anti-CD3 Proliferation Research Articles

Expansion of human bone marrow-derived mesenchymal stromal cells with enhanced immunomodulatory properties

BackgroundMesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD.MethodsHuman bone marrow-derived MSCs were expanded at high-confluency (MSCHC) and low-confluency state (MSCLC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSCHC or MSCLC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45+ blood count and histopathological examination.ResultsAs compared to MSCLC, MSCHC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSCHC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSCHC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSCLC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSCHC as well as enriched pathways including cytokine–cytokine receptor interactions, cell adhesion and PI3K-AKT signalling. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSCHC showed greater suppression of inflammation and improved outcomes compared to MSCLC and saline controls.ConclusionOur study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.

New immunosuppressive secondary metabolites from the endophytic fungus Aspergillus sp.

Six new metabolites, including two diphenolic derivatives (1 and 2), one pseurotin (3), one butenolide derivative (4), one benzopyran (5) and one isochromane lactone (6), together with ten known compounds (7–16) were isolated from an endophytic fungus Aspergillus sp. Their planar structures and absolute configurations were established based on techniques of MS, NMR, IR, UV, [Rh2(OCOCF3)4] complex-induced ECD, quantum chemical electronic circular dichroism (ECD) calculations, and single crystal X-ray diffraction. Structurally, compound 2 represents the first example of diphenolic derivative possessing an unusual 1-oxaspiro[2.4]heptane core bearing a 5/3 bicyclic skeleton; compound 3 represents the first example of pseurotin type natural products that only one hydroxy group is substituted at side chain. In bioassay, compounds 3, 7 and 8 exhibited potential inhibitory effect on the proliferation of anti-CD3/anti-CD28 monoclonal antibodies (mAbs) induced murine T cells, with IC50 values of (7.81 ± 0.71), (8.25 ± 0.78) and (8.84 ± 0.81) μM, respectively.

Activation of EL-4 T-cells by irradiation with atmospheric oxygen plasma

The activation of EL-4 T-cells is investigated when the T-cells are sensitized with monoclonal antibodies against CD3/CD28 followed by irradiation with atmospheric oxygen plasma. The proliferation of anti-CD3/CD28-sensitized EL-4 T-cells was evaluated as a function of the oxygen plasma irradiation duration. The plasma irradiation promotes cell proliferation: when plasma irradiation was applied to sensitize T-cells for 40 s, the cell number increased by five times over 24 h compared with that in a cell population that was not exposed to irradiation. Also, the plasma irradiation increases the production of interferon gamma in T-cells by a factor of two. Gene expression analysis of T-cell supports results of the proliferation and the immune function enhancements.

Induction of Regulatory T Cells and Its Regulation with Insulin-like Growth Factor/Insulin-like Growth Factor Binding Protein-4 by Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) are multipotent and exert anti-inflammatory effects, but the underlying mechanism remains to be elucidated. In the current study, we investigated the regulatory mechanism of regulatory T cell (Treg) induction through the growth factors released by human MSCs. Human naive CD4+ T cells were stimulated with anti-CD3/28 Abs and cocultured with human MSC culture supernatant for 48 h. The proliferation and cytokine production of CD4+ T cells and surface molecule expression on CD4+ T cells were evaluated. The proliferation of anti-CD3/28 Abs-stimulated CD4+ T cells was suppressed by the addition of human MSC culture supernatant; in addition, the production of IL-10 and IL-4 increased. The human MSC culture supernatant induced CD4+FOXP3+ Tregs that expressed CD25, CTLA-4, glucocorticoid-induced TNFR-related protein, insulin-like growth factor (IGF)-1R, and IGF-2R, showing antiproliferative activity against CD4+ T cells. In addition, the induction of Tregs by human MSC culture supernatant was enhanced by the addition of IGF and suppressed by the inhibition of IGF-1R. In contrast, a significant amount of IGF binding protein (IGFBP)-4, an inhibitor of IGF action, was detected in the human MSC culture supernatant. After neutralization of IGFBP-4 in the human MSC culture supernatant by anti-IGFBP-4 Ab, Treg numbers increased significantly. Thus, our results raise the possibility that human MSC actions also involve a negative-regulatory mechanism that suppresses Treg proliferation by releasing IGFBP-4. The results of this study suggest that regulation of IGF may be important for treatments using human MSCs.

