anti-CD3 Antibody Activation Research Articles

S-15 in combination of Akt inhibitor promotes the expansion of CD45RA\u2212CCR7+ tumor infiltrating lymphocytes with high cytotoxic potential and downregulating PD-1+Tim-3+ cells as well as regulatory T cells

BackgroundAutologous tumor-infiltrating lymphocytes (Tils) immunotherapy is a promising treatment in patients with advanced hepatocellular cancer. Although Tils treatment has shown great promise, their persistence and the efficacy after adoptive-transfer are insufficient and remain a challenge. Studies have demonstrated that IL-15 and Akt inhibitor can regulate T cell differentiation and memory. Here, we constructed S-15 (Super human IL-15), a fusion protein consisting of human IL-15, the sushi domain of the IL-15 receptor α chain and human IgG-Fc. Herein we compared the effects of S-15 with IL-2 or in combination with Akti on the expansion and activation of Tils.MethodsHepatocellular cancer tissues were obtained from 6 patients, Tils were expanded using IL-2, IL-2/S-15, IL-2/Akti or in combination IL-2/S-15/Akti. At day 10, anti-CD3 antibody was added to the culture media and expanded to day 25. The composition, exhaustion and T-cell differentiation markers (CD45RA/CCR7) were analyzed by flow cytometry.ResultsWe found that IL-2/S-15/Akti expanded Tils and showed the highest percentage of central memory CD45RA−CCR7+ phenotype prior to anti-CD3 antibody activation and after anti-CD3 antibody activation. T cells cultured with IL-2/S-15/Akti exhibited a mixture of CD4+, CD8+, and CD3+CD4−CD8− T cells; S-15 in combination with Akt inhibitor downregulated the expression of PD-1+Tim-3+ on Tils and decreased the Tregs in Tils. Additionally, the Tils expanded in the presence of the Akt inhibitor and S-15 showed enhanced antitumor activity as indicated by the increase in IFN-γ producing tumor infiltrating CD8+ T cells and without comprising the Tils expansion.ConclusionOur study elucidates that IL-2/S-15/Akti expanded Tils and represent a viable source for the cellular therapy for patients with hepatocellular cancer.

TU-100 (Daikenchuto) and ginger ameliorate anti-CD3 antibody induced T cell-mediated murine enteritis: microbe-independent effects involving Akt and NF-κB suppression.

The Japanese traditional medicine daikenchuto (TU-100) has anti-inflammatory activities, but the mechanisms remain incompletely understood. TU-100 includes ginger, ginseng, and Japanese pepper, each component possessing bioactive properties. The effects of TU-100 and individual components were investigated in a model of intestinal T lymphocyte activation using anti-CD3 antibody. To determine contribution of intestinal bacteria, specific pathogen free (SPF) and germ free (GF) mice were used. TU-100 or its components were delivered by diet or by gavage. Anti-CD3 antibody increased jejunal accumulation of fluid, increased TNFα, and induced intestinal epithelial apoptosis in both SPF and GF mice, which was blocked by either TU-100 or ginger, but not by ginseng or Japanese pepper. TU-100 and ginger also blocked anti-CD3-stimulated Akt and NF-κB activation. A co-culture system of colonic Caco2BBE and Jurkat-1 cells was used to examine T-lymphocyte/epithelial cells interactions. Jurkat-1 cells were stimulated with anti-CD3 to produce TNFα that activates epithelial cell NF-κB. TU-100 and ginger blocked anti-CD3 antibody activation of Akt in Jurkat cells, decreasing their TNFα production. Additionally, TU-100 and ginger alone blocked direct TNFα stimulation of Caco2BBE cells and decreased activation of caspase-3 and polyADP ribose. The present studies demonstrate a new anti-inflammatory action of TU-100 that is microbe-independent and due to its ginger component.

