Donor-Derived B Cells Produce Potent AML-Specific Antibodies That Recognize Novel Tumor-Specific Antigens and Mediate Graft-Versus-Leukemia Immunity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3146-3146 ◽  
Author(s):  
Marijn Aletta Gillissen ◽  
Martijn Kedde ◽  
Etsuko Yasuda ◽  
Sophie E. Levie ◽  
Arjen Q. Bakker ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
B Cells ◽  
Stem Cell Transplantation ◽  
Tumor Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Allogeneic Hsct ◽  
Graft Versus Leukemia ◽  
Specific Antigens

Abstract Unleashing the tumor-specific immune response by immunotherapies such as checkpoint inhibitors or allogeneic stem cell transplantation can result in long lasting tumor regression. While the role of T cells in such graft versus leukemia (GvL) immune responses has been established, the contribution of B cells to GvL responses is less clear. Using SEREX and other techniques, the presence of antibodies directed against established tumor antigens following allogeneic hematopoietic stem cell transplantation (HSCT) has been demonstrated. The function of these antibodies remains to be established though. Here we tested the hypothesis that B cells contribute to GvL. We selected three patients with high-risk AML who mounted potent GvL responses after allogeneic HSCT. Of these patients we established antibody-producing clonal B cell lines following transduction of memory B cells from the peripheral blood of these patients with Bcl-6 and Bcl-xL and screened those for producing antibodies specifically binding to surface antigens on AML cell lines and AML blasts. A number of antibodies were identified that recognized primary AML blasts isolated from newly diagnosed AML patients, but did not bind to healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. Target identification analyses revealed two novel, AML-specific surface proteins as primary targets of these antibodies. These newly identified AML-specific antigens are widely expressed on different types of AML, including blasts of the last 17 AML patients that entered our clinic of whom diagnosis material was made available for this project. Strikingly, antibody binding to one of these targets induced direct cell death of cultured AML cell lines and of primary AML blasts. Cytotoxicity of the antibodies was rapid, occurred both at 37o C and 4o C and could be prevented by Cytochalasin D, an actin polymerization inhibitor that stabilizes the cytoskeleton. This, in combination with the observation that cell death could not be prevented by apoptosis inhibitors indicated that the tumor cells were killed through a necrotic pathway like oncosis. Other antibodies induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent-cytotoxicity (CDC). Together these data indicate that tumor selective antibodies are elicited following allogeneic HSCT in AML patients with a strong GvL response. The direct and indirect cytotoxic activities against tumor cells of these antibodies suggest that they contribute to the GvL response. Disclosures No relevant conflicts of interest to declare.

Cytotoxicity of Genetically Engineered Fusion Protein with CD154 Peptide Mimetic (OmpC-CD154p) on B-NHL Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4525-4525
Author(s):  
Bernardo Martinez-Miguel ◽  
Melisa A. Martinez-Paniagua ◽  
Sara Huerta-Yepez ◽  
Rogelio Hernandez-Pando ◽  
Cesar R. Gonzalez-Bonilla ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Fusion Protein ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Genetically Engineered ◽  
B Cell Malignancies ◽  
Peptide Mimetic

