Recovery Of Myeloid Derived Suppressor Cell Subsets Following Allogeneic Hematopoietic Stem/Progenitor Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4617-4617
Author(s):  
Qingdong Guan ◽  
Anna Blankstein ◽  
Anjos Karla ◽  
Oleksandra Synova ◽  
Marie Tulloch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Airway Inflammation ◽  
Progenitor Cell ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Inflammatory Conditions ◽  
Hla Dr ◽  
Healthy Donors ◽  
Immature Myeloid Cells

Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that expand during many inflammatory conditions and malignancies. MDSCs may play an important role following allogeneic hematopoietic stem/progenitor cell transplant (HSCT). MDSCs suppress T-cell, B-cell and dendritic cell responses by a number of mechanisms, including promoting regulatory T cell expansion and producing soluble mediators such as Arginase 1 (Arg-1) and iNOS. MDSCs are divided into two subsets: monocytic (M-MDSCs) and granulocytic (G-MDSCs). MDSC morphology and function differ in various tissues under different inflammatory conditions. In a murine asthma model, M-MDSCs inhibit airway inflammation, but the other subset of MDSCs exacerbated airway inflammation. In a sepsis model, MDSCs exaggerated inflammation in the early stage, but suppressed inflammation in the later stage of sepsis. As the early post-transplant period is characterized by the rapid expansion of immature myeloid cells, we postulated this time period may also be a time when MDSCs might play a major role in modulating immune recovery post-transplant, and aid in the development of immune regulatory networks potentially important in the pathophysiology of graft-versus-host disease (GVHD). In nine patients undergoing allogeneic HSCT, peripheral blood was drawn on the day prior to the start of conditioning, days +4-5, +7-9, +14-16, +21-23, +27-29 and +80-100 post HSCT. White blood cells were quantified, red cell depleted using HetaSep (Stem Cell Technologies), then stained with fluorescence-labelled antibodies against CD45, CD15, CD14, HLA-DR, CD33 and CD66b and analyzed by flow cytometry for MDSC subsets. The soluble mediators iNOS and Arg-1were evaluated by intracellular staining for iNOS and Arg-1 and analyzed by flow cytometry. Four of the nine patients developed acute GVHD (II-IV) and/or extensive chronic GVHD. Early recovery of CD33+CD14+HLA-DR-/low M-MDSCs and CD33+CD15+CD66b+ G-MDSCs was seen post-transplant. Compared to healthy donors, the percentage of M- and G-MDSCs was increased by 3 weeks post-transplant. Interestingly, the patients who went on to develop GVHD had lower percentage and number of M-MDSCs, but inversely had higher numbers of G-MDSCs by day +27-29 and day +80-100 post-transplant (Fig. 1). When compared with healthy donors, the expression of Arg-1 in G-MDSCs, a measure of activation of MDSCs, was increased in patients pre- and post-HSCT, especially at day +80-100; while there was no difference seen iNOS expression in G-MDSCs (Fig 2). The expression of Arg-1 and iNOS in M-MDSCs was increased pre-transplant but fell by day +80-100 post HSCT (Fig 2). Taken together, our pilot data indicates that both M- and G- MDSCs recover early post HSCT and may contribute to the pathophysiology of GVHD. Patients with lower numbers of M-MDSCs and higher numbers of G-MDSCs at earlier time points post-transplant might be at greater risk for developing GVHD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals T Cell Activation By OKT3 Is a Function of the Percent Monocytes in PBMC of Healthy Donors and MDS Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4870-4870
Author(s):  
Alison Tarke ◽  
Valentina Ferrari ◽  
Hannah Fields ◽  
Luca Ferrari ◽  
Franco Ferrari ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Transplant ◽  
Activation Markers ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Healthy Donors