Lactobacillus isolates from healthy volunteers exert immunomodulatory effects on activated peripheral blood mononuclear cells

As probiotics in the gut, Lactobacilli are believed to play important roles in the development and maintenance of both the mucosal and systemic immune system of the host. This study was aimed to investigate the immuno-modulatory function of candiate lactobacilli on T cells. Lactobacilli were isolated from healthy human feces and the microbiological characteristics were identified by API 50 CHL and randomly amplified polymorphic DNA (RAPD) assays. Anti-CD3 antibody activated peripheral blood mononuclear cells (PBMCs) were treated by viable, heat-killed lactobacilli and genomic DNA of lactobacilli, and cytokine profiles were tested by ELISA. Isolated lactobacilli C44 and C48 were identified as L. acidophilus and L. paracacei, which have properties of acid and bile tolerance and inhibitor effects on pathogens. Viable and heat-killed C44 and C48 induced low levels of proinflammatory cytokines (TNF-α, IL-6 and IL-8) and high levels of IFN-γ and IL-12p70 in PBMCs. In anti-CD3 antibody activated PBMCs, viable and heat-killed C44 increased Th2 cytokine levels (IL-5, IL-6 and IL-10), and simultaneously enhanced Th1 responses by inducing IFN-γ and IL-12p70 production. Different from that of lactabacillus strains, their genomic DNA induced low levels of IL-12p70, IFN-γ and proinflammatory cytokines in PBMCs with or without anti-CD3 antibody activation. These results provided in vitro evidence that the genomic DNA of strains of C44 and C48, especially C44, induced weaker inflammation, and may be potentially applied for treating allergic diseases.

Classification of CD4 + T Helper Cell Clones in Human Melanoma

We have previously described the generation of tumor-infiltrating lymphocyte (TIL) clones from renal cell cancer by solid-phase anti-CD3 antibody activation and expansion in 100 IU/ml IL-2 plus irradiated allogeneic B cells. These culture conditions did not select for a particular T cell subset. Using these culture conditions, we report here the generation of 66 CD4 + and 36 CD8 + TIL clones from five patients with melanoma. Eighty-five percent of the CD4 + TIL clones were not cytolytic (<30% lysis, E:T 20:1) as determined by antibody-redirected lysis (ARL), whereas all CD8 + clones showed strong ARL activity (> 30% lysis). Clones were further tested for production of IL-2, IL-4, and IFN-γ after activation for 48 hr by solid-phase anti-CD3. CD8 + clones produced significant amounts of IFN-γ little IL-2, and no IL-4. CD4 + clones were classified as Th0, Th1, or Th2, analogous to the classification of T helper cells in the mouse. Sixty-six percent existed as Th0, producing IL-2, IL-4, and IFN-γ. Only 15% existed as Th1, producing IL-2 and IFN-γ, and 19% as Th2, producing IL-4 but no IL-2 or IFN-γ. In all cases, unstimulated clones or clones stimulated with the allogeneic B cell line did not produce detectable amounts of cytokines. Solid-phase anti-CD3 activation was compared to activation with autologous melanoma cells. Five of nine CD8 + clones produced low amounts of IL-2 (<200 pg/ml/10 6 cells) in response to autologous tumor, but none of the CD8 clones produced IFN-γ or IL-4. Also, 5/7 Th0 clones from one patient produced similar amounts of IL-2 after stimulation with anti-CD3 or autologous tumor. The other two clones produced only 10% or less of the amount produced in response to anti-CD3. No IL-4 or IFN-γ could be detected in response to autologous tumor. In contrast, none of the 12 T helper clones from two other patients produced any cytokines after stimulation with autologous tumor cells. Together these data suggest that the T cell infiltrate in melanoma consists primarily of IL-2-producing Th0 cells, but few of those are triggered by autologous tumor cells.

Detection of individual interleukin 2-producing cells after anti-CD3 antibody activation

A new intracytoplasmic immunofluorescence staining technique to detect and quantify human interleukin-2 (IL-2)-producing lymphocytes is described. Blood mononuclear cells (MNC) were cultured and stimulated by mitogenic anti-CD3 antibody to produce IL-2. The cells were then fixed and subsequently permeabilized in suspension by the detergent saponin. Cytoplasmic IL-2 could then be demonstrated using polyclonal IL-2 specific antibodies and indirect immunofluorescence staining. A characteristic morphology of the IL-2 staining was noted with a local circle-shaped accumulation in the cytoplasm in a perinuclear position, probably reflecting the presence of the lymphokine in the Golgi stacks. A rapid, but transient IL-2 production peak 6 h after initiation of the cultures was observed. Only 1% of the cells produced IL-2, but each IL-2 stained cell was very bright, indicating a low capacity of anti-CD3 antibody to induce IL-2 production rather than an insensitivity of our detection system. Of the IL-2-producing cells 90% were CD4-positive T cells and 10% were CD8-positive T cells as revealed by two-colour staining of cell surface antigens and cytoplasmic IL-2.