scholarly journals CD31 Expression Defines Heterogeneity of Donor Plasmacytoid Dendritic Cell Populations That Home to the Thymus: Implications for Control of GvHD

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3325-3325
Author(s):  
Alina Ulezko Antonova ◽  
Yitong Wang ◽  
Mojibade Hassan ◽  
Yiwen Li ◽  
Edmund K. Waller
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Epithelial Cells ◽  
Research Funding ◽  
Flow Cytometric Analysis ◽  
Thymic Epithelial Cells ◽  
Specific Marker ◽  
Flow Cytometric ◽  
Ccr9 Expression ◽  
Cd31 Expression

Abstract Introduction: Plasmacytoid dendritic cells (pDC) are known to possess tolerogenic properties in allogeneic bone marrow transplantation (allo-BMT), but the relationship between pDC lineage and their modulation of graft-versus-host disease (GvHD) has not been defined. Recently, we have shown that murine treatment with the pleiotropic cytokine FMS-like Tyrosine Kinase 3 Ligand (Flt3L) increases pDC content in the marrow, and that allo-BMT of Flt3L-treated marrow (FBM) grafts leads to higher overall survival and reduced GvHD in lethally irradiated hosts. Furthermore, FBM pDC have increased potency in preventing GvHD on a cell-by-cell basis (Hassan, EBMT, 2018). Interestingly, FBM pDC expressed CD11b, a myeloid-specific marker that is not expressed on currently defined pDC. Lineage ontogeny of pDC can be tracked based upon exclusive expression of CD31 and Ly6C on myeloid-progenitor derived pDC, while pDC from lymphoid progenitors express RAG1 and Siglec H. We hypothesized that treatment of murine bone marrow donors with Flt3L, which enhances their GvHD-reducing activity, modifies the distribution of lineage-specific precursors of pDC in bone marrow, and that lineage-associated pDC will have distinct biological activity in allo-BMT. Methods and Results: To assess the effect of Flt3L on BM pDC, we treated C57BL/6 mice with Flt3L (CDX-301, 300 ug/kg) on days -1 and -4 relative to bone marrow harvest. Using flow cytometric analysis, we show that FBM pDC over-express myeloid-specific markers CD31, Ly6C and CD11b, but down-regulate the lymphoid-specific marker Siglec H (Figure 1). Moreover, we observed similar phenotypic trends for myeloid-specific markers in pDC from the marrow of RAG1KO mice, confirming the existence of a unique myeloid-derived population of pDC. To determine the effect of pDC linage on their gene expression, we performed Illumina RNA Sequencing on human-derived pDC from Flt3L-mobilized peripheral blood and from human bone marrow. Interestingly, we found that Flt3L-treated pDC overexpress PRSS16, which is known to be exclusively expressed on cortical thymic epithelial cells. Furthermore, using GFP-transgenic mice as a source of donor pDC in B6 -> B10.BR allo-BMT, we show via confocal microscopy that donor pDC selectively home to the recipient thymus (Figure 2). Moreover, flow cytometric analysis revealed that homing of FBM pDC to the thymus is not impaired, in spite of decreased CCR9 expression on FBM pDC in comparison to BM pDC. Because it is currently believed that CCR9 expression on pDC is necessary for their homing to the thymus, these observations denote that FBM pDC use a CCR9-independent homing strategy. Since CD31 expression is upregulated in FBM pDC with enhanced immune-regulatory activity in allo-BMT models, these data suggest that local expression of CD31 by donor pDC in the thymic microenvironment may be relevant to their ability to favorably regulate thymopoiesis and limit the development of alloreactive T cells. Conclusions: Our data poses a link between pDC lineage and control of post-transplant GvHD, possibly via regulation of positive and negative selection of donor-derived T cells in the thymus. Specifically, we identify a CD31+ myeloid-specific population of pDC that emerges in the marrow upon treatment with Flt3L and exhibits a genetic profile that resembles thymic epithelial cells as a candidate for adoptive cell therapy to prevent GvHD. Disclosures Waller: Pharmacyclics: Other: Travel Expenses, EHA, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kalytera: Consultancy; Celldex: Research Funding; Cambium Medical Technologies: Consultancy, Equity Ownership.