Abstract The interaction between CD40, a member of the tumor necrosis factor super family, and its ligand CD154 is essential for the development of humoral and cellular immune responses. Selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically-based diseases and malignancies. CD40 is expressed primarily on dendritic cells, macrophages and B cells. Engagement of CD40-CD154 induces activation and proliferation of B lymphocytes and triggers apoptosis of carcinoma and B lymphoma cells. Agonist CD40 antibodies mimic the signal of CD154-CD40 ligation on the surface of many tumors and mediate a direct cytotoxic effect in the absence of immune accessory molecules. CD40 expression is found on nearly all B cell malignancies. Engagement of CD40 in vivo inhibits B cell lymphoma xenografts in immune compromised mice. Several clinical trials have been reported targeting CD40 in cancer patients using recombinant CD154, mAbs and gene therapy, which were well tolerated and resulted in objective tumor responses. In addition to these therapies, CD54 mimetics have been considered with the objective to augment and potentiate the direct cytotoxic anti-tumor activity and for better accessibility to tumor sites. This approach was developed by us and we hypothesized that the genetic engineering of a fusion protein containing a CD154 peptide mimetic may be advantageous in that it may have a better affinity to CD40 on B cell malignancies and trigger cell death and the partner may be a carrier targeting other surface molecules expressed on the malignant cells. This hypothesis was tested by the development of a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Trp140-Ser149 amino acid strand (Vega et al., Immunology2003; 110: 206–216). This OmpC-CD154p fusion protein binds CD40 and triggers the CD40 expressing B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth and proliferation of the B-NHL cell lines Raji and Ramos. In addition, significant apoptosis was achieved and the extent of apoptosis was a function of the concentration used and time of incubation. The anti-tumor effect was specific as treatment with OmpC alone had no effect. These findings establish the basis of the development of new fusion proteins with dual specificity (targeting the tumor cells directly or targeting the tumor cells and immune cells). The advantages of this approach over conventional CD40-targeted therapies as well as the mechanism of OmpC-CD154p-induced cell signaling and cell death will be presented.

scholarly journals Granulocyte Colony-Stimulating Factor Effectively Mobilizes TCR γδ and NK Cells Providing an Allograft Potentially Enhanced for the Graft-Versus-Leukemia Effect for Allogeneic Stem Cell Transplantation

2021 ◽  
Vol 12 ◽  
Author(s):  
Lia Minculescu ◽  
Henrik Sengelov ◽  
Hanne Vibeke Marquart ◽  
Lars Peter Ryder ◽  
Anne Fischer-Nielsen ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Cell Compartment ◽  
Effector Cells ◽  
Graft Versus Leukemia ◽  
Hla Dr

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potential cure for patients with hematological malignancies but substantial risks of recurrence of the malignant disease remain. TCR γδ and NK cells are perceived as potent innate effector cells in HSCT and have been associated with post-transplant protection from relapse in clinical studies. Immunocompetent cells from the donor are crucial for patient outcomes and peripheral blood stem cells (PBSC) are being increasingly applied as graft source. G-CSF is the preferential mobilizing agent in healthy donors for PBSC grafts, yet effects of G-CSF on TCR γδ and NK cells are scarcely uncovered and could influence the graft composition and potency of these cells. Therefore, we analyzed T and NK cell subsets and activation markers in peripheral blood samples of 49 donors before and after G-CSF mobilization and—for a subset of donors—also in the corresponding graft samples using multicolor flowcytometry with staining for CD3, CD4, CD8, TCRαβ, TCRγδ, Vδ1, Vδ2, HLA-DR, CD45RA, CD197, CD45RO, HLA-DR, CD16, CD56, and CD314. We found that TCR γδ cells were mobilized and harvested with an efficiency corresponding that of TCR αβ cells. For TCR γδ as well as for TCR αβ cells, G-CSF preferentially mobilized naïve and terminally differentiated effector (TEMRA) cells over memory cells. In the TCR γδ cell compartment, G-CSF preferentially mobilized cells of the nonVδ2 types and increased the fraction of HLA-DR positive TCR γδ cells. For NK cells, mobilization by G-CSF was increased compared to that of T cells, yet NK cells appeared to be less efficiently harvested than T cells. In the NK cell compartment, G-CSF-stimulation preserved the proportion of CD56dim NK effector cells which have been associated with relapse protection. The expression of the activating receptor NKG2D implied in anti-leukemic responses, was significantly increased in both CD56dim and CD56bright NK cells after G-CSF stimulation. These results indicate differentiated mobilization and altering properties of G-CSF which could improve the effects of donor TCR γδ and NK cells in the processes of graft-versus-leukemia for relapse prevention after HSCT.

