Expanded t cell population

Abstract

The present invention provides methods of producing, from malignant pleural effusion, expanded populations of tumour tissue-resident T cell memory (Trm) like CD103+CD8+ T cells based on expression of GPR183 and CXCR4 or expression of LITAF, JUNB and PI3KR1. Also provided are uses of said cells as a medicament and as a prognostic marker.

Description

[0001] Expanded T Cell Population

[0002] The present invention provides methods of producing, from malignant pleural effusion, expanded populations of tumour tissue-resident T cell memory (Trm) like CD103+CD8+ T cells based on expression of GPR183 and CXCR4 or expression of LITAF, JUNB and PI3KR1. Also provided are uses of said cells as a medicament and as a prognostic marker.

[0003] Malignant pleural effusion (MPE) is the accumulation of fluid comprising malignant cells in the pleural space due to cancer. MPE affects up to 15% of all cancer patients, and current guidelines indicate a median survival ranging from 3-12 months, depending on underlying disease and patient factors. For approximately 15% of newly diagnosed cancer patients, MPE is the first indication of malignancy. MPE can result from primary disease, most commonly mesothelioma, or advanced disseminated disease, most commonly lung cancer. However, other cancer types, such as breast cancer, can lead to MPE.

[0004] MPE is enriched in immune infiltrate and previous studies have described the role of helper and regulatory CD4+ T-cells in the pathogenesis of MPE, but the composition and function of CD8+ T cells in MPE, and their role in cancer progression, remains elusive. Surface expression of integrin aE (gene ITGAE encoding CD103) with or without the type II C-lectin receptor CD69 (gene CD69) are key markers of tissue-resident memory (Trm) T-cells. In cancer, Trm CD103+ T cells are phenotypically diverse, and have been shown to be clonally expanded and drive anti-tumoral responses. However, the study of human Trm CD8+ T cells has been significantly restrained by highly exhausted cell states induced by the chronic antigen stimulation of the tumour microenvironment (TME). Additionally, conventional study of Trm CD8+ T cells requires highly precious fresh ex vivo tissue sections, which are essential and prioritised for patients’ clinical diagnostics. After clinical diagnostics samples are taken, a significant volume of fluid remains and is typically wasted. The pleural microenvironment is immunosuppressive and hypoxic, and the effect this plays on tumour- reactive Trm CD8+ T cells is uncharacterised.

[0005] The treatment of malignancies mostly frequently causing MPE (mesothelioma and lung cancer) are commonly treated using single or combined immune checkpoint blockade (ICB) targeting T-cell immunoinhibitory receptors, programmed cell death protein-1 / ligand-1 (PD- 1 / PD-L1) and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) with or without chemotherapy. ICB has shown durable survival benefits in advanced and relapsed disease. However, upon disease progression and induction of MPE, treatment focus often shifts toward palliative care that largely aims to relieve symptoms through successive fluid drainage and pleural fluid control.

[0006] Accordingly, there is a need to provide novel diagnostic and therapeutic tools for treating cancer patients with malignant pleural effusion (MPE). The present invention aims to address this need at least in part.

[0007] In addition, there is a need to provide improved methods for expanding and producing Trm cell populations from MPE that can be used as medicaments.

[0008] In addition, there is a need to provide improved methods of predicting a subject's response to treatment with immune checkpoint therapies.

[0009] Brief summary of the disclosure

[0010] The invention is based on the surprising finding that immune infiltrate in the pleural cavity is topographically sequestered away from the chronic antigen stimulation of the tumour bed. It has been found that CD8+ T cells, in particular, tumour tissue-resident T cell memory (Trm) like CD103+CD8+ T cells based on the expression of GPR183 and CXCR4 or expression of LITAF, JUNB and PI3KR1, in MPE are rested and have better preserved survival, proliferation and effector function which may be converted from an anergistic state to an energised state by expanding the tumour tissue-resident T cell memory (Trm) like CD103+CD8+ T cells.

[0011] The inventors have comprehensively characterised CD8+ T cells in MPE. The inventors have uncovered that Trm CD8+CD103+ T-cells are enriched in pleural fluid and the inventors use pleural fluid as a module to study underlying Trm CD8+ T cell biology. Furthermore, the inventors describe how the pleural microenvironment induces new cell states that are unique to the pleura that may have favourable characteristics for potential cell therapy. The inventors isolated tumour reactive T-cells from ex vivo MPE and showed for the first time that the Trm CD8+CD103+ compartment in MPE harbours cytotoxic tumour- reactive T-cell clones that have robust in vitro killing capacity against autologous primary cancer cell lines. This work provides, for the first time, an in-depth characterisation of cytotoxic lymphocytes in MPE and alters the current understanding by revealing MPE is a valuable source of tumour-reactive T-cells for cell therapy.

[0012] In first aspect, there is provided a method of expanding tumour tissue-resident T cell memory (Trm) like CD103+CD8+ T cells expressing at least:

[0013] GPR183 and CXCR4 (late unique to pleura cells); and / or LITAF, JUNB and PI3KR1 (early unique to pleura cells); the method comprising: a) enriching CD8+cytotoxic T cells from malignant pleural effusion (MPE); b) co-culturing the CD8+ cytotoxic T cell enriched MPE with irradiated feeder cells obtained from a subject for a first time period under conditions to allow the T cells of the MPE to expand, wherein co-culturing comprises supplementing with one or more cytokines; c) washing the expanded T cells and stimulating the expanded T cells with irradiated cancer cells from the subject; d) co-culturing the expanded T cells and irradiated cancer cells for a second time period under conditions to allow the expanded T cells to further expand; e) co-culturing the further expanded T cells with live cancer cells obtained from the subject; f) sorting the co-cultured further expanded T cells based on a level of expression of : i. one or more tumour reactive markers comprising CD103, CD8, and one or more of 4-1BB, CD25, and / or CD154; ii. memory markers; and iii. exhaustion markers; and g) determining which of the sorted T cells express or have increased expression of GPR183 and CXCR4 and / or LITAF, JUNB and PI3KR1.

[0014] In certain embodiments, the method further comprises step (h) isolating the T cells expressing GPR183 and CXCR4 and / or LITAF, JUNB and PI3KR1.

[0015] In certain embodiments, the method further comprises step (i) maintaining and / or further expanding the T cells expressing GPR183 and CXCR4 and / or cells expressing LITAF, JUNB and PI3KR1.

[0016] In certain embodiments, the first time period is 14 days.

[0017] In certain embodiments, the second time period is at least 14 days.

[0018] In certain embodiments, steps (a), (c) and (d) comprise co-culturing the MPE, expanded T cells and / or further expanded T cells at a 1:1 EffectorTarget cell ratio. In certain embodiments, step (a) comprises supplementing with one or more of IL-2, IL7 and / or IL15 every three days from starting co-culturing.

[0019] In certain embodiments, the IL-2 is at a concentration of 100U / ml;

[0020] In certain embodiments, memory markers comprise one or more of CCR7, CD45RA and / or CD45RO.

[0021] In certain embodiments, exhaustion markers comprise one or more of PD1, Tim3 and / or NKG2a.

[0022] In certain embodiments, steps (e) and (f) of claim 1 comprise activation-induced marker cell sorting.

[0023] In certain embodiments, determining comprises bulk or single cell RNA sequencing analysis.

[0024] In a second aspect, there is provided a population of cells comprising an expanded population of Trm like CD103+CD8+ T cells expressing at least:

[0025] GPR183 and CXCR4 (late unique to pleura cells); and / or

[0026] LITAF, JUNB and PI3KR1 (early unique to pleura cells); produced by the method according to the first aspect.

[0027] In a third aspect, there is provided a population of cells, comprising an expanded population of Trm like CD103+CD8+ T cells expressing at least:

[0028] GPR183 and CXCR4 (late unique to pleura cells); and / or

[0029] LITAF, JUNB and PI3KR1 (early unique to pleura cells).

[0030] In a fourth aspect, there is provided a pharmaceutical composition comprising a population of cells as described herein.

[0031] In a fifth aspect, there is provided a population of cells or a pharmaceutical composition as described herein for use as a medicament.

[0032] In a sixth aspect, there is provided a population of cells or a pharmaceutical composition as described herein for use in preventing or treating cancer.

[0033] In a seventh aspect, there is provided a method of preventing or treating cancer in a subject in need thereof, the method comprising administering an effective amount of a population of cells or a pharmaceutical composition as described herein to the subject.

[0034] In an eighth aspect, there is provided a method of preventing or treating a disease or condition in a subject in need thereof, the method comprising administering an effective amount of a population of cells or a pharmaceutical composition as described herein to the subject.

[0035] In certain embodiments of any aspect described herein, the expanded late unique to pleura cells and / or early unique to pleura cells are de-anergised (energised).

[0036] In certain embodiments of any aspect described herein, the expanded late unique to pleura cells and / or early unique to pleura cells have increased expression of genes indicative of memory and / or a proliferative phenotype in comparison to reference T cells;

[0037] In certain embodiments of any aspect described herein, genes indicative of memory and / or a proliferative phenotype comprises expression of one or more of TCF7, STAT4, PIK3R1, and IL7R.

[0038] In certain embodiments of any aspect described herein, the expanded late unique to pleura cells express or have increased expression of one or more of LMNA, FAM107B, TUBA1A, YPEL5, DDX3X, DENND4 and / or DDX21 in comparison to early unique to pleura cells and / or canonical Trm cells.

[0039] In certain embodiments of any aspect described herein, the expanded late unique to pleura cells express or have increased expression of LMNA, GPR183, FAM107B, CXCR4, TUBA1A, YPEL5, DDX3X, DENND4A, and DDX21 in comparison to early unique to pleura cells and / or canonical Trm cells.

[0040] In certain embodiments of any aspect described herein, the expanded late unique to pleura cells have reduced expression of SOCS3, DUSP2, PD-1, NKG2A, and / or Tim-3 in comparison to early unique to pleura cells and / or canonical Trm cells.

[0041] In certain embodiments of any aspect described herein, the expanded late unique to pleura cells have a relatively high score for a gene signature comprising the genes LMNA, GPR183, FAM107B, CXCR4, TUBA1A, CLDND1, YPEL5, DDX3X, DENND4A, and DDX21 in comparison to early unique to pleura cells and / or canonical Trm cells.

[0042] In certain embodiments of any aspect described herein, the expanded early unique to pleura cells express or have increased expression of one or more of ZFP36L2, EEF1A1, PABPC1, CLDND1, DUSP2, SOCS3 and / or FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells.

[0043] In certain embodiments of any aspect described herein, the expanded early unique to pleura cells express or have increased expression of ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells. In certain embodiments of any aspect described herein, the expanded early unique to pleura cells have reduced expression of RGCC, FAM107B, TUBA1A, PD-1, NKG2A, and / or Tim-3 in comparison to late unique to pleura cells and / or canonical Trm cells.

[0044] In certain embodiments of any aspect described herein, the expanded early unique to pleura cells have a relatively high score for a gene signature comprising the genes ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells.

[0045] In certain embodiments of any aspect described herein, the late unique to pleura cells have an expression profile indicative of a good response to immune checkpoint inhibitor blockade in comparison to a reference T cell.

[0046] In certain embodiments of any aspect described herein, the early unique to pleura cells have an expression profile indicative of a good response to immune checkpoint inhibitor blockade in comparison to a reference T cell.

[0047] In certain embodiments of any aspect described herein, the reference T cell is a canonical T cell obtained from the subjects MPE.

[0048] In certain embodiments of any aspect described herein, the population of cells comprises at least 10% 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% de-anergised late unique to pleura cells and / or early unique to pleura cells.

[0049] In certain embodiments of any of the fourth to eighth aspects, the method or use according to the fourth and fifth aspects comprises administering one or more checkpoint inhibitors.

[0050] In a ninth aspect, there is provided a method of predicting a subject’s response to one or more immune checkpoint inhibitors, the method comprising: providing a sample of the subject’s MPE; and determining the presence of Trm like CD103+CD8+ T cells expressing at least:

[0051] GPR183 and CXCR4 (late unique to pleura cells); and / or

[0052] LITAF, JUNB and PI3KR1 (early unique to pleura cells) in the sample; and wherein the presence of late unique to pleura cells and / or early unique to pleura cells is indicative of a positive response to the one or more immune checkpoint inhibitors.

[0053] In certain embodiments of the ninth aspect the method further comprises: generating a diagnostic report based on the presence or absence of the late unique to pleura cell and / or early unique to pleura cells. In certain embodiments, the diagnostic report is provided to a medical professional for providing guidance on selection of a cancer treatment to be administered.

[0054] In certain embodiments the method further comprises administering to the subject one or more immune checkpoint inhibitors.

[0055] In a tenth aspect, there is provided a method of treating cancer in a subject in need thereof, comprising administering an effective amount of one or more immune checkpoint inhibitors to the subject, wherein:

[0056] Trm like CD103+CD8+ T cells expressing at least:

[0057] GPR183 and CXCR4 (late unique to pleura cells); and / or

[0058] LITAF, JLINB and PI3KR1 (early unique to pleura cells); have been detected in a sample of the subject’s MPE.

[0059] In an eleventh aspect, there is provided one or more immune checkpoint inhibitors for use in a method of treating cancer in a subject in need thereof, comprising administering an effective amount of the one or more immune checkpoint inhibitors to the subject, wherein:

[0060] Trm like CD103+CD8+ T cells expressing at least:

[0061] GPR183 and CXCR4 (late unique to pleura cells); and / or

[0062] LITAF, JLINB and PI3KR1 (early unique to pleura cells); have been detected in a sample of the subject’s MPE.

[0063] In certain embodiments the late unique to pleura cells and / or early unique to pleura cells are as described herein.

[0064] In certain embodiments the method comprises administering an effective amount a population of cells or a pharmaceutical composition according are as described herein to the subject.

[0065] In a twelfth aspect there is provided a method of predicting a subject’s overall survival (OS), wherein the subject suffers from a cancer, the method comprising: providing a sample of the subject’s MPE; and determining the presence of Trm like CD103+CD8+ T cells expressing at least:

[0066] LITAF, JUNB and PI3KR1 (early unique to pleura cells) in the sample; determining a level of expression of CD8A, CD3E and ITGAE of the early unique to pleura cells; wherein the presence of early unique to pleura cells expressing or having increased expression of CD8A, CD3E and ITGAE in comparison to a reference T cell is indicative of an improved OS in comparison to a subject lacking early unique to pleura cells and / or early unique to pleura cells without increased or decreased expression of CD8A, CD3E and ITGAE in comparison to the reference T cell.

[0067] In certain embodiments, the early unique to pleura cells express or have increased expression of one or more of ZFP36L2, EEF1A1, PABPC1, CLDND1, DUSP2, SOCS3 and / or FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells.

[0068] In certain embodiments, the early unique to pleura cells express or have increased expression of ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells.

[0069] In certain embodiments, the early unique to pleura cells have reduced expression of RGCC, FAM107B, TUBA1A, PD-1, NKG2A, and / or Tim-3 in comparison to late unique to pleura cells and / or canonical Trm cells.

[0070] In certain embodiments, the early unique to pleura cells have a relatively high score for a gene signature comprising the genes ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3, FXYD5, CD8A, CD3E and ITGAE in comparison to late unique to pleura cells and / or canonical Trm cells.

[0071] In certain embodiments, the early unique to pleura cells the method further comprises: generating a diagnostic report based on the presence or absence of the late unique to pleura cell and / or early unique to pleura cells. In certain embodiments, the diagnostic report is provided to a medical professional for providing guidance on selection of a cancer treatment to be administered. In certain embodiments, a cancer treatment is administered to the subject.

[0072] In certain embodiments, the early unique to pleura cells are as described herein.

[0073] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0074] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0075] Various aspects of the invention are described in further detail below.

[0076] Brief description of the Figures

[0077] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0078] Figure 1 shows ex vivo MPE is enriched for Tissue Resident (TRM) effector memory (TEM) CD103+CD8+T cells, a Representative FACS plots for gating for different cell subsets b Frequency comparison of CD8+, CD103+CD8+and CD69+CD103+CD8+T cells between PBMC, MPE and biopsy, c Expression of memory markers (CCR7 and CD45RA) on CD8+, CD103+CD8+and CD69+CD103+CD8+T cells d Frequency comparison of CD39+CD103+CD8+T cells between PBMC, MPE and biopsy, and the expression of memory markers. Central memory (TCM) (CCR7+CD45RA_); Naive (CCR7+CD45RA+); Effector Memory+RA (TEMRA) (CCR7'CD45RA+); Effector Memory (TEM) (CCR7+CD45RA_). Bar graph data is presented as median ± interquartile range. Data in the pie chart is presented as mean. Kruskal-Wallis test followed by Dunn’s post hoc test was used for multiple comparisons. Two tailed p values were calculated.

[0079] Figure 2 shows ex vivo MPE-derived CD103+CD8+T cells show increased expression of surface and combined immune checkpoint molecules, a Representative FACS plots of gating for different immune checkpoint molecules on TEM CD103±CD8+T cells, b Single expression of TIM3 (n=53), NKG2a (n=55), PD1 (n=55) and TIGIT (n=42) and c co-expression of TIM3+NKG2a+(n=53), TIM-3+PD1+(n=53), NKG2a+PD1+(n=55), TIGIT+TIM3+(n=40), TIGIT+NKG2a+(n=42) and TIGIT+PD1+(n=42) surface immune checkpoint molecules on paired TEM CD103'CD8+and TEM CD103+CD8+T cells from ex vivo MPE. Bar graph data is presented as median ± interquartile range. A paired, two-tailed, Wilcoxon matched-pairs signed-rank test was used for the analysis.

[0080] Figure 3 shows MPE harbors tumor-reactive cytotoxic CD103+CD8+T cell clones that have robust in vitro killing capacity against autologous primary cancer cell lines, a Cytokine production of tumor-reactive CD103+CD8+T cell clones when co-cultured with autologous primary cancer cell line, and b killing capacity of CD103+CD8+T cell clones against autologous primary cancer cell line was assessed by Incucyte. Green Object Integrated Intensity reflects CellTox™ Green Dye binding DNA released from lysed cells over time, c Example killing assay of Clone B5 when co-cultured with autologous primary cancer cells at 24h, 48h and 72h assessed by Incucyte.

[0081] Figure 4 shows ex vivo M PE-derived CD8+T cells harbor a robust TRM compartment defined by ZNF683, GZMA and GZMB. a LIMAP of total CD8+T cells (7,802 cells) isolated from ex vivo MPE (n=3) have a diverse transcriptomic profile with 11 clusters. Cluster 5 is enriched for cytotoxic TRM cells, b Heatmap displaying the top 5 differentially expressed genes per cluster, c Proportion of cells expressing surface CD103 from each cluster. CD103 expression was measured by Antibody-derived Tag (ADT) via CITE-Seq. d Comparison of overall gene expression (Volcano plot, left) and ZNF683 expression (violin plot, right) between CD103+CD8+(6228 cells) and CD103'CD8+(1574 cells), e Violin plots showing scores for effector (top left panel) and integrin gene sets (bottom left panel). Expression of individual genes within the gene sets are shown by dot plot (right panel), d and e An unpaired, two- tailed, Wilcoxon rank-sum test was used for the analysis, f Dynamic relationships among all 11 clusters analyzed by STARTRAC. Heatmap shows transition index between clusters.

[0082] Figure 5 shows that CD103+CD8+T cells are transcriptionally diverse and have clonally expanded subsets, a A LIMAP of ex vivo CD103+CD8+T cells (15,308 cells) isolated from MPE (n=11) forming 13 clusters, b Heatmap displaying the top 3 DEGs per cluster, c UMAPs showing regions corresponding to Memory and Effector function T cells, d UMAPs showing regions corresponding to CD8-G / Progenitor exhausted (top panel) and CD8-B / Terminally exhausted T cells (bottom panel), e TVRB Shannon diversity index of Effector function and Memory T cells. Each point represents one patient. A paired, two-tailed, Wilcoxon matched- pairs signed-rank test was used for the analysis, f and g Cells stratified by TRVB plus amino acid sequence clonal expansion with small, medium, large and hyper-expanded clonotypes. Each dot represents one cell (f), and their corresponding cytotoxicity gene signature scores. An unpaired, two-tailed, Wilcoxon rank-sum test was used for the analysis. P values were adjusted for multiple comparisons by using Bonferroni correction (g).

[0083] Figure 6 shows a subset of ex vivo CD103+CD8+T cells have novel cell states “Unique to the pleura", a Query mapping M PE-derived CD103+CD8+T cells to a previously published reference dataset of 96,057 CD8+T cell TILs identified three cell states: “Canonical”, ‘Early pleura” and “Late pleura”. Pseudo-time analysis is overlaid, b and c Heatmap displaying differentially expressed genes between “Canonical”, “Early pleura” and “Late pleura” (b), and the corresponding expression of three individual genes in violin plots (c). d comparison of gene scores for metabolic (fatty acid oxidation (FAO), glycolysis, and oxidative phosphorylation (Oxphos)), co-inhibitors, sternness and potential responsiveness to immune checkpoint blockade (ICB) between “Canonical”, “Early pleura” and “Late pleura”. Wilcoxon rank-sum test was used for the analysis. P values were adjusted for multiple comparisons by using Bonferroni correction.

[0084] Figure 7 shows gene signature from “Early pleura" stratifies mesothelioma patients for significantly enhanced overall survival (OS), a Beeswarm plot displaying the distribution of cell neighborhoods across CD103+CD8+T cell clusters from Fig. 5a that are enriched for “Early pleura" cells. Neighborhoods that lack differential abundance are colored grey, b Neighborhood graph of the results from Milo differential abundance testing showing the position of cell neighborhoods that are enriched for cells scoring high for the “Early pleura" gene signature on the LIMAP. Neighborhoods that lack differential abundance are colored white. Neighborhoods with differential abundance of cells scoring high in the “Early pleura" gene signature at FDR 10% are colored indicating (red = down, blue = up). Sizes correspond to the number of cells in each neighborhood, c ‘Early pleura” gene signature scores for cells with different cell status (left) and individual genes in the “Early pleura” gene signature are shown on the right. Wilcoxon rank-sum test was used for the analysis. values were adjusted for multiple comparisons by using Bonferroni correction, d Kaplan-Meier survival curve of patients with mesothelioma using TCGA dataset. Patient groups: (i) patients scoring high for a signature comprised of CD8A, CD3E and ITGAE plus the “Early pleura” signature (G1 , light red, n=9), and (ii) patients scoring low (G2, light blue, n=53). Cox proportional hazards P values (two-sided) are presented.

[0085] Figure 8 shows gating strategies used in this study, a Representative FACS plots of gating used to identify CD8+T cells, b Representative FACS plots of gating used in intracellular cytokine staining, c Representative FACS plots of gating used to sort CD8+T cells for the 10X genomics single-cell RNAseq workflow, d Representative FACS plots of gating used to sort CD8+CD8103+T cells for 10X genomics single-cell RNAseq.

[0086] Figure 9 shows validation of tumour-reactive CD8+T cells in MPE. Novel in vitro expansion and isolation method to single cell sort tumour-reactive CD8+T cell clones from MPE.

[0087] Figure 10 shows the identification of TRM-like CD103+CD8+T cells, a Surface CD103 protein CITE-seq antibody expression cut-offs used to label CD8+T cells for patients D057, D060 and D061. b Expression of ITGAE (gene encoding CD103).

[0088] Figure 11 shows the immunophenotype of M PE-derived CD8+CD103+T cells, a Expression of key genes encompassing the CD8_Good gene signature, b Expression of key genes encompassing CD8_Bad gene signature.

[0089] Figure 12 shows cell states identified in the inventors analysis stratify TGCA mesothelioma dataset. “Canonical” and “Late pleura" gene signatures stratifies TGCA mesothelioma patients for reduced overall survival probability, a Patient groups: (i) patients scoring high for a signature comprised of CD8A, CD3E and ITGAE plus the “Canonical gene signature (G1 , light red), and (ii) patients scoring low (G2, light blue), b Patient groups: (i) patients scoring high for a signature comprised of CD8A, CD3E and ITGAE plus the “Late pleura" gene signature (G1 , light red), and (ii) patients scoring low (G2, light blue). Cox proportional hazards values (two-sided) are presented (P=0.00065 and P <0.0001 , respectively).

[0090] Figure 13 shows single-cell multi-omic profiling of M PE-derived CD8+T cells delineates a TRM CD103+CD8+compartment with concordant transcriptional and surface- protein features indicating high effector capacity, (a) LIMAP of 7,802 MPE- derived CD8+T cells coloured by cluster identity, (b) LIMAP showing surface CD103 protein (ITGAE) expression, demarcating the TRM-enriched region Cluster 5. (c) Heatmap show surface protein expression split by clusters. Cluster 5 shows co-expression of CD103, CD49a, CD39, PD-1 , TIM-3; hereby renamed “TRM” with all other cells grouped as “Non-TRM”. (d) Proportional composition of CD103+ cells split by cluster, (e) Differential gene expression (DEG) between CD103+and CD103- CD8+T cells displayed as a volcano plot; top 10 DEGs are annotated revealing key TRM- associated transcripts (e.g., ZNF683, ITGAE, CXCR6, ITGA1) in CD103+ cells alongside recirculation markers (e.g., CCR7, SELL) in CD103' cells, (f-g) Expression of HAVCR2 (TIM-3 transcript) and PD-1 co-localises to TRM cluster 5. (h,j) Violin plots for top differentially expressed RNA transcripts and ADT surface proteins in CD103+and TRM cells when compared to CD103' Non-TRM cells, highlighting checkpoint molecules (PD-1 , TIM-3) and residency-associated integrins in CD1037TRM cells. (i,k) Clonal expansion as calculated by scRepertoire indicating CD103VTRM cells have increased clonal expansion. Violin plots are presented as median ± interquartile range. Wilcoxon test followed by Bonferonie post hoc test for multiple comparisons. Two tailed p values were calculated (P<0.0001 for all shown).

