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CELL THERAPY METHODS

20220306989 · 2022-09-29

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention is in the field of cell therapy and provides compositions and methods for treating cancer and/or viral infections in patients. The invention provides lymphocytes comprising a synthetic polynucleotide encoding at least one iron regulatory protein and, optionally, a chimeric antigen receptor. The invention further provides methods for producing these lymphocytes and administering them to patients.

    Claims

    1. A lymphocyte comprising a synthetic polynucleotide encoding at least one iron regulatory protein (IRP), wherein the at least one iron regulatory protein is IRP1 (SEQ ID NO: 1) and/or IRP2 (SEQ ID NO: 2-6).

    2. The lymphocyte according to claim 1, wherein the synthetic polynucleotide encodes IRP2 as set forth in SEQ ID NO:2.

    3. The lymphocyte according to claim 1, wherein the lymphocyte is a T cell or a natural killer (NK) cell.

    4. The lymphocyte according to claim 3, wherein the lymphocyte is a tumor infiltrating lymphocyte, a modified T cell or a virus specific T cell.

    5. The lymphocyte according to claim 1, wherein the at least one iron regulatory protein is constitutively expressed.

    6. The lymphocyte according to claim 1, wherein the synthetic polynucleotide encoding the at least one iron regulatory protein is under control of a constitutive promoter.

    7. The lymphocyte according to claim 6, wherein the constitutive promoter is an EF-1α promoter.

    8. The lymphocyte according to claim 1, wherein the lymphocyte further comprises a chimeric antigen receptor (CAR).

    9. The lymphocyte according to claim 8, wherein the CAR comprises an antigen binding domain, a transmembrane domain, a co-stimulatory signaling region and a signaling domain.

    10. The lymphocyte according to claim 9, wherein the antigen binding domain is an antibody or an antigen-binding fragment thereof.

    11. The lymphocyte according to claim 10, wherein the antigen-binding fragment is a Fab or an scFv.

    12. The lymphocyte according to claim 9, wherein the antigen binding domain specifically binds a tumor antigen or a viral antigen.

    13. The lymphocyte according to claim 12, wherein the tumor antigen is present on the surface of cells of a target cell population or tissue.

    14. The lymphocyte according to claim 8, wherein the CAR is encoded by a polynucleotide, wherein the polynucleotide encoding the CAR is transcriptionally linked to the synthetic polynucleotide encoding IRP1 and/or IRP2.

    15. The lymphocyte according to claim 14, wherein the polynucleotide encoding the CAR and the synthetic polynucleotide encoding IRP1 and/or IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

    16. The lymphocyte according to claim 15, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

    17. The lymphocyte according to claim 15, wherein the self-cleaving peptide is T2A.

    18. A viral vector comprising at least one polynucleotide encoding IRP1 (SEQ ID NO: 1) and/or IRP2 (SEQ ID NO: 2-6).

    19. The viral vector according to claim 18, wherein the viral vector comprises a polynucleotide encoding IRP2 as set forth in SEQ ID NO:2.

    20. The viral vector according to claim 18, wherein the viral vector is derived from a lentivirus, an adeno-associated virus (AAV), an adenovirus, a herpes simplex virus, a retrovirus, an alphavirus, a flavivirus, a rhabdovirus, a measles virus, a Newcastle disease virus or a poxvirus.

    21. (canceled)

    22. The viral vector according to claim 18, wherein the at least one polynucleotide encoding IRP1 and/or IRP2 is under control of a constitutive promoter.

    23. The viral vector according to claim 22, wherein the constitutive promoter is an EF-1α promoter.

    24. The viral vector according to claim 18, wherein the viral vector comprises a further polynucleotide encoding a CAR.

    25. The viral vector according to claim 24, wherein the polynucleotide encoding the CAR is transcriptionally linked to the polynucleotide encoding IRP1 and/or IRP2.

    26. The viral vector according to claim 25, wherein the polynucleotide encoding the CAR and the polynucleotide encoding IRP1 and/or TRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

    27. The viral vector according to claim 26, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

    28. The viral vector according to claim 26, wherein the self-cleaving peptide is T2A.

    29. A pharmaceutical composition comprising the lymphocyte according to claim 1 and a pharmaceutically acceptable carrier.

    30-35. (canceled)

    36. A method for treating a subject having cancer or for preventing and/or treating a viral infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the lymphocyte according to claim 1.

    37. The method according to claim 36, wherein the cancer is a hematologic cancer or a solid tumor, in particular wherein the hematologic cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia or multiple myeloma and wherein the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma or sarcoma.

    38. The method according to claim 36, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenoviruses, polyomaviruses, influenza virus or human herpesvirus, in particular wherein the human herpesvirus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), Varizella-Zoster virus (VZV) or human herpesvirus 8 (HHV8).

    39. A method for producing the lymphocyte according to claim 1, the method comprising the steps of: a) providing a lymphocyte obtained from a subject; b) introducing a synthetic polynucleotide encoding at least one iron regulatory protein into the lymphocyte of step (a), wherein the iron regulatory protein is IRP1 (SEQ ID NO:1) and/or IRP2 (SEQ ID NO:2-6); and c) expressing the at least one iron regulatory protein encoded by the synthetic polynucleotide that has been introduced into the lymphocyte in step (b).

    40. The method according to claim 39, wherein a second synthetic polynucleotide encoding a chimeric antigen receptor (CAR) is introduced into the lymphocyte in step (b).

    41. The method according to claim 40, wherein the synthetic polynucleotide encoding the CAR is: (a) combined with the synthetic polynucleotide encoding IRP1 and/or IRP2 or (b) transcriptionally linked to the synthetic polynucleotide encoding IRP1 and/or IRP2.

    42. (canceled)

    43. The method according to claim 40, wherein the synthetic polynucleotide encoding the CAR and the polynucleotide encoding IRP1 and/or IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

    44. The method according to claim 43, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

    45. The method according to claim 43, wherein the self-cleaving peptide is T2A.

    46. The method according to claim 39, wherein the one or more synthetic polynucleotide is introduced into the lymphocyte by viral transduction.

    47. (canceled)

    48. The method according to claim 39, wherein the lymphocyte is activated before or after the one or more synthetic polynucleotide is introduced into the lymphocyte.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0261] FIG. 1: Naive and cytokine-enhanced NK cells similarly rely on glycolysis for IFN-γ production

    [0262] (A) Schematic of the experiment used to generate CE NK cells. (B) IFN-γ production by NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=18 donors). (C) GMFI of CD69 expression on NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=13 donors). (D) PCA of the transcriptome data, depicting the group relationships in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. The proportion of component variance is indicated as percentage (n=5 donors). (E) Heatmap of relative expression of mRNA encoding for glycolysis genes from NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 of the transcriptome data (n=5 donors). (F) Upper panel: Representative mitochondrial perturbation assay of NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Glycolysis (extracellular acidification rate—ECAR) was measured “in Seahorse” after injection of oligomycin, FCCP, and rotenone. Lower panel: Basal and maximal rate of ECAR in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 analyzed by mitochondrial perturbation assay (mean±SEM, n=12 donors). (G) Upper panel: Representative histogram of NBDG uptake in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Lower panel: GMFI of NBDG uptake in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=15 donors). (H) Expression of IFNG mRNA in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+2-DG. Transcript levels were determined relative to 18S mRNA levels and normalized to unstimulated (no stim) NV NK cells (mean±SEM, n=6 donors). (I) Upper panel: IFN-γ production by NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+2-DG (mean±SEM, n=6 donors). Lower panel. IFN-γ production by NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 in 10 mM glucose and in 2 mM glucose (mean±SEM, n=5 donors). Statistical significance was assessed by paired two-tailed Student's t-test (C, F, H, I) or linear-regression analysis (B, G, H, 1). *p<0.05, **p<0.01, *** p<0.001, ns, not significant.