Unique and Potent Tumor Specific Antibodies in Graft Versus Leukemia Responses

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3826-3826
Author(s):  
Marijn A. Gillissen ◽  
Etsuko Yasuda ◽  
Sophie Levie ◽  
Arjen Q Bakker ◽  
Martijn Kedde ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
High Risk ◽  
B Cell ◽  
Cell Lines ◽  
Surface Antigens ◽  
Antibody Responses ◽  
Hematopoietic Stem ◽  
Specific Antibodies ◽  
Graft Versus Leukemia

Abstract Introduction Acute myeloid leukemia (AML) is a high-risk malignancy with a poor prognosis. Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative if it induces a potent graft versus leukemia (GvL) response. GvL responses and graft versus host disease (GvHD) are typically considered T cell mediated, because T cell depletion from hematopoietic stem cell grafts reduces the risk of GvHD at the cost of leukemia relapse. In addition, depletion of B-lymphocytes with rituximab has led to amelioration of GvHD in a number of studies suggesting that B cells are also important in the pathophysiology of GvHD and, in analogy, in GvL responses. However, the characteristics of the antibodies produced by these leukemic specific B cells have not yet been studied. Methods We selected three patients with high-risk myelomonocytic leukemia who remained disease free years after allogeneic HSCT, from whom we established clonal human B cell lines, using a unique and innovative technology that was developed in our laboratory (Kwakkenbos ea, Nat Med 2010). These B cell lines, that concomitantly express immunoglobulin on their membranes and secrete antibodies, were used to select antibodies specific for cell surface antigens on AML cell lines with similar morphologic and immunophenotypic characteristics as the patients’ leukemic blasts, using a FACS based assay. Results From each patient, several AML specific B cell clones were retrieved. Their antibodies recognized surface antigens on primary AML blasts derived from multiple patients and on AML cell lines, but not on healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. The majority of the antibodies were of the IgG3 isotype. Approximately 40% of the AML-specific antibodies induced direct death of cultured AML cell lines and of primary AML blasts. The cell death pathway induced by these cytotoxic antibodies was oncotic rather than apoptotic. Conclusion Our data demonstrate that high-risk AML patients with a potent GvL response mount robust antibody responses against surface antigens that are specifically expressed on tumor cells and binding of these antibodies induces direct cell death. The targets recognized by the recovered antibodies are also expressed on leukemic blast from other patients suggesting evidence for a common immune mechanism responsible for AML clearance. Our findings suggest that antibody responses are important in GvL and open up new ventures for specific antibody treatment of AML patients. Disclosures No relevant conflicts of interest to declare.

Expansion of Large Granular Lymphocytes Following Allogeneic Stem Cell Transplantation: Its Association with Graft-Versus-Host Disease and Graft-Versus-Leukemia Effect.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2253-2253
Author(s):  
Dong Hwan (Dennis) Kim ◽  
Ghuzayel Al-Dawsari ◽  
Chul Won Jung ◽  
Chang Hong ◽  
Suzanne Kamel-Reid ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Graft Versus Host Disease ◽  
Peripheral Blood ◽  
Chronic Gvhd ◽  
Large Granular Lymphocytes ◽  
Graft Versus Host ◽  
Graft Versus Leukemia ◽  
Cmv Reactivation ◽  
Granular Lymphocytes