[0091] Figure 14 shows PD-1+TIM-3+CD103+CD8+TRM cells from MPE are tumour-reactive (a) Ex vivo MPE harbours a population of CD103+CD8+TRM T cells that were bulk sorted for PD- 1+TIM-3+surface expression, (b) Fold expansion of TRM TIM-3+PD-1+CD103+CD8+buIk T cells in vitro demonstrating scalability for manufacturing. Data is plotted as median with 95% confidence intervals, (c) Up-regulation of CD137 on MPE- derived bulk PD-1+TIM- 3+CD103+CD8+ T cells after co-culture with live autologous primary cancer cells versus negative control, (d) Bulk PD-1+TIM-3+CD103+CD8+T-cell cytotoxicity quantified on the Incucyte platform: near-infrared (NIR) object integrated intensity over time for target-only, T- cells-only and co-culture conditions at indicated effector: target (E:T) ratios, (e) Time-lapse Incucyte image analysis of bulk PD-1+TIM-3+CD103+CD8+ T cells co-cultured with autologous primary cancer cell lines (0, 24, 48, 72 h) with cell-masking and percentage NIR live / dead categorisation illustrating progressive target-cell death at E:T 1 :1 and 10:1 ; target- only wells shown as baseline, (f) Intracellular cytokine staining of single-cell-derived TRM CD103+CD8+T cell clones following short-term co-culture with live autologous primary cancer cell lines: representative biaxial plots for IFN-y and TNF-a. (g) Single-cell-derived CD103+CD8+T-cell clone killing tested against autologous primary cancer cell lines (aCC) at 1 :1 E:T in the Incucyte platform. Time courses demonstrate selective killing compared with T-cells-only and aCC-only controls, (h) Example Incucyte images indicating efficient cytolysis of matched autologous primary cancer cell line over time, (i) Frequency of TEM PD-1+TIM- 3+CD103+CD8+T cells in our cohort. Patients for which we have isolated tumour-reactive PD-1+TIM-3+CD103+CD8+ bulk-lines or CD103+CD8+ T cell clones are highlighted by filled circles.

[0092] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0093] Various aspects of the invention are described in further detail below.

[0094] Detailed Description

[0095] The methods described herein include expanding cell populations obtained from malignant pleural effusion. Malignant pleural effusion (MPE) is generally defined by the presence of malignant cells in the pleural fluid or pleural tissue. It is very common among patients with malignant tumours at later stage of disease (see Kaifi JT, et al., Journal Surg Oncol 2012 ; 105: 731-8). Common symptoms associated with MPE are dyspnea, cough, fatigue, weight loss and pleuritic pain.

[0096] A classic view of MPE formation is due to pleural fluid retention resulting from lymphatic obstruction of tumour development in pleural space. These pleural-based tumour metastases block pleural fluid drainage, while increasing pleural fluid production via enhanced plasma extravasation into the pleural space. It is now believed that MPE results from a complex biological interplay between the host vasculature and tumour cells, involving the mediators of the host immune system (see Stathopoulos GT, et al., Am J Respir Crit Care Med 2012 ; 186: 487-92).

[0097] The frequency of MPE is about 500 new cases per million population per year. It is predominantly found with adenocarcinomas of the lungs and other organs, signalling incurability, shortened life expectancy, and severely compromised quality of life (see Light RW, et al,., Textbook of pleural diseases. Philadelphia: Lippincott, Williams and Wilkins ; 2001. 4 ; Antunes G, et al., Thorax 2003 ; 58: ii29— ii38) . The prognosis of patients with MPE is very limited, with a mean survival of approximately 6 to 8 months. In addition to limited survival, patients also suffer significantly reduced quality of life, with frequent medical interventions usually as a result of recurring pleural effusions (see Antunes G, et al., Thorax 2003 ; 58: ii29— ii38) . Therefore, the primary aim of treatment for patients with pleural carcinosis is usually palliative care in the form of a reduction in clinical complaints (see Neragi-Miandoab S, Lung Cancer 2006 ; 54: 1-9).

[0098] Because of the lungs’ close anatomical proximity to the pleurae, MPE is seen in about 8-15 % of lung cancer patients, accounting for approximately 40%of all MPE cases. The second- most common cause is metastatic breast cancer (accounting for approximately 25%of MPE), followed by lymphoma (approximately 10%), ovarian cancer (approximately 5%), and gastrointestinal cancers (approximately 5%) (see Michael Ried, et al., Dtsch Arztebl Int 2013

[0099] ; 110 (18) ; Antony VB, et al., Eur Respir J 2001 ; 18: 402-19 ; Putnam JB, Surg Clin North Am 2002 ; 82: 867-83. 7-9).

[0100] As such, in some examples, the methods include obtaining MPE from a subject suffering from or suspected as suffering from cancer. In some examples, the methods include providing MPE obtained from a subject suffering from or suspected as suffering from cancer.

[0101] In some examples, the MPE may be obtained from a sample that has previously been obtained from a subject. For example, the MPE may be obtained from a subject and then stored before being used in the methods described herein. As such, in some examples, the methods do not include the steps required for obtaining the MPE.

[0102] MPE may be obtained from a sample of pleural fluid from a subject, “a sample of pleural fluid”, as used herein, refers to a pleural fluid sample obtained from a subject. Frequently the sample will be a “clinical sample” which is a sample derived from a subject with MPE or suspected of having MPE (a “patient”). Methods of obtaining MPE will be well known by the person skilled in the art. For example, thoracocentesis. In thoracocentesis, a needled catheter is introduced into the pleural space through an incision in the chest cavity and fluid is positively drawn out through the catheter using a syringe or a vacuum source. In some examples, a second syringe may be used. Once pleural fluid aspirates into the first needle, a larger needle is inserted to drain the fluid more efficiently. Other approaches to removing fluid from the pleural space include surgically implanting a chest tube or using a special catheter device that can be implanted in the pleural space for extended periods of time (see U.S. Pat. No. 5,484,401). After collection, the pleural fluids are processed according to standard procedure. For example, in some methods, the pleural fluids are collected into polypropylene tubes and fractionated by centrifugation. The samples are then stored, for example frozen, until further use.

[0103] In some examples, the MPE provided is obtained from a stored (for example frozen) pleural fluid sample.

[0104] Expanded T cell populations

[0105] The cells expanded by the methods described herein are tumour tissue-resident T cell memory (Trm) like cells.

[0106] As used herein, the term “memory” T cells refer to a subset of T cells that have previously encountered and responded to their cognate antigen. The term is synonymous with “antigen- experienced” T cells. Memory T cells can be effector memory T cells or central memory T cells. The memory T cells are tissue-resident memory T cells. As used herein, the term “tissue-resident memory T cells” or “Trm cells” refers to a lineage of T cells that occupy tissues (e.g., skin, lung, gastrointestinal tract) without recirculating. Trm cells are transcriptionally, phenotypically and functionally distinct from central memory and effector memory T cells which recirculate between blood, the T cell zones of secondary lymphoid organs, lymph and nonlymphoid tissues. One of the roles of Trm cells is to provide immune protection against infection in extra lymphoid tissues.

[0107] Trm T cells provide antigen-specific protection from pathogens and viruses in peripheral tissues. Characterized by their residency in tissues and distinct inability to recirculate, Trm have been identified in lung, skin, liver, brain, intestinal, and mucosal tissues. While initial studies focused on their persistence at sites of previous infection and their long-lived role in combating reinfection (through immediate effector function and accelerated recruitment of circulating immune cells), Trm have also been reported to accumulate in the tumour microenvironment, including those of epithelial (ovarian, pancreatic, colorectal, and lung) and nonepithelial (malignant glioma and melanoma) origin.

[0108] Trm cells may be key players in the tumour microenvironment, capable of initiating and maintaining antitumor responses due to their high levels of expression of inhibitory receptors such as CD39, PD-1, TIM-3, TIGIT, and / or NKG2a. In some examples, high levels of expression of LAG-3, CTLA-4, and / or CD73. In some examples, high levels of expression of LAG-3, CTLA-4, CD73, CD39, PD-1, TIM-3, TIGIT, and / or NKG2a. For example, in comparison to other cells of the MPE. Potential mechanisms of action include rapid and local antigen-specific proliferative responses, with expression of effector molecules to promote inflammation and immune cell recruitment and differentiation (IFNy, TNFa, IL-2, IL-17) and to direct target-cell lysis (perforin, granzyme B). Therefore, increased activation of Trm is thought to be a fruitful strategy for enhancing current immunotherapy approaches and vaccination efficacy.

[0109] Trm and Trm like cells may be defined by expression of CD8 and CD103. Therefore, in some examples, the cells sorted, expanded, further expanded and / or maintained by the methods described herein may be Trm like CD8+ CD103+ T cells.

[0110] Human TRM are typically associated with the expression of the surface markers CD69, CD49a (integrin a1), and CD103 (integrin aE); these markers, along with CD44. Although CD69 is a marker of early T cell activation, most Trm express CD69 under steady-state conditions, without expression of other activation markers such as CD25, CD38, and H LA- DR. CD69 expression may therefore be used in helping distinguish TRM in most tissues. The CD69+Trm population is phenotypically and transcriptionally distinct from recirculating CD69- memory T cells in both tissues and blood, each having a defined gene expression signature that includes molecules associated with adhesion, migration, and regulation (see Behr FM, Chuwonpad A, Stark R et al. (2018) Front Immunol 9:1770. PMID 30131803 and Kumar BV, Ma W, Miron M et al. (2017) Cell Rep 20:2921-2934. PMID 28930685).

[0111] CD49a (integrin a1) may also be expressed in some TRM, as it acts with CD29 (integrin pi) to form the heterodimeric molecule VLA-1, which can bind collagen and laminin and promotes tissue residency (see for example, Corgnac S, Boutet M, Kfoury M et al. (2018) Front Immunol 9:1904. PMID 30158938 and Ray SJ, Franki SN, Pierce RH et al. (2004) Immunity 20:167-179. PMID 14975239).

[0112] CD103 (integrin aE) expression in TRM is variable. CD103 is upregulated after exposure to TGF-p, and it complexes with integrin p7 on the T cell surface to allow adherence through binding to CD324 (E-cadherin) on epithelial cells. CD103 expression is restricted to CD8+TRM . TRM that exist outside of epithelial tissues generally lack CD103 expression, although they may express other adhesion molecules such as LFA-1, which is a heterodimeric integrin composed of CD11a (LFA-1a) and CD18 (LFA-1 P).

[0113] CD44 is a C-lectin-containing glycoprotein that is expressed on leukocytes and other cell types and serves as a receptor for hyaluronic acid (HUA), which is an extracellular matrix component produced by vascular endothelial cells and other immune cells. CD44 also binds to other matrix proteins like fibronectin, laminin, and collagen. In mice, CD44 is considered a core marker with a functional role in TRM biology. Its expression, however, does not distinguish the TRM subset from other CD8+T cell populations, and it is mainly used as a marker of previous T cell activation, as it also labels TCM and TEM. The role of CD44 in TRM may include regulation of cell-cell interactions, cell adhesion, migration, or lymphocyte activation.

[0114] Additional markers that may be used to identify and / or define Trm and / or Trm like T cells include one or more of CD69, CD49a (integrin a1), CD137 (4-1 BB), CD101, CRTAM (CD355), CD186 (CXCR6), CLA, MKP3 (DUSP6), IL-10, CD45, CD69, CD279 (PD-1),

[0115] CCR8, CX3CR1 , S1PR1 , CD19 (negative), CD45RA (negative), CCR7 (negative), and / or CD62L (negative). The term “negative” refers to the decreased expression of absence of the specified marker.

[0116] Table 5 below provided examples of markers and suitable antibodies that may be used to detect said markers. For example, when using fluorescent activated cell sorting (FACS) or other cell sorting techniques.

[0117] Table 5 - Markers for Trm cells and antibodies for detecting said markers (adapted from https: / / www.thermofisher.com / uk / en / home / references / newsletters-and- journals / bioprobes-journal-of-cell-biology-applications / bioprobes-81 / phenotyping- flow-cytometry-antibodies-tissue-resident-memory-t-cells.html).

[0118] In addition, Trm and / or Trm like T cells may have increased expression of one or more transcription factors selected from Hobit, Blimpl , Runx3; Notch / RBPj enriched on lung TRM.

[0119] In some examples, the Trm like T cells sorted, expanded, further expanded and / or maintained by the methods described herein may selected by based on expression levels of one or more tumour reactive markers, memory markers and / or exhaustion markers.

[0120] Tumour reactive markers may include, for example, CD103 and CD8. In addition, tumour reactive markers may include one or more of 4-1 BB, CD25, and / or CD154. For example, sorted, expanded, further expanded and / or maintained by the methods described herein may be sorted based on expression levels of CD103, CD8, 4-1 BB, CD25, and / or CD154. For example, cells having an increased level of expression of CD103 and CD8 and one or more of 4-1 BB, CD25, and / or CD154 in comparison to a reference T cell may be sorted for further expansion. For example, in comparison to a canonical Trm cell obtained from MPE or circulating T cell. Memory markers refers to markers indicative of memory T cells and may include one or more of the markers noted above. In some examples, sorted, expanded, further expanded and / or maintained by the methods described herein are sorted based on a level of expression of one or more memory markers selected from CCR7, CD45RA and / or CD45RO. For example, cells sorted, expanded, further expanded and / or maintained by the methods described herein may have increased expression of one or more of CCR7, CD45RA and / or CD45RO in comparison to a reference T cell. For example, in comparison to a canonical Trm cell obtained from MPE or circulating T cell.

[0121] Exhaustion markers include any markers that are indicative of T cell exhaustion as described herein. For example, exhaustion markers may include any one or more of CD7, CD160, RGS1 , CXCR6, GZMA, CCL3, CCL4, NR4A2, LAG3, 2900026A02RIK, PLAC8, ABI3, SH2D2A, PTGER4, AW112010, ISG15, CD3G, SERPINA3G, LAX1, PDCD1, GZMK, GZMB, MBNL1 , GLRX, ID2, RGS3, ARL6IP1, CD8A, EFHD2, FASL, PTPN22, VMP1, DUSP2, IFI47, GIMAP7, ITPKB, TAPBPL, STAT1 , and / or SHISA5. In some examples, cells sorted, expanded, further expanded and / or maintained by the methods described herein may have decreased expression of one or more exhaustion markers as described herein in comparison to a canonical Trm cell obtained from MPE. In some examples, cells sorted, expanded, further expanded and / or maintained by the methods described herein may have a low score for a gene signature indictive of terminal exhaustion. In some examples, the gene signature includes one or more genes selected from CD7, CD160, RGS1 , CXCR6, GZMA, CCL3, CCL4, NR4A2, LAG3, 2900026A02RIK, PLAC8, ABI3, SH2D2A, PTGER4, AW112010, ISG15, CD3G, SERPINA3G, LAX1 , PDCD1 , GZMK, GZMB, MBNL1, GLRX, ID2, RGS3, ARL6IP1 , CD8A, EFHD2, FASL, PTPN22, VMP1, DUSP2, IFI47, GIMAP7, ITPKB, TAPBPL, STAT1 , and / or SHISA5. In some examples, the gene signature comprises or consist of CD7, CD160, RGS1 , CXCR6, GZMA, CCL3, CCL4, NR4A2, LAG3, 2900026A02RIK, PLAC8, ABI3, SH2D2A, PTGER4, AW112010, ISG15, CD3G, SERPINA3G, LAX1, PDCD1, GZMK, GZMB, MBNL1, GLRX, ID2, RGS3, ARL6IP1 , CD8A, EFHD2, FASL, PTPN22, VMP1 , DUSP2, IFI47, GIMAP7, ITPKB, TAPBPL, STAT1 , and SHISA5.

[0122] In some examples, cells sorted, expanded, further expanded and / or maintained by the methods described herein may have a low score for a gene signature indicative of progenitor exhaustion. In some examples, the gene signature includes one or more genes selected from XCL1, MS4A4C, ID3, CXCL10, SLAMF6, TCF7, IL7R, TNFSF8, CTLA2A, RPS20, LTB, CD9, RPL36A, SOCS3, RPL35, RPS2, RPS19, JUN, RPS17, GPR183, RPLPO, RPL13, TRAF1 , RPL31 , ITGB1 , MT-CYTB, RPS18, RPL10-PS3, RPL12, GM8730, RPS28, EMB, RPL21 , RPS29, RPS26, RPSA, RPS7, GM10073, and / or MT-CO3. In some examples, the gene signature comprises or consist of XCLI, MS4A4C, ID3, CXCL10, SLAMF6, TCF7, IL7R, TNFSF8, CTLA2A, RPS20, LTB, CD9, RPL36A, SOCS3, RPL35, RPS2, RPS19, JUN, RPS17, GPR183, RPLPO, RPL13, TRAF1, RPL31 , ITGB1, MT-CYTB, RPS18, RPL10-PS3, RPL12, GM8730, RPS28, EMB, RPL21, RPS29, RPS26, RPSA, RPS7, GM10073, and / or MT-CO3.

[0123] In some examples, cells sorted, expanded, further expanded and / or maintained by the methods described herein may be sorted based on the level of expression of one or more of PD1 , Tim3 and / or NKG2a. For example, cells sorted, expanded, further expanded and / or maintained by the methods described herein may have reduced or decreased expression of one or more of PD1, Tim3 and / or NKG2a in comparison to a canonical Trm cell. For example, cells expanded by the methods described herein may express PD1, Tim3 and / or NKG2a but to a lower level than other Trm cells present in MPE.

[0124] PD-1 encodes programmed cell death protein 1 (PDCD1), an immune-inhibitory receptor expressed in activated T cells; it is involved in the regulation of T-cell functions, including those of effector CD8+ T cells. In addition, this protein can also promote the differentiation of CD4+ T cells into T regulatory cells. PDCD1 is expressed in many types of tumours including melanomas, and has been demonstrated to play a role in anti-tumour immunity. Moreover, this protein has been shown to be involved in safeguarding against autoimmunity, however, it can also contribute to the inhibition of effective anti-tumour and anti-microbial immunity.

[0125] NKG2A encodes Killer Cell Lectin Like Receptor C1. NKG2 also known as CD159 (Cluster of Differentiation 159) is a receptor for natural killer cells (NK cells). There are 7 NKG2 types: A, B, C, D, E, F and H. NKG2D is an activating receptor on the NK cell surface. NKG2A dimerizes with CD94 to make an inhibitory receptor (CD94 / NKG2). Inhibitory NKG2 molecules containing ITIMs recruit the Src homology 2 domain containing phosphatases SHP-1 and SHP-2, which leads to the inhibition of cytotoxicity. ITAMs, included in DAP-12, on the other hand, recruite the Src homology domain containing kinases Syk (spleen tyrosine kinase) or Zap70 (Zeta-chain-associated protein kinase 70). Kinase activation is followed by NK cell degranulation and transcription of cytokine and chemokine genes.

[0126] Hepatitis A virus cellular receptor 2 (HAVCR2), also known as T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), is a protein that in humans is encoded by the HAVCR2 (T / / W-3)gene. HAVCR2 is a cell surface receptor implicated in modulating innate and adaptive immune responses. Generally accepted to have an inhibiting function. Reports on stimulating functions suggest that the activity may be influenced by the cellular context and / or the respective ligand. HAVCR2 regulates macrophage activation and inhibits T-helper type 1 lymphocyte (Thl)-mediated auto- and alloimmune responses and promotes immunological tolerance. In CD8+ cells HAVCR2 attenuates TCR-induced signalling, specifically by blocking NF-kappaB and NFAT promoter activities resulting in the loss of IL-2 secretion. The function may implicate its association with LCK proposed to impair phosphorylation of TCR subunits, and / or LGALS9-dependent recruitment of PTPRC to the immunological synapse. In contrast, HAVCR2 has been shown to activate TCR-induced signalling in T-cells probably implicating ZAP70, LCP2, LCK and FYN. HAVCR2 expressed on Treg cells can inhibit Th17 cell responses. HAVCR2 is also a receptor for LGALS9. Binding to LGALS9 is believed to result in suppression of T-cell responses; the resulting apoptosis of antigen-specific cells may implicate HAVCR2 phosphorylation and disruption of its association with BAG6. Binding to LGALS9 is proposed to be involved in innate immune response to intracellular pathogens. HAVCR2 is also expressed on Th1 cells and interacts with LGALS9 expressed on Mycobacterium tuberculosis-infected macrophages to stimulate antibactericidal activity including IL-1 beta secretion and to restrict intracellular bacterial growth. However, the function as receptor for LGALS9 has been challenged. HAVCR2 has also been reported to enhance CD8+ T-cell responses to acute infections such as Listeria monocytogenes. HAVCR2 may be a receptor for phosphatidylserine (PtSer); PtSer-binding is calcium-dependent. HAVCR2 may recognise PtSer on apoptotic cells leading to their phagocytosis. HAVCR2 also mediates the engulfment of apoptotic cells by dendritic cells. HAVCR2 is expressed on T-cells and promotes conjugation but not the engulfment of apoptotic cells. HAVCR2 expressed on dendritic cells (DCs) positively regulates innate immune response and in synergy with Toll-like receptors promotes secretion of TNF-alpha. In tumour-infiltrating DCs, HAVCR2 suppresses nucleic acid-mediated innate immune responses through interaction with HMGB1 and interferes with nucleic acid-sensing and trafficking of nucleic acids to endosomes. HAVCR2 expressed on natural killer (NK) cells acts as a coreceptor to enhance IFN-gamma production in response to LGALS9. In contrast, HAVCR2 has been shown to suppress NK cell-mediated cytotoxicity. HAVCR2 negatively regulates NK cell function in LPS-induced endotoxic shock.

[0127] That is to say that cells that may be sorted from MPE in order to provide the expanded cell populations described herein may be sorted based on the above markers. After further expanding and / or maintaining the cells, expression of the above markers may increase or decrease (except for those mentioned below in respect of late pleura cells and / or early pleura cells).

[0128] In addition to the markers above, the Trm cells that are sorted, expanded, further expanded and / or maintained express or have increased expression of GPR183 and CXCR4 or LITAF, JUNB and PI3KR1. For example in comparison to a reference T cell such as a canonical T cell.

[0129] Trm like cells that express or have increased expression of GPR183 and CXCR4 may be referred to herein as late pleura cells or late unique to pleura cells. Trm like cells that express or have increased expression of LITAF, JUNB and PI3KR1 may be referred to herein as early pleura cells or early unique to pleura cells.

[0130] GPR183 encodes the G-protein coupled receptor 18 (GPR18) which is abundantly expressed in lymphocytes, with particularly high expression in CD8aa y<5T intraepithelial lymphocytes (lELs). GPR183 acts as a chemotactic receptor for B-cells, T-cells, splenic dendritic cells, monocytes / macrophages and astrocytes. GPR183 is a receptor for oxysterol 7-alpha,25-dihydroxycholesterol (7-alpha,25-OHC) and other related oxysterols; mediates cell positioning and movement of a number of cells by binding the 7-alpha,25-OHC ligand that forms a chemotactic gradient. GPR183 acts as a chemotactic receptor for some T-cells upon binding to 7-alpha,25-OHC ligand. GPR183 promotes follicular helper T (Tfh) cells differentiation by positioning activated T-cells at the follicle-T-zone interface, promoting contact of newly activated CD4 T-cells with activated dendritic cells and exposing them to Tfh-cell-promoting inducible costimulator (ICOS) ligand. Expression in splenic dendritic cells is required for their homeostasis, localization and ability to induce B- and T-cell responses.

[0131] CXCR4 encodes a CXC chemokine receptor specific for stromal cell-derived factor-1. The protein has 7 transmembrane regions and is located on the cell surface. CXCR4 controls homing of naive and CM CD8+ T cells and promotes homeostatic self-renewal of CM CD8+ T cells.

[0132] LITAF encodes lipopolysaccharide-induced tumour-necrosis factor. LITAP. plays a role in endosomal protein trafficking and in targeting proteins for lysosomal degradation; plays a role in targeting endocytosed EGFR and ERGG3 for lysosomal degradation, and thereby helps down-regulate downstream signalling cascades; helps recruit the ESCRT complex components TSG101, HGS and STAM to cytoplasmic membranes; may play a role in regulating protein degradation via its interaction with NEDD4; may also contribute to the regulation of gene expression in the nucleus; binds DNA (in vitro) and may play a synergistic role with STAT6 in the nucleus in regulating the expression of various cytokines; regulate the expression of numerous cytokines, such as TNF, CCL2, CCL5, CXCL1, I L1 A and IL10.

[0133] JUNB encodes the JUNB Proto-Oncogene, AP-1 Transcription Factor Subunit. JUNB enables sequence-specific double-stranded DNA binding activity and is involved in the positive regulation of transcription by RNA polymerase II.

[0134] PIK3R1 encodes a subunit of phosphatidylinositol 3-kinase (PI3K). The primary function of the subunit is to regulate the enzyme's activity. PI3K is a kinase, which is important for many cell activities, including cell growth and division (proliferation), movement (migration) of cells, production of new proteins, transport of materials within cells, and cell survival. PI3K is activated downstream of many receptors important for regulating immune cell activation and differentiation, including the T and B cell antigen receptors (TCR / BCR), costimulatory molecules (e.g., CD40, CD28), cytokine receptors, and TLRs.

[0135] Markers of Trm cells include one or more of those listed in Table 6 below (adapted from Samji, Tasleem, and Kamal M Khanna. “Understanding memory CD8+ T cells.” Immunology letters vol. 185 (2017): 32-39. doi:10.1016 / j.imlet.2017.02.012):

[0136] Table 6 - Genes highly expressed in Trm cells

[0137] Expanded populations

[0138] The expanded cell populations described herein may be defined by the altered (i.e. increased or decreased) expression of various genes. The genes may be part of a gene signature. The genes of a gene signature may be up or down regulated in comparison to a reference cell (such as a canonical Trm cell, early pleura cell (not expended by the methods described herein), late pleura cell (not expended by the methods described herein), expanded early pleura cell (expended by the methods described herein), expanded late pleura cell (expended by the methods described herein), a circulating T cell, or any other cells present in a sample of MPE) or in comparison to a housekeeping gene. A gene signature as used herein, may thus refer to any set of up- and down-regulated genes between different cells or cell (sub)populations derived from a gene-expression profile. For example, a gene signature may comprise a list of genes differentially expressed in a population of cells of interest. It is to be understood that also when referring to proteins (e.g. differentially expressed proteins), such may fall within the definition of “gene” signature.

[0139] A gene signature score may be allocated to cells based on the expression levels of genes in comparison to the reference cell or in comparison to a baseline score previously determined for the gene signature. For example, a high score may be assigned when the expression levels of the genes in the cell of interest correlate or have a relatively high correlation with the level of expression for the baseline score. In some examples, a high score may be assigned when the genes upregulated in the gene signature are also upregulated in the cell of interest to the same or a greater extent than the baseline. In some examples, a high score may be assigned when the genes downregulated in the gene signature are also downregulated in the cell of interest to the same or a greater extent than the baseline. As such, a low gene signature score may be assigned to a cell when the expression levels of the genes of the gene signature in the cell of interest do not correlate or have a relatively low correlation with the level of expression for the baseline score. In some examples, a low score may be assigned when the genes upregulated in the gene signature are not upregulated in the cell of interest to the same or a lesser extent than the baseline. In some examples, a high score may be assigned when the genes downregulated in the gene signature are not downregulated in the cell of interest to the same or a greater extent than the baseline.

