Autoreactive T Cells From Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Specifically Recognize Glycosyl- Phosphatidyl-Inositol (GPI)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 647-647
Author(s):  
Lucia Gargiulo ◽  
Michela Sica ◽  
Maria Papaioannou ◽  
Giulia Talini ◽  
Aristeidis Chaidos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Median Frequency ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
Alpha Chain

Abstract Abstract 647 PNH is caused by somatic mutations in the PIG-A gene in hematopoietic stem cells leading to disruption of the synthesis of GPI, a glycolipid used for post-translational modification of proteins. PNH is classically characterized by the triad of intravascular hemolysis, severe acquired thrombophilia and multiple cytopenias. Whereas the first two components are the consequence of increased susceptibility to activated complement, due to the deficiency on red cells of GPI-linked CD55 and CD59, cytopenias are an expression of bone marrow failure. This fact, together with strong clinical evidence associating PNH with aplastic anemia, has led to the notion that a T cell-mediated attack underlies both conditions whereby in PNH the PIG-A mutant clone is able to escape the attack. Consistent with this, we previously found skewing of the spectratyping pattern of T cells, and recurrent TCR beta chain sequences. Here, using complementary assays, we tested the hypothesis that PIG-A mutant cells escape an auto-immune T cell attack targeted to the GPI molecule itself, which would be presented by CD1d. First, CD8+ T cells from PNH patients and from normal donors were co-cultured with C1R-CD1d B cells pulsed with either trypanosomal GPI (t-GPI) or with solvent. Reactive T cells producing interferon gamma (IFNG) were then enumerated by ELISPOT technology. We found that in the presence of t-GPI-pulsed C1R-CD1d cells, the frequency of reactive T cells was significantly increased (up to 5-fold) in patients on each of days 1, 7, 14 but not in controls. To confirm that T cell reactivity to GPI depended on the presence of CD1d, when similar experiments were carried out with the untransfected, CD1d-negative C1R cells, no such increase was seen. Similar results were obtained using as antigen presenting cells (APC) K562 cells and appropriate derivatives. Next, we used for antigen presentation, in addition to the C1R-CD1d cell line, a derivative that lacks the ability to synthesize GPI (GPI- C1R-CD1d). In these experiments, reactive (IFNG-producing) T cells were counted by intracellular staining and flow-cytometry:in PNH patients, but not in controls, we found a 3-fold increase in the frequency of reactive CD8+ T cells when the APC were GPI+ C1R-CD1d compared to when they were GPI- C1R-CD1d. This increase was not seen when APC were the C1R cells. By contrast, with GPI- C1R-CD1d the increase was restored by the addition of exogenous human GPI (h-GPI). Next, we tested the reactivity of T cells from 11 PNH patients using as APC mature dendritic cells (DC) obtained from autologous peripheral blood monocytes. By selecting CD14-neg cells – that are the majority in PNH patients - we took advantage of the fact that in this way we had APC that were naturally GPI negative. In the absence of added GPI we could not demonstrate reactive T cells; however, in 7 out of 11 patients there was a significant increment when the DC were loaded with h-GPI (p =0,0149; Mann-Whitney test). Since CD1d is required for development and function of iNKT cells, immunoregulatory, glycolipid-specific T cells characterized by an invariant TCRValpha24Jalpha18 chain, we tested the hypothesis that CD1d-restricted, GPI-specific T cells might also possess an invariant TCR alpha chain. Global TCR alpha chain repertoire analysis of flow-sorted dimer CD1d/GPI+ T cells identified a novel TCRValpha21Jalpha31 invariant chain (9/9 clones) in 1 out of 3 patients tested. Further analysis of the TCRValpha21 repertoire of patients and controls by next generation sequencing (GS Junior, Roche) revealed that the invariant TCRValpha21Jalpha31 chain was present but not expanded in 5/8 controls (frequency<0.5% of TCRValpha21-Calpha T cell repertoire); by contrast, it was expanded in 6/11 patients (median frequency: 1.86%, range: 1.05–75% of TCRValpha21-Calpha T cell repertoire). In conclusion, we provide the first direct evidence that GPI-specific, CD1d-restricted T cells, enriched in a novel invariant TCR alpha chain sequence, are increased in patients with PNH but not normal controls. Disclosures: No relevant conflicts of interest to declare.

