted as on IL-12 antagonist as it inhibited interferon γ (IFN-γ) secretion, suppressed proliferation, and increased apoptosis of LPMNC from patients with CD. However, in concentrations >10-6 M, IL-12(p40)-IgG2b increased IFN-γ secretion and lymphocyte proliferation thereby acting as on IL-12 agonist. In TNBS colitic mice, IL-12(p40)-IgG2b decreased mortality (10% v 68%), prevented body weight loss, reduced tumour necrosis factor α, and increased IL-10 secretion. Conclusions: The IL-12(p40)-IgG2b fusion protein has dichotomic properties as a specific IL-12 antagonist and selective repressor of mucosal inflammation at low concentration and as an IL-12 agonist at high concentration. [ABSTRACT FROM AUTHOR]

MUC1 mucin is aberrantly glycosylated and overexpressed in a number of epithelial malignancies and is therefore a promising tumour-associated antigen for target-directed immunotherapy of a panel of malignant diseases. In MUC1-positive tumours, MHC class I expression is frequently downregulated and MUC1-specific cytotoxic T cells (CTLs) are either not available or in a state of anergy allowing tumour growth without limitation by CTL control. To activate lymphocytes and natural killer (NK) cells, we here generated an anti-MUC1-scFv-IL2 fusion protein (C595scFv-Fc-IL2) that contains the C595 single-chain antibody for MUC1 binding, the human IgG1 CH2CH3 domain for protein dimerisation, and interleukin-2 (IL2) for activation of immunological effector cells. The fusion protein binds to MUC1-derived peptides and to MUC1-positive tumour cells with the same specificity as does the C595 monoclonal antibody. Bound to MUC1, the C595scFv-Fc-IL2 fusion protein stimulates proliferation of human activated lymphocytes in vitro. Upon binding to MUC1-positive MCF7 breast carcinoma cells, moreover, the fusion protein activates resting NK cells to tumour cell lysis. These properties make the C595scFv-Fc-IL2 fusion protein a suitable candidate for the immunotherapy of MUC1-positive tumours. [ABSTRACT FROM AUTHOR]

In early stages of cutaneous T cell lymphoma (Sézary syndrome) both CD4[SUP+]CD7[SUP-] and CD4[SUP+]CD7[SUP+] T cells clonally expand whereas in late stages of the disease CD7[SUP-] cells are predominant in number, giving rise to the question whether CD7[SUP-]T cells have a survival advantage in the skin. Galectin-1, a cell-bound lectin, was recently reported to trigger apoptosis in activated CD7[SUP+] T cells. Here, we demonstrate that in contrast to activated CD7[SUP+] T cells, quiescent and activated CD69[SUP+]CD7[SUP-] T cells from healthy donors and from Sézary patients are resistant to galectin-1-mediated cell death. CD7[SUP-] T cells are apoptosis-resistant even during coculture with IFN-γ-stimulated endothelial cells that constitutively express galectin-1 in high amounts. These data imply that resistance of CD7[SUP-] T cells to galectin-1-induced apoptosis may contribute to the accumulation of CD7[SUP-] Sézary T cells during progression of the disease. [ABSTRACT FROM AUTHOR]

T cells can be directed to antigen-specific, MHC-independent target ceil lysis by grafting with a recombinant receptor with antibody-like specificity. Here, we asked whether T cells from the peripheral blood of a patient with cutaneous T cell lymphoma can be recruited for an immune response against autologous tumor cells. Lymphoma cells with a CD3[sup -] CD4[sup +] CD30[sup +] phenotype and clonal TCR-Vβ7 rearrangement were isolated from a cutaneous lesion. The lymphoma lesion additionally harbored CD3[sup +] CD25[sup +] activated normal T cells despite ongoing tumor progression. Peripheral blood-derived T cells from the lymphoma patient were retrovirally engrafted with a recombinant anti-CD30scFv-γ receptor. Upon cocultivation with autologous CD30[sup +] lymphoma cells, grafted T cells increase IFN-γ secretion and lyse specifically lymphoma cells with high efficiency, even at an effector to target cell ratio of as low as 1:20. Our data demonstrate that the recombinant anti-CD30-γ receptor overcomes T cell tolerance for tumor cells and directs T cells specifically against autologous lymphoma cells. [ABSTRACT FROM AUTHOR]

The CD7[sup -] subset of CD4[sup +] T cells reflects a stable differentiation state of post-thymic helper T cells with CD45R0[sup +]CD45RA[sup -] 'memory' phenotype. Here we report that CD4[sup +]CD7[sup -] T cells are prone to increased spontaneous apoptosis in vitro compared to CD4[sup +]CD7[sup +] T cells. Spontaneous apoptosis is prevented by IL-15, but not by IL-2. Moreover, IL-15 increases Bcl-2 and decreases CD95/Fas expression of CD7[sup -], but not of CD7[sup +] T cells. Because IL-15 is physiologically not secreted but expressed in a membranebound form, we cocultured T cells with TNF-α stimulated fibroblasts that expose membrane IL-15. TNF-α stimulated fibroblasts rescue CD4[sup +]CD7[sup -] Tcells from apoptosis whereas unstimulated fibroblasts do not. Rescue from apoptosis requires cell-cell contact and is abolished by addition of neutralizing antibodies to IL-15. We conclude that membrane IL-15 prevents accelerated apoptosis of CD4[sup +]CD7[sup -] T cells. This mechanism may contribute to accumulation of CD7[sup -] T cells in chronic inflammatory skin lesions. [ABSTRACT FROM AUTHOR]

