Thalidomide (TM) has been reported to have anti-cancer and anti-inflammatory properties, and dexamethasone (DX) is known to reduce inflammation and inhibit production of inflammatory cytokines. Many studies have reported that combinatorial therapy with TM and DX is clinically used to treat multiple myeloma and lupus nephritis, but the mechanism responsible for its effects has not been elucidated. In this study, we determined that TM and DX co-treatment had an enhanced immune-modulatory effect on T cells through regulating the expression of co-stimulatory molecules. Splenic naive T cells from C57BL/6 mice were sort-purified and cultured for CD4 T cell proliferation and regulatory T (Treg) cell conversion in the presence of TM and/or DX. Following incubation with the drugs, cells were collected and OX40, 4-1BB, and glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR) expression was quantified by flow cytometry. TM (1 or 10 μm) decreased CD4 T cell proliferation in a dose-dependent manner, whereas TM/DX (0·1 or 1 nm) co-treatment further decreased proliferation. Treg cell populations were preserved following drug treatment. Furthermore, expression of co-stimulatory molecules decreased upon TM/DX co-treatment in effector T (Teff) cells and was preserved in Treg cells. Splenic CD4 T cells isolated from TM- and DX-treated mice exhibited the same patterns of Teff and Treg cell populations as observed in vitro. Considering the selective effect of TM on different T cell subsets, we suggest that TM may play an immunomodulatory role and that TM/DX combinatorial treatment could further enhance these immunomodulatory effects by regulating GITR, OX40, and 4-1BB expression in CD4 T cells.
During virus infection, T cells must be adapted to activation and lineage differentiation states via metabolic reprogramming. Whereas effector CD8 + T cells preferentially use glycolysis for their rapid proliferation, memory CD8 + T cells utilize oxidative phosphorylation for their homeostatic maintenance. Particularly, enhanced AMP-activated protein kinase (AMPK) activity promotes the memory T cell response through different pathways. However, the level of AMPK activation required for optimal memory T cell differentiation remains unclear. A new metformin derivative, IM156, formerly known as HL156A, has been reported to ameliorate various types of fibrosis and inhibit in vitro and in vivo tumors by inducing AMPK activation more potently than metformin. Here, we evaluated the in vivo effects of IM156 on antigen-specific CD8 + T cells during their effector and memory differentiation after acute lymphocytic choriomeningitis virus infection. Unexpectedly, our results showed that in vivo treatment of IM156 exacerbated the memory differentiation of virus-specific CD8 + T cells, resulting in an increase in short-lived effector cells but decrease in memory precursor effector cells. Thus, IM156 treatment impaired the function of virus-specific memory CD8 + T cells, indicating that excessive AMPK activation weakens memory T cell differentiation, thereby suppressing recall immune responses. This study suggests that metabolic reprogramming of antigen-specific CD8 + T cells by regulating the AMPK pathway should be carefully performed and managed to improve the efficacy of T cell vaccine.
Purpose In organ transplantation, the need for immune modulation rather than immune suppression has been emphasized. In this study, we investigated whether combinatorial treatments of with thalidomide (TM) and dexamethasone (DX) might be new approaches to induce systemic immunomodulation on T cells and other immune cells that regulate the expression of co-inhibitory molecules. Materials and Methods Naïve splenic T cells from C57BL/6 mice were sort-purified and cultured in vitro for CD4 + T cell proliferation and regulatory T cell (Treg) conversion in the presence of TM or/and DX. Expression of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1) in proliferated and converted T cells was quantified by flow cytometry. We also quantified in vivo expression of CTLA-4 and PD-1 on splenic CD4 + T cells and other immune cells isolated from TM- or/and DX-treated mice. Mixed lymphocytes reactions (MLR) were performed to evaluate the capacity of immune cells in carrying out immune responses. Results CTLA-4 expressions in effector T cells in vivo and in Tregs in vivo/vitro significantly increased upon TM/DX combinatorial treatment. Corresponding to increased CTLA-4 expression in T cells, the expression of ligand molecules for CTLA-4 significantly increased in splenic dendritic cells in TM/DX-treated groups. In addition, MLR results demonstrated that splenocytes isolated from TM/DX-treated mice significantly suppressed the proliferation of T cells isolated from other strains. Conclusion Based on these results, we suggest that TM/DX combinatorial treatments might be efficient immunomodulatory methods for regulating T cell immunity.
