Bone marrow-derived mesenchymal stromal/stem cells (MSCs) are nonhematopoietic cells that are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In addition, they are known to participate in niche formation for hematopoietic stem cells and to display immunomodulatory properties. Conventionally, these cells are functionally isolated from tissue based on their capacity to adhere to the surface of culture flasks. This isolation procedure is hampered by the unpredictable influence of secreted molecules, the interactions between cocultured hematopoietic and other unrelated cells, and by the arbitrarily selected removal time of nonadherent cells before the expansion of MSCs. Finally, functionally isolated cells do not provide biological information about the starting population. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSCs based on the selective expression, or absence, of surface markers. In this report, we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow- and amnion-derived MSCs.

The association between blood cell development and progressive cell death in the brain of Parkinson's patients should be further investigated as a potential dynamic biomarker and indicator of disease progression.

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Dendritic cells (DCs) arise from hematopoietic stem cells and develop into a discrete cellular lineage distinct from other leucocytes. Mainly three phenotypically and functionally distinct DC subsets are described in the human peripheral blood (PB): plasmacytoid DCs (pDCs), which express the key marker CD303 (BDCA-2), and two myeloid DC subsets (CD1c+ DC (mDC1) and CD141+ DC (mDC2)), which express the key markers CD1c (BDCA-1) and CD141 (BDCA-3), respectively. In addition to these primary cell subsets, DCs can also be generated in vitro from either CD34+ stem/progenitor cells in the presence of Flt3 (Fms-related tyrosine kinase 3) ligand or from CD14+ monocytes (monocyte-derived DCs (mo-DCs)) in the presence of granulocyte–macrophage colony-stimulating factor+interleukin-4 (GM-CSF+IL-4). Here we compare the reactivity patterns of HLDA10 antibodies (monoclonal antibody (mAb)) with pDCs, CD1c+ DCs and CD141+ DCs, as well as with CD14+-derived mo-DCs cultured for 7 days in the presence of 100 ng/ml GM-CSF plus 20 ng/ml IL-4. A detailed profiling of these DC subsets based on immunophenotyping and multicolour flow cytometry analysis is presented. Using the panel of HLDA10 Workshop mAb, we could verify known targets selectively expressed on discrete DC subsets including CD370 as a selective marker for CD141+ DCs and CD366 as a marker for both myeloid subsets. In addition, vimentin and other markers are heterogeneously expressed on all three subsets, suggesting the existence of so far not identified DC subsets.

We observed that the immune checkpoint protein B7-H3 is overexpressed in acute myeloid leukemia (AML) patients with poor treatment outcomes. Inhibition of B7-H3 expression or blocking of its activity using a novel monoclonal antibody (T-1A5) in AML cells significantly enhanced NK cell-mediated cytotoxicity in AML cells in vitro and in vivo. Moreover, human-mouse chimera of this antibody (ChT-1A5) induced antibody-dependent cell-mediated cytotoxicity (ADCC) in B7-H3+ primary AML cells, but not in normal hematopoietic cells, suggesting the specify of this antibody for AML cells. Epitope mapping studies identified that both T-1A5 and ChT-1A5 antibodies bind to the FG-loop region of B7-H3, which is known to regulate the immunosuppressive function of B7-H3. Furthermore, treatment with ChT-1A5 in combination with human NK cells significantly prolonged survival in AML patient-derived xenograft models. Our results suggest that ChT-1A5 antibody can inhibit the immunosuppressive function of B7-H3 protein as well as induce ADCC in B7-H3+ AML.

The results of the study indicate that localized PC is associated with early specific changes of the RANKL pathway in serum and bone marrow (BM). These changes might be part of the pre-metastatic niche of PC and implicate a potential benefit of RANKL inhibition in patients with localized PC.

Acute myeloid leukemia (AML) is the most common and aggressive acute leukemia found in adults. Immune checkpoint inhibition has led to important clinical advances in cancer therapy in recent years due to superior cure rates compared with standard therapy. We hypothesize that B7-H3 (CD276) an immune checkpoint protein is overexpressed in AML cells and targeting B7-H3 activates immune cells against AML cells. We analyzed B7-H3 expression in peripheral blood (PB) and bone marrow (BM) mononuclear cells from AML patients (n=65) and healthy donors (n=10) at MD Anderson Cancer Center. Cell surface expression analysis by flow cytometry revealed that the cells of ~60% of the patients were positive for B7-H3 and its expression was 2- to 3-fold higher in AML cells than in healthy donor cells. B7-H3 expression is relatively higher in CD34+ AML cells than in CD34- AML cells (p<0.01). In contrast, no difference was observed between CD34+ and CD34- cells from healthy donors. The Cancer Genome Atlas RNA sequencing data revealed that patients with high B7-H3 expression had significantly lower overall and disease-free survival durations than did patients with low B7-H3 expression (p=0.024). To investigate the role of B7-H3 in immunomodulation, we stably knocked down B7-H3 in OCI-AML3 and co-cultured them with or without human PB-derived NK cells at a 2:1 ratio and measured apoptosis induction in AML cells by annexin-v binding approach. We found that knockdown of B7-H3 induced NK cell-mediated apoptosis in AML cells 3-fold compared to control AML cells. These data indicate that inhibition of B7-H3 in AML cells enhances NK cell-mediated apoptosis in AML cells. To target B7-H3, we have generated four monoclonal antibodies: B1, B2, B3 and B4 (codenamed to protect IP). To investigate whether these novel anti-B7-H3 monoclonal antibodies are able to block B7-H3 immunomodulatory function and activate NK cells, we performed a co-culture experiment with GFP-expressing OCI-AML3 cells and PB-derived NK cells in the presence or absence of anti-B7-H3 antibodies. Apoptosis induction was measured by real-time annexin-v binding using IncuCyte live cell imaging system. The addition of anti-B7-H3 monoclonal antibodies, B1, B2, B3 and B4 at 25μg/ml enhanced NK cell-induced apoptosis 3-fold in OCI-AML3 cells. These data indicate that anti-B7-H3 antibodies block the immunomodulatory function of B7-H3 and induce NK cell-mediated apoptosis in AML cells. In vivo testing of these antibodies against AML-PDX models is currently ongoing. In conclusion, we found that B7-H3 is overexpressed in AML cells and its expression is associated with bad prognosis in AML patients. Knockdown or antibody-mediated blocking of B7-H3 enhanced NK cell-induced apoptosis in AML cells. These data indicated that B7-H3 is a novel immune-checkpoint protein in AML and patients could potentially benefit from anti-B7-H3 therapies. Citation Format: Stanley Ly, Bin Yuan, Sabrina Grimm, Michael Andreeff, Hans-Jörg Bühring, Venkata Lokesh Battula. B7-H3, an immune checkpoint protein is overexpressed in AML and the blocking monoclonal antibodies enhance NK cell-mediated apoptosis in AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3248.

