Single-cell RNA sequencing identifies distinct transcriptomic signatures between PMA/ionomycin- and αCD3/αCD28-activated primary human T cells

Immunologists have activated T cells in vitro using various stimulation methods, including phorbol myristate acetate (PMA)/ionomycin and αCD3/αCD28 agonistic antibodies. PMA stimulates protein kinase C, activating nuclear factor-κB, and ionomycin increases intracellular calcium levels, resulting in activation of nuclear factor of activated T cell. In contrast, αCD3/αCD28 agonistic antibodies activate T cells through ZAP-70, which phosphorylates linker for activation of T cell and SH2-domain-containing leukocyte protein of 76 kD. However, despite the use of these two different in vitro T cell activation methods for decades, the differential effects of chemical-based and antibody-based activation of primary human T cells have not yet been comprehensively described. Using single-cell RNA sequencing (scRNA-seq) technologies to analyze gene expression unbiasedly at the single-cell level, we compared the transcriptomic profiles of the non-physiological and physiological activation methods on human peripheral blood mononuclear cell–derived T cells from four independent donors. Remarkable transcriptomic differences in the expression of cytokines and their respective receptors were identified. We also identified activated CD4 T cell subsets (CD55+) enriched specifically by PMA/ionomycin activation. We believe this activated human T cell transcriptome atlas derived from two different activation methods will enhance our understanding, highlight the optimal use of these two in vitro T cell activation assays, and be applied as a reference standard when analyzing activated specific disease-originated T cells through scRNA-seq.

Intratracheal inoculation of human varicella zoster virus (VZV; MAV strain) vaccine successfully induced VZV IgG antibodies in rhesus monkeys / 한국실험동물학회지

Background@#The objective of this study was to investigate whether the use of live attenuated varicella zoster virus (VZV) MAV vaccination can efficiently induce VZV antibody production in naive rhesus monkeys as an approach to prevent simian varicella virus (SVV) reactivation in animals immunosuppressed for transplantation studies. @*Results@#Clinically available human VZV vaccine was used to induce the production of anti-VZV antibodies in rhesus monkeys. A vial of the vaccine was subcutaneously injected at 0 week, and the second and third vaccination was performed at 5 and 6 weeks by intratracheal inoculation. The titer of anti-VZV IgG was assessed at 0, 2, 4, 6, and 7 weeks. At 2 weeks, 3/16 were seropositive for VZV IgG. At 6 weeks, 9/16 were shown to be seropositive. At 7 weeks, 16/16 were found to be seropositive. @*Conclusions@#The VZV vaccine via intratrachael inoculation was shown to induce VZV IgG humoral immunity in rhesus monkeys and may be important immunosuppressed macaques for transplantation studies. Although the humoral immunity produced is an important finding, further studies will be necessary to confirm possible protection and it could protect probably against SVV infection in rhesus monkey.

Suppression of Fibrotic Reactions of Chitosan-Alginate Microcapsules Containing Porcine Islets by Dexamethasone Surface Coating

Background@#The microencapsulation is an ideal solution to overcome immune rejection without immunosuppressive treatment. Poor biocompatibility and small molecular antigens secreted from encapsulated islets induce fibrosis infiltration. Therefore, the aims of this study were to improve the biocompatibility of microcapsules by dexamethasone coating and to verify its effect after xenogeneic transplantation in a streptozotocin-induced diabetes mice. @*Methods@#Dexamethasone 21-phosphate (Dexa) was dissolved in 1% chitosan and was cross-linked with the alginate microcapsule surface. Insulin secretion and viability assays were performed 14 days after microencapsulation. Dexa-containing chitosan-coated alginate (Dexa-chitosan) or alginate microencapsulated porcine islets were transplanted into diabetic mice. The fibrosis infiltration score was calculated from the harvested microcapsules. The harvested microcapsules were stained with trichrome and for insulin and macrophages. @*Results@#No significant differences in glucose-stimulated insulin secretion and islet viability were noted among naked, alginate, and Dexa-chitosan microencapsulated islets. After transplantation of microencapsulated porcine islets, nonfasting blood glucose were normalized in both the Dexa-chitosan and alginate groups until 231 days. The average glucose after transplantation were lower in the Dexa-chitosan group than the alginate group. Pericapsular fibrosis and inflammatory cell infiltration of microcapsules were significantly reduced in Dexa-chitosan compared with alginate microcapsules. Dithizone and insulin were positive in Dexa-chitosan capsules. Although fibrosis and macrophage infiltration was noted on the surface, some alginate microcapsules were stained with insulin. @*Conclusion@#Dexa coating on microcapsules significantly suppressed the fibrotic reaction on the capsule surface after transplantation of xenogenic islets containing microcapsules without any harmful effects on the function and survival of the islets.