scholarly journals CD4+ T Cell Fate in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4494-4494
Author(s):  
Rachel Elizabeth Cooke ◽  
Jessica Chung ◽  
Sarah Gabriel ◽  
Hang Quach ◽  
Simon J. Harrison ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Flow Cytometric Analysis ◽  
Advisory Committees ◽  
Mitochondrial Mass ◽  
Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Donor CD8+ T Cells Facilitate Graft-Versus-Tumor Effect Via Alloantigen Rather Than Tumor-Specific Antigen Recognition Following Murine Myeloablative BMT.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3051-3051
Author(s):  
Asha B. Pillai ◽  
Pearline A. Teo ◽  
Aditi Mukhopadhyay ◽  
Samuel Strober
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Specific Antigen ◽  
Flow Cytometric Analysis ◽  
Antigen Recognition ◽  
Wild Type ◽  
Flow Cytometric ◽  
Tumor Specific Antigen

Abstract Previous data from our group and others has shown that donor CD8+ Tcells mediate graft-versus-tumor (GVT) function in murine myeloablative bone marrow transplantation (BMT) via Perforin/Fas ligand-dependent mechanisms. However, there has to date been no analysis of the mechanism of tumor recognition (i.e allo- versus tumor-specific antigen recognition) by donor CD8+ T cells following myeloablative MHC-mismatched BMT. In order to test the hypothesis that donor CD8 T-cells require allo-antigen recognition to maintain graft-versus-tumor effect, we developed stable full chimeras by transplanting T-cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) donor mice into myeloablated BALB/c (H-2d) hosts given 800 cGy total body irradiation (TBI) and evaluated the in vivo ability of CD8+ TCR+ splenocytes from these donors to kill the BCL1 tumor (a BALB/c-derived B-cell lymphoma carrying a detectable tumor-specific idiotype which can be monitored via peripheral blood flow cytometric analysis). These chimeras showed complete donor chimerism in myeloid, B- and T-lymphocytic lineages by day +100 following transplantation, and splenocytes from these chimeras exhibited tolerance to host-type but not third-party alloantigens. BALB/c hosts were given 800 cGy TBI on day -1, followed on day 0 by intravenous administration of 500 BCL1 tumor cells and infusion of 0.3x 106 CD8+ T cells of C57 origin (H-2b+) sorted by flow cytometric analysis from the either spleens of the chimeric mice or from the spleens of untreated wild-type C57BL/6 mice. All hosts were given 5 x 106 T cell-depleted wild-type C57 bone marrow cells. All mice were observed for clinical signs of graft-versus-host disease (GVHD) and mortality through day +100. Autopsy was performed at death to assess for sub-clinical target organ involvement with GVHD or tumor. Donor chimerism and BCL1 status was assessed at day +28 and day +100 by 3-color flow cytometric analysis for donor-specific MHC versus T-, B- and myeloid lineage markers as well as tumor-specific idiotype in the peripheral blood (or in the spleen at time of death for animals dying prior to day +100). All animals receiving BCL1 tumor cells and sorted CD8+T cells from wild-type untreated C57 donors cleared tumor idiotype but succumbed to GVHD. All animals receiving tumor cells and sorted chimeric C57 CD8+ T cells remained free of clinical or pathologic evidence of GVHD, but died with tumor progression. Control myeloablated animals given C57 TCD BM alone with BCL1 tumor cells all succumbed to tumor, whereas those receiving C57 whole bone marrow with tumor demonstrated tumor survival without GVHD. The data indicate that chimeric donor peripheral CD8+ T cells, which lose their capacity to induce lethal GVHD in BALB/c hosts, also lose the capacity to eradicate BALB/c-type lymphoma cells. We conclude that CD8+ T cell-induced GVT effect in this model is dependent upon alloantigen rather than tumor-specific antigen recognition.

