scholarly journals Pro-inflammatory CD8 T Cells Contribute to Age-Related Large Artery Stiffening

2020 ◽  
Vol 4 (Supplement_1) ◽  
pp. 134-134
Author(s):  
Daniel Trott ◽  
Sunita Sharma
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Large Artery ◽  
Cd8 Cells ◽  
Age Related ◽  
Tnf Α ◽  
Artery Stiffness ◽  
Ifn Γ ◽  
Old Mice

Abstract We have shown that T cells accumulate around the aorta with advanced age and that both pharmacologic and genetic deletion of total T cells result in improved large artery stiffness in old mice. The purpose of this study was to test the hypothesis that CD8 T cells specifically mediate age-related large artery stiffening. We randomized old (22-24 month) C57Bl6 mice (n=5/group) to treatment with an anti-CD8 or isotype control antibody (100µg every 5 days) for 28 days. We assessed large artery stiffness using pulse wave velocity (PWV) before and after antibody treatment. We found that PWV was similar between isotype (301±14 cm/s) and anti-CD8 (292±18 cm/s) before treatment. Following treatment, anti-CD8 treated mice exhibited lower PWV (272±11 cm/s) compared to the isotype (315±14 cm/s). Following euthanasia, we assessed aortic T cell infiltration by flow cytometry we found that anti-CD8 treated mice exhibited blunted aortic total CD3+ (262±42 vs. 1400±94 cells per aorta) and CD8+ (52±19 vs. 565±139 cells per aorta) cells compared to isotype controls. In a separate cohort of mice we compared interferon (IFN)-γ and tumor necrosis factor (TNF)-α production of aortic infiltrating CD8 cells from young (4-6 months, n=5) and old mice (n=8) using flow cytometry. A greater proportion of CD8 cells from old aortas produced IFN-γ (70±3% vs. 46±6%) compared to young. Similarly, a greater proportion of aortic infiltrating CD8 cells from old mice produced TNF-α (35±6% vs. 17±3%) compared to young. Collectively, these results suggest that pro-inflammatory CD8 cells contribute to cell non-autonomous arterial aging.

scholarly journals Type 1 Cytotoxic T Cells Increase in Placenta after Intrauterine Inflammation

2021 ◽  
Vol 12 ◽  
Author(s):  
Jin Liu ◽  
Yang Liu ◽  
Snigdha Panda ◽  
Anguo Liu ◽  
Jun Lei ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Cd8 T Cells ◽  
Cytotoxic T Cells ◽  
Cd8 T Cell ◽  
Tnf Α ◽  
Ifn Γ

CD8+ T cells recognize non-self antigen by MHC class I molecules and kill the target cells by the release of proinflammatory cytokines such as interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). Our group previously reported an increase of CD8+ T‐cell trafficking in the placenta with exposure to Lipopolysaccharides (LPS). CD8+ cytotoxic T cells have been classified into distinct subsets based upon cytokine production: Tc1 cells produce IFN-γ, Tc2 cells produce interleukin 4 (IL-4). Accordingly, the purpose of this research is to analyze the subsets of placenta CD8+ T cells. We hypothesized that LPS injection would induce a change of properties of CD8+ T cell and Tc1/Tc2 ratio. We investigated the subsets of CD8+ T cell infiltration to placenta and their specific function in response to LPS-induced inflammation in a mouse model. At embryonic (E) day 17, pregnant CD-1 dams received an intrauterine injection of 25 µg LPS in100 μl PBS or 100 μl of PBS only. Flow cytometry was used to quantify CD8+ T cells, evaluate the phenotype and subtypes, and detect markers of Tc1 and Tc2 cells in placenta, at 6 hours and 24 hours post injection (hpi). Intracellular staining and flow cytometry were performed to characterize cytokines produced by CD8+ T cells. Standard statistical analysis were employed. After 6 and 24 hours of LPS injection, total CD8 T cells increased (P<0.05). Tc1 cells expanded (P<0.05) in LPS-treated dams compared with the PBS group. The Tc1/Tc2 ratio was significantly higher in the LPS group than the PBS group (P<0.05). The expression of TNF-α and IFN-γ were increased in LPS group both at 6hpi and 24 hpi (P<0.05). We identified functional placental CD8+ T cell subtypes and found a significant increase ratio of Tc1/Tc2. Following IUI, CD8+ T cells induced inflammatory response in the placenta primarily via the production of Type 1 cytokines such as IFN-γ and TNF-α. We have provided evidence of a Tc1-bias response and cytokines in the mouse model of IUI.

scholarly journals Peritoneal Cavity Cells Ameliorate Graft-Versus-Host Disease of Mice after MHC Mismatched Bone Marrow Transplantation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5675-5675
Author(s):  
Zhigang Liu ◽  
Wanhua Zhang ◽  
Xinchuan Chen ◽  
Chuan He ◽  
Jie Ji ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Survival Time ◽  
Peritoneal Cavity ◽  
Inflammatory Factors ◽  
Cd8 Cells ◽  
Graft Versus Host ◽  
Tnf Α ◽  
Ifn Γ

Abstract Background: Peritoneal cavity cells are a group of isolated cells with unique functions, they have great anti-inflammatory, immune modulation and tissue recovering abilities. Several studies have independently reported the therapeutic function of peritoneal cavity cells on mice models with ulcerative colitis or other inflammatory bowel diseases. In the current study, we further investigated the effect of peritoneal cells on treatment of GVHD on mice models. Methods: We selected a dose of 7.0Gy of TBI for BABL/c mice. And 4 hours after TBI, BABL/c mice were infused with 5*106BMCs cells or 1*106spleen cells of C57BL/6 mice through tail veins to construct MHC mismatched myeloablative allogeneic hematopoietic stem cell transplantation model. The mice were randomly separated into two groups, which were infused with normal saline and 5* 106peritoneal cavity cells on day 0, 3, and 5. Survival time, weight changes, GVHD score were evaluated. Peritoneal cavity cells of GFP C57BL/6 transgenic mice were injected into mice recipients at day 5 after transplantation to observe the distribution of GFP positive cells in mice recipients. Flow cytometry was used to test proportion of CD4/CD8 cells and proportions of effector T cells and Naïve T cells in CD4 and CD8 cells of spleen and bone marrow in BMT group and peritoneal cavity cells group at different time points after transplantation. TNF-α, IFN-γ, IL-2, IL-17, IL-15, IL-4, IL-10 and TGF-β in plasma were studied with the method of Luminex at different time points in two groups. Expression of IL-10 and TGF-β in colons, intestines, and livers were assessed with immunofluorescence staining. Results: Mice in peritoneal cavity cells group had significant longer survival time and rapidly weight loss recover. Mice in peritoneal group had better performance in activity, unhairing, and skin changes as well. Counts of blood cells and chimeric status at day 7, 14, 21, 28 after transplantation showed that blood count recovered and stable chimerism in both groups. Small living animal imaging technology found that peritoneal cavity cells concentrate in colons and intestines after injection of GFP C57BL/6 transgenic mice peritoneal cavity cells. Fluorescence microscope showed that large amounts of green fluorocyte distributed mostly in colons and intestines, with few in liver. Flow cytometry proved that many GFP positive cells in intestines and colons (30%, and 15%, respectively), and a few in livers and lungs (approximately 5%), while negative in control group. We analyzed the lymphocyte subsets of spleen and bone marrow in two groups with flow cytometry and found that peritoneal cells treatment could increase the proportion of CD4 cells and decrease CD8 cells. In CD4 subsets, proportion of effective T cell decreased apparently 3 weeks after transplantation, and count of naïve T cells increased, which is not found in BMT group. Flow cytometry also showed that proportion of Treg cells, Th2 cells and NK cells were significantly higher in peritoneal cavity cells group, while proportion of Th1 cells were lower. TGF-β, IL-10 and IL-4 were significantly higher in peritoneal cavity cell treatment group, while TNF-α, IFN-γ, IL-15, IL-2 were lower. Immunofluorescence staining also showed that TGF-β and IL-10 were strongly expressed in colons and intestines, but not in BMT group. Conclusion: These results demonstrate that peritoneal cavity cells could ameliorate graft-versus-host disease of mice after MHC mismatched bone marrow transplantation. Survival time was prolonged, and weight loss, GVHD score, and pathologic injuries to tissues improved after infusion of 5* 106peritoneal cavity cells into BMT mice. Peritoneal cavity cells injected to BMT mice concentrate mainly in colons and intestines, which functioned as anti-inflammation and tissue repairing cells. These cells could modulate differentiation of T lymphocytes of mice recipients, decrease proportion of CD8 cells and increase CD4 cells, increase proportion of Tn cells and decrease that of Te cells in CD4 subsets, and increase proportion of Tregs, Th2 and NK cells and decrease that of Th1 cells. Peritoneal cavity cells could influence levels of cytokines by increasing anti-inflammatory factors including TGF-β, IL-10, IL-4, and significantly decreasing inflammatory factors like IFN-γ, TNF-α, and IL-15. Figure. Figure. Disclosures Liu: West China Hospital of Sichuan University: Employment.

scholarly journals The Immunomodulatory Effect of Triptolide on Mesenchymal Stem Cells in Vitro

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5011-5011
Author(s):  
Haiping He ◽  
Atsuko Takahashi ◽  
Yuki Yamamoto ◽  
Akiko Hori ◽  
Yuta Miharu ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Complete Medium ◽  
Surface Markers ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ

Background: Mesenchymal stromal cells (MSC) are known to have the immunosuppressive ability and have been applied in clinic to treat acute graft-versus-host disease (GVHD), as one of severe complications after hematopoietic stem cells transplantation (HSCT) in Japan. However, MSC are activated to suppress the immune system only upon the stimulation of inflammatory cytokines and the clinical results of MSC therapies for acute GVHD are varied. It is ideal that MSC are primed to be activated and ready to suppress the immunity (=priming) before administration in vivo. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb - Tripterygium Wilfordii Hook F (TWHF). It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aim to use TPL as the activator for umbilical cord-derived MSC (UC-MSC) to entry stronger immunosuppressive status. Methods: The proliferation of UC-MSC with TPL at the indicated concentrations for different time of 24, 48, 72, and 96 hours. Cell counting kit-8(CCK-8) was added in the culture medium to detect cell toxicity and the absorbance was measured using microplate reader. Flow cytometry was used to identify the MSC surface markers expression. TPL-primed UC-MSC were once replaced with fresh medium and co-culture with mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and irradiated allogenic dendritic cell line (PMDC05) in RPMI 1640 medium supplemented with 10 % FBS (complete medium). IDO-1, SOD1, and TGF-β gene expression in TPL-primed UC-MSC and UC-MSC induced by 10 ng/ml IFN-γ and/or 15 ng/ml TNF-α were evaluated by RT-PCR. PDL1 and PDL2 expression in TPL-primed UC-MSC and UC-MSC in response to IFN-γ and/or TNF-α were checked by Flowjo. Results: Exposure of TPL for UC-MSC for 72hour at the concentration above 0.1 μM resulted in the cell damage significantly. Therefore, we added TPL in UC-MSC at 0.01μM of TPL for up to 48 hours, then washed thourouphly for the following culture for experiments. To evaluate the influence of TPL on the surface markers of UC-MSC, we cultured UC-MSC for 4 hours in complete medium following culture with 0.01μM of TPL for 20 hours (TPL-primed UC-MSC). TPL-primed UC-MSC revealed positive for CD105, CD73, and CD90, negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules as same as the non-primed UC-MSC. In MLR suppression by UC-MSC, the TPL-primed UC-MSC activity revealed stronger anti-proliferative effect on the CD4+ and CD8+ T cells activated by allogeneic DC than those of non-primed UC-MSC in MLR. Furthermore, the TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β in response to IFN-γ+/-TNF-α by RT-PCR and enhanced the expression of PD-L1 by FACS analysis. Discussion:In this study, we found the TPL-primed UC-MSC showed stronger antiproliferative potency on CD4+ and CD8+ T cells compared with non-primed UC-MSC. TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β stimulated by IFN-γ+/-TNF-α, although TPL alone did not induce these factors. Furthermore, we found that the PD1 ligand (PD-L1) was induced in TPL-primed UC-MSC, likely IFN-γ enhanced the PD-L1 expression, evaluated by flowcytometry. These results suggested that TPL-primed UC-MSC seemed more sensitive to be activated as the immunosuppressant. Here, we firstly report the new function of TPL to induce the upregulation of immunosuppressive effect, although the mechanisms of TPL inhibition to MSC need to be explore. Conclusively, TPL-primed UC-MSC might be applied for the immunosuppressive inducer of MSC. Figure Disclosures He: SASAGAWA Medical Scholarship: Research Funding; IMSUT Joint Research Project: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding. Nagamura-Inoue:AMED: Research Funding.

Immunosenescence of Lymphocytes T and Implications for Age-Related Disorders

2020 ◽  
Author(s):  
Anna Tylutka ◽  
Barbara Morawin ◽  
Artur Gramacki ◽  
Agnieszka Zembron-Lacny
Keyword(s):  
T Cells ◽  
Elderly People ◽  
Cd8 T Cells ◽  
The Elderly ◽  
Health Concern ◽  
Female Group ◽  
Major Health ◽  
Cd8 Cells ◽  
Age Related ◽  
Sex Disorders

Abstract Background. The decrease in immunity with age is still a major health concern as elderly people are more susceptible to infections and increased incidence of autoimmunity. Consequently, there is an increasing interest in immunosenescence and changes in immunology cells like T cells. The aim of our study was to find a disproportion in subpopulation of T cells as well as CD4/CD8 ratio depending on the age, gender or comorbidities. Results. In the present study, a flow cytometry was used to indicate the differences between age, sex, disorders and fat content in the CD4+ and CD8+ T cells population divided into naïve and memory cells as well as CD4/CD8 ratio in people aged 71.9± 5.8 years (females n=83, males n=16) compared to young people aged 20.6 ± 1.1 years (females n=12, males n=19). The percentage of naïve CD4+ and CD8+ cells was found to be statistically significantly lower in the elderly compared to the young. In addition, gender was observed to play an important role in the outcomes in the analysed subpopulations and in female group, who live statistically longer than males, our older group of Polish women demonstrated a significantly higher percentage of naïve lymphocytes in both the CD4+ and CD8+ populations compared to men. The CD4/CD8 ratio increases with age, which can be considered one of the markers determining longevity. Elderly people with age-related diseases (hypertension) also show an increased level of CD4/CD8 ratio as well as CD4+. Conclusion. We demonstrated that changes in the T cells population, including naïve cell population as well as CD4/CD8 ratio, are important markers which can be predictive of healthy status. In order to accurately determine longevity, gender or age-associated diseases should be taken into account.

scholarly journals Complementary Dendritic Cell–activating Function of CD8+ and CD4+ T Cells

2002 ◽  
Vol 195 (4) ◽  
pp. 473-483 ◽  
Author(s):  
Robbie B. Mailliard ◽  
Shinichi Egawa ◽  
Quan Cai ◽  
Anna Kalinska ◽  
Svetlana N. Bykovskaya ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Class I ◽  
Chronic Infections ◽  
Cd40 Ligation ◽  
Activation Level ◽  
Tnf Α ◽  
Ifn Γ

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of “T helper (Th)” signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood– or peripheral blood–isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-γ at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I–presented epitopes by antigen-specific CD8+ T cells results in the TNF-α– and IFN-γ–dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I–restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I–presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections.

scholarly journals The Level of Viral Antigen Presented by Hepatocytes Influences CD8 T-Cell Function

2007 ◽  
Vol 81 (6) ◽  
pp. 2940-2949 ◽  
Author(s):  
Adam J. Gehring ◽  
Dianxing Sun ◽  
Patrick T. F. Kennedy ◽  
Esther Nolte-'t Hoen ◽  
Seng Gee Lim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Viral Antigen ◽  
Cell Function ◽  
Cd8 T Cell ◽  
T Cell Function ◽  
Tnf Α ◽  
Antiviral Function ◽  
Ifn Γ

ABSTRACT CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-γ and TNF-α production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes.

Identification of the AAV2 Capsid CD8+ T Cell Epitope in C57BL/6 Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3188-3188
Author(s):  
Denise E. Sabatino ◽  
Federico Mingozzi ◽  
Haifeng Chen ◽  
Peter Colosi ◽  
Hildegund C.J. Ertl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Capsid Protein ◽  
Cd8 T Cells ◽  
Cell Epitope ◽  
T Cell Response ◽  
Cd8 Cells ◽  
Media Control ◽  
Ifn Γ

Abstract Recently, a clinical trial for adeno-associated virus serotype 2 (AAV2) mediated liver directed gene transfer of human Factor IX to subjects with severe hemophilia B revealed that two patients developed transient asymptomatic transaminitis following vector administration. Immunology studies in the second patient demonstrated a transient T cell response to AAV2 capsid peptides suggesting that the immune response to the AAV capsid may be related to the transient transaminitis. We hypothesized that the observations made in the human subjects were due to a CD8 T cell response to AAV2 capsid protein. Preclinical studies in mice and dogs, which are not naturally infected by wild type AAV2 viruses, did not predict these findings in the clinical study. Thus, we developed a mouse model in which we were able to mimic this phenomenon (Blood 102:493a). In an effort to further characterize the immune responses to AAV2 capsid proteins in this mouse model, we identified the T cell epitope in the AAV capsid protein recognized by murine C57Bl/6 CD8 T cells. A peptide library of AAV2 VP1 capsid peptides (n=145) that were synthesized as 15mers overlapping by 10 amino acids were divided into 6 pools each containing 24–25 peptides. C57Bl/6 mice were immunized intramuscularly with an adenovirus expressing AAV2 capsid protein. Nine days later the spleen was harvested and intracellular cytokine staining (ICS) was used to assess release of IFN-γ from CD8 T cells in response to 6 AAV2 capsid peptide pools. ICS demonstrated CD8 cells from mice immunized with Ad-AAV2 produced IFN-γ (3.5% of the CD8 cells) in response to Pool F (amino acid 119–145) while no IFN-γ release in CD8 cells was detected with Pool A to E (mean 0.28%±0.25%) compared to the media control (0.16%). This detection of IFN-γ release from CD8 T cells indicates a specific proliferation to a peptide(s) within this peptide pool (Pool F). A matrix approach was used to further define which peptide(s) contained the immunodominant epitope. Eleven small peptide pools of Pool F were created in which each peptide was represented in 2 pools. ICS of splenocytes from immunized (Ad-AAV2 capsid) C57Bl/6 mice demonstrated IFN-γ response from CD8 cells to 3 of the matrix pools corresponding to peptide 140 (PEIQYTSNYNKSVNV) and 141 (TSNYNKSVNVDFTVD) compared with media controls. To determine the exact peptide sequence that binds to the MHC Class I molecule, 9 amino acid peptides (n=7) were created that overlap peptide 140 and 141. Peptide SNYNKSVNV showed positive staining for both CD8 and IFN- γ(3.2%) compared with the six other peptides (0.14%±0.08%), media control (0.08%) and mice that were not immunized (0.11%). This epitope lies in the C terminus of the AAV2 VP1 capsid protein. Current studies using strains of mice with different MHC H2 haplotypes will allow us to determine which of the C57Bl/6 MHC alleles the epitope binds. These findings will provide us with a powerful tool for assessing immune responses to AAV capsid in the context of gene therapy. Specifically, they will allow us to determine how long immunologically detectable capsid sequences persist in an animal injected with AAV vectors. This in turn will provide a basis for a clinical study in which subjects are transiently immunosuppressed, from the time of vector injection until capsid epitopes are no longer detectable by the immune system.

Prophylactic Amotosalen-Treated Donor T-Cells Prevent Late CMV Infection in Allogeneic Bone Marrow Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3024-3024
Author(s):  
Mohammad S. Hossain ◽  
John D. Robak ◽  
Edmund K. Waller
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Viral Infection ◽  
Cd8 T Cells ◽  
Immune Reconstitution ◽  
Chronic Gvhd ◽  
Mcmv Infection ◽  
Tnf Α ◽  
Donor T Cells ◽  
Ifn Γ

Abstract A major problem in allogeneic BMT is post transplant immunodeficiency leading to opportunistic infection and relapse. Previously we showed that amotosalen-treated allogeneic donor T cells given at the time of BMT and experimental murine cytomegalovirus (MCMV) infection could prevent lethal MCMV disease without producing GvHD. In this study we have focused on a more clinically applicable model where prophylactic amotosalen-treated allogeneic donor splenocytes are given at the time of BMT, followed by MCMV infection 100 days later. We observed that amotosalen-treated donor T-cells significantly expanded and responded well in presence of viral infection without inducing any GvHD, protected recipients against viral disease, and were associated with significantly improved hematopoietic engraftment and immune reconstitution. Methods: Using a parent to F1 mouse BMT model, splenocytes (3x106 untreated or 10x106 amotosalen-treated) from MCMV immunized C57BL/6 donors were transplanted along with 5x106 T-cell depleted bone marrow (TCD BM) from naïve congeneic mice into lethally irradiated (11Gy) CB6F1 recipients (C57BL/6 x Balb/C). Recipient mice were infected i.p. with a sublethal dose (5x104 pfu per mouse) of MCMV 100 days or more after transplant. Clinical chronic GvHD was monitored by weight loss, hair loss, ruffled fur, diarrhea, and decreased activity. Flow cytometry was used to quantitate T cell chimerism (in recipient PBMC, spleen, liver and thymus) and MCMV-peptide specific CD8+ T-cells (tetramer+ and IFN-γ producing). Serum IFN-γ and TNF-α were determined by ELISA. Liver and spleen viral loads were determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: Recipients of untreated control donor splenocytes suffered from chronic GvHD within 100 days of transplant, while those that received amotosalen-treated splenocytes experienced no GvHD. In response to MCMV infection at 100 days post transplant, residual amotosalen-treated donor T-cells rapidly expanded over 25-fold within 10 days, but did not cause lethality or detectable GvHD. Expanded amotosalen-treated T-cells showed activated anti-viral responses and developed a memory phenotype at late phases of viral infection. PBMC, spleen and liver showed elevated levels of MCMV specific tetramer+, IFN-γ+, and TNF-α+ CD8+ T-cells that were associated with accelerated viral clearance within day 3 after viral infection. While expansion and generation of amotosalen-treated donor T-cells mostly occurred in the liver, the generation of donor bone marrow-derived new T-cells occurred through both the thymus and the liver. In contrast, recipients of untreated donor splenocytes had reduced thymic function, resulting in severely impaired immune reconstitution and decreased anti-viral immunity. Conclusion: Prophylactically administered amotosalen-treated allogeneic donor T cells 1) were almost completely devoid of GvHD activity, 2) promoted hematopoietic engraftment and improved immune reconstitution, and 3) persisted long-term (>100 days) and successfully protected recipients from sublethal MCMV infection. Thus, infusion of amotosalen-treated donor T-cells at the time of transplantation is a clinically-attractive approach to adoptive anti-viral immunotherapy without chronic GvHD following hematopoietic progenitor cell transplantation.

Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4099-4099
Author(s):  
Zhenhua Qiao ◽  
Xiujuan Zhao
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Hematopoietic Cell ◽  
Control Group ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Tgf Β1

Abstract Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA). Methods: MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen. Results: There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality. Conclusions: morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.

scholarly journals Itacitinib, a JAK1 Selective Inhibitor Preserves Graft-Versus-Leukemia (GVL), Enhances Survival and Is Highly Efficacious in a MHC-Mismatched Mouse Model of Acute GvHD

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4522-4522 ◽  
Author(s):  
Ashish Juvekar ◽  
Bruce Ruggeri ◽  
Sindy Condon ◽  
Andrew Borkowski ◽  
Reid Huber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Inflammatory Cytokine ◽  
Acute Gvhd ◽  
Colon Tissue ◽  
Graft Versus Leukemia ◽  
Tnf Α ◽  
Dosing Regimens ◽  
Ifn Γ

Abstract Introduction: Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK1/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (GRAVITAS-301: NCT03139604; GRAVITAS-119: NCT03320642). Methods: Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2Kd) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells from allogeneic cell transfer from donor C57BL/6 (H-2Kb) mice. Animals were dosed orally with vehicle or the selective JAK1 inhibitor, itacitinib (60 mg/kg or 120 mg/kg twice daily). Engraftment was analyzed for the proportion of donor and host leukocytes (CD45+, H-2Kb, and H-2Kd). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analysis. Colon samples were sectioned and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3 and CD3+phosphoSTAT3 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of itacitinib on preservation of Graft-versus-Leukemia (GVL) were evaluated by injecting BALB/c mice with A20 lymphoma cells that are of H-2Kd phenotype along with combination of splenocytes and T cell depleted bone marrow from C57BL/6 (H-2Kb) mice. Results: Itacitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss and improving GvHD scores. Itacitinib did not significantly impact donor engraftment as determined by CD45+/H-2Kb quantification by flow cytometry. Similar efficacy was observed with 60 mg/kg versus 120 mg/kg twice daily dosing regimens. Oral itacitinib administration achieved JAK1 IC50 coverage for 4 h and 12 h at 60 mg/kg twice daily and 120 mg/kg twice daily, respectively. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Itacitinib (120 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4+ T cells and CD8+ T cells in blood and spleen with itacitinib treatment, but significant reductions were detected in CD4+ T cells and CD8+ T cells in the inflamed colon tissue along with significant JAK1/STAT3 inhibition as measured by reductions in normalized pSTAT3 in T cells and colonic epithelial cells. Itacitinib treatment did not negatively impact GVL responses, as evidence by T cell mediated reduction of tumor burden. Furthermore, itacitinib treatment enhanced the survival of the recipient BALB/c mice in comparison to the vehicle treated animals. Conclusions: Itacitinib, a selective JAK1 inhibitor ameliorated GvHD severity when administered prophylactically or therapeutically and had no detrimental effects on engraftment and preservation of GVL. Furthermore, itacitinib inhibited JAK1/STAT3 activation in diseased colon tissue and infiltrating T-cells, and reduced disease burden and improved survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD. Disclosures Juvekar: Incyte Corporation: Employment. Ruggeri:Incyte Corporation: Employment. Condon:Incyte Corporation: Employment. Borkowski:Biomodels LLC: Employment. Huber:Incyte Corporation: Employment. Smith:Incyte Corporation: Employment.

Sign in / Sign up

Export Citation Format

Share Document