Alloantigen-activated (AAA) CD4+ T cells reinvigorate host endogenous T cell immunity to eliminate pre-established tumors in mice

Background Cancer vaccines that induce endogenous antitumor immunity represent an ideal strategy to overcome intractable cancers. However, doing this against a pre-established cancer using autologous immune cells has proven to be challenging. “Allogeneic effects” refers to the induction of an endogenous immune response upon adoptive transfer of allogeneic lymphocytes without utilizing hematopoietic stem cell transplantation. While allogeneic lymphocytes have a potent ability to activate host immunity as a cell adjuvant, novel strategies that can activate endogenous antitumor activity in cancer patients remain an unmet need. In this study, we established a new method to destroy pre-developed tumors and confer potent antitumor immunity in mice using alloantigen-activated CD4+ (named AAA-CD4+) T cells. Methods AAA-CD4+ T cells were generated from CD4+ T cells isolated from BALB/c mice in cultures with dendritic cells (DCs) induced from C57BL/6 (B6) mice. In this culture, allogeneic CD4+ T cells that recognize and react to B6 mouse-derived alloantigens are preferentially activated. These AAA-CD4+ T cells were directly injected into the pre-established melanoma in B6 mice to assess their ability to elicit antitumor immunity in vivo. Results Upon intratumoral injection, these AAA-CD4+ T cells underwent a dramatic expansion in the tumor and secreted high levels of IFN-γ and IL-2. This was accompanied by markedly increased infiltration of host-derived CD8+ T cells, CD4+ T cells, natural killer (NK) cells, DCs, and type-1 like macrophages. Selective depletion of host CD8+ T cells, rather than NK cells, abrogated this therapeutic effect. Thus, intratumoral administration of AAA-CD4+ T cells results in a robust endogenous CD8+ T cell response that destroys pre-established melanoma. This locally induced antitumor immunity elicited systemic protection to eliminate tumors at distal sites, persisted over 6 months in vivo, and protected the animals from tumor re-challenge. Notably, the injected AAA-CD4+ T cells disappeared within 7 days and caused no adverse reactions. Conclusions Our findings indicate that AAA-CD4+ T cells reinvigorate endogenous cytotoxic T cells to eradicate pre-established melanoma and induce long-term protective antitumor immunity. This approach can be immediately applied to patients with advanced melanoma and may have broad implications in the treatment of other types of solid tumors.

Immune Privileged Features of Multipotent Mesenchymal Stromal Cells Are Lost after Co-Cultivation with Allogeneic Lymphocytes in Vitro

Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed. The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics. Materials and methods MSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2. After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA). Data are presented as mean ± standard error. Statistical analysis was performed using Student's t-test (considered reliable p <0.05). Results The number of cells in cultures of MSCs and IFN-g-MSCs did not differ significantly during the 4 days of observations. The presence of non-activated lymphocytes had no effect on the parameters of growth and viability of MSCs. Co-cultivation of activated lymphocytes with MSCs resulted in a reduction of viable MSCs, to 51.6 ± 5.5% compared with IFN-g-MSCs (68,1 ± 6,2%, p = 0.02). The viability of MSCs cultures without lymphocytes was 74.5% ± 4.8 on day 4 from the beginning of the experiment. HLA-DR expression level gradually increased in IFN-g-MSCs (see table). Co-culturing MSCs with lymphocytes also leads to a gradual increase in HLA-DR expression on MSCs apparently due to IFN-g, produced by lymphocytes. However, in IFN-g-MSCs co-cultured with lymphocytes HLA-DR MFI was significantly lower (p = 0.05) than without lymphocytes. HLA-DR MFI increased about 10 times in MSCs co-cultured with activated lymphocytes regardless of their IFN-g pretreatment The data obtained indicate that IFN-g-MSCs did not change in their growth characteristics and had increased immunomodulating properties. IFN-g-MSCs have a greater resistance to activated lymphocytes, which makes them more effective for cell therapy. The results suggest that injected to patient MSCs could have increased level of HLA-DR expression regardless of their initial treatment with IFN-g. The increase of HLA-DR expression on MSCs co-cultured with lymphocytes indicates a possibility of loss of immune privilege by these cells when injected to patient. Disclosures No relevant conflicts of interest to declare.

Human Articular Chondrocytes Induce Interleukin-2 Nonresponsiveness to Allogeneic Lymphocytes