    [0263] FIG. 2: Activated CE NK cells are characterized by high levels of cell-surface CD71 and rapid cell proliferation

    [0264] (A) Upper panel: Representative histogram of CD98 expression on NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Lower panel: MFI of CD98 expression on NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=8 donors). (B) Upper panel: Representative histogram of CD71 expression on NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Lower panel. GMFI and percentage of CD71 expression on NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=14 donors). (C) Left panel: Representative Western blot of total CD71 expression in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Right panel: Total CD71 expression normalized to actin in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean & SEM, n=13 donors). (D) Left panel. GMFI of CD71 expression on NV and CE NK cells unstimulated (no stim) or stimulated with K562 (mean±SEM, n=6). Right panel: Percentage of CD71+NK cells on NV and CE NK cells unstimulated (no stim) or stimulated with K562 (mean±SEM, n=6 donors). (E) Upper panel: Representative histogram of Tf-488 uptake in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Lower panel: GMFI of Tf-488 uptake in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=10 donors). (F) Upper panel: Schematic of the experiment used to analyze CFSE dilution in NV and CE NK cells. Middle panel: Representative histogram of CFSE dilution in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Lower panel: Percentage of proliferated NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 analyzed by CFSE dilution (mean±SEM, n=13 donors). (G) Heatmap of relative expression of mRNA encoding for cell cycle genes (GO:0006098) in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (n=5 donors). (H) Percentage of proliferated NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+BIP (1, 10 and 50 μM) analyzed by CFSE dilution (mean±SEM, n=11 donors for unstimulated, IL-12/IL-18 and IL-12/IL-18+BIP 10 μM stimulation, n=8 donors for IL-12/IL-18+BIP 1 μM stimulation, n=3 donors IL-12/IL-18+BIP 50 μM stimulation). (I) GMFI of CD69 expression on NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+BIP 100 μM (mean±SEM, n=5 donors). (J) Upper panel: Heatmap of relative expression of mRNA encoding for PPP genes (GO:0006098) in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (n=5 donors). Lower panel: Percentage of proliferated cells in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+6AN 50 μM analyzed by CFSE dilution (mean f SEM, n=6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (F, H, I, J) or linear-regression analysis (A, B, C, E). *p<0.05, **p<0.01, *** p<0.001, ns, not significant.

    [0265] FIG. 3: CD71-mediated iron uptake and dietary iron availability impact NK cell function (A) Upper panel: Schematic of the experiment used to analyze CFSE dilution in WT and TfrcY20H/Y20H NK cells from spleen. Lower left panel: Representative histogram of CFSE dilution in WT and TfrcY20H/Y20H NK1.1+NK cells from spleen with IL-12/IL-18 stimulation. Lower right panel: Percentage of proliferated WT and TfrcY20H/Y20H NK1.1+NK cells from spleen in IL-15 LD or stimulated with IL-12/IL-18 analyzed by CFSE dilution (mean±SEM, n=5 for WT NK cells, n=6 for TfrcY20H/Y20H NK cells). (B) Upper panel: Schematic of MCMV infection experiment of mice fed +/− iron feed for 6 weeks. Lower panel: Serum levels of iron, ferritin, UIBC, TIBC; and hematocrit from mice fed +/− iron feed for 6 weeks (mean±SEM, n=8-18 for iron, ferritin, UIBC and TIBC and n=3 for hematocrit). (C) Left panel. Percentage of NK1.1+NK cells in spleen of mice fed +/− iron feed for 6 weeks (mean±SEM, n=5). Right panel: Percentage of CD8+, CD4+, CD19+ cells in spleen of mice fed +/− iron feed for 6 weeks (mean±SEM, n=5). (D) Left panel: Percentage of CD27+CD 11b−, CD27+CD 11b+, CD27-CD11b+ on NK1.1+NK cells in spleen of mice fed +/− iron feed for 6 weeks (mean±SEM, n=5). Right panel: Percentage of KLRG1+ and CD62L+ on NK1.1+NK cells in spleen of mice fed +/− iron feed for 6 weeks (mean±SEM, n=5). (E) Left panel: Viral titer in liver and spleen of WT MCMV-infected mice fed +/− iron feed for 6 weeks 3 dpi (each dot represents data from cells isolated from one mouse, data displayed as fold change difference normalized to mice fed + iron feed, horizontal line indicates median, n=10). Right panel: Viral titer in liver and spleen of Δm157 MCMV infected-mice fed an +/− iron feed for 6 weeks 3 dpi. (each dot represents data from cells isolated from one mouse, data displayed as fold change difference normalized to mice fed + iron feed, horizontal line indicates median, n=9-10). (F) Percentage of IFN-Y+ in NK1.1+NK cells in liver and spleen of WT MCMV-infected mice fed +/−feed for 6 weeks 1.5 dpi (mean f SEM, n=4-5). Statistical significance was assessed by unpaired two-tailed Student's t-test (A, B, C, D, E, F). *p<0.05, **p<0.01, *** p<0.001, **** p<0.0001, ns, not significant.

    [0266] FIG. 4: CD71 supports NK cell proliferation and optimal effector function during viral infection