OR42 Expanded thymic regulatory T cells (Tregs) have low class I HLA expression levels and are HLA-DR negative – Potential for ‘Off-The-Shelf’ Cellular Therapy?

Aim Tregs modulate immune responses against alloantigens and are a promising therapeutic tool in organ transplantation. We previously showed that abundant CD25 + FOXP3 + Tregs can be isolated and expanded from discarded human thymuses, routinely removed during pediatric cardiac surgery, indicating potential use of thymic Tregs as an ‘off-the-shelf’ tolerogenic therapy. Immunogenicity of allogenic therapeutic Tregs, however, is unclear. Here we studied HLA-ABC and -DR expression on expanded thymic Tregs compared to blood Tregs. Methods Thymocytes were obtained from infant thymuses (age n = 3) by mechanical dissociation; FOXP3 + CD25 + Tregs were isolated by magnetic-bead cell separation. Tregs were also isolated from infant peripheral blood mononuclear cells (PBMCs; n = 3) autologous to the thymus and PBMCs from healthy adults ( n = 3). After expansion with artificial antigen-presenting cells loaded with anti-CD3, IL-2, and rapamycin, Treg characteristics were confirmed by analyzing FOXP3 expression and suppressive capacity of proliferation of anti-CD3/28-stimulated T cells. HLA-ABC (clone W6/32) and HLA-DR (clone LN3) expression before and after expansion was assessed by flow cytometry. Results Before expansion, HLA-ABC expression levels were similar between thymic, infant blood and adult blood Tregs (Median Fluorescence Intensity (MFI) range: 3673–6059 vs. 3841–3830 vs. 6355–7952, respectively). Few thymic Tregs or infant blood Tregs expressed HLA-DR ( Conclusions Expanded thymic Tregs had lower class I HLA expression levels and contained fewer HLA-DR + cells compared to expanded blood Tregs, suggesting that thymic Tregs may be less immunogenic than blood Tregs. These findings indicate that discarded human thymus is potentially an excellent source of Tregs for ‘off-the-shelf’ tolerogenic therapy.

Enhanced cell cycle activation in response to stimulation in RGC-32-/- T lymphocytes (115.3)

Abstract We have previously identified Response Gene to Complement (RGC)-32 as a factor induced by complement activation, growth factors and cytokines. To study the in vivo function of RGC-32, we have generated mice carrying a null allele of the gene. Mice lacking RGC-32 were generated by replacing most of the second exon of the RGC-32 gene with sequences encoding neomycin resistance. These mice develop normally, have a similar size as wild type (WT) mice and a normal life span. Although RGC-32 was found to be involved in cell cycle regulation in vitro and is considered a tumor suppressor gene, RGC-32-/- mice do not spontaneously develop overt tumors. Little is known about the roles played by RGC-32 in T-cell proliferation. The effect of the lack of RGC-32 on cell cycle activation was investigated in CD4 cells isolated from spleen. Proliferation of anti-CD3/anti-CD28 stimulated CD4 cells from RGC-32-/- mice measured by [3H]-thymidine incorporation was significantly increased when compared with WT mice. In addition, by flow cytometry, both CD4 and CD8 T-cells from RGC-32-/- mice displayed a significant increase in the percentage of proliferating Ki-67 cells upon TCR stimulation. Furthermore, Akt phosphorylation was induced significantly in CD4 cells of RGC-32-/- when compared with WT mice. Our data indicate that in T-cells, RGC-32 plays an important role in inhibiting cell cycle activation by suppressing Akt activation.

Anti-inflammatory action ofCudrania tricuspidataon spleen cell and T lymphocyte proliferation

This study examined whether an extract of Cudrania tricuspidata shows anti-proliferative effects in anti-CD3/CD28-mediated spleen and CD4+CD25- T cells and decreases the production of the proinflammatory cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in anti-CD3/CD28-mediated CD4+CD25- T cells. The proliferation of anti-CD3/CD28-mediated spleen cells and CD4+CD25- T cells was effectively suppressed by C. tricuspidata. This extract, however, did not show cytotoxicity in spleen cells under conditions where the antigen was not stimulated using CCK-8 analysis. C. tricuspidata also decreased the production of the pro-inflammatory cytokines IL-2 and IFN-gamma by selective inhibition of this extract on proliferating cells in anti-CD3/CD28-mediated CD4+CD25- T cells. These results suggest that C. tricuspidata may be useful in the treatment of autoimmune diseases and organ transplantation through the inhibitory action of T cells in inflammation.