Abstract Abstract 2253 Poster Board II-230 Background: Expansion of large granular lymphocytes (LGLs) following allogeneic hematopoietic stem cell transplantation (HSCT) has been reported infrequently, and data from large scaled population is still lacking. Expansion of LGLs following HSCT has been suggested to be associated either with the development of graft-versus-host disease (GVHD) associated with graft-versus-leukemia (GVL) effect or with viral infection such as CMV or VZV reactivation. Methods: A total of 500 consecutive patients who received HSCTs from related or unrelated donors between January 2000 and October 2007 at the Princess Margaret Hospital, Toronto, Canada were included in this retrospective study. LGL expansion has been defined as 1) increasing number of peripheral blood lymphocyte counts ≥ 3.0×109/L for at least 3 months, and 2) the predominance of LGLs in the peripheral blood smears. Cases with LGL expansion were investigated prospectively for immunophenotypic profiles and T-cell receptor RT-PCR for T-cell monoclonality. Results: Out of 500 recipients, 77 cases (15.4%) showed LGL expansion after HSCT. The median onset of LGL expansion was 312 days (95% C.I. 233-390 days). The 1-, 2- and 3-year(s) incidence of LGL expansion was 12.8±1.8%, 20.6±2.4%, and 25.7±2.9%, respectively. Compared to the patients without LGL expansion, improved transplant outcomes were observed in patients with LGL expansion: better overall survival (OS; 90% vs 46%, p<0.001), lower non-relapse mortality (NRM; 3% vs 38%, p<0.001), lower relapse incidence (10% vs 31%, p<0.001) and higher GVHD-specific survival (GSS; 97% vs 64%, p<0.001). Three risk factors were identified for the development of LGL expansion such as CMV serostatus of recipient, CMV reactivation, and occurrence of chronic GVHD. Higher incidence of LGL expansion was noted 1) in CMV seropositive recipients (CMV-R+) compared to CMV seronegative recipients (CMV-R-) regardless of CMV serostatus of the donor (p<0.001); 2) in patients experiencing CMV reactivation (p<0.001); 3) in patients developing chronic GVHD (p=0.007). Of interest, when dividing patients according to the recipient's CMV serostatus and the occurrence of chronic GVHD, the group with CMV-R+ and cGVHD+ showed the highest incidence of LGL expansion up to 46%, while those with CMV-R- and cGVHD+ showed intermediate risk of LGL expansion with 25% incidence, and those with CMV-R+/cGVHD- or with CMV-R-/cGVHD- showed only 3-6% incidence of LGL expansion. Conclusion: Expansion of LGLs following allogeneic HSCT is not uncommon, and strongly associates with favorable transplant outcomes including a lower relapse rate suggesting that LGLs may be involved in the mediation of a GVL effect. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Memory B-cell reconstitution following allogeneic hematopoietic stem cell transplantation is an EBV-associated transformation event

Blood ◽  
2015 ◽  
Vol 126 (25) ◽  
pp. 2665-2675 ◽  
Author(s):  
David M. Burns ◽  
Rose Tierney ◽  
Claire Shannon-Lowe ◽  
Jo Croudace ◽  
Charlotte Inman ◽  
...  
Keyword(s):  
Stem Cell ◽  
B Cells ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
B Cell ◽  
Memory B Cells ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Key Points ◽  
High Level

Key PointsCD19+CD27+ memory B cells are detectable at supranormal frequencies in patients with high-level EBV DNAemia following allogeneic HSCT. These memory B cells are frequently positive for EBV genomes and bear many of the hallmarks of lymphoblastoid transformation.

scholarly journals GSTM1 and GSTT1 double null genotypes determining cell fate and proliferation as potential risk factors of relapse in children with hematological malignancies after hematopoietic stem cell transplantation

2021 ◽  
Author(s):  
Simona Jurkovic Mlakar ◽  
Chakradhara Rao Uppugunduri Satyanarayana ◽  
Tiago Nava ◽  
Vid Mlakar ◽  
Hadrien Golay ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Purpose This study aimed to retrospectively evaluate the genetic association of null variants of glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with hematological malignancies (HMs) undergoing busulfan (BU)- containing allogeneic hematopoietic stem cell transplantation (HSCT) and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized lymphoblastoid cell lines (LCLs) and tumor THP1 GST gene-edited cell models. Methods GSTM1- and GSTT1-null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1-null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell death studies were conducted in GSTs- null and non-null LCLs and CRISPR–Cas9 gene-edited THP1 leukemia cell lines. Results Carrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76–15.42; p = 1.9 × 10–5]). BU-induced cell death preferentially in THP1GSTM1(non−null) and LCLsGSTM1(non−null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. Conclusion The clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation.

scholarly journals A Human Bone Marrow Failure Syndrome Caused By a Homozygous Mutation in MYSM1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1204-1204 ◽  
Author(s):  
Ehsan Bahrami ◽  
Tomas Racek ◽  
Maximilian Witzel ◽  
Jacek Puchalka ◽  
Naschla Greif-Kohistani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
B Cells ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Cell Lines ◽  
Bone Marrow Failure ◽  
Bone Marrow Failure Syndrome ◽  
Hematopoietic Stem