Prospective Molecular Identification of Alloreactive CTL Clones in Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4972-4972
Author(s):  
Christine L. O’Keefe ◽  
Ronald Sobecks ◽  
Alexander Rodriguez ◽  
Julie Curtis ◽  
Elizabeth Kuckowski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Molecular Analysis ◽  
Cd8 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Activation Markers ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Donor Cells

Abstract The process of immune recovery after allogeneic HSCT can be characterized by an often profound oligoclonality of the TCR spectrum which may reflect: 1) A decreased diversity within the T cell population or 2) Expansion of individual clones that may be caused by specific antigenic drive exerted by pathogens (e.g., CMV) or alloantigens during the process of GvHD. Novel technologies based on the molecular analysis of the TCR repertoire can be applied to study clonal responses, including multiplex amplification of rearranged TCR VB chains followed by sequencing and quantitation of their contribution to the entire T cell repertoire. We initially studied the T cell repertoire after allogeneic HSCT in sibling (N=20) and matched unrelated (N=9) transplants. VB spectratyping was performed on CD8+ T cells in 22 patients; of the expanded VB families tested, 61.2% (30 of 49) were mono- or oligoclonal by genotyping. The clonal size and structure was determined by sequencing. Immunodominant clones contributed up to 5.4% (avg. 1.4%; range 0.1–5.4%) of all CD8+ T cells, indicating that certain stimuli may drive expansion of immunodominant clones. We originally hypothesized that these expanded clones were allospecific and likely played a role in GvHD; however, we found no correlation between the presence of significant expansions and grade III/IV GvHD. Therefore, in order to identify alloreactive CTL clones and their clonotypic markers, an alternative approach was devised. The proposed technique utilizes an allostimulation step: recipient cells serve as targets to induce activation of allospecific donor cells. Donor alloreactive cells are identified by their expression of activation markers, such as CD25 or CD69. After sorting, allospecific T cells are used as a source of cDNA for identification and quantitation of allospecific clonotypes. In this fashion, we have analyzed patients undergoing allogeneic sibling and matched unrelated donor grafting (N=7). Prior to transplant, allostimulation was performed and alloreactive CD8-derived clonotypes were subjected to molecular analysis. VB families represented within alloresponsive CTL populations that were oligoclonal by genotyping were subcloned and a large number of CDR3 clones were sequenced to identify the immunodominant clonotypes. Sequences have been derived from activated CD8+ donor cells in 6 cases; an average of 4 (range 1–7) VB families per pair have been characterized.. Although the presence of multiple VB families with a diversified CDR3 spectrum suggests the polyclonal nature of alloresponsive clones, immunodominant clones were identified. A total of 13 immunodominant clonotypes have been identified in 5 patients. Five such clones were identified in one donor/recipient pair; in each pair at least one immunodominant clonotype was isolated. Up to 18 clones per VB family were sequenced, and the average expansion contributed 56% to the entire VB family (range 15–100%). Clonotype-specific primers have been designed from two expanded clones and used to detect the allospecific clones in post-transplant blood samples in one patient/donor pair. In sum, molecularly defined marker clonotypes indicative of alloresponsive CTLs in HSCT can be individually and prospectively isolated. Such clonotypes may find application in tissue and blood diagnosis of GvHD.

scholarly journals Characterization of human cytomegalovirus peptide–specific CD8+ T-cell repertoire diversity following in vitro restimulation by antigen-pulsed dendritic cells

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 213-223 ◽  
Author(s):  
Karl Peggs ◽  
Stephanie Verfuerth ◽  
Arnold Pizzey ◽  
Jenni Ainsworth ◽  
Paul Moss ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Viral Replication ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Repertoire Diversity ◽  
Clonal Composition