Anti-inflammatory action of alpha-melanocyte stimulating hormone (α-MSH) in anti-CD3/CD28-mediated spleen and CD4+CD25− T cells and a partial participation of IL-10

α-Melanocyte stimulating hormone (α-MSH) has been shown to inhibit the production and the effects of pro-inflammatory cytokine by inflammatory cells in innate immunity. We have determined whether α-MSH inhibits anti-CD3/CD28-mediated spleen cells and CD4+CD25− T cells proliferation and its mechanism of action. The proliferation of anti-CD3/CD28-mediated spleen cells and CD4+CD25− T cells markedly were suppressed by 50–100nM and 5–100nM α-MSH, respectively. α-MSH (100nM) increased the production of the anti-inflammatory cytokine IL-10 and decreased the production of the pro-inflammatory cytokine IL-2 and IFN-γ from CD4+CD25− T cells. Moreover, anti-IL-10 blocking Ab decreased the inhibitory effects of anti-CD3/CD28-mediated spleen cells and CD4+CD25− T cells proliferation by α-MSH, indicating a partial participation of IL-10 in its mechanism of inhibitory action. These results suggest that α-MSH may be useful for treatment of autoimmune diseases and transplantation involving innate and adaptive immunity.

Ceramide-independent CD28 and TCR signaling but reduced IL-2 secretion in T cells of acid sphingomyelinase-deficient mice.

Ceramide generated by lysosomal acid sphingomyelinase (aSMase) has been proposed to contribute to CD28 co-stimulatory signaling pathways. We used an aSMase-deficient mouse line (asmase-/-) to elucidate the role of the aSMase in splenocytes stimulated with either a combination of anti-CD3 and anti-CD28 antibodies, the lectin concanavalin A (Con A) or the superantigen staphylococcal enterotoxin B. All stimuli were shown to induce IL-2 expression, Con A additionally triggered the expression of high-affinity IL-2 receptor. However, in asmase-/- mice secretion of IL-2 was significantly reduced, whereas the intracellular IL-2 levels were elevated. Proliferation of anti-CD3/anti-CD28 or Con A-stimulated aSMase-deficient splenocytes was reduced up to 50% after 72 h in comparison to wild-type cells. We conclude that ceramide generated by aSMase is not involved in CD28 signal transduction, but rather a perturbation of the secretory system is responsible for the impaired proliferation of aSMase-deficient splenocytes.

Anti-interleukin-10 antibody restores burn-induced defects in T-cell function

Background. Studies have shown that susceptibility to sepsis after severe injury correlated with reduced production of T-helper 1 (Th1) cytokines (interleukin-2 [IL-2] and interferon-γ [IFN-γ]) and a persistence of T-helper 2 (Th2) cytokines (IL-4 and IL-10). The mechanisms responsible for this effect are not clear. We used a T-dependent antigen to study both the effect of burn injury on antigen-specific Th functions in vivo and the effect of anti-IL-10 antibody on these functions. Methods. Male A/J mice were anesthetized and given a 25% scald burn or a sham burn. On day 0 all mice were immunized with 100 μg trinitrobenzene sulfonic acid (TNP) haptenated ovalbumin (TNP-OVA) in complete Freund's adjuvant. Mice (10 per group) were given 250 μg monoclonal rat antimurine IL-10 antibody (anti-IL-10 MAB) or control rat immunoglobin G (IgG) on day 0 and 100 μg anti-IL-10 MAB or IgG on day 2. On day 10 the mice were killed to obtain serum and spleen cells. TNP-specific serum antibody isotype titers were determined by enzyme-linked immunosorbent assay (ELISA). Splenocyte proliferation and cytokine production in response to TNP-OVA or to anti-CD3 MAB were determined by tritiated thymidine incorporation and by ELISA, respectively. Results. Burn injury resulted in depressed levels of the TNP-specific IgG2a antibody isotype (Th1 dependent), whereas TNP-specific IgG1 and IgE (Th2 dependent) levels were not decreased in burn versus sham burn mice. Anti-IL-10 MAB but not IgG restored the IgG2a response. Burn injury also resulted in reduced TNP-OVA-specific proliferation of splenocytes, whereas anti-CD3 proliferation was equivalent in burn and sham mice. TNP-OVA-specific IL-2 and IFN-γ production were significantly reduced by burn injury. Anti-IL-10 MAB restored TNP-OVA-specific proliferation and antigen-specific IL-2 and interferon-γ production by splenocytes from burn mice. Conclusions. Burn injury induces the loss of antigen-specific Th1 cell function, and IL-10 acts as a trigger to down-regulate Th1 activity after injury.