Background: Myelodysplastic Syndromes (MDS) are a heterogeneous hematologic malignancy characterized by bone marrow failure and cytopenias. The median survival rate for patients with higher-risk MDS who fail standard-of-care chemotherapy with hypomethylating agents (HMAs) is less than 6 months, and the only curative treatment for these patients is hematopoietic stem cell transplant (HSCT). Over the past 10 years, immunotherapy as a cancer treatment has achieved variable levels of success in different tumor types. There are currently 22 active clinical trials of immunotherapies for MDS (www.clinicaltrials.gov; 7/30/19), including our phase I clinical trial with a personalized adoptive cellular therapy targeting MDS patient neoantigens (NCT 03258359). Because MDS patients are frequently monocytopenic and the existing literature is inconsistent regarding the ability of MDS patients' monocytes to support T cell activation, we compared the activation of MDS T cells with those of healthy donors in the presence of autologous monocytes. Methods: Peripheral blood mononuclear cells (PBMC) from 5 healthy donors and 7 higher-risk MDS patients were cryopreserved after Ficoll separation. These PBMC were thawed and aliquoted into 6 replicate wells of 200,000 cells in 96-well u-bottom plates in R-10 culture medium. Half of the wells were treated with 25 ng/mL OKT3 and 200 U/mL IL-2. After 48 hours at 37˚C with 5% CO2, the wells were collected for analysis by flow cytometry. Beads were used to detect T cell activation induced secretion of IFNg, TNFa, IL-4, IL-10, and IL-17 in the supernatant and fluorescent antibodies were used to phenotype viable cells for CD3, CD4, CD8, and the T cell activation markers, CD69, CD25, CTLA-4, PD-1, and HLA-DR. Results: We measured a higher release of IFNg and TNFa in donor PBMC compared to MDS patients after OKT3/IL-2 activation, p < 0.01 and 0.04, respectively by 2-way ANOVA. The expression of CD69, CD25, HLA-DR, and CTLA4 increased variably on activated T cells from donors or MDS patients, but expression of CD4+CD25+ was more frequent on donor T cells after activation (p = 0.03). Activation also resulted in a higher frequency of PD-1 expression on donor CD4+ and CD8+ T cells than on MDS T cells (p < 0.01 and < 0.01, respectively). Interestingly, on both MDS and normal T cells the percentage of CD8+PD1+ activated cells correlated strongly with the percent of CD14+ monocytes present in the PBMC (R2 = 0.92 and 0.60 respectively; Fig 1a and 1b). We designed further experiments to test whether this was a patient intrinsic phenomenon, or if the absolute number of CD14+ monocytes in the PBMC was associated with different levels of PD1 expression upon T cell activation. First, we separated CD14+ cells from the PBMC of a patient with MDS using magnetic beads. Then CD14+ cells were added back to the CD14-depleted PBMC at a final percent of 0.5, 5, 10, 20, 35, 70, or 100% of the original amount. Unmodified PBMC was included as a control and all cells were stimulated with OKT3 and IL-2 or left in R-10 medium without stimulus. After 24, 48, and 70 hours, samples were collected to analyze by flow cytometry for CD3, CD4, CD8, CD14, and PD1 expression. The results show that an increasing percent of monocytes corresponded to the increased expression of PD1 on CD8+ and CD4+ T cells. Conclusion: Our results show that there are variable reductions in markers of T cell activation and cytokine secretion in MDS patients compared to healthy donors. We also observed that the fold increase in activation induced PD-1 expression was well correlated with the percent of CD14+ monocytes in the PBMC of both MDS patients and healthy donors. Direct experimentation revealed that this correlation is a cause-effect relationship. We are continuing to investigate the role of monocytes in T cell activation in MDS patients. Disclosures Bejar: Celgene: Consultancy; Takeda Pharmaceuticals: Research Funding; AbbVie/Genentech: Consultancy, Honoraria; Astex/Otsuka: Consultancy; Modus Outcomes: Consultancy; Daiichi-Sankyo: Consultancy. Lane:PersImmune, Inc.: Employment.

scholarly journals Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2770-2778 ◽  
Author(s):  
AH Galy ◽  
D Cen ◽  
M Travis ◽  
S Chen ◽  
BP Chen
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Cell Activity ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Hla Dr ◽  
Adult Bone

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin-ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA-cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.

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.

Myeloid Impairment Contributes to Immunoparesis in Multiple Myeloma but Not MGUS

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1831-1831
Author(s):  
Alessandra Romano ◽  
Nunziatina Parrinello ◽  
Calogero Vetro ◽  
Piera La Cava ◽  
Annalisa Chiarenza ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
T Cell ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Healthy Subjects ◽  
Conflicts Of Interest ◽  
Myeloid Suppressor Cells ◽  
Hla Dr ◽  
Healthy Donors

