Polarized CTL Responses Detected in Patients with Autoimmune Neutropenia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1459-1459
Author(s):  
Marcin W. Wlodarski ◽  
Yadira Narvaez ◽  
Alexander Rodriquez ◽  
Jaroslaw P. Maciejewski
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Significant Proportion ◽  
Diagnostic Algorithm ◽  
Diagnostic Methods ◽  
Cytotoxic T Cell ◽  
Autoimmune Neutropenia ◽  
Pcr Cloning ◽  
Myeloid Progenitor Cells ◽  
Lgl Leukemia

Abstract Drugs and intrinsic bone marrow diseases can explain most of the cases of neutropenia, and true autoimmune neutropenia (AIN) is a diagnosis of exclusion. Anti-neutrophil antibodies are not reliable, and their absence does not preclude the diagnosis of AIN. Lineage-restricted cytopenias, including neutropenia, were associated with T cell Large Granular Lymphocyte leukemia (T-LGL), but the diagnosis of this condition involves positive TCR rearrangement and flow cytometric identification of a pathologic cytotoxic T cell (CTL) population. These routinely applied methods have a limited sensitivity and rely on the presence of a high frequency of clonal cells in the sample. AIN, similar to T-LGL, may be related to a CTL-mediated process. We hypothesize that AIN, in a portion of patients, is a CTL-mediated disease in which myeloid progenitor cells are the targets. Consequently, in those patients, polarized expansions of CTL clones may be detected if efficient and sensitive diagnostic methods will be applied. Previously, we developed a diagnostic algorithm for the identification and quantification of clonal expansions in T-LGL based on the molecular analysis of TCR- utilization pattern. We studied a cohort of patients with various degrees of neutropenia (N=23) that was unexplained by clinical grounds and standard laboratory testing. Anti-neutrophil antibodies were found in 6 of these patients; in 3 patients, serum-mediated inhibition (>20%) of colony formation by normal hematopoietic progenitor was found, but there was no correlation between antibodies and serum inhibition. For detection of CTL expansions in AIN, VB typing and VB specific RT-PCR were applied followed by PCR cloning and sequencing of a large number of clones, and determination of expanded CDR3 clonotypes. When no expansion was detected by flow cytometry, multiplex PCR was used to amplify the whole VB spectrum. If identical CDR3 regions were detected by sequencing of at least 22 clones, CDR3 fragments of appropriate VB families were subcloned and sequenced, and immunodominant (identical clones occurring repetitively) were identified. Using this approach, we found only 2 expanded clones in 24 healthy donors. Those expanded clones accounted for 20% of a given VB family, or 0.7% of the CD8+ repertoire (as calculated by multiplication of clonal expansion within VB family by VB family contribution to the whole CTL population). In AIN we found expansion in 9 of 21 patients (3 of them were not detected by VB flow cytometry). Clonal frequency was 40%± 13% of a given VB family or 13%± 14% of the total CD8+ population. The presence of expanded CTL did not correlate with anti-neutrophil antibodies of serum-mediated colony inhibition. By comparison, CTL clones found in patients with T-LGL leukemia (N=75) comprised 68% of a given VB family, or 43% of the entire VB repertoire. We conclude that, using sensitive approaches, CTL expansions can be detected in a significant proportion of patients with AIN. These cases may represent minor variants of an autoimmune process that operates in T-LGL leukemia. The antigens that trigger these expansions likely may be shared. Clinically, detection of the CTL-mediated process in neutropenia may point toward rational immunosuppressive therapy aimed at T cells.