[0140] Methods of determining levels of expression of genes of a gene signature will be known and described below.

[0141] During expanding or before expanding, further expanding and / or maintenance of late pleura cells and / or early pleura cells may be anergised cells. T cell anergy is a tolerance mechanism in which the lymphocyte is intrinsically functionally inactivated following an antigen encounter, but remains alive for an extended period of time in a hyporesponsive state. Models of T cell anergy affecting both CD4(+) and CD8(+) cells fall into two broad categories. One, clonal anergy, is principally a growth arrest state, whereas the other, adaptive tolerance or in vivo anergy, represents a more generalized inhibition of proliferation and effector functions. The former arises from incomplete T cell activation, is mostly observed in previously activated T cells, is maintained by a block in the Ras / MAP kinase pathway, can be reversed by IL-2 or anti-OX40 signalling, and usually does not result in the inhibition of effector functions. The latter is most often initiated in naive T cells in vivo by stimulation in an environment deficient in co-stimulation or high in co-inhibition. Adaptive tolerance can be induced in the thymus or in the periphery. The cells proliferate and differentiate to varying degrees and then downregulate both functions in the face of persistent antigen. The state involves an early block in tyrosine kinase activation, which predominantly inhibits calcium mobilization, and an independent mechanism that blocks signalling through the IL-2 receptor. Adaptive tolerance reverses in the absence of antigen.

[0142] In some examples, the late pleura cells and / or early pleura cells are clonally anergistic. Without being bound by theory some ex vivo CD8+CD103+ cells from the pleura are observed to have an altered metabolic prolife which is modulated towards an increased use of glycolysis. This is indicative of the effects of the hypoxic microenvironment in the pleura, that may induce clonal anergy.

[0143] The methods provided herein may energise or de-anergise the late pleura cells and / or early pleura cells.

[0144] In some examples, at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 87, 98, or 99% of the cells in the population of expanded late pleura cells and / or early pleura cells are energised or de-anergised cells.

[0145] Energised or de-anergised cells may have an increased level of expression of genes indicative of a memory cell phenotype. Energised or de-anergised cells may have an increased level of expression of genes indicative of a proliferative cell phenotype. Energised or de-anergised cells may have an increased level of expression of genes indicative of a memory cell and proliferative cell phenotype. For example, increased expression in comparison to canonical Trms. For example, increased expression in comparison to anergised Trms such as anergised late pleura cells and / or early pleura cells. For example, in comparison to an unexpanded late pleura cell, unexpanded early pleura cell, a canonical Trm cell and / or other cells of the MPE.

[0146] The methods of expanding the late unique to pleura and / or early pleura cells may help reduce the expression of suppressive factors compared to the cells when present in the MPE.

[0147] Examples of genes that are indictive of a proliferative phenotype include ABCB1A, KAT6A, RRAS, ABCB1 B, KLF4, SALL4, ALPL, LDHA, SATB1, ANTXR2, LDHB, SETBP1, ARL4C, LDHC, SETDB1, ATR, LDHD, SKIL, BAALC, LECT1, SMARCAD1, BASP1 , LPIN1, SOX2, BCL6, LY6A, SPON1, BUB1 , LY6E, STAT3, CCR7, MAP3K8, TBX21, CCR9, MAPK12, TBX3, CD27, MCM3AP, TCF3, DOCK9, MCOLN2, TCL1 , DUSP9, MYC, TDGF1 , EOMES, NANOG, TERT, ESRRB, NCOR2, TIGIT, EVL, NR0B1, TNFAIP2, FAS, NR1D2, TNFRSF1 B, FGF2, P2RY14, TRAF1, FUT4, PAX6, TRAF4, GZMK, PCGF2, TRIB2, HAND1 , PLEKHA5, TXNIP, HESX1 , PODXL , IER3, POU5F1, ZFP42, IL2RB, POU6F1, ZFX, IL7R, PRKCE, ZIC3, IRF4, REST, IRF8, RIF1 , JARID2, and / or RNF138.

[0148] In some examples, the expanded late unique to pleura and / or early pleura cells may have a relatively high score for a gene signature comprising expression levels of one or more genes selected from ABCB1A, KAT6A, RRAS, ABCB1 B, KLF4, SALL4, ALPL, LDHA, SATB1, ANTXR2, LDHB, SETBP1 , ARL4C, LDHC, SETDB1 , ATR, LDHD, SKIL, BAALC, LECT1, SMARCAD1, BASP1 , LPIN1 , SOX2, BCL6, LY6A, SPON1, BUB1, LY6E, STAT3, CCR7, MAP3K8, TBX21, CCR9, MAPK12, TBX3, CD27, MCM3AP, TCF3, DOCK9, MCOLN2, TCL1 , DUSP9, MYC, TDGF1 , EOMES, NANOG, TERT, ESRRB, NCOR2, TIGIT, EVL, NR0B1, TNFAIP2, FAS, NR1D2, TNFRSF1 B, FGF2, P2RY14, TRAF1 , FUT4, PAX6, TRAF4, GZMK, PCGF2, TRIB2, HAND1 , PLEKHA5, TXNIP, HESX1 , PODXL , IER3, POU5F1, ZFP42, IL2RB, POU6F1, ZFX, IL7R, PRKCE, ZIC3, IRF4, REST, IRF8, RIF1 , JARID2, and / or RNF138. In some examples, the gene signature comprises or consist of ABCB1A, KAT6A, RRAS, ABCB1B, KLF4, SALL4, ALPL, LDHA, SATB1, ANTXR2, LDHB, SETBP1, ARL4C, LDHC, SETDB1, ATR, LDHD, SKIL, BAALC, LECT1, SMARCAD1, BASP1, LPIN1 , SOX2, BCL6, LY6A, SPON1, BUB1 , LY6E, STAT3, CCR7, MAP3K8, TBX21 , CCR9, MAPK12, TBX3, CD27, MCM3AP, TCF3, DOCK9, MCOLN2, TCL1 , DUSP9, MYC, TDGF1 , EOMES, NANOG, TERT, ESRRB, NCOR2, TIGIT, EVL, NR0B1, TNFAIP2, FAS, NR1D2, TNFRSF1B, FGF2, P2RY14, TRAF1, FUT4, PAX6, TRAF4, GZMK, PCGF2, TRIB2, HAND1 , PLEKHA5, TXNIP, HESX1 , PODXL , IER3, POU5F1 , ZFP42, IL2RB, POU6F1, ZFX, IL7R, PRKCE, ZIC3, IRF4, REST, IRF8, RIF1, JARID2, and / or RNF138.

[0149] Examples of genes that may be indicative of a memory cell and / or proliferative cell phenotype include one or more of TCF7, STAT4, PIK3R1, FAM177A1, F0XP1 and IL.7R. In some examples, the expanded late unique to pleura and / or early pleura cells may express or have increased expression of one or more of TCF7, STAT4, PIK3R1, FAM177A1, F0XP1 and IL7R in comparison to a reference cell (such as canonical Trm cell).

[0150] In some examples, the expanded late unique to pleura and / or early pleura cells may express or have increased expression of one or more of TCF7, STAT4, PIK3R1, and IL7R in comparison to a reference cell (such as canonical Trm cell).

[0151] In some examples, the expanded late unique to pleura and / or early pleura cells may not express or have decreased expression of one or more genes associated with glycolysis. For example, the expanded late unique to pleura and / or early pleura cells may have a low score for a gene signature indicative of glycolysis. For example, the gene signature may include any one or more of the genes selected from ACSS1, ACSS2, ADH1A, ADH1B, ADH1C, ADH4, ADH5, ADH6, ADH7, AKR1A1 , ALDH1A3, ALDH1B1, ALDH2, ALDH3A1 , ALDH3A2, ALDH3B1, ALDH3B2, ALDH7A1 , ALDH9A1, ALDOA, ALDOB, ALDOC, BPGM, DLAT, DLD, ENO1 , ENO2, ENO3, FBP1, FBP2, G6PC, G6PC2, GALM, GAPDH, GCK, GPI, HK1 , HK2, HK3, LDHA, LDHAL6A, LDHAL6B, LDHB, LDHC, PCK1 , PCK2, PDHA1, PDHA2, PDHB, PFKL, PFKM, PFKP, PGAM1 , PGAM2, PGAM4, PGK1, PGK2, PGM1, PGM2, PKLR, PKM, and TPI1.

[0152] In some examples, the gene signature comprises or consist of ACSS1 , ACSS2, ADH1A, ADH1 B, ADH1C, ADH4, ADH5, ADH6, ADH7, AKR1A1 , ALDH1A3, ALDH1B1, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1 , ALDH3B2, ALDH7A1, ALDH9A1 , ALDOA, ALDOB, ALDOC, BPGM, DLAT, DLD, ENO1, ENO2, ENO3, FBP1, FBP2, G6PC, G6PC2, GALM, GAPDH, GCK, GPI, HK1 , HK2, HK3, LDHA, LDHAL6A, LDHAL6B, LDHB, LDHC, PCK1 , PCK2, PDHA1 , PDHA2, PDHB, PFKL, PFKM, PFKP, PGAM1 , PGAM2, PGAM4, PGK1, PGK2, PGM1 , PGM2, PKLR, PKM, and TPI1.

[0153] TCF7 encodes a member of the T-cell factor / lymphoid enhancer-binding factor family of high mobility group (HMG) box transcriptional activators. This gene is expressed predominantly in T-cells and plays a critical role in natural killer cell and innate lymphoid cell development.

[0154] The encoded protein forms a complex with beta-catenin and activates transcription through a Wnt / beta-catenin signalling pathway. Mice with a knockout of this gene are viable and fertile, but display a block in T-lymphocyte differentiation.

[0155] STAT4 encodes a member of the STAT family of transcription factors. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein is essential for mediating responses to IL12 in lymphocytes, and regulating the differentiation of T helper cells.

[0156] IL7R encodes receptor for interleukin 7 (IL7). The function of this receptor requires the interleukin 2 receptor, gamma chain (IL2RG), which is a common gamma chain shared by the receptors of various cytokines, including interleukins 2, 4, 7, 9, and 15. This protein has been shown to play a critical role in V(D)J recombination during lymphocyte development.

[0157] Expanded late pleura cells (i.e. after expansion, further expansion or during maintenance) may express one or more of LMNA, GPR183, FAM107B, CXCR4, TUBA1A, YPEL5, DDX3X, DENND4 and / or DDX21 . For example, late pleura cells may have increased or greater expression of one or more of LMNA, GPR183, FAM107B, CXCR4, TUBA1A, YPEL5, DDX3X, DENND4 and / or DDX21 in comparison to a reference cell. For example, late pleura cells may have increased or greater expression of one or more of LMNA, GPR183, FAM107B, CXCR4, TUBA1A, YPEL5, DDX3X, DENND4 and DDX21 in comparison to a reference cell. For example, in comparison to an unexpanded late pleura cell, an expanded or unexpanded early pleura cell, a canonical Trm cell or other cells of the MPE.

[0158] That is to say that late pleura cells may express or have increased expression of GPR183 and CXCR4 and in addition express or have increased expression of LMNA, FAM107B, TUBA1A, YPEL5, DDX3X, DENND4 and / or DDX21 in comparison to a reference cell such as an unexpanded late pleura cell, an expanded or unexpanded early pleura cell, a canonical Trm cell or other cells of the MPE.

[0159] In some examples, late pleura cells may have a relatively high score for a gene signature including the genes LMNA, GPR183, FAM107B, CXCR4, TUBA1A, YPEL5, DDX3X, DENND4 and DDX21 in comparison to an unexpanded late pleura cell, an expanded or unexpanded early pleura cell, a canonical Trm cell or other cells of the MPE.

[0160] LMNA encodes part of the nuclear lamina, a two-dimensional matrix of proteins located next to the inner nuclear membrane. The lamin family of proteins make up the matrix and are highly conserved in evolution. During mitosis, the lamina matrix is reversibly disassembled as the lamin proteins are phosphorylated. Lamin proteins are thought to be involved in nuclear stability, chromatin structure and gene expression. Lamins are induced in T cells upon antigen recognition. Lamins enhance T cell responses by coupling the plasma membrane to the nucleus via the linker of nucleoskeleton and cytoskeleton (LING) complex and the actin cytoskeleton. Lamin A expression after T cell antigen recognition occurs in order to coordinate the process of T cell activation and that the subsequent decrease in lamin A expression may facilitate T cell proliferation and differentiation. In this sense, a low degree of differentiation and / or high levels of proliferation have been associated with a reduction in the expression of A-type lamins during homeostasis and in human malignancies, including leukemias and lymphomas. Lamins A / C proteins might also be critical regulators in the coordination of T cell migration, an essential process during immune responses.

[0161] FAM107B encodes Family With Sequence Similarity 107 Member B. FAM107B inhibition has been shown to lead to increased proliferation and migrating abilities of cells.

[0162] TUBA1A encodes Tubulin Alpha 1a (a-tubulin). a-tubulin is part of the tubulin family of proteins that form and organize microtubules. Microtubules are integral for maintaining cell structure, intracellular transport, and mitosis (cell division). In the context of CD8 T cells, microtubules play an essential role in the generation of cell structure, that provides the foundation of T cell effector function. Specifically, microtubules enable cell migration and facilitate the formation of the immune synapse for cytotoxic T cells to engage and eliminate target cells.

[0163] YPEL5 encodes Yippee Like 5. YPEL5 is predicted to enable metal ion binding activity and to be involved in cell population proliferation.

[0164] DDX3X encodes a member of the large DEAD-box protein family, that is defined by the presence of the conserved DEAD motif, and has ATP-dependent RNA helicase activity. This protein has been reported to display a high level of RNA-independent ATPase activity, and unlike most DEAD-box helicases, the ATPase activity is thought to be stimulated by both RNA and DNA. This protein has multiple conserved domains and is thought to play roles in both the nucleus and cytoplasm. Nuclear roles include transcriptional regulation, mRNP assembly, pre-mRNA splicing, and mRNA export. In the cytoplasm, this protein is thought to be involved in translation, cellular signalling, and viral replication.

[0165] DENND4 encodes a DENN domain-containing protein that may function as a guanine nucleotide exchange factor that specifically activates ras-related protein Rab-10.

[0166] DDX21 encodes DExD-Box Helicase 21.

[0167] Expanded late pleura cells (i.e. after expansion, further expansion or during maintenance) may have decreased expression of or do not express one or more of SOCS3, DLISP2, CLDND1, PD-1, NKG2A, and / or Tim-3 in comparison to a reference T cell. For example, in comparison to an unexpanded late pleura cell, an expanded or unexpanded early pleura cell, a canonical Trm cell or other cells of the MPE.

[0168] SOCS3 encodes a member of the STAT-induced STAT inhibitor (SSI), also known as suppressor of cytokine signalling (SOCS), family. SSI family members are cytokine-inducible negative regulators of cytokine signalling. The expression of this gene is induced by various cytokines, including IL6, IL10, and interferon (IFN)-gamma. The protein encoded by this gene can bind to JAK2 kinase, and inhibit the activity of JAK2 kinase. SOCS3 is expressed in peripheral T cells, and overexpression of SOCS3 has been reported to modulate antigen- and / or costimulation-induced T-cell activation. SOCS3-deficient CD8 T cells showed greater proliferation than wild-type cells in response to T-cell receptor (TCR) ligation despite normal activation of signalling pathways downstream from TCR or CD28 receptors. Socs3-deficient CD8+ T cells, IL-6 upregulates the expression of type I interferon (IFN)-regulated genes and enhances the anti-tumour effector function of T cells

[0169] DLISP2 encodes a member of the dual specificity protein phosphatase subfamily. These phosphatases inactivate their target kinases by dephosphorylating both the phosphoserine / threonine and phosphotyrosine residues. They negatively regulate members of the mitogen-activated protein (MAP) kinase superfamily (MAPK / ERK, SAPK / JNK, p38), which are associated with cellular proliferation and differentiation. Different members of the family of dual specificity phosphatases show distinct substrate specificities for various MAP kinases, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. This gene product inactivates ERK1 and ERK2, is predominantly expressed in hematopoietic tissues, and is localized in the nucleus. DLISP2 has been identified as a negative regulator of immune checkpoint inhibitor response within CD8 T cells. Downregulation of DLISP2, may increase cytotoxicity of human CD8 T cells. DLISP2 overexpression, may decrease release of effector molecules (Granzyme B and Interferon-gamma) from human CD8 T cells. Expanded early pleura cells (i.e. after expansion, further expansion or during maintenance) may express of have increased expression of one or more of ZFP36L2, EEF1A1, PABPC1, CLDND1, DUSP2, SOCS3 and / or FXYD5. .

[0170] For example, expanded early pleura cells may have increased or greater expression of one or more of ZFP36L2, EEF1A1, PABPC1, CLDND1, DUSP2, SOCS3 and / or FXYD5 in comparison to a reference cell. For example, early pleura cells may have increased or greater expression of one or more of ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to a reference cell. For example, in comparison to an unexpanded early pleura cell, an expanded or unexpanded late pleura cell, a canonical Trm cell or other cells of the MPE.

[0171] That is to say that early pleura cells may express or have increased expression of LITAF, JUNB and PIK3R and in addition express or have increased expression of ZFP36L2, EEF1A1, PABPC1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to a reference cell such as late pleura cells, canonical Trm cells or other cells of the MPE.

[0172] In some examples, early pleura cells may have a relatively high score for a gene signature including the genes ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to an unexpanded early pleura cell, an expanded or unexpanded late pleura cell, a canonical Trm cell or other cells of the MPE.

[0173] FXYD5 encodes a member of a family of small membrane proteins that share a 35-amino acid signature sequence domain, beginning with the sequence PFXYD and containing 7 invariant and 6 highly conserved amino acids. The approved human gene nomenclature for the family is FXYD-domain containing ion transport regulator. Mouse FXYD5 has been termed RIC (Related to Ion Channel). FXYD2, also known as the gamma subunit of the Na,K-ATPase, regulates the properties of that enzyme. FXYD1 (phospholemman), FXYD2 (gamma), FXYD3 (MAT-8), FXYD4 (CHIF), and FXYD5 (RIC) have been shown to induce channel activity in experimental expression systems. Transmembrane topology has been established for two family members (FXYD1 and FXYD2), with the N-terminus extracellular and the C-terminus on the cytoplasmic side of the membrane. This gene product, FXYD5, is a glycoprotein that functions in the up-regulation of chemokine production, and it is involved in the reduction of cell adhesion via its ability to down-regulate E-cadherin. FXYD5 expression has been positively correlated with immune checkpoint genes, DNA mismatch repair genes, MSI (microsatellite instability) and TMB (tumour mutational burden) across various cancers.

[0174] PABPC1 encodes a poly(A) binding protein. The protein shuttles between the nucleus and cytoplasm and binds to the 3' poly(A) tail of eukaryotic messenger RNAs via RNA- recognition motifs. The binding of this protein to poly(A) promotes ribosome recruitment and translation initiation; it is also required for poly(A) shortening which is the first step in mRNA decay. The gene is part of a small gene family including three protein-coding genes and several pseudogenes.

[0175] EEF1A1 encodes an isoform of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.

[0176] ZFP36L2 encodes a member of the TIS11 family of early response genes. Family members are induced by various agonists such as the phorbol ester TPA and the polypeptide mitogen EGF. The encoded protein contains a distinguishing putative zinc finger domain with a repeating cys-his motif. This putative nuclear transcription factor most likely functions in regulating the response to growth factors. ZFP36L2 may block translation of pre-formed cytokine encoding mRNA in quiescent memory T cells. ZFP36L1 may limit the rate of differentiation of activated naive CD8+ T cells.

[0177] CLDND1 encodes a transmembrane protein. Differential display studies revealed CLDND1 is differentially expressed between anergic and activated T-cells. Over-expression of CLDND1 has revealed that CLDND1 is a potential negative regulator of T-cell activation and proliferation, and CLDND1 can induce a hypo-proliferative T cell phenotype. Silencing of the CLDND1 gene may result in increased T-cell proliferation, compared to control cells.

[0178] Expanded early pleura cells (i.e. after expansion, further expansion or during maintenance) may have decreased expression of or do not express one or more of RGCC, FAM107B, TLIBA1A, PD-1, NKG2A, and / or Tim-3 in comparison to a reference T cell. For example, in comparison to an unexpanded early pleura cell, an expanded or unexpanded late pleura cell, a canonical Trm cell or other cells of the MPE.

[0179] RGCC encodes Regulator Of Cell Cycle. RGCC is induced by p53 in response to DNA damage, or by sublytic levels of complement system proteins that result in activation of the cell cycle. The encoded protein localizes to the cytoplasm during interphase and to centrosomes during mitosis. The protein forms a complex with polo-like kinase 1. The protein also translocates to the nucleus in response to treatment with complement system proteins, and can associate with and increase the kinase activity of cell division cycle 2 protein. In different assays and cell types, overexpression of this protein has been shown to activate or suppress cell cycle progression.

[0180] In some examples, the expanded late unique to pleura and / or early unique to pleura cells have an expression profile indicative of a good response to immune checkpoint inhibitor blockade in comparison to a reference T cell. For example, see Sade-Feldman, Moshe, et al. "Defining T cell states associated with response to checkpoint immunotherapy in melanoma." Cell 175.4 (2018): 998-1013.

[0181] In some examples, the expanded late unique to pleura and / or early pleura cells express one or more AIM1, BTG2, CD55, CSRNP1, EGR1, FAM177A1, FAM65B, FOSL2, F0XP1, GPR183, IFRD1, IL7R, LMNA, MGAT4A, MYADM, NEU1, NR4A1, NR4A3, PDE4B, PER1, PFKFB3, PIK3R1, REL, RGCC, RGPD5, SKIL, STAT4, TC2N, TCF7, TSC22D2, TSPYL2, USP36, YPEL5, and / or ZFP36L2 at a level indicative of a good response to ICB.

[0182] In some examples, the expanded late unique to pleura and / or early pleura cells express one or more of LMNA, RGCC, MYADM, YPEL5, GPR183, IL7R, REL, FAM177A1, CD55, NR4A3, NEU1, PER1 , TSPYL2, NR4A1 , CSRNP1, STAT4, IFRD1, SKIL, PDE4B, BTG2, EGR1 , FOSL2, TSC22D2, TCF7, CRYBG1, PFKFB3, FOXP1, PIK3R1 , USP36, MGAT4A, TC2N, ZFP36L2, RGPD5, and RIPOR2 at a level indicative of a good response to ICB.

[0183] In some examples, the expanded late pleura and / or early pleura cells have increased expression of LMNA, IL7R, FAM177A1, STAT4, TCF7, FOXP1 , RIPOR2, RGCC, MYADM, YPEL5, REL, CD55, NR4A3, NEU1 , PER1, TSPYL2, IFRD1, SKIL, PDE4B, BTG2, FOSL2, TSC22D2, PFKFB3, USP36, MGAT4A, TC2N and / or ZFP36L2 in comparison to a reference T cell, such as a canonical T cell, such as a canonical Trm cell. In some examples, the expanded late pleura and / or early pleura cells have increased expression of LMNA, IL7R, FAM177A1, STAT4, TCF7, FOXP1, RIPOR2, RGCC, MYADM, YPEL5, REL, CD55, NR4A3, NEU1 , PER1, TSPYL2, IFRD1, SKIL, PDE4B, BTG2, FOSL2, TSC22D2, PFKFB3, USP36, MGAT4A, TC2N and ZFP36L2 in comparison to a reference T cell, such as a canonical T cell, such as a canonical Trm cell.

[0184] In some examples, the expanded late unique to pleura and / or early pleura cells have decreased expression of NR4A1, CSRNP1 and / or CRYBG1 in comparison to a reference T cell. For example, in comparison to a canonical T cell.

[0185] In some examples, the expanded late unique to pleura and / or early pleura cells have a high score for a good response to ICB gene signature in comparison to a reference T cell. For example, a high score for a gene signature comprising the genes AIM1, BTG2, CD55, CSRNP1, EGR1, FAM177A1 , FAM65B, FOSL2, FOXP1, GPR183, IFRD1, IL7R, LMNA, MGAT4A, MYADM, NEU1, NR4A1 , NR4A3, PDE4B, PER1 , PFKFB3, PIK3R1 , REL, RGCC, RGPD5, SKIL, STAT4, TC2N, TCF7, TSC22D2, TSPYL2, USP36, YPEL5, and ZFP36L2. As described above, a high score refers to the cumulative expression of genes within the gene signature for cells in a cell state (e.g. unique late to pleura cells) that is significantly increased when compared to another cell state (e.g. “early pleura cells” and “canonical T cells”). In some examples, the expanded late unique to pleura and / or early pleura cells have a high score for a good response to ICB gene signature in comparison to a reference T cell. For example, a high score for a gene signature comprising the genes LMNA, IL7R, FAM177A1, STAT4, TCF7, FOXP1, RIPOR2, RGCC, MYADM, YPEL5, REL, CD55, NR4A3, NEU1, PER1, TSPYL2, IFRD1 , SKIL, PDE4B, BTG2, FOSL2, TSC22D2, PFKFB3, USP36, MGAT4A, TC2N and ZFP36L2.

[0186] As described above, expression of certain markers described herein may be evaluated in comparison to one or more reference cells. In some examples, the reference cell is a canonical Trm cell. Canonical Trm cell refers to CD8+CD103+ Trm cells that may be defined by expression of cytotoxic genes such as IFNG, GZMB and GZMH. Canonical Trm cells may also have enriched expression of genes associated with GOBP Cell Killing, GOMF MHC protein complex binding and GOMF Cytokine activity. Genes associated with GOBP Cell Killing are provided at https: / / www.gsea- msigdb.org / gsea / msigdb / cards / GOBP_CELL_KILLING (version 2024.1.Hs: Updated to GO Release 2024-04-24.). Genes associated with GOMF MHC protein complex binding are provided at https: / / www.gsea- msigdb.org / gsea / msigdb / human / geneset / GOMF_MHC_PROTEIN_COMPLEX_BINDING.ht ml (version 2024.1.Hs: Updated to GO Release 2024-04-24.). Genes associated with GOMF Cytokine activity are provided at https: / / gsea- msigdb.org / gsea / msigdb / human / geneset / GOM F_CYTOKINE_ACTIVITY.html (version 2024.1.Hs: Updated to GO Release 2024-04-24.).

[0187] In some examples, canonical Trm cells may be defined by expression or increased expression (in comparison to early or late pleura cells or other cells of the MPE) of any one or more of CCL4, GZMB, CCL4L2, GZMH, ACTB, ALOX5AP, IFNG, GZMA, TUBB, and / or CLIC1.

