Enhanced Anti-tumor Immunity through T Cell Immunotherapy

Abstract: T cells play an important role in adaptive immunity, anti-tumor and anti-viral reaction. After vaccination or acute infection, T cells can be differentiated into effector cells to form memory cells. During chronic infection or cancer, T cell exhaustion easily happens. Exhausted T cells are heterogeneous, some of which are stem cell-like(e.g. self-renewal and differentiation potential). These features are important for maintaining persistent anti-tumor immunity and immunotherapy. Recent researches deeply reveal regulation of exhausted T cells and stemness-related molecules, providing new T cell immunotherapy strategies via intervening with these pathways.

Keywords: Anti-tumor Immunity, T Cell Immunotherapy, T Cell Exhaustion, Tumor Microenvironment, Flow Cytometry

1. Heterogeneity of Exhausted T Cells (TEX)

This figure shows differentiation of T cells stimulated by acute and chronic antigens. Acute antigens stimulate differentiation of naive T cells into effector T cells(TEFF, CD127hiKLRG1hi) and memory precursor cells(TMP, CD127+KLRG1lo). The latter grows into memory T cells further(TMEM, CD62L+CD127+). During persistent antigen exposure(e.g. chronic infection or tumor), T cells enter exhaustion process. Naive T cells are differentiated into early TEX and stem-like exhausted T cells(TSL, TOX+TCF1+). TSL can be self-renewed and differentiated into TEX-Trans and terminally exhausted t cells(TEX-Term, PD-1hiTim3+CD101+) or Effector-like exhausted T cells(Eff-like TEX). Stemness features of TSL are important for maintaining long-term anti-tumor immunity.

2. Flow Cytometry Markers for T Cell Subsets

T cells are highly heterogeneous under different immune conditions. The subsets can be distinguished via specific surface markers. In chronic infection or tumor microenvironment, TEX includes multiple functions: TSL/TPEX expresses TCF1, PD-1int, TIM3−, CD101−, and also co-expresses CXCR5 and Ly108(SLAMF6). Some of them also highly expresses CD62L. TEX-Trans and TEX-Term are present as CD101−TIM3+ and CD101+TIM3+CD69+ respectively. eff-like TEX highly expresses KLRG1 and CX3CR1. By contrast, TEFF and TMP in acute infection are KLRG1hiCD127lo and KLRG1loCD127hi respectively. Memory T cells include stem-cell memory T cells(TSCM, CD62L+CD45RA+CCR7+IL-7Rα+), central memory T cells(TCM, CD62L+CD45RA−), effector memory T cells(TEM, CD62L−CD45RA−), TEMRA(CD62L−CD45RA+) and tissue-resident memory T cells(TRM, CD69+CD103+). These marker combinations provide key evidences for analysis of T cell fate and optimization of immunotherapy.

3. Key Regulation Role of TCF1

TCF1 is the key transcription factor for regulating stemness and differentiation of T cells, including two subsets: p33 and p45. Roles in T cell development, memory formation and exhaustion process are significant. TCF1 establishes T-cell identity and maintains stemness via remodeling chromatin structure and histone acetylation. In acute infection, TCF1(especially for p45) restricts excessive effect differentiation, and promotes generation of memory T cells. Regulation of pathways(e.g. EOMES, BCL-2) maintains secondary immune response. In chronic infection or tumor microenvironment, TSL expresses CXCR5 and Ly108, excluding terminal markers(e.g. TIM3/CD39). Self-renewal capability is sensitive for PD-1 immune checkpoint inhibitor therapy. Researches show TCF1 inhibits premature terminal differentiation and also maintains TSL cell fate via pathways(e.g. Wnt/β-catenin). Roles in coupling of T cell stemness, memory formation and anti-tumor immune response are significant.

4. Effects of T cell Exhaustion on Tumor Immune Microenvironment

T cell exhaustion obviously reduces anti-tumor immunity, and remodels immunosuppressive tumor microenvironment(TME). Exhausted T cells show decrease of effector function, secretion of cytokines(e.g. IFN-γ, TNF-α, IL-2), and cytotoxicity. Persistently high expression of inhibitory receptors(e.g. PD-1, TIM-3, LAG-3) inhibits self-function. Ligand-activated MDSCs and regulatory T cells aggravate immunosuppression. Besides, damaged mitochondrial function and poor metabolic adaptation are disadvantageous in nutrient-deficient TME. Insufficient amount or differentiation blockade of TCF1+ TSL cells can't continuously supplement effector T cells. Rapid exhaustion of immune response promotes phenotypic formation of cold tumor. Thus, improvement of immunotherapy effects depends on restoration of T cell stemness or reinvigoration of exhaustion.

5. T Cell Exhaustion Markers in Flow Cytometry

Multicolor flow cytometry panel design accurately analyzes T cell status according to surface and functional markers: Classical exhaustion phenotypes include CD8⁺PD-1⁺TIM3⁺LAG-3⁺TIGIT⁺. Stemness-exhaustion hierarchy depends on comparison among TCF1⁺TOX⁺PD-1int(stem-like) and TCF1⁻TOX⁺TIM3⁺(terminal exhaustion). Addition of CXCR6 localizes T cells near dendritic cells in tumor microenvironment. Intracellular staining is very important for detecting transcription factors(e.g. TCF1, TOX, EOMES, T-BET) and phosphorylated signal proteins(p-STAT5/3). Besides, flow cytometry can simultaneously evaluate proliferation(Ki-67/CFSE), cytokine secretion(e.g. decreased IFN-γ, TNF-α, IL-2) and mitochondrial function(MitoTracker), comprehensively reflecting dysfunction of exhausted T cells.

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