The Use of Clinical Grade Magnetic Beads to Deplete Immune Suppressive Elements from Autologous Peripheral Blood Hematopoietic Stem Cell Grafts Enhances T Cell Immune Responses.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2311-2311
Author(s):  
Zaid S Al-Kadhimi ◽  
Haider Mahdi ◽  
Moshrik Abdalamir ◽  
Elyse Paul ◽  
Voravit Ratanatharathorn ◽  
...  
Keyword(s):  
Immune Response ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Magnetic Beads ◽  
T Cell Proliferation ◽  
Clinical Grade ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Autologous Grafts

Abstract Background: Despite the demonstrated efficacy of high dose chemotherapy and autologous hematopoietic stem cell transplantation in Myeloma and relapsed Lymphoma, relapse continues to be a problem. Improving post transplant immune reconstitution in general and tumor specific immune response can reduce relapse rates. Pre-clinical and clinical data suggest that the abundance of CD4+/CD25+ regulatory T-cells (T-regs) and CD14+ monocytes in the autologous graft can contribute to the well described immune suppression state in the early months post transplant. We hypothesized that it is feasible to deplete both of these cellular inhibitory elements from the autologous grafts using clinical grade CD25 and CD14 magnetic beads from Miltenyi. We also hypothesized that the depletion of these populations would improve the proliferative capacity of T-cells in these grafts. Methods: Samples of peripheral blood hematopoietic stem cell grafts from 9 patients (4 lymphomas and 5 myelomas) undergoing autologous stem cell transplantation, with G-CSF mobilized grafts. We depleted T regs and monocytes, using CD25 and CD14 beads from these graft samples. Then we compared T cell reactivity between the manipulated graft versus the unmanipulated graft samples, in a mixed lymphocyte reaction. Results: We show that it is feasible to deplete both populations based on phenotype analysis that confirmed selective depletion of CD4+/CD25+/CD127-T reg population (70–80%) and CD14 population (74%.). The positively selected population was enriched for T regs CD4+CD25+CD127-(50–85%) and monocytes CD14+ (75%+). Furthermore in 9 out of 9 patients T cell proliferation measured by 3H thymidine uptake was close to two fold or higher in the double depleted grafts compared to the unmanipulated grafts (P=0.004 Wilcoxon Signed Rank Test). Conclusion: Double depletion of T regs and monocytes from autologous grafts using clinical grade magnetic beads, leads to enhanced T cell proliferation. This work will lay the ground for a subsequent clinical trial of regulatory T-cell/Monocyte depleted autologous transplantation using the same strategy, to enhance immune recovery post transplant and achieve better anti-lymphoma, or anti-myeloma immune response. Figure Figure

Case Report: Haploidentical Stem Cell Transplantation In a 78 Year-Old Patient

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5468-5468
Author(s):  
Thiago Xavier Carneiro ◽  
André Domingues Pereira ◽  
Theodora Karnakis ◽  
Celso Arrais Rodrigues
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
High Dose ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Post Transplant

