Immune Response In Bone Marrow Research Articles

Reduced bone formation and increased bone resorption drive bone loss in Eimeria infected broilers

Coccidiosis is an economically significant disease in the global poultry industry, but little is known about the mechanisms of bone defects caused by coccidiosis; thus, the study focused on effects of coccidiosis on the bone homeostasis of young broiler chickens. A total of 480 male Cobb500 broilers were randomly allocated into four treatment groups, including an uninfected control consuming diet ad libitum, two infected groups were orally gavaged with two different concentrations of sporulated Eimeria oocysts, and an uninfected pair-fed group fed the same amount of feed as the high Eimeria-infected group consumed. Growth performance and feed intake were recorded, and samples were collected on 6 days post infection. Results indicated that coccidiosis increased systemic oxidative status and elevated immune response in bone marrow, suppressing bone growth rate (P < 0.05) and increasing bone resorption (P < 0.05) which led to lower bone mineral density (P < 0.05) and mineral content (P < 0.05) under Eimeria infection. With the same amount of feed intake, the uninfected pair-fed group showed a distinguished bone formation rate and bone resorption level compared with the Eimeria infected groups. In conclusion, inflammatory immune response and oxidative stress in broilers after Eimeria infection were closely associated with altered bone homeostasis, highlighting the role of inflammation and oxidative stress in broiler bone homeostasis during coccidiosis.

Bone Marrow CD8 T Cells Express High Frequency of PD-1 and Exhibit Reduced Anti-Leukemia Response in AML Patients

Acute myeloid leukemia (AML) is a devastating blood cancer with 5- year survival of only 25%. Optimizing anti-leukemia immune response to improve clinical outcome is a promising strategy for novel effective leukemia therapeutics. However due to limited accessibility, the majority of studies have been restricted to evaluate samples from peripheral blood. The immune response in bone marrow of AML is poorly understood. In this study, we aim to investigate the T cell immune response in bone marrow samples derived from AML patients.Samples collected from a cohort of patients with newly diagnosed AML (n=19) were used in this study. Mononuclear cells of bone marrow and peripheral blood were isolated from clinical samples before multi-channel flow cytometry analysis. We observed a comparable frequency of CD3+ T cells among lymphocytes in bone marrow and peripheral blood. Similarly there is no difference of CD4, CD8 T cell frequency, or CD4/CD8 ratio. However, when T cell subsets for each differentiation stage were analyzed based on the expression of CD45RA and CCR7, we found significantly increased effector memory T cells (CD45RA-CCR7-) among CD8 T cells in bone marrow compared with that in peripheral blood (43.88±5.012% vs. 35.15±5.104%, P =0.0088). Whereas the percentage of terminally differentiated effector cells (CD45RA+CCR7-) was significantly lower in bone marrow (20.36±5.025% vs. 28±5.947%, P =0.0481). No difference was found in the frequency of naive (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) subsets.Several studies including ours have demonstrated an involvement of the inhibitory receptors PD-1, TIM-3 and TIGIT in AML progression. Most of the studies were performed using clinical samples from peripheral blood. To evaluate whether the effect of inhibitory pathways is different between bone marrow and peripheral blood, we assessed the expression of these inhibitory receptors on T cells derived from both sites. We observed comparable expression of TIGIT and TIM-3 in bone marrow and peripheral blood. The frequency of PD-1 expression on CD8 T cells was however significantly higher in bone marrow (39.55±3.809% vs. 32.8±3.512%, P =0.0058). We further dissected the intracellular expression of Eomesodermin (Eomes) and T-bet, the two T box transcription factors that are crucial in regulating T cell function. We found that the frequency of Eomes+T-betint CD8 T cells was significantly higher in bone marrow than in peripheral blood (17.01±1.843% vs. 14.95±1.682%, P =0.0405), indicating a predominance for late stage of T cell exhaustion status in bone marrow. This result suggests a relatively more suppressive environment in bone marrow compared with that of peripheral blood.We next examined the functional status of CD8 T cells. Intracellular IFN-γ and TNF-α production was assessed upon in vitro stimulation with anti-CD3/CD28. Intriguingly no significant difference between bone marrow and peripheral blood was detected. Consistently we observed no difference between the two sites in term of the expression of Ki67, Granzyme B, and Perforin, suggesting a comparable proliferation and killing capacity of CD8 T cells between bone marrow and peripheral blood. We further investigated the function of leukemia-reactive CD8 T cells by testing T cell response against a WT-1 peptide. WT-1 is a well known tumor-associated antigen, in which HLA-A*0201 restricted WT-1126-134 is one of the mostly studied epitope in AML. Purified CD8 T cells derived from bone marrow or blood of newly diagnosed HLA-A*0201 AML patients were co-cultured with T2 cells (used as antigen presenting cells) pulsed with WT-1126-134. We found a significantly lower production of IFN- γ by CD8 T cells from bone marrow compared with that from peripheral blood (0.4325±0.1612% vs, 0.7425±0.2809%, P =0.0472). These data demonstrate that although the total CD8 T cells in bone marrow remain functional, the leukemia-reactive CD8 T cells are less functional in bone marrow compared with that in peripheral blood.Collectively, our data demonstrate that bone marrow CD8T cells from AML patients express higher frequency of PD-1 and exhibit reduced anti-leukemia response. Our study suggests that bone marrow is a unique anatomical location fostering negative immune response in AML, thus targeting the negative regulatory pathways in bone marrow is of great potential to develop novel effective leukemia treatment. DisclosuresNo relevant conflicts of interest to declare.

