Weekly Digests
‹ Back to July

Neoantigen-specific CD4+ T cells are powerful helpers

July 19, 2023

CD4+ T cells have been shown time and time again to play roles in antitumor immunity, but the exact mechanism and factors that influence their effects remain unclear. Previous research in mice identified a specific CD4+ T cell response to CLTCH129>Q – a validated neoantigen target expressed by the MHC-II-deficient squamous cell carcinoma tumor model (SCC VII). Furthering this research, Brightman et al. recently identified four distinct TCR clonotypes recognizing this target with different avidities, and evaluated and optimized the capacity of CLTCH129>Q-specific CD4+ T cells to contribute to antitumor immunity. Their results were published in Nature Immunology.

To begin, Brightman et al. used mice that were vaccinated with irradiated SCC VII cells and adjuvant polyI:C and were protected from SCC VII live tumor challenge to isolate an expanded pool of CLTCH129>Q-specific CD4+ T cells. From there, the researchers identified and validated four distinct TCRs consisting of 3 TCR β clonotypes paired with four α-chains. TCR 1 and TCR 2 shared the same TCR β-chain and nearly identical α-chains that differed by only a single amino acid. Further, TCRs 1 and 2 showed notably (~10x) higher avidities than TCRs 3 and 4, though unlike results previously seen in CD8+ T cells, this was not found to correlate with differences in proximal TCR signaling.

To study the expansion and activation of CLTCH129>Q-specific CD4+ T cells in vivo, the researchers transferred equal numbers of retrovirally transfected, labeled CD4+ T cells expressing either TCR 1 or TCR 3 into naive mice. When the mice were subsequently challenged with SCC VII, both subsets responded to antigen recognition, expanding equally, expressing similar levels of the acute activation marker CD69, and upregulating PD-1 to a similar extent.

While SCC VII does not express MHC-II, even under inflammatory conditions, adoptive transfer of activated CD4+ T cells expressing TCR 1 protected mice from tumor challenge 1 day later. This protection was antigen-specific, as the same protection was not observed when mice were treated with activated polyclonal non-specific CD4+ T cells. Mice that were protected from the initial challenge were also protected from rechallenge at 30 days, suggesting that CLTCH129>Q-specific CD4+ T cells mediates durable immune memory. These results were dependent on the number of cells transferred, as mice that received lower cell doses showed a reduced survival benefit compared to those receiving higher cell doses.

Given that CLTCH129>Q-specific CD4+ T cells cannot directly recognize neoantigens on tumor cells due to the lack of MHC-II on SCC VII tumor cells, the researchers hypothesized that the observed antitumor effects were likely mediated through CD8+ T cell help. Indeed, CD8+ T cell depletion prior to the adoptive transfer of neoantigen-specific CD4+ T cells abrogated their protective effect. Based on recent evidence that CD4+ T cells provide help through CD40L-dependent licensing of cDC1s, the researchers evaluated CD4+ T cells expressing TCR 1 for CD40L expression and found that it was upregulated upon in vitro exposure to peptide-pulsed splenocytes. In vivo, CD40L-blocking antibodies administered on the day of tumor implantation and again 2 days later abrogated antitumor immunity induced by neoantigen-specific CD4+ T cells. Again, there was no difference between TCR 1 and TCR 3.

Next, Brightman et al. investigated the potential efficacy of CLTCH129>Q-specific CD4+ T cells against large, established tumors. In this setting, neoantigen-specific CD4+ T cells proliferated in tdLNs and infiltrated tumors, but failed to improve survival. In an effort to enhance their potency, the researchers tried culturing the cells in IL-15 and IL-7 prior to transfer, rather than the standard IL-2, as this strategy has been shown to induce a less differentiated stem cell memory-like state and enhance ACT in studies with CD8+ T cells. CLTCH129>Q-specific CD4+ T cells cultured in IL-15/IL-7 showed much greater expansion in peripheral blood and accumulation in tumors and tdLNs compared to cells cultured in IL-2, and effectively delayed tumor growth and enhanced survival. Results were similar between CD4+ T cells expressing TCR 1 or TCR 3.