Abstract Inherited bone marrow failure syndromes comprise a heterogeneous group of genetic disorders characterized by dysfunction of hematopoietic stem or progenitor cells. We present a consanguineous pedigree with two siblings with early-onset, progressive bone marrow failure (neutropenia, anemia, thrombocytopenia) associated with trigonocephaply, hyperplastic gingiva, cataract, accessory mammilla, dental changes and mild neurocognitive developmental delay. Pancytopenia at birth as well as non-compaction cardiomyopathy was observed in one of the patients. Bone marrow histology showed marked hypocellularity with decrease of all hematopoietic cell lines but without cellular atypia, monocytosis, blast excess or fibrosis. Fanconi anemia was excluded by mitomycin C induced chromosomal breakage studies. Immunophenotyping of peripheral blood cells showed a reduction of mature B cells, reduced switched memory B cells, whereas the level of activated B-cells and plasma blasts were increased. T cell proliferation upon PHA and specific antigens was normal. Using whole exome sequencing in patients and both parents we could identify a single potentially disease causing homozygous stop codon mutation (NM_001085487: c.1168G>T: p.E390*) in MYSM1 (Myb-Like, SWIRM And MPN Domain-Containing Protein 1). A family with two affected patients carrying the same mutation has been reported previously (Alsultan A et al, Blood 122:3844, 2013). MYSM1 is a Histone H2A deubiquitinase that has previously been implicated in controlling hematopoietic stem cells, progenitor B cells, and NK cells in mice (Nijnik A et al, Blood 119:1370, 2012). MYSM1 protein was absent in patients' EBV-transformed B cell lines (EBV-LCLs). MYSM1-deficient EBV-LCLs had elevated γ-H2AX levels, indicative of increased genomic instability. Upon exposure to UV light, we observed a sustained expression of p53 and phospho-p53 as well as p38 MAPK and phospho-p38 MAPK in patients' fibroblasts in comparison to fibroblasts from healthy individuals. In view of the progressive pancytopenia, both siblings underwent allogeneic hematopoietic stem cell transplantation from 10/10 HLA-matched family donors after reduced intensity conditioning with alemtuzumab, fludarabine and treosulfan at 2 and 4 years of age, respectively. The procedure was well tolerated, and both siblings exhibit full donor hematopoietic reconstitution at 21 and 26 months after transplant. In summary, we here identify a novel human bone marrow failure syndrome caused by MYSM1-deficiency that can be cured by allogeneic hematopoietic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

scholarly journals GSTM1 and GSTT1 Double Null Genotypes Determining Cell Fate and Proliferation as Potential Risk Factors of Relapse in Children with Hematological Malignancies after Stem Cell Transplantation.

2021 ◽  
Author(s):  
Simona Jurkovic Mlakar ◽  
Chakradhara Rao Uppugunduri Satyanarayana ◽  
Tiago Nava ◽  
Vid Mlakar ◽  
Hadrien Golay ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Leukemia Cell ◽  
Hematopoietic Stem ◽  
Glutathione S Transferases ◽  
The Impact

Abstract Background: Relapse is the major cause of treatment failure in children with hematological malignancies (HMs) undergoing busulfan (BU)- based allogeneic hematopoietic stem cell transplantation (HSCT). Glutathione S-transferases (GSTs) isoforms that participate in BU detoxification and protect cells against stress and cell death may be linked to post-HSCT outcomes. This study aimed to retrospectively evaluate the genetic association of null variants of Glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with HMs undergoing BU- containing allogeneic HSCT and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized and tumor lymphoblastoid cell lines (LCLs).Methods: GSTM1- and GSTT1- null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1- null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell-death studies were conducted in GSTs- null and non-null LCLs and CRISPR-Cas9 gene-edited THP1 leukemia cell lines. Results: Carrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76 - 15.42; p= 1.9 x 10-5]). BU induced cell death preferentially in THP1GSTM1(non-null) and LCLsGSTM1(non-null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. Conclusion: The clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation. Trial registration: ClinicalTrials.gov identifier: NCT01257854, Registered February 2008 – retrospectively registered.

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