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1199 Identifying a source of dysfunctional CAR T cells incorporating with PBMC scRNA-seq data in patients with Non-Hodgkin Lymphoma
  1. Changjin Hong1,
  2. Yoon Ho Choi1,
  3. David Wald2 and
  4. Tae Hyun Hwang1
  1. 1Mayo Clinic, Jacksonville, FL, USA
  2. 2Case Western Reserve University, Shaker Heights, OH, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background The CAR T cell therapy initiated as a treatment for CD19+ malignant B cell has rapidly expanded to many other cancers including solid tumors. Among multiple key factors to improve efficacy while minimizing adverse effects, it is critical to understand the mechanisms of T cell exhaustion and harness it. Upregulation of TIGIT is a hallmark of exhausted T cells in non-responders among non-Hodgkin’s lymphoma (NHL) patients under CD19+ CAR T therapy.1 However, we have not paid attention to PBMC that can (in)directly interact with CAR+T cells and its synergistic role in the treatment result. Furthermore, it is desirable to identify transcriptional factors (TFs) facilitating the unexpected T cell status. Ultimately, we can target them with TIGIT for better outcomes.

Methods We evaluate 27 scRNA-seq data for CAR+T (n=12) and its matched PBMC (n=15) in two sequential time points after the infusion from 8 relapsed/refractory NHL patients; 6 responders (R) and 2 non-responders (NR).1

Results TIGIT is upregulated in post-infusion PBMC samples. The TIGIT is overexpressed across CD8+T, NK, and most significantly Treg population. DEG analysis reveals that more NR cells activate TIGIT whereas more R cells activate CD226. Furthermore, NECTIN2 is mostly expressed in a monocyte subpopulation dominated by the NR group. CellphoneDB analysis3 demonstrates that KLRC1 in an inflammatory T population in CAR+T interacts with HLA-E detected across PBMC cells (potentially originated from malignant B cells). These suggest that more prevalent immune checkpoints in NR trigger immune evasion.4–7 Next, we investigate how the dysfunctional T state was acquired. A gene set analysis uncovers that it is due to chronic antigen exposure (CAE).2 NK-like T cell populations resemble dysfunctional tumor-infiltrated lymphocytes (TILs) under CAE. Among monocyte populations, the NR-dominant monocytes upregulate TFs represented into CAE TILs. A comprehensive TF analysis reveals that TSC22D3, ZFP36, and DUSP1 known for anti-inflammatory response8 are strikingly overexpressed in the NR group. A higher interaction of TSC22D3 with AP-1 proteins results in a lack of AP-1 to form a binding with NFAT required for T cell effectors. Even worse, NR patients upregulate NR4A2 expression. The gene potentially interferes with the binding of NFAT/AP-1 and remodels chromatin accessibility to facilitate T cell exhaustion.9

Conclusions In conclusion, dysregulated TSC22D3 and NR4A2 compromise the effector function of CAR+T, hijack AP-1 proteins, and impede eradicating the antigens, and thus CAR+T cells transit to exhausted T cells represented by overexpressed TIGIT.


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