Weekly Digests
‹ Back to May

Tumor-reactive T cells engineered to not get exhausted after adoptive transfer

May 10, 2023

T cell exhaustion in response to chronic activation of effector CD8+ T cells limits the efficacy of particular adoptive cell transfer (ACT) therapies. Corria-Osora et al. hypothesized that T cells could be rewired by engineering them to express orthogonal cytokines to acquire and maintain a functional state upon chronic stimulation. The researchers assessed various engineered T cells to research this hypothesis in murine models, from which the data were recently published in Nature Immunology.

The researchers engineered OT-1 T cells to express an IL-2 variant (IL-2v) that does not engage with the IL-2 receptor α-chain, as well as IL-33, which serves as a pro-inflammatory alarmin. Furthermore, a secreted PD-1 decoy (PD1d – a fusion molecule of the ectodomain of mouse PD-1 with the Fc region of human IgG4) was also included. Cells engineered with individual molecules, PD1d/IL-2v, PD1d/IL-33, or PD1d/IL-2v/IL-33 were shown to secrete the relevant transgenes.

Mice with advanced non-immunogenic B16-OVA tumors were treated with ACT with various OT-1 variants without preconditioning lymphodepletion. Untransduced, PD1d, IL-2v, or PD1d/IL-2v-transduced OT-1 cells had minimal impact on tumor growth. Transduction with IL-33 alone or PD1d/IL-33 had limited effects on tumor growth. However, PD1d/IL-2v/IL-33 OT-1 cells induced tumor regression and resulted in an objective response rate (ORR) of 85.7%, compared to 0-9% for all other cell variants. When ACT was administered earlier, therapy cured the mice.

There was a significant increase in CD8+ T cells in the tumor in response to PD1d/IL-2v/IL-33 OT-1 cell ACT, as compared to ACT with the other OT-1 variants. Phenotypically, when IL-2v was included in the cells, it resulted in expansion of TCF1+ OT-1 cells. High numbers of TCF1- effector CD8+ TILs, necessary for tumor regression, were only detected after ACT with PD1d/IL-2v/IL-33 OT-1 cells, suggesting that IL-33 was necessary for TCF1+ precursor cells to transition to effector cells.

Total CD8+ TILs were isolated from B16-OVA tumors treated with each ACT subset. Single-cell RNAseq revealed five distinctive clusters. PD1d/IL-2v/IL-33 cells clustered separately (C5) from canonical T cell clusters, and were characterized by low levels of exhaustion markers and high levels of cytotoxicity genes, including Gzmb and, uniquely, Gzmc. Projecting these clusters on a reference atlas suggested cells in cluster C5 were projected to canonical Tex. To further assess the C5 TILs, they were subjected to an independent higher-dimension analysis. This separated the C5 TILs from the reference Tex TILs, as the C5 cells had a unique cytotoxic effector-like program, with granzymes, perforin, and effector machinery genes and prosurvival factors. These data suggested these cells were different from the TOX+ exhausted phenotype, as these cells did not express TOX, and had downregulated expression of checkpoint molecules and various exhaustion-associated chemokines and transcription factors.

Based on these data, the researchers suggest that the C5 cells represented a novel T-synthetic effector (Tse) state. When OT-1 and endogenous CD44+CD8+ TILs were analyzed separately after PD1d/IL-2v/IL-33 ACT, two new clusters were detected (C6 and C7). The endogenous TILs mostly clustered in the C4 and C2 clusters during tumor regression after PD1d/IL-2v/IL-33 ACT, and the C5 Tse cells were specific for OT-1 TILs. This C5 state was gradually acquired during tumor regression. Most OT-1 cells on day 5 after ACT were distributed in the C2, C6, and C7 clusters, with minimal in C5, while on day 8, most of these cells clustered in C5 and C6, and by day 12, almost all clustered in C5.

After building a new TIL reference map using the obtained data, the researchers assessed whether this novel effector state was detectable in other T cell datasets. They did not detect this profile in various tumor cohorts, datasets obtained after ICB, or CD19 CAR-T datasets. Tse TIL were also different from previously described CD8+ “better effector” (Tbe) TILs, confirming that the C5 Tse might be a novel and synthetic effector state.

To further characterize the C5 Tse state, TILs were phenotyped by gating on TOX and Gzmc. In untreated mice, CD8+ TILs had a TOX+Gzmclo-neg exhausted phenotype. During tumor regression after PD1d/IL-2v/IL-33 ACT, OT-1 cells expressed PD-1 and were enriched in TOX-Gzmc+ cells, with most Gzmc+ cells being TCF1-. These cells were polyfunctional effector cells, expressing Gzmb, Gzma, perforin, TNF, IFNγ, and Ki67. Furthermore, they were enriched for genes associated with energy production, effector molecule synthesis, and cell killing, suggestive of metabolic and effector fitness.