Identification of a Costimulation Blockade Resistant Mechanism of Bone Marrow Allograft Rejection Induced by Infection with a Murine EBV Homologue.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1461-1461
Author(s):  
Jonathan Beus ◽  
Kelly Hamby ◽  
Allan D Kirk ◽  
Christian P Larsen ◽  
Samuel H Speck ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Adhesion Molecules ◽  
Cell Population ◽  
Latent Infection ◽  
Memory T Cells ◽  
Transplant Rejection ◽  
Flow Cytometric Analysis ◽  
Costimulation Blockade ◽  
Flow Cytometric

Abstract Abstract 1461 Latent herpesvirus infections are ubiquitous and are important causes of transplant complications mediated by both direct viral pathology and anti-viral-related immune dysregulation. An emerging paradigm, heterologous immunity, suggests that alloreactivity arises in a subset of anti-viral memory T cells during infection, putting patients at increased risk for rejection. Murine γ-herpesvirus 68 (MHV68) provides a rodent model of human herpesvirus infections, including EBV, allowing the study of herpesvirus infections in transplantation. We have previously shown that latent infection with wild type (WT) MHV68 induces resistance to engraftment in a non-myeloablative, costimulation-blockade- (CoB) based bone marrow transplant model. Resistance to donor-engraftment was not observed in mice infected with a mutant virus lacking the viral M1 gene. (Stapler, et al. J Immunol 2008). Based on these findings, we sought to determine the mechanism(s) by which MHV68 induces heterologous alloimmunity and transplant rejection. B6 mice were infected i.p. with WT MHV68 or M1 mutant MHV68 and viral latency was defined as 6–8 weeks post infection (p.i.) after which some mice received allogeneic (BALB/c) BMT or skin grafts (SG). Transplanted mice received 0.5 mg each of anti-CD154 and CTLA4-Ig on days 0, 2, 4, and 6 relative to transplantation. BMT mice additionally received 0.6 mg busulfan on the day before transplant. Analysis of graft survival and multicolor flow cytometric analysis of immune phenotype was then performed. In addition to inducing rejection of donor bone marrow, as we previously observed, our new results indicate that latent infection with WT MHV68 decreases the length of allogeneic SG survival. While SG in non-infected, CoB-treated recipients have a mean survival time (MST) of 26 days (n = 5), MHV68 decreases this to 13 days (n=10, p=.0004*), similar to that in non-CoB treated recipients (11 days, n=5). This suggests a broad impact of latent MHV68 infection on transplant rejection. Flow cytometric analysis has revealed a unique cell population that may increase the risk of transplant rejection in MHV68-infected mice. MHV68 infection is associated with the expansion of a distinct CD8 T cell population with decreased per-cell CD8 expression and a unique phenotype, CD8Int. In non-infected mice, CD8Int are uncommon (11% of CD8s), but are highly induced by WT MHV68 (76%) and less so by an M1 mutant strain (35%, p<.0001*, Fig 1). CD8Int appear by 7 days p.i., and have been observed through >165 days p.i. In infected mice, T cells specific for two MHV68 epitopes (p56 and p79) are exclusively CD8Int by tetramer staining, identifying the CD8Int compartment as containing anti-viral T cells. Given the correlation of high-level induction of CD8Int and costimulation blockade-resistant transplant rejection, we sought to determine the mechanisms by which these cells might heterologously induce graft rejection. After infection with WT MHV68, CD8Int are significantly more activated vs CD8Nml (CD8 T cells with unchanged CD8 expression) as assessed by increased expression of CD44 (mean fluorescence intensity [MFI] 2746 vs 1640, p=.0025*) and KLRG1 (2553 vs 1003, p=.004*). Despite high KLRG1 expression, a significant fraction of CD8Int also express the memory marker CD127 suggesting that CD8Int consists of both effector and effector memory T cells. CD8Int exhibit decreased expression of the costimulatory molecules ICOS (MFI Int vs Nml, 171 vs 329, p<.0001*), 4-1BB (26 vs 84, p<0.0001*), and CD28 (416 vs 556, p=.078), consistent with a role for these cells in CoB-resistant rejection. Importantly, CD8Int demonstrate increased expression of the adhesion molecules LFA-1 (MFI Int vs Nml, 12965 vs 5630, p=.0001*) and VLA-4 (1511 vs 805, p=.002*). The specific upregulation of these molecules on activated T cells has been correlated with increased rejection risk in other models of transplant rejection. This observation thus raises the possibility that the CD8Int subpopulation may use heightened expression of adhesion molecules to subvert costimulation-blockade, and suggests that adjuvant therapy with clinically available agents blocking either the LFA-1 or VLA-4 pathways may be a therapeutic strategy to overcome herpesvirus-induced heterologous alloimmunity and rejection. We are currently testing these hypotheses directly in our murine models of MHV68-mediated rejection of both skin and bone marrow allografts. Disclosures: No relevant conflicts of interest to declare.