Under conditions of impaired T-cell immunity, human cytomegalovirus (HCMV) can reactivate from lifelong latency, resulting in potentially fatal disease. A crucial role for CD8+ T cells has been demonstrated in control of viral replication, and high levels of HCMV-specific cytotoxic T-lymphocytes are seen in immunocompetent HCMV-seropositive individuals despite very low viral loads. Elucidation of the minimum portion of the anti-HCMV T-cell repertoire that is required to suppress viral replication requires further study of clonal composition. The ability of dendritic cells to take up and process exogenous viral antigen by constitutive macropinocytosis was used to study HCMV-specific T-cell memory in the absence of viral replication. The specificity and clonal composition of the CD8+ T-cell responses were evaluated using HLA tetrameric complexes and T-cell receptor β chain (TCRBV) spectratypic analyses. There was a skewed reactivity toward the matrix protein pp65, with up to 40-fold expansion of CD8+ T cells directed toward a single peptide-MHC combination. Individual expansions detected on TCRBV spectratype analysis were HCMV-specific and composed of single or highly restricted numbers of clones. There was preferential TCRBV gene usage (BV6.1/6.2, BV8, and BV13 in HLA-A*0201+ individuals) but lack of conservation of CDR3 length and junctional motifs between donors. While there was a spectrum of TCR repertoire diversity directed toward individual MHC-peptide combinations between donors, a relatively small number of clones appeared to predominate the response in each case. These data provide further insight into the range of anti-HCMV responses and will aid the design and monitoring of adoptive immunotherapy protocols.

Reconstitution of the T Cell Repertoire Following Treatment with Alemtuzumab in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2986-2986
Author(s):  
Mohammad R. Rezvany ◽  
Mahmood J. Tehrani ◽  
Claes Karlsson ◽  
Jeanette Lundin ◽  
Hodjattallah Rabbani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Long Term Follow Up

Abstract Background and Methods: B-cell chronic lymphocytic leukemia (B-CLL) occurs as a result of clonal accumulation of functionally abnormal B cells. Alemtuzumab is a humanized monoclonal antibody specific for the CD52 antigen, which is highly expressed on both B-CLL cells and normal lymphocytes, but not on hematopoietic (CD34) stem cells. Alemtuzumab has been shown to effectively deplete the blood and bone marrow of lymphocytes, including CD4 and CD8 T cells, which may lead to profound immunosuppression and make patients more susceptible to infections. We and others have previously shown that the CD4 T cells in B-CLL patients may be clonally distinct from the normal population in that they present a more clonal pattern of the T-cell receptor (TCR) repertoire (Rezvany et al, Blood2003;101:1063–1070). It is therefore of interest to study the T cell repertoire following alemtuzumab administration as well as factors affecting T cell reconstitution following CD52 targeted therapy. In this study, we evaluated in depth the T-cell receptor-beta-variable sequence (TCR BV) in CD4 and CD8 T cells by real-time PCR, before and repeatedly after/during long term follow-up, in 5 B-CLL patients who had received alemtuzumab as first-line therapy (Lundin et al, Blood2002;100:768–773). Also, an analysis was conducted of CDR3 length polymorphism to describe changes in the clonality pattern. Results: A decline in most of BV genes either in CD4 or CD8 T cells was observed shortly after alemtuzumab treatment, which was followed by a gradual increase in most of the BV genes during long-term follow up. CDR3 length polymorphism analysis shortly after treatment revealed an even more highly restricted pattern in CD4 T cells compared to baseline with a shift towards a monoclonal/oligoclonal pattern regardless of increased or decreased BV usage. Furthermore, in the analysis of the clonal spectrum that was expressed shortly after alemtuzumab therapy, the number of peaks was significantly reduced in CD4 (P &lt;0.01) but not in CD8 T cells, which was followed by a gradual increase in diversity towards a polyclonal repertoire during long-term follow up. Conclusions: These results indicate that perturbations in the T cell repertoire following alemtuzumab are complex, and are not reflected by changes in CD4/CD8 T cell numbers only. The restricted CDR3 pattern present prior to therapy became even more restricted after end of treatment, followed by a normalization of CDR3 patterns in CD4 T-cells during long-term follow-up. These results further suggest a regulatory role for T cells in relation to the malignant B cell clone in patients with B-CLL.