    [0267] (A) Left panel: Percentage and absolute numbers of NK1.1+NK cells in liver of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=10). Right panel: Percentage and absolute numbers of NK1.1+NK cells in spleen of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=17-22). (B) Upper left panel: Percentage and absolute numbers of CD8+ cells in liver of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=10). Upper right panel: Percentage and absolute numbers of CD4+ cells in liver of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=4). Lower panel: Percentage and absolute numbers of CD19+ cells in liver of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=10). (C) Upper left panel: Percentage and absolute numbers of CD8+ cells in spleen of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=9-19). Upper right panel: Percentage and absolute numbers of CD4+ cells in spleen of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=11-22). Lower panel: Percentage and absolute numbers of CD19+ cells in spleen of Tfrcf/f and Tfrcfl/flNcr1Cre mice (mean±SEM, n=12-22). (D) Upper panel: Percentage of CD27+CD11b−, CD27+CD11b+, CD27-CD11b+ on NK1.1+ NK cells in liver of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=10). Lower panel. Percentage of CD62L+ and Ly6C+ on NK1.1+NK cells in liver of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=6). (E) Upper panel: Percentage of CD27+CD11b−, CD27+CD11b+, CD27-CD11b+ on NK1.1+NK cells in spleen of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=5). Lower panel: Percentage of KLRG1+, CD62L+ and Ly6C+ on NK1.1+NK cells in spleen of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=5). (F) Percentage of Ly49H+ on NK1.1+NK cells in liver and spleen of Tfrcfl/fl and Tfrcfl/flNcr1Cre mice (mean±SEM, n=5-6). (G) Upper left panel: Schematic of adoptive transfer experiment into Klra8−/− recipients to track expansion of WT and Tfrcfl/fl NK cells upon MCMV infection. Upper right panel: Representative flow plot gated on adoptively transferred CD45.1+ and CD45.2+(Ly49H+NK1.1+) NK cells in liver of WT MCMV-infected recipients 7 dpi. Lower panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) and Tfrcfl/flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in liver, spleen, lung and blood of WT MCMV-infected recipients 7 and 30 dpi (each dot represents data from cells isolated from one mouse, bars indicate t SEM, two independent experiments, 1st n=5 for 7 dpi and n=3-5 for 30 dpi, 2nd n=4 for 7 dpi and n=2 for 30 dpi). (H) Left panel: Schematic of adoptive transfer experiment into Rag2−/−IL2rg−/− recipients to track expansion of WT and Tfrcfl/fl NK cells. Right panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) and Tfrcfl/flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in liver, spleen, lung and blood 6 dpt (each dot represents data from cells isolated from one mouse, bars indicate±SEM, n=3-4). (1) Upper left panel: Schematic of adoptive transfer experiment into Klra8−/− recipients to analyze CFSE dilution in WT and Tfrcfl/fl NK cells upon WT MCMV infection. Upper right panel: Representative histogram of CFSE dilution in adoptively transferred WT and Tfrcfl/flNcr1Cre NK1.1+NK cells in liver of WT MCMV-infected recipients 3.5 dpi. Lower panel: GMFI of CFSE of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) and Tfrcfl/flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in liver and spleen of WT MCMV-infected recipients 3.5 dpi (mean±SEM, two independent experiments, 1st n=5, 2nd n=4). (J) Percentage of proliferated Tfrcfl/fl and Tfrcfl/flNcr1Cre NK1.1+NK cells from spleen in IL-15 LD or stimulated with IL-12/IL-18 analyzed by CFSE dilution (mean±SEM, n=3-4). (K) Upper panel: Schematic of MCMV infection experiment of Tfrc/fl and Tfrcfl/flNcr1cre mice. Middle panel: Percentage and absolute number of NK1.1+NK cells in liver of WT MCMV-infected Tfrcfl/fl and Tfrcfl/flNcr1cre mice 3.5 and 5.5 dpi (mean±SEM, n=4-6). Lower panel: Percentage and absolute number of NK1.1+NK cells in spleen of WT MCMV-infected Tfrcfl/fl and Tfrc/flNcr1cre mice 3.5 and 5.5 dpi (mean±SEM, n=3-6). (L) Viral titer in liver and spleen of WT MCMV-infected Tfrcfl/fl and Tfrcfl/flNcr1cre mice 3.5 dpi (each dot represents data from cells isolated from one mouse, horizontal line indicates median, n=5). (M) Percentage of IFN-γ+ in NK 1.1+NK cells in liver and spleen of WT MCMV-infected Tfrcfl/fl and Tfrcfl/flNcr1cre mice 1.5 dpi (mean±SEM, n=4). (N) Left panel: Percentage of CD27+CD11b−, CD27+CD11b+, CD27-CD11b+ on NK1.1+NK cells in spleen of WT MCMV-infected Tfrcfl/fl and Tfrcfl/flNcr1Cre mice 5.5 dpi (mean±SEM, n=3-5). Right panel: Percentage of KLRG1+ on NK1.1+NK cells in spleen of WT MCMV-infected Tfrcfl/fl and Tfrcfl/flNcr1Cre mice 5.5 dpi (mean±SEM, n=3-5). Statistical significance was assessed by unpaired two-tailed Student's t-test (A, B, C, D, E, F, I, J, K, L, M, N). *p<0.05, **p<0.01, *** p<0.001, **** p<0.0001, ns, not significant.

    [0268] FIG. 5: Glycolysis is required for induction of CD71 in activated NK cells (A) Upper panel: Representative Western blot of total CD71 expression in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+ActD (1 and 10 μM) and IL-12/IL-18+CHX (10 and 100 μg/ml). Lower left panel: Total CD71 expression normalized to actin in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+ActD 10 μM (mean±SEM, n=3 donors). Lower right panel: Total CD71 expression normalized to actin in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+CHX 100 μg/ml (mean±SEM, n=2 donors). (B) Expression of TFRC mRNA in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+2-DG. Transcript levels were determined relative to 18S mRNA levels and normalized to unstimulated (no stim) NV NK cells (mean±SEM, n=6 donors). (C) Upper left panel: GMFI of CD71 expression on NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+2-DG (mean±SEM, n=6 donors). Upper right panel: Representative Western blot of total CD71 expression in NV and CE NK cells unstimulated (no stim) or stimulated with TL-12/IL-18, TL-12/IL-18+2-DG. Lower panel: Total CD71 expression normalized to actin in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+2DG (mean±SEM, n=5 donors). (D) GMFI of CD71 expression on NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 in 10 mM glucose and in 2 mM glucose (mean±SEM, n=5 donors). (E) GMFI of Tf-488 uptake in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18, IL-12/IL-18+2-DG (mean±SEM, n=5 donors). (F) Upper panel: Representative Western blot of total c-Myc expression in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Lower panel: Total c-Myc expression normalized to actin in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, B, C, D, E, F) or linear-regression analysis (B, C, E). *p<0.05, **p<0.01, *** p<0.001, ns, not significant.

    [0269] FIG. 6: Cytokine priming induces the IRP/IRE regulatory system

    [0270] (A) Upper panel: Expression of ACO1 and IREB2 mRNA in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 of transcriptome data (n=5 donors). Middle panel: Representative Western blot of total IRP1 and IRP2 expression in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Lower panel. Total IRP1 and IRP2 expression normalized to actin in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=7 donors for IRP1, n=6 donors for IRP2). (B) Heatmap of relative expression of mRNAs encoding for genes harboring IREs in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (n=5 donors). (C) Upper left panel. Expression of EIF4E mRNA in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 of transcriptome data (n=5 donors). Upper right panel: Representative Western blot of total eIF4E expression in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Lower panel: Total eIF4E expression normalized to actin in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean t SEM, n=6 donors). (D) Upper panel: Representative histogram of HPG incorporation in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Lower panel: GMFI of HPG incorporation in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=5 donors). (E) Left panel: Representative Western blot of total ferritin heavy chain 1 expression in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18. Right panel: Total ferritin heavy chain 1 expression normalized to actin in NV and CE NK cells unstimulated (no stim) or stimulated with IL-12/IL-18 (mean±SEM, n=4 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, C, E) or linear-regression analysis (D). *p<0.05, **p<0.01, ns, not significant.

    [0271] FIG. 7: The IRP/ARE regulatory system orchestrates CD71 expression in NK cells (A) Expression of TFRC mRNA in NV and CE NK cells no stim vs. IL-12/IL-18, data derived from transcriptome data (n=5). (B) Expression of FTH1 mRNA in NV and CE NK cells no stim vs. IL-12/IL-18 of transcriptome data (n=5). (C) Representative Western blot of total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA. Total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA (n=7). (D) Representative Western blot of total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA. Total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA (n=6). (E) Left panel: Representative histogram of CD71 expression on NK92 cells transfected with control, ACO1 or IREB2 siRNA. Right panel: GMFI of CD71 expression on NK92 cells transfected with control, ACO1 or IREB2 siRNA (n=4-5). (F) Left panel: Representative Western blot of total FTH1 expression in NK92 cells transfected with control, Aco1 and IREB2 siRNA. Right panel: Total FTH1 expression in NK92 cells transfected with control, Aco1 and IREB2 siRNA (n=5). (G) Left panel: Representative Western blot of total IRP1 expression in NKL cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in NKL cells transfected with control or Aco1 siRNA (n=6). (H) Left panel: Representative Western blot of total IRP2 expression in NKL cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in NKL cells transfected with control or IREB2 siRNA (n=7). (1) Left panel: Representative histogram of CD71 expression on NKL cells transfected with control, ACO1 or IREB2 siRNA. Right panel: GMFI of CD71 expression on NKL cells transfected with control, ACO1 or IREB2 siRNA (n=5). (J) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and Aco1 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and Aco1 siRNA (n=6). (K) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and IREB2 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and IREB2 siRNA (n=5). All averaged data are presented as mean±s.e.m. and were analyzed using an unpaired two-tailed Student's t-test (a,b,d,e) or ANOWA (c). Asterisks indicate significance between groups. *p<0.05, **p <0.01, ns, not significant. (L) Left panel: Representative Western blot of total IRP2 expression in NK92 cells transfected with control or IREB2 sgRNA. Right panel: Total IRP2 expression in NK92 cells transfected with control or IREB2 sgRNA (n=3). (M) Left panel: GMFI of CD71 expression on NK92 cells transfected with control or IREB2 sgRNA (n=5). Right panel: Number of NK92 cells transfected with control or IREB2 sgRNA (n=4). All averaged data are presented as mean t s.e.m. and were analyzed using a two-tailed Student's t-test (A-B), unpaired two-tailed Student's t-test (C, D, G, H, J, K, L, M) or ANOWA (E, F, I). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ***p<0.001, ns, not significant.