Taking a closer look at CLTCH129>Q-specific CD4+ T cells in different locations, the researchers found through gene expression analysis that cells in the tdLNs differentially expressed markers of memory T cells, while those in tumors differentially expressed genes associated with effector functions, including TH1 cytokines, TCR signaling pathway components, and cytotoxicity genes. In line with this, flow cytometry revealed that while tumor populations contained mostly effector memory T cells, tdLNs contained various memory subpopulations, including effector memory, central memory, and stem cell memory-like populations. Notably, a unique memory population was observed when cells were cultured in IL-15/IL-7 that was not observed when cells were treated with IL-2. These results suggest that the lymph nodes act as a reservoir for transferred TSCM cells, which give rise to more differentiated subsets that traffick to tumors.

To evaluate the impact of therapeutically transferred CLTCH129>Q-specific CD4+ T cells (cultured in IL-15/IL-7) on CD8+ T cell immunity, the researchers measured PD-1 expression. While there was no change in the percentage of host CD8+ T cells expressing PD-1 in the tumor, the levels of PD-1 were lower, suggesting reduced terminal exhaustion. In tdLNs, the researchers found a significant increase in PD-1+CD8+ T cells, which correlated with the number of transferred cells, suggesting increased priming. When CLTCH129>Q-specific CD4+ T cells were transferred into tumor-bearing mice that were recently depleted of CD8+ T cells, some initial antitumor effects were observed, but longer-term tumor growth delay was lost. Together, these results suggests that neoantigen-specific TSCM-like CD4+ T cells are actively involved in the priming of tumor-specific CD8+ T cells in tdLNs.

Overall, these findings suggest that CLTCH129>Q-specific CD4+ T cells induce antitumor immunity against SCC VII through CD40L, resulting in enhanced priming of CD8+ T cells. Antitumor effects were apparent regardless of TCR avidity, and could be enhanced by culturing engineered neoantigen-specific T cells in a cocktail of IL-15/IL-7 to induce a stem cell memory-like phenotype that enhances in vivo T cell expansion and induces durable tumor control.

Write-up and image by Lauren Hitchings

Meet the researcher

This week, first author Spencer Brightman and lead author Stephen Schoenberger answered our questions.

What was the most surprising finding of this study for you?
SB: We were surprised to see that CD4+ T cells expressing the T cell receptors with moderate avidity were similarly functional to those expressing higher-avidity receptors in vivo in terms of expansion, antitumor efficacy, and surface marker phenotype. We were also surprised to find that the therapeutically transferred T cells maintained their stem-like phenotype in the tumor-draining lymph node several days after transfer.
SS: I've been working on the mechanism of CD4+ T help for CD8+ T cells for a number of years and so was gratified, rather than surprised, that it also regulates neoantigen-specific responses. I was, however, surprised that there appears to be little difference in the efficacy of high- versus moderate-affinity CD4+ T cells, and that their therapeutic capacity involves CD8-dependent and -independent activities.

What is the outlook?
SB: We hope that these findings will influence the field by increasing the appetite for immunotherapies focusing on CD4+ T cells in addition to cytotoxic CD8+ T cells, which have been the main focus of immunotherapies to date. Further studies to advance these findings ought to determine the broad cellular and transcriptomic alterations to the tumor microenvironment induced by adoptive transfer with stem-like neoantigen-specific CD4+ T cells.
SS: Ah, this is where it gets interesting! Current adoptive cell therapy involves engineered T cells specific for a single target antigen expressed by the tumor, and it's been observed in patients that this kind of approach can be rendered ineffective if the tumor loses or modifies its presentation of that one antigen. Our work shows that effective adoptive immunotherapy can be achieved by targeting antigen-presenting cells that cross-present tumor antigen, and this enhances activation, not killing. These activated APC can then launch an army of cytotoxic CD8+ T cells specific for many tumor antigens rather than only one. We are ready to translate this to the clinic now, and if successful, this approach could inform a new approach to immunotherapy.

What was the coolest thing you’ve learned (about) recently outside of work?
SS: I've recently become an "empty nester" and have been enjoying all the opportunities that change has compelled: more travel, less shopping, and a quieter life.


Brightman SE, Becker A, Thota RR, Naradikian MS, Chihab L, Zavala KS, Griswold RQ, Dolina JS, Cohen EEW, Miller AM, Peters B, Schoenberger SP. Neoantigen-specific stem cell memory-like CD4+ T cells mediate CD8+ T cell-dependent immunotherapy of MHC class II-negative solid tumors. Nat Immunol. 2023 Jul 3.

In the Spotlight...