[0188] In some examples, canonical Trm cells may be defined by expression or increased expression (in comparison to early or late pleura cells or other cells of the MPE) of any one or more of CCL4, GZMB, CCL4L2, GZMH, ACTB, ALOX5AP, IFNG, GZMA, TUBB, and CLIC1.

[0189] In some examples, canonical Trm cells may may have a high score of expression (in comparison to early or late pleura cells or other cells of the MPE) for a gene signature comprising the gene CCL4, GZMB, CCL4L2, GZMH, ACTB, ALOX5AP, IFNG, GZMA, TUBB, and CLIC1.

[0190] In some examples, the reference T cell is a late unique to pleura or early pleura cell that has not been expanded, not been further expanded or not been maintained as described herein. For example, for comparison to further expanded late unique to pleura or early pleura cells a late unique to pleura or early pleura cell may be sorted and collected before being expanded or further expanded and used to compare changes in expression of genes that occur during or as a consequence of the expansion methods described herein.

[0191] In some examples, the references cell is an expanded late unique to pleura or expanded early unique to pleura cell. For example, expanded late unique to pleura cells may be compared to expanded early unique to pleura cells or vice versa.

[0192] Expanded cell populations of the invention may be characterized with respect to certain cell types being positive or negative for a protein or other marker of interest.

[0193] For the purposes of the present disclosure, references to “positive” (or “+”) cells, or to cells “expressing” a protein or other marker, should be interpreted as encompassing both cases in which an expressed protein is detectable in respect of a cell, and cases in which increased expression of a recited gene (or gene encoding a recited protein) is detectable in respect of a cell.

[0194] In the case of a detectable protein, this may be confirmed by a suitable approach such as antibody labelling. In the case of increased expression of a gene, this may be confirmed by the presence of elevated levels of mRNA, which can be detected by any appropriate RNA analysis approach. Elevation of mRNA levels can be determined by normalization with respect to an appropriate control, such as a housekeeping gene.

[0195] References to cells being “negative” (or should be construed with the same considerations in mind.

[0196] In some examples, there is provided a population of cells including an expanded population of late pleura cells. In some examples, the late pleura cells make up at least 5% of the cells. For example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60. 65. 70, 75, 80, 85, 90, 95 % or more of the cells in the population of cells.

[0197] In some examples, there is provided a population of cells including an expanded population of early pleura cells. In some examples, the early pleura cells make up at least 5% of the cells. For example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60. 65. 70, 75, 80, 85, 90, 95 % or more of the cells in the population of cells.

[0198] In some examples, there is provided a population of cells including an expanded population of the late pleura cells and early pleura cells. In some examples, the early pleura cells and the late pleura cells make up at least 5% of the cells. For example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60. 65. 70, 75, 80, 85, 90, 95 % or more of the cells in the population of cells.

[0199] In some examples, the population of cells are provided as a pharmaceutical composition. A pharmaceutical composition may comprise a a population of cells or composition thereof, described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0200] Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.

[0201] As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e. , the material may be administered to an individual along with the population of cells as described herein without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0202] Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a nucleic acid molecule, vector, or modified cell as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility.

[0203] Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0204] Adjuvants are pharmacological and / or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.

[0205] Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

[0206] Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art. Methods

[0207] The methods provided herein includes the steps of: a) enriching CD8+cytotoxic T cells from malignant pleural effusion (MPE); b) co-culturing the CD8+ cytotoxic T cell enriched MPE with irradiated feeder cells obtained from a subject for a first time period under conditions to allow the T cells of the MPE to expand, wherein co-culturing comprises supplementing with one or more cytokines; c) washing the expanded T cells and stimulating the expanded T cells with irradiated cancer cells from the subject; d) co-culturing the expanded T cells and irradiated cancer cells for a second time period under conditions to allow the expanded T cells to further expand; e) co-culturing the further expanded T cells with live cancer cells obtained from the subject; f) sorting the co-cultured further expanded T cells based on a level of expression of : i. one or more tumour reactive markers comprising CD103, CD8, and one or more of 4-1BB, CD25, and / or CD154; ii. memory markers; and iii. , exhaustion markers; and g) determining which of the sorted T cells express or have increased expression of GPR183 and CXCR4 and / or UTAF, JUNB and PI3KR1.

[0208] As mentioned above, the MPE may be or have been obtained from a subject suffering from or suspected of suffering from cancer. After MPE is obtained the cells of the MPE and the steps of the method described herein are carried out in vitro or ex vivo. It will be understood that the steps of enriching, culturing, sorting and determining are carried out in vitro or ex vivo. As such, the methods described herein may be in vitro methods. As such, the methods described herein may be ex vivo methods.

[0209] After provision of the MPE, CD8+ cytotoxic T cells are enriched. Enriching cells refers to increasing the percentage of cells characterized by a specific marker expression signature in a heterogeneous population of cells. Methods of enriching cells are known to those skilled in the art and include, for example, flow cytometry and the use of columns containing molecules that bind specific marker (i.e. bind to CD8). In some examples, the concentration of T cells is increased by incubating the isolated cells with a molecule that binds T cells and then separating T cells from non-T cells by flow cytometry. In such examples, the molecules that bind T cell markers can be labelled with a detectable marker such as, for example, a florescent dye or a radiolabel. Suitable detectable markers are known to those skilled in the art. Without being bound by theory enrichment may help with expansion of autologous tumour-reactive tissue-resident T cell memory (Trm) like CD103+CD8+ T cells in preparation for isolation. Enrichment may increase the relative frequency and overall abundance of tumour-reactive Trm T cells from ex vivo MPE samples in an autologous system.

[0210] In some examples, the MPE is enriched for CD8+ T cells using magnetic cell isolation microbeads. Magnetic cell isolation using microbeads may be utilised to purify CD8+ T cells from heterogeneous cell suspensions. The procedure involves incubating the cell suspension with microbeads conjugated to antibodies. This can either be performed to positively select CD8+ T cells with increased specificity, or negatively select CD8+ T cells without activating the cells.

[0211] Positive selection utilises antibodies that specifically bind the CD8 antigen expressed on the surface of CD8+ T cells. Once labelled with antibodies and microbeads, the cell suspension is passed through a magnetic column placed in a magnetic field. CD8+ T cells, now magnetically labelled, are retained within the column, while unlabelled cells are washed away. Following this, the magnetic field is removed, allowing the enriched CD8+ T cells to be eluted for further use. This method may provide for high specificity and purity of cells, and may help ensure the cells remain functionally viable for downstream analyses.

[0212] Negative selection utilises a cocktail of antibodies that specifically bind a wide diversity of antigens that are expressed on cells other than CD8 T cells. This includes CD4 (helper T cells), CD19 (B cells), CD14 (monocytes), CD56 (NK cells), and others. Once labelled, the sample is passed through a magnetic column, where the magnetically labelled non-CD8 cells are retained, while the CD8+ T cells pass through the column unbound. This method is gentler, as the CD8+ T cells remain untouched by the magnetic particles, which may be beneficial when functional assays or sensitive downstream applications are required.

[0213] After enriching, the enriched MPE is co cultured with irradiated feeder cells. The feeder cells may have been obtained or are obtained from a subject. In some examples, the feeder cells have been obtained or are obtained from the same subject as the MPE has been obtained from (for example, autologous feeder cells). In some examples, the feeder cells include or are obtained from the MPE. In some examples, the feeder cells include one or more of cancer cells obtained from the same subject as the MPE )e.g. autologous cancer cells such as primary cancer cells), healthy peripheral blood mononuclear cells (PBMCs) from leukocyte cone, or healthy PBMCs from peripheral blood. Healthy cells may be or have been obtained from a healthy donor. For example, a donor who does not have cancer.

[0214] The cell culture medium that may be used in the methods described herein may be any suitable cell culture medium. The cell culture medium may be selected depending on the type of cell cells being cultured. Examples of culture mediums that may be used include minimal essential medium (MEM, Sigma, St. Louis, Mo); Dulbecco’s modified Eagle medium (DMEM, Sigma); Ham F10 medium (Sigma); Cell culture media (HyClone, Logan, Utah); RPMI-1640 culture media (Sigma); and chemical-defined (CD) culture media (which are formulated for individual cell types), such as CD-CHO culture media (Invitrogen, Carlsbad, Calif). The culture solution described above can be supplemented with auxiliary components or contents as needed. This includes any component of the appropriate concentration or amount required or desired.

[0215] The culture medium described above can be supplemented with auxiliary components or contents as needed. The culture medium may include one or more additives such as antibiotics, proteins, amino acids and / or sugars.

[0216] “Medium” and “cell culture medium” refer to a nutrient source used for growing or maintaining cells. As is understood by a person of skill in the art, the nutrient source may contain components required by the cell for growth and / or survival or may contain components that aid in cell growth and / or survival. Vitamins, essential or non-essential amino acids, trace elements, and surfactants (e.g., poloxamers) are examples of medium components. Any media provided herein may also be supplemented with any one or more of insulin, plant hydrolysates and animal hydrolysates.

[0217] “Culturing” a cell refers to contacting a cell with a cell culture medium under conditions suitable for the viability and / or growth and / or proliferation of the cell.

[0218] In some examples, the feeder cells are produced by culturing MPE in a high adhesion culturing vessel with a culture medium that includes foetal bovine serum. For example, 5 to 15% FBS. In some examples, 10% FBS. For example, feeder cells may be prepared by culturing MPE with a DMEM medium including 10% FBS. The feeder cells may be cultured at a temperature above ambient or room temperature, for example about 37°C. The feeder cells may be cultured at 37°C until attached, then washed with culture medium to remove any unattached cells. The feeder cells may be then cultured until passage 5.

[0219] In some examples, the feeder cells include cancer cells. In some examples, the feeder cells include primary cancer cells. When the feeder cells include or are primary cancer cells, the cancer phenotype of the cells may be validated. For example, by any suitable pathology methods. For example, validation of cancer cells may include staining cultured cells of the MPE (e.g. cultured to the fifth passage) with Papanicolaou and May-Gruwald Giemsa dyes. The stained cells may then be reviewed by an independent and blinded medical pathologist to confirm malignant nature. The feeder cells may then be irradiated and seeded into a suitable culturing vessel, such a well of a well plate. Other suitable culturing vessels will be known.

[0220] CD8+ cytotoxic T cell enriched MPE is then added to the culture vessel including the irradiated feeder cells. Before addition to the culture vessel, CD8+ cytotoxic T cell enriched MPE may be washed using a culture medium including human serum. For example, the CD8+ cytotoxic T cell enriched MPE may be washed using a culture medium including 10% human serum. For example, the culture medium may be Roswell Park Memorial Institute (RPMI) medium. RPMI medium contains the reducing agent glutathione and high concentrations of vitamins. RPMI medium contains biotin, vitamin B12, and PABA. In addition, the vitamins inositol and choline are present in very high concentrations. Other suitable medias that may be used include DMEM, MEM, Ham’s F-10 nutrient mix, McCoy's 5A, Ham’s F-12K, Medium 199, BME, Leibovitz's L-15, IMDM and combinations thereof.

[0221] The CD8+ cytotoxic T cell enriched MPE may be added to the irradiated feeder cells to provide a ratio of effector to target ratio of 1 : 1.

[0222] The CD8+ cytotoxic T cell enriched MPE and irradiated feeder cells may be cultured for a first time period. In some examples, the first time period is at least 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, or more days. In some examples, the first time period is about 10 to 14 days. In some examples, the first time period is 14 days. The CD8+ cytotoxic T cell enriched MPE and irradiated feeder cells may be cultured using a culture medium including 10% human serum. For example, RPMI medium including 10% human serum. Other suitable medias that may be used include DMEM, MEM, Ham’s F-10 nutrient mix, McCoy's 5A, Ham’s F-12K, Medium 199, BME, Leibovitz's L-15, IMDM and combinations thereof.

[0223] Co-culturing of the CD8+ cytotoxic T cell enriched MPE and irradiated feeder cells may be carried out at a temperature above ambient or room temperature, for example about 37°C.

[0224] Co-culturing of the CD8+ cytotoxic T cell enriched MPE and irradiated feeder cells may include supplementing with one or more cytokines. For example, one or more cytokines may be added to the co-culture. In some examples, one or more cytokines are added every three days of co-culturing. For example, after 3 days of co-culturing, the one or more cytokines are added to the co-culture. On days 6, 9 and 12 from starting co-culturing additional amounts of the one or more cytokines are added to the co-culture.

[0225] In some examples, the one or more cytokines include IL-2, IL7 and / or IL15. In some examples, IL-2 is added to provide a final concentration of 100U / ml. In some examples, IL7 is added to provide a final concentration of 1 to 10 ng / pL. For example 5 ng / pL. In some examples, I L15 is added to provide a final concentration of 1 to 10 ng / pL. For example 5 ng / pL.

[0226] After co-culturing of the CD8+ cytotoxic T cell enriched MPE and irradiated feeder cells the expanded cells produced are washed. For example, washed in culture medium. For example, washed using RPMI media including human serum (for example 10% human serum). Washing may help remove dead cell debris including dead cancer cell debris and T cells debris. Washing may also help remove cytokine supplements.

[0227] The washed expanded cells may then be rested in culture medium (such as RPMI media including human serum). For example, the expanded cells may maintained in culture media for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more hours. For example, the expanded cells may be rested overnight. The expanded cells may be rested at a temperature greater than ambient or room temperature. For example, the expanded cells may be rested at a temperature above ambient or room temperature, for example about 37°C.

[0228] After resting the expanded cells, the expanded cells may be stimulated by contacting the expanded cells with irradiated cancer cells (or additional irradiated cancer cells if the feeder cells include caner cells). The irradiated cancer cells may be derived from the subject from which the MPE has been obtained (i.e. autologous irradiated cancer cells). In some examples, the irradiated cancer cells are primary cancer cells. In some examples, the irradiated cancer cells have been obtained as described above.

[0229] The expanded cells may be contacted with the irradiated cancer cells at a ratio of effector to target of 1 : 1.

[0230] The expanded cells and irradiated cancer cells may be contacted in culture medium (such as RPMI media including human serum) and then co-cultured with the irradiated cancer cells. The second co-culture provides further expanded cells.

[0231] The expanded cells and irradiated cancer cells may be cultured for a second time period. In some examples, the second time period is at least 1 , 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, or more days. In some examples, the second time period is about 10 to 14 days. In some examples, the second time period is 14 days. The expanded cells and irradiated cancer cells may be cultured using a culture medium including 10% human serum. For example, RPMI medium including 10% human serum. Other suitable media that may be used include DMEM, MEM, Ham’s F-10 nutrient mix, McCoy's 5A, Ham’s F-12K, Medium 199, BME, Leibovitz's L-15, IM DM and combinations thereof. Co-culturing of the expanded cells and irradiated cancer cells may be carried out at a temperature above ambient or room temperature, for example about 37°C.

[0232] Co-culturing of the expanded cells and irradiated cancer cells may include supplementing with one or more cytokines. For example, one or more cytokines may be added to the coculture. In some examples, one or more cytokines are added every three days of co- culturing. For example, after 3 days of co-culturing, the one or more cytokines are added to the co-culture. On days 6, 9 and 12 from starting co-culturing additional amounts of the one or more cytokines are added to the co-culture.

[0233] In some examples, the one or more cytokines include IL-2, IL7 and / or IL15.

[0234] In some examples, IL-2 is added to provide a final concentration of 100U / ml. In some examples, IL7 is added to provide a final concentration of 1 to 10 ng / pL. For example 5 ng / pL. In some examples, IL15 is added to provide a final concentration of 1 to 10 ng / pL. For example 5 ng / pL.

[0235] After co-culturing of the expanded cells and irradiated cancer cells the further expanded cells produced are washed. For example, washed in culture medium. For example, washed using RPMI media including human serum (for example 10% human serum).

[0236] The washed further expanded cells may then be rested in culture medium including human serum (such as RPMI media including 10% human serum). For example, the further expanded cells may maintained in culture media for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more hours. For example, the further expanded cells may be rested overnight. The further expanded cells may be rested at a temperature greater than ambient or room temperature. For example, the further expanded cells may be rested at 37°C.

[0237] The rested further expanded cells may then be co-cultured (third co-culture) with live cancer cells (i.e. not irradiated cancer cells). The live cancer cells may be derived from the subject from which the MPE has been obtained (i.e. autologous live cancer cells). In some examples, the live cancer cells are primary cancer cells. In some examples, the live cancer cells have been obtained as described above. In some examples, prior to the third co- culturing, live cancer cells may be seeded into a culture vessel (such as well of a well plate) in a culture medium including FBS. For example, 10% FBS. For example, cu-culturing in DM EM with 10% FBS. Live cancer cells may then be cultured as described above. For example, the live cancer cells may be cultured at a temperature above ambient or room temperature, for example 37°C. The live cancer cells may be cultured at a temperature above ambient or room temperature, for example, at 37°C until attached, then washed with culture medium to remove any unattached cells. For example, the live cancer cells may be cultured for at least 24 hours. After culturing the live cancer cells in the culture vessel, prior to the third co-culture (i.e. at most 5, 4, 3, 2, 1 hours or less before the third co-culture) the live cancer cells may be washed with phosphate-buffered saline and then resuspended in a culture media including FBS (such as RPMI with 10% FBS).

[0238] The rested further expanded cells may be added to the reaction vessel and co-cultured with the liver cancer cells at a ratio of effector to target of 1 : 1.

[0239] The third co-culture may include co-culturing the live cancer cells and rested further expanded cells in culture medium including human serum (such as RPMI media including 10% human serum).

[0240] The third co-culture may include co-culturing the live cancer cells and rested further expanded cells for about 20 to 24 hours at a temperature above ambient or room temperature, for example, about 37°C.

[0241] The third co-culture may include supplementing with one or more cytokines. For example, one or more cytokines may be added to the co-culture. In some examples, one or more cytokines are added every three days of co-culturing. For example, after 3 days of co- culturing, the one or more cytokines are added to the co-culture. On days 6, 9 and 12 from starting co-culturing additional amounts of the one or more cytokines are added to the coculture.

[0242] In some examples, the one or more cytokines include IL-2, IL7 and / or IL15.

[0243] In some examples, IL-2 is added to provide a final concentration of 100U / ml. In some examples, IL7 is added to provide a final concentration of 1 to 10 ng / pL. For example 5 ng / pL. In some examples, IL15 is added to provide a final concentration of 1 to 10 ng / pL. For example 5 ng / pL.

[0244] After the third co-culture, the further expanded cells may then be sorted to identify and separate out further expanded cells that express the markers CD103 and CD8. In addition, cells may be sorted based on the expression levels of one or more of 4-1 BB, CD25, CXCR4, CCR7, PD-1 , NKG2A, Tim-3, CD45RA, and / or CD45RO. The further expanded cells may be sorted based on the expression of any one or more of the markers provided in Table 5 and / or Table 6.

[0245] In some examples, the further expanded cells that express or have increased expression of 4-1BB, CD25, and / or CD154, memory markers as described herein (e.g. CCR7, CD45RA and / or CD45RO) and exhaustion markers (e.g. PD1, Tim3 and / or NKG2a); are sorted and separated from other cells of the MPE. For example, the further expanded cells may be sorted by any suitable cell sorting method in order to separate out Trm cells, in particular, late unique to pleura and / or early pleura cells.

[0246] In some examples, cells are sorted using florescent based sorting methods such as flow cytometry methods such as fluorescent activated cell sorting (FACS).

[0247] Fluorescence activated cell sorting or FACS refers to a method by which the individual cells of a sample are analyzed and sorted according to their optical properties (e g., light absorbance, light scattering and fluorescence properties, etc.) as they pass in a narrow stream in single file through a laser beam. Fluorescence-activated cell sorting is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. In a typical FACS system, the cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet. Just before the stream breaks into droplets the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems the charge is applied directly to the stream and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off. The fluorescent labels for FACS technique depend on the lamp or laser used to excite the fluorochromes and on the detectors available. The most commonly available lasers on single laser machines are blue argon lasers (488 nm). Fluorescent labels workable for this kind of lasers include, but not limited to, 1) for green fluorescence (usually labelled FL1): FITC, Alexa Fluor 488, GFP, CFSE, CFDA-SE, and DyLight 488; 2) for orange fluorescence (usually FL2): PE, and PI; 3) for red fluorescence (usually FL3): PerCP, PE-Alexa Fluor 700, PE-Cy5 (TRI-COLOR), and PE-Cy5.5; and 4) for infra-red fluorescence (usually FL4; in some FACS machines): PE- Alexa Fluor 750, and PE- Cy7. Other lasers and their corresponding fluorescent labels include, but are not limited to, 1) red diode lasers (635 nm): Allophycocyanin (APC), APC- Cy7, Alexa Fluor 700, Cy5, and Draq-5; and 2) violet lasers (405 nm): Pacific Orange, Amine Aqua, Pacific Blue, 4 ,6- diamidino-2-phenylindole (DAPI), and Alexa Fluor 405.

[0248] Accordingly, FACS typically involves use of a panel of binding partners specific for some cell surface markers of interest (e.g. those described in Table 5). The binding partners are thus conjugated to the fluorescent labels as described above. The binding partners may be antibodies that may be polyclonal or monoclonal, preferably monoclonal. In another example, the binding partners may be a set of aptamers. Antibodies or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Monoclonal antibodies or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally; the human B-cell hybridoma technique; and the EBV-hybridoma technique.

[0249] For example, FACS may be carried out using the following antibody reporter conjugates: CD3-FITC (BD Biosciences), CD8-PerCP.Cy5.5 (BD Biosciences), CD103-APC (BD Biosciences), CD39-PE_Dazzle 594 (Biolegend).

[0250] Finally, once the single cells are sorted, they are individually deposited in a multi-well container. Preferably, the container consists of a 96-well plate.

[0251] Other suitable cell sorting methods such as magnetic-activated cell sorting (MACS) may be used.

[0252] Given the third co-culture step, the method of sorting may be referred to as activation- induced marker (AIM) assay cell sorting. AIMs is a cytokine-independent technique to identify antigen-specific T cells based on the upregulated expression of activation markers after antigen restimulation (i.e. with the live cancer cells). For example, see WO2024023522A1 and Eugster et al., 2013; Dan et al., 2016. AIM assays seek to identify antigen-responsive CD4+ or CD8+ T cells by proliferation and activation marker expression. AIM assays typically utilise combinations of CD25, CD69, CD134 (0X40), CD137 (4-1 BB) and CD154 (CD40L) to identify recently activated antigen-responsive T cells.

[0253] After sorting, the sorted cells that possess the desired markers are analysed for expression of specific markers to identify late unique to pleura and / or early pleura cells. For example, expression levels of GPR183 and CXCR4 are determined to identify late pleura cells. For example, expression levels of LITAF, JUNB and PI3KR1 are determined to identify early pleura cells. Identification of genes that undergo a change in expression may be carried out by any suitable method known in the art. “Differentially expressed" refers generally to a protein or nucleic acid (RNA, e.g. mRNA) that is overexpressed (upregulated) or underexpressed (downregulated) in one cell compared to at least one other cell. For example, in comparison to the expression level of one or more housekeeping genes in a control sample, in comparison to canonical Trm cells, or in comparison to other cells found in the MPE.

[0254] The terms "overexpress", "overexpression", "overexpressed", “increased expression” “enriched expression”, “enriched for” (in reference to genes or proteins), or “upregulated” interchangeably refer to a protein or nucleic acid (RNA) that is translated or transcribed at a detectably higher level, in comparison to a control. The term includes overexpression due to changes in transcription, post-transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelle, cytoplasm, nucleus, cell surface), RNA stability, protein stability, etc as compared to a control sample. Overexpression can be detected using conventional techniques for detecting RNA (i.e. , RT-PCR, PCR, hybridization, RNA-Sequencing, NGS) or proteins (i.e., ELISA, immunohistochemical techniques). Overexpression can be an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to the control. In certain instances, overexpression is an increase of 1-fold, 2-fold, 3-fold, 4-fold, or more in comparison to the control.

[0255] The terms "underexpress", "underexpression", "underexpressed" “decreased expression” or "downregulated" interchangeably refer to a protein or nucleic acid (RNA) that is translated or transcribed at a detectably lower level, in comparison to a control. The term includes underexpression due to changes in transcription, post-transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelle, cytoplasm, nucleus, cell surface), RNA stability and / or protein stability, as compared to a control sample. Underexpression can be detected using conventional techniques for detecting RNA (i.e., RT- PCR, PCR, hybridization, RNA-Sequencing, NGS) or proteins (i.e., ELISA, immunohistochemical techniques). Underexpression can be a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to the control. In certain instances, underexpression is a decrease of 1-fold, 2-fold, 3-fold, 4-fold, or more in comparison to the control.

[0256] Differential expression of genes may be determined by performing RNA expression analysis. RNA may be extracted from samples of sorted cells and the level of RNA may be quantified by hybridisation of probes to provide a gene count. The level of expression or gene count, of each gene may then be normalised based on the expression levels of a number of housekeeping genes by subtracting the average counts of the housekeeping genes from the counts of the gene of interest. The counts for the gene of interest may be expressed as log 10 normalized gene counts in order to provide less skewed data.

[0257] In some examples differential expression is evaluated by determining a magnitude of change in nucleic acid molecule or protein expression, to determine if gene or protein expression is up- or down- regulated. For example, a relative value of expression can be determined. In some examples, a decrease in the relative value of expression indicates that the gene or protein is downregulated, while an increase in the relative value of expression indicates that the gene or protein is upregulated.

[0258] Differential expression of genes, and the expression levels of genes may be determined by any known methods. For example, using RNAseq based methods such as DESeq , edgeR , NBPSeq, TSPM, baySeq, EBSeq, NOISeq, SAMseq and ShrinkSeq.

[0259] In some examples, determining comprises RNA sequencing analysis.

[0260] In some examples, determining comprises bulk RNA sequencing analysis. Bulk RNA sequencing analysis measures the average expression level of individual genes across hundreds to millions of input cells. Bulk RNA sequencing analysis refers to a group of technologies used to sequence the entire set of messenger RNA (mRNA) molecules, or transcripts, which are expressed at a given time, in a given tissue or heterogenous population of cells. Sequence reads are mapped to a reference genome, or assembled de novo, and expression of each transcript is quantified by various software algorithms, such as HTSeq, FeatureCounts, Rcount, Maxcounts, FIXSEQ, Cuffquant, DEseq, and edgeR.

[0261] The basic steps of bulk RNA sequence analysis include RNA extraction, RNA fragmentation, cDNA generation, library amplification, and sequencing on an NGS platform to get strings of continuous sequence data in “reads”. The most common approach is short-read sequencing (read lengths < 300 bp).