Intratracheal inoculation of human varicella zoster virus (VZV; MAV strain) vaccine successfully induced VZV IgG antibodies in rhesus monkeys / 한국실험동물학회지

Background@#The objective of this study was to investigate whether the use of live attenuated varicella zoster virus (VZV) MAV vaccination can efficiently induce VZV antibody production in naive rhesus monkeys as an approach to prevent simian varicella virus (SVV) reactivation in animals immunosuppressed for transplantation studies. @*Results@#Clinically available human VZV vaccine was used to induce the production of anti-VZV antibodies in rhesus monkeys. A vial of the vaccine was subcutaneously injected at 0 week, and the second and third vaccination was performed at 5 and 6 weeks by intratracheal inoculation. The titer of anti-VZV IgG was assessed at 0, 2, 4, 6, and 7 weeks. At 2 weeks, 3/16 were seropositive for VZV IgG. At 6 weeks, 9/16 were shown to be seropositive. At 7 weeks, 16/16 were found to be seropositive. @*Conclusions@#The VZV vaccine via intratrachael inoculation was shown to induce VZV IgG humoral immunity in rhesus monkeys and may be important immunosuppressed macaques for transplantation studies. Although the humoral immunity produced is an important finding, further studies will be necessary to confirm possible protection and it could protect probably against SVV infection in rhesus monkey.

Contrasting Prognostic Effects of Tumor-Infiltrating Lymphocyte Density in Cardia and Non-cardia Gastric Adenocarcinomas

Purpose@#This study sought to investigate the prognostic significance of tumor-infiltrating lymphocytes (TILs) in relation to tumor location within the stomach. @*Materials and Methods@#The densities and prognostic significance of TIL subsets were evaluated in 542 gastric cancer patients who underwent gastrectomy. Immunohistochemical staining for CD3, CD4, CD8, forkhead/winged helix transcription factor (Foxp3), and granzyme B was performed. @*Results@#Cardia cancer was associated with significantly lower densities of CD8 T-cells and higher densities of Foxp3 and granzyme B T-cells than non-cardia tumors. Multivariate analysis showed that advanced age (hazard ratio [HR], 1.023; 95% confidence interval [CI], 1.006–1.040), advanced T classification (HR, 2.029; 95% CI, 1.106–3.721), lymph node metastasis (HR, 3.319; 95% CI, 1.947–5.658), low CD3 expression (HR, 0.997; 95% CI, 0.994–0.999), and a high Foxp3/CD4 ratio (HR, 1.007; 95% CI, 1.001–1.012) were independent predictors of poor overall survival in cardia cancer patients. In non-cardia cancer patients, total gastrectomy (HR, 2.147; 95% CI, 1.507–3.059), advanced T classification (HR, 2.158; 95% CI, 1.425–3.266), lymph node metastasis (HR, 1.854; 95% CI, 1.250–2.750), and a low Foxp3/CD4 ratio (HR, 0.978; 95% CI, 0.959–0.997) were poor prognostic factors for survival. @*Conclusions@#The densities and prognostic effects of TILs differed in relation to the location of tumors within the stomach. The contrasting prognostic effects of Foxp3/CD4 ratio in cardia and non-cardia gastric cancer patients suggests that clinicians ought to consider tumor location when determining treatment strategies.

