Targeting of MDSCs is a major clinical challenge in the era of immunotherapy. Antibodies which deplete MDSCs in murine models can reactivate T cell responses. In humans such approaches have not developed due to difficulties in identifying targets amenable to clinical translation. RNA-sequencing of M-MDSCs and G-MDSCs from cancer patients was undertaken. Flow cytometry and immunohistochemistry of blood and tumours determined MDSC CD33 expression. MDSCs were treated with Gemtuzumab ozogamicin and internalisation kinetics, and cell death mechanisms determined by flow cytometry, confocal microscopy and electron microscopy. Effects on T cell proliferation and CAR-T cell anti-tumour cytotoxicity were identified in the presence of Gemtuzumab ozogamicin. RNA-sequencing of human M-MDSCs and G-MDSCs identified transcriptomic differences, but that CD33 is a common surface marker. Flow cytometry indicated CD33 expression is higher on M-MDSCs, and CD33+ MDSCs are found in the blood and tumours regardless of cancer subtype. Treatment of human MDSCs leads to Gemtuzumab ozogamicin internalisation, increased p-ATM, and cell death; restoring T cell proliferation. Anti-GD2-/mesothelin-/EGFRvIII-CAR-T cell activity is enhanced in combination with the anti-MDSC effects of Gemtuzumab ozogamicin. The study identifies that M-MDSCs and G-MDSCs are transcriptomically different but CD33 is a therapeutic target on peripheral and infiltrating MDSCs across cancer subtypes. The immunotoxin Gemtuzumab ozogamicin can deplete MDSCs providing a translational approach to reactivate T cell and CAR-T cell responses against multiple cancers. In the rare conditions of HLH/MAS gemtuzumab ozogamicin provides a novel anti-myeloid strategy. FUND: This work was supported by Cancer Research UK, CCLG, Treating Children with Cancer, and the alumni and donors to the University of Birmingham.

Metabolic pathways that regulate T-cell function show promise as therapeutic targets in diverse diseases. Here, we show that at rest cultured human effector memory and central memory CD4+ T-cells have elevated levels of glycolysis and oxidative phosphorylation (OXPHOS), in comparison to naïve T-cells. Despite having low resting metabolic rates, naive T-cells respond to TCR stimulation with robust and rapid increases in glycolysis and OXPHOS. This early metabolic switch requires Akt activity to support increased rates of glycolysis and STAT5 activity for amino acid biosynthesis and TCA cycle anaplerosis. Importantly, both STAT5 inhibition and disruption of TCA cycle anaplerosis are associated with reduced IL-2 production, demonstrating the functional importance of this early metabolic program. Our results define STAT5 as a key node in modulating the early metabolic program following activation in naive CD4+ T-cells and in turn provide greater understanding of how cellular metabolism shapes T-cell responses.

Linking immunometabolic adaptation to T-cell function provides insight for the development of new therapeutic approaches in multiple disease settings. T-cell activation and downstream effector functions of CD4+ and CD8+ T-cells are controlled by the strength of interaction between the T-cell receptor (TCR) and peptides presented by human leukocyte antigens (pHLA). The role of TCR-pHLA interactions in modulating T-cell metabolism is unknown. Here for the first time we explore the relative contributions of the main metabolic pathways to functional responses in human CD4+ and CD8+ T-cells. Increased expression of hexokinase II accompanied by higher basal glycolysis is demonstrated in CD4+ T-cells; cytokine production in CD8+ T-cells is more reliant on oxidative phosphorylation. Using antigen-specific CD4+ and CD8+ T cell clones and altered peptide ligands we demonstrate that binding affinity tunes the underlying metabolic shift. Overall this study provides important new insight into how metabolic pathways are controlled during antigen-specific activation of human T-cells.

Cancer cells take up amino acids from the extracellular space to drive cell proliferation and viability. Similar mechanisms are applied by immune cells, resulting in the competition between conventional T cells, or indeed chimeric antigen receptor (CAR) T cells and tumor cells, for the limited availability of amino acids within the environment. We demonstrate that T cells can be re-engineered to express SLC7A5 or SLC7A11 transmembrane amino acid transporters alongside CARs. Transporter modifications increase CAR T-cell proliferation under low tryptophan or cystine conditions with no loss of CAR cytotoxicity or increased exhaustion. Transcriptomic and phenotypic analysis reveals that downstream, SLC7A5/SLC7A11–modified CAR T cells upregulate intracellular arginase expression and activity. In turn, we engineer and phenotype a further generation of CAR T cells that express functional arginase 1/arginase 2 enzymes and have enhanced CAR T-cell proliferation and antitumor activity. Thus, CAR T cells can be adapted to the amino acid metabolic microenvironment of cancer, a hitherto recognized but unaddressed barrier for successful CAR T-cell therapy.

