Tim-3 and PD-1 blocking cannot restore the functional properties of natural killer cells in early clinical stages of chronic lymphocytic leukemia: An in vitro study.

Background: Programmed death-1 (PD-1) and T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) are two major immune checkpoint receptors expressed on immune cells and their expression is related to the exhaustion phenotype. In the present in vitro study, blocking of PD-1 and Tim-3 molecules was performed on isolated natural killer (NK) cells from patients with chronic lymphocytic leukemia (CLL) to restore their functional properties. Materials and Methods: NK cells fraction was positively isolated from fresh peripheral blood of 18 CLL patients, treated with anti-PD-1 and anti-Tim-3 blocking monoclonal antibodies and co-cultured with K562 target cells to evaluate their apoptosis induction by Annexin V-PI method. Blocked NK cells were also incubated with anti-CD107a antibody to assess their degranulation properties by flow cytometry. The level of secreted tumor node factor-alpha (TNF-α) and interferon-gamma (IFN-γ) by NK cells was also measured by ELISA. Results: Our results showed similar functional properties in terms of degranulation and apoptosis of K562 target cells by isolated NK cells from CLL patients in PD-1/Tim-3 blocked and control groups. It was also shown that blocking of PD-1 and Tim-3 could not improve the production of pro-inflammatory TNF-α and IFN-γ cytokines by isolated NK cells from CLL patients. Conclusion: Altogether, our results indicated that pretreatment of NK cells with anti-PD-1 and anti-Tim-3 blocking antibodies in CLL patients at early clinical stages cannot improve their functional properties. Besides many other malignancies, the application of checkpoint inhibitors in CLL needs more investigations and complementary studies.

Combined Blockade Of PD-1 and TIGIT is not Sufficient to Improve the Function Of CD8+ T-Cells in Chronic Lymphocytic Leukemia.

BACKGROUND AND OBJECTIVE: Blockade of immune checkpoint receptors in the treatment of cancers has been mentioned in several studies. Here, we investigated the efficacy of combined blockade of two inhibitory receptors, PD-1 and TIGIT, in restoring functional features of CD8+ T-cells in CLL. METHODS: CD8+ T-cells were separated from the peripheral blood of 11 CLL patients and targeted with malignant B-cells isolated from the same patients. Cells were then stimulated with anti-CD3/CD28 and PMA/ionomycin to assess their proliferative response and cytotoxic activity using MTT and CD107a degranulation assays, respectively. Cytokine production of isolated CD8+ T-cells was also determined using ELISA. RESULTS: There were no significant differences in proliferation and cytotoxic activity of CD8+ T-cells co-blocked with anti-PD-1/TIGIT compared to those single blocked with anti-PD-1, anti-TIGIT, or the control antibody. There was no significant difference in cytokine production of mentioned groups, either. CONCLUSIONS: Collectively, combined blockade of PD-1 and TIGIT failed to restore the proliferation and function of CD8+ T-cells isolated from CLL patients.

miR-34a mimic or pre-mir-34a, which is the better option for cancer therapy? KatoIII as a model to study miRNA action in human gastric cancer cells.

BACKGROUND: Aberrantly expressed microRNAs play important roles in gastric tumorigenesis. However, use of miRNAs as a therapeutic option in gastric cancer still remains as a challenging problem. METHODS: We performed transient transfection of miR-34a-5p mimic and stable transfection of pre-mir-34a into KatoIII cells. Then, we evaluated the effect of transfected miRNAs on numerous cellular and molecular processes. RESULTS: Following transient transfection of miR-34a-5p mimic at 25 nM-a commonly used concentration-into KatoIII cells, inhibition of two target genes expression, namely Notch1 and ß-catenin, was not observed, but a non-significant marginal increase of these genes was detected. No changes were detected in the percentage of apoptotic cells as well as in CD44 + and EpCAM + cells after 25 nM miR-34a-5p mimic transfection. Interestingly, stable transfection of pre-mir-34a into KatoIII cells (named as KatoIII-pGFPC1-34a cells) caused a significant repression in ß-catenin protein and Notch1 mRNA levels (p < 0.05 and p < 0.01, respectively) relative to equivalent control (KatoIII- pGFPC1-empty cells). The percentage of CD44 + cells in the KatoIII-pGFPC1-34a cells (< 40%) was significantly lower than that in control cells (~ 95%) (p < 0.05). An increase of ~ 3.5% in apoptotic cells and a slower proliferation rate were detected in KatoIII-pGFPC1-34a cells. CONCLUSIONS: Our study revealed that the effect of miR mimic in target gene repression can be dependent to its concentration as well as to the cell type. Meanwhile, our findings further support a regulatory function for pre-miRNAs in target repression and will help to develop effective therapeutic strategies in cancer treatment.