Galectin-4 Interaction with CD14 Triggers the Differentiation of Monocytes into Macrophage-like Cells via the MAPK Signaling Pathway

Galectin-4 (Gal-4) is a β-galactoside-binding protein mostly expressed in the gastrointestinal tract of animals. Although intensive functional studies have been done for other galectin isoforms, the immunoregulatory function of Gal-4 still remains ambiguous. Here, we demonstrated that Gal-4 could bind to CD14 on monocytes and induce their differentiation into macrophage-like cells through the MAPK signaling pathway. Gal-4 induced the phenotypic changes on monocytes by altering the expression of various surface molecules, and induced functional changes such as increased cytokine production and matrix metalloproteinase expression and reduced phagocytic capacity. Concomitant with these changes, Gal-4-treated monocytes became adherent and showed elongated morphology with higher expression of macrophage markers. Notably, we found that Gal-4 interacted with CD14 and activated the MAPK signaling cascade. Therefore, these findings suggest that Gal-4 may exert the immunoregulatory functions through the activation and differentiation of monocytes.

Implications of Calcineurin/NFAT Inhibitors' Regulation of Dendritic Cells and Innate Immune Cells in Islet Xenotransplantation

Calcineurin inhibitors, such as cyclosporine and tacrolimus (FK506) are broadly used in organ transplantations as immune suppressants. As the calcineurin/NFAT signaling pathway has been identified as critical pathway in the interleukin-2 (IL-2) production of T cells, inhibition of T-cell derived IL-2 has been considered the major mechanism of calcineurin inhibitors. However, there is increasing evidence that NFAT transcription factor is involved in multiple functions of dendritic cells and innate immune cells as well. NFAT expression is not restricted to T cells, and IL-2 can be produced in dendritic cells and macrophages through the calcineurin/NFAT pathway. Furthermore, it has been discovered that NFAT regulates expressions of several inflammatory mediators, including TNF-α and cyclooxygenase-2 in innate immune cells. Therefore, calcineurin inhibitors may have much broader effects in the transplant recipients than previously being considered. In this review, we reviewed recently discovered roles of NFAT pathway in dendritic cells and innate immune cells, and discussed positive and negative implications of calcineurin inhibitors' broader effects with a focus on islet xenotransplantation.

Association between Chemotherapy-Response Assays and Subsets of Tumor-Infiltrating Lymphocytes in Gastric Cancer: A Pilot Study

PURPOSE: The purpose of this pilot study was to evaluate the association between adenosine triphosphate-based chemotherapy response assays (ATP-CRAs) and subsets of tumor infiltrating lymphocytes (TILs) in gastric cancer. MATERIALS AND METHODS: In total, 15 gastric cancer tissue samples were obtained from gastrectomies performed between February 2007 and January 2011. Chemotherapy response assays were performed on tumor cells from these samples using 11 chemotherapeutic agents, including etoposide, doxorubicin, epirubicin, mitomycin, 5-fluorouracil (5-FU), oxaliplatin, irinotecan, docetaxel, paclitaxel, methotrexate, and cisplatin. TILs in the tissue samples were evaluated using antibodies specific for CD3, CD4, CD8, Foxp3, and Granzyme B. RESULTS: The highest cancer cell death rates were induced by etoposide (44.8%), 5-FU (43.1%), and mitomycin (39.9%). Samples from 10 patients who were treated with 5-FU were divided into 5-FU-sensitive and -insensitive groups according to median cell death rate. No difference was observed in survival between the two groups (P=0.216). Only two patients were treated with a chemotherapeutic agent determined by an ATP-CRA and there was no significant difference in overall survival compared with that of patients treated with their physician's choice of chemotherapeutic agent (P=0.105). However, a high number of CD3 TILs was a favorable prognostic factor (P=0.008). Pearson's correlation analyses showed no association between cancer cell death rates in response to chemotherapeutic agents and subsets of TILs. CONCLUSIONS: Cancer cell death rates in response to specific chemotherapeutic agents were not significantly associated with the distribution of TIL subsets.