[0262] For more details of RNA sequence analysis see, for example, Ozsolak, Fatih, and Patrice M. Milos. "RNA sequencing: advances, challenges and opportunities." Nature reviews genetics 12.2 (2011): 87-98, Li, Xinmin, and Cun-Yu Wang. "From bulk, single-cell to spatial RNA sequencing." International journal of oral science 13.1 (2021): 36, Stark, Rory, Marta Grzelak, and James Hadfield. "RNA sequencing: the teenage years." Nature Reviews Genetics 20.11 (2019): 631-656 and Wang, Zhong, Mark Gerstein, and Michael Snyder.

[0263] "RNA-Seq: a revolutionary tool for transcriptomics." Nature reviews genetics 10.1 (2009): 57- 63.

[0264] In some examples, determining comprises single cell RNA sequencing analysis. Single-cell RNA-sequencing refers to a novel technique for high throughput sequencing and analysis of RNA at the single cell level, unlike the results obtained from conventional tissue or cell population sequencing (which is simply the average expression level of a large number of cells), single cell sequencing is capable of determining expression levels in each individual cell analysed. For example, see WO2021252375A1 and Haque, A., Engel, J., Teichmann,

[0265] 5.A. et al. A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications. Genome Med 9, 75 (2017). https: / / doi.org / 10.1186 / s13073-017-0467-4.

[0266] In some examples, singe cell sequencing may include the steps of in-drop lysis of cells and reverse transcription. The cDNA generated by reverse transcription may be generated alongside barcodes or UMIs in each droplet in order to allow identification of the cell of origin. cDNA may then be amplified and used to construct a nucleic acid library for downstream analysis.

[0267] After determining expression of genes identifying late unique to pleura and / or early pleura cells, the late unique to pleura and / or early pleura cells may be isolated or separated from other cells.

[0268] Therefore, in some examples, the cell populations provided herein are populations of isolated expanded cells.

[0269] In some examples, the sorted cells may not be isolated but may be pooled to provide a cell population enriched for late unique to pleura and / or early pleura cells (e.g. a cell population comprising at least 10, 20, 30, 40 ,50 , 60, 70, 80 or 90% late unique to pleura and / or early pleura cells).

[0270] In some examples, the sorted cells or isolated cells may be further expanded and / or maintained. For example, by culturing the sorted or isolated cells may be cultured in RPMI medium including 10% human serum at a temperature greater than ambient or room temperature (for example at 37°C). Other suitable media that may be used include DMEM, MEM, Ham’s F-10 nutrient mix, McCoy's 5A, Ham’s F-12K, Medium 199, BME, Leibovitz's L- 15, IM DM and combinations thereof.

[0271] The further expansion and / or maintenance may include supplementing with one or more cytokines. For example, one or more cytokines may be added to the co-culture. In some examples, one or more cytokines are added every three days of co-culturing. For example, after 3 days of co-culturing, the one or more cytokines are added to the co-culture. On days

[0272] 6, 9 and 12 from starting co-culturing additional amounts of the one or more cytokines are added to the co-culture.

[0273] In some examples, the one or more cytokines include IL-2, IL7 and / or IL15. In some examples, IL-2 is added to provide a final concentration of 100U / ml. In some examples, IL7 is added to provide a final concentration of 1 to 10 ng / pL. For example 5 ng / pL. In some examples, IL15 is added to provide a final concentration of 1 to 10 ng / pL. For example 5 ng / pL.

[0274] In some examples, the methods include further expanding isolated late unique to pleura and / or early pleura cells.

[0275] Prognosis Methods

[0276] As late unique to pleura and / or early pleura cells express genes that are indicative of a good response to checkpoint inhibitor blockade (ICB) treatment (see markers described above), detection of late unique to pleura and / or early pleura cells in a subject may be used as an indicator that the subject may respond positively to treatment with checkpoint inhibitors.

[0277] Therefore, also provided herein are methods of predicting a subject’s response to one or more immune checkpoint inhibitors, the method comprising: providing a sample of the subject’s MPE; and determining the presence of Trm like CD103+CD8+ T cells expressing at least:

[0278] GPR183 and CXCR4 (late pleura cells); and / or

[0279] LITAF, JUNB and PI3KR1 (early pleura cells) in the sample; and wherein the presence of late pleura cells and / or early pleura cells is indicative of a positive response to the one or more immune checkpoint inhibitors.

[0280] Methods of determining the presence of late pleura cells and / or early pleura cells may be carried out using the method steps described above. For example, by a) enriching CD8+cytotoxic T cells from malignant pleural effusion (MPE); b) co-culturing the CD8+ cytotoxic T cell enriched MPE with irradiated feeder cells obtained from a subject for a first time period under conditions to allow the T cells of the MPE to expand, wherein co-culturing comprises supplementing with one or more cytokines; c) washing the expanded T cells and stimulating the expanded T cells with irradiated cancer cells from the subject; d) co-culturing the expanded T cells and irradiated cancer cells for a second time period under conditions to allow the expanded T cells to further expand; e) co-culturing the further expanded T cells with live cancer cells obtained from the subject; f) sorting the co-cultured further expanded T cells based on a level of expression of : i. one or more tumour reactive markers comprising CD103, CD8, and one or more of 4-1 BB, CD25, and / or CD154; ii. memory markers; and iii. , exhaustion markers;; and g) determining which of the sorted T cells express or have increased expression of GPR183 and CXCR4 and / or UTAF, JUNB and PI3KR1.

[0281] In some examples, the method is a method of stratifying subjects into groups. For example, into groups defined by the likelihood of a positive response to immune checkpoint inhibitor treatment as indicated by the level of late pleura cells and / or early pleura cells.

[0282] Also provided herein is a method of predicting overall survival (OS) in a cancer patient. The method includes: providing a sample of the subject’s MPE; and determining the presence of Trm like CD103+CD8+ T cells expressing at least:

[0283] UTAF, JUNB and PI3KR1 (early unique to pleura cells) in the sample; determining a level of expression of CD8A, CD3E and ITGAE of the early unique to pleura cells; wherein the presence of early unique to pleura cells expressing or having increased expression of CD8A, CD3E and ITGAE in comparison to a reference T cell is indicative of an improved OS in comparison to a subject lacking early unique to pleura cells and / or early unique to pleura cells without increased or decreased expression of CD8A, CD3E and ITGAE in comparison to the reference T cell.

[0284] The early unique to pleura cells may be early unique to pleura cells as described above. For example, they may express or have increased expression of one or more of ZFP36L2, EEF1A1, PABPC1 , CLDND1, DLISP2, SOCS3 and / or FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells.

[0285] The early unique to pleura cells may express or have increased expression of ZFP36L2, JUNB, EEF1A1 , LITAF, PABPC1 , PIK3R1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells. The early unique to pleura cells may have reduced expression of RGCC, FAM107B, TUBA1A, PD-1 , NKG2A, and / or Tim-3 in comparison to late unique to pleura cells and / or canonical Trm cells.

[0286] The early unique to pleura cells may have a relatively high score for a gene signature comprising the genes ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3, FXYD5, CD8A, CD3E and ITGAE in comparison to late unique to pleura cells and / or canonical Trm cells.

[0287] CD8A encodes the CD8 alpha chain. CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The CD8 antigen acts as a coreceptor with the T-cell receptor on the T lymphocyte to recognize antigens displayed by an antigen presenting cell in the context of class I MHC molecules. The coreceptor functions as either a homodimer composed of two alpha chains or as a heterodimer composed of one alpha and one beta chain. Both alpha and beta chains share significant homology to immunoglobulin variable light chains.

[0288] CD3E encodes CD3-epsilon polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha / beta and gamma / delta heterodimers, forms the T-cell receptor- CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The genes encoding the epsilon, gamma and delta polypeptides are located in the same cluster on chromosome 11. The epsilon polypeptide plays an essential role in T-cell development.

[0289] ITGAE encodes an l-domain-containing alpha integrin that undergoes post-translational cleavage in the extracellular domain, yielding disulfide-linked heavy and light chains. In combination with the beta 7 integrin, this protein forms the E-cadherin binding integrin known as the human mucosal lymphocyte-1 antigen.

[0290] Overall survival refers to the patient remaining alive for a defined period of time, such as 1 year, 5 years, etc from the time of diagnosis or treatment.

[0291] In some examples, the method may predict overall survival in a subject suffering from mesothelioma.

[0292] The methods of predicting or stratifying of subjects described herein may provide a medical practitioner with a diagnostic report based on the patient’s stratification, prognosis and / or disease underlying biology. As such, the methods provided herein may further include generating a diagnostic report based on the prediction, underlying disease biology and / or stratification. In certain examples, the diagnostic report is provided to a medical professional (such as a medical doctor) for providing guidance on the selection of a treatment to be administered.

[0293] In some examples, the methods further comprise administering to the subject a treatment.

[0294] In some examples, the methods further comprise administering to the subject a treatment regimen based on the prediction, stratification and / or underlying disease biology determined by the methods described herein.

[0295] The methods described herein can further comprise selecting, and optionally administering, a treatment regimen for the subject based on the prediction, underlying disease biology or stratification. Treatment can include, for example, surgery, therapy (e.g., radiation, hormone, ultrasound, chemotherapy, immunotherapy, targeted therapy), or combinations thereof. In some examples, the treatment is an immune checkpoint therapy.

[0296] In some examples, subjects may not be provided a treatment based on the prediction and / or stratification determined using the methods described herein. Thus avoiding over treatment (e.g. when treatment is not necessary). For example, if a subject does not have late pleura cells and / or early pleura cells present in their MPE, they may not be treated with an immune checkpoint therapy. In some examples, if a subject does not have late pleura cells and / or early pleura cells present in their MPE, they may be treated with an alternative of different therapy or by surgery. In some examples, if a subject does not have late pleura cells and / or early pleura cells present in their MPE they may be monitored.

[0297] As such, there is provided herein a method of determining a treatment regimen for a subject based up stratification and / or prediction using the methods as described herein.

[0298] As used herein, the terms “active surveillance”, “monitoring” and “watchful waiting” are used interchangeably herein to mean closely monitoring a subject’s condition without giving any treatment until symptoms appear or change.

[0299] Medical Uses

[0300] Given the above, there is provided herein a method of treating a subject suffering from cancer, the method comprising: a. stratifying a subject based on the presence or level of late pleura cells and / or early pleura cells; b. determining a prognosis for the subject based on the stratification of the subject; c. generating a diagnostic report based on the prognosis and / or underlying disease biology; d. administering a treatment to the subject based on the prognosis and / or underlying disease biology.

[0301] There is also provided herein a treatment for use in a method of treating a subject suffering from cancer, the method comprising: a. stratifying a subject as described herein; b. determining a prognosis for the subject based on the stratification of the subject; c. generating a diagnostic report based on the prognosis and / or underlying disease biology; and d. administering a treatment to the subject based on the prognosis and / or underlying disease biology.

[0302] In some examples, the treatment is an immune checkpoint therapy (such as one or more immune checkpoint inhibitors).

[0303] As such, there is also provided herein one or more immune checkpoint inhibitors for use in a method of treating cancer in a subject in need thereof, comprising administering an effective amount of the one or more immune checkpoint inhibitors to the subject, wherein:

[0304] Trm like CD103+CD8+ T cells expressing at least:

[0305] GPR183 and CXCR4 (late pleura cells); and / or

[0306] UTAF, JUNB and PI3KR1 (early pleura cells); have been detected in a sample of the subject’s MPE.

[0307] As such there is also provided a method of treating cancer in a subject in need thereof, comprising administering an effective amount of one or more immune checkpoint inhibitors to the subject, wherein:

[0308] Trm like CD103+CD8+ T cells expressing at least:

[0309] GPR183 and CXCR4 (late pleura cells); and / or

[0310] UTAF, JUNB and PI3KR1 (early pleura cells); have been detected in a sample of the subject’s MPE

[0311] Immune checkpoint therapies as described herein may be used as a neoadjuvant therapy. Neoadjuvant therapies are administered to a subject prior to treatment with a primary therapy, such as surgery or radiation therapy. The object of neoadjuvant therapy in treating cancer is to reduce the size or extent of the subject’s tumour before the primary therapy, preferably improving the likelihood of successful outcome and / or decreasing the adverse effects of more extensive treatment that would be required in the absence of neoadjuvant therapy. Neoadjuvant treatment may also target micrometastases that may be unaffected by the primary therapy.

[0312] Immune checkpoint therapies as described herein may be used as a first line therapy. First line therapy refers to the first treatment given for a disease. It may be part of a set of treatments e.g. second line therapy, third line therapy etc., such as surgery followed by chemotherapy and / or radiation therapy. First line therapies may also be known as induction therapy, primary therapy, or primary treatment. Immune checkpoint therapies as described herein may be used as a first line therapy. If the first line therapy is used alone it is referred to as a monotherapy. Monotherapy refers to therapy that uses one type of treatment, such as an immune checkpoint inhibitor or immunostimulant, to treat a certain disease or condition such as cancer. For example, in the case of a drug or therapeutic agent monotherapy refers to the use of a single drug to treat a disease or condition.

[0313] As used herein, an “immune checkpoint inhibitor” means an agent that inhibits proteins or peptides (e.g. immune checkpoint proteins) which are blocking the immune system, e.g., from attacking cancer cells. In some examples, the immune checkpoint protein blocking the immune system prevents the production and / or activation of T cells. An immune checkpoint inhibitor can be an antibody or antigen-binding fragment thereof, a protein, a peptide, a small molecule, or a combination thereof. Typically, the inhibitor interacts directly to a target immune checkpoint protein (or its ligand, where appropriate) and thereby disrupts its function / biological activity. For example, it may bind directly to a target immune checkpoint protein (or its ligand, where appropriate). In one example, direct binding to a target immune checkpoint protein (or its ligand, where appropriate) inhibits, prevents or reduces the formation of protein complexes which are needed for immune checkpoint protein function / biological activity.

[0314] A review describing immune checkpoint pathways and the blockade of such pathways with immune checkpoint inhibitor compounds is provided by Pardoll in Nature Reviews Cancer (April, 2012), pages 252-264. Immune checkpoint inhibitor compounds display anti-tumour activity by blocking one or more of the endogenous immune checkpoint pathways that downregulate an anti-tumour immune response. The inhibition or blockade of an immune checkpoint pathway typically involves inhibiting a checkpoint receptor and ligand interaction with an immune checkpoint inhibitor compound to reduce or eliminate the signal, resulting in diminishment of the anti-tumour response. The immune checkpoint inhibitor compound may inhibit the signalling interaction between an immune checkpoint receptor and the corresponding ligand of the immune checkpoint receptor. The immune checkpoint inhibitor compound can act by blocking activation of the immune checkpoint pathway by inhibition (antagonism) of an immune checkpoint receptor (some examples of receptors include CTLA-4, PD-1, and NKG2A) or by inhibition of a ligand of an immune checkpoint receptor (some examples of ligands include PD-L1 and PD-L2). In such examples, the effect of the immune checkpoint inhibitor compound is to reduce or eliminate down regulation of certain aspects of the immune system's anti-tumour response in the tumour microenvironment.

[0315] In some examples, the immune checkpoint inhibitor inhibits the CTLA-4 pathway or the PD- L1 / PD1 pathway. In some examples, the immune checkpoint inhibitor is an antibody. In some examples, the immune checkpoint inhibitor comprises an antibody that inhibits CTLA- 4, PD1, or PD-L1. Immune checkpoint inhibitors, immune checkpoint inhibitors and examples thereof are provided in, e.g., WO 2016 / 062722.

[0316] In some examples, the immune checkpoint inhibitor is an anti-CTLA-4 antibody or derivative or antigen-binding fragment thereof. In one example, the anti-CTLA-4 antibody selectively binds a CTLA-4 protein or fragment thereof. Examples of anti-CTLA-4 antibodies and derivatives and fragments thereof are described in, e.g., US 6,682,736; US 7,109,003; US 7,123,281 ; US 7,411 ,057; US 7,807,797; US 7,824,679; US 8,143,379; US 8,491 ,895, and US 2007 / 0243184. In some examples, the anti-CTLA-4 antibody is tremelimumab or ipilimumab.

[0317] The immune checkpoint receptor cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is expressed on T-cells and is involved in signalling pathways that reduce the level of T-cell activation. It is believed that CTLA-4 can downregulate T-cell activation through competitive binding and sequestration of CD80 and CD86. In addition, CTLA-4 has been shown to be involved in enhancing the immunosuppressive activity of TReg cells.

[0318] In some examples, the immune checkpoint inhibitor is an anti-PD-L1 antibody or derivative or antigen-binding fragment thereof. In some examples, the anti-PD-L1 antibody or derivative or antigen-binding fragment thereof selectively binds a PD-L1 protein or fragment thereof. Examples of anti-PD-L1 antibodies and derivatives and fragments thereof are described in, e.g., WO 01 / 14556, WO 2007 / 005874, WO 2009 / 089149, WO 2011 / 066389, WO 2012 / 145493; US 8,217,149, US 8,779,108; US 2012 / 0039906, US 2013 / 0034559, US 2014 / 0044738, and US 2014 / 0356353. In some examples, the anti-PD-L1 antibody is MEDI4736 (durvalumab), MDPL3280A, 2.7A4, AMP-814, MDX-1105, atezolizumab (MPDL3280A), or BMS-936559. The immune checkpoint receptor programmed death 1 (PD-1) is expressed by activated T- cells upon extended exposure to antigen. Engagement of PD-1 with its known binding ligands, PD-L1 and PD-L2, occurs primarily within the tumour microenvironment and results in downregulation of anti-tumour specific T-cell responses. Both PD-L1 and PD-L2 are known to be expressed on tumor cells. The expression of PD-L1 and PD-L2 on tumors has been correlated with decreased survival outcomes.

[0319] In some examples, the anti-PD-L1 antibody is MEDI4736, also known as durvalumab. MEDI4736 is an anti-PD-L1 antibody that is selective for a PD-L1 polypeptide and blocks the binding of PD-L1 to the PD-1 and CD80 receptors. MEDI4736 can relieve PD-L1 -mediated suppression of human T-cell activation in vitro and can further inhibit tumor growth in a xenograft model via a T-cell dependent mechanism. MEDI4736 is further described in, e.g., US 8,779,108. The fragment crystallizable (Fc) domain of MEDI4736 contains a triple mutation in the constant domain of the IgG 1 heavy chain that reduces binding to the complement component C1q and the Fey receptors responsible for mediating antibodydependent cell-mediated cytotoxicity (ADCC).

[0320] In some examples, the immune checkpoint inhibitor is an anti-PD-1 antibody or derivative or antigen-binding fragment thereof. In some examples , the anti-PD-1 antibody selectively binds a PD-1 protein or fragment thereof. In some examples , the anti-PD1 antibody is nivolumab, pembrolizumab, or pidilizumab.

[0321] NKG2A receptors are inhibitory receptors binding to HLA-E and expressed on tumor infiltrating cytotoxic NK and CD8 T lymphocytes. By expressing HLA-E, cancer cells can protect themselves from killing by NKG2A+ immune cells. HLA-E is frequently up-regulated on cancer cells of many solid tumors or hematological malignancies. Monalizumab (IPH2201), a humanized lgG4, blocks the binding of NKG2A to HLA-E allowing activation of NK and cytotoxic T cell responses. Examples of anti-NKG2A antibodies and derivatives and fragments thereof are described in WO 2016 / 041947, the content of which is hereby incorporated by reference in its entirety including, but not limited to, the sequence listings.

[0322] In some examples, the immune checkpoint inhibitor compound is a small organic molecule (molecular weight less than 1000 daltons), a peptide, a polypeptide, a protein, an antibody, an antibody fragment, or an antibody derivative. In some examples , the immune checkpoint inhibitor compound is an antibody. In some examples , the antibody is a monoclonal antibody, specifically a human or a humanized monoclonal antibody.

[0323] Monoclonal antibodies, antibody fragments, and antibody derivatives for blocking immune checkpoint pathways can be prepared by any of several methods known to those of ordinary skill in the art, including but not limited to, somatic cell hybridization techniques and hybridoma, methods. Hybridoma generation is described in Antibodies, A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Publications, New York. Human monoclonal antibodies can be identified and isolated by screening phage display libraries of human immunoglobulin genes by methods described for example in U.S. Patent Nos. 5223409, 5403484, 5571698, 6582915, and 6593081. Monoclonal antibodies can be prepared using the general methods described in U.S. Patent No. 6331415 (Cabilly).

[0324] As an example, human monoclonal antibodies can be prepared using a XenoMouse™ (Abgenix, Freemont, CA) or hybridomas of B cells from a XenoMouse. A XenoMouse is a murine host having functional human immunoglobulin genes as described in U.S. Patent No.6162963 (Kucherlapati).

[0325] Methods for the preparation and use of immune checkpoint antibodies are described in the following illustrative publications. The preparation and therapeutic uses of anti-CTLA-4 antibodies are described in U.S. Patent Nos. 7229628 (Allison), 7311910 (Linsley), and 8017144 (Korman). The preparation and therapeutic uses of anti-PD-1 antibodies are described in U.S. Patent No. 8008449 (Korman) and U.S. Patent Application No. 2011 / 0271358 (Freeman). The preparation and therapeutic uses of anti-PD-L1 antibodies are described in U.S. Patent No. 7943743 (Korman). The preparation and therapeutic uses of anti-TIM-3 antibodies are described in U.S. Patent Nos. 8101176 (Kuchroo) and 8552156 (Tagayanagi). The preparation and therapeutic uses of anti-LAG-3 antibodies are described in U.S. Patent Application No. 2011 / 0150892 (Thudium) and International Publication Number W02014 / 008218 (Lonberg). The preparation and therapeutic uses of anti-KIR antibodies are described in U.S. Patent No. 8119775 (Moretta). The preparation of antibodies that block BTLA regulated inhibitory pathways (anti-BTLA antibodies) are described in U.S. Patent No. 8563694 (Mataraza).

[0326] In some examples, the immune checkpoint inhibitor compound is a CTLA-4 inhibitor, a PD-1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, or a KIR inhibitor. In some examples, the immune checkpoint inhibitor compound is an inhibitor of PD-L1 or an inhibitor of PD-L2.

[0327] In some examples, the immune checkpoint inhibitor compound is an inhibitor of the PD- L1 / PD-1 pathway or the PD-L2 / PD-1 pathway. In some examples, the inhibitor of the PD- L1 / PD-1 pathway is MEDI4736.

[0328] In some examples, the immune checkpoint inhibitor compound is an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-BTLA antibody, an anti-KIR antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody. In some examples, the anti-CTLA-4 receptor antibody is ipilimumab or tremelimumab. In some examples, the anti-PD-1 receptor antibody is lambrolizumab, pidilizumab, or nivolumab. In some examples, the anti-KIR receptor antibody is lirilumab.

[0329] Immune checkpoint inhibitors that may be administered to a subject include but are not limited to an anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-KLRB1 antibody, anti-LILRB2 antibody, anti-LILRB4 antibody, anti-LILRB2 and LILRB4 antibody and / or anti-TIM-3 antibody. Examples of immune checkpoint inhibitors include atezolizumab, ipimilumab, pembrolizumab, lambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), pidilizumab (CT-011, CURETECH LTD) and tislelizumab. Exemplary anti-PD-L1 antibodies include MDX-1105 (MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB). Other examples include LILRB2 and LILRB4 antibodies described in US20190194327A1.

[0330] The inhibitor need not be an antibody, but can be a small molecule or other agent or compound. If the inhibitor is an antibody it may be a polyclonal, monoclonal, fragment, single chain, or other antibody variant construct. Inhibitors may target any immune checkpoint protein known in the art, including but not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7- H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and the B-7 family of ligands. Combinations of inhibitors for a single target immune checkpoint or different inhibitors for different immune checkpoints may be used. In particular the immune checkpoint therapy may be an inhibitor of one or more of CD274 (PD- L1), PDCD1 LG2 (PD-L2), TIGIT, HAVCR2 (TIM-3), LAG-3, KLRB1, LILRB2 and / or LILRB4.

[0331] Breast cancer treatments include treatment by surgery, radiation therapy, or a combination of both, as well as systemic treatment by chemotherapy, endocrine therapy, checkpoint inhibitor therapy (or immunotherapy), or a combination thereof. Examples of drugs used for breast cancer chemotherapy include: Cytoxan®(Cyclophosphamide), Methotrexate, 5- Fluorouracil (5-FU), Adriamycin® (Doxorubicin), Prednisone, Nolvadex® (Tamoxifen), Taxol® (Paclitaxel), Leucovorin, Oncovin® (Vincristine), Thioplex® (Thiotepa), Arimidex® (Anastrozole), Taxotere® (Docetaxel), Navelbine®, (Vinorelbine tartrate), Gemzar® (Gemcitabine).

[0332] Examples of combination chemotherapy include the following: CMF (cyclophosphamide, methotrexate, and 5-fluorouracil); classic CMF (oral cyclophosphamide plus methotrexate and 5-fluorouracil); CAF or FAC (cyclophosphamide, Adriamycin® (doxorubicin), and 5- fluorouracil); AC (Adriamycin® and cyclophosphamide); ACT (Adriamycin® plus cyclophosphamide and tamoxifen); AC taxol (Adriamycin® plus cyclophosphamide and paclitaxel (Taxol®)); FACT (5-fluorouracil plus Adriamycin®, cyclophosphamide, and tamoxifen); A-CMF or Adria / CMF (4 cycles of Adriamycin® followed by 8 cycles of CMF); CMFP (CMF plus prednisone); CMFVP (CMF plus vincristine and prednisone); CAFMV (CAF plus methotrexate and vincristine); CMFVATN (CMF plus vincristine, Adriamycin®, thiotepa, and tamoxifen); MF (methotrexate plus 5-fluorouracil and leucovorin).

[0333] Medicines used to relieve side effects caused by chemotherapy include anti-nausea drugs (e.g., reglan), anti-anaemia drugs (e.g., epoetin alfa [Procrit®, Epogen®]), and cellprotecting drugs (e.g., amifostin [Ethyol®]).

[0334] Examples of additional anticancer drugs that can be used in breast cancer therapy include: alkylating agents including cyclophosphamide (Cytoxan®), ifosphamide (Ifex®), melphalan (L-Pam®), thiotepa (Thioplex®), cisplatin (Cisplatinum®, Platinol®), carboplatin (Paraplatin®), and carmustine (BCNll; BiCNll®); antimetabolites including 5-Fluorouracil (5- Fll) methotrexate and edatrexate; antitumor antibiotics including doxorubicin (Adriamycin®) and mitomycin C (Mutamycin®); cytotoxics including mitoxantrone (Novantrone®); vinca alkaloids including vincristine (Oncovin®), vinblastine (Velban®) and vinorelbine (Navelbine®); taxanes including paclitaxel (Taxol®) and docetaxel (Taxotere®); retinoids including fenretinide, corticosteroids including prednisone; antiestrogens including tamoxifen (Nolvadex®); male hormones including fluoxymesterone (Halotestin®); topoisomerase-l compounds including topotecan, irinotecan, 9-amino-camptothecin [9-AC]; anthrapyrazoles including biantrazole and losoxantrone; epidophylotoxins including etoposide and teniposide and angiogenesis inhibitors including compounds that block growth promoting receptors (e.g., PDGF-R and VEGF-R) such as sunitinib (Sutent®).