Blockade of PD-1 and TIM-3 immune checkpoints fails to restore the function of exhausted CD8+ T cells in early clinical stages of chronic lymphocytic leukemia.

Blocking antibodies targeting immune checkpoint molecules achieved invaluable success in tumor therapy and amazing clinical responses in a variety of cancers. Although common treatment protocols have improved overall survival in patients with chronic lymphocytic leukemia (CLL), they continue to relapse and progress. In the present in vitro study, the application of anti-PD-1 and anti-TIM-3 blocking antibodies was studied to restore the function of exhausted CD8+ T cells in CLL. CD8+ T cells were isolated from peripheral blood of 20 patients with CLL, treated with blocking antibodies, and cocultured with mitomycin-frozen non-CD8+ T cell fraction as target cells. Cultures were stimulated with anti-CD3/CD28 antibodies to assess the proliferation of CD8+ T cells by MTT and stimulated with PMA/ionomycin to measure the levels of CD107a expression and cytokine production by flow cytometry and ELISA, respectively. Our results showed that the blockade of PD-1 and TIM-3 does not improve the proliferation of CD8+ T cells in CLL patients. No significant difference was found between control and blocked groups in terms of degranulation properties and production of IFN-γ, TNF-α, IL-2, and IL-10 by CD8+ T cells. We observed that pre-treatment of CD8+ T cells with blocking antibodies in CLL patients at early clinical stages had no effects on restoring their functional properties. Further in vitro and in vivo complementary studies are required to more explore the utility of checkpoint inhibitors for CLL patients.

A comparative study on immunophenotypic characterization and osteogenic differentiation of human mesenchymal stromal cells derived from periodontal ligament and gingiva.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from periodontal ligament (PDL) and gingiva can be used for the development of cell-based regenerative approaches in dentistry and medicine. The purpose of this investigation was to establish a method for isolation of human stem cells from the PDL and gingiva, multilineage differentiation of those cells, and comparison of periodontal ligament mesenchymal stem cells (PDLMSCs) and gingival mesenchymal stem cells (GMSCs). METHODS: PDL and gingival tissues of third molar teeth were digested enzymatically and the proliferative potential of human PDLMSCs and GMSCs was compared by MTT assay. The expression of cell surface epitopes was analyzed by flow cytometry. To investigate the multilineage differentiation capacity of these stem cells, osteogenic and adipogenic differentiation was achieved. The specific staining of nodules was performed to evaluate differentiation, whereas the expression of alkalin phosphatase (ALP) and collagen A I (COL I) genes was analyzed by quantitative real-time polymerase chain reaction. RESULTS: The outgrown cells derived from PDL and gingival tissues were similar, fibroblast-like, and spindle-shaped. Further, the proliferation potential of GMSCs was greater than PDLMSCs. Both types of stem cells expressed MSC precursor markers, including CD73, CD90, and CD105, whereas they were negative for hematopoietic markers, including CD34 and CD45. PDLMSCs demonstrated more osteogenic potential compared to GMSCs with strong mineral noduls, and significantly greater expression of up-regulated bone-related markers ALP and COL I. CONCLUSION: MSCs derived from PDL and gingiva demonstrated multipotent characteristics, suggesting new therapeutic approaches in tissue engineering and PDLMSCs are more appropriate candidates for this purpose.

Cellular apoptosis: An alternative mechanism of action for caspofungin against Candida glabrata.

BACKGROUND AND PURPOSE: Although the mechanism of action for echinocandins is known, the physiological mechanisms by which these antifungal agents cause cell death via the classical apoptotic pathways are not well-defined yet. Regarding this, the present study aimed to evaluate the mechanisms of caspofungin-induced Candida glabrata cell death. MATERIALS AND METHODS: For the purpose of the study, the minimum inhibitory concentration (MIC) of caspofungin against C. glabrata (ATCC 90030) was determined using the broth microdilution reference method (CLSI M27-A2 and M27-S4). The annexin V and propidium iodide staining was performed to determine the way through which caspofungin acts against C. glabrata (i.e., through the induction of apoptosis and/or necrosis). Additionally, the possible effect of caspofungin on inducing the expression of two apoptotic genes, namely MCA1 and NUC, was studied using the real-time polymerase chain reaction assay. RESULTS: According to the obtained MIC value (0.5 µg/mL), C. glabrata, exposed to 0.25, 0.5, and 1 µg/mL of caspofungin, exhibited the features of late apoptosis/necrosis after 18 h of incubation. Furthermore, the use of 0.25, 0.5, and 1 µg/ml caspofungin induced apoptosis (early/late) in 14.67%, 17.04%, and 15.89% of the cells, respectively. The results showed a significant difference between the percentages of early-apoptotic cells at the three concentrations (P<0.05). In addition, the rate of necrosis was significantly greater than that of apoptosis in response to caspofungin. Accordingly, necrosis occurred in 71.26%, 71.26%, and 61.26% of the cells at the caspofungin concentrations of 0.25, 0.5, and 1 µg/mL, respectively (P<0.05). The analysis of the data in the REST software demonstrated a significant increase in the expression of MCA1 and NUC1 genes (P<0.05). CONCLUSION: As the findings of the present study indicated, caspofungin promoted both necrosis and apoptosis of C. glabrata cells at concentrations higher than or equal to the MIC value.

Cell Density Counts of the Intestinal Intraepithelial Lymphocytes in the Celiac Patients.

BACKGROUND: Increased number of intestinal intraepithelial lymphocytes (IELs) is a key histological finding in the diagnosis of celiac disease (CD); however, the number of IELs in celiac patients and healthy subjects may vary from one region to another. Additionally, there are some seronegative celiac patients with a borderline histology. OBJECTIVE: To determine the number of the CD3+ and CD8+ IELs T-cells in the celiac patients and healthy subjects (controls) in Isfahan. METHODS: The duodenal biopsies were obtained from the celiac patients (n=15) and the controls (n=19). The total number of IELs/100 epithelial cells (ECs) were counted using the hematoxylin-eosin (H&E) staining method, and that of CD3+ and CD8+ IELs/100 ECs were counted using the immunohistochemistry (IHC) staining method. RESULTS: This study defined the upper normal limit for each variable as mean + 2SD. Accordingly, the upper normal limits of the total IELs, CD3+ IELs, and CD8+ IELs/100 ECs were calculated as 37 (95% confidence intervals, CI: 33-41), 22 (95% CI: 19-25) and 12 (95% CI: 10-14), respectively. In 3 clinically CD diagnoses, the total IELs counts/100 ECs were below the upper normal limit, and the histopathological and serologic assays were negative. Nevertheless, the CD8+ IELs T-cells counts/100 ECs showed borderline values. Interestingly, these patients responded to a gluten-free diet (GFD). CONCLUSIONS: The study findings suggest that in the clinically diagnosed celiac disease, IELs count/100 ECs below the upper normal limit as well as negative histopathological and serologic assays and the cell density counts of the CD8+ IELs T-cells/100 ECs could be a useful parameter in CD diagnosis and make a decision to put them on a GFD.

Expression analysis of PD-1 and Tim-3 immune checkpoint receptors in patients with vitiligo; positive association with disease activity.

The contribution of immune checkpoint receptors in the immunopathogenesis of various autoimmune diseases has been addressed in previous reports. In this study,  the expression profile of T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) and programmed cell death-1 (PD-1) checkpoint molecules was investigated in CD8+ T cells of Vitiligo patients. The association of Tim-3 and PD-1 expression with disease activity was also explored. The frequency of Tim-3+ /PD-1+ /CD8+ T cells in 30 patients with vitiligo and 30 sex- and age-matched controls was determined by flow cytometry. CD8+ T cells were then positively isolated by magnetic beads, and the mRNA expression of PD-1 and Tim-3 was determined by TaqMan-based real-time PCR. To measure the cytokines production, PBMCs were stimulated with PMA/ionomycin and concentrations of IL-4, IFN-γ and TNF-α were measured in culture supernatants by ELISA. Disease activity of patients with vitiligo was determined using the Vitiligo Area Severity Index. Patients with vitiligo have significantly shown more expression of Tim-3 and PD-1 on their CD8+ T cells compared with controls. Expression analysis of Tim-3 mRNA, but not PD-1, confirmed the results obtained from flow cytometry. While the production levels of TNF-α and IFN-γ were found higher by patients with vitiligo, IL-4 production was lower in patients compared with controls. A direct association was observed between the Tim-3 and PD-1 expression and also the production of pro-inflammatory cytokines with disease activity of patients with vitiligo. Our results indicate that Tim-3 and PD-1 are involved in immune dysregulation mechanisms of CD8+ T cells in vitiligo and may introduce as potential biomarkers for disease progression and targeted immunotherapy.

SDF-1α Reduces Human Natural Killer Cell Cytotoxicity against Chronic Myelogenous Leukemia K562 Cells.

Stromal cell-derived factor-1 alpha (SDF-1α) has been shown to be up-regulated in a variety of malignancies. So that, its expression is associated with poor prognosis and invasiveness. Natural killer (NK) cells are important effector cells against virus-infected and transformed cells. Especially they play a key role in tumor immune surveillance. Whereas it was not well understood whether SDF-1α modulates anti-tumor immune response or not, the purpose of the present study was to investigate the effect of SDF-1α on the cytotoxic properties of peripheral blood NK cells. Human peripheral blood NK cells were freshly isolated using MACSxpess system and cultured in the presence or absence of recombinant human SDF-1α or SDF-1α plus CXCR4 antagonist, AMD3100. CD107a degranulation assay was conducted through the co-culture of NK cells with K562 cells. The percentage of CD107a positive cells was assessed by flowcytometry. Effect of SDF-1α was also examined on the mRNA levels of NKG2A and NKG2D as indicator examples of NK cell inhibitory and activating receptors, respectively. SDF-1α significantly decreased the degranulation activity of NK cells (p=0.04). The mRNA content of NKG2D was down-regulated under the influence of SDF-1α (p=0.03). Moreover, AMD3100 exhibited a trend in recovering the NKG2D mRNA level to its un-treated state (p=0.05).  The present study reveals that SDF-1α has a negative impact on NK cell activity and might is involved in tumor immune-suppression. Thus, it can be concluded that microenvironment manipulations targeting SDF-1α may reinforce current cancer therapies by disturbing one of the immune-suppressive axes in the cancerous milieu.

Analysis of PD-1 and Tim-3 expression on CD4+ T cells of patients with rheumatoid arthritis; negative association with DAS28.