Characteristics of Immune Reconstitution by Mixed Umbilical Cord Blood Transplantation In Mice: Predominant Engraftment of Major Histocompatibility Complex (MHC)-Matched Umbilical Cord Blood Stem Cell In Recipients' Bone Marrow.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3721-3721
Author(s):  
Akira Nakano ◽  
Hideaki Sato ◽  
Ayumi Wakayama ◽  
Nobukata Shinohara ◽  
Kyoko Ito ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Survival Rates ◽  
Flow Cytometric Analysis ◽  
Antibody Responses ◽  
Flow Cytometric ◽  
Alloreactive T Cell

Abstract Abstract 3721 Background: Currently, umbilical cord blood cells (UCBCs) are used as the primary source of hematopoietic stem cells (HSCs) for transplantation instead of bone marrow cells (BMCs). UCBC transplantation has several advantages over BMC transplantation, including the much larger size of the available donor pool, the rich proportion of hematopoietic progenitor cells, and the low content of mature T cells that might cause a graft-versus-host reaction. However, the limited quantity of cord blood samples that can be obtained from a pregnant woman is considered to be a disadvantage of UCBC transplantation. To overcome quantity limitations, the use of mixed cord blood might be necessary. Due to the lack of appropriate animal models, the analyses of the differentiation capacity of mixed UCBCs in recipients have been limited to in vivo xenogeneic experiments and clinical observations. In this study, we evaluated the reconstitution and functioning of immune systems induced by mixed UCBC transplantation in mice. Method: To deplete NK cells, female C57BL/6 (B6) [H-2b] recipient mice were intraperitoneally administered rabbit anti-asialo GM1 antibody 1 day before transplantation. On the following day, the B6 recipients irradiated with a radiation dose of 9 Gray were transplanted with 3 different combinations of mixed UCBC {[1] GFP-Tg B6 (H-2b) and C3H (H-2k); [2] GFP-Tg B6 (H-2b) and BALB/c (H-2d); and [3] C3H (H-2k) and BALB/c (H-2d)}, with each combination containing equal number of cells. Engraftment of cells in the recipients' peripheral blood was detected by flow cytometric analysis using a specific antibody against lineage markers such as CD3e (T cells), CD45R/B220 (B cells), CD11b (macrophages), or Ly-6G (granulocytes) at intervals of 4 weeks for 16 weeks. In addition, the donor origin of each lineage population was determined by H-2Kk and/or H-2Kd antibody staining. GFP+ lineage cells were found to have B6 donor origin. The dermis samples harvested from the B6, C3H, and BALB/c mice were simultaneously skingrafted on the shaved backs of the recipients at over 16 weeks after transplantation. After the recipients rejected the skin graft, alloreactive T cell and antibody responses were examined to evaluate the functional maturity of the reconstituted immune system in the recipients. Result: The survival rates of the recipient mice at 16 weeks after transplantation were as follows: (1), 73%, 8/11; (2), 92%, 12/13; and (3), 100%, 1/1. Flow cytometric analysis showed that cells of all lineages were reconstituted by only GFP+ cells in almost all the B6 recipients transplanted with mixed UCBCs: (1), 50%, 4/8 and (2), 100%, 12/12. This findings indicated predominant bone marrow engraftment of UCBC-HSCs from MHC-matched B6 donors. Furthermore, the allogeneic UCBCs in the recipients transplanted with combination [3] were eliminated from the recipients, and their lineage cells (GFP− H-2Kk− H-2Kd−) in peripheral blood were derived from the B6 recipient's own X-ray-resistant HSC in the bone marrow. The recipient mice in which the immune system was reconstituted by UCBC-HSCs from the B6 donors accepted the skin graft from the B6 donors, but completely rejected the skin grafts from the C3H and BALB/c donors. This finding indicates that both CD8+ killer and CD4+ helper T cells were functionally mature in the recipients. Furthermore, the functional competence of both cellular and humoral immunity in the recipients that rejected the skin grafts from the C3H and BALB/c donors was determined by evaluating alloreactive T cell and antibody responses against H-2k and H-2d in an in vitro experiment. Conclusion: This study showed the promising potential of transplantation of mixed UCBCs for achieving high survival rates and functional immune reconstitution. We found that only MHC-matched HSCs were involved in hematopoiesis in the recipients' bone marrow. This finding suggests the presence of a surveillance system in bone marrow with the ability to distinguish self from non-self with different MHC antigens. Disclosures: No relevant conflicts of interest to declare.