[0335] Hormonal medications also may be used in treatment. If the patient is ER / PR-negative, then chemotherapy usually is given without hormone therapy, however, hormone therapy may be suitable for patients who are in poor health or who have a short projected survival time. In addition to tamoxifen (Nolvadex®), such drugs include: aromatase inhibitors including anastrozole (Arimidex®) and aminoglutethimide (Cytadren®); luteinizing hormone-releasing hormone-inhibiting compounds including goserelin (Zoladex®) and leuprolide (Lupron®); progestins including megestrol acetate (Megace®) and medroxyprogesterone acetate (Provera®); and androgens including fluoxymesterone (Halotestin®), testolactone (Teslac®), and testosterone enanthate (Delatestryl®).

[0336] For tumours that are c-erbB2 (HER2) positive, trastuzumab (Herceptin®), a humanized monoclonal antibody against the extracellular domain of HER2, can be used.

[0337] When the disease is prostate cancer the treatment may include one or more of surgery (e.g., radical proctectomy, pelvic lymphadenectomy, radical prostatectomy, transurethral resection of the prostate (TURP), excision, dissection, and tumour biopsy / removal), radiation therapy, hormone therapy (e.g., using GnRH antagonists, GnRH agonists, antiandrogens such as Goserelin (Zoladex®), Leuprorelin acetate (Prostap® or Lutrate®), Triptorelin (Decapeptyl® or Gonapeptyl Depot®), Buserelin acetate (Suprefact®), Histrelin (Vantas®), Degarelix (Firmagon®), Bicalutamide (Casodex®), Cyproterone acetate (Cyprostat®), Flutamide (Drogenil®), Abiraterone acetate (Zytiga®), or Nilutamide (Nilandron®)) , ultrasound, chemotherapy (e.g. Docetaxel (Taxotere®), Cabazitaxel (Jevtana®), Strontium-89 (Metastron®), Samarium-153 (Quadramet®), Enzalutamide (Xtandi®), Radium-223 dichloride (Xofigo®), or Apalutamide (Erleada®)), Steroids (e.g. Prednisolone, Dexamethasone, Hydrocortisone); Sipuleucel-T (Provenge®) (to treat advanced, recurrent prostate cancer), or Ketoconazole, optionally in combination with a treatment selected from the group consisting of: radical prostatectomy, external beam radiotherapy / Brachytherapy (with or without hormone therapy), High Intensity Focused Ultrasound (HIFU), Cryotherapy and Trans-urethral resection of the prostate (TURP), Monoclonal antibody therapies (e.g. Pembrolizumab (keytruda), Avastin (bevacizumab), Erbitux (cetuximab), Rituxan (rituximab) and Herceptin (trastuzumab)). or combinations thereof.

[0338] When the disease is endometrial cancer the treatment may include one or more of the treatments described in W02016071520A1. Examples include hysterectomy, bilateral salpingo-oophorectomy, radical hysterectomy, mTOR inhibitor therapy, and / or Lenvatinib therapy.

[0339] Chemotherapy and radiation therapy may both be used either sequentially and / or simultaneously. Use of both therapies or one of chemotherapy or radiation therapy may be referred to as (chemo)radiation therapy. Use of both therapies may be referred to as chemoradiation therapy.

[0340] In some examples, the methods or immune checkpoint inhibitors for use as described are administered with an expanded population of cells or pharmaceutical composition thereof as described herein.

[0341] In some examples, there is provided herein a population of cells or pharmaceutical composition thereof as described herein (i.e. comprising late unique to pleura and / or early pleura cells) for use in methods of treating a disease. For example, for use a medicament.

[0342] In some examples, there is provided herein a population of cells or pharmaceutical composition thereof as described herein (i.e. comprising late unique to pleura and / or early pleura cells) for use in methods of treating cancer.

[0343] The methods include administering and effective amount of the population of cells or pharmaceutical composition thereof to the subject. An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response. The effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient / subject. For example, the suitable dosage of the population of cells or pharmaceutical composition thereof as described herein for a given patient / subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the population of cells or pharmaceutical composition thereof as described herein for example severity and type of disease (e.g. cancer), body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. The dosages and schedules may be varied according to the particular condition, disorder or symptom the overall condition of the patient / subject. Effective dosages may be determined by either in vitro or in vivo methods.

[0344] As used herein, the terms “treat”, “treating” and "treatment" are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom (i.e. in this case a haematological malignancy). Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom. “Treatment” therefore encompasses a reduction, slowing or inhibition of the amount or concentration of malignant cells, for example as measured in a sample obtained from the subject, of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to the amount or concentration of malignant cells before treatment.

[0345] As used here in the term “subject” refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom. The subject may be a patient i.e. a subject in need of treatment. The subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the population of cells or pharmaceutical composition thereof as described herein.

[0346] The population of cells or pharmaceutical composition thereof as described herein can be administered to the subject by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be by infusion or by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration. The population of cells or pharmaceutical composition thereof as described herein may be in any form suitable for the above modes of administration. For example, the population of cells or pharmaceutical composition thereof as described herein may be in any form suitable for infusion. As further examples, suitable forms for parenteral injection (including, subcutaneous, intramuscular, intravascular or infusion) include a sterile solution, suspension or emulsion; suitable forms for topical administration include an ointment or cream; and suitable forms for rectal administration include a suppository. Alternatively, the route of administration may be by direct injection into the target area, or by regional delivery or by local delivery. The identification of suitable dosages of the population of cells or pharmaceutical composition thereof as described herein is well within the routine capabilities of a person of skill in the art.

[0347] The cancer may be any cancer which leads to the subject having MPE. For example, the subject may be a subject with or suspected of having cancer and suffers from MPE.

[0348] In some examples, the method may be a method of cell therapy, the term “cell therapy” refers to a therapy comprising the in vivo administration of at least a therapeutically effective amount of cells to a subject in need thereof. The cells administered to the patient may be allogenic or autologous. In some examples, the methods are autologous cell therapy methods. For example, the expanded population of late unique to pleura and / or early pleura cells have been obtained from a subject’s MPE and are then administered (after expansion) to the same subject.

[0349] In some examples, the cancer is glioblastoma, prostate cancer, pancreatic cancer, non-small cell lung carcinoma, melanoma, breast cancer, gastric cancer; a head and / or neck cancer, a cancer related to viral infection or colorectal cancer. In some examples, the cancer is glioblastoma. In some examples, the cancer is prostate cancer. In some examples, the cancer is pancreatic cancer. In some examples, the cancer is non-small cell lung carcinoma. In some examples, the cancer is melanoma. In some examples, the cancer is breast cancer. In some examples, the cancer is gastric cancer. In some examples, the cancer is a head and / or neck cancer. In some examples, the cancer is a cancer related to viral infection. In some examples, the cancer is colorectal cancer. In some examples, the cancer is a metastatic cancer. In some examples, the cancer is lung adenocarcinoma. For example, stage 2 lung adenocarcinoma. For example, metastatic lung adenocarcinoma. In some examples, the cancer is ovarian cancer. For example, metastatic ovarian cancer. In some examples, the cancer is oesophageal adenocarcinoma. For example, metastatic oesophageal adenocarcinoma. In some examples, the cancer is breast cancer. For example, metastatic breast cancer. In some examples, the cancer is epithelioid mesothelioma. In some examples, the cancer is epithelioid mesothelioma. In some examples, the cancer is mesothelioma. In some examples, the cancer is biphasic mesothelioma. In some examples, the cancer is endometrial adenocarcinoma. For example, metastatic endometrial adenocarcinoma. In some examples, the cancer is endometrial stromal sarcoma. For example, metastatic endometrial stromal sarcoma. In some examples, the cancer is myxofibrosarcoma. For example, metastatic myxofibrosarcoma. In some examples, the cancer is myxofibrosarcoma. For example, metastatic myxofibrosarcoma. In some examples, the cancer is sarcomatoid mesothelioma. In some examples, the cancer is small cell lung cancer. For example, metastatic small cell lung cancer. In some examples, the cancer is pancreatic cancer. For example, metastatic pancreatic cancer. For example, metastatic pancreatic adenocarcinoma. In some examples, the cancer is renal cancer. For example, renal cell cancer. For example, metastatic renal cell cancer.

[0350] “Glioblastoma” refers to a tumour of the brain and / or spinal cord, originating from cell populations in the brain such as glial cells, astrocytes, oligodendrocytes, neural stem cells, or cells of an existing astrocytoma.

[0351] “Prostate cancer” refers to any cancer that originates in the prostate. Prostate cancer is classified as an adenocarcinoma, or glandular cancer, that begins when normal semensecreting prostate gland cells mutate into cancer cells.

[0352] "Gastric cancer" refers to malignant tumours occurring in the stomach, including gastric adenocarcinoma occurring in the gastric mucosal epithelium and malignant lymphoma, myosarcoma, and stromal tumours occurring in the submucosa, but is not limited thereto.

[0353] “Pancreatic cancer” refers to “locally advanced pancreatic cancer” and “metastatic pancreatic cancer.” “Locally advanced pancreatic cancer” refers to tumours that arise in pancreatic exocrine or neuroendocrine tissue, but distant metastases are absent. In contrast, “metastatic pancreatic cancer” refers to cancer spreading from the site from which it originates in the pancreas to involve another part of the body, for example, the liver.

[0354] “Non-small cell lung carcinoma” or “non-small cell lung cancer” refers to any type of epithelial lung cancer other than small cell lung cancer (SCLC). The most common types of non-small cell lung cancer are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma.

[0355] “Melanoma” refers to a condition characterized by the growth of a tumour arising from the melanocytic system of the skin and other organs. Most melanocytes occur in the skin, but are also found in the meninges, digestive tract, lymph nodes and eyes.

[0356] “Breast cancer” refers to any malignancy of the breast tissue, including, for example, carcinomas and sarcomas. “Head and / or neck” cancer refers to other malignancies, except brain cancer, located in the head and neck region. In some examples, head and / or neck cancer may be oral cancer, nasopharyngeal carcinoma, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, sinus cancer, salivary gland cancer or the like. Head and neck cancer commonly occurs in the oral cavity, nasal cavity, throat, sinus, salivary gland, larynx and the like.

[0357] “Colorectal cancer” refers to any cancer of the large bowel, which includes the colon (the large intestine from the cecum to the rectum) and the rectum.

[0358] “Cancer related to viral infection” refers to any cancer that may be caused or the result of a viral infection. Some examples of pathogenic viruses causing infections that may be related to or cause cancers include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV- 6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

[0359] In some examples, the subject has or is suspected of having cancer and is undergoing treatment with one or more additional therapeutics as described herein. In some examples, the subject has been previously or is undergoing treatment with adoptive T cell therapy. In some examples, the additional therapeutic is an immune checkpoint inhibitor as described herein.

[0360] In some examples, the additional therapeutic is a chimeric antigen receptor therapeutic. Chimeric antigen receptor therapeutics typically include chimeric antigen receptor cells, which may be chimeric antigen receptor T cells, chimeric antigen receptor NK cells, and the like. The term "chimeric antigen receptor" (CAR), as used herein, refers to a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular domain. The "chimeric antigen receptor (CAR)" is sometimes called a "chimeric receptor", a "T-body", or a "chimeric immune receptor (CIR) " The "extracellular domain capable of binding to an antigen" means any oligopeptide or polypeptide that can bind to a certain antigen. The "intracellular domain" or "intracellular signalling domain" means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell. In certain examples, the intracellular domain may comprise, alternatively consist essentially of, or yet further comprise one or more costimulatory signalling domains in addition to the primary signalling domain. The "transmembrane domain" means any oligopeptide or polypeptide known to span the cell membrane and that can function to link the extracellular and signalling domains. A chimeric antigen receptor may optionally comprise a "hinge domain" which serves as a linker between the extracellular and transmembrane domains.

[0361] Examples of CAR therapeutics Abecma®, Breyanzi ®, Kymriah ®, Tecartus ®, Yescarta ®, and Carvykti ®. Other examples of CAR therapeutics can be found in, for example, WO2019220109A1, US11034750B2, W02013123061A1, US20130287748A1 , WO2014055668A1, WO2014138704A1, WO2015075468A1 , and WO2017216561A1.

[0362] In some examples, the methods may include prior, simultaneous and / or subsequent administration of one or more of the therapeutic agents as described herein and the population of cells or pharmaceutical compositions thereof as described herein.

[0363] In some examples, there is provided use of a population of cells or pharmaceutical composition thereof as described herein in the manufacture of a medicament for treating or preventing cancer as described herein.

[0364] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0365] Aspects of the invention are demonstrated by the following non-limiting examples.

[0366] EXAMPLES

[0367] Materials and Methods Ethics and clinical data collection

[0368] Cancer patients with malignant pleural effusion diagnosed by NHS consultant pathologist were recruited to the study by the Oxford Pleural Unit, Oxford University Hospitals NHS Trust between 2019 and 2023. All participants provided written informed consent. All samples were collected for Oxford Radcliffe Pleural Biobank (Ethical approval reference: 19 / SC / 0173, South Central - Oxford C Research Ethics Committee) and the study was approved by the Oxford Radcliffe Biobank Tissue Access Committee (reference number: 19 / A022). The clinical data were collected from the Electronic Patient Records (EPR), Oxford University Hospitals, NHS Foundation Trust, by two blinded clinicians. All clinical data were stored and analyzed in an anonymized and non-identifiable manner in accordance with the Human Tissue Act policy and guidelines.

[0369] Specimen collection and processing

[0370] Patient derived specimens including pleural fluid, blood, and pleural biopsies were collected at the Pleural Clinic, John Radcliffe Hospital, Oxford University Hospitals, NHS Foundation Trust. The samples were transferred to the lab within one hour after collection. Pleural fluid was centrifuged at 500g for 20 mins and cell pellets were resuspended in red-blood-cell lysis buffer for 5 mins. Cells were washed in R10 (RPMI + 10% FBS + 100 U / ml penicillin, and 100 mg / ml streptomycin + 2mM L-glutamine) and counted. Fresh isolated cells were then taken for ex vivo FACS immuno-phenotyping and for generation of autologous primary cancer cell lines. The remaining cells were cryopreserved. Peripheral blood was processed as previously described (45). Briefly, whole blood was layered on top of Lymphoprep (STEMCELL technology) and centrifuged at 500g for 20 mins with the brake off. The PBMC buffy coat was isolated via pasture pipette and resuspended in red-blood-cell lysis buffer for 5 mins. Granulocytes were taken for gDNA isolation and HLA-typing. PBMCs were washed in R10 and counted. An aliquot was taken for ex vivo FACS immuno-phenotyping and the remaining cells were cryopreserved. Pleural biopsy was dissected into 2-4mm2pieces and transferred into an aliquoted human Tumour Dissociation enzyme mix (Miltenyi Biotec) for 30 mins at 37°C under continuous rotation. The single cell suspension was then washed with R10, and all cells used for ex vivo FACS immuno-phenotyping.

[0371] Generation of autologous primary cancer cell lines

[0372] Autologous primary cancer cell lines were generated as previously described (46). Briefly, freshly isolated cells from MPE were cultured in high adhesion tissue culture flasks with D10 (DMEM + 10% FBS + 100 ll / ml penicillin, and 100 mg / ml streptomycin + 2mM L-glutamine) at 37°C in 5% CO2. Upon reaching passage five, cytospin preparations of the cells stained with Papanicolaou and May-Gruwald Giemsa dyes were reviewed by an independent and blinded NHS consultant pathologist to confirm their malignant nature.

[0373] Set up short-term tumor-reactive T cell lines with two rounds of expansion

[0374] To isolate tumor-reactive T cells from MPE, primary cancer cell lines were used as stimulus to establish short-term T cell lines from autologous MPE in a two-round expansion protocol (Fig. 9a) cryopreserved cells isolated from MPE were thawed, washed and rested in H10 for 1 hr. Autologous primary cancer cell lines were irradiated at 2000rads and seeded into a 96 well flat-bottomed plate, which was co-cultured with cells from MPE at a 1 :1 E:T ratio for 14- days in Expansion Round-1. On day 14, cells after Expansion Round-1 were washed in H10 and rested overnight at 37°C, then stimulated with additional irradiated autologous primary cancer cell line at 1 :1 E:T for Expansion Round-2 and cultured as above. On day 29, cells after these two rounds of expansion were washed in H10 and rested overnight at 37°C before single-cell sorting for cloning. Cells were cultured as previously described and supplemented with IL-2 every 3 days (45).

[0375] Single-cell sorting with Activation-induced markers (AIMs) for T cell cloning

[0376] Tumor-reactive short-term T cell lines obtained above were co-cultured with autologous cancer cell lines at an E:T ratio of 1 :1 for 20-24 hrs (47). In addition, co-culture of T cell lines without autologous primary cancer cell lines were set up as negative controls, and stimulation with PHA at 20 pg / ml was used as a positive control. The next day, cells were then washed in PBS and first incubated with LIVE / DEAD aqua dye (Invitrogen, L34965) for 20 mins. Cells were then washed and subsequently stained with surface markers CD3-FITC, CD8- PerCP.Cy5.5, CD25-BV650, CD137-PE, CD103-APC and CD39-PE_Dazzel. After staining with surface antibodies at 4°C for 30mins, cells were washed with FACS washing buffer and resuspended in H10 for FACS sorting. Activated CD8+ T cells (CD25+CD137+CD8+T cells) after stimulation with autologous cancer cell lines were single-cell sorted into 96-well U-bottom plates on FACS Aria III, with index of all the markers including CD103 and CD39 expression.

[0377] Generating tumor-reactive T cell clones Flow cytometry sorted single CD25+CD137+CD8+T cells were expanded with feeders cells containing the irradiated PBMCs from three healthy donors as previously described (22). Tumor-reactivity of each T cell clone was either confirmed by positive staining with peptide- MHC Class I tetramer or cytokine production assessed using intracellular cytokine staining (ICS) (22).

[0378] Ex vivo immunophenotyping using flow cytometry

[0379] A total of 0.5-1x106live freshly isolated cells derived from MPE, PBMC and pleural biopsy were washed and resuspended in PBS. Cells were first incubated with LIVE / DEAD marker (Invitrogen, L34965), then washed and stained for surface markers CD14-BV510, CD16- BV510, CD19-BV510, CD3-BUV395 or CD3-BV786, CD8-PerCP.Cy5.5, CD4-APC-R700 or CD4-BUV737 or CD4-AF700, CD56-PE, CD69-APC-H7, NKG2a-BV421 or NKG2a-APC, CD103-BUV737 or CD103-BV786 or BV711 , CD39-PE_Dazzel, PD-1-BV650, TIM-3-BB515, TIGIT-AF700 or TIGIT-BV786 or TIGIT-FITC or TIGIT-APC-R700, CD45-RA-BV711 or CD45- RA-APC-H7 or CD45-RA-BV605, CCR7-AF647 or CCR7-BV421 , and CD27-PE-Cy7. After 20mins incubation at 4°C, cells were washed, and then fixed by cell fixation buffer (BD Bioscience). All samples were acquired on a BD LSRFortessa X50 (BD Biosciences) flow cytometer and analyzed using FlowJov.10.

[0380] Intracellular cytokine staining (ICS) with T cell clones

[0381] ICS was performed as described previously (45). Briefly, 50,000 in vitro cultured CD8+T cell clones were co-cultured with either 50,000 autologous cancer cells as described or BLCs with peptide stimulation, together with GolgiPlug, GolgiStop and PE-anti-CD107a for 5h at 37°C. Dead cells were labelled with LIVE / DEAD Fixable Aqua dye (Invitrogen). Cells were stained for surface markers with the following antibodies: CD8-BV421 (BioLegend). Cells were washed, fixed with Cytofix / Cytoperm and stained with IFNy-FITC (BD Biocsience), TNFa-APC (eBioscience) and MIP1P-APC-H7 (BioLegend). After 30mins incubation at 4°C, cells were washed with 1X Perm / Washing buffer before resuspended in 1X Fixation buffer (BD Bioscience). Negative controls were comprised of CD8+T cell clones co-cultured with irrelevant H LA-mismatched cancer cells or target cells without peptide. All samples were acquired on Attune NxT flow Cytometer (software v.3.2.1) and analyzed using FlowJov.10 software (FlowJo LLC). T cell cytotoxicity assay with Incucyte

[0382] CD8+T cell clones were isolated as above, and killing capacity was evaluated in autologous co-culture experiments as per the manufacture’s instruction (48). Briefly, 25,000 autologous primary cancer cells as described above, or 25,000 H LA-mismatched cancer cells were plated overnight in D10 in a 96-well flat-bottomed plate. After 12 h, cancer cells were washed with PBS and cultured in R10 for co-culture. CD8+T cell clones in R10 (25,000 cells per well) were added to the co-culture wells (n=3 wells for each density). CellTox™ Green Cytotoxicity Assay reagent (Catalog number: G8741) was added to each well as per the manufacture’s instruction, and cell killing was measured by evaluating the Total Green Object Integrated Intensity (GCU x / zm2 / Well) where increased levels correspond to killing.

[0383] FACS sorting for sing-cell RNASeq

[0384] Cryopreserved ex vivo cells isolated from MPE were thawed, washed and rested in H10 (RPMI1640+10% human AB serum) for 1 hr at 37°C and 5% CO2. Cells were then washed with FACS washing buffer and stained with CD3-FITC, CD8-BV421 ,CD4-APC or CD103-APC or TotalSeqTM-C0145 anti human CD103 antibody (Biolegend) for 20mins at 4°C. Before FACS sorting, cells were washed again with FACS washing buffer and then resuspended in H10 (RPMI-1640+10% human serum) with Propidium Iodide dye (eBioscience). Live CD3+CD8+CD4' T cells or live CD3+CD8+CD103+T cells were sorted into Eppendorf tubes with PBS-BSA buffer.

[0385] 10x Genomics library preparation and sequencing

[0386] ScRNA-seq of ex vivo sorted CD8+T cells and CD103+CD8+T cells was performed using the 10x Genomics 5' Single Cell V(D)J+5' Gene Expression Reagent kits v2 for Immune Profiling with Feature Barcode Technology. Libraries were generated as per the manufacturer’s instructions. In brief, cells were subjected to in-drop lysis and reverse transcription, generating cDNA derived from mRNA in each cell and from the oligonucleotide-tagged antibodies, bearing bead-specific sequences to identify the cell of origin. cDNA was then amplified and cleaned using SPRIselect beads (Beckman Coulter) and used in downstream VDJ and gene expression library generation. 2 pl of amplified cDNA product was used to enrich for the TCR cDNA for the VDJ libraries. Subsequently, both the gene expression and enriched VDJ libraries were then subjected to enzymatic fragmentation, end repair, A-tailing, ligation to adaptors, and sample indexing by PCR. Cell surface protein libraries underwent sample indexing PCR. All samples were subsequently cleaned with SPRI select beads and quantified on the Tapestation using a High sensitivity D5000 tape. Gene expression, feature barcode and TCR libraries were pooled at a ratio of 10:1 :1 to a final molarity of 20nM and sequenced by Azenta Life Sciences on the NovaSeq 6000 as paired end 26bp+90bpreads.

[0387] 10X Genomics scRNA / TCRseq data processing

[0388] Cell Ranger count (1 Ox Genomics; v3.1.0) was used for the gene expression and ADT data to align FASTQ sequencing reads to the GRCh38 reference human genome and the antibody barcodes respectively, to create filtered gene / barcode matrices for downstream analysis. Cell Ranger was also used to generate consensus TCR alpha and beta sequences for each cell using the ‘vdj’ command.

[0389] QC, normalization and integration

[0390] QC was performed on each patients datasets individually, and cells that passed the following thresholds were retained: for the RNA modality on a per patient basis cells that surpassed between 400 to 1 ,800 UM Is, exhibited <7% to 15% of reads to mitochondrial regions and >10% of reads to ribosomal regions were retained (Table 3). Furthermore, cells with more than 2 TCRa and / or 2 TCR / ? chains were classed as doublets and filtered out. Once cells were filtered for read count, mitochondrial and ribosomal proportions, and the number of TCRa / / ? chains, cells were scored for the cell cycle an. Subsequently, individual patient samples were nomalized with SCTransform (vst.flavor = "v2") with "percent.mt", "percent. ribo", and "CC. Difference" variables regressed and integrated with Seurat integration via SelectlntegrationFeatures (selection, method = "vst", nfeatures = 2000), FindlntegrationAnchors (normalization. method = "SCT") and IntegrateData (normalization. method = "SCT") functions.

[0391] Table 3. Quality control parameters

[0392] The ADT assay was normalized using the NormalizeData function (normalization. method = "LogNormalize") followed by the ScaleData function. Cells were classed as CD103+or CD103' based on the distribution of cells displayed by the RidgePlot function (Fig. 10a). FACS sorted CD103+CD8+T cells were combined with the CD103+CD8+fraction for patients D057 and D060 identified in the total CD8+T cell analysis (Fig. 10a).

[0393] Clustering and visualization

[0394] Total CD8+T cells: An integrated dataset of 7,802 cells from 3 patients entered principal component analysis. LIMAP visualization was performed using the RunllMAP command in Seurat (dims = 1 :30), constructed nearest neighbour graph using FindNeighbors (dims = 1 :30) function and clustering using the FindClusters (resolution = 0.8) function in Seurat. Based on RNA expression cells were grouped into 11 clusters by unsupervised hierarchical clustering. To identify cluster marker genes FindAIIMarkers (test. use = 'MAST') and adjusting for patient ID using the latent. vars argument). Top marker genes were visualized on a heatmap using DoHeatmap function.

[0395] CD103+CD8+T cells: An integrated dataset of 15,306 cells from 11 patients entered principal component analysis. LIMAP visualization was performed using the RunllMAP command in Seurat (dims = 1 :30), constructed nearest neighbour graph using FindNeighbors (dims = 1 :30) function and clustering using the FindClusters (resolution = 0.8) function in Seurat. Based on RNA expression cells were grouped into 13 clusters by unsupervised hierarchical clustering. To identify cluster marker genes FindAIIMarkers (test. use = 'MAST') and adjusting for patient ID using the latent. vars argument). Top marker genes were visualized on a heatmap using DoHeatmap function.