Abstract An older chronologic age has been a consistent predictor of poor outcomes in hematopoietic stem cell transplantation (HSCT), mainly due to non-relapse mortality (NRM). Therefore, non-curative treatment strategies are commonly adopted for these patients. However, mortality and treatment toxicity has decreased as a result of improved supportive measures, such as reduced intensity conditioning regimens and optimized infection management. T-cell replete haploidentical HSCT emerged as a feasible alternative for leukemia patients without substantial differences in outcomes when compared to fully matched related donor. We report an old adult woman treated with haploidentical HSCT. A 78 year-old female patient presented with anemia, leukocytosis, thrombocytopenia and blasts in the peripheral blood. Diagnosis of acute myelogenous leukemia was established. Conventional cytogenetic demonstrated chromosome eight trisomy, and FISH was negative for other common MDS/AML cytogenetic abnormalities. FLT3-ITD and NPM1 mutations were negative. Her medical history was negative except for heavy smoking. Considering the patients advanced age, the first attending physician chose not to administer intensive treatment and started on decitabine 20 mg/m2 for 5 days. She was refractory to the first-line treatment with persistent cytopenias and blasts in the peripheral blood four weeks after treatment was started. Comprehensive geriatric assessment was performed. She was considered independent for Basic Activities of Daily Living (ADL score 6) and Instrumental Activities of Daily Living (IADL score 27), without cognitive impairment in mini-mental state examination (MMSE score 30), at risk of malnutrition in mini nutritional assessment (MNA 9). As she was considered fit, we decided to perform high-dose chemotherapy with idarubicin and cytarabine, but, once more, the disease was refractory. A rescue regimen was attempted with high-dose cytarabine and mitoxantrone, again, with no response. After discussing pros and cons with the patient and the family, we decided to start Another regimen consisting of topotecan and high dose cytarabine immediately followed by allogeneic hematopoietic stem cell transplantation (HSCT). At day 14, she had 3% blasts in the BM aspirate a T-cell replete haploidentical HSCT using her 52 year-old son as donor and mobilized peripheral blood as stem cell source was performed. Conditioning regimen consisted of fludarabine, cyclophosphamide, TBI 2Gy and post-transplant cyclophosphamide. Graft versus host disease (GVHD) prophylaxis consisted of mycophenolate mofetil and cyclosporine. She had neutrophil engraftment with complete donor chimerism at day+15 and platelet engraftment at day+17. At day+48, she had mild (stage II) skin acute GVHD resolved with topical steroids. Cyclosporine was withdrawn at day+ 93. Due to high relapse risk, the patient was started on monthly post-transplant azacitidine 36 mg/m2. At day+100 the patient remained in complete remission, complete donor chimerism in peripheral blood and bone marrow. Functionality was preserved (ADL score 6 and IADL score 24), presented discrete cognitive impairment (MMSE 28) and malnoutrition (MNA 5). She is now at day+182, doing well and performing again all usual daily activities. To the best of our knowledge, this is the oldest patient treated with haploidentical HSCT. Post transplant cyclophosphamide as T cell depletion strategy in haploidentical HSCT is well tolerated and widely available, being therefore an excellent alternative for patients without conventional donors who require immediate transplant. Older adults with hematologic malignancies are a heterogeneous group and decisions based on chronological age alone are clearly inappropriate. Recently, geriatric assessment proved to be an important prognostic tool in acute leukemia and may be useful in HSCT. In experienced centers, haploidentical HSCT in older adults may be a safe procedure and more accurate pre-transplantation risk stratification tools should be developed. Figure 1 Timeline of main events during hematopoietic stem cell transplant. Figure 1. Timeline of main events during hematopoietic stem cell transplant. Disclosures: No relevant conflicts of interest to declare.

Unique Patterns of Lymphocyte Recovery Induced By Hyperbaric Oxygen Therapy Given Prior to Stem Cell Transplantation: A Case Series Report on Three Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5488-5488
Author(s):  
Haitham Abdelhakim ◽  
Leyla Shune ◽  
Da Zhang ◽  
Omar S. Aljitawi
Keyword(s):  
Clinical Trial ◽  
Flow Cytometry ◽  
Stem Cell ◽  
T Cell ◽  
Partial Response ◽  
Hematopoietic Stem Cell ◽  
Case Series ◽  
Antigen Expression ◽  
Hematopoietic Stem ◽  
Post Transplant