    [0272] FIG. 8: Enforced IRP expression is a molecular module also supporting T cell proliferation. (A) Left panel: Representative Western blot of total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA (n=5). (B) Left panel: Representative Western blot of total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA (n=4). (C) Left panel: Representative histogram of CD71 expression on Jurkat cells transfected with control, ACO1 or IREB2 siRNA. Right panel: GMFI of CD71 expression on Jurkat cells transfected with control, ACO1 or IREB2 siRNA (n=7). (D) Left panel: Representative Western blot of total FTH1 expression in Jurkat cells transfected with control, Aco1 or IREB2 siRNA. Right panel: Total FTH1 expression in Jurkat cells transfected with control, Aco1 and IREB2 siRNA (n=5). (E) Representative Western blot of total IRP2 expression in IRP2 knockout (ko) Jurkat cells transduced with control vector coding for mCherry (LV-mCherry) or for IREB2 (LV-IREB2). (F) Upper panel: Representative histogram of CD71 expression on IRP2 ko Jurkat cells transduced with LV-mCherry or LV-IREB2. Lower left panel: GMFI of CD71 expression on IRP2 ko Jurkat cells transduced with LV-mCherry vs. LV-IREB2 (n=4). Lower right panel: Number of IRP2 ko Jurkat cells transduced with LV-mCherry vs. LV-IREB2 (n=3). (G) Left panel: Representative histogram of CD71 expression on primary CD4.sup.+ T cells transduced with LV-mCherry vs. LV-IREB2. Right panel: GMFI of CD71 expression on primary CD4.sup.+ T cells transduced with LV-mCherry vs. LV-IREB2 (n=2). (H) Left panel: Representative histogram of CD71 expression on primary CD8.sup.+ T cells transduced with LV-mCherry vs. LV-IREB2. Right panel: GMFI of CD71 expression on primary CD8.sup.+ T cells transduced with LV-mCherry vs. IREB2 (LV-IREB2) (n=2). (I) Representative Western blot of total IRP2 expression in untransduced CD4.sup.+ T cells (UTD), PSMA-specific CAR CD4 T cells (CAR) and PSMA-specific CAR CD4.sup.+ T cells co-expressing IRP2 (CAR-IREB2). (J) Upper panel: Representative histogram of CD71 expression on unstimulated UTD, CAR and CAR-IREB2 transduced CD4.sup.+ T cells. MFI of CD71 expression on unstimulated UTD, CAR and CAR-IREB2 transduced CD4.sup.+ T cells (n=3). Lower panel: Representative histogram of CD71 expression on Fab-stimulated UTD, CAR and CAR-IREB2 transduced CD4.sup.+ T cells. MFI of CD71 expression on Fab-stimulated UTD, CAR and CAR-IREB2 transduced CD4′ T cells (n=3). (K) Upper panel: Percentage of unstimulated UTD, CAR and CAR-IREB2 transduced CD4.sup.+ T cells having entered 0, 1 and 2 cycles of cell proliferation (n=3). Lower panel: Percentage of Fab-stimulated UTD, CAR and CAR-IREB2 transduced CD4.sup.+ T cells having entered 0, 1 and 2 cycles of cell proliferation (n=3). All averaged data are presented as mean±s.e.m. and were analyzed using an unpaired two-tailed Student's t-test (a,b,f) or ANOWA (c,d). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant.

    EXAMPLES

    Example 1: Naive and Cytokine-Enhanced NK Cells Similarly Rely on Glycolysis for IFN-γ Production

    [0273] Enhanced recall responses of cytokine-enhanced (CE) NK cells reflect a promising feature for immune cell therapy against cancer. If and how CE NK cell metabolism underpins cytokine production, target cell clearance and proliferation remains unknown. To elucidate these key features of CE NK cells, the inventors used an established in vitro CE NK cell model that allowed comparison of naive (NV) vs. CE NK cells. Briefly, the inventors primed freshly isolated human NK cells with IL-12 and IL-18 (IL-12/IL-18) for 16 h, followed by a rest period in low dose IL-15 (IL-15 LD) to support survival. After 7 days of rest, features of NV vs. CE NK cells upon stimulation were compared (FIG. 1A). In line with previous data, priming of NK cells with IL-12/IL-18 augmented their capacity to produce IFN-γ upon re-stimulation (FIG. 1B). Of note, NK cells were similarly activated upon stimulation, as indicated by CD69 expression (FIG. 1C). To explore how cellular metabolism relates to the function of NV vs. CE NK cells at the transcriptional level, RNA sequencing (RNA-seq) was performed using unstimulated and cytokine-stimulated cells. Both unstimulated as well as activated NV and CE NK cells clustered separately in the principal component analysis (PCA). Activation was, however, a much stronger overall discriminating factor, indicating a relative similarity between the transcriptomes of NV and CE NK cells (FIG. 1D).

    [0274] Rapid upregulation of aerobic glycolysis is a metabolic hallmark of activated lymphocytes, including NK cells. Unexpectedly, both NV and CE NK cells similarly upregulated gene transcripts encoding for glycolytic enzymes upon stimulation, with the exception of HK2 which was higher in CE than in NV NK cells (FIG. 1E). In line with the transcriptome data, metabolic flux assays showed increased basal and maximal glycolytic rates of activated compared to unstimulated cells, yet no difference between NV and CE NK cells was observed (FIG. 1F).

    [0275] Likewise, uptake of the glucose analogue 2-NBDG was not different between unstimulated and activated NV and CE NK cells (FIG. 1G). To assess whether increased glycolytic metabolism was linked to the capacity of NV and CE NK cells to produce IFN-γ, the inventors stimulated NV and CE NK cells with IL-12/IL-18 in the presence of the hexokinase inhibitor 2-deoxy-d-glucose (2-DG). Inhibition of glycolysis during cytokine stimulation similarly reduced IFNG mRNA abundance and IFN-γ secretion in NV and CE NK cells (FIGS. 1H and 1I, left panel). Likewise, culturing NK cells in low glucose reduced production of IFN-γ in both subsets (FIG. 1I, upper panel). Together these data identified a similar increase in basal and maximal glycolytic activity upon activation of NV and CE NK cells, which was in both subsets required for efficient production of the key inflammatory cytokine IFN-γ.