Increase in Anti-Gal IgM Level is Associated With Early Graft Failure in Intraportal Porcine Islet Xenotransplantation

BACKGROUND: Anti-Gal is a major antibody induced in non-human primates (NHPs) after xenotransplantation. To understand the mechanism of graft rejection, we investigated the association between anti-Gal responses and graft failure in NHP recipients of porcine islet transplantation (PITx). METHODS: Intraportal PITx was performed in 35 diabetic NHPs, and graft function was monitored. Early graft failure (EGF) was defined as loss of graft function within a month after PITx. Seven, 19, nine NHPs received immunosuppression (IS) without CD40 pathway blockade (Group I), with anti-CD154 (Group II), and with anti-CD40 (Group III), respectively. The anti-Gal levels on day 0 and day 7 of PITx were measured by ELISA. RESULTS: The frequency of EGF was significantly lower in Group II (26.3%) than in Group I (100%, P=0.0012) and Group III (77.8%, P=0.0166). While levels of anti-Gal IgG in Group I and anti-Gal IgM in Group III increased on day 7 compared with day 0 (P=0.0156 and 0.0273), there was no increase in either on day 7 in Group II. The ratio of anti-Gal IgM or IgG level on day 7 to that on day 0 (Ratio7/0) was significantly higher in recipients with EGF than without EGF (P=0.0009 and 0.0027). ROC curve analysis of anti-Gal IgM Ratio7/0 revealed an area under the curve of 0.789 (P=0.0003). CONCLUSIONS: IS with anti-CD154 suppressed anti-Gal responses and prevented EGF in PITx. Anti-Gal IgM Ratio7/0, being associated with EGF, is a predictive marker for EGF.

Analysis of env Subtypes of Porcine Endogenous Retrovirus in SNU Miniature Pigs

All xenografts from pigs impose infection risk by porcine endogenous retrovirus (PERV). The purpose of this study was to investigate the env constructs with the comparison of the ratio of the competent form to the defective one of env in subtypes, PERV-A, PERV-B and PERV-C in different pig breeds. The results of PCR amplification of env represented that all env subtypes had more than two defective forms which cannot bind to host cells due to the absence of binding regions of env in miniature pigs, SNU and PWG, and farm pig breeds, Duroc, Yorkshire and Landrace. In addition, comparing the full sequences with the defective ones in three subtypes demonstrated that the present percentages of env sequences in defective PERV-A, PERV-B and PERV-C were approximately 50%, 38~45% and 4~11%, respectively, in SNU and PWG pigs whereas PERV-A and PERV-B occupied around 40 to 60% but PERV-C was not detected in farm pigs. Quantitative real-time PCR assays with primers and probes targeted to proline-rich region (PRR) of each env subtype were done to measure the copy numbers of each env subtype. When the reference was set with copy number of PERV-A, the ratio of those of PERV-B and PERV-C to the reference were 1.5 to 6.0 folds high in SNU and PWG pigs while 1.0 or less in farm pigs. These contradictory results of PERV-C constructs and copy numbers in SNU pigs suggests that many truncated or short defective sequences of PERV-C might be present in them.

Erratum: Correction of the Grant Number

We found an error in our published article.

Erratum: Correction of the Grant Number

We found an error in our published article.

Role of Regulatory T Cells in Transferable Immunological Tolerance to Bone Marrow Donor in Murine Mixed Chimerism Model

Constructing a bone marrow chimera prior to graft transplantation can induce donor-specific immune tolerance. Mixed chimerism containing hematopoietic cells of both recipient- and donor-origin has advantages attributed from low dose of total body irradiation. In this study, we explored the mechanism of mixed chimerism supplemented with depletion of Natural Killer cells. Mixed chimerism with C57BL/6 bone marrow cells was induced in recipient BALB/c mice which were given 450 cGy of gamma-ray irradiation (n = 16). As revealed by reduced proliferation and cytokine production in mixed leukocyte reaction and ELISpot assay (24.6 vs 265.5), the allo-immune response to bone marrow donor was reduced. Furthermore, the induction of transferable immunological tolerance was confirmed by adoptive transfer and subsequent acceptance of C57BL/6 skin graft (n = 4). CD4+FoxP3+ regulatory T cells were increased in the recipient compartment of the mixed chimera (19.2% --> 33.8%). This suggests that regulatory T cells may be therapeutically used for the induction of graft-specific tolerance by mixed chimerism.