To assess the role PD-1 functionality plays in the Tse TILs, the researchers added anti-PD-L1 treatment to PD1d/IL-2v/IL-33 ACT, and found that it did not improve tumor control in response to ACT. Furthermore, removing the PD-1 ectodomain from the PD1d did not impact tumor control of IL-2v/IL-33-transduced OT-1 cells, nor did it impair accumulation of PD-1+Gzmc+TCF1- OT-1 TIL. These data suggest that the Tse TIL were not dependent on PD-1-mediated inhibition.

Gzmc+ cells were not detected in the tumor-draining lymph nodes (TDLN) or spleens after ACT, suggesting the Tse state was specific to tumors. As Tse TIL did not express the IL-33 receptor, the contribution of IL-33 was likely through indirect reprogramming of the tumor microenvironment.

The researchers found that the C6 cluster cells were characterized by precursor features, expression of Pdcd1 and stemness-related markers, and overexpression of Gzmc and other effector molecules, while cells were negative for TOX. Therefore, these cells might represent a Tse precursor state (Tsp). Trajectory and pseudotime analyses suggested that the OT-1 cells transitioned from memory-like and effector memory states to Tsp and then Tse.

To assess the role IL-2v played in the development of these various T cell states, OT-1 TILs obtained 12 days after IL-2v OT-1 ACT were analyzed. These cells mostly clustered in the C7 memory-like cluster, which was characterized by high expression of T cell activation and stemness-related markers, low expression of checkpoint molecules, and a lack of TOX and Gzmc expression. Therefore, IL-2v may play a role in preparing cells for the superior effector function of Tse and Tsp cells. Transcription factor analysis revealed that IL-2v was involved in the regulation of two sets of transcription factors – one that was active in the C7 TILs in the absence of IL-33, associated with sustaining cell expansion and persistence, stemness, and memory/readiness, and a second that was active across all synthetic states, involved in metabolic support.

Together, these data suggest that by engineering T cells to express IL-2v and IL-33, with or without the PD-1 decoy PD1d, a new functional subset of T cells can be obtained that is activated in the tumor microenvironment and effectively targets murine tumors, but without antigen-driven exhaustion. If these data can be reproduced in human TILs, this may be a strategy to overcome some of the current limitations of ACT therapy in patients.

Written by Maartje Wouters, image by Lauren Hitchings

Meet the researcher

This week, first author Jesus Corria-Osorio answered our questions.

On the left first author Jesus Corria-Osorio; on the right lead author George Coukos

What was the most surprising finding of this study for you?
That IL-2v/IL-33-engineered T cells evade TOX+ canonical exhaustion and acquire an improved functional effector state in tumors, achieving high levels of engraftment and driving tumor regression without the need for prior lymphodepletion or exogenous cytokine support post-adoptive T cell therapy (ACT). This novel PD-1+TOX- T cell synthetic effector (TSE) state was not functionally restrained by the PD-1/PD-L1 axis, and has seemingly never been observed naturally in mice or humans to date.

What is the outlook?
This discovery offers new insights about the plasticity of CD8+ TIL states and unlocks the opportunity to rationally develop novel “synthetic” T cell states through genetic engineering. These cells were not only very potent, but expanded in a cell-autonomous manner, engrafted the host, and expanded in tumors without the need for lymphodepletion or IL-2 support. If we reproduced this TSE state with engineered T cells in the clinic, we would have a more effective ACT approach for solid tumors, and it could lead to substantial improvement of toxicities and clinical care costs associated with ACT, particularly for ACT-based TILs, in which profound lymphodepletion and adjuvant IL-2 administration are responsible for most toxicities.

What was the coolest thing you’ve learned (about) recently outside of work?
I recently discovered a very cool sport called Paddle that is very similar to Tennis. I love it. It makes me super happy these days.


Corria-Osorio J, Carmona SJ, Stefanidis E, Andreatta M, Ortiz-Miranda Y, Muller T, Rota IA, Crespo I, Seijo B, Castro W, Jimenez-Luna C, Scarpellino L, Ronet C, Spill A, Lanitis E, Romero P, Luther SA, Irving M, Coukos G. Orthogonal cytokine engineering enables novel synthetic effector states escaping canonical exhaustion in tumor-rejecting CD8+ T cells. Nat Immunol. 2023 May 24.

In the Spotlight...