Clonotype Switching Indicates Propensity for Clonal Outgrowth From Diverse Components of the T Cell Repertoire In T Cell Large Granular Lymphocyte Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1171-1171
Author(s):  
Michael J. Clemente ◽  
Marcin W. Wlodarski ◽  
Aaron D. Viny ◽  
Mohammed Shaik ◽  
Nelli Bejanyan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Autoimmune Disease ◽  
Significant Proportion ◽  
Surrogate Marker ◽  
The Body ◽  
Large Granular Lymphocyte ◽  
Large Granular Lymphocyte Leukemia ◽  
Flow Cytometric ◽  
Lgl Leukemia

Abstract Abstract 1171 T cell large granular lymphocyte leukemia (T-LGLL) is characterized by the chronic proliferation of cytotoxic T lymphocytes (CTL) and is often associated with lineage-restricted cytopenias, autoimmune disease and B cell dyscrasias. In many patients clinical features of a true leukemia are absent. Lack of transformation, absence of progressive increase in blood counts, and a paucity of recurrent chromosomal defects or mutations clearly distinguish LGL from typical leukemic development. Thus, some forms of T-LGLL resemble more of a reactive process and possibly represent an extreme pole of oligo- or polyclonal immune reactions to viruses, autoantigens, or perhaps even exaggerated immune tumor surveillance. We have shown that a highly skewed T cell receptor (TCR) variable beta (Vβ) repertoire is strongly associated with monoclonality of TCR CDR3 regions by sequencing and therefore, detection of a Vβ expansion by flow cytometric clonotyping can serve as a surrogate marker for the presence of a clonal CTL process. Flow cytometric Vβ typing offers an opportunity to study the dynamics of the CTL clonal progression during therapy and throughout the clinical course of disease. We studied 124 patients who not only met the WHO guidelines for the diagnosis of T-LGLL but also show skewing of the Vβ spectrum by flow cytometry consistent with the mono/oligoclonal process persistent for over 6 months; 100% of cases demonstrated both TCRγ rearrangement and abnormal CTL population by flow cytometry. LGL count >900/μL was present in 73% of patients (mean 2513±3571 cells/μL). A pathologic clonal Vβ expansion was defined as > mean + 2SD of controls (n=65). Expansions identified by the Vβ panel were present in 92% (mean clone size 55±28%) and 2% had a borderline expansion (within 20% of 2SD) and 3% a δ/γ CD8+ TCR expansion. In 6% the Vβ expanded clone expressed CD4. Absolute clone count (ACC) was calculated by the Vβ contribution multiplied by the absolute CD8 (or CD4) count per μL of blood. ACC correlated well with LGL count (p<.0001, R2=.58). Clinically, patients presented with anemia (25%), neutropenia (9%), pancytopenia (19%), thrombocytopenia (3%), or multi-lineage cytopenia (29%), and 15% of patients were asymptomatic. In order to assess clonal kinetics, 62 of these patients were available for serial Vβ measurements with a follow up range of 0.5–8 years. Paired statistical analysis of clonal dynamics pre and post therapy (cyclophosphamide, alemtuzumab, methotrexate or cyclosporine) demonstrated a significant decrease in ACC between responders and refractory patients (p=.024). Unexpectedly, some patients displayed a change in the dominant clone as demonstrated by a switch in the major clonal Vβ T cell population, i.e. “clonotype switch.” Overall, 32% exhibited a clonotype switch during the study period, while others exhibited the persistence of multiple clones (22%); in 46% the initially diagnosed Vβ monoclonal expansion persisted. Those with multiple clones were more likely to change clonal dominance (p=.05). Clonotype switch was observed in both in relapsed and in refractory patients, and also was frequently accompanied by a change in clinical hematologic features. Significant absolute lymphocytosis (>4000 lymphocytes/μL) was present in 34/124 (27%) patients while 66 had normal lymphocyte counts and 24 were lymphopenic. Of the patients followed serially who clonotype switched, only 3/20 (15%) had absolute lymphocytosis, suggesting that 2 distinct subtypes of T-LGLL may exist. Patients with high lymphocyte and LGL counts may represent a true leukemic process, are less likely to have associated autoimmune disease or second malignancy, and their dominant clone is stable, suggestive of a single transformed precursor. In contrast, in patients with clonal CTL expansions not associated with absolute lymphocytosis, clonotype switching is difficult to reconcile with a true autonomous leukemic process. In sum, our results suggest that in a significant proportion of patients with T-LGL leukemia, the propensity for clonal CTL dominance is not inherent to an aberrant molecular event within the abnormal CTL clone but may rather be related to extrinsic chronic antigenic drive and immune dysregulation. Furthermore, our results lend credence to the body of evidence suggesting that T-LGL leukemia may not, in many instances, be a true leukemia, and may best be classified as T-LGL lymphoproliferation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals No cell is an island: circulating T cell:monocyte complexes are markers of immune perturbations