Intraperitoneal (IP) anesthesia is commonly used for laboratory animal experiments including rat islet isolation. However, the direct effects of anesthetics on pancreatic islets have been neglected. This study compared the islet function and recovery yield from rats that were anesthetized using IP and intramuscular (IM) injection. In addition, the lag time required to lose deep pain was measured in the following anesthetics combinations. Lewis rats were anesthetized using ketamine and xylazine (K/X) or zoletil and xylazine (Z/X). A glucose challenge test was performed on each group of prepared islets. The effect of the anesthetic agents (e.g., ketamine, zoletil, xylazine alone, and the combination of K/X and Z/X) on cell lines (rat insulinoma; RIN-5F) was investigated by determining their effect on the cell viability, the amount of insulin, and insulin mRNA expression levels of RIN-5F. The time needed for deep anesthesia in IM anesthesia was significantly shortened in comparison to IP [K/X (IM: 313 ± 66 s, IP: 371 ± 84 s) and Z/X (IM: 206 ± 76 s, IP: 245 ± 92 s)]. In addition, number of isolated islet yield by IM anesthesia was significantly improved [K/X (IM: 1530 ± 242, IP: 1245 ± 149) and Z/X (IM: 1136 ± 226, IP: 511 ± 154)]. The functions of fresh islets, indicated by the stimulation index, acquired under IM anesthesia was better preserved than that of IP. The viability and the insulin secretion of RIN-5F were decreased at 24 and 48 h. Insulin gene expression levels were decreased at 24 h as well. Anesthetics may be absorbed through the pancreas surface to the islets and have a direct effect, resulting in islet exposure and deterioration during isolation. In conclusion, for rodent islet isolation, IM anesthesia is simpler and safer in comparison to IP anesthesia.
Purpose The immunomodulatory effects of thalidomide (TM) and dexamethasone (DX) on immune cells and their co-stimulatory, co-inhibitory molecules in vitro and in vivo have been previously reported. The current study investigated the effects of TM and the combinatorial treatment with DX on immune cells using a murine cardiac allograft transplantation model. Materials and Methods Intraabdominal transplant of cardiac allografts from BALB/c (H-2 d ) donors to C57BL/6 (H-2 b ) recipients was performed. After transplantation, mice were injected daily with TM or DX or a combination of both TM and DX (TM/DX) by intraperitoneal route until the time of graft loss. CD4 + T cell subsets and CD11c + cells in the peripheral blood mononuclear cells and spleen were examined and quantified with flow cytometry. Serum IL-6 levels were measured by enzyme-linked immunosorbent assay on day 7. Results The mean graft survivals were 6.86 days in the untreated group, and 10.0 days in the TM/DX group ( p <0.001). The TM/DX treatment affected the CD4 + T cell subsets without suppressing the total CD4 + T cell population. The CD4 + FOXP3 + /CD4 + CD44 hi T cell ratio increased. Increase in cell counts and median fluorescence intensity on CD11c + CD85k + with TM/DX were observed. The inhibition of pro-inflammatory cytokine interleukin-6 was also observed. Conclusion These outcomes suggest the immunomodulating effect of the TM/DX combinatorial treatment. In conclusion, TM/DX combination may be a promising immunomodulatory approach for preventing allograft rejection and improving graft survival by inducing tolerance in transplantation.
Background: Predicting tumor response for neoadjuvant chemotherapy (NAC) is critical for evaluating prognosis and deciding the treatment strategy in patients with breast cancer; however, there are no reliable circulating biomarkers that can assess therapeutic responses appropriately and effectively. Therefore, we aimed to validate the clinical feasibility of extracellular vesicles (EV) collected through liquid biopsy as biomarkers for predicting tumor response after NAC. Methods: Drug-resistant sublines were generated from a panel of triple-negative breast cancer (TNBC) cell lines, including HCC1395, MDA-MB-231, and MDA-MB-468. Comparison with parental cells suggested candidate biomarkers to detect resistance to chemotherapeutic drugs. EVs were isolated from cultured cells and plasma samples collected before NAC from breast cancer patients (N=36). The EVs were then coupled to immuno-beads to allow the quantification of drug resistance-related EV markers by flow cytometry. Results: Drug-resistant TNBC cell lines were stably established showing specific morphology and rapidly growing features. Among the differentially expressed gene profiles between parental and drug-resistant cell lines, drug efflux transporters, such as multidrug resistance protein 1 (MDR1), MDR-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP) were highly expressed in resistant cell lines. Drug efflux transporters were identified in not only cell lines but also EVs released from parental cells with immuno-affinity-based EV isolation. Among 20 TNBC patients, the expression of drug-resistant markers in EVs from the patients with the residual disease was relatively high compared to that of patients with pathological complete response (pCR). The optimal combination of drug-resistant EV markers was significantly efficient in predicting patients showing resistance to NAC and represented the best performance with 93% sensitivity, 80% specificity, and 0.93 area under the receiver operating characteristic curve. Conclusions: The combination of drug-resistant EV markers is effective in predicting the therapeutic response of breast cancer patients who are treated with NAC.
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