Abstract Abstract 1831 Introduction In Multiple Myeloma (MM), but not in the monoclonal gammopathy of unknown significance (MGUS), the immune function is impaired as consequence of an immunologically hostile microenvironment and cellular defects, including reduction of immuno-surveillance and T-cell immunoparesis. We conducted an study focused on the myeloid compartment in MM, and its role in the progression from MGUS to MM. Methods Between January 2009 and April 2011 peripheral blood obtained from 60 consecutive newly diagnosed MM and 70 MGUS plus 30 healthy subjects was studied for evaluation of myeloid subpopulations and lymphoid paresis. Myeloid dysfunction was evaluated as percentage and absolute count of circulating myeloid suppressor cells (MDSC) in peripheral blood assessed by flow cytometry as follows: im-MDSC (CD34+/CD11b+/CD13+/CD14-/ HLA-DR-/CD45+), neutrophilic-like N-MDSC (CD11b+/CD13+/CD15+/CD14-/HLA-DR-/Lin-) and monocytic-like mo-MDSC (CD14+/HLA-DRlow/-). Myeloid function was evaluated by phagocytic activity using a commercially available kit (Phagotest R). Further, we investigated whether MM-neutrophils were able to induce anergy in T-cells. Neutrophils isolated from healthy donor (N=6), MGUS (N=3) or MM (N=6) peripheral blood (PB) were co-cultured with T-lymphocytes obtained from healthy donors. Expression of markers of activation in response to stimulation with PHA-P for 2 hours was assessed by flow cytometry as antigen density expressed as normalized mean of fluorescence intensity (N-MFI) of CD71 at 48 hours. Results The capability of phagocytosis of in neutrophils and monocytes from MM patients at diagnosis was significantly reduced compared to healthy subjects (p<0.001) and MGUS (p<0.0001). While the mature suppressive N-MDSC subset was not increased in MGUS and MM patients, the mo-MDSC subpopulation showed an increasing trend from healthy donors through MM (p=0.06) and the im-MDSC subset was significantly higher in MM vs healthy (p=0.002) and MGUS (p=0.001). After PHA-P stimulation, expression of CD71 (a marker of activation) in normal T-lymphocytes was increased (2954 ± 240.6 arbitrary units, au), and it was reduced (751.3 ± 30.48 au, p=0.0001) when co-coltured with MM-neutrophils, while no differences were evident in co-colture with MGUS- (2783 ± 206.1 au, p=0.61) or healthy donors-neutrophils (2588 ± 135.4, p=0.38). Conclusion Taken together, our findings suggest that in MM but not in MGUS there is a myeloid cell dysfunction that is correlated to impairment of T- cell arm. These alterations may have a role in the development of MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Multiplex, Droplet Digital PCR Assay for the Detection of T-Cell Receptor Excision Circles and Kappa-Deleting Recombination Excision Circles

2019 ◽  
Vol 66 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Tracie Profaizer ◽  
Patricia Slev
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Receptor ◽  
Reference Gene ◽  
Cell Receptor ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Blood Samples ◽  
Healthy Donors ◽  
Excision Circles

Abstract BACKGROUND T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count &lt;20 copies/μL and 17.7% had a KREC count &lt;20 copies/μL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.

scholarly journals Absolute Lymphocyte Count (ALC) Less Than 100 Predicts Adverse Transplant Outcomes with Rabbit Thymoglobulin in Patients Undergoing Matched Unrelated Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4527-4527
Author(s):  
Dipenkumar Modi ◽  
Malini Surapaneni ◽  
Seongho Kim ◽  
Lois Ayash ◽  
Asif Alavi ◽  
...  
Keyword(s):  
T Cell ◽  
Relapse Rate ◽  
Peripheral Blood ◽  
Conditioning Regimen ◽  
Advisory Board ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Transplant Outcomes ◽  
Gvhd Prophylaxis