Cross Talk between the Immune Compartment and the Tumor Cells in Myelodysplastic Syndromes (MDS)

Background: Abnormalities of the immune system and innate immune signaling have been described in patients (pts) with MDS, however the interaction between the MDS tumor cells and the immune compartment is poorly defined. Immune modulatory therapies are being explored in MDS and a better understanding of the interaction and potential cross-talk with immune components and MDS tumor cells is critical in developing new therapeutic strategies. Methods: We developed a multiscale immune profiling strategy to map the immune environment of treatment naïve pts with lower- and higher-risk MDS, using mass cytometry by time of-flight (CyTOF) combined with Olink proteomics analyses. We obtained bone marrow (BM) and peripheral blood (PB) from 33 pts with MDS, (17 higher-risk; 16 lower-) and 10 healthy donors. Pts were representative of the MDS distribution across age, gender, mutational status and histologic subtype. PB and BM were freshly processed and stained with an antibody panel for myeloid cells. For the lymphoid panel, all the pts' samples were stained simultaneously to avoid batch effect. We processed the BM and the PB plasma for Olink. Results: We profiled myeloid cells, NK cells and T cells in pts with MDS to determine differences in their constituents. Phenograph clustering across all pts revealed distinct T lymphoid metaclusters, that corresponded to known immune cell populations. We observed a robust immune response in the BM from MDS patients. T cells and myeloid cells were found in equal proportion in the BM from MDS and healthy patients. However, the B cell population were present at significantly lower frequency in the MDS BM (p&lt;0.05), whereas gamma delta (gd)T cells and NK cells were at significantly higher frequency in the MDS pts (p &lt;0.05). Paired mass cytometric analysis revealed a distinct composition and phenotype of T cell subsets. Among the T cell compartment, regulatory T cells (Treg) (p&lt;0.001), cytolytic CD8 T cells (expressing perforin (p&lt;0.01) or granzyme (p&lt;0.001)) and exhausted CD8 T cells (expressing PD-1 (p= 0.09), LAG-3 (p&lt;0.001) or TIM-3) seemed to accumulate in the MDS BM. Compared to healthy BM Treg in MDS BM expressed higher levels of TIGIT, TIM3, CD27, CD25, CD28 and OX40. Cytolytic NK cells accumulate in MDS BM (p&lt;0.05). In addition to measuring adaptive lymphocyte responses to tumors, we also analyzed the distribution of innate lymphocytes and in particular NK cells in MDS BM. In the NK cell population, the cytotoxic NK cells (CD56dim CD16+) were found in a higher proportion (p&lt;0.05) than the cytokinic counterpart (CD56bright) in the MDS BM. NK cells that accumulate in the MDS BM expressed higher level of CD45RA, CD38, granzyme B, CD56 suggesting their superior cytotoxic potential at a critical developmental stage of retaining enhanced sensitivity to pro-inflammatory cytokines derived from myeloid cells. We also analyzed a panel of 92 unique cytokines, chemokines and inflammatory markers produced in the tumor milieu using OLink Proteomics proximity extension assay. Many chemokines and cytokines were differently expressed in the MDS BM compared to the healthy BM, suggesting an important inflammatory reaction. Conclusion: These data demonstrate new findings suggesting interactions and cross-talk between Tregs, NK cells, CD8 T cells and gdT cells in the BM of pts with untreated MDS. The data also suggest that there is a strong immune response in the MDS BM, particularly an intense cytotoxic response with the accumulation of cytotoxic CD8 T cells, NK cells and gdT cells. These results are are contrary to those observed in most solid tumors. This cytotoxic response seemed to be counteracted by the presence of Treg. Each of the subgroups of T cells (as well as NK cells) have been implicated as possibly promoting immune tolerance and tumor growth in different model systems and other tumor types. NK cells and gdT cells are known to respond to virus-infected and transformed cells rapidly and without the need for prior sensitization. However, anecdotal evidence from numerous independent studies may suggest that a strong but prolonged NK cell- and gdT cell-mediated antiviral response may result in dysregulation and promote tumorigenesis and/or aggravate pathology. Additional studies are underway to further characterize the immune landscape in those untreated and treated and to study the interaction of the immune components with tumor cells. Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents &amp; Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding.