A distinct stimulatory cDC1 subpopulation amplifies CD8+ T cell responses in tumors for protective anticancer immunity

Meiser and Knolle et al. showed that a distinct population of immunostimulatory MHC-IIhiCCR7neg cDC1 through the CXCL9/CXCL10–CXCR3 axis formed clusters with TCF1+CD8+ T cells at the stroma–tumor interface. These niches resulted in cDC1-mediated TCF1+CD8+ T cell activation and subsequent effector T cell differentiation that enabled protective anti-cancer immunity. Spatial organization of cDC1-CD8+ T cell interactions was conserved in human cancers, and higher expression of the CXCL9+CCR7neg cDC1 gene signature and their clustering with intratumoral CD8+ T cells positively correlated with survival across multiple cancers and with response to ICB.

Contributed by Shishir Pant

Hypoxic niches attract and sequester tumor-associated macrophages and cytotoxic T cells and reprogram them for immunosuppression

Sattiraju et al. demonstrated that host immunocompetence status influences progressive vascular alterations during GBM progression – including expansion and maturation of hypoxic zones/pseudopalisades – and spatial patterning of CD68+ TAMs in mouse and human GBM. Single-cell RNAseq revealed a distinct in vivo GBM hypoxia gene signature that correlated with recurrence and worse prognosis in GBM patients. Hypoxic GBM cells induced CCL8 and IL-1β expression in TAMs, and promoted trafficking/sequestration of TAMs and CTLs in hypoxic zones and the maturation of pseudopalisades. Targeting hypoxic niches disrupted TAM spatial patterning.

Contributed by Shishir Pant

T cell receptor signaling strength establishes the chemotactic properties of effector CD8+ T cells that control tissue-residency

Using an IFNγ-YFP reporter system, Abdelbary et al. showed that epicutaneous VacV infection induced some skin-infiltrating effector CD8+ T cells (TEFF), but not circulating memory T cell precursors to express IFNγ in the presence of antigen. Levels of IFNγ and induction of tissue-resident CD8+ T cell (TRM) differentiation depended on TCR signal strength and expression of the transcriptional repressor Blimp1. Blimp1 was upregulated in TEFF re-exposed to antigen in the periphery, and regulated chemotaxis. Low-affinity TCR agonism bolstered CXCR6-mediated tissue retention, but strong agonism also hindered the complementary pathway of S1P-mediated TEFF egress.

Contributed by Paula Hochman

Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma

Patterson et al. used scRNAseq and monocyte fate-mapping methods to study crosstalk between macrophages (Mψ) and tumor-specific CD4+ T cells in pancreatic ductal adenocarcinoma (PDA). In an orthotopic mouse model, tumor-specific CD4+, but not CD8+ T cells drove monocyte differentiation into antitumor MHC-IIhi macrophages through cognate antigen presentation by Mψ and downstream IFNγ and CD40:CD40L pathways. Most Mψ subsets in the PDA TME were derived from monocyte differentiation. Loss of either MHC-II or CD4+ T cells promoted monocyte differentiation toward alternatively activated states that stimulated tumor progression.

Contributed by Katherine Turner

Combination of cancer vaccine with CD122-biased IL-2/anti-IL-2 Ab complex shapes the stem-like effector NK and CD8+ T cells against tumor

In combination with a WT1 vaccine in a model off advanced leukemia, Shimizu, Ueda, and Kawamura, et al. tested two IL-2/anti-IL-2 mAb complexes (IL-2Cxs) – S4B6, which is biased towards CD122 and expands NK cells and memory CD8+ T cells, and JES6, which is biased towards CD25 and expands Tregs. The combination with S4B6, but not JES6, induced 100% survival, dependent on T and NK cells. Treatment enhanced activation of NKT1 cells, expanded NK cells at immature and semi-mature stages, and increased distinct subsets of NK and CD8+ T cells with stem-like phenotypes. Memory CD8+ T cells capable of potent antitumor protection were maintained long-term.

Contributed by Lauren Hitchings

Fecal microbiota transplantation plus anti-PD-1 immunotherapy in advanced melanoma: a phase I trial

In a phase 1 trial, 20 patients with advanced melanoma received a first-line treatment of fecal microbiota transplant (FMT) from a healthy donor followed by anti-PD-1 therapy. Treatment was safe; adverse events were low-grade and mainly ICB-related. Overall response rate was 65%, with four complete responses. Patient microbiome diversity increased after FMT and trended towards donor composition, but this change was only sustained in anti-PD-1 responders, who also increased blood ICOS+CD8+ T cells and histidine levels. Mice recolonized with patient samples and treated with FMT and anti-PD-1 recapitulated clinical antitumor responses.

Contributed by Alex Najibi

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.