2018 ◽  
Author(s):  
Julie G. Burel ◽  
Mikhail Pomaznoy ◽  
Cecilia S. Lindestam Arlehamn ◽  
Daniela Weiskopf ◽  
Ricardo da Silva Antunes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Immune Responses ◽  
Human Blood ◽  
Significant Proportion ◽  
Complex Frequency ◽  
The Common ◽  
First Time ◽  
High Resolution Microscopy

AbstractOur results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artefacts and thus ignored in data acquisition and analysis, are the result of true biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell:monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be revisited.

Effect of MMP9 inhibition on effector T-cell responses in a PD1-axis refractory model.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 104-104
Author(s):  
Victoria Smith ◽  
Vladi Juric ◽  
Amanda Mikels-Vigdal ◽  
Chris O'Sullivan ◽  
Maria Kovalenko ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cell Activation ◽  
Immune Cell ◽  
Single Agent ◽  
Fold Increase ◽  
Cytotoxic T Cell

104 Background: Matrix metalloproteinase 9 (MMP9) acts via diverse mechanisms to promote tumor growth and metastasis, and is a key component of the immune-suppressive myeloid inflammatory milieu. We developed a monoclonal antibody (AB0046) that inhibits murine MMP9 and assessed its mechanism of action in immunocompetent mice as a single agent, or in combination with a murine anti-PDL1 antibody. Methods: An orthotopic, syngeneic tumor model (NeuT), which models MMP9-positive myeloid infiltrate, was utilized for efficacy and pharmacodynamic studies involving RNA and T cell receptor (TCR) sequencing, and flow cytometry. Enzymatic analyses were performed on T cell chemoattractant CXCR3 ligands (CXCL9, CXCL10, and CXCL11) which were subsequently evaluated in chemotaxis assays. Results: Anti-MMP9 treatment alone or in combination with an anti-PDL1 antibody decreased primary tumor growth as compared to IgG control-treated animals (56% vs 335% tumor growth increase, p = 0.0005) or anti-PDL1 alone. Profiling of tumors by RNA sequencing revealed that inhibition of MMP9 resulted in elevated expression of genes associated with immune cell activation pathways (Hallmark Interferon Gamma Response, FDR p < 0.001). Treatment with anti-MMP9 and anti-PDL1 antibodies decreased TCR clonality, with evidence of a more diverse TCR repertoire (p = 0.005). Immunophenotyping of tumor-associated T cells by flow cytometry showed that anti-MMP9 and anti-PDL1 co-treatment promoted a 2.8-fold increase in CD3+ cells in tumors (p = 0.01), which was associated with an increase in CD4+ T cells (3.2-fold increase; p = 0.006) and CD8+ T cells (2.8-fold increase; p = 0.013). In contrast, anti-MMP9 and combination treatment resulted in a decrease in tumor-associated regulatory T cells (CD25+ FoxP3+ cells, p = 0.04). MMP9 cleavage of T cell chemoattractant ligands in vitro rendered them functionally inactive for recruitment of activated primary human effector T cells. Conclusions: Inhibition of MMP9 reduces tumor burden and promotes cytotoxic T cell infiltration in a PD1-axis refractory mouse model. The combination of nivolumab and GS-5745, a humanized anti-MMP9 inhibitory antibody, is currently being evaluated in gastric cancer (NCT02864381).

scholarly journals Neutropenia Associated With T-Cell Large Granular Lymphocyte Leukemia: Long-Term Response to Cyclosporine Therapy Despite Persistence of Abnormal Cells