Application of the Multiplex Cytokine Analysis to Monitor Xenogeneic Immune Responses to the Porcine Islet Graft in Non-Human Primate

Non-human primate studies must be conducted prior to the clinical trial of xenotransplantation. In order to develop clinically applicable immune-modulatory regimen through non-human primate studies, close monitoring of xenogeneic immune responses is required. We adopted multiplex cytokine analysis in assessment of the immune status during the course of pig-to-non-human primate islet transplantation. This study aimed to assess the feasibility of this multiplex cytokine assay in the development of immune-modulatory regimen. Using this assay, we were able to detect different cytokines with a minimal usage of blood samples, and this allowed us to detect various immunological situations in the recipients. Detection of TNF-alpha surge (347.8 pg/mL) guided us to block TNF-alpha in the early phase of transplantation. Supportive information for in vivo efficacy of cytokine neutralizing antibody could be speculated by in vitro neutralization assay (1,250 pg/mL --> 0 pg/mL). In addition, periodic monitoring of cytokines in peripheral blood allowed the detection of the infection episode prior to other routine assays. These benefits of multiplex cytokine assay may be generally applied to other pre-clinical research, which is a prerequisite for clinical trials.

Functional Characteristics of C-terminal Lysine to Cysteine Mutant Form of CTLA-4Ig

CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation.

Analysis of reference interval and age-related changes in serum biochemistry and hematology in the specific pathogen free miniature pig / 한국실험동물학회지

We analyzed 72 items related to serum biochemistry and hematology in 85 specific pathogen-free (SPF) Seoul National University (SNU) miniature pigs aged 1- to 36-months which originated from a Minnesota miniature pig. Almost all examined items were similar between male and female pigs. However, some items such as Cr level, B/C ratio, C.R.F, LDH, LAP and T4 were significantly different between male and female pigs (P<0.05). Thirty four examined items showed age-related changes, and the significant changes were observed in animals less than six months old. The values for BUN, K, uric acid, Ca, Ca++, and Pi were significantly higher in pigs younger than six months of age, which might reflect poor kidney function in young pigs. Additionally, TIBC, UIBC and RDW were significantly higher in young pigs, and RBC, Hb, HCT, MCHC and MCV were significantly lower in young pigs, thus indicating a similar physiology of iron deficiency anemia. These age-related specific phenotypes seemed to be normal, but it should be considered in the long-term experiment using the young pigs. In conclusion, in this study, we defined the normal reference intervals for SPF SNU miniature pigs, and we also determined that there are some physiological differences between the pig genders and ages. This study provides fundamental data for use in experiments involving SPF SNU miniature pigs.

Heavy metal concentrations in hair of newly imported China-origin rhesus macaques (Macaca mulatta) / 한국실험동물학회지

Macaque monkeys are good sentinel to humans for environmental pollutions because their similarities in genetic and physiological characteristics. So, their reference values about exposures to heavy metals are required for proper data interpretation. Here, we report several heavy metals concentrations in the hair of rhesus monkeys which are widely used in biomedical research. The hair of 28 imported rhesus monkeys from an animal farm in southwest China were examined for the presence of eight heavy metals (Arsenic, Beryllium, Cadmium, Chromium, Iron, Lead, Mercury, and Selenium). The analyzed data in parts per million (ppm) for hair concentrations of heavy metals in rhesus monkeys were as follow: As (0.654+/-0.331), Be (0.005+/-0.003), Cd (0.034+/-0.022), Cr (11.329+/-4.259), Fe (87.106+/-30.114), Pb (0.656+/-0.613), Hg (0.916+/-0.619), and Se (3.200+/-0.735). The concentrations of Be, Cr, and As showed significant higher in females than in males (P<0.05). We present here the reference values of several heavy metals in healthy China-origin rhesus monkeys. These data may provide valuable information for veterinarians and investigators using rhesus monkeys in experimental studies.