Abstract Hyperbaric oxygen therapy (HBO) is being studied at our institution to improve hematopoietic stem cell homing and engraftment. Pre-clinical experiments have demonstrated that HBO therapy to the recipient prior to umbilical cord blood CD34+ cell infusion induces a low Erythropoietin (EPO) environment that results decreased erythroid differentiation and instead favored early bone marrow retention of CD34 cells with positive impact on engraftment in animal experiments1. We hereby report a case series of 3 patients who received HBO on ongoing clinical trials and demonstrated unique patterns for lymphocyte recovery associated with unique clinical presentations. Patients were exposed to 100% oxygen at 2.5 atmosphere pressure for total of 2 hours prior to hematopoietic stem cell infusion. Case 1: A 64 year old male with IgG kappa multiple myeloma who relapsed post tandem cycles of high-dose Melphalan and autologous transplants. The patient received salvage therapy with Carfilzomib and Dexamethasone and achieved a partial response following which he underwent a preparative regimen of Carmustine, Etoposide, Cytarabine and Melphalan (BEAM) with subsequent autologous transplantation on an HBO clinical trial. Post-transplant course was complicated by fever, rigors and extensive skin rash. These findings coincided with a rapid rise in white blood cell count predominantly composed of lymphocytes (Fig.2). Peripheral blood flow cytometry revealed atypical T-cell population with lymphocytes comprising 94% of total events. The majority were T cells (76%) with CD4 to CD8 ratio of 1:1 and co-expression of CD15, CD38 and HLA-DR. An associated infectious etiology was ruled out and the patient improved over a period of several days. Case 2: A 33 year old male with refractory nodular sclerosis Hodgkin Lymphoma. He initially received Doxorubicin, Bleomycin, Vinblastine and Dacarbazine (ABVD) for 2 cycles with partial response. He was then switched to Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine, Procarbazine and Prednisone (BEACOPP). After 2 cycles of BEACOPP, a repeat PET scan showed partial response. He was then salvaged with Brentuximab followed by BEAM and autologous transplant on HBO clinical trial. The patient achieved neutrophil engraftment by day +11, of note his blood counts showed early increase in lymphocytes (86%) (Fig. 3). Post-transplant he was restarted on Brentuximab for two cycles with achievement with complete response, but his course was complicated by uveitis. An aqueous chamber fluid sample was sent for flow cytometry which showed Lymphocytes comprising 13% of total events. Flow cytometry of Cerebrospinal fluid revealed lymphocytes comprising 97% of total events. Both aqueous chamber and cerebrospinal fluids showed majority of T cells with normal CD4:CD8 ratio and antigen expression. His vision improved with topical steroid eye drops. Unfortunately, his disease progressed again off therapy. Case 3: A46 year old female with T cell large granular lymphoma who was initially treated with weekly methotrexate progressed to hepatosplenic gamma-delta T cell lymphoma and was treated with 4 cycles of hyper CVAD. Patient then received a reduced intensity preparative regimen of Fludarabine, Cytoxan, Total body irradiation and single unit umbilical cord blood transplantation on HBO clinical trial. The patient had an unremarkable post-transplant course and achieved neutrophil engraftment by day +25. Of note the patient demonstrated persistent peripheral lymphocytosis ranging between 42-64% of total white blood cell counts (Fig. 4). Bone marrow biopsies at multiple milestones showed her to be in complete remission and to be 100% donor by molecular tests. Conclusions: Three unique patterns of lymphocyte recovery are seen in association with HBO therapy given prior to hematopoietic stem cell transplantation. We hypothesize that these patterns of lymphocyte recovery are secondary to engraftment kinetics caused by HBO and low EPO environment at the time of stem cell infusion. We suspect the early robust lymphocytosis may have played a role enhancing the inflammatory milieu resulting in fevers, rigors and extensive skin rash in case one, uveitis with CSF and aqueous chamber lymphocytosis in case two and persistent peripheral lymphocytosis in case three. In all three cases, the T cell exhibited normal antigen expression on flow cytometry. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Eosinophils from Hematopoietic Stem Cell Recipients Suppress Allogeneic T Cell Proliferation

2014 ◽  
Vol 20 (12) ◽  
pp. 1891-1898 ◽  
Author(s):  
Jennie Andersson ◽  
Julia Cromvik ◽  
Madeleine Ingelsten ◽  
Christine Lingblom ◽  
Kerstin Andersson ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
T Cell Proliferation ◽  
Hematopoietic Stem

scholarly journals IMMUNITY TO INFECTIONS AFTER HAPLOIDENTICAL HEMATOPOIETIC STEM CELL TRANSPLANTATION

2016 ◽  
Vol 8 ◽  
pp. 2016057 ◽  
Author(s):  
Franco Aversa ◽  
Lucia Prezioso ◽  
Ilenia Manfra ◽  
Federica Galaverna ◽  
Angelica Spolzino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Conditioning Regimen ◽  
Hematopoietic Stem ◽  
Post Transplant

The advantage of using a Human Leukocyte Antigen (HLA)-mismatched related donor is that almost every patient who does not have a HLA-identical donor or who urgently needs hematopoietic stem cell transplantation (HSCT) has at least one family member with whom shares one haplotype (haploidentical) and who is promptly available as a donor. The major challenge of haplo-HSCT is intense bi-directional alloreactivity leading to high incidences of graft rejection and graft-versus-host disease (GVHD). Advances in graft processing and in pharmacologic prophylaxis of GVHD have reduced these risks and have made haplo-HSCT a viable alternative for patients lacking a matched donor. Indeed, the haplo-HSCT  has spread to centers worldwide even though some centers have preferred an approach based on T cell depletion of G-CSF-mobilized peripheral blood progenitor cells (PBPCs), others have focused on new strategies for GvHD prevention, such as G-CSF priming of bone marrow and robust post-transplant immune suppression or post-transplant cyclophosphamide (PTCY). Today, the graft can be a megadose of T-cell depleted PBPCs or standard dose of unmanipulated bone marrow and/or PBPCs.  Although haplo-HSCT modalities are based mainly on high intensity conditioning regimens, recently introduced reduced intensity regimens (RIC)   showed promise in decreasing early transplant-related mortality (TRM), and extending the opportunity of HSCT to an elderly population with more comorbidities. Infections are still mostly responsible for toxicity and non-relapse mortality due to prolonged immunosuppression related, or not, to GVHD. Future challenges lie in determining the safest preparative conditioning regimen, minimizing GvHD and promoting rapid and more robust immune reconstitution.