Interaction of IL-15 with the shared IL-2 receptor beta and gamma c subunits. The IL-15/beta/gamma c receptor-ligand complex is less stable than the IL-2/beta/gamma c receptor-ligand complex.

This study was designed to compare the interactions of IL-2 and IL-15 with the IL-2R beta and IL-2R gamma c subunits, as differences in receptor interactions between IL-2 and IL-15 might contribute to the functional differences between these two cytokines. The results suggest the existence of a human IL-15R alpha subunit, although physical evidence of this molecule was not obtained. Proliferation of anti-CD3 (OKT3)-stimulated human PBL was compared for responsiveness to IL-2, IL-15, and F42K, and IL-2 mutant that does not bind the IL-2R alpha chain. F42K was more potent than IL-15 in activating a dose-dependent response. This fact, along with Scatchard binding analyses of IL-15 on OKT3 blasts and YT cells revealing both high and intermediate affinity receptors, supports the existence of IL-15R alpha on these cells. Additional characterization of the IL-15R utilized covalent cross-linking to affinity label IL-2R and IL-15R on YT cells and OKT3 blasts. Consistent with previously reported functional data, IL-2R alpha was not co-precipitated from the [125I]IL-15 receptor-ligand complex, demonstrating that IL-15 does not interact physically with the IL-2R alpha subunit. While IL-2R alpha did co-precipitate with IL-2R beta and IL-2R gamma c in the presence of IL-2, IL-15R alpha did not co-precipitate with the IL-2R beta/gamma c complex. Finally, YT cells equilibrated with IL-2 and then precipitated through IL-2R beta showed that IL-2R beta and IL-2R gamma c co-precipitate in a 1:1 ratio, while only IL-2R beta was found in the immunoprecipitates of YT cells equilibrated with IL-15. This indicates that IL-15 creates a less stable bridge between the IL-2R beta and IL-2R gamma c chains than does IL-2 on YT cells. This result was identical for both surface-iodinated YT cells and immunoprecipitates that were probed for IL-2R beta and IL-2R gamma c on Western blots.

Anti-CD3 + interleukin-2 stimulation of marrow and blood: comparison of proliferation and cytotoxicity