Introduction We previously showed that articular chondrocytes (ACs) have immune privilege and immunomodulatory functions like those of mesenchymal stem cells. To elucidate these mechanisms, we focused on interleukin-2 (IL-2), which plays critical roles in lymphocyte mitogenic activity. The purpose of this study was to explore whether ACs affect the role of IL-2 underlying immunomodulatory functions. Material and Methods Irradiated human ACs from osteoarthritis donors were used. Third-party ACs were added to the mixed lymphocyte reaction (MLR) with or without recombinant human IL-2 (rhIL-2), and the levels of IL-2 and the soluble form of the IL-2 receptor α (sIL-2Rα) protein in supernatant were measured by enzyme-linked immunosorbent assay. Recombinant human IL-2 (rhIL-2) was also added to the MLR. To detect the expression of IL-2 receptor α (CD25) on lymphocytes in the MLR, flow cytometric analysis was performed. Last, ACs and allogeneic activated CD4+ T cell were co-cultured, and the expression of CD25 on activated T cells was examined by flow cytometry. Results Third-party ACs significantly inhibited the MLR and reduced the level of sIL-2Rα in a dose-dependent manner, but did not affect the concentration of IL-2. Exogenous rhIL-2 accelerated MLR but did not rescue the inhibitory effect of ACs. ACs inhibited the expression of CD25 on activated CD4+ T cells. Discussion Our results showed that third-party ACs inhibited the proliferation of allogeneic activated lymphocytes, thereby inhibiting production sIL-2Rα, although ACs did not affect IL-2 secretion from lymphocytes. Also, ACs inhibited CD25 expression on activated CD4+ T cells. Thus, ACs inhibited the immune response of allogeneic lymphocytes by inducing IL-2 nonresponsiveness.

Does a Chronic Inflammatory Process Underlie Clonal Progression In Aplastic Anemia?- In Vitro and In Vivo Evidence That Inflammation Produces Aneuploidy for Chromosomes 7 and 8 In Replicating Cells

Abstract 641 Inflammatory conditions such as ulcerative colitis or Barrett's esophagus predispose to chromosomal instability, microsatellite instability, gene hypermethylation, loss of tumor suppressor genes such as p53 leading to malignant transformation. Aplastic anemia (AA) also shows some features of chronic inflammation – immunosuppressive treatment (IST) responders show persisting activation of Th1 lymphocytes (Sloand et al Blood 2002) and it is well recognized that AA patients can develop clonal hematologic disorders. Bone marrow (BM) at presentation may show clonal abnormalities by fluorescence in situ hybridization (FISH) or SNP array analysis. We postulated that chromosomal abnormalities in severe AA may be a consequence of genomic damage caused by effector T-cells as well as telomere shortening resulting from accelerated cell turnover. Selective survival of aneuploid cells such as trisomy 8 and monosomy 7 in an inflammatory environment may occur subsequently due to upregulation of anti-apoptotic genes. We examined the relationship between inflammation and clonal expansion in bone marrow (BM) by culturing normal bone marrow mononuclear cells (BMMNCs) for two days with mis-matched allogeneic lymphocytes, autologous lymphocytes or interferon gamma (IFN γ) prior to short-term methylcellulose culture. After two weeks, colonies were picked, pooled, and stained with annexin; annexin-negative cells were subjected to FISH and spectral karyotyping (SKY). Telomere length was determined by qPCR. To assess the role of free-radicals in the inflammatory setting, we performed separate studies using the free radical scavengers, alpha-tocopherol, and desferrioxamine. We also cultured BMMNCs in low oxygen (5%) to decrease free radical generation. P53 gene was examined by FISH, sequenced, and protein expression measured by immunoblot. We also examined the in vivo effect of inflammation in the AA mouse model (Bloom ML et al Exp Hematol 2004). Luminex for cytokines was performed on the supernatants of all samples. Annexin negative human BMMNCs showed significant aneuploidy after short-term culture with allogeneic lymphocytes or with IFNγ (Fig 1; N=10; p<0.01). SKY showed aneuploidy in chromosomes 7 and 8. Lymphocytes preincubated with 200ng/mL of cyclosporine prior to co-cultivation with BMMNCs did not induce aneuploidy, nor did autologous lymphocytes or allogeneic lymphocytes separated from bone marrow by a semi-permeable membrane. Aneuploidy-induced by allogeneic lymphocytes was ameliorated by alpha-tocopherol and desferrioxamine in a dose-dependent manner (Fig 2) and eliminated by MHC blocking antibody. BMMNCs cultured in 5% O2 had significantly less aneuploidy after culture with allogeneic lymphocytes than BMMNCs cultured in 21% O2 (mean 4% and 15% aneuploidy respectively N=3;). BMMNCs, the samples cultured with CD8 cells but not CD4 cells developed aneuploidy (N=3; mean 10% and 3% respectively) and had a 36% decrease in telomere length compared to control. P53 protein expression was decreased in BMMNCs cultured with mis-matched lymphocytes, but was upregulated ten-fold by alpha-tocopherol and low O2. Aneuploidy was also detected in samples exposed to H2O2 (N=4) suggesting a role for free radicals. No abnormalities in p53 gene sequence could be detected in any of the cultured BMMNC. Haploinsufficiency for p53 was noted in 70% of the aneuploid cells but none of the diploid cells (N=3). CXCL5 and IL10, IFNγ and TFNα concentrations were greatest in the supernants of BMMNCs cultured with lymphocytes. We assessed whether lymphocyte-infusion induced aneuploidy in the murine model of AA. Mice receiving lymphocyte infusions but not controls showed clonal populations of aneuploid cells by SKY. Chromosomes 11 (containing p53 gene), 19 and 7 clones were most frequently affected. These data suggest that inflammatory changes can facilitate clonal evolution in AA by causing DNA damage, telomere erosion and selection of cells resistant to apoptosis. The relationship of inflammatory changes to clonal progression in AA deserves further study. Disclosures: No relevant conflicts of interest to declare.