# Improving engineered TCR-T cell therapy for solid tumors by targeting Tcf1+ stem-like reservoirs in tumor-draining lymph nodes

> **NIH NIH F31** · FRED HUTCHINSON CANCER CENTER · 2024 · $44,154

## Abstract

PROJECT SUMMARY
 Adoptive transfer of engineered T cells targeting tumor antigens is ineffective against most solid tumors,
due in-part to poor persistence and the rapid onset of dysfunction. Recent studies of endogenous tumor-
specific T cells (Endog-T) have described a population of stem-like PD-1+Tcf1+ progenitor exhausted T cells
(Tpex) that can self-renew and differentiate into effector-like T cells in the tumor. Tpex preferentially reside in
tumor-draining lymph nodes (TdLNs), where they are maintained through activation by dendritic cells (DCs).
Importantly, Tpex in TdLNs are necessary for promoting persistent and functional T cell responses, as well as
conferring response to PD-L1 blockade. However, it is not clear whether engineered T cells form PD-1+Tcf1+
populations in TdLNs, and how this impacts their long-term persistence and function in the tumor.
 To test this, I adapted the clinically relevant KrasG12D-LSL/+;p53fl/fl murine model (KP) of lung adenocarcinoma
to overexpress the model neoantigen Ovalbumin (Ova) to compare Ova-specific engineered T cells (TCR-T)
and Endog-T cells. While the majority of Endog-T cells in TdLNs were PD-1+Tcf1+, TCR-T cells were
predominantly PD-1-Tcf1+, markers associated with lack of activation and maintenance of a pre-exhausted
central-memory phenotype. Consistent with these phenotypes, PD-L1 blockade failed to increase numbers of
TCR-T cells and enhance tumor control in comparison to Endog-T. Thus, I hypothesize that factors unique to
TCR-T therapy, such as their in vitro pre-activation and/or infusion at a high cell number, promote their
formation of PD-1-Tcf1+ reservoirs in TdLNs instead of PD-1+Tcf1+. I also hypothesize that formation of PD-1-
Tcf1+ reservoirs in the TdLN is actually an advantage for TCR-T therapy, since the PD-1-Tcf1+ population may
possess superior functional potential that can be mobilized through vaccination targeting DCs in the TdLN.
 In this project, I seek to determine 1) why TCR-T cells form PD-1-Tcf1+ reservoirs in TdLNs instead of PD-
1+Tcf1+ like what is described for Endog-T, as well as 2) whether the PD-1-Tcf1+ TCR-T cells in the TdLN have
stem-like characteristics and functional potential that can be unleashed with vaccination. In Aim 1, I will use
fluorescently-labeled tumors in the KP model, flow cytometry, and high-parameter fluorescence microscopy to
determine how infusion of a large number of cells and/or in vitro pre-activation affect TCR-T cell formation of
PD-1+Tcf1+ populations in TdLNs, and how this correlates with their localization near DCs. In Aim 2, I will use
RNA-seq, ATAC-seq, and ex vivo assays to describe the stem-ness and functional potential of the PD-1-Tcf1+
TCR-T cells in the TdLN compared to PD-1+Tcf1+ Endog-T. I will also evaluate whether a novel T cell
vaccination approach targeting DCs in TdLNs can activate PD-1-Tcf1+ TCR-T cells to promote tumor control.
 Through this work, I will define the mechanisms by which TCR-T cells form Tcf1+ stem-li...

## Key facts

- **NIH application ID:** 10997222
- **Project number:** 1F31CA294918-01
- **Recipient organization:** FRED HUTCHINSON CANCER CENTER
- **Principal Investigator:** William Samuel Nutt
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $44,154
- **Award type:** 1
- **Project period:** 2024-08-16 → 2026-08-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10997222

## Citation

> US National Institutes of Health, RePORTER application 10997222, Improving engineered TCR-T cell therapy for solid tumors by targeting Tcf1+ stem-like reservoirs in tumor-draining lymph nodes (1F31CA294918-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10997222. Licensed CC0.

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