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3372-3378 ◽  
Author(s):  
Raman Sood ◽  
Carleton C. Stewart ◽  
Peter D. Aplan ◽  
Hiroyuki Murai ◽  
Pamela Ward ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Large Granular Lymphocyte ◽  
Severe Neutropenia ◽  
Multiparameter Flow Cytometry ◽  
Gene Rearrangements ◽  
Aortofemoral Bypass ◽  
Blood Samples ◽  
Abnormal Cells ◽  
Lgl Leukemia

Abstract T-cell large granular lymphocyte (T-LGL) leukemia is clinically indolent, but is associated with severe neutropenia in approximately 50% of cases. The pathogenesis of the neutropenia is unclear. We report reversal of severe neutropenia associated with T-LGL leukemia in five patients treated with cyclosporine (CSA). All five had persistent neutrophil counts below 0.5 × 109/L, two had agranulocytosis, and four had recurrent infections. Increased populations of LGL were present in blood and marrow, with a T-LGL immunophenotype (CD3+CD8+CD16±CD56±CD57+) shown by multiparameter flow cytometry, and clonal T-cell receptor (TCR) gene rearrangements in two of two pretreatment blood samples studied. CSA was initiated at doses of 1 to 1.5 mg/kg orally every 12 hours, with subsequent dose adjustments based on trough serum levels. Four patients attained normal neutrophil counts with CSA alone; one required addition of low-dose granulocyte-macrophage colony-stimulating factor. Time to attainment of 1.5 × 109/L neutrophils ranged from 21 to 75 days. Attempts to taper and withdraw CSA resulted in recurrent neutropenia. Three patients have maintained normal neutrophil counts on continued CSA therapy for 2, 8, and 8.5 years. Two patients died 1.7 and 4.6 years after initiation of CSA despite normal neutrophil counts—one of metastatic melanoma and one of complications after aortofemoral bypass surgery. Despite resolution of neutropenia, increased populations of T-LGL cells have persisted in all patients during CSA therapy, as shown by morphology and flow cytometry and by the presence of clonal TCR gene rearrangements in four patients' posttreatment blood samples. We conclude that CSA is an effective therapy for neutropenia associated with T-LGL leukemia, and that resolution of neutropenia despite persistence of abnormal cells implies that CSA may inhibit T-LGL secretion of yet unidentified mediators of neutropenia.

Skewing of the T-Cell Receptor Repertoire in Patients Receiving Rituximab after Allogeneic Hematopoietic Cell Transplantation: What Lies Beneath?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3962-3962
Author(s):  
Apostolia Papalexandri ◽  
Maria Karypidou ◽  
Evangelia Stalika ◽  
Michail Iskas ◽  
Anna Vardi ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Transplantation ◽  
T Cell Receptor ◽  
Hematopoietic Cell Transplantation ◽  
Late Onset ◽  
Cell Receptor ◽  
Hematopoietic Cell ◽  
Cytotoxic T Cell ◽  
Gene Repertoire ◽  
Lgl Leukemia