    Example 2: Activated CE NK Cells are Characterized by High Levels of Cell-Surface CD71 and Rapid Cell Proliferation

    [0276] To further characterize the metabolic profile of NV vs. CE NK cells, the inventors analyzed surface expression of the nutrient transporters CD98 and CD71 reported to be upregulated on activated NK cells. Upon stimulation, a slight and comparable increase in CD98 expression on both NK cell subsets was observed (FIG. 2A). In contrast, upregulation of the transferrin receptor CD71 was much greater on CE vs. NV NK cells, both when expressed as GMFI and percentage of positive cells (FIG. 2B). Increased cell surface expression of CD71 was reflected by an overall greater cellular abundance of CD71 protein as assessed by immunoblot analysis of whole cell lysates (FIG. 2C). To test whether differential cell surface expression of CD71 could also be driven by NK cell stimulation via activating receptors, both subsets were stimulated with HLA-deficient target cells (K562 cell line). Similar to cytokine stimulation, upregulation of CD71 was more prominent on K562-exposed CE than NV NK cells (FIG. 2D). To assess the functional capacity of increased CD71 expression, the inventors used fluorescently labeled transferrin to monitor transferrin uptake in NV and CE NK cells. These experiments revealed increased transferrin uptake in activated CE as compared to NV NK cells (FIG. 2E).

    [0277] Expression of CD71 and rates of proliferation have previously been linked in neoplastic cells. To test whether this association also applied to NK cells, proliferation of NV and CE NK cells was monitored using CFSE dilution assays. Under both steady-state conditions and upon stimulation, CE NK cells proliferated to a greater extent than their NV counterparts (FIG. 2F). In line with differential proliferation rates, stimulated CE NK cells clustered when testing abundance of transcripts encoding for cell-cycle progression genes (FIG. 2G). To elucidate if increased transferrin uptake was linked to increased cell proliferation, the inventors used the intracellular iron chelator 2,2′-bipyridyl (BIP). These experiments revealed that BIP inhibited NK cell proliferation in a dose-dependent manner in both NV and CE NK cells (FIG. 2H). Of note, BIP had minimal effects on cell viability (data not shown), and no effect on NK cell activation as assessed by CD69 expression (FIG. 2I).

    [0278] The pentose phosphate pathway (PPP), providing ribose 5-phosphate and NADPH for nucleotide synthesis and reducing equivalents, respectively, supports cell proliferation. In line with the increased proliferation observed in CE NK cells, cytokine stimulation increased mRNA abundance of several PPP-related genes more prominently in CE than NV NK cells (FIG. 2J, upper panel). The PPP inhibitor 6-aminonicotinamide (6AN) prevented expansion of cytokine-stimulated NK cells, further supporting the relevance of the PPP in promoting proliferation of both NV and CE NK cells (FIG. 2J, lower panel). Together, these experiments identified (i) preferential upregulation of CD71 on activated CE vs. NV NK cells, and (ii) increased proliferation of activated CE over NV NK cells, which relied on PPP activity.

    Example 3: CD71-Mediated Iron Uptake and Dietary Iron Availability Impact NK Cell Function

    [0279] Recently, a mutation in the TFRC gene (TFRCY20H/Y20H) has been shown to impair B and T cell function, causing a primary immunodeficiency (PID). The mutation affects receptor-mediated endocytosis and compromises CD71-mediated iron uptake both in human cells and when introduced into mice. NK cell numbers in patients harboring this mutation are normal, however, functional properties have not been previously assessed. To test whether CD71 function and NK cell proliferation are linked, the inventors assessed CFSE dilution in IL-1S LD and IL-12/IL-18-stimulated wild type (WT) and TfrcY20H/Y20H murine NK cells, ex vivo. These experiments revealed a striking lack of IL-15 LD and IL-12/IL-18-induced proliferation among NK cells harboring the Tfrc mutation (FIG. 3A).

    [0280] Given this strong phenotype, the inventors wondered whether mild iron deficiency might be sufficient to cause NK cell dysfunction. To explore this notion, the inventors first established systemic iron deficiency in a mouse model (FIG. 3B, upper panel). As expected, mice maintained on an iron-deficient diet for 6 weeks displayed reduced iron, ferritin and hematocrit levels in peripheral blood, while the unsaturated iron-binding capacity (UIBC) and the total iron-binding capacity (TIBC) increased as compared to mice kept on control diet (FIG. 3B, lower panel). While splenic T and B cell numbers were normal in mice with iron deficiency, NK cell numbers tended to be lower, possibly indicating selective sensitivity of these cells to systemic iron abundance (FIG. 3C). No impact of iron deficiency on the NK cell maturation phenotype was observed (FIG. 3D). However, upon MCMV infection, splenic NK cell-mediated viral control and IFN-γ production by NK cells tended to be reduced in mice maintained on iron-deficient diet, indicating impaired NK cell function (FIG. 3E. left panel. and 8F). Noteworthy, replication of Am157 MCMV evading NK cell-mediated control was unaffected by reduced iron levels (FIG. 3E, right panel). Together, these data established that CD71-mediated iron uptake had an important role in regulating NK cell proliferation. Further, reduced systemic iron levels significantly impaired immune control of MCMV infection in vivo, possibly by reducing NK-cell function. It remains to be elucidated, whether impaired NK cell-mediated immunity resulted from NK cell intrinsic or extrinsic factors.

    Example 4: CD71 Supports NK Cell Proliferation and Optimal Effector Function During Viral Infection

    [0281] In order to test the functional importance of CD71-mediated iron uptake for NK cells, the inventors generated mice specifically lacking CD71 in NK cells, by crossing Ncr1Cre mice with Tfrcfl/fl mice (Tfrcfl/flNcr1Cre). Under homeostatic conditions, percentage and absolute numbers of NK cells in Tfrcfl/flNcr1Cre mice were slightly reduced in both liver and spleen as compared to Tfrcfl/f littermate controls (FIG. 4A). Percentage and absolute numbers of CD8+ and CD4+ T cells, as well as CD19+ B cells, were unaffected by NK cell specific deletion of CD71 (FIGS. 4B and 4C). Further, expression of terminal NK cell maturation markers was comparable between Tfrcfl/flNcr1Cre and Tfrcfl/fl mice (CD27, CD11b, KLRG1, CD62L and Ly6C) (FIGS. 4D and 4E). Likewise, the NK cell activating receptor, Ly49H, which is important for controlling MCMV infection, was equally expressed on Tfrcfl/flNcr1Cre and Tfrcfl/fl NK cells (FIG. 4F).

    [0282] NK cell activation by MCMV drives proliferation of Ly49H+NK cells. To examine whether deletion of CD71 affects antigen-specific NK cell expansion in vivo, the inventors co-transferred congenic Ly49H+WT and Tfrcfl/flNcr1Cre NK cells into Ly49H-deficient (Klra8−/−) recipients (FIG. 4G, upper panel). The inventors then infected recipient mice with MCMV and tracked expansion of transferred NK cells. WT NK cells robustly expanded in liver, spleen, lung and blood, constituting 80-90% of the Ly49H+NK cell pool at 7 and 30 days post-infection (dpi) when compared to Tfrcfl/flNcr1Cre NK cells (FIG. 4G, lower panel). The inventors next addressed whether expansion of NK cells in lymphopenic hosts, which is driven by the availability of common-g-chain-dependent cytokines, was also dependent on CD71. To this end, the inventors transferred WT and Tfrcfl/flNcr1Cre NK cells at equal ratios into Rag2−/−IL2rg−/− recipient mice (FIG. 411, left panel). Similar to the infection experiment, at 6 dpi frequencies of Tfrcfl/flNcr1Cre NK cells were much lower than those of WT cells (FIG. 4H, right panel). Reduced numbers of Tfrcfl/flNcr1Cre NK cells in both adoptive transfer experiments could have resulted from either a lack of expansion, increased cell death or a combination of both. To assess how deletion of CD71 in NK cells related to their proliferation in vivo, WT and 7frcfl/flNcr1Cre NK cells were labeled with CFSE and transferred into recipient mice at equal ratios (FIG. 4I, upper panel). Recipients were then infected with MCMV and donor cells harvested at 3.5 dpi. CFSE dilution, and hence proliferation, of adoptively transferred Tfrcfl/flNcr1Cre NK cells in liver and spleen was significantly lower than that of WT cells (FIG. 4I, lower panel). These findings were further confirmed in in vitro proliferation studies, in which IL-15 LD and IL-12/IL-18-stimulated Tfrcfl/flNcr1Cre NK cells showed reduced proliferation compared to control cells (FIG. 4J).