Application of Regulatory T Cells in Transplantation Field / 대한이식학회지

The development of immunosuppressant treatments has enabled remarkable progress in the tissue and organ transplantation field by helping to prevent acute graft rejection. However, complications related to transplantation, such as infection by bacteria and viruses, and the occurrence of cancers resulting from prolonged immune suppression are major obstacles to overcome. Therefore, transplantation immunology research efforts should focus on the induction of donor-specific immune tolerance which preserves patient immune competence which promotes infection and cancer surveillance. Additionally, lifelong administration of immunosuppressants should be forgone in preference to short term therapies. In the 1990s, Dr. Shimon Sakaguchi identified the CD4+CD25+ regulatory T cells which develop in the thymus, and demonstrated that these cells play crucial roles in the maintenance of immune self tolerance. Studies which followed proved that these regulatory T cells are important to the control of autoimmune disease and prevention of graft rejection. Regulatory T cells have also been found to induce immune tolerance in rodent models. In this review, we discuss several considerations for the use of regulatory T cell therapy in the clinical transplantation field.

Influence of Interferon-gamma Deficiency in Immune Tolerance Induced by Male Islet Transplantation

BACKGROUND: Traditionally, interferon-gamma (IFN-gamma) was regarded as a pro-inflammatory cytokine, however, recent reports suggested role of IFN-gamma in immune tolerance. In our previous report, we could induce tolerance to male antigen (HY) just by male islet transplantation in wild type C57BL/6 mice without any immunological intervention. We tried to investigate the influence of IFN-gamma deficiency on tolerance induction by male islet transplantation. METHODS: To examine the immunogenicity of male tissue in the absence of IFN-gamma, we transplanted male IFN-gamma knock-out (KO) skin to female IFN-gamma KO mice. Next, we analyzed male IFN-gamma KO islet to streptozotocin-induced diabetic female IFN-gamma KO mice. And, we checked the functionality of grafted islet by graft removal and insulin staining. RESULTS: As our previous results in wild type C57BL/6 mice, female IFN-gamma KO mice rejected male IFN-gamma KO skin within 29 days, and did not reject male IFN-gamma KO islet. The maintenance of normal blood glucose level was dependent on the presence of grafted male islet. And the male islet recipient did not reject 2nd challenge of male islet graft also. CONCLUSION: Deficiency of IFN-gamma does not have influence on the result of male skin graft and male islet transplantation. Conclusively, male islet transplantation induced T cell tolerance is not dependent on the presence of IFN-gamma.

Shigella flexneri infection in a newly acquired rhesus macaque (Macaca mulatta) / 한국실험동물학회지

A 3.4 year-old rhesus macaque weighing 4.5 kg, was suffering from anorexia, acute mucous and bloody diarrhea. On physical examination, the monkey showed a loss of activity, hunched posture, abdominal pain, dehydration, mild gingivitis and unclean anus with discharge. Whole blood was collected for the examination of electrolytes, hematology and serum chemistry; fresh stool was also collected for bacterial culture. Blood profiles showed leukocytosis (14.5 K/microL) and neutrophilia (11.0 K/microL) on complete blood cell count and imbalanced electrolytes associated with diarrhea. As a result of bacterial culture, Shigella flexneri was identified through Mac/SS, IMVIC test, TCBS and VITEK II. Based on these results, this monkey was diagnosed as having acute enteritis caused by Shigella flexneri. Treatment was performed with enrofloxacin prior to the isolation of Shigella flexneri to prevent the transmission of disease. Fortunately, mucus and bloody diarrhea did not persist and general conditions fully recovered. Our results show that the use of enrofloxacin is effective in controlling Shigella flexneri infection in newly acquired rhesus monkeys.