Development Of Mature T Lymphocytes Requires Alpha-Synuclein

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3490-3490
Author(s):  
Afshin Shameli ◽  
Wenbin Xiao ◽  
Clifford Harding ◽  
Howard Meyerson ◽  
John Sumodi ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Bone Marrow ◽  
T Cells ◽  
T Lymphocytes ◽  
Blood Cell ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Alpha Synuclein ◽  
Lymphoid Organs ◽  
Flow Cytometric

Abstract Synucleins (including α-, β- and γ-synucleins) are a group of proteins that are expressed at high levels in the central nervous system. The physiologic function of these proteins is unknown. Alpha-synuclein has been implicated in the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body dementia, as it is highly expressed in the Lewy bodies from both disorders. The expression of α-synuclein in hematopoietic system has been shown in erythroid precursors and megakaryocytes in bone marrow, as well as erythrocytes and platelets in peripheral blood. Moreover, some studies demonstrated the expression of α-synuclein on peripheral blood mononuclear cells (PBMC), including B and T lymphocytes, NK cells and monocytes; and its expression is shown to be higher in PBMCs of individuals with Parkinson's disease compared to healthy controls. In order to study the role of α-synuclein in development of different hematopoietic elements, we compared bone marrow, peripheral blood and lymphoid organs of age and sex-matched α-synuclein knock-out (KO) mice and wild type (WT) animals of the same genetic background (n=10). Flow cytometric analysis of bone marrow elements did not show differences in the percentages and absolute numbers of erythroid, megakryocytic and myeloid lineages (data not shown). However, differential complete blood cell count (CBC) showed statistically significant decrease in red blood cell (RBC) count, hemoglobin (Hb) and hematocrit (Hct) in KO mice compared to WT mice. No difference was noted in other RBC indices (Table 1). However, platelets were smaller in KO mice as measured by the mean platelet volume (MPV). There was no difference in the number of platelets and white blood cell (WBC) counts. There was a significant reduction in the percentage of circulating lymphocytes, and associated increase in the percentage of neutrophils and monocytes in KO mice compared to WT mice, although the difference in the number of lymphocytes did not reach statistical significance (Table 1). Flow cytometric analysis of T lymphocytes in thymus and peripheral lymphoid organs demonstrated marked defect in development of mature T cells. There was a significant increase in the number of double negative thymocytes in KO mice associated with significant decrease in the number of single positive T cells. Furthermore, splenic CD4+ and CD8+ T cells were markedly decreased in KO mice, indicating that α-synuclein is required for T cell development (Table 2). In summary, our findings indicate an absolute requirement for α-synuclein in development of mature T lymphocytes. The underlying mechanism for this function is subject of future studies. Moreover, while α-synuclein-deficiency does not affect the development of myeloid lineage and platelets, lack of this protein is associated with lower number of erythrocytes, suggesting its role in development and/or survival red blood cells. Disclosures: No relevant conflicts of interest to declare.