T Cell Receptor Repertoire of Patients with Chronic Graft-Versus Host Disease.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5174-5174
Author(s):  
Olga Y. Azhipa ◽  
Scott D. Rowley ◽  
Michele L. Donato ◽  
Robert Korngold ◽  
Thea M. Friedman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Flow Cytometric Analysis ◽  
T Cell Responses ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Cell Responses ◽  
Flow Cytometric ◽  
Complexity Index

Abstract Chronic GVHD (cGVHD) is a major risk factor in patients receiving allogeneic hematopoietic cell transplantation (HCT), and is a complicated syndrome with a combination of autoimmune-like features and a range of multiorgan manifestations. Currently, efforts are being made to standardize the criteria for diagnosis and staging of cGVHD, but there is little understanding of the pathogenesis of the disease, associated biomarkers, and the immune perturbations that may result. Reconstitution of the T cell repertoire after allo-HCT often takes several months to a year, and may be significantly impaired or skewed in patients who develop cGVHD. We thus sought to assess the immune T cell status of cGVHD patients by TCR Vβ CDR3-size spectratype analysis. A cohort of 9 patients who underwent allo-HCT (PBMC n=7; BM n=2) were enrolled in the study. The underlying diseases in these patients were CML (n=1), AML (n=4), ALL (n=1), CLL (n=1), and MM (n=2). Patients received either reduced intensity or myeloablative conditioning before transplantation, and 8 of the 9 had a previous history of acute GVHD. Furthermore, the patients did not have evidence of infectious disease. PBMC was collected from each patient at one time point ranging from 2 wk to 3 yr from the time they were diagnosed with cGVHD. The onset of cGVHD ranged from 100 d to 3 yr post-HCT (median of 5 mo). Flow cytometric analysis was performed on peripheral blood lymphocytes from 7 of the 9 patients to analyze recovery of different subpopulations. PCR amplification of the CDR3 region of 21 TCR Vβ genes was used to analyze the diversity of the T cell repertoire. The PCR products were run on a sizing gel to separate the CDR3-lengths, and further analyzed by ABI GeneMapper software. Flow cytometric analysis revealed diverse percentages of CD4+ and CD8+ T cells among the 7 patients tested, which were correlated with the post-HCT period. Two patients who received HCT, 4 and 9 months before blood sampling, had only 3% and 4% CD4+ and 3% and 9% CD8+ T cells in their PBMC sample, respectively. On the other hand, the remaining 5 patients, who were all at later time points post-HCT, had CD4+ and CD8+ T cell percentages within normal range. One patient had a ratio close to the normal 2:1 CD4/CD8 ratio, two patients had a 1:1 ratio, and four had inverse CD4/CD8 ratios. Based on CDR3-size spectratype analysis, we determined the recipient TCR-Vβ complexity index within each resoluble family, which represented the percentage of the number of peaks found for each Vβ relative to that found in the average corresponding Vβ family of 10 healthy donors. We considered Vβ to be fully complex if the complexity index exceeded 85%. The results indicated that 41 to 88% of resolved Vβ in all 9 patients were fully complex, with the lower range corresponding to those patients sampled early post-HCT. Vβ 1, 2, 4, 6, 8, 12, and 13 families revealed the best recovery in all patients, even in patients after 4-mo post-HCT. Importantly, extensive skewing of the repertoire within most of the TCR Vβ families were found in all 9 recipients, suggesting that there were active heterogenous T cell responses in those patients with cGVHD. As to what these T cell responses were directed to remains to be seen, and could theoretically involve autoantigens, alloantigens, tumor antigens, or sub-detectable infectious agents. In any case, the presence of a wide-ranging T cell response in these patients may serve as an important new diagnostic indicator for cGVHD.

Evolution of the Donor T Cell Repertoire In Allogeneic Stem Cell Transplant Recipients In the Second Decade After Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 831-831
Author(s):  
Robert Q. Le ◽  
J. Joseph Melenhorst ◽  
Brenna Hill ◽  
Sarfraz Memon ◽  
Minoo Battiwalla ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cell Receptor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Significant Difference

Abstract Abstract 831 After allogeneic stem cell transplantation (SCT), donor T lymphocyte immune function is slowly re-established in the recipient through reconstruction of the donor's post-thymic T cell repertoire and from T cell neogenesis in the thymus. Although long-term survivors from SCT appear healthy, their immune repertoire and differences from that of their donors have not been characterized. We studied 38 healthy patients surviving more than 10 years from a myeloablative SCT for hematological malignancy (median follow-up 12 years, range 10–16 years). T cell and natural killer (NK) cell repertoires in these patients were compared with cells from their stem cell donors cryopreserved at time of transplant and from the same donors at 10 year after SCT. The median age of both recipients and their sibling donors at time of transplant was identical (36 years). Patients received cyclosporine GVHD prophylaxis and delayed add-back of donor lymphocytes 30–90 days post transplant. Only one patient was on continued immunosuppressive treatment at the time of study. Compared with the donor pre-transplant counts there was no significant difference in the absolute lymphocyte, neutrophil, monocyte, CD4+ and CD8+ T cell, NK cell, and B cell subset counts. However, compared to their donors, recipients had a) significantly fewer naïve CD4+ and CD8+ T cells; b) lower T cell receptor excision circles levels; c) fewer CD4+ central memory T cells; d) more effector CD8+ T cells; e) and more FOXP3+ regulatory T cells. These data suggest that the patient had a persistent deficiency on T cell neogenesis. Molecular examination of the T cell receptor Vbeta (TCRBV) repertoire by spectratype analysis showed that there was no significant difference in total complexity score, defined as the sum of the number of discrete peaks for each Vbeta subfamily, between the patients and their donors. TCRBV subfamily spectratyping profiles of patients and donors, however, had diverged, with both gains and losses of peaks identifiable in both patient and donor. In conclusion, patients surviving 10 or more years after allogeneic SCT still show a T cell repertoire that reflects expansion of the donor-derived post thymic T cell compartment, with a limited contribution by new T cell generation and persistently increased Tregs. It therefore appears that a diverse TCRBV repertoire predominantly derived from the memory T cell pool is compatible with good health. Disclosures: No relevant conflicts of interest to declare.

Anti-Thymocyte Globulin (ATG) Differentially Depletes naïve and Memory T Cells and Permits Memory-Type Regulatory T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4670-4670
Author(s):  
Chang-Qing Xia ◽  
Anna Chernatynskaya ◽  
Clive Wasserfall ◽  
Benjamin Looney ◽  
Suigui Wan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Memory T Cells ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem

Abstract Abstract 4670 Anti-thymocyte globulin (ATG) has been used in clinic for the treatment of allograft rejection and autoimmune diseases. However, its mechanism of action is not fully understood. To our knowledge, how ATG therapy affects naïve and memory T cells has not been well investigated. In this study, we have employed nonobese diabetic mouse model to investigate how administration of anti-thymocyte globulin (ATG) affects memory and naïve T cells as well as CD4+CD25+Foxp3+ regulatory T cells in peripheral blood and lymphoid organs; We also investigate how ATG therapy affects antigen-experienced T cells. Kinetic studies of peripheral blood CD4+ and CD8+ T cells post-ATG therapy shows that both populations decline to their lowest levels at day 3, while CD4+ T cells return to normal levels more rapidly than CD8+ T cells. We find that ATG therapy fails to eliminate antigen-primed T cells, which is consistent with the results that ATG therapy preferentially depletes naïve T cells relative to memory T cells. CD4+ T cell responses post-ATG therapy skew to T helper type 2 (Th2) and IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) are less sensitive to ATG depletion and remain at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory-like immunophenotype is significantly increased in ATG-treated animals, which might play an important role in controlling effector T cells post ATG therapy. In summary, ATG therapy may modulate antigen-specific immune responses through modulation of naïve and memory T cell pools and more importantly through driving T cell subsets with regulatory activities. This study provides important data for guiding ATG therapy in allogenieic hematopoietic stem cell transplantation and other immune-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Significant mobilization of both conventional and regulatory T cells with AMD3100

Blood ◽  
2011 ◽  
Vol 118 (25) ◽  
pp. 6580-6590 ◽  
Author(s):  
Leslie S. Kean ◽  
Sharon Sen ◽  
Olusegun Onabajo ◽  
Karnail Singh ◽  
Jennifer Robertson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Effector Memory ◽  
Cell Populations ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
Macaque Model ◽  
The Impact

AbstractIn this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4+/CD25high/CD127low/FoxP3+ Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8+ T cells were mobilized to a greater extent than CD4+ T cells, with accumulation of 3.7 ± 0.4-fold more total CD8+ T cells and 6.2 ± 0.4-fold more CD8+ effector memory T cells in the leukapheresis product compared with G-CSF alone. Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation.

scholarly journals Development of T-Cell Therapy Targeting Hematopoietic Minor Histocompatibility Antigen HA-1

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5749-5749
Author(s):  
Artem Pilunov ◽  
Dmitrii Romaniuk ◽  
Savely Sheetikov ◽  
Alexandra Khmelevskaya ◽  
Anton Shmelev ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Significant Proportion ◽  
Published Data ◽  
Functional Screening ◽  
Hematopoietic Stem ◽  
Normalized Frequency

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only curative therapy for hematological malignancies yet in nearly one-third of patients, it is followed by a relapse of the disease contributing to high mortality. In fully HLA-matched allo-HSCT graft versus leukemia reaction is driven by the recognition of the minor histocompatibility antigens (MiHAs) - endogenous polymorphic peptides presented by MHC. Particularly, HA-1 MiHA is a promising target for immunotherapy. HA-1 is presented by frequent among Caucasians HLA allele - A*02:01. The single nucleotide variation in ARGHAP45 gene which generates the MiHA has the optimal allelic distribution, thus immunogenic mismatch occurs in 30% of allo-HSCT. Also, ARGHAP45 is overexpressed in certain types of leukemia. Here we aim to develop HA-1-specific T-cells for post-transplant relapse therapy. To obtain the sequences of HA-1-specific T-cell receptors (TCRs), naive CD8+ T-cells from 3 HLA-A*02:01 positive and HA-1 negative donors were expanded in vitro using autologous dendritic cells pulsed with HA-1 peptide. Antigen-specific cells were enriched by CD137 marker expression or by HLA-tetramer staining, RNA from positive and negative fractions was isolated for cDNA library preparation. The α and β TCR-repertoires were sequenced using the Illumina MiSeq system. The representative enrichment plot is shown in Figure 1 (A - α chains, B - β chains). Each circle represents a unique TCR. The vertical axis shows the normalized frequency in enriched fraction, the horizontal axis shows the normalized frequency in tetramer or CD137 negative flowthrough. HA-1-specific TCRs are denoted by green filled circles.TCRs were considered to be HA-1-specific if they were significantly enriched in CD137+ or tetramer+ fraction (exact Fisher test, p=0.05). In total, 49 α and 80 β chains were described. To determine the degree of similarity between HA-1-specific TCRs Levenstein distance was calculated between amino acid sequences of complementarity-determining region 3 for both chains. Sequences of previously published HA-1-specific TCRs were also included in the analysis (Verdijk et.al., Haematologica, 2002; Bleakley et.al., 2017, WO2018058002A1). α chains demonstrated low degree of mutual similarity, the majority of sequences did not belong to any cluster (Figure 2A, sequences with the Levenstein distance <3 are connected). In contrast, a significant proportion of β chains were organized in a few clusters containing sequences from all 3 donors and previously published data (Figure 2B). We selected 14 α and 12 β HA-1-specific TCR chains (marked by the black dots in Figure 2). Clones were picked to represent separate clusters of similarity to Levenstein metrics, and unique sequences. Selected α and β-chains were cloned for subsequent functional screening in different combinations. Besides, we developed the modular lentiviral backbone for manufacturing HA-1 specific transgenic CD8+ T-cells. Our approach utilizes Golden Gate Cloning, which allows rapid assembly of lentiviral backbone carrying any combination of TCR α and β chains fused with the selective marker for sorting via p2A peptides. We used truncated CD34 as a transduced cell surface marker for the rapid separation of transduced cells by clinical-grade antibodies and subsequent expansion. In order to prevent the mispairing of transgenic TCR with endogenous one, CRISPR/Cas9 knockout strategy of endogenous TCR chains was developed. We used guide RNAs specific to TRAC,TRBC1 and TRBC2 genes and recombinant Cas9. The efficiency was demonstrated on Jurkat E6-1 cell line, the knockout was confirmed both by flow cytometry and genotyping of the modified cells using fragment analysis. Constant regions of the transgenic TCRs were modified to prevent cleavage by Cas9, the resistance was confirmed by in vitro Cas9 digestion assay. Moreover additional cysteines were introduced in the constant regions of transgenic TCRs for increased transgenic TCR stability. Cytotoxic activity of modified cells will be confirmed on lymphoblastoid cell lines and patient leukemia samples, cytokine secretion of modified cells will be detected using ELISPOT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. Disclosures No relevant conflicts of interest to declare.

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