Accumulation of Myeloid-Derived Suppressor Cells in Bone Marrow in Multiple Myeloma Induces Tumor-Specific Immune Suppression and Promotes Tumor Growth

Abstract 3954Multiple myeloma (MM) is an incurable malignancy of clonal plasma cells in bone marrow (BM). Accumulation of MM cells in BM is associated with a corresponding dysfunction of local immune responses. The mechanisms involved in the immune suppression remains poorly understood. Myeloid-derived suppressor cells (MDSC) are heterogeneous group of relatively immature myeloid cells characterized by potent immune suppressive activity. However, their role in regulation of immune responses in BM in MM remains unclear.To determine MDSC accumulation in MM patients, BM and blood samples were obtained from newly diagnosed patients and healthy donors. We observed a significant increase in CD11b+ CD33+ CD14− HLA-DRlo MDSCs, but not in CD11b+ CD33+ CD14+ HLA-DRhi monocytes, in the blood and BM of patients as compared to cells with the same phenotype in healthy donors. MDSC isolated from patient BM suppressed T cell responses as measured by IFN-g Elispot assays of mixed leukocyte reactions. Immature myeloid cells with the same phenotype from BM of healthy donor did not inhibit T cells.The possible role of MDSC in MM progression was further evaluated in mouse model of MM. Three different MM cell lines (BCM, DP42 and ATLN) obtained from Dr Van Ness (University of Minnesota) were used. MM tumors were established by i.v. injection of syngeneic mice. We observed an expansion of CD11b+ GR1+ MDSCs (up to 70%) in the BM of tumor-bearing (TB) mice within one week after injection of tumor cells. MDSC purified from BM of TB mice at that time point potently suppressed antigen-specific T cell proliferation and IFN-g production. As tumor progressed, CD138+ MM cells expanded (up to 90%) in the BM replacing normal BM cells including MDSC. However, at later time points (2–3 weeks), MDSC expansion was seen in spleen and lymph nodes of TB mice.To evaluate the possible role of MDSC in MM progression, we used S100A9 knockout (KO) mice. S100A9 plays an important role in MDSC accumulation in cancer and S100A9 KO TB mice had reduced accumulation of these cells. When wild-type (WT) and S100A9 KO mice were inoculated with DP42 cells no differences in tumor growth and survival were observed. S100A9 KO mice had a decrease in MDSC accumulation in the BM of TB mice compared to WT mice. Additionally, MDSC from S100A9 KO mice failed to suppress antigen-specific T cells ex vivo, whereas MDSC from WT TB mice did. To evaluate the development of antigen-specific immune responses in MM, we generated a MM cell line over-expressing the chicken ovalbumin protein (DP42-OVA). S100A9 KO mice injected with the immunogenic DP42-OVA cell line showed significantly delayed tumor growth and improved survival as compared with WT mice. Further analysis of T cell responses in vivo showed increased accumulation of antigen-specific T cells within the BM of S100A9 KO mice, but not WT mice, as early as one week post-tumor inoculation. Antigen-specific T cells in spleens were not detected at that time point. These cells were seen in spleens on S100A9 KO mice only 2–3 weeks after tumor injection.Thus, our study demonstrated that MM is associated with rapid accumulation of MDSC with potent immune suppressive activity against tumor-specific T cells. Our data directly suggest that targeting MDSC in MM may improve antitumor immune response and clinical outcome of the disease. Disclosures:No relevant conflicts of interest to declare.

Nonplasmacytoid, High IFN-α–Producing, Bone Marrow Dendritic Cells

Plasmacytoid dendritic cells (pDC) are the producers of type I IFNs in response to TLR9 ligands. However, we have found that when bone marrow is depleted of pDC, the IFN-α produced in response to TLR9 ligands is not fully removed. We assign the source of this non-pDC IFN-α as a newly described DC type. It displays the high IFN-α producing activity of pDC but to a more limited range of viruses. Unlike pDC, the novel DC display high T cell stimulation capacity. Moreover, unlike mouse pDC, they are matured with GM-CSF and are less prone to apoptosis upon activation stimuli, including viruses. We propose that these DC constitute a novel bone marrow inflammatory DC type, ideally geared to linking innate and adaptive immune responses in bone marrow via their potent IFN-α production and high T cell stimulatory capacity.

Experiment Therapy of Aplastic Anemia Using Cyclosporin A Combined with IDO Agonist.