Promoter IV of the class II transactivator gene is essential for positive selection of CD4+ T cells

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3550-3559 ◽  
Author(s):  
Jean-Marc Waldburger ◽  
Simona Rossi ◽  
Georg A. Hollander ◽  
Hans-Reimer Rodewald ◽  
Walter Reith ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Epithelial Cells ◽  
Positive Selection ◽  
Negative Selection ◽  
Cd4 T Cell ◽  
Thymic Epithelial Cells ◽  
Mhcii Expression ◽  
Selection Of

Major histocompatibility complex class II (MHCII) expression is regulated by the transcriptional coactivator CIITA. Positive selection of CD4+ T cells is abrogated in mice lacking one of the promoters (pIV) of the Mhc2ta gene. This is entirely due to the absence of MHCII expression in thymic epithelia, as demonstrated by bone marrow transfer experiments between wild-type and pIV−/− mice. Medullary thymic epithelial cells (mTECs) are also MHCII− in pIV−/− mice. Bone marrow–derived, professional antigen-presenting cells (APCs) retain normal MHCII expression in pIV−/− mice, including those believed to mediate negative selection in the thymic medulla. Endogenous retroviruses thus retain their ability to sustain negative selection of the residual CD4+ thymocytes in pIV−/− mice. Interestingly, the passive acquisition of MHCII molecules by thymocytes is abrogated in pIV−/−mice. This identifies thymic epithelial cells as the source of this passive transfer. In peripheral lymphoid organs, the CD4+T-cell population of pIV−/− mice is quantitatively and qualitatively comparable to that of MHCII-deficient mice. It comprises a high proportion of CD1-restricted natural killer T cells, which results in a bias of the Vβ repertoire of the residual CD4+ T-cell population. We have also addressed the identity of the signal that sustains pIV expression in cortical epithelia. We found that the Jak/STAT pathways activated by the common γ chain (CD132) or common β chain (CDw131) cytokine receptors are not required for MHCII expression in thymic cortical epithelia.

Fhl2 Regulates The Function Of Hematopoietic Stem/Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2414-2414
Author(s):  
Yu Hou ◽  
Zhijian Qian
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Progenitor Cells ◽  
Flow Cytometric Analysis ◽  
Myeloid Cells ◽  
Dependent Manner ◽  
Chimeric Mice ◽  
Hematopoietic Stem ◽  
Flow Cytometric