Abstract The development of CD3+/CD8+/CD57+ cytotoxic cell expansions after allogeneic hematopoietic cell transplantation (allo-HCT) driven by antigenic stimulation, viral or associated with chronic graft-versus-host disease (cGvHD), has been suggested as related with favorable outcome. Rituximab, an anti-CD20 humanized monoclonal antibody, has been linked to the development of oligo- or even monoclonal expansions of CD3+/CD8+/CD57+ T-large granular lymphocytes (T-LGLs) that can manifest with neutropenia of delayed origin in relation to Rituximab administration. We have recently reported remarkable skewing of the T-cell receptor (TR) gene repertoire in two allo-HCT transplanted patients with delayed neutropenia associated with T-LGL expansions developing in a context of GvHD and Rituximab administration for EBV reactivation. Prompted by these preliminary findings, we here extend our immunogenetic studies of the TR repertoire in patients receiving Rituximab post allo-HCT. The study group was comprised of 9 patients (including the two previously reported) aged 14-50 years (median 41) who were subjected to myeloablative allo-HCT (4 from matched related, 3 from matched unrelated donors), haplo-identical transplantation (1) or Reduced Intensity Conditioning-allo-HCT from sibling donor (1), all for hematological malignancies. All patients received Rituximab consecutively between 2010-2013 either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. In all patients TR gene repertoire analysis was performed at least one year after the transplantation (range 12-72 months), when immune reconstitution normally would have been achieved, and 5-24 months after the first treatment with Rituximab. Each patient received a mean of 7 cycles of Rituximab (range, 1-14). TRBV-TRBD-TRDJ gene rearrangements were PCR-amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data was interpreted using the IMGT/V-QUEST tool. A total of 164 sequences were analyzed (9-25/case, median=18) revealing 106 productive TRBV-TRBD-TRBJ rearrangements. Among the 29 TRBV functional genes identified only three accounted for 48% of cases: (i) TRBV27*01 (25%), (ii) TRBV6-5*01 (13%), (iii) TRBV6-2*01 (10%). Of note, TRBV27*01 has been reported as the most frequent TRBV gene in Rituximab-related late-onset neutropenia in CLL. All cases were found to carry clusters of identical (>=2) rearrangements corresponding to clonotypes. In the majority of cases (5/9), 2-4 (median 3) immunodominant clonotypes accounted for over 30% of the analyzed sequences (frequency of immunodominant clonotype/case 13-40%). Lymphocyte subpopulation analysis by flow cytometry in 6 patients revealed T-LGL expansion. Samples from additional time points (spanning a period of 10 years), pre- and post- Rituximab, were studied in one patient. Analysis of 71 sequences demonstrated progressive expansion of a certain clonotype overtime, associated with the emergence of steroid-refractory autoimmune hemolytic anemia in a context of CD3+CD8+CD57+ lymphoproliferation. This particular clonotype dominated the repertoire by far, thus establishing a diagnosis of T-LGL leukemia. which, remarkably, proved to be of donor origin (97% and 30% donor chimerism in T lymphocytes and total hematopoeisis, respectively). No association of oligoclonality to stronger GvL effect could be found among the rest of the patients. However, a strong correlation with cGvHD (100% vs 25% among polyclonal cases) was identified. Late-onset neutropenia was documented in 4/9 patients, regardless of the composition of the repertoire i.e whether it was polyclonal or oligo(mono)clonal. In conclusion, we report frequent development of oligoclonal cytotoxic T-cell populations after Rituximab treatment post allo-HCT likely of multifactorial evidence. Direct evidence of the anti-leukemic effect of this phenomenon could not be provided, however, the observed association of oligoclonality with GvHD and the development of a possible “T-LGL leukemia vs leukemia” effect in one patient is noteworthy and merits further investigation. Finally, the observed skewing of the TR gene repertoire strongly implicates antigen selection in the development of cytotoxic T-cell expansions after allo-HCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Extracellular Vesicles Derived from Cytokine-Activated CD8+ T Cell Exhibit Broad Anticancer Activity

2021 ◽  
Author(s):  
Lin Zhang ◽  
Yang An ◽  
Xuena Yang ◽  
Wenwen Yu ◽  
Baihui Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cancer Cells ◽  
Extracellular Vesicles ◽  
Cd8 T Cells ◽  
Imaging System ◽  
Nano Particles ◽  
Cytotoxic T Cell ◽  
Resting Cells