    [0283] To address whether deletion of CD71 affects NK cell-mediated viral control, the inventors challenged Tfrcfl/fl and Tfrcfl/flNcr1Cre mice with MCMV (FIG. 4K, upper panel). In line with the competitive transfer assays described above, a significant reduction in both percentage and absolute numbers of NK cells at 3.5 and 5.5 dpi was observed in both liver and spleen of Tfrcfl/flNcr1Cre mice (FIG. 4K, middle and lower panel). Insufficient expansion of NK cells was associated with higher splenic viral titers among Tfrcfl/flNcr1Cre mice at 3.5 dpi, with a similar trend observed in the liver (FIG. 4L). In addition, IFN-γ production of splenic and liver infiltrating Tfrcfl/flNcr1Cre NK cells was reduced upon MCMV infection (FIG. 4M). Of note, despite poor expansion and reduced effector capacity, CD71 deficiency did not impair terminal maturation of MCMV-challenged CD71 deficient NK cells, as indicated by CD27, CD11b and KLRG1 expression (FIG. 4N). Altogether, these data indicated a critical role of CD71 in NK cell proliferation both during infection and in a lymphopenic environment.

    Example 5: Glycolysis is Required for Induction of CD71 in Activated NK Cells

    [0284] Our experiments established (i) iron uptake via CD71 as a critical metabolic checkpoint controlling NK cell proliferation; and (ii) highly preferential upregulation of CD71 on activated CE vs. NV NK cells. These findings prompted the inventors to ask how CD71 per se was regulated in NV and CE NK cells. To address this question, the inventors first assessed whether induction of CD71 relied on NK cell transcriptional activity. As in previous experiments, CD71 was induced to a greater extent in cytokine-stimulated CE than NV NK cells (FIG. 5A). In both subsets inhibition of transcription (using Actinomcyin D) entirely prevented stimulation-induced upregulation of CD71, as did blocking of translation (using cycloheximide) (FIG. 5A). Thus, transcription and translation were similarly required in both cell subsets. Glycolytic reprogramming has previously been demonstrated to drive transcription in activated NK cells (72). As glycolysis was similarly triggered in activated NV and CE NK cells (FIG. 1F), the inventors examined the possibility that glycolytic metabolism may differentially impact TFRC (which encodes CD71) transcription between NV and CE NK cells. TFRC mRNA abundance was indeed higher in activated CE than NV NK cells, yet similarly reduced when inhibiting glycolysis with 2-DG (FIG. 5B). Cell surface expression of CD71 and total CD71 levels followed the same pattern when exposing cells to 2-DG (FIG. 5C). Dependence on glucose to induce CD71 expression was recapitulated upon activation of NK cells in low glucose medium and translated into reduced transferrin uptake in 2-DG treated NK cells (FIGS. 5D and 5E). Glycolysis thus enabled transcription and translation of CD71. However, no evidence was found that glycolysis regulated the differential abundance of CD71 in activated CE vs. NV NK cells.

    [0285] c-Myc has been established as a key regulator of TFRC transcription in various immune cells. Given the increased abundance of TFRC mRNA among activated CE over NV NK cells (FIG. 5B), preferential c-Myc induction in CE NK cells could explain differential regulation of CD71 between activated CE and NV NK cells. Yet, c-Myc was robustly but equally induced in both NK cell subsets (FIG. 5F). Together these data established a symmetric need in activated NV and CE NK cells for (i) continuous transcription and translation to support expression of CD71 and (ii) glycolytic reprogramming as a metabolic requirement for CD71 expression.

    Example 6: Cytokine Priming Induces the IRP/IRE Regulatory System

    [0286] Many genes involved in cellular iron homeostasis contain iron responsive elements (IREs) in the 5′ or 3′UTR of their mRNA. Iron regulatory proteins 1 and 2 (IRP1 and IRP2) bind IREs, thereby controlling mRNA stability and translation. TFRC mRNA contains 5 IREs in the 3′UTR; binding of IRPs stabilizes the mRNA and facilitates translation. As described, this would occur under iron-deficient conditions. Hence, the inventors hypothesized that increased abundance of IRPs, selectively in CE NK cells, could be a possible mechanism regulating enhanced CD71 expression in activated CE NK cells. At the mRNA level, abundance of both IRP transcripts, ACO1 and IREB2, was similar in NV and CE NK cells (FIG. 6A, upper panel). Protein abundance of IRP1 and IRP2 was, however, higher in quiescent and activated CE NK cells (FIG. 6A, lower panel). This finding was compatible with a novel role for IRPs, generating a pseudo iron deficient state in a cell subset-specific manner, thereby post-transcriptionally controlling abundance of a distinct set of proteins.

    [0287] To extend this observation, the inventors analyzed transcript abundance of known IRE containing mRNAs expressed in NK cells (FIG. 6B). The inventors included the critical eukaryotic translation initiation factor 4E (eIF4E) in the list of IRE containing mRNAs, since searching for ironresponsive elements (SIRE) algorithm revealed an IRE-like motif in the 3′UTR of the EIF4E mRNA (data not shown). This analysis prompted the inventors to assess the transcriptional and translational pattern for EIF4E. The pattern observed for CD71 was somewhat recapitulated, activated CE NK cells expressed more EIF4E transcript and clearly more eIF4E protein (FIG. 6C). Yet, already quiescent CE NK cells expressed increased levels of eIF4E compared to NV NK cells. Of note, despite higher abundance of eIF4E, global protein translation was not discernibly different between activated NV and CE NK cells, as assessed by using L-homopropargylglycine (HPG) incorporation assays (FIG. 6D). In addition, the inventors noted increased Fr1l mRNA abundance in activated CE NK cells (FIG. 6B). FTH1 mRNA contains an IRE in the 5′UTR and binding of IRPs to 5′UTRs IREs inhibits translation. This constellation enabled the inventors to test the hypothesis of a pseudo iron deficiency driven by selective increase in IRPs abundance in CE NK cells. Indeed, despite higher transcript levels, protein abundance of ferritin heavy chain 1 (the gene product of FTH1) was, if anything, lower in both unstimulated and activated CE NK cells (FIG. 6E). This finding was highly suggestive of IRPs, with their CE and NV NK cell specific abundance, being involved in regulating FTH1 mRNA translation. Together these data established a regulatory axis, selectively induced in CE NK cells, in which pseudo iron deficiency enables increased translation of CD71—and hence proliferation—of activated CE NK cells.