Abstract Fhl2 is a member of four and one half LIM domain protein family. It acts as a transcriptional modulator, regulating proliferation and apoptosis in a cell-context dependent manner. The role of endogenous Fhl2 in hematopoietic stem/progenitor cells (HSCs/HPCs) is still unknown. To determine the in vivo function of Fhl2 in hematopoiesis, we characterized the HSCs/HPCs in Fhl2-null mice. At age of 3 months, Fhl2-null mice revealed normal hematopoietic parameters. As shown by flow cytometric analysis, Fhl2-null mice had a normal distribution of Mac-1+Gr+ myeloid cells, B220+ B cells and CD3+ T cells in bone marrow and spleen, suggesting that Fhl2 loss does not affect mature cell differentiation in young mice. The frequency and total number of stem cell enriched population LSK (Lin-Sca+c-Kit+) and Long-term HSCs (LT-HSCs) (Lin-Sca+c-Kit+CD48-CD150+) were marginally increased in Fhl2-/- mice as compared to wildtype mice, whereas the frequency and the number of hematopoietic progenitor cells was reduced in Fhl2-/- mice as compared to Fhl2+/+ mice. Transplantation exposes HSCs to various stresses including replicative and oxidative stresses. To examine whether Fhl2 regulates the function of HSCs/HPCs under stress, we transplanted bone marrow cells from Fhl2-/- or control mice into lethally-irradiated wildtype mice to generate chimeric mice. 7 months after transplantation, the Fhl2-/- chimeric mice developed MDS-like disease with a significant decrease in platelet, red blood cell (RBC) counts and Hemoglobin (Hb) level as compared to control chimeric mice. The number of LT-HSC, LSK and HPCs in Fhl2-/- chimeric mice were significantly less than they were in control chimeric mice, as determined by flow cytometric analysis. However, the frequency of Mac-1+Gr-1+ myeloid cells, B220+ B cells and CD3+ T cells were comparable in Fhl2-/- and Fhl2+/+ chimeric mice, suggesting that Fhl2 loss reduces repopulation capacity of LT-HSCs but does not affect HPCs differentiation in recipient mice. As evidenced by the reduced G0 population of HSCs and LSKs in Fhl2-null chimeric mice as compared to control chimeric mice, Fhl2 may regulate HSCs self-renewal under stress by controlling HSC quiescence. To further determine the function of HSCs, we performed the competitive repopulation assay. In agreement with the observation that Fhl2-/- HSCs have a reduced repopulation capacity, the ratio of Fhl2-/- derived total Peripheral Blood (PB) cells vs. competitor-derived PB cells was significantly decreased as compared to Fhl2+/+-derived recipients at secondary transplantation, and the ratio gradually decreased up to 4-5-fold as compared to Fhl2+/+-derived recipients at tertiary transplantation. Previously, we showed that FHL2 is down-regulated in subsets of Therapy-related Myelodyplastic Syndrome or Acute Myeloid Leukemia (t-MDS/t-AML patients). Together, these data suggest that Fhl2 is required for maintenance of HSCs/HPCs, and its downregulation may contribute to the development of t-MDS/t-AML in patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gene Expression Profile of Circulating Myeloma Cells Reveals CD44 and CD97 (ADGRE5) Overexpression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5639-5639
Author(s):  
Tereza Sevcikova ◽  
Fedor Kryukov ◽  
Lucie Brozova ◽  
Jana Filipova ◽  
Zuzana Kufova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Research Funding ◽  
Plasma Cells ◽  
Flow Cytometric Analysis ◽  
Differentially Expressed ◽  
Paired Samples ◽  
Flow Cytometric

Abstract Introduction: Release of the aberrant plasma cells (PC) from the bone marrow (BM) and their presence in the peripheral blood (PB) is a maker of disease progression and worse survival in multiple myeloma (MM) (Nowakowski et al., 2005). Circulating plasma cells (cPCs) are able to survive without homing microenvironment, evade the original tumor and colonize other bone marrow niche. Detailed analysis of various surface proteins showed that cPCs display decreased levels of integrins, adhesion molecules N-CAM (CD56) and the stem cell factor receptor (Paiva et al., 2013). Comprehensive analysis of the genome-wide gene expression profiling that could provide deeper insight into the expression patterns of cPCs of MM is still lacking. Aims: To identify differentially expressed genes in paired samples of aberrant plasma cells from BM and PB and to describe potential biomarkers of cPCs in MM. Material and methods: Ten patients with multiple myeloma (seven new diagnoses and three relapses) have been included in the study after signing the informed consent form. Paired samples of aberrant plasma cells from bone marrow and peripheral blood were obtained from each patient. Aberrant plasma cells (aPCs) were sorted according to the immunophenotype as CD45dim/CD38+/CD19-/CD56-/+ cells. Gene expression profiling (GEP) was performed on paired samples using Affymetrix GeneChip Human Gene ST 1.0 array. RMA normalized data at gene level were analyzed using Wilcoxon paired test with Benjamini-Hochberg multiple testing correction. Results: The median infiltration of aberrant PC in the BM was 27.5% (range 1.1 - 93%) and 1.2% (range 0.19 - 2.8%) for cPCs in the PB. The median level of M-protein was 32.35 g/l (range 18.6 - 62.2 g/l). GEP analysis of paired BM and PB samples revealed 1001 significantly changed genes in cPCs (adjusted p-value<0.05). Gene ontology analysis did not reveal any significantly affected pathways. Nevertheless, two genes upregulated in cPCs, ADGRE5 and CD44, can be suggested as biologically relevant potential biomarkers of cPCs (Figure 1). Conclusion: The infiltration of aPCs in the bone marrow does not correlate with the amount of cPCs (p=0.16). Among differentially expressed genes, two surface markers upregulated in cPCs are of particular interest: CD44 and ADGRE5 (CD97). The CD44 antigen is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. Moreover, CD44 contribute to lenalidomide resistance in multiple myeloma (Bjorklund et al., 2014). CD97 is encoded by ADGRE5 gene and belongs to the EGF-TM7 subgroup of adhesion G-protein-coupled receptors. The expression of CD97 has been linked to invasive behavior in thyroid and colorectal cancer. Moreover, higher CD97 expression levels have been detected in 54% (208/385) of primary AML samples based on flow cytometric analysis (Wobus et al., 2015). Nevertheless, neither ADGRE5 nor CD97 expression were described in plasma cell dyscrasia previously. Thus, despite non-systemic changes of gene expression at the whole transcriptome level, cPCs in MM likely represent distinct biological entity with specific expression profile underlying advanced PC malignant transformation. To confirm the results, flow cytometric analysis on the bigger cohort will be performed. Acknowledgment: This study was supported by Institutional Development Plan of University of Ostrava (IRP201550) and The Ministry of Education, Youth and Sports (Specific university research of the Faculty of Medicine, University of Ostrava) project no. SGS03/LF/2015-2016, Ministry of Health Czech Republic RVO-FNOs/2014/17P and RVO-FNOs/2016/21. Figure 1 Genes of interest differentially expressed in the bone marrow (BM) versus peripheral blood (PB) aberrant plasma cells. Figure 1. Genes of interest differentially expressed in the bone marrow (BM) versus peripheral blood (PB) aberrant plasma cells. Disclosures Hajek: BMS: Honoraria; Onyx: Consultancy; Novartis: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

