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915 CD8+ TEMRA Clones Cause Platelet Lysis in Immune Thrombocytopenia

Program: Oral and Poster Abstracts
Type: Oral
Session: 311. Disorders of Platelet Number or Function: Clinical and Epidemiological: Mechanisms and Treatment of ITP
Hematology Disease Topics & Pathways:
Research, Bleeding and Clotting, Translational Research, platelet disorders, Diseases, thrombocytopenias, immune mechanism, immunology, Biological Processes
Monday, December 12, 2022: 5:00 PM

Amna Malik, PhD1*, Michelle MH Tan, MSc1*, Anwar A Sayed, PhD, MBBS, MSc1,2, Panpan Han3,4*, Eleanor Watt5*, Adela Constantinescu-Bercu1*, Ahmad Khoder, MD, PhD, MRCP, ASCP1*, Alexander TH Cocker6,7*, Emma Thorley, MB BChir, BA8*, Ariam Teklemichael, BSc8*, Yunchuan Ding, PhD1*, Rocel C. Saputil, MSc1*, Alice C J Hart, MBBS, BSc, MRCP, FRCPath1,9*, Haiyu Zhang4*, Wayne A. Mitchell, PhD10*, Nesrina Imami, PhD7*, James TB Crawley, PhD1*, Isabelle Salles-Crawley1*, James B. Bussel, MD11, James L Zehnder, MD12, Stuart Adams, PhD5*, Bing Melody Zhang4* and Nichola Cooper, MD1

1Centre For Haematology, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom
2Department of Medical Microbiology and Immunology, Taibah University, Medina, Saudi Arabia
3Department of Hematology, Qilu Hospital, Shandong University, Jinan, Shandong, China
4Department of Pathology, Stanford University School of Medicine, Stanford
5SIHMDS-Haematology, Great Ormond Street Hospital for Children, London, United Kingdom
6Department of Structural Biology, Stanford University School of Medicine, Stanford
7Centre For Immunology and Vaccinology, Imperial College London, London, United Kingdom
8Imperial College Healthcare NHS Trust, Hammersmith Hospital, London, United Kingdom
9Imperial College Healthcare NHS Trust, Hammermsith Hospital, London, United Kingdom
10Department of Immunology and Inflammation, Imperial College London, London, United Kingdom
11Department of Pediatrics, Weill Cornell Medicine, New York, NY
12Department of Pathology, Stanford University School of Medicine, Stanford, CA

Background
Immune thrombocytopenia (ITP) is traditionally thought of as an antibody-mediated disease. However, there are a number of features to suggest alternative mechanisms of platelet destruction. In this study we use a multi-dimensional approach to explore the role of terminally differentiated effector memory CD8+ T cells (TEMRA).

Methods
We characterised 83 adults with chronic ITP and 30 age-matched controls using immunophenotyping, next generation sequencing of T cell receptor (TCR), single cell (sc) RNA sequencing and functional assays. Next generation DNA sequencing of TCRβ was carried out using the Illumina MiSeq platform. Analysis of the raw TCR sequences was performed using MiXCR. Single cell immune profiling (Gene expression and TCR) was done using Chromium Single Cell V(D)J and 5' Library kits from 10x Genomics. The constructed library was sequenced on the HiSeq platform (Illumina). Seurat v.2.3.4 was used for gene expression analysis. Interactions between CD8+ T cells and platelets were measured by co-culturing CD8+ T cells with autologous platelets overnight and by perfusing them on the autologous platelet covered VenaFluoro8+ microchip channel.

Results
We found an expansion of CD8+ CD45RA+ CD62L- TEMRA cells expressing IFN gamma, TNF alpha and granzyme B, with no evidence of physiological exhaustion (failure to upregulate TIM-3 or PD-1 expression) in ITP patients which correlated with disease activity. DNA TCR sequencing revealed that expansion of TEMRA cells in ITP was associated with reduced T cell repertoire diversity (p ≤ 0.0001) and lower Simpson’s diversity index (p ≤ 0.05). Patients with ITP had a higher number of T cell clones occupying more than 5% of the T cell repertoire when compared to age matched controls (p ≤ 0.05). Expanded clones were not shared across ITP patients and displayed no known antigen specificities when compared to available databases of TCR sequences, providing evidence for the lack of virally driven clonal expansion.

Clonally expanded CD8+ T cells persisted over many years in ITP patients and characterised disease severity. Longitudinal samples from 9 patients showed expanded clones and reduced T cell repertoire diversity during active disease (platelet count of less than 30 x 109/L) compared to stable disease (platelet count greater or equal to 30 x 109/L) (p ≤ 0.01; r = 0.56 – Figure 1 shows an example patient).

To better characterise CD8+ T cells, we performed paired scRNA and scTCR sequencing on isolated whole CD8+ T cells from patients with active and stable disease. Paired sequencing enables transcriptomic characterisation of T cell clones. ScRNA sequencing revealed four distinct clusters in CD8+ T cells: naïve, TEMRA, central memory (CM) and natural killer T cells (NKT). By mapping scTCR CD8+ sequencing to single cell gene expression, we found that the largest expanded clones showed an aggregative distribution, indicating transcriptional homogeneity and were almost exclusively comprised of TEMRA cells (Figure 2). Consistent with immunophenotyping and DNA TCR sequencing, expanded TEMRA cell clones were expanded in active disease compared to stable disease.

To assess the impact of these CD8+ T cells on platelets in ITP, PBMCs from patients with ITP and HC were passed across a platelet layer at flow rates consistent with venous blood flow. CD8+ T cells in ITP patients interacted with platelets 4x more frequently than HCs. Isolated CD8+ T cells were cultured over night with autologous platelets to further explore these interactions: CD8+ T cell:platelet aggregates were identified more frequently in the co-cultures from patients with ITP (p ≤ 0.05) and were inhibited by blocking MHC Class I (p ≤ 0.01). CD8+ T cells within the aggregates had increased CD107a expression compared to non-aggregated CD8+ T cells (p ≤ 0.01), indicating cytolytic activity and release of Granzyme and Perforin. Correspondingly, platelets in the aggregates showed increased CD62P expression, a marker of activation and impending death (p ≤ 0.001).

Conclusions
Here we demonstrate clonal expansion of disease-associated TEMRA CD8+ T cells in ITP. These cells bind to platelets and cause TCR-mediated platelet activation and death providing an antibody-independent mechanism of platelet destruction. This could serve as biomarker to direct treatment in patients with refractory ITP and targeting specific T-cell clones could be a novel therapeutic option in refractory ITP.

Disclosures: Bussel: Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rallybio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Other: Data and Safety Monitoring Board; Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; UCB: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Data and Safety Monitoring Board; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cooper: Sanofi, Principia, Novartis, Griffols, Sobi, Argenyx, UCB, Rigel: Consultancy, Honoraria, Research Funding.

*signifies non-member of ASH