Background: The immune checkpoint molecule B7-H3 (CD276) is overexpressed in various solid tumors and hematological malignancies; however, its expression is limited in normal tissue, which makes it an attractive therapeutic target in cancer. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in acute myeloid leukemia (AML) remains unexplored. Here, we hypothesized that targeting B7-H3 using mAbs alters the immunomodulatory function of B7-H3 and enhances NK cell-mediated cytotoxicity against AML cells. Methods: B7-H3 protein expression was analyzed in the peripheral blood (PB) and bone marrow of 100 patients with AML and 20 healthy donors by flow cytometry and tested for associations with multiple clinical parameters and disease outcomes. To investigate B7-H3's role in immunomodulation, we stably knocked down B7-H3 in AML cell lines including OCI-AML3, MV4-11, and U937 and co-cultured them with activated human NK cells. NK cell-induced apoptosis was measured by annexin-v binding assay using an IncuCyte live-cell imaging system. B7-H3-blocking mAbs (clones T-1A5, HEK5-1B3, and 58B1) were tested for their effect on NK-cell-mediated cytotoxicity in AML cell lines using live-cell imaging. AML xenograft (OCI-AML3) or patient-derived xenograft (PDX) models were used to determine the effect of anti-B7-H3 antibodies on AML growth. In vivo AML growth was monitored by measuring human CD45 positive cells with flow cytometry. A human-mouse chimeric (ch) antibody was generated based on sequences from the T-1A5 antibody, and its binding site on the B7-H3 protein was characterized by epitope mapping. Further, we evaluated the effect of chT-1A5 on NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in primary AML cells and healthy donor PB-derived mononuclear cells (PBMCs). Results: Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p < 0.01) and was higher in CD34 positive than in CD34 negative AML cells (p < 0.01). High B7-H3 expression was associated with poor overall survival (p = 0.04) and prognostic risk scores (p = 0.05). NK cell-mediated apoptosis was 3-fold higher in all 3 B7-H3-knockdown AML cell lines than in scrambled control cells, suggesting that B7-H3 is an important immunomodulator of NK cells. Moreover, we observed a significant increase in NK cell-mediated killing of AML cells in the presence of anti-B7-H3 mAbs (p < 0.01). In vivo, anti-B7-H3 antibodies significantly inhibited AML growth and extended survival in PDX models compared to IgG control treatment. Among PDX-bearing mice treated with the three anti-B7-H3 antibodies, the T-1A5 antibody-treated group survived for longer than the other groups. In combination with NK cells, T-1A5 treatment also significantly increased the survival of AML xenograft-bearing mice compared to NK + IgG control treatment. These data suggest that the T-1A5 antibody blocks B7-H3 and enhances NK cell-mediated cytotoxicity in AML cells in vivo. Next, we found that a chT-1A5 antibody induced NK cell-mediated ADCC in primary AML cells and cell lines in a dose-dependent manner. In contrast, it did not induce any ADCC activity in healthy donor-derived PBMCs, suggesting that chT-1A5 is not toxic to healthy cells. Moreover, the chT-1A5 antibody combined with human NK cells dramatically inhibited leukemia growth and extended survival in B7-H3 positive AML PDX models (p < 0.001) compared to the control (rituximab-treated) group. Finally, epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region of B7-H3 as the binding site for the T-1A5 antibody, which may be involved in the immunomodulatory function of B7-H3. Conclusion: B7-H3 is overexpressed in AML cells, and its expression is associated with poor overall survival. Anti-B7-H3 antibodies block B7-H3's immunomodulatory function and inhibit AML growth in vivo. A chT-1A5 antibody in combination with NK cells induced ADCC in primary AML cells in vitro and in vivo but had no effect on PBMCs from healthy donors. Therefore, targeting B7-H3 could benefit AML patients, specifically those with a poor clinical prognosis. Disclosures Battula: Tolero Pharmaceuticals: Research Funding.