Re-arrangement and expression of the immunoglobulin (Ig) genes in B cells occurs under an ordered developmental control. The sequential model of Ig light chain exclusion predicts that only after non- productive re-organization or deletion of both Ig kappa alleles would re-arrangement of Ig lambda gene segments occur. To prove this model, we asked whether expression of Ig lambda light chains is always associated with rearrangement and/or deletion of both Ig kappa alleles in human cells. Therefore, we established human diploid B-cell clones in vitro that produce Ig lambda light chains. Southern blot analysis of the Ig kappa alleles revealed that three Ig lambda expressing cell lines (out of six Ig lambda+ cell lines tested) harbour one Ig kappa allele in germline configuration. Furthermore, a 1.5 kb RNA derived from the germline Ig kappa locus was detected by Northern blot hybridizations. The results implicate that the mechanism of Ig light chain exclusion is not precisely sequential and that it does not necessarily need re-arrangement or deletion of both Ig kappa alleles as a prerequisite for Ig lambda light chain expression in human cells. [ABSTRACT FROM AUTHOR]

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7- T cells express the CD4CD45RO+CD45RA- memory phenotype. We analysed CD4+D45RA- peripheral blood lymphocytes that were separated into CD7+ and CD7- for their in vitro cytokine secretion in response to different stimuli. The CD4+ CD7- subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4+ CD7+ subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion. IL-4 and interferon-gamma (IFN-γ) secretion was not significantly different in CD7+ and CD7- T cells upon stimulation in vitro. The data indicate that the CD+ CD7- T cell represents the majority of IL-5-secreting cells within the population of CD4+CD45RA- memory T cells. Since CD4+CD7- T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7- T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases. [ABSTRACT FROM AUTHOR]

Three brothers from one family died of Hodgkin disease and a fourth brother from a diffuse malignant non-Hodgkin lymphoma. This patient exhibited a constant deficiency of serum immunoglobulins and elevated antibody titers to Epstein-Barr viral antigens. Epstein-Barr virus DNA sequences were detected in DNA isolated from lymph node biopsies from two of the patients. Initially, no abnormalities in the numbers of B and T cells could be detected. Peripheral blood lymphocytes of the patients did not react in the mixed lymphocyte culture assay. We suggest that an immune deficiency to Epstein-Barr virus may favor the proliferation of malignant lymphocytes after Epstein-Barr viral infection. Monoclonal lymphoid B cell lines established spontaneously in vitro from a lymph node biopsy specimen and from peripheral blood specimens from two of the patients. The cells harbor Epstein-Barr viral DNA sequences in multiple genome equivalents and express Epstein-Barr nuclear antigen. The cells contain a tenfold increased level of c-fgr-related RNA transcripts compared with peripheral blood lymphocytes of healthy adults. No obvious amplifications or translocations of the c-myc, c-abl, or c-fgr gene could be detected. [ABSTRACT FROM AUTHOR]

Adoptive therapy with engineered T cells shows promising results in treating patients with malignant disease, but is challenged by incomplete responses and tumor recurrences. Here, we aimed to direct the tumor microenvironment in favor of a successful immune response by local secretion of interleukin (IL-) 12 and IL-18 by sadministered T cells. To this end, we engineered T cells with a melanoma-specific T cell receptor (TCR) and murine IL-12 and/or IL-18 under the control of a nuclear-factor of activated T-cell (NFAT)-sensitive promoter. These T cells produced IL-12 or IL-18, and consequently enhanced levels of IFNγ, following exposure to antigen-positive but not negative tumor cells. Adoptive transfer of T cells with a TCR and inducible (i)IL-12 to melanoma-bearing mice resulted in severe, edema-like toxicity that was accompanied by enhanced levels of IFNγ and TNFα in blood, and reduced numbers of peripheral TCR transgene-positive T cells. In contrast, transfer of T cells expressing a TCR and iIL-18 was without side effects, enhanced the presence of therapeutic CD8+T cells within tumors, reduced tumor burden and prolonged survival. Notably, treatment with TCR+iIL-12 but not iIL-18 T cells resulted in enhanced intra-tumoral accumulation of macrophages, which was accompanied by a decreased frequency of therapeutic T cells, in particular of the CD8 subset. In addition, when administered to mice, iIL-18 but not iIL-12 demonstrated a favorable profile of T cell co-stimulatory and inhibitory receptors. In conclusion, we observed that treatment with T cells engineered with a TCR and iIL18 T cells is safe and able to skew the tumor microenvironment in favor of an improved anti-tumor T cell response. [ABSTRACT FROM PUBLISHER]