Expression of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) and programmed cell death-1 (PD-1) was studied on CD4+ T cells of patients with rheumatoid arthritis (RA). Association of Tim-3 and PD-1 expression with disease activity of RA patients was also addressed. A total of 37 RA patients and 31 sex- and age-matched healthy controls were included in this study. Disease activity of RA patients was determined by Disease Activity Score of 28 joints scoring system (DAS28). A three-color flow cytometry method was applied to determine the frequency of Tim-3+/PD-1+/CD4+ T cells. To measure the cytokine production, peripheral blood mononuclear cells (PBMCs) were stimulated with PMA/ionomycin. Concentrations of IL-17, IL-10, IFN-γ, and TNF-α were measured in culture supernatants by ELISA. The frequency of PD-1+/CD4+ and Tim-3+/PD-1+/CD4+ T cells was significantly higher in patients with RA compared to that in controls (p = 0.0013 and p = 0.050, respectively). The percentage of Tim-3+/CD4+ T cells was similar in patients and controls (p = 0.4498). The RA patients have produced significant higher levels of TNF-α, IL-17, and IFN-γ than those of healthy controls (p = 0.0121, p = 0.0417, and p = 0.0478, respectively). Interestingly, an inverse correlation was found between the frequency of Tim-3+/CD4+ cells and DAS28 of RA patients (r = - 0.4696, p = 0.0493). Similarly, the percentage of Tim-3+/PD-1+/CD4+ T cells was also revealed an inverse correlation with DAS28 (r = - 0.5268, p = 0.0493). Moreover, significant positive correlations were detected between the concentrations of TNF-α (r = 0.6418, p = 0.0023) and IL-17 (r = 0.4683, p = 0.0373) with disease activity of RA patients. Our results indicate that Tim-3 and PD-1 are involved in immune dysregulation mechanisms of rheumatoid arthritis and could be considered as useful biomarkers for determination of disease activity and progression.

Elevated Expression of Tim-3 and PD-1 Immune Checkpoint Receptors on T-CD4+ Lymphocytes of Patients with Asthma.

Asthma is a chronic disorder of the airways characterized by reversible airflow obstruction, inflammation and bronchial hyperresponsiveness. Different immune cells and molecules have been attributed to involve in pathogenesis of asthma. In the current case-control study, the expression of T cell Ig and mucin domain-containing molecule-3 (Tim-3) and programmed death-1 (PD-1) was studied on CD4+ T cells of patients with asthma and normal controls. The frequency of Tim-3+/PD-1+/CD4+ T cells was determined by a three color flow cytometry method in 37 patients with asthma and 32 healthy controls. To evaluate the Th1/Th2 ratio, peripheral blood mononuclear cells were isolated from all samples and stimulated with phorbol 12- myristate 13- acetate ( PMA)/ionomycin for 18 h. IFN-γ) and Interleukin-4 (IL-4) were measured in culture supernatants by-(ELISA). Serum total immunoglobulin E (IgE) was also measured in all samples. Significant increase in percentage and absolute count of Tim-3+/PD-1+/CD4+, Tim-3+/CD4+ and PD-1+/CD4+ T cells was found in asthmatic patients compared to healthy controls (p=0.02 and p=0.003, respectively). The IFN-γ/IL-4 ratio (Th1/Th2 ratio) was significantly higher in healthy controls than that of asthmatic patients (p=0.029). Our data regarding the increased expression of PD-1 and Tim-3 on CD4+ T cells of patients with asthma suggest the potential roles of these immune checkpoint receptors in immune dys-regulation of asthma.

