Immune checkpoint inhibitors targeting receptors on T cells, such as cytotoxic T-lymphocyte associated protein 4 (CTLA‑4) and programmed death receptor 1 (PD‑1), have demonstrated long term anti-tumour effects, but only in a subset of patients. These therapies are of little benefit to the majority of treated patients, expensive and often result in toxicity so focus is on the identification of sensitive and specific biomarkers of response. A number of biomarkers currently in use, such as PD‑L1, tumour mutation burden, gene expression profiles of the tumour microenvironment and the extent of tumour infiltrating immune cells are limited in their efficacy/sensitivity such that no accurate biomarker of response in multiple cancers is available.
In this study, the authors suggest that CD8+ cytotoxic T lymphocytes (CTL) could offer a biomarker of response to immune checkpoint inhibitors. Activated CTLs in the tumour microenvironment are suppressed when inhibitory checkpoint signalling through the PD‑1/PD‑L1 pathway is activated, resulting in decreased tumour killing. Immune checkpoint inhibition has been demonstrated to change the characteristics of CTLs, including their phenotype, function and frequency and TCR sequencing has demonstrated clonal proliferation of CTLs in tumours and associated stroma.
The authors used a mouse model bearing tumours derived from monoclonal cancer cell lines. In response to anti‑CTLA‑4 and anti‑PD‑L1 ICT, they either had complete regression in a few days or did not respond to therapy. A T‑cell transfer model was also used in which T‑cells specific for a MHC‑I restricted HA533‑541 antigen form CL4xThy1.1 mice was transferred into BALB/c mice and inoculated with a HA expressing tumour cell line to measure frequency and phenotype of tumour antigen-specific CD8+ T‑cells. These models were analysed by FACS, bulk RNA and bulk TCRβ sequencing.
In the T‑cell transfer model, immune checkpoint inhibitors did not change the frequency of CD8+Thy1.1+ and CD8+Thy1.1- T‑cells compared to control cells. CD8+Thy1.1+ T‑cells increased in the tumours of treated mice compared to controls.
In lymph nodes, recipient and donor CD8+ T‑cells in all treatments had low expression of Granzyme B. Immune checkpoint therapy increased the number of CD8+Thy1.1+GrB+ TILs suggesting an increase in cytotoxic function of HA-specific CTLs.
The number of HA-specific CTLs was variable between animals, with some having 20% CD8+ TIL populations in their tumours and others as low as 5%.
In mice bearing two monoclonal cell line tumours, one was resected prior to immune checkpoint inhibitor treatment (Day 0) and the other tumour tracked to day 7. At day 0, there were no detectable differences in tumour size, total cell count and CD45+ cell proportions. At day 7, total cell counts and CD45+ cell proportions were similar in responders and non-responders, however non-responding tumours were significantly greater in size than responders. In non-responders, some tumours reached 100mm2, whereas there were complete regressions observed in responders.
In response to immune checkpoint inhibition, tumours and draining lymph nodes from responders had increased HA-specific CD8+ TILs and reduced Tregs compared to non-responders.
Bulk TCRβ sequencing demonstrated significantly increased CL4 TCRβ sequences in responders compared to non-responders. Responders also had significantly reduced diversity in TCRβ repertoires, suggesting that one clone, the CL4 clone, expanded in the responders and this correlates with response. The CL4 clone was the most frequent clone in all responders and the majority of non-responders.
The top 50 most abundant TCRβ clones from each animal were arranged into a network based on the TCRβ CDR3 amino acid sequence similarities. The majority of the 560 were unrelated with only 57 clones forming networks.
Although most TCRβ CDR3 sequences were randomly distributed throughout the networks, in the responding tumours two clones with highly similar CDR3 sequences were detected. Only 2-3 mice from the responders had these clones representing only 0.6% of the TCRβ repertoire suggesting each animal established a private and highly diverse tumour-infiltrating CD8+ TCRβ repertoire.
Day 7 T‑cells were analysed for the expression of differentiation and memory-associated markers. CD8+Thy1.1+ T‑cells treated with immune checkpoint inhibitors had upregulated IL‑7Rα, killer cell lectin-like receptor subfamily G1 (KLRG1) and transcription factor T‑box (T‑bet) suggesting TILs had developed an effector memory phenotype. This phenotype had higher frequencies in responders compared to non-responders.
Some non-responding mice demonstrated CD8+Thy1.1+ T‑cells (>10%) infiltration in tumours, however, expression of CD127 was much higher in responders. No difference in KLRG1 expression was observed between responders and non-responders.
RNAseq of CD8+Thy1.1+ TILs did not detect many differences in immune-related gene sets between responders and non-responders except for genes in the WNT/b‑catenin signalling pathway being upregulated in non-responders.
Both responders and non-responders had tumour infiltrating HA-specific CTLs with a memory-like phenotype that demonstrated cytotoxic function, however animals were much more likely to be responders if there were much higher frequencies of memory-type HA-specific CTLs.
In animals with complete regression of tumours, re-challenging these animals with the original cell line of an AB1‑HA cell line demonstrated protection with CD8+Thy1.1+ T‑cells still detectable after the original tumours had regressed.
In this study, the authors used a mouse bilateral tumour model to determine if infiltrating T‑cells could be used as a biomarker for response to immune checkpoint inhibitor therapies. Whilst immune checkpoint inhibitors induced increased CTL infiltration, these effects were variable between animals, however in tumours that responded to therapy an increased frequency of effector memory type T‑cells was observed driven by the IL‑7Rα receptor. The authors speculate that further research into the distribution of TCRβ clones may yield potential biomarkers of response.
Principe et al. Tumor Infiltrating Effector Memory Antigen-Specific CD8+ T Cells Predict Response to Immune Checkpoint Therapy. Front. Immunol., 11:584423, 2020.