scholarly journals Interleukin 7 Transgenic Mice Develop Chronic Colitis with Decreased Interleukin 7 Protein Accumulation in the Colonic Mucosa

1998 ◽  
Vol 187 (3) ◽  
pp. 389-402 ◽  
Author(s):  
Mamoru Watanabe ◽  
Yoshitaka Ueno ◽  
Tomoharu Yajima ◽  
Susumu Okamoto ◽  
Tatsuhiko Hayashi ◽  
...  
Keyword(s):  
T Cells ◽  
Epithelial Cells ◽  
Transgenic Mice ◽  
Chronic Inflammation ◽  
Colonic Mucosa ◽  
Flow Cytometric Analysis ◽  
Receptor Expression ◽  
Protein Accumulation ◽  
Chronic Colitis ◽  
Interleukin 7

We have demonstrated that intestinal epithelial cells produce interleukin 7 (IL-7), and IL-7 serves as a potent regulatory factor for proliferation of intestinal mucosal lymphocytes expressing functional IL-7 receptor. To clarify the mechanism by which locally produced IL-7 regulates the mucosal lymphocytes, we investigated IL-7 transgenic mice. Here we report that transgenic mice expressing murine IL-7 cDNA driver by the SRα promoter developed chronic colitis in concert with the expression of SRα/IL-7 transgene in the colonic mucosa. IL-7 transgenic but not littermate mice developed chronic colitis at 4–12 wk of age, with histopathological similarity to ulcerative colitis in humans. Southern blot hybridization and competitive PCR demonstrated that the expression of IL-7 messenger RNA was increased in the colonic mucosal lymphocytes but not in the colonic epithelial cells. IL-7 protein accumulation was decreased in the goblet cell–depleted colonic epithelium in the transgenic mice. Immunohistochemical and cytokine production analysis showed that lymphoid infiltrates in the lamina propria were dominated by T helper cell type 1 CD4+ T cells. Flow cytometric analysis demonstrated that CD4+ intraepithelial T cells were increased, but T cell receptor γ/δ T cells and CD8α/α cells were not increased in the area of chronic inflammation. Increased IL-7 receptor expression in mucosal lymphocytes was demonstrated in the transgenic mice. These findings suggest that chronic inflammation in the colonic mucosa may be mediated by dysregulation of colonic epithelial cell–derived IL-7, and this murine model of chronic colitis may contribute to the understanding of the pathogenesis of human inflammatory bowel disease.

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