Background: Melanoma antigen gene (MAGE)-type antigens are promising targets for cancer immunotherapy as they are expressed in cancer cells but not in normal tissues, except for male germline cells. The mouse P1A antigen shares this MAGE-type expression pattern and has been used as a target antigen in preclinical tumor models aiming to induce antitumor CD8⁺ T-cell responses. However, so far only one MHC I-restricted P1A epitope has been identified. It is presented by H-2L^d in mice of the H-2^d genetic background such as DBA/2 and BALB/c. Given the availability of multiple genetically altered strains of mice in the C57BL/6 background, it would be useful to define P1A T-cell epitopes presented by the H-2^b haplotype, to facilitate more refined mechanistic studies. Methods: We employed a heterologous prime-boost vaccination strategy based on a chimpanzee adenovirus (ChAdOx1) and a modified vaccinia Ankara (MVA) encoding P1A, to induce P1A-specific T-cell responses in C57BL/6 mice. Vaccine-induced responses were measured by intracellular cytokine staining and multiparameter flow cytometry. We mapped the immunogenic CD8 epitope and cloned the cognate T-cell receptor (TCR), which we used for adoptive cell therapy. Results: ChAdOx1/MVA-P1A vaccination induces a strong P1A-specific CD8⁺ T-cell response in C57BL/6 mice. This response is directed against a single 9-amino acid peptide with sequence FAVVTTSFL, corresponding to P1A amino acids 43–51. It is presented by H-2D^b. P1A vaccination, especially with ChAdOx1 administered intramuscularly and MVA delivered intravenously, protected mice against P1A-expressing EL4 (EL4.P1A) tumor cell challenge. We identified and cloned four TCRs that are specific for the H-2D^b-restricted P1A_43-51 peptide. T cells transduced with these TCRs recognized EL4.P1A but not MC38.P1A and B16F10.P1A tumor cells, likely due to differences in the proteasome subtypes present in these cells. Adoptive transfer of these T cells in mice bearing EL4.P1A tumors reduced tumor growth and increased survival. Conclusions: We identified the first CD8⁺ T-cell epitope of the MAGE-type P1A tumor antigen presented in the H-2^b background. This opens new perspectives for mechanistic studies dissecting MAGE-type specific antitumor immunity, making use of the wealth of genetically altered mouse strains available in the C57BL/6 background. This should facilitate the advancement of specific cancer immunotherapies.

Glioblastoma is a lethal brain tumor that is unresponsive to current cancer immunotherapeutic approaches, including immune checkpoint blockade (ICB). This suggests that initial priming of T cells, rather than their expansion and licensing as effectors, is a restricting feature in this tumor setting. To overcome the limited initiation of CD8 ⁺ T cell responses, we employed a strong heterologous prime-boost vaccination with the simian adenovirus ChAdOx1 and poxvirus modified vaccinia Ankara (MVA). Vaccination conferred therapeutic efficacy against orthotopic, immune checkpoint-blockade (ICB)-refractory SB28 murine glioblastoma. Vaccination was effective against both the murine tumor antigen, P1A, and a newly identified glioblastoma-associated antigen, Gpr149. Additional treatment with ICB provided no additional benefit. Systemic ChAdOx1/MVA vaccination induced robust infiltration of antigen-specific T cells in tumor-challenged brains, the majority of which exhibited a CD103 ⁺ CD69 ⁺ CD8 ⁺ tissue-resident memory (TRM)-like phenotype. These cells were polyfunctional, durable in brains with sustained tumor control, and mediated tissue-specific immunological memory. Moreover, intracranial adoptive transfer of glioblastoma-derived antigen-specific TRM-like cells was sufficient to protect naïve recipients from subsequent orthotopic tumor challenge. Together, these findings establish that viral vector vaccination can generate tumor-specific TRM-like cells that mediate effective anti-glioblastoma immunity, providing a rationale for clinical evaluation of ChAdOx1/MVA-based strategies in glioblastoma.