[0396] Module scores Module scores were generated using the AddModuleScore function. Gene lists used can be found in Tables 2a-d.

[0397] Table 2a

[0398]

[0399] Table 2b

[0400]

[0401] Table 2c

[0402] Table 2d

[0403] Reference mapping

[0404] A reference dataset was generated from three previously published 10x Genomics datasets of CD8+T cell tumor-infiltrating lymphocytes (TILs) from selected stage-2 non-small cell lung cancer samples (28-30). Samples with less than 200 cells were excluded. Individual patient samples were nomalized with SCTransform (vst.flavor = "v2") with "percent.mt", "percent.ribo", and "CC. Difference" variables regressed and integrated into one dataset with Seurat integration via SelectlntegrationFeatures (selection. method = "vst", nfeatures = 3000), FindlntegrationAnchors (normalization. method = "SCT") and IntegrateData (normalization. method = "SCT", k.weight was set to the cell number of the smallest sample) functions. The MapQuery and merge function was used in Seurat to project the CD8+CD103+ T cell dataset onto the combined reference dataset.

[0405] Pseudotime analysis

[0406] Pseudotime analysis was performed using the Monocle 3 algorithm (31-33). Pseudotime was chosen to start in the center of the cells that mapped to the reference dataset as to identify the trajectories from cells that mapped to the reference dataset to those that didn’t map. Cells with a pseudotime values less than 7, between 7 and 12, and greater than 12 were categorized as “Canonical’’, “Early pleural" and “Late pleural" respectively. “Early pleural" and “Late pleural" cells were considered to be “Unique to the pleura’’. Cells with an infinite pseudotime value (n=19) were excluded from further analysis. To identify marker genes between cells states, FindAIIMarkers (test.use = 'MAST') was used, the patient ID was accounted for using the latent. vars argument) and cells were visualized using the DoHeatmap function.

[0407] Milo differential abundance analysis

[0408] Milo differential abundance testing was used to identify differential abundance of “Early pleural” cells between clusters (35) and to analyze a kNN graph generated from RNA-seq data. The precomputed shared nearest neighbor graph (‘snn’) was first used as input required for Milo using the ‘buildFromAdjacency’ function (k = 20, d = 30). Cells were grouped into neighborhoods using the ‘makeNhoods’ function (refined=TRUE, prop=0.1 , refinement_scheme = ‘graph’), and counted using the ‘countCells’ function. To test for differential abundance, the ‘testNhoods’ function was used (fdr.weighting = ‘graph-overlap’) with design = cell state, and neighborhoods with SpatialFDR < 0.1 were classed as statistically significant and colored.

[0409] TCR analysis

[0410] After single-cell TCR sequences were associated with cell barcodes using Cell Ranger vdj, TCRs that were full length and productive were maintained. The TCRp complementaritydetermining region 3 (CDR3) sequences in conjunction with the cell barcodes were added to the TCRb sequences as ‘metadata’ in the Seurat objects for downstream analysis. Unique TCRp CDR3 sequences were used to denote individual T cell clones. The expression of TCRs and clonality across samples was leveraged for analysis of differentiation as described below. Clonotype sharing between Seurat clusters was calculated using the STARTRAC package (24) with default parameters. The scRepertoire package (49) was used to combine gene expression and TCR data. Clonal expansion of the TCR-beta chain clonotype (TRBV plus CDR3-beta) was calculated using the following thresholds ("Single (0 < X <= 1)", "Small (1 < X <= 5)", "Medium (5 < X <= 20)", "Large (20 < X <= 100)", "Hyperexpanded (100 < X <= Inf)". TCR repertoire diversity was calculated using Shannon Diversity Index.

[0411] Kaplan-Meier survival curve analysis

[0412] RNA expression datasets were downloaded from the TCGA database (https: / / portal.gdc.cancer.gov / ) using RTCGA (version 1.18.0) (https: / / rtcga.github.io / RTCGA / ). Patient clinical metadata for associated datasets were downloaded from cBioPortal (https: / / www.cbioportal.org / ). The datasets used were the Mesothelioma (TCGA, PanCancer Atlas). A score was calculated for the patients based on the expression of CD8A, CD3E, ITGAE and the “Early Pleural" signature, and the surv_cutpoint function from the survminer R package was used to objectively determined the optimal cut-off point for the score using the following arguments: time = ’Months. of.disease.specific.survival’, event = ’Disease. specific.Survival. status’. These patients were used to plot Kaplan-Meier survival curves between score high and score low patients (survminer version 0.4.9, publicly available coding). P values were determined by Cox proportional hazard model.

[0413] Statistical analysis

[0414] All statistical analysis was carried out in R (4.3.0) or GraphPad Prism software (version 10). In GraphPad Prism a two-sided Kruskal-Wallis test followed by Dunn’s post hoc test was used for multiple comparisons, and a two-tailed Wilcoxon signed-rank test was used for comparison of matched-pairs. In R (4.3.0) a two-tailed Wilcoxon rank-sum test with Bonferroni corrections was used for unpaired multiple comparisons, a two-tailed Wilcoxon signed-rank test was used for comparison of matched-pairs, and a Cox proportional hazard model was used for survival analysis.

[0415] Results

[0416] Study participants

[0417] A total of 61 malignant pleural effusion patients were recruited to the study by the Oxford Pleural Unit, Oxford University Hospitals NHS Trust between 2018 and 2023. Malignancies causing MPE were predominantly primary pleural mesothelioma (33.87%) followed by metastatic lung adenocarcinoma (27.42%) and metastatic breast cancer (14.52%). Participant characteristics are summarized in Table 1.

[0418] Table 1. Cohort characteristics. Age (years, mean ±sd), sex, and pathologies of malignant pleural effusion patients for the cohort

[0419] MPE is enriched for TRM-like CD103+CD8+T cells. MPE is recognized for its abundant immune infiltrate, but the specific composition of the CD8+T cell population is uncharacterized. To address this, the inventors examined the expression of memory markers (CCR7 and CD45RA), TRM markers (CD103 and CD69) on paired and unpaired T cells from pleural fluid, PBMC and pleural biopsy (Fig. Fig. 1a and Fig. 8a). While the percentage of total CD8+T cells was lower in MPE (23.44%) compared to PBMC (38.28%) (P=0.0008) and pleural biopsy (34.94%) (P=0.0036) (Fig 1b), TRM-like CD8+T cells defined by surface expression of CD103+and CD103+CD69+’ were more frequently detected in MPE (10.60% and 3.83%, respectively), than in the blood (1.87% and 0.11%, respectively) (P<0.0001 and P<0.0001 , respectively) and displayed an intermediate percentage between PBMC and pleural biopsy (18.40% and 12.34%, respectively) (Fig. 1b). The majority of TRM- like CD8+T cells (69.87% of CD103+and 80.85% of CD103+CD69+) are CCR7 CD45RA’ effector memory cells (TEM), and the TEM compartment is more enriched in TRM-like cells than total CD8+T cells (47.27%) (Fig.1c). Interestingly, CD39+CD103+CD8+T cells, a population that has been described as being enriched with tumor-reactive T cells (20) was also found to be enriched (P<0.0001) in MPE (4.34%) when compared to PBMC (0.29%) and displayed an intermediate frequency between PBMC and pleural biopsy (8.69%) (Fig. 1d). Furthermore, the CD39+CD103+CD8+T cell compartment also displayed a dominant TEM phenotype (77.33%), suggesting that MPE contains a high proportion of tumor-reactive TRM-like cells.

[0420] Collectively, the inventors data showed that M PE-derived CD8+T cells contain TRM-like CD103+cells with a predominant effect memory phenotype that likely harbors tumor-reactive T cells.

[0421] CD103+CD8+T cells have increased surface expression of immunoinhibitory molecules

[0422] To further explore the enriched CD103+CD8+TRM-like compartment in MPE, the inventors compared the expression of surface immunoinhibitory molecules between CD103+and CD103- CD8+TEM cells using ex vivo flow cytometry (Fig. 2a). The individual expression of TIM3, NKG2a, PD-1 and TIGIT on CD103+CD8+T cells was found to be significantly increased (P<0.0001 ; P<0.0001 ; P<0.0001 ; P=0.0002, respectively) when compared to CD103+CD8' T cells (Fig. 2b). Furthermore, a greater proportion of CD103+CD8+T cells also exhibited the combined expression of TIM3+NKG2a+, TIM3+PD-1+, NKG2a+PD-1+, TIGIT+TIM3+, TIGIT+NKG2a+and TIGIT+PD1+when compared to CD103'CD8+T cells (Fig. 2c; all P<0.0001). Collectively, the increased expression of immunoinhibitory molecules on CD103+CD8+T cells in MPE suggested a strongly antigen-experienced compartment.

[0423] MPE harbors tumor-reactive cytotoxic CD103+CD8+T cell clones with robust killing against autologous cancer cell lines.

[0424] To explore whether MPE harbors tumor-reactive immune infiltrate, the inventors developed an assay to isolate and clone tumor-reactive CD8+T cells from ex vivo MPE using autologous primary cancer cell lines (Fig. 9a). This method was able to isolate tumor-reactive CD103+CD8+T cell clones from MPE from two metastatic patients who presented with metastatic esophageal cancer and metastatic ovarian cancer respectively. M PE-derived CD103+CD8+T cell clones were expanded, and their specificity was validated by cytokine production against autologous primary cancer cell lines (Fig. 3a). Furthermore, tumor-reactive CD103+CD8+T cell clones also displayed robust in vitro killing capacity against live autologous primary cancer cell lines when co-cultured and induced tumor clearance within 48 h (Fig. 3b, c) In summary, strong evidence that M PE-derived CD103+CD8+TRM-cell harbor tumor-reactive T cells with potent killing capacity against cancer cells is shown, indicating the potential of M PE as a source of cell therapy.

[0425] The inventors also developed an assay to isolate and clone tumor-reactive CD8+T cells from ex vivo MPE using autologous primary cancer cell lines (Fig. 9b). In addition to the CTA- specific CD8+T cell clones isolated from primary disease, the inventors isolated tumor-reactive CD103+CD8+T cell clones from MPE from two metastatic patients who presented with metastatic esophageal cancer and metastatic ovarian cancer respectively. M PE-derived CD103+CD8+T cell clones were expanded, and their specificity was validated by cytokine production against autologous primary cancer cell lines (Fig. 3a). Furthermore, tumor-reactive CD103+CD8+T cell clones also displayed robust in vitro killing capacity against live autologous primary cancer cell lines when co-cultured and induced tumor clearance within 48 h (Fig. 3b).

[0426] In summary, the inventors showed strong evidence that MPE-derived CD103+CD8+TRM-cell harbor tumor-reactive T cells with potent killing capacity against cancer cells, indicating the potential of MPE as a source of cell therapy. scRNAseq identifies a robust CD8+TRM-Hke compartment with enriched ZNF683, GZMA and GZMB expression

[0427] To characterize transcriptional features of CD8+T cells in MPE, the inventors performed single-cell RNAseq analysis with flow cytometry sorted CD8+T cells (Fig. 8c). Analysis of gene expression revealed that CD8+T cells in MPE contain very diverse cell populations (Fig. 4a). Unsupervised cluster analysis identified 11 clusters, including the different populations identified from the flow cytometry analysis: population of Naive T cell cluster 0 (SEZ_Z_+), central memory T cell cluster 1 (TUBA4A+) and cluster 4 (HSPA1A+), effector memory T cell cluster 6 (CCZ_4+) and cluster 7 (GNLY*), and TRM-like T cells at cluster 5 defined by ZNF683+(Fig. 4b). This TRM-like cluster was confirmed by CITE-Seq antibody analysis with surface expression of CD103 and was also found to largely encompass ITGAE gene expression (the transcript encoding CD103) (Fig. 10b).

[0428] To further investigate the functionality of TRM-like T cells, the inventors defined CD103+ / _expression based on adt_CD103 expression. Differential gene expression analysis revealed CD103+CD8+T cells are defined by ZNF683+(Fig. 4c), the gene encoding the key TRM transcription factor Hobit. Additionally, the inventors generated module scores of genes known to be involved in CD8+T cell activation and adhesion and used them to score CD103+ / 'CD8+T cells (Table 2). Analysis of module scores revealed CD103+CD8+T cells showed significantly higher gene signatures associated with T cell effector function and integrin expression (Fig. 4d; all P < 0.0001), indicating greater effector and migration potential when compared to CD103'CD8+T cells.

[0429] Combining with T cell receptor data analysis, the inventors then evaluated dynamics of CD8+T cells in MPE. STARTRAC pairwise transition analysis (24) based on TCR sharing showed that central memory cluster 1 TUBA4A+(TCM1) and cluster 4 JUNB+(TCM2) exhibited the greatest developmental connectiveness to CCL4+effect memory cluster 6 (TEM1). Subsequently, both the central memory cluster 4 JUNB+(TCM2) and effector memory cluster 6 CCL4+(TEM1) displayed further developmental connectiveness to the TRM cluster 5 ZNF683+, as indicated by their significant sharing of TCR clonotypes. This suggests a differentiation trajectory that results in TRM T cells (Fig. 4e).

[0430] TRM-Hke CD103+CD8+T cells are diverse and contain clonally expanded, cytotoxic subsets with an effector phenotype

[0431] To further characterize CD103+CD8+T cells at the transcript level and explore their potential role in cancer progression, the inventors enriched the CD103+CD8+T cell compartment by FACS sorting (Fig. 8d) and performed single-cell analysis using 10x Genomics for eleven patients.

[0432] Analysis of scRNA-seq data with LIMAP visualization and unbiased clustering revealed a diverse cell population that was stratified into 13 clusters (Fig. 5a) with distinct gene expression profiles (Fig. 5b). Consistent with the inventors ex vivo flow cytometry phenotype data, the majority of CD103+CD8+T cells expressed genes for defining “Memor ’, and “Effector function" cells (25) (Fig. 5c; Table 2). Sade-Feldman et al. and Miller et al. have identified sets of genes that identify antitumor responsive CD8+T cells, namely “CD8-G” and “Progenitor exhausted” (26, 27). Analysis of these gene signatures revealed subsets of CD103+CD8+T cells from MPE that scored highly for genes associated with cell activation, survival and memory such as F0XP1, STAT4, IL7R, TCF7, and are predicted to provide subsequent anti-tumoral immunity (Fig. 5c; Fig. 11a). Conversely, “CD8-B" and “Terminally exhausted" gene signatures revealed populations scoring highly for T cell exhaustion (e.g., HAVCR2, PDCD1, LAG3, CTLA4, CD38, ENTPD1) (Fig. 5d; Fig. 11b). Unsurprisingly, these cells are more clonally expanded with lower TCR diversity compared to “memory” cell subset, indicating tumor-reactive T cells in this compartment underwent repeated antigenstimulation and have an exhaustive phenotype that may lack the capacity to provide continued anti-tumoral immunity (Fig. 5e). Clonal expansion amongst TRM-like CD103+CD8+T cells was further investigated. The frequency of unique TRVB plus amino acid sequences was used to group cells into single (n=1), small (n=2-5), medium (6-20), large (n=21-100) and hyperexpanded (n=101-inf) subsets (Fig. 5f). Interestingly, cell subsets that displayed increased clonal expansion also scored highest for “Effector1’ phenotypes and “CD8-B" I “Terminally exhausted" gene signatures (Fig. 5c, f). In addition, the inventors observed a progressive increase in the level of “cytotoxicity’ for cells grouped with increased clonal expansion, where Large and Hyperexpanded subsets displayed the highest level of cytotoxicity (Fig. 5g).

[0433] Collectively, the inventors data showed that TRM-like CD103+CD8+T cells in MPE are diverse and contain clonally expanded, cytotoxic subsets with Effector function phenotype.

[0434] Sc-RNAseq reveals a subset of CD103+CD8+T cells with novel cell states that are unique to the pleural microenvironment.

[0435] To investigate the effects of the pleural microenvironment on CD103+CD8+T cells, the inventors used the MapQuery function in Seurat to project the inventors dataset onto a reference dataset of three previously published 10x Genomics datasets of CD8+T cell tumorinfiltrating lymphocytes (TILs) (Fig. 6a) (28-30). Unsurprisingly, the inventors observed a subset of M PE-derived CD103+CD8+T cells accurately map to the reference dataset and are labeled as “Canonical Trm”. Interestingly, most CD103+CD8+T cells did not align with the reference dataset from TILs. This suggests they have unique transcriptional characteristics that are specific to the pleural microenvironment. To delve deeper into these cells, which are “Unique to the pleura", a pseudotime analysis was conducted. The Monocle 3 algorithm (SI- 33) reveals a trajectory from “Canonical’ to “Unique to the pleura" cells, through an “Early pleura" cluster that had partial cell state overlap with the reference dataset, to “Late pleura" cells (Fig. 6a). To identify marker genes defining each cell state, differential gene expression analysis was performed on M PE-derived “Canonical”, “Early pleura" and “Late pleura” CD103+CD8+T cells (Fig. 6b). “Canonical” cells are defined by cytotoxic gene expression, IFNG, GZMB and GZMH. “Early pleura” cells are defined by the expression of LITAF, JUNB, and PI3KR1. “Late pleura" cells have upregulated expression of GPR183 and CXCR4 (Fig.6b), which may have been attracted to pleural fusion with high expression of CXCL12 (SDF-1) (34).

[0436] To further explore the different cell states, the inventors generated module scores using gene signatures derived from the literature and used them to score CD103+CD8+T cells. “Canonical” cells scored highest for metabolic gene signatures (FAO, Glycolysis, and Oxidative phosphorylation) and expression of immunoinhibitory molecules. The inventors observed a progressive reduction for these gene signatures through cells “Unique to pleura” from “Early pleura" to “Late pleura" cells, suggesting the pleural microenvironment progressively reduces the metabolic capacity of CD103+CD8+T cells (Fig. 6d). Interestingly, the opposite pattern was observed for “Sternness” and a predictive “good response to immune checkpoint blockade”, whereby “Late pleura" cells scored highest (Fig. 6c) (26). This may suggest that CD103+CD8+T cells draining into pleural effusion and becoming topographically isolated from the chronic antigen stimulation of the tumor microenvironment may have preserved T cell effector function, such as proliferation and survival; in turn, this may allow these cells to launch successive anti-tumoral immune responses in disease and during immunotherapy.

[0437] Taken together, these findings highlight the dichotomy of MPE. MPE is enriched for clonally expanded TRM-like cells that display highly cytotoxic, effector function gene sets, and cell states indicative of tumor-reactivity. However, the pleural microenvironment progressively modulates such cells into “Late pleura" cell states that are indicative of T cell anergy.

[0438] A subset of CD103+CD8+T cells “Unique to the pleura” stratify mesothelioma patients for enhanced overall survival (OS) probability.

[0439] To explore the cell state transition from “Canonical’ cells to those “unique to the pleura", the inventors used the MiloR package to investigate which clusters “Early pleura" cells predominantly reside (35). The inventors found “Early pleura" cells are significantly enriched in neighborhoods within cluster 6 (+FOS) and cluster 12 (+FTHT) (Fig. 7a, b) that score highly for memory and anti-tumor responsiveness (Fig. 6c). Furthermore, “Early pleura" cells are found to be absent from neighborhoods in cluster 4 (+HSPA1A) and cluster 2 t+GZMA') (Fig. 7a, b), which are predominantly composed of effector and highly exhausted cells (Fig. 6c).

[0440] As “Early pleura" cells are enriched in clusters scoring highly for memory and are defined by genes with reported CD8+T cell memory function (JUNB and PIK3R1), the inventors generated an “ Early pleura" gene signature of the top ten differentially expressed genes. When scoring the inventors’ dataset, this gene signature robustly stratified ‘Early pleura” cells from the other cell states, and the expression of its individual gene constituents was largely restricted to “Early pleura" cells (Fig. 7c).

[0441] Since the inventors identified an enrichment of M PE-derived CD39+CD103+CD8+T cells in the inventors flow cytometry data, and isolated tumor-reactive CD8+T cell clones from MPE caused by primary and metastatic disease, the inventors wanted to explore the clinical relevance of these cells on overall survival (OS) (20). The inventors used the “Early pleura" gene signature to stratify a cohort of mesothelioma patients from The Cancer Genome Atlas (TCGA) database (36, 37). The inventors found that the mesothelioma patients who scored high for a signature comprised of CD8A, CD3E and ITGAE plus the “Early pleura" signature exhibited better overall survival (OS) probability compared to patients who scored low ( = 0.019, Fig. 7d).

[0442] Similarly, the inventors generated “Canonical” and “Late pleura" gene signatures and used these to stratify the TGCA mesothelioma dataset. Interestingly, patients who scored high for both the “Canonical” and “Late pleura" gene signatures had reduced OS probability (Fig. 12a, b).

[0443] Collectively, the inventors reveal that “Early pleura" CD103+CD8+T cells in MPE display a memory phenotype with an intermediate metabolic profile and encompass a gene signature that acts as a cellular biomarker to stratify mesothelioma patients for enhanced OS probability.

[0444] Discussion

[0445] This is the first in-depth analysis of CD8+T cell populations in human malignant pleural effusion. The inventors comprehensively analyzed CD103+CD8+T cells in MPE at single-cell resolution caused by different cancer types, including primary mesothelioma, metastatic lung cancer, and metastatic breast cancer. The inventors identified novel TRM CD103+CD8+T cell states “unique to the pleura" that are independent of cancer of origin, and the inventors demonstrated that ‘Early pleura” T cell subsets are associated with improved clinical outcomes. The inventors revealed tissue-resident CD103+CD8+T cells in MPE contain a tumor-reactive compartment, which may be a potential resource for cellular immunotherapy, and used in pleural-specific and metastatic treatment options. The inventors data has allowed us to explore pan-cancer factors that contribute to anti-tumoral immunity in TRM T cells and M PE-associated mortality.

[0446] As ICB predominantly acts through TRM T cells, the inventors investigated whether CD103+CD8+T cell subsets that have previously been associated with improved clinical outcomes for primary disease also played a role in MPE (38-40). The inventors firstly performed immune phenotyping T cells in MPE using multi-parameter flow cytometry. The inventors identified TRM-like CD103+CD8+T cells, with predominant effector memory, are enriched in MPE, comprising an average of 10.60% of CD8+T cells. Although they expressed a higher percentage of inhibitory receptors when compared to PBMC, their expression of inhibitory receptors is lower than that found on cells from tissue biopsy, indicating they are less exhausted. Interestingly, in MPE, the inventors observed enriched CD39+CD103+CD8+T cells, a cell subset that has been reported to contain tumor-reactive T cells in primary tumors (20), suggesting that MPE is a valuable source of tumor-reactive T cells. This is further confirmed by the isolation of tumor-reactive T-cell clones in vitro. These tumor-reactive T cells are functional, capable of killing primary tumor cells, and can be expanded in vitro. All of these data suggest an important role of the CD103+CD8+T cell subset in MPE in cancer control and its potential use for cell therapy.

[0447] Single-cell RNA-Seq has been used in several studies to characterize the molecular description of immune cells in human MPE at the single-cell level and has provided a global overview of the phenotype of immune cells in MPE (13, 19). However, due to limited cell numbers of CD8+T cells in previous studies, the composition and functionality of CD8+T cells and the impact of the MPE microenvironment has not been fully explored. In the inventors’ study, the inventors enriched CD8+T cells and CD103+CD8+T cells by FACS and performed single-cell RNA sequencing. This enrichment enabled us to conduct a comprehensive analysis of this specific T cell population (TRM cells) in MPE and to assess the impact of the pleural microenvironment on tumor-reactive T cells. Consistent with flow-cytometry immune phenotype data, at the transcription level, CD103+CD8+T cells in MPE are diverse and contain clonally expanded, cytotoxic subsets with effector phenotypes. T cell receptor analysis indicated a range of CD103+CD8+T cell clonal expansion and expanded clonotypes contributed the greatest proportion of cells in the most cytotoxic clusters, which expressed “Effector function" phenotypes indicative of their tumor reactivity.

[0448] By comparing the inventors single-cell dataset on CD103+CD8+T cells from MPE to a reference dataset of three previously published 10x Genomics datasets of CD8+T cell tumorinfiltrating lymphocytes (TILs) from stage-2 non-small cell lung cancer (28-30), the inventors identified “Canonical TRM” cells and cells “Unique to the pleura" that have novel cell states. The inventors sub-stratified cells “Unique to the pleura" into two cell statuses, which the inventors have here labeled as “Early Pleura" and “Late Pleura” CD103+CD8+T cells. "Canonical TR " cells represent a subset of typical tissue-resident memory T cells characterized by cytotoxic gene expression, including IFNG, GZMB, and GZMH, and display broad clonal expansion. “Early Pleura” cells are defined by the expression of memory marker genes such as LITAF, JUNB and PI3KR1. “Late Pleura" cells upregulate expression of GPR183 and CXCR4. The ligand of CXCR4, SDF-1a, or CXCL12 has been reported to upregulate in pleural effusion from lung cancer patients (34) and in the tissues that were the preferred destination for tumor metastases in NSCLC and breast cancer (41-43). Given that CXCL12 is the only known ligand for CXCR4, it is likely that CXCR4-expressing CD103+CD8+ T cells, which display a "Late Pleura" phenotype have migrated from the tumor-bed to the pleura due to the high levels of CXCL12 in the pleural microenvironment (34). Furthermore, these CXCF?4-expressing T cells possess a migration advantage to tumor metastasis sites, enhancing their role in controlling tumor progression and underscoring their potential value in cell-based immunotherapy.

[0449] It has been shown that M2 macrophages and F0XP3+Tregs are enriched in malignant pleural effusion when compared to effusion without cancer, indicating an immunosuppressive microenvironment (13), which likely results in less exhausted T cells than seen within pleural biopsy. Evidence from studies on ascites (44) has shown that malignant fluid sites are less immunosuppressive when compared to the tumor microenvironment (TME), with fewer Tregs and PD1+CD8+T cells. Consistently, the inventors flow cytometry data showed that CD103+CD8+T cells in MPE express lower levels of inhibitory receptors such as PD-1 , Tim3 and NKG2a and are less exhausted compared to pleural biopsy. Another reason why T cells, in particular tumor-reactive T cells, are less exhausted in MPE is that T cells in the pleural cavity are topographically isolated from the tumor bed and, in turn, avoid constant antigen stimulation, unlike in the tumor. Hence, sequestration of tumor-reactive T cells in the pleura from the chronic antigen stimulation of the tumor microenvironment may better preserve tumor-reactive T cell effector function and sternness. This is further supported by ex vivo CD103+CD8+cells in the “Late Pleura” population scoring highest for the “good response” gene signature to ICB and in vitro isolation and expansion of high functional avidity tumor- reactive T cell clones from MPE.