Aplastic anemia(AA) is an unusual hematologic disease and the paradigm of the human bone marrow failure syndromes. Abnormal immunological responses have been found in most aplastic patients. Activated type 1 cytotoxic T cells may be the main contributing factor of pathogenesis. IDO is a rate-limiting enzyme in Tryptophan(Trp) metabolism. The proliferation of Th1 cells is specifically inhibited by the over-expression of IDO, which can degrade the Trp in local environment, hamper the conductioin of the activating signal in T cell and induce immune tolerance. 3,4-DAA is an IDO agonist and can activate IDO. In our experiment, Balb/c mice were irradiated (5.0Gy of 60Co), and then infused with 5×106 lymph node(LN) cells from DBA/2 mice in 4 hours. Blood count was monitored and marrow damage was assessed by histogical study. Concentrations of serum IFN-γ were measured by ELISA. The levels of Tryptophan and kynurenine were evaluated by high performance liquid chromatography (HPLC). CD4+CD25+ T cells in spleen were analyzed by flow cytometry. The level of Foxp3 in CD4+CD25+ T cell was measured by RT-PCR. Irradiation and infusion of LN cells led to rapid development of severe pancytopenia and bone marrow hypoplasia. Bone marrow of affected mice showed lymphocyte infiltration. Serum IFN-γ concentration increased 3.7 fold at d6 post infusion. The recipient mice were divided into 4 different treating groups as follow:1.0.9% Sodium Chloride as control;2.Cyclosporin A (CsA) (50ug/g/d ×5d, peritoneal injection);3.3,4-DAA(5mg/d, orally daily);4.the combination of CsA and 3,4-DAA.The effects of CsA, IDO agonist and CsA combined IDO agonist were analyzed at day 6,10,14,21,24 and 28 after LN cells infusion. The white cell and the platelet recovered to near normal, respectively (4.2±0.32)×109, (937±190.47)×1012 at d21 in the combination group. Early stage treatment with CsA can improve periphery blood cells and BM nucleated cells, but long term effect was not remarkable. In contrast, the 3,4-DAA group exhibited slow and gradually enhanced role. Periphery blood cells and BM nucleated cells were improved remarkably in the combination group. The number and function of CD4+CD25+T cells also increased remarkably. In the treatment of AA, abnormal immune response in bone marrow was inhibited by CsA, meanwhile immune tolerance was induced through up-regulating the regulatory T cells(Treg) by 3,4-DAA. In this way, the balance of immune in bone marrow could be reestablished quickly. CsA combined IDO agonist could provide a new strategy for the management of AA.

Mesenchymal Stem Cells (MSC) Exhibit Antigen Presenting (APC) and Phagocytic Properties: Implicatins to Bnone Marrow Failure during Inflammation.

Mesenchymal stem cells (MSC) mostly surround the vasculature system of bone marrow (BM). MSC have been shown to exhibit immune suppressive properties. Since MSC express MHC Class II antigen, the question is whether these cells can act as APC. To this end, we hypothesize that MSC have the ability to present non-self antigens while acting as immune modulators. These dual roles of MSC prevent exacerbated inflammatory responses in the BM, thereby preventing hematopoietic dysfunction.A ‘dampened' immune response in BM during insults by foreign agents could cause protection of the barrier that separates BM cavity with the periphery. The phagocytic role of MSC was shown by confocal microscopy and fluoresbrite plain YG 1.0-micron microspheres. APC property was demonstrated by challenging MSC with C. albicans (pulsed MSC), followed by exposure to CD4+ cells. The latter was obtained by immunoselection from peripheral blood mononuclear cells (PBMC) cultured for 5 days with C. albicans (10 mg/ml). Proliferation of the CD4+ cells (3H-thymidine incorporation and cell counts) proved APC properties of MSC, at efficiency comparable to macrophages. Overall, the studies show that the window between APC function and the period at which MSC could become immune suppressive is critical, since activated T-cells could destroy the endothelial barrier between BM and lymphatics/peripheral circulation. These studies show that MSC could be key cells in regulating immune responses in BM, and thereby protect BM from failure.

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A rabbit anti- Rana anti-DNP antiserum was used in order to estimate changes in frequency of bone marrow (BM) cells containing cytoplasmic anti-DNP antibodies (cId +) for eight weeks following a single injection. We found that (i) cId + cells increased from 0.2 to 4.3 percent of total mononuclear cells (MNC's) during the first 8 weeks, (ii) BM granulocytes increased in frequency up to week 2, then gradually decreased to week 8, and (iii) serum anti-DNP antibody levels, evaluated by ELISA, increased to week 8. Our observations (i) support those which suggest that Rana BM resembles mammaliam BM as a source of antibody-producing cells, (ii) indicate that specific immune responses in BM may result in a 20-fold increase in the frequency of antigen-related plasma cells among total MNC's, and (iii) suggest that BM B cell clonal expansion, maturation and secretion may result in an increase in specific antibody levels in serum.