Generation and Signaling Function of CD19 Chimeric Antigen Receptor Modified CD8+ T Cells Derived From Virus-Specific Central Memory Cells for Adoptive Therapy After Allogeneic Hematopoietic Stem Cell Transplant

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2978-2978
Author(s):  
Seitaro Terakura ◽  
Tori N. Yamamoto ◽  
Rebecca A. Gardner ◽  
Cameron J. Turtle ◽  
Michael C. Jensen ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
B Cell ◽  
Hematopoietic Stem Cell ◽  
Adoptive Transfer ◽  
Clinical Grade ◽  
Donor Blood ◽  
Adoptive Therapy ◽  
Hematopoietic Stem

Abstract Abstract 2978 Allogeneic hematopoietic stem cell transplantation (allo-HCT) is the most effective post-consolidation therapy for high-risk B cell acute lymphocytic leukemia (ALL) in pediatric and adult patients. However, relapse after allo-HCT remains a common cause of failure, and treatment of ALL that has recurred after transplant, either with additional chemotherapy or with donor lymphocyte infusions to enhance a graft-versus-leukemia effect is mostly unsuccessful and can cause graft-versus-host disease (GVHD). The adoptive transfer of donor T cells that have been genetically modified to recognize leukemia could prevent or treat leukemia relapse after allo-HCT. However, adoptive therapy after allo-HCT should be performed with T cells that have a defined endogenous T cell receptor (TCR) specificity to avoid GVHD, and with T cells that have the capacity to persist in vivo to ensure leukemia eradication. Our lab has previously shown that donor virus-specific T cells can be adoptively transferred without causing GVHD, and identified a role for cell intrinsic properties of central memory T (TCM) cells in determining cell persistence after adoptive transfer. Thus, we developed a strategy for deriving virus-specific T cells from CD45RA−CD62L+CD8+ TCM cells purified from donor blood with clinical grade reagents, and redirecting their specificity to the B-cell lineage marker CD19 through lentiviral transfer of a gene encoding an anti-CD19 ectodomain, a CD3 ζ and CD28 endodomain, and a truncated epidermal growth factor receptor as a transduction marker downstream of a T2A sequence. CD8+ TCM were enriched by first depleting CD4+, CD14+, and CD45RA+ cells from PBMC using clinical grade monoclonal antibodies (mAbs) conjugated to paramagnetic beads, and then positively selecting CD62L+ cells from the depleted subset with a biotinylated anti-CD62L mAb and clinical grade anti-biotin beads. After this two-step selection, the enriched virus-specific TCM were selectively transduced by exposure to the CD19-CAR lentivirus after stimulation with viral peptides. This procedure resulted in a selective expansion of bi-specific (CD19-CAR+/virus-specific) T cells that could be readily expanded by stimulation with CD19+ EBV-transformed lymphoblastoid cells (LCL). The bi-specific subset was further enriched to high purity using reversible class I MHC Streptamers (Stage Cell Therapeutics) folded with viral peptides. The frequency of tetramer positive and CD19-CAR+ cells in the final cell product was ∼90% (range, 80.7–96.3%) and ∼85% (range, 77.9–95.3%) respectively, and this high frequency of bi-specific T cells was maintained through additional in vitro expansion. With this procedure, large numbers of highly pure bi-specific TE cells that are derived from CD8+ TCM precursors and engineered to express a CD19-CAR could be readily generated for adoptive immunotherapy of allo-HCT recipients in ∼30–35 days from 400 ml of donor blood. CAR binding to its ligand is structurally distinct from that of a TCR, and a direct comparison of effector functions and signaling through an introduced CAR and the endogenous TCR on the same cell could reveal differences that might affect cellular functions and therapeutic efficacy. To compare functions after CAR- and TCR-stimulation, we transduced K562 tumor cells with the costimulatory molecules CD80 and CD86, and with CD19 or HLA molecules to serve as antigen presenting cells. Activation of bi-specific T cells through the CAR or the virus-specific TCR elicited comparable lytic function and induced equivalent phosphorylation of downstream signaling molecules (CD3 ζ, Zap70, p38, ERK, JNK) with similar kinetics. Signaling through either CAR or TCR induced comparable production of IL-2, IFN- γ, and TNF- α as assessed by intracellular cytokine staining and Luminex assay, and T cell proliferation as assessed by CFSE dye dilution and absolute cell count. These studies identify a strategy for tumor-specific immunotherapy with CAR-modified T cells after allo-HCT, and for comparative studies of signaling through the TCR and CARs derived from antibodies of different affinities and containing distinct signaling domains. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document