Tim-3 Up-regulation in Patients with Gastric Cancer and Peptic Ulcer Disease

Background: T-cell immunoglobulin and mucin domain protein-3 (Tim-3), an inhibitory immunoregulatory receptor, has been recently implicated in tumor biology and tumor-associated immune suppression. In the present study, expression of Tim-3 was evaluated in gastric cancer (GC) and peptic ulcer disease (PUD) at both mRNA and protein levels. Methods: A total of 133 gastric tissue biopsies, comprising 43 from GC cases, 48 from PUD and 42 from non-ulcer dyspepsia (NUD) serving as controls were collected. Additionally, non-neoplastic adjacent tissue biopsies were also obtained from 6 patients with GC. Infection with Helicobacter pylori was determined by the rapid urease test for all participants and H&E staining was conducted for GC and PUD patients. Tim-3 relative mRNA expression was determined by SYBR Green based Real-Time PCR using ß-actin as a reference gene. Tim-3 protein expression was also studied by immunohistochemistry in 7 GC, 7 PUD and 10 NUD tissue samples. Results: Tim-3 was expressed at higher levels in GC (p=0.030) and PUD (p=0.022) cases compared to he NUD group. Among paired samples obtained from gastric cancer patients, tumor tissues showed elevated Tim-3 expression (p=0.019) in comparison with adjacent non-neoplastic biopsies. Tim-3 mRNA findings were supported by detection of more Tim-3 protein in cancerous (p=0.002) and ulcerative (p=0.01) tissues than in controls. Tim-3 was similarly expressed in H. pylori positive and negative cases.Conclusion: Higher Tim-3 expression in patients with gastric cancer and peptic ulcer implies that it might be involved in immune regulation and establishment of these gastrointestinal diseases. Targeted immunotherapy by blocking of inhibitory receptors like Tim-3 could be a promising approach for gastric cancer treatment.

Frequency and functional characterization of exhausted CD8+ T cells in chronic lymphocytic leukemia.

OBJECTIVES: The phenotypic and functional properties of Tim-3+ /PD-1+ /CD8+ cells as exhausted T cells were investigated in chronic lymphocytic leukemia (CLL). METHODS: Frequency of CD8+ /Tim-3+ /PD-1+ exhausted cells was determined by flow cytometry. For functional analysis, magnetic beads-isolated CD8+ T cells were stimulated with PHA and PMA/ionocymin to assess their proliferative responses and cytokine production by MTT and ELISA, respectively. Cytotoxic activity of isolated CD8+ T cells was determined using CD107a degranulation assay. RESULTS: The proportion of exhausted CD8+ T cells was significantly higher in CLL compared to controls. Isolated CD8+ T cells from CLL showed functional defects in proliferation, degranulation, and cytokines production. While IL-2, TNF-α, and IFN-γ were significantly lower in CLL patients, IL-10 was higher in the patients group. Patients with progressive clinical stages showed higher frequency and dysfunction of exhausted CD8+ T cells. CONCLUSION: Targeting immune inhibitory receptors to restore the function of tumor surrounding T cells could be helpful for immunotherapy of CLL.

Uterine Natural Killer Cell and Human Leukocyte Antigen-G1 and Human Leukocyte Antigen-G5 Expression in Vaginal Discharge of Threatened-Abortion Women: A Case-Control Study.

The immunotolerant human leukocyte antigen-G (HLA-G) molecules have a major role in fetal-maternal tolerance during pregnancy. Interaction between these molecules and uterine natural killer (uNK) cells inhibitory receptors prevents NK cell invasion against fetus trophoblast cells. The aim of this study was to evaluate the percentages of uNK cells and HLA-G1 and HLA-G5 isoforms expression in vaginal discharge of threatened-abortion women in comparison with control. In a case-control study, we investigated 30 threatened-abortion women with bleeding or spotting less than 20 weeks of pregnancy as compared to 30 normal pregnant women. uNK cells percentage was assessed by flow cytometry. Furthermore, we evaluated HLA-G1 and HLA-G5 isoforms expression by Real-Time PCR in these groups. The results of this study showed that threatened-abortion women had increased uNK cells and decreased T cells percentage in vaginal discharge in comparison with normal pregnant women (p = 0.01, p = 0.003, resp.). In addition, HLA-G1 isoform had lower expression in threatened-abortion women in comparison with control group (p = 0.0001). The increase of uNK cells level with the decrease of HLA-G expression in vaginal discharge of threatened-abortion pregnant women is an indicator of mother's immune dysregulation. It is concluded that HLA-G expression level with uNK cells percentage can be determined as a diagnostic marker for threatened-abortion women.