The proliferation and in vitro cytolytic activity of interleukin-2 (IL- 2)-activated and anti-CD3 + IL-2-stimulated marrow mononuclear cells (MMC) and peripheral blood mononuclear cells (PBMC) were studied. Samples from 8 normal donors, 15 patients with acute lymphoblastic leukemia (ALL), and 7 patients with non-Hodgkin's lymphoma (NHL) in remission were cultured in IL-2 (100 U/mL) or IL-2 (100 U/mL) plus anti- CD3 (10 ng/mL). MMC as well as PBMC samples demonstrated significant synergy between IL-2 and anti-CD3 in the promotion of proliferation as measured by 3H thymidine incorporation on day 5 (P less than .001) or fold increase in cell number on day 14. Cryopreserved marrow specimens had equally rapid proliferation as fresh MMC when cultured in the presence of anti-CD3 + IL-2. Anti-CD3 concentrations of 3, 11, 33, and 100 ng/mL augmented proliferation similarly in the presence of IL-2 (0.1 to 100 U/mL). Mean fold increases in cell number of both marrow- and blood-derived cultures after 14 days were significantly higher for anti-CD3 + IL-2-stimulated cultures compared with cultures stimulated with IL-2 only (50- to 200-fold increase in cell number; P = .01). Comparison of remission MMC and PBMC from ALL and NHL patients with normal controls showed equivalent growth rates of activated cultures at 7, 14, and 21 days. Marrow purging with immunotoxin anti-CD19 pokeweed antiviral protein plus 4HC had no significant effect on proliferation of anti-CD3 + IL-2-stimulated MMC cultures in patients with ALL. Cytolytic activity of IL-2- and IL-2 + anti-CD3-activated PBMC and MMC cultures was assessed in 51Cr release assays using K562 (natural killer ([NK]-sensitive), Daudi (Burkitt's lymphoma-, NK-resistant), and Nalm-6 (ALL-, lymphokine-activated killer [LAK]-resistant) cell lines and cryopreserved ALL blasts. Cytolytic activity on a per-cell basis (percent cytotoxicity at an effector:target ratio of 30:1) was similar in IL-2-activated PBMC- and MMC-derived cultures from ALL patients. MMC activated with anti-CD3 plus IL-2 killed Daudi significantly less well than IL-2-activated cultures on days 12 and 19 (P = .03); no significant differences were observed in lysis of LAK-resistant Nalm-6 or cryopreserved ALL blast targets. Dose response of anti-CD3 augmentation of Daudi and Nalm-6 killing was different in IL-2- and IL- 2 + anti-CD3-stimulated cultures.

Anti-CD3 + Interleukin-2 Stimulation of Marrow and Blood: Comparison of Proliferation and Cytotoxicity

The proliferation and in vitro cytolytic activity of interleukin-2 (IL-2)-activated and anti-CD3 + IL-2-stimulated marrow mononuclear cells (MMC) and peripheral blood mononuclear cells (PBMC) were studied. Samples from 8 normal donors, 15 patients with acute lymphoblastic leukemia (ALL), and 7 patients with non-Hodgkin's lymphoma (NHL) in remission were cultured in IL-2 (100 U/mL) or IL-2 (100 U/mL) plus anti-CD3 (10 ng/mL). MMC as well as PBMC samples demonstrated significant synergy between IL-2 and anti-CD3 in the promotion of proliferation as measured by 3H thymidine incorporation on day 5 (P less than .001) or fold increase in cell number on day 14. Cryopreserved marrow specimens had equally rapid proliferation as fresh MMC when cultured in the presence of anti-CD3 + IL-2. Anti-CD3 concentrations of 3, 11, 33, and 100 ng/mL augmented proliferation similarly in the presence of IL-2 (0.1 to 100 U/mL). Mean fold increases in cell number of both marrow- and blood-derived cultures after 14 days were significantly higher for anti-CD3 + IL-2-stimulated cultures compared with cultures stimulated with IL-2 only (50- to 200-fold increase in cell number; P = .01). Comparison of remission MMC and PBMC from ALL and NHL patients with normal controls showed equivalent growth rates of activated cultures at 7, 14, and 21 days. Marrow purging with immunotoxin anti-CD19 pokeweed antiviral protein plus 4HC had no significant effect on proliferation of anti-CD3 + IL-2-stimulated MMC cultures in patients with ALL. Cytolytic activity of IL-2- and IL-2 + anti-CD3-activated PBMC and MMC cultures was assessed in 51Cr release assays using K562 (natural killer ([NK]-sensitive), Daudi (Burkitt's lymphoma-, NK-resistant), and Nalm-6 (ALL-, lymphokine-activated killer [LAK]-resistant) cell lines and cryopreserved ALL blasts. Cytolytic activity on a per-cell basis (percent cytotoxicity at an effector:target ratio of 30:1) was similar in IL-2-activated PBMC- and MMC-derived cultures from ALL patients. MMC activated with anti-CD3 plus IL-2 killed Daudi significantly less well than IL-2-activated cultures on days 12 and 19 (P = .03); no significant differences were observed in lysis of LAK-resistant Nalm-6 or cryopreserved ALL blast targets. Dose response of anti-CD3 augmentation of Daudi and Nalm-6 killing was different in IL-2- and IL-2 + anti-CD3-stimulated cultures.(ABSTRACT TRUNCATED AT 400 WORDS)