Introduction: Rabbit thymoglobulin, an in-vivo T-cell depleting agent, is widely used as a part of GVHD prophylaxis regimen. Current dosing of thymoglobulin is often weight based and does not consider patient related factors. This results in highly variable exposure of thymoglobulin. Although higher doses (>7mg/kg) of thymoglobulin have shown to reduce the risk of GVHD, it is associated with increased rate of opportunistic infections and disease recurrence. Conversely, lower dose (2.5mg/kg) of thymoglobulin is associated with increased risk of GVHD. Thus, optimum dosing of thymoglobulin remains undefined. We hypothesized that recipient peripheral blood ALC on the first day of thymoglobulin infusion would interact with the dose of thymoglobulin administered and predict post-transplant outcomes. We plan to identify association of thymoglobulin dose with the ALC on the first day of thymoglobulin. Methods: We retrospectively evaluated clinical outcomes of adult patients (pts) who underwent matched unrelated donor AHSCT and received tacrolimus, mycophenolate (cellcept) and thymoglobulin as GVHD prophylaxis. Thymoglobulin was given at a total dose of 4.5mg/kg in divided fashion (0.5mg/kg on day -3, 1.5mg/kg on day -2 and 2.5mg/kg on day -1). The objectives were to determine rate of GVHD, overall survival (OS), relapse-free survival (RFS), relapse rate and non-relapse mortality (NRM) following AHSCT using Cox proportional hazard regression and competing risk models. Results: Between January 2005 and December 2017, 217 pts underwent AHSCT. The most common indications for AHSCT were AML (n=95, 44%), MDS (n=57, 26%), non-Hodgkin's lymphoma (n=23, 11%), and ALL (n=22, 10%). Median age of pts was 60 years (range, 18-79). All pts received peripheral blood stem cells. Ninety-eight pts (45%) received full intensity conditioning regimen and 119 pts (55%) received reduced intensity regimen. The median ALC on the first day of thymoglobulin administration was 200 K/cubic millimeter. The 6-month cumulative incidence rate (CIR) of grade III-IV acute GVHD was 14.8% and the 2-year CIR of chronic extensive GVHD was 35.4%. With a median follow up of 3.82 years for surviving patients, the 2-year RFS, OS, relapse and NRM were 50%, 57.1, 20.1%, and 30.2%, respectively. CMV and EBV reactivation rates were 37% and 11%, respectively. Four pts developed CMV disease. By our lowest ALC cutoff of 100 K/cubic millimeter, pts were divided into two groups (ALC ≤ 100 vs. ALC > 100). Multivariable analysis revealed that ALC > 100 was associated with significantly superior OS (HR 0.51, 95% CI 0.33-0.79, p=0.002), RFS (HR 0.49, 95% CI 0.33-0.74, p=0.001) and lower NRM (SHR 0.57, 95% CI 0.34-0.97, p=0.038) and marginally lower relapse rate (SHR 0.57, 95% CI 0.31-1.05, p=0.070). In addition, higher infused total nucleated cells was associated with higher NRM (SHR 1.70, 95% CI 1.02-2.83, p=0.041). No impact of disease risk index, KPS, conditioning regimen, infused CD34 cells on NRM, relapse, RFS or OS was observed. Conclusion: Our study indicates that ALC ≤ 100 is associated with adverse post-transplant outcomes when thymoglobulin dose of 4.5mg/kg is used for in-vivo T cell depletion. This finding may indicate that in pts with lower ALC, thymoglobulin dose may need to be adjusted to optimize its efficacy and avoid toxicities. In the future prospective studies, which evaluate dose reduction of thymoglobulin in pts with low ALC need to be planned to confirm these results. Disclosures Deol: Agios: Other: Advisory board; Novartis: Other: Advisory board; Kite: Other: Advisory board.

scholarly journals CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1054-1054 ◽  
Author(s):  
Hongxing Liu
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Lymph Nodes ◽  
Conflicts Of Interest ◽  
Sh2 Domain ◽  
Janus Kinase ◽  
Gingival Hyperplasia ◽  
Hematopoietic Stem ◽  
T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals Impact of Different T Cell Depletion Protocols on Immune Reconstitution Following Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-44
Author(s):  
Amandine Pradier ◽  
Adrien Petitpas ◽  
Anne-Claire Mamez ◽  
Federica Giannotti ◽  
Sarah Morin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Cell Depletion ◽  
Unrelated Donor ◽  
T Cell Depletion ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact

Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapeutic modality for a variety of hematological malignancies and congenital disorders. One of the major complications of the procedure is graft-versus-host-disease (GVHD) initiated by T cells co-administered with the graft. Removal of donor T cells from the graft is a widely employed and effective strategy to prevent GVHD, although its impact on post-transplant immune reconstitution might significantly affect anti-tumor and anti-infectious responses. Several approaches of T cell depletion (TCD) exist, including in vivo depletion using anti-thymocyte globulin (ATG) and/or post-transplant cyclophosphamide (PTCy) as well as in vitro manipulation of the graft. In this work, we analyzed the impact of different T cell depletion strategies on immune reconstitution after allogeneic HSCT. Methods We retrospectively analysed data from 168 patients transplanted between 2015 and 2019 at Geneva University Hospitals. In our center, several methods for TCD are being used, alone or in combination: 1) In vivo T cell depletion using ATG (ATG-Thymoglobulin 7.5 mg/kg or ATG-Fresenius 25 mg/kg); 2) in vitro partial T cell depletion (pTCD) of the graft obtained through in vitro incubation with alemtuzumab (Campath [Genzyme Corporation, Cambridge, MA]), washed before infusion and administered at day 0, followed on day +1 by an add-back of unmanipulated grafts containing about 100 × 106/kg donor T cells. The procedure is followed by donor lymphocyte infusions at incremental doses starting with 1 × 106 CD3/kg at 3 months to all patients who had received pTCD grafts with RIC in the absence of GVHD; 3) post-transplant cyclophosphamide (PTCy; 50 mg/kg) on days 3 and 4 post-HSCT. Absolute counts of CD3, CD4, CD8, CD19 and NK cells measured by flow cytometry during the first year after allogeneic HSCT were analyzed. Measures obtained from patients with mixed donor chimerism or after therapeutic DLI were excluded from the analysis. Cell numbers during time were compared using mixed-effects linear models depending on the TCD. Multivariable analysis was performed taking into account the impact of clinical factors differing between patients groups (patient's age, donor type and conditioning). Results ATG was administered to 77 (46%) patients, 15 (9%) patients received a pTCD graft and 26 (15%) patients received a combination of both ATG and pTCD graft. 24 (14%) patients were treated with PTCy and 26 (15%) patients received a T replete graft. 60% of patients had a reduced intensity conditioning (RIC). 48 (29%) patients received grafts from a sibling identical donor, 94 (56%) from a matched unrelated donor, 13 (8%) from mismatched unrelated donor and 13 (8%) received haploidentical grafts. TCD protocols had no significant impact on CD3 or CD8 T cell reconstitution during the first year post-HSCT (Figure 1). Conversely, CD4 T cells recovery was affected by the ATG/pTCD combination (coefficient ± SE: -67±28, p=0.019) when compared to the T cell replete group (Figure 1). Analysis of data censored for acute or chronic GVHD requiring treatment or relapse revealed a delay of CD4 T cell reconstitution in the ATG and/or pTCD treated groups on (ATG:-79±27, p=0.004; pTCD:-100±43, p=0.022; ATG/pTCD:-110±33, p&lt;0.001). Interestingly, pTCD alone or in combination with ATG resulted in a better reconstitution of NK cells compared to T replete group (pTCD: 152±45, p&lt;0.001; ATG/pTCD: 94±36, p=0.009; Figure 1). A similar effect of pTCD was also observed for B cells (pTCD: 170±48, p&lt;.001; ATG/pTCD: 127±38, p&lt;.001). The effect of pTCD on NK was confirmed when data were censored for GVHD and relapse (pTCD: 132±60, p=0.028; ATG/pTCD: 106±47, p=0.023) while only ATG/pTCD retained a significant impact on B cells (102±49, p=0.037). The use of PTCy did not affect T, NK or B cell reconstitution when compared to the T cell replete group. Conclusion Our results indicate that all TCD protocols with the only exception of PTCy are associated with a delayed recovery of CD4 T cells whereas pTCD of the graft, alone or in combination with ATG, significantly improves NK and B cell reconstitution. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals Heterogeneity in a lymphoid tumor: coexpression of T and B surface markers

Blood ◽  
1982 ◽  
Vol 60 (2) ◽  
pp. 373-380 ◽  
Author(s):  
RW Schroff ◽  
KA Foon
Keyword(s):  
Monoclonal Antibodies ◽  
T Cell ◽  
B Cell ◽  
Progenitor Cell ◽  
Leukemic Cells ◽  
Cytotoxic T Cell ◽  
Surface Markers ◽  
Lymphoid Tumor ◽  
Surface Immunoglobulin ◽  
Hla Dr

Abstract Heterogeneity of leukemic cells was defined in a case of lymphoma. Four phenotypically distinct subpopulations of leukemic cells were identified. One subpopulation was observed to simultaneously express B- and T-cell characteristics. B-cell characteristics included monoclonal IgM (lambda) surface immunoglobulin, HLA-DR antigens, and expression of the B-cell antigen identified by the BA-1 monoclonal antibody. T-cell characteristics included E-rosette formation, expression of the pan-T- associated antigens recognized by the Leu-1 and OKT-11 monoclonal antibodies, and expression of the suppressor cytotoxic T-cell- associated antigen recognized by the Leu-2 and OKT-8 monoclonal antibodies. In addition to this subpopulation, three other phenotypically distinct subpopulations were identified, two of which expressed monoclonal IgM (lambda) surface immunoglobulin. The results of this investigation indicates that three phenotypically distinct lymphoid subpopulations bearing B-cell characteristics, and probably a fourth T-cell subgroup, were derived from a common lineage. These findings suggest that the malignancy involved a lymphoid progenitor cell that may possess diverse maturational capacity.

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