Immune mechanisms shape the clonal landscape during early progression of prostate cancer

To model tumor heterogeneity in prostate cancer, Tschering and Luo et al. used multicolor lineage tracing in GEMMs, and found that slow-growing tumors had a more evenly distributed multiclonal (“Mx”) tumor landscape, while more advanced or more aggressive tumors had fewer tumor clones, which were either dominant and minor (“D/M”), and a more disorganized immune landscape associated with differential editing. Minor clones were marked by increased IFNγ response genes, Cxcl9, Cxcl11, and MHC-II. IFNγ or Cxcl9 blockade stabilized the limited clonal dominance and rescued minor clones, which were associated with better outcomes in patients.

Contributed by Lauren Hitchings

Simultaneous targeting of PD-1 and IL-2Rβγ with radiation therapy inhibits pancreatic cancer growth and metastasis

In mouse models of PDAC, radiation therapy (RT) plus PD1–IL2v (a bispecific that blocks PD-1 and activates IL-2β/IL-2γ, but not IL-2α on Tregs) induced durable local and systemic antitumor immunity marked by transcriptionally and metabolically active antigen-specific CD8+ T cells and enhanced T cell polyfunctionality, activation, and immune memory across tumors, lymph nodes, and blood. It also increased and activated systemic NK cells and reduced the number and function of tumor Tregs. The addition of anti-CD25 blunted immunity due to elimination of an active CD25+ CD8+ T cell population. In patients with PDAC, RT responders upregulated the molecular targets of PD1–IL2v.

Contributed by Lauren Hitchings

Prior anti-CTLA-4 therapy impacts molecular characteristics associated with anti-PD-1 response in advanced melanoma

Campbell and Amouzgar et al. aggregated and harmonized whole exome sequencing (WES) and RNAseq data from biopsies across seven checkpoint blockade-treated clinical cohorts comprising 514 patients with melanoma. Harmonization enhanced the statistical power to correlate genomic and expression-based features with clinical responses to different ICB regimens. Using this resource, the team found that in anti-CTLA-4-experienced (but not anti-CTLA-4 naive) patients, immune cell signatures were enhanced, and inflammatory signatures, altered cell cycle processes, and tumor mutation burdens (TMB) were associated with response to anti-PD-1.

Contributed by Lauren Hitchings

Neoadjuvant immune checkpoint blockade triggers persistent and systemic T(reg) activation which blunts therapeutic efficacy against metastatic spread of breast tumors

Blomberg, Kos, and Spagnuolo et al. investigated the impact of neoadjuvant ICB therapy on mechanisms of tumor immunosuppression. In ICB-refractory primary and metastatic breast mouse models mimicking the poor ICB response in patients with breast cancer, combined anti-PD-1/anti-CTLA-4 treatment stimulated Treg proliferation and activation in the tumor, tumor-draining lymph nodes, and blood. Treg depletion during neoadjuvant ICB resulted in sustained increases in activated CD8+ T and NK cells, which significantly prolonged survival due to metastases, without inhibiting primary tumor growth, suggesting Treg targeting could improve neoadjuvant ICB.

Contributed by Katherine Turner

Early immune pressure initiated by tissue-resident memory T cells sculpts tumor evolution in non-small cell lung cancer

Weeden et al. found higher levels of CD8+ resident memory T cells (Trm) in the healthy lungs of ever-smokers (ES) than in those of never-smokers (NS). These Trm in ES were activated earlier in progression from non-malignant to early- and late-stage cancer. Mouse lung cancer models showed that pre-existing Trm cells, independent of Ag specificity, increased T cell recruitment and activation in tumors, leading to early immune evasion and resistance to checkpoint blockade. Human lung cancer datasets confirmed these data, suggesting the “soil” in which tumors arise and the timing of immune pressure impact tumor evolution.

Contributed by Maartje Wouters

CD103+ regulatory T cells underlie resistance to radio-immunotherapy and impair CD8+ T cell activation in glioblastoma

Investigating the sequencing of radiotherapy (RT) and ICB in preclinical glioblastoma models, Van Hooren and Handgraaf et al. found that RT increased T cells, and that administration of anti-PD-1 at the peak of RT-induced T cell infiltration enhanced survival over concurrent RT + anti-PD-1. However, anti-PD-1 induced an accumulation of CD103+ Tregs with upregulated lipid metabolism, which repressed CD8+ T cell activation and restrained antitumor responses. Adding anti-CD25 to deplete most Tregs increased the formation of TLSs and the priming and frequency of CD4+ and CD8+ T cells, improving responses to RT + anti-PD-1.

Contributed by Lauren Hitchings

Everything New this Week In...

Close Modal

Small change for you. Big change for us!

This Thanksgiving season, show your support for cancer research by donating your change.

In less than a minute, link your credit card with our partner RoundUp App.

Every purchase you make with that card will be rounded up and the change will be donated to ACIR.

All transactions are securely made through Stripe.