[0450] The “Early pleura” gene signature significantly stratifies mesothelioma patients for enhanced OS probability. When compared to the “Canonical” and “Late pleura" gene signatures defined by effector molecules (IFNG and GZMB) and inflammatory responsive genes (GPR183), respectively, the “Early pleura" signature is enriched for memory markers (JUNB and PIK3R1). This suggests sustained memory recall is essential for anti-tumor immunity within MPE. Conversely, the diminished OS probability observed with the “Canonical” signature likely results from a lack of sustained memory, indicating highly cytotoxic short-lived effector cells are insufficient to eliminate tumors, but instead require continued replenishment by a proliferative memory pool for efficient anti-tumoral immunity. Furthermore, progressive modulation by the pleural microenvironment observed by the “Late pleura" gene signature may restrain CD8+T cell metabolism and function to induce an anergic cell state. This would likely reduce the pool of functional CD8+T cells available for anti-tumoral immunity in vivo. However, the inventors ex vivo discovery and in vitro characterization of proliferative and highly functional M PE-derived tumor-reactive CD8+T cells indicate these cells are recoverable and, therefore, a valuable source of tumor-reactive T cells for immune therapy. In conclusion, this study reveals that MPE contains a robust TRM CD8+T cell compartment with cell states that are “Unique to the pleura" and can be used as a biomarker for prognosis. M PE-derived CD8+T cells are perfectly poised to be harnessed in immune cell therapy, as they display a unique combination of tumor-reactivity and rested cell-state which may allow CD8 T cells from MPE to be isolated, expanded, and reinfused in anti-tumor cell therapy, particularly for patients with advanced mesothelioma and metastatic disease.

[0451] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0452] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0453] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0454] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

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[0482] 28. Liu B, Hu X, Feng K, Gao R, Xue Z, Zhang S, et al. Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer. Nat Cancer. 2022;3(1): 108-21.

[0483] 29. Gueguen P, Metoikidou C, Dupic T, Lawand M, Goudot C, Baulande S, et al. Contribution of resident and circulating precursors to tumor-infiltrating CD8+ T cell populations in lung cancer. Science Immunology. 2021;6(55):eabd5778.

[0484] 30. Caushi JX, Zhang J, Ji Z, Vaghasia A, Zhang B, Hsiue EH, et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1 -treated lung cancers. Nature.

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[0503] EXAMPLE 2

[0504] Malignant pleural effusion (MPE) is the pathological accumulation of fluid in the pleural space due to cancer. It affects approximately 15-20% of patients with malignancy and is a frequent complication in thoracic and metastatic cancers, and is associated with significant symptoms and reduced survival (1, 2). Standard care relies on therapeutic thoracentesis and, for recurrent effusions, indwelling pleural catheters that enable serial outpatient drainage. Single-session thoracentesis commonly removes up to about 1-1.5 litres to limit the risk of re-expansion pulmonary oedema. Larger volumes can be drained safely when pleural pressures are monitored. These procedures provide repeated access to large volumes of pleural fluid (3). MPE fluid is highly cellular and typically lymphocyte predominant. Malignant exudative effusions typically contain at least 1 xio6total nucleated cells per millilitre (1M / mL). Lymphocytes often comprise approximately 50-70% of leukocytes, with absolute CD4+and CD8+T-cell counts in the 104to 105cells per millilitre range in malignant effusions Hence, a 1 -litre drain can therefore contain billions of leukocytes, with a substantial fraction constituting T cells (1, 4).

[0505] This work uses these clinical and biological principles to reveal M PE-derived CD8+T cells are a source of tumour-reactive T cells that can be used in autologous cell therapy. We first characterised malignant pleural effusion by ex vivo multiparameter flow cytometry to define the composition and checkpoint landscape of CD8+T cells, and then utilised single-cell RNA sequencing to profile the transcriptional landscape. Guided by these data, we prospectively enriched antigen-experienced, tissue-resident-like CD103+CD8+CD3+T cells using the surface phenotype PD-1+TIM-3+. We reveal the population of interest is present at readily isolatable frequencies across donors within a lymphocyte-rich input, and provides a sufficient starting material for downstream work. We evaluated tumour reactivity using two complementary approaches: bulk-sorted PD-1+TIM-3+CD103+CD8+CD3+T cells and singlecell sorted CD103+CD8+CD3+T-cell clones. In both instances the readouts are performed against patient- matched primary tumour cell lines and autologous CD103+CD8+CD3+T cells were isolated using flow cytometry and expanded. Functional assays against autologous primary cancer cell lines measured antigen-dependent activation by CD137+up-regulation by flow cytometry, and direct cytotoxicity at effector-to-target ratios of 1 :1 and 10:1 by Incucyte in real time. The workflow revealed the effector population is suitable for translational utilisation, since the input is abundant, the selection is phenotype-guided, and we observed sufficient expansion and phenotype maintenance. Together, these features position MPE as a scalable source for generating tumour-reactive CD8+T-cell products to be used in autologous reinfusion cell therapy.

[0506] MPE compartment is a rich, preferential source of CD103+, antigen- experienced CD8+T cells

[0507] Across our cohort (MPE n=56; PBMC n=21), MPE contained a significantly higher proportion of Tissue Resident memory (TRM) CD103+CD8+T cells compared with blood (P<0.0001). The pleural compartment is anatomically proximate to the pleural tumour bed yet topographically separated from densely antigenic tumour nests, thereby enabling a reduced level of T-cell receptor engagement, and reduced metabolic stress. Within this niche we observe an enrichment of TRM CD103+CD8+T cells with an effector-memory (TEM) (CCR7“CD45RA“) phenotype, a profile consistent with tissue residency and rapid recall capacity. Immune checkpoint receptor profiling revealed that individual expression of TIM-3 and PD-1 , alongside TIM-3+PD-1+ co-expression was enriched on CD103+versus CD103“CD8+TEM cells (all P<0.0001). These data indicate MPE contains a compartment of antigen experienced TRM CD8+ T cells that may have retained capacity to respond to tumour rather than terminal dysfunction. Taken together, these features suggest that MPE harbours tumour-proximal, checkpoint-high, TRM CD103+CD8+T cells that have been partially insulated from relentless antigenic drive, thereby preserving cytotoxic fitness for manufacture. Practically, thoracentesis yields high cell numbers with patient-matched specificity, enabling robust immunophenotyping and providing a scalable starting material for autologous T-cell therapy development.

[0508] CD103+T EM cells are checkpoint-high (PD-1, TIM-3), nominating a tumour-proximal phenotype and a sort-able clinical product

[0509] To define the molecular identity and therapeutic potential of CD8+T cells within MPE, we undertook 10x Genomics single-cell RNA / TCR seguencing with parallel surface-protein (ADT) profiling. This approach enabled us to map the transcriptional and phenotypic landscape of thousands of MPE-derived CD8+T cells at single-cell resolution, providing insight into their functional state and relevance for cell-therapy manufacture. Our flow cytometry data indicated the TEM CD103+CD8+compartment exhibited markedly higher freguencies of TIM-3+and PD-1+expression, and greater PD-1+TIM-3+co-expression than paired CD103- counterparts (all P < 0.0001). This demonstrates a clear phenotypic separation by surface markers compatible with clinical-grade isolation. The checkpoint-high signature is indicative of T-cell receptor engagement at the tumour-pleural interface, suggesting enrichment for tumour-reactive cells within the MPE compartment.

[0510] Unsupervised clustering of single-cell RNA-seguencing data from MPE-derived CD8+T cells (n = 3 donors) resolved nine discrete clusters (Figure 13 a). Cells expressing surface CD103 were not evenly distributed but instead predominantly localised to cluster 5, representing over 68 per cent of the population (Figure 13 b-d). Differential gene-expression analysis revealed that CD103+cells were defined by the residency-associated transcription factor ZNF683 (HOBIT), chemokine receptors linked to tissue retention (CXCR6, ITGA1), and cytotoxic effector genes (GZMB, PRFT) (Figure 2e). Concordant with the flow-cytometry findings above, checkpoint molecules PDCD1 (PD-1) and HAVCR2 (TIM-3) were coenriched within the CD103+population, and UMAP feature overlays demonstrated spatial co-localisation of PD-1 and TIM-3 transcripts and proteins (Figure 13 f,g). Further differential-expression analysis confirmed coordinated up-regulation of tissue- residency and effector modules (ZNF683, ITGAE, ITGA1, GZMB, IFNG) alongside checkpoint regulators (PDCD1, HAVCR2, LAG3) in CD103+CD8+T cells (Figure 13 e,h). These features indicate that CD103+cells are antigen-experienced and functionally capable, positioned topographically close to the tumour yet sequestered within the effusion space and shielded from chronic antigenic overstimulation that drives exhaustion in the tumour core. Paired surface-protein (ADT) data corroborated these transcriptional findings, showing PD-1 and TIM-3 enrichment within the CD103+domain of the UMAP (Figure 13 c) together with other residency-associated proteins (CD103, CD49a, PD-1 , CD39, CD69) (Figure 13 h). Expression of HAVCR2 RNA and TIM-3 protein closely mirrored one another, demonstrating strong RNA-protein concordance.

[0511] This unified molecular and phenotypic signature defines a checkpoint-high, residency- competent CD103+subset that is clonally expanded (Figure 13 i) and can be isolated using only surface markers, offering a practical and reproducible route for therapeutic manufacturing. A prominent TRM signature centred on cluster 5 therefore establishes MPE as a rich source of tumour-proximal, antigen-experienced CD8+T cells that are both biologically and operationally suited for development as an autologous cell-therapy product.

[0512] To further characterise this compartment, differential-expression analysis focused specifically on the TRM cluster 5 revealed a clonally expanded cluster with an identical pattern of up-regulated surface proteins (CD103, CD49a, PD-1 , CD39, CD69), alongside a set of transcripts consistent with high functional capacity (ZNF683, CD74, GZMA, GZMB, GZMH) (Figure 13 j,k). This reinforces cluster 5 as the transcriptional and phenotypic core of the TRM population, comprising clonally expanded, checkpoint-high effector cells (PD- 1+TIM-3+CD103+CD8+) that represent a biologically privileged and therapeutically exploitable resource within the MPE.

[0513] MPE-derived PD-1+TIM-3+CD103+CD8+T cells show robust, dose- dependent cytotoxicity against autologous tumour

[0514] Having defined a CD103+TRM-enriched cluster that concentrates PD-1 and TIM-3 expression and exhibits clonal expansion, we next sought to determine whether this phenotype marks functional tumour reactivity. To this end, we prospectively isolated PD- 1+TIM-3+CD103+CD8+T cells from MPE and evaluated both bulk-sorted lines and single- cell-derived clones against autologous primary cancer cell lines, with unrelated cancer cell lines used as specificity controls. Real-time killing, cytokine production, and activation- induced CD137 (4-1 BB) up-regulation were used as orthogonal readouts of antigen-specific function, together with assessment of ex vivo expandability to support manufacturability.

[0515] Ex vivo MPE was bulk sorted for CD103+CD8+T cells up-regulating TIM-3 with or without PD-1 (Figure 14a) and cells were expanded in vitro using feeder cells recording substantial (median:>100) fold expansions (Figure 14b) revealing their proliferative capacity. After expansion, bulk MPE- derived PD-1+TIM-3+CD103+CD8+T cells were co-culture with live autologous primary cancer cell which elicited a marked up-regulation of CD137, whereas negative controls showed minimal activation (Figure 14c). This rapid and antigen-dependent response confirms functional engagement of the prospectively enriched population. Realtime Incucyte imaging demonstrated potent, E:T ratio-dependent tumour-cell killing, with progressive near-infrared (NIR+) target-cell death over 24-72 hours, while target-only wells remained intact (Figure 14d,e).

[0516] To support the finding that MPE contains a population of tumour-reactive CD8+ T cells that upregulate CD137 in response to autologous cancer cell lines, we profiled the total CD103+CD8+T cell compartment in MPE at single cell resolution for tumour-reactive T cells. Ex vivo MPE cultures were expanded through two rounds of stimulation using irradiated autologous primary cancer cells as targets to enrich for tumour-reactive T cells within the total CD103+CD8+pool. Activation-induced marker (AIM) assays were subsequently performed using live autologous tumour targets, followed by surface selection on CD103+CD8+CD3+T cells. AIM+events were defined by up-regulation of CD137 (4-1 BB) with or without CD25 following short-term co-culture (24h, 48, 72h), permitting isolation of viable, cytokine-independent responders by flow sorting. Single CD103+CD8+T cells were subsequently flow-sorted and expanded clonally using feeder cells, generating stable longterm T-cell lines. Functional validation of single- cell-derived CD103+clones demonstrated robust, antigen-specific responses upon re-challenge with autologous primary cancer cell lines. Representative clones produced IFN-y and TNF-a by intracellular cytokine staining, with a sizeable fraction of double-positive cells confirming polyfunctional activation at the single-clone level (Figure 14 f). Collectively, these data show that MPE contains a durable compartment of tumour-reactive CD103+CD8+T-cell clones with substantial proliferative capacity, capable of generating effector pools exceeding 106cells. Furthermore, single-cell- derived CD103+CD8+clones retained specificity and effector competence throughout expansion, producing IFN-y and TNF-a and efficiently eliminating autologous primary cancer cell lines in Incucyte killing assays, while sparing unrelated cancer cell lines (Figure 14 g-h). This antigen-dependent response confirms that MPE-derived CD103+CD8+T cells include a clonally expanded, polyfunctional, tumour-reactive subset that can be prospectively isolated using PD-1 , TIM-3, and CD103 as surface identifiers. Across the wider cohort, PD-1+TIM- 3+CD103+CD8+T cells were consistently detectable in ex vivo MPE, with variable but quantifiable frequencies between patients (Figure 14 i). Notably, all donors from whom we successfully established tumour-reactive bulk lines or single-cell clones possessed a discernible population of these checkpoint-high TRM-like cells. This association suggests that the abundance of PD-1+TIM-3+CD103+CD8+T cells may serve as a practical biomarker for identifying individuals most likely to generate potent, tumour-specific products. In translational terms, simple pre- manufacture immunophenotyping of MPE could therefore inform patient selection, streamline process predictability, and maximise the likelihood of therapeutic success. The presence of this population provides both a biological rationale and a measurable criterion for prioritising MPE- derived material as a high-value starting point for autologous T-cell therapy development. Together, these findings demonstrate that MPE harbours a reservoir of tumour-reactive TRM-like CD8+T cells with high cytotoxic potential, antigen specificity, and scalable expansion characteristics, thereby fulfilling key biological and practical criteria for an autologous T-cell therapy product.

[0517] Discussion

[0518] MPE provides a practical, enriched source of CD103+, checkpoint-high, antigen-experienced CD8+T cells that are prospectively isolatable, GMP-compatible, and functionally potent and specific against autologous tumour. These cells have expansion capacity and maintain their functional capacity. These attributes justify advancing MPE-derived CD103+CD8+T cells into process development and early clinical testing with built-in potency and safety analytics.

[0519] References

[0520] 1. Gonnelli F, Hassan W, Bonifazi M, Pinelli V, Bedawi EG, Porcel JM, et al. Malignant pleural effusion: current understanding and therapeutic approach. Respiratory Research. 2024;25(1):47.

[0521] 2. Kulandaisamy PC, Kulandaisamy S, Kramer D, McGrath C. Malignant Pleural Effusions-A Review of Current Guidelines and Practices. J Clin Med. 2021 ; 10(23).

[0522] 3. Kasmani R, Irani F, Okoli K, Mahajan V. Re-expansion pulmonary edema following thoracentesis. Cmaj. 2010;182(18):2000-2.

[0523] 4. Awadallah SF, Bowling MR, Sharma N, Mohan A. Malignant pleural effusion and cancer of unknown primary site: a review of literature. Ann Transl Med. 2019;7(15):353.

Claims

Claims1. A method of expanding tumour tissue-resident T cell memory (T rm) like CD103+CD8+ T cells expressing at least:GPR183 and CXCR4 (late unique to pleura cells); and / orLITAF, JUNB and PI3KR1 (early unique to pleura cells); the method comprising: a) enriching CD8+cytotoxic T cells from malignant pleural effusion (MPE); b) co-culturing the CD8+ cytotoxic T cell enriched MPE with irradiated feeder cells obtained from a subject for a first time period under conditions to allow the T cells of the MPE to expand, wherein co-culturing comprises supplementing with one or more cytokines; c) washing the expanded T cells and stimulating the expanded T cells with irradiated cancer cells from the subject; d) co-culturing the expanded T cells and irradiated cancer cells for a second time period under conditions to allow the expanded T cells to further expand; e) co-culturing the further expanded T cells with live cancer cells obtained from the subject; f) sorting the co-cultured further expanded T cells based on a level of expression of : i. one or more tumour reactive markers comprising CD103, CD8, and one or more of 4-1BB, CD25, and / or CD154; ii. memory markers; and iii. exhaustion markers; and g) determining which of the sorted T cells express or have increased expression of GPR183 and CXCR4 and / or LITAF, JUNB and PI3KR1.

2. The method of claim 1, wherein the method further comprises step (h) isolating the T cells expressing GPR183 and CXCR4 and / or LITAF, JUNB and PI3KRT, and / or step (i) maintaining and / or further expanding the T cells expressing GPR183 and CXCR4 and / or cells expressing LITAF, JUNB and PI3KR1.

3. The method of claim 1 or 2, wherein: a) the first time period is 14 days;b) the second time period is at least 14 days; c) in steps (a), (c) and (d) of claim 1 , comprise co-culturing the MPE, expanded T cells and / or further expanded T cells at a 1:1 EffectorTarget cell ratio; d) step (a) of claim 1 comprises supplementing with one or more of IL-2, IL7 and / or IL15 every three days from starting co-culturing, optionally wherein the IL-2 is at a concentration of 100U / ml; e) memory markers comprise one or more of CCR7, CD45RA and / or CD45RO; and / or f) exhaustion markers comprise one or more of PD1, Tim3 and / or NKG2a.

4. The method of any of claims 1 to 3, wherein steps (e) and (f) of claim 1 comprise activation-induced marker cell sorting.

5. The method of any of claims 1 to 4, wherein determining comprises bulk or single cell RNA sequencing analysis.

6. A population of cells comprising an in vitro expanded population of Trm like CD103+CD8+ T cells expressing at least:GPR183 and CXCR4 (late unique to pleura cells); and / orLITAF, JUNB and PI3KR1 (early unique to pleura cells); produced by the method according to any one of claims 1 to 5.

7. A population of cells, comprising an in vitro expanded population of Trm like CD103+CD8+ T cells expressing at least:GPR183 and CXCR4 (late unique to pleura cells); and / orLITAF, JUNB and PI3KR1 (early unique to pleura cells).

8. A pharmaceutical composition comprising a population of cells according to claim 6 or 7.

9. A population of cells according to claims 6 or 7 or a pharmaceutical composition according to claim 8 for use as a medicament.

10. A population of cells according to claims 6 or 7 or a pharmaceutical composition according to claim 8, for use in preventing or treating cancer.

11. A method of preventing or treating cancer in a subject in need thereof, the method comprising administering an effective amount of a population of cells according to claims 6 or 7 or a pharmaceutical composition according to claim 8 to the subject.

12. A method of preventing or treating a disease or condition in a subject in need thereof, the method comprising administering an effective amount of a population of cells according to claims 6 or 7 or a pharmaceutical composition according to claim 8 to the subject.

13. The population of cells according to claims 6 or 7, a pharmaceutical composition according to claim 8, population of cells or pharmaceutical composition for use according to claim 9 or 10, or the method according to claim 11 or 12, wherein the expanded late unique to pleura cells and / or early unique to pleura cells are de- anergised (energised).

14. The population of cells according to any of claims 6, 7, or 13, a pharmaceutical composition according to claim 8 or 13, the population of cells or pharmaceutical composition for use according to any of claims 9, 10, or 13 or the method according to any of claims 11, 12, or 13 wherein: a) the expanded late unique to pleura cells and / or early unique to pleura cells have increased expression of genes indicative of memory and / or a proliferative phenotype in comparison to reference T cells; optionally wherein genes indicative of memory and / or a proliferative phenotype comprises expression of one or more of TCF7, STAT4, PIK3R1 , and IL7R; and / or b) the expanded late unique to pleura cells: i. express or have increased expression of one or more of LMNA, FAM107B, TUBA1A, YPEL5, DDX3X, DENND4 and / or DDX21 in comparison to early unique to pleura cells and / or canonical Trm cells; and / or ii. express or have increased expression of LMNA, GPR183, FAM107B, CXCR4, TUBA1A, YPEL5, DDX3X, DENND4A, and DDX21 in comparison to early unique to pleura cells and / or canonical Trm cells; and / or iii. have reduced expression of SOCS3, DUSP2, PD-1, NKG2A, and / or Tim-3 in comparison to early unique to pleura cells and / or canonical Trm cells; and / or iv. have a relatively high score for a gene signature comprising the genes LMNA, GPR183, FAM107B, CXCR4, TUBA1A, CLDND1, YPEL5, DDX3X, DENND4A, and DDX21 in comparison to early unique to pleura cells and / or canonical Trm cells; and / orc. the expanded early unique to pleura cells: v. express or have increased expression of one or more of ZFP36L2, EEF1A1, PABPC1, CLDND1, DUSP2, SOCS3 and / or FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells; and / or vi. express or have increased expression of ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells; and / or vii. have reduced expression of RGCC, FAM107B, TUBA1A, PD-1 , NKG2A, and / or Tim-3 in comparison to late unique to pleura cells and / or canonical Trm cells; and / or viii. have a relatively high score for a gene signature comprising the genes ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells.

15. The population of cells according to any of claims 6, 7,13, and 14, a pharmaceutical composition according to claim 8 or 13, population of cells or pharmaceutical composition for use according to any of claims 9, 10, 13 or 14 or the method according to any of claims 11 to 14, wherein: a. the late unique to pleura cells have an expression profile indicative of a good response to immune checkpoint inhibitor blockade in comparison to a reference T cell; and / or b. the early unique to pleura cells have an expression profile indicative of a good response to immune checkpoint inhibitor blockade in comparison to a reference T cell.

16. The population of cells according to claim 14 or 15, a pharmaceutical composition according to claim 14 or 15, population of cells or pharmaceutical composition for use according to claim 15 or the method according to claim 14 or 15, wherein the reference T cell is a canonical T cell obtained from the subjects MPE.

17. The population of cells according to any of claims 6 or 7 and 13 to 16, a pharmaceutical composition according to any of claims 8 and 13 to 16, population of cells or pharmaceutical composition for use according to any of claims 9, 10, and 13 to 16 or the method according to any of claims 11 to 16, wherein the population ofcells comprises at least 10% 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% de- anergised late unique to pleura cells and / or early unique to pleura cells.

18. The population of cells or pharmaceutical composition for use according to any of claims 9, 10, and 13 to 17 or the method according to any of claims 11 to 17, wherein the method comprises administering one or more checkpoint inhibitors.

19. A method of predicting a subject’s response to one or more immune checkpoint inhibitors, the method comprising: providing a sample of the subject’s MPE; and determining the presence of Trm like CD103+CD8+ T cells expressing at least:GPR183 and CXCR4 (late unique to pleura cells); and / orLITAF, JLINB and PI3KR1 (early unique to pleura cells) in the sample; and wherein the presence of late unique to pleura cells and / or early unique to pleura cells is indicative of a positive response to the one or more immune checkpoint inhibitors.

20. The method of claim 19, wherein the method further comprises: generating a diagnostic report based on the presence or absence of the late unique to pleura cell and / or early unique to pleura cells, optionally wherein the diagnostic report is provided to a medical professional for providing guidance on selection of a cancer treatment to be administered.

21. The method of claim 19 or 20, wherein the method further comprises administering to the subject one or more immune checkpoint inhibitors.

22. A method of treating cancer in a subject in need thereof, comprising administering an effective amount of one or more immune checkpoint inhibitors to the subject, wherein:Trm like CD103+CD8+ T cells expressing at least:GPR183 and CXCR4 (late unique to pleura cells); and / orLITAF, JLINB and PI3KR1 (early unique to pleura cells); have been detected in a sample of the subject’s MPE.

23. One or more immune checkpoint inhibitors for use in a method of treating cancer in a subject in need thereof, comprising administering an effective amount of the one or more immune checkpoint inhibitors to the subject, wherein:Trm like CD103+CD8+ T cells expressing at least:GPR183 and CXCR4 (late unique to pleura cells); and / orLITAF, JLINB and PI3KR1 (early unique to pleura cells); have been detected in a sample of the subject’s MPE.

24. The method of claim 22, or the one or more immune checkpoint inhibitors for use according to claim 23, wherein the late unique to pleura cells and / or early unique to pleura cells are according to any of claims 13 to 15; optionally wherein the method comprises administering an effective amount of a population of cells according to any of claims 6, 7 and 13 to 17, or a pharmaceutical composition according to any of claims 8 and 13 to 17 to the subject.

25. A method of predicting a subject’s overall survival (OS), wherein the subject suffers from a cancer, the method comprising: providing a sample of the subject’s MPE; and determining the presence of Trm like CD103+CD8+ T cells expressing at least:LITAF, JUNB and PI3KR1 (early unique to pleura cells) in the sample; determining a level of expression of CD8A, CD3E and ITGAE of the early unique to pleura cells; wherein the presence of early unique to pleura cells expressing or having increased expression of CD8A, CD3E and ITGAE in comparison to a reference T cell is indicative of an improved OS in comparison to a subject lacking early unique to pleura cells and / or early unique to pleura cells without increased or decreased expression of CD8A, CD3E and ITGAE in comparison to the reference T cell.

26. The method of claim 25, wherein early unique to pleura cells: / . express or have increased expression of one or more of ZFP36L2, EEF1A1, PABPC1, CLDND1, DUSP2, SOCS3 and / or FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells; and / or / / . express or have increased expression of ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3 and FXYD5 in comparison to late unique to pleura cells and / or canonical Trm cells; and / or iii. have reduced expression of RGCC, FAM107B, TUBA1A, PD-1 , NKG2A, and / or Tim-3 in comparison to late unique to pleura cells and / or canonical Trm cells; and / orhave a relatively high score for a gene signature comprising the genes ZFP36L2, JUNB, EEF1A1, LITAF, PABPC1, PIK3R1, CLDND1, DUSP2, SOCS3, FXYD5, CD8A, CD3E and ITGAE in comparison to late unique to pleura cells and / or canonical Trm cells; optionally, wherein the method further comprises: generating a diagnostic report based on the presence or absence of the late unique to pleura cell and / or early unique to pleura cells, optionally wherein the diagnostic report is provided to a medical professional for providing guidance on selection of a cancer treatment to be administered.

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