Abstract Background: Extracellular vesicles (EVs) are the nano-sized membrane particles secreted by various cell types, which are involved in many important cellular processes. Recently, EVs originating from immune cells, such as dendritic cells, chimeric antigen receptor T cells (CAR-T) and natural killer cells, have attracted much attention because of their known direct and indirect antitumor activity. Here, we reported another EV released by cytokine-activated CD8+ T cells (caCD8-EVs) and its phenotype and function. Methods: CaCD8-EVs were purified by ultracentrifugation and phenotypically characterized by flow cytometry. The cytotoxicity induced by caCD8-EVs was assessed using the CellTiter-Glo Viability assay in vitro and xenograft tumor established in vivo. The uptake of caCD8-EVs was detected by a Live Cell Imaging System and flow cytometry. Mass spectrometry and protein bioinformatics analysis, together with western blotting and real-time PCR, were used to investigate the effect of caCD8-EVs on the cancer cells.Results: CaCD8 cells released EVs following stimulation of CD8+ T cells with an anti-CD3 antibody and a cytokines cocktail ex vivo. Phenotype analysis showed that caCD8-EVs carried cytotoxic T cell membrane molecules (CD3, CD8 and TCRs) and partially inherited NKG2D molecule which was inductively expressed on the caCD8 T cells. In contrast, the nano-particles were devoid of checkpoint proteins on their surface. Notably, caCD8-EVs displayed extensive cytotoxicity against cancer cells, but limited effects on non-cancerous cell lines. Uptake assessment showed that actively dividing tumor cells were more likely to capture caCD8-EVs compared with resting cells. Mechanism analysis demonstrated for the first time that caCD8-EVs contained not only typical cytotoxic proteins (i.e., granzyme B and perforin) but also IFNg that participated in EVs-induced cytotoxicity.Conclusion: Our data reveal the characteristics of caCD8-derived EVs and support the use of these EVs as a novel potential approach for cancer therapy.

scholarly journals Ex Vivo Modeling of Multiple Myeloma Provides Basis for Studying Treatment Combinations and Immunotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2113-2113
Author(s):  
Michael P. Chu ◽  
Christopher P. Venner ◽  
Irwindeep Sandhu ◽  
Eva Baigorri ◽  
Jitra Kriangkum ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Ex Vivo ◽  
3D Culture ◽  
Active Immunotherapy ◽  
Cytotoxic T Cell ◽  
Checkpoint Inhibition ◽  
Effector Cells ◽  
Culture Model ◽  
Tissue Culture Model

Abstract Background Multiple myeloma (MM) remains incurable despite treatment advances. While passive immunotherapy such as anti-CD38 antibodies is highly effective, active immunotherapy may provide long-lasting remissions by virtue of triggering memory. A phase 1 nivolumab study, an antibody targeting programmed death-1 (PD1), was unable to yield any responses in multiply relapsed MM patients. Conversely, preliminary trial data of lenalidomide combined with pembrolizumab, a different anti-PD1 antibody, found significantly higher response rates. These two differing outcomes reflect our limited understanding of checkpoint inhibition and immunotherapy in MM. There is a paucity of preclinical models to guide therapeutic studies. Cell lines and xenografted murine models are incapable of exploring active immunotherapy due to a lack of microenvironment and endogenous immune cell signals. Furthermore, malignant cells responsive to drugs in 2-dimensional (2D) cultures are known to display a more resistance in 3D. We have previously demonstrated that B-cell malignancies can be accurately studied using a 3D culture system of patient bone marrow mononuclear cells (BMCs) and can better inform translational trials. Herein we describe an ex vivo, 3D tissue culture model of patient-derived MM samples to more accurately test therapeutics including checkpoint inhibition using ipilimumab, a monoclonal antibody targeting cytotoxic T-lymphocyte antigen 4 (CTLA) which is crucial in co-stimulatory signaling of effector T-cells. Methods A 3D extracellular matrix was created using matrigel in 12-well plates. BMCs were isolated from marrow aspirates of 5 MM patients at time of diagnosis and individually cultured. Each patient sample was tested for sensitivity against increasing concentrations of ipilimumab (1X, 3X, and 10X clinical doses) added into supportive medium. Plates were monitored visually by microscopy followed by harvest on day 21 using enzymatic degradation. Unique clonotypic heavy chain immunoglobulin rearrangement (IgH VDJ) from each sample was sequenced, validated and used for semi-quantitative PCR. Semi-QT PCR with clone-specific primers estimated malignant cell survival after harvest. Flow cytometry was used to define cell populations present in culture and to correlate with clonotypic PCR data. T-cell mediated activity was examined by reverse transcription of trizol-extracted, T-cell RNA after harvest. Results All samples were successfully cultured, followed for 21 days and harvested. Flow cytometry confirmed presence of T-cell subsets, B-cells, NK cells and dendritic cells before and after culture in 3D. Minimal depletion of clonotypic cells was observed at 3x clinical levels of drug. At 10x simulated clinical therapeutic levels, 3 MM samples demonstrated >90% death of clonotypic MM cells while the other 2 demonstrated 62% and 72% death, respectively, compared to untreated control cultures. The extent to which the drug diffuses into the matrigel is as yet unknown. Flow cytometry of harvested cells suggest that T-cells demonstrate a modest shift toward CD4 and CD8 effector cells. Preliminary mechanistic data from one MM sample using trizol-extracted RNA and reverse transcriptase PCR harvested at 21 days from 3D culture suggests that anti-malignant, cytotoxic T-cell effect may be driven by granzyme B expression. Expanded data from the remaining samples will be presented. Conclusions We demonstrate that an ex vivo 3D tissue culture model of MM is both feasible and informative in studying immunotherapy. By culturing unselected BMCs which include stromal cells, immune cells and malignant populations, the 3D culture more closely mimics the tumor microenvironment with both the patient's immune system present as well as stromal supportive signals. In this study, we show that in the presence of active immune effector cells, ipilimumab has activity against patient-derived MM cells. The data suggests the importance of targeted cytotoxic T-cell activation as a primary mechanism of action. We have previously studied standard MM therapeutics such as cytotoxic chemotherapy, immunomodulatory drugs, and proteasome inhibitors in the same way. Consequently, this model is well positioned to study other immunotherapies such as other checkpoint inhibitors, cellular therapy, and combinations. Further testing with therapeutics targeting PD1/PDL1, and adenosine receptors are underway. Disclosures Venner: Takeda: Honoraria; Celgene: Honoraria, Research Funding; J+J: Research Funding; Janssen: Honoraria; Amgen: Honoraria. Belch:Celgene: Honoraria; Janssen: Honoraria; Amgen: Honoraria; Takeda: Honoraria.

scholarly journals Oligoclonality and subpopulation structure of bone marrow T-cells in patients with aplastic anaemia

2020 ◽  
Vol 65 (4) ◽  
pp. 417-430
Author(s):  
A. V. Abramova ◽  
I. V. Galtseva ◽  
E. A. Mikhailova ◽  
N. M. Kapranov ◽  
Yu. O. Davydova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Cell Subpopulation ◽  
Cytotoxic T Cell ◽  
Double Negative ◽  
Cell Subpopulations

Introduction. The main pathogenetic mechanism of the development of aplastic anemia (AA) is a violation of the immune regulation of hematopoiesis.Aim: to study of the subpopulation composition of T-cells and the repertoire of the T-cell receptor in AA patients.Patients and Methods. The study included AA patients (n = 40) without prior immunosuppressive therapy in 2018–2020. The T-cell subpopulation structure and T-cell receptor Vβ-family (TCR-Vβ) oligoclonality were studied in samples of bone marrow using flow cytometry.Results. We report characteristic properties of T-cell subpopulations of bone marrow in all AA patients: elevated counts of cytotoxic T-cells, effector CD4+ and CD8+ cells, CD4+ memory cells, which may suggest a long-term antigenic stimulation with subsequent activation of these cell subpopulations resulting in hyperexpression of pro-inflammatory cytokines. Diminishing of naive CD4+ and CD8+ cells, regulatory and double negative T-cells may indicate a relaxing control of cytokine-producing T-cells. A relationship has been established between the AA severity and counts of effector, regulatory, double negative and PD-1 positive T-cells. A highest count of potentially cytokine-producing T-cells and lowest count of cells involved in T-cell activity regulation were observed in very severe AA patients. Studies of the TCR-Vβ repertoire revealed oligoclonal expansion in the cytotoxic T-cell subpopulation.Conclusion. Enrichment in selected Vβ families suggests autoreactive T-cell clonality and attests to the immune nature of AA. A dynamic TCR-Vβ repertoire assay may be recommended in the disease monitoring. Flow cytometry helps identify valuable biomarkers for T-cell clone monitoring in AA and a better assessment of the disease progression.

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