    Example 7: Expression of IRPs in CAR T Cells

    CAR T Cell Generation

    [0288] Sequences of the antigen binding domain, the transmembrane domain, the CD3ζ domain, and the CD28 costimulatory domain of the CAR and IRP1 and/or IRP2 are cloned into a corresponding lentiviral vector. If necessary, the IRPs and the CAR sequences are cloned into separate lentiviral packaging vectors. Lentiviral production is performed in a suitable cell line. CD8+ and/or CD4+ T cells are isolated from peripheral blood mononuclear cells from the patient or a healthy donor and activated with anti-CD3 and anti-CD28 (soluble or bead-bound) in the presence of IL-2. One to two days post-activation, soluble antibodies or beads are removed and cells are transduced with the lentivirus for approximately 18 hours and media is replaced with fresh media supplemented with IL-2. CAR and IRP expression will be confirmed by flow cytometry at indicated time points.

    Optional:

    [0289] CD8+ and/or CD4+ T cells are isolated from peripheral blood mononuclear cells from the patient or a healthy donor and activated with anti-CD3 and anti-CD28 (soluble or bead-bound) in the presence of IL-2. One day post-activation cells are transduced with the lentivirus for approximately 48 hours and media is replaced with fresh media supplemented with IL-2. Five days post activation beads are removed and replaced with media containing IL-15 and IL-7. CAR and IRP expression will be confirmed by flow cytometry at indicated time points.

    Proliferation of CAR T Cells In Vitro

    [0290] To analyze cell proliferation of CAR T cells, cells are loaded prior to activation with the cell-proliferation dye carboxyfluorescein succinimidyl ester (CFSE, 1 sM, Molecular probes, USA) and seeded in 96-well plates. A fixable live-dead cell stain (Fixable Viability Dye, eBioscience or Zombie Aqua, Biolegend) is used to exclude dead cells prior to sample acquisition. CFSE dilution is analyzed at various time points post-stimulation by flow cytometry.

    Proliferation of CAR T Cells In Vivo

    [0291] To analyze cell proliferation of CAR T cells in vivo, CAR T cells overexpressing at least one IRP and CAR T cells not overexpressing any IRP are adoptively transferred into a corresponding murine tumor model and the frequency and number of transferred cells is analyzed at various time points. In certain experiments, CAR T cells overexpressing at least one IRP and CAR T cells not overexpressing any IRP are loaded with cell proliferation dye CFSE prior to transfer to analyze the proliferation in vivo.

    Mouse Tumor Models

    [0292] CAR T cells overexpressing at least one IRP and CAR T cells not overexpressing any IRP are adoptively transferred into a corresponding murine tumor model. Depending on the tumor model, in certain experiments the tumor diameter is measured. Depending on the tumor model, in certain experiments lungs are dissected and fixed in the corresponding buffer and numbers of nodules are counted using a microscope. Depending on the tumor model, in certain experiments the survival rate is analyzed.

    Example 8: Materials and Methods

    Mice

    [0293] Animal experiments performed at the University of Rijeka, Faculty of Medicine, were approved by Ethical Committee of the Faculty of Medicine, University of Rijeka, and Ethical Committee at the Croatian Ministry of Agriculture, Veterinary and Food Safety Directorate (UP/1-322-01/18-01/44). Mice were strictly age- and sex-matched within experiments and were held in SPF conditions. Animal handling was in accordance with the guidelines contained in the International Guiding Principles for Biomedical Research Involving Animals.

    [0294] Wild-type C57BL/6J (B6, strain 000664), B6 Ly5.1 (strain 002014), Tfrcfl/fl (strain 028363) and Rag2−/−yc−/− (strain 014593) mice were purchased from the Jackson laboratory. Ncr1Cre mice were kindly provided by V. Sexl (Vienna, Austria) and B6.Ly49h−/− were kindly provided by Silvia M. Vidal (Montreal, Canada). In some experiments mice were put on iron-deficient diet and corresponding control diet for 6 weeks (C1038 and C1000, Altromin).

    [0295] Animal experiments at the University of Basel were performed in accordance with local rules for the care and use of laboratory animals. Mice were strictly age- and sex-matched within experiments and were held in SPF conditions. Wild-type C57BL/6J (B6, strain 000664) mice were purchased from Jackson Laboratories (USA) and TfrcY20H/Y20H mice were kindly provided by R. Geha (Boston, USA).

    Hematologic Analyses

    [0296] The serum iron, ferritin, unsaturated iron binding capacity (UIBC) and total iron binding capacity (TIBC) were determined using AU5800 Analyzer (Beckman Coulter). Hematocrit was determined using hematology analyzer DxH500 (Beckman Coulter). Measurements were conducted at the Clinical Institute of Laboratory Diagnostics (Clinical Hospital Center, Rijeka, Croatia).

    Viruses

    [0297] The bacterial artificial chromosome-derived murine cytomegalovirus (BAC-MCMV) strain pSM3fr-MCK-2fl clones 3.3 has previously been shown to be biologically equivalent to MCMV Smith strain (VR-1399; ATCC) and is herein after referred to as wild-type (WT) MCMV231. pSM3fr-MCK-2fl clone3.3 and Am157 were propagated on mouse embryonic fibroblasts (MEFs)232. Animals were infected intravenously (i.v.) with 2×10.sup.5 plaque forming units (PFU). Viral titers were determined on MEFs by standard plaque assay.

    Adoptive Transfer Experiments

    [0298] Adoptive co-transfer studies were performed by transferring splenocytes from WT B6 (CD45.1) and Tfrcfl/flNcr1Cre (CD45.2) mice in an equal ratio into B6Ly49h−/− and, respectively, into Rag2−/−yc−/− recipients 1 day prior to MCMV infection. For cell proliferation assays in vivo, splenocytes were loaded, prior to transfer, with cell proliferation dye carboxyfluorescein succinimidyl ester (5 μM CFSE, Molecular probes, USA).

    Human NK Cell Isolation and Cell Culture

    [0299] Blood samples were obtained from healthy donors after written informed consent. Peripheral blood mononuclear cells were isolated by standard density-gradient centrifugation protocols (Lymphoprep; Fresenius Kabi). NK cells were negatively selected using EasySep negative NK cell isolation kit (Stemcell). Human NK cells were maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated human AB serum, 50 U/ml penicillin (Invitrogen) and 50 μg/ml streptomycin (Invitrogen) (R10AB). To generate CE NK cells, isolated NK cells were primed in R10AB containing IL-12 (10 ng/ml, R&D systems), IL-15 (1 ng/ml, PeproTech) and IL-18 (50 ng/ml, R&D systems) over-night. The next day cells were washed twice with PBS and maintained in R10AB containing IL-15 (1 ng/ml) until stimulation. Every 2-3 days 50% of the medium was replaced with fresh IL-IS (1 ng/ml). After 7 days cells were stimulated in R10AB containing IL-12 (10 ng/ml), IL-15 (1 ng/ml) and IL-18 (50 ng/ml) or with K562 leukemia targets (effector: target ratio, 5:1) for 6 hours. When indicated, cells were pre-incubated with 2-deoxy-D-glucose (10 mM, Sigma-Aldrich), Actinomycin D (1 and 10 μM, Sigma-Aldrich), Cycloheximide (10 and 100 μg/ml, Sigma-Aldrich), 2,2′-Bipyridyl (1, 10, 50 and 100 μM, Sigma-Aldrich) or 6-aminonicotinamide (50 μM, Sigma-Aldrich) for 30 min and then stimulated in R10AB containing IL-12 (10 ng/ml), IL-1S (1 ng/ml) and IL-18 (50 ng/ml) for 6 hours.

    [0300] The NK cell lines, NK92 and NKL were maintained in R10AB supplemented with IL-2 (50 U/ml). Jurkat and K562 cell lines were maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated human fetal bovine serum (FBS), 50 U/ml penicillin (Invitrogen) and 50 μg/ml streptomycin (Invitrogen) (R10FBS). 293T human embryonic kidney (HEK-293T) cells were maintained in DMEM medium (Invitrogen) supplemented with 10% heat-inactivated human fetal bovine serum (FBS), 50 U/ml penicillin (Invitrogen) and 50 μg/ml streptomycin (Invitrogen).

    Flow Cytometry Analysis of Human Cells

    [0301] For surface staining NK cells were stained for 30 min at 4° C. with saturating concentrations of antibodies. Following antibodies were used: anti-human CD71 (clone CY1G4, Biolegend), antihuman CD69 (clone FN50, Immunotools), anti-human CD98 (clone MEM-108, Biolegend). Samples were acquired using a BD AccuriC6 or a CytoFLEX flow cytometer (Beckman Coulter). Data were analyzed with Flowjo®_V10.5 (Tree Star, USA).

    [0302] For cell proliferation assays, NK cells were loaded prior to activation with the cell-proliferation dye CFSE (1 μM, Molecular probes, USA) and seeded in 96-well plates. Cells were washed twice and maintained in R10AB with IL-15 (1 ng/ml), when indicated in the presence of inhibitors. A fixable live-dead cell stain (Fixable Viability Dye, eBioscience or Zombie Aqua, Biolegend) was used to exclude dead cells prior to sample acquisition. CFSE dilution was analyzed 65 hours post-stimulation by flow cytometry. Samples were acquired using a BD AccuriC6 or a CytoFLEX flow cytometer (Beckman Coulter). Data were analyzed with Flowjo®_V 10.5 (Tree Star, USA).

    Flow Cytometry Analysis of Murine Cells

    [0303] Lymphocytes from spleens were isolated by meshing organs and filtering them through a 100-100 μm strainer. To isolate lymphocytes from liver, the tissue was meshed and filtered through a 100 μm strainer and purified using a discontinuous gradient of 40% over 80% Percoll. Red blood cells in spleen and liver were lysed using erythrocyte lysis buffer. Cells were pretreated with Fc block (clone 2.4G2) and a fixable live-dead cell stain (Fixable Viability Dye, eBioscience) was used to exclude dead cells. Cells were stained for 30 min at 4° C. with saturating concentrations of antibodies. Following antibodies purchased from Thermo Fisher Scientific were used: anti-mouse CD8α (clone 53-6.7), anti-mouse CD45.2 (clone 104), anti-mouse CD4 (clone RM4-5), anti-mouse CD69 (clone H1.2F3), anti-mouse CD45.1 (clone A20), anti-mouse CD3e (clone 145-2C11), anti-mouse CD19 (clone 1D3), anti-mouse NK1.1 (clone PK136), antimouse NKp46 (clone 29A1.4), anti-mouse CD62L (clone MEL-14), anti-mouse Ly6c (clone HK1.4), anti-mouse KLRG1 (clone 2F1), anti-mouse Ly49H (clone 3D10), anti-mouse CD11b (clone M1/70) and anti-mouse CD27 (clone 0323). Samples were acquired using a BD FACSAria. Data were analyzed with Flowjo®_V10.5 (Tree Star, USA).

    [0304] For intracellular cytokine staining upon MCMV infection, lymphocytes from spleen and liver of MCMV-infected mice were isolated as indicated above. Cells were resuspended in RPMI-1640 medium supplement with 10% fetal bovine serum (Thermo Fisher Scientific), 50 U/ml penicillin (Invitrogen), 50 μg/ml streptomycin (invitrogen) and 50 μM 2-mercaptoethanol (Thermo Fisher Scientific) (R10FBS) in the presence of IL-2 (500 IU/ml). Cells were incubated at 37° C. in the presence of brefeldin A (eBioscience) for 5 hours. Cells were surface-stained, followed by fixation and permeabilization according to the manufacturer's protocol (BD Biosciences). Intracellular cytokines were stained using mouse-anti IFN-γ (clones XMG1.2, Thermo Fisher Scientific). Samples were acquired using a BD FACSAria. Data were analyzed with Flowjo®_V10.5 (Tree Star, USA).

    [0305] For cell proliferation assays, lymphocytes were loaded prior to activation with the cellproliferation dye CFSE (1 μM, Molecular probes, USA) and seeded in U-bottom 96-well plates (5×10.sup.5 cells/well). Cells were stimulated in R10FBS containing IL-12 (10 ng/ml, PeproTech), IL-15 (10 ng/ml, PeproTech) and IL-18 (50 ng/ml, R&D Systems) for 16 hours. Cells were washed twice and maintained in R10FBS containing IL-15 (10 ng/ml). CFSE dilution was analyzed 65 hours post-stimulation by flow cytometry. A fixable live-dead cell stain (Fixable Viability Dye, eBioscience or Zombie Aqua, Biolegend) was used to exclude dead cells. Samples were acquired using a BD FACSAria or CytoFLEX flow cytometer (Beckman Coulter). Data were analyzed with Flowjo®_V10.5 (Tree Star, USA).

    Seahorse Metabolic Flux Analyzer

    [0306] A Seahorse XF-96e extracellular flux analyzer (Seahorse Bioscience, Agilent) was used to determine the metabolic profile of cells. NK cells were plated (3×10.sup.5 cells/well) onto Celltak (Corning, USA) coated cell plates. Mitochondrial perturbation experiments were carried out by sequential addition of oligomycin (1 μM, Sigma), FCCP (2 μM, Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, Sigma), and rotenone (1 μM, Sigma). Oxygen consumption rates (OCR, pmol/min) and extracellular acidification rates (ECAR, mpH/min) were monitored in real time after injection of each compound.

    2-NBDG Uptake

    [0307] NK cells were seeded in U-bottom 96-well plates (2×10.sup.5 cells/well). When indicated cells were pre-incubated for 30 min with inhibitors and stimulated in R10AB containing IL-12 (10 ng/ml), IL-15 (1 ng/ml) and IL-18 (50 ng/ml) for 6 hours. Cells were then incubated in medium containing 20 μM 2-NBDG (Invitrogen) for 15 min and analyzed by flow cytometry. Samples were acquired using a BD AccuriC6 flow cytometer. Data were analyzed with Flowjo®_V10.5 (Tree Star, USA).

    IFN-γ Measurement in Human NK Cells

    [0308] NK cells were seeded in U-bottom 96 well plates (2×10.sup.5 cells/well) using R10AB. When indicated cells were pre-incubated for 30 min with inhibitors and stimulated in R10AB containing IL-12 (10 ng/ml), IL-15 (1 ng/ml) and IL-18 (50 ng/ml) for 6 hours. Cell supernatants were harvested after stimulation and IFN-γ was measured using a human Th1 cytokine bead-based immunoassay (Legendplex, Biolegend) according to manufacturer's protocol.

    Transferrin Uptake Assay

    [0309] NK cells were seeded in U-bottom 96-well plates (2×10.sup.5 cells/well). When indicated cells were pre-incubated for 30 min with inhibitors and stimulated in R10AB containing IL-12 (10 ng/ml), IL-15 (1 ng/ml) and IL-18 (50 ng/ml) for 4 hours. Cells were then stimulated in RPMI-1640 medium containing 5% BSA and IL-12 (10 ng/ml), IL-15 (1 ng/ml) and IL-18 (50 ng/ml) for 2 hours. After stimulation cells were washed with RPMI-1640 containing 0.5% BSA before incubation with transferrin-alexa488 conjugate (Tