Characterisation of PD1-expressing CMV-specific CD8+ T-cells in healthy donors and cancer patients.

Butler, Megan ORCID: 0000-0001-8719-3180 (2025). Characterisation of PD1-expressing CMV-specific CD8+ T-cells in healthy donors and cancer patients. University of Birmingham. Ph.D.

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Abstract

Cytomegalovirus (CMV) is a prevalent herpesvirus that establishes lifelong infection and is highly immunogenic. Viral replication is controlled by persistent immune surveillance that includes the establishment of a large virus-specific CD8+ T-cell response that can comprise up to 20% of the total systemic CD8+ T-cell repertoire. As such there is considerable interest in how viral replication is controlled whilst minimising potential inflammatory tissue damage.
An important protein that is expressed on CMV-specific CD8+ T-cells is the immune checkpoint regulator PD1. Due to the remarkable success of antibody-mediated PD1 inhibition in the treatment of cancer this is one of the most important proteins in medical research at the current time. These therapeutic successes have arisen due to the presence of PD1 on tumour-specific T-cells that undergo chronic antigen stimulation from cancer cells but become functionally exhausted within the tumour microenvironment. However, this success is only present in a subset of patients, and it is vital to define determining factors in treatment success.
Despite the expression of PD1 on many CMV-specific CD8+ T-cells, the functional profile of PD1+ CD8+ T-cells and their broader profile of checkpoint co-expression are unknown. Furthermore, the profile of PD1 expression on CMV-specific T-cells within the tumour microenvironment and their response to therapeutic PD1 inhibition are uncertain.
Using MHC-I tetramer technology, I identified CMV-specific CD8+ T-cells in blood samples from healthy donors, within the tumour microenvironment of patients with ovarian cancer, and in blood of patients undergoing PD1 blockade for the treatment of lung cancer. EBV is another prevalent herpesvirus that leads to lifelong infection, and in many of these setting I was able to compare my findings in CMV-specific CD8+ T-cells to EBV-specific CD8+ T-cells.
These studies show that PD1 is expressed on a significant portion of CMV-specific T-cells and is often co-expressed with the checkpoint proteins 2B4 and TIGIT. Unexpectedly, PD1+ CMV-specific CD8+ T-cells showed enhanced cytokine production in response to antigen presentation revealing little evidence of functional exhaustion. Despite this, cytokine responses increased further in the setting of PD1 inhibition, indicating that the function of the PD1+ cells is somewhat constrained by this inhibitory interaction.
PD1+ CMV-specific T-cells were the dominant viral phenotype in the tissue of ovarian cancer patients. However, in this setting I identified two distinct cellular subsets based on the relative intensity of PD1 expression. In particular, a PD1MID population which corresponded with cells seen in peripheral blood whilst a PD1HIGH subset was only seen within tumour. Importantly, PD1HIGH cells exhibited higher expression of the checkpoint proteins TIM3, LAG3 and the ectoenzyme CD39 in comparison to the PD1MID subset. This work suggests that PD1MID cells within tumour are unlikely to be functionally exhausted, possibly increasing interest in their potential to be ‘redirected’ against tumour. In contrast, PD1HIGH subsets may have undergone localised functional exhaustion but it is now critical that experiments are undertaken to assess this using cells isolated directly from tissue samples.
In my final studies I was able to assess how CMV-specific T-cells were modulated by in vivo PD1 checkpoint blockade. Overall, rather minimal changes were observed over several months of therapy although a transient modulation was seen with the first cycle, with CMV-specific CD8+ T-cells exhibiting increased expression of TIGIT and granzyme B.
The efficacy of PD1 blockade in the treatment of cancer is suboptimal due to the small subset of patients it affects and needs further optimisation. The study of PD1 function in the setting of persistent viral infection will be helpful in uncovering insights into the physiological function of this protein. My work has uncovered a range of novel observations relating to the pattern and functional correlates of PD1. These findings emphasise the need for further research in this area. One important aim of this will be to guide the use of checkpoint blockade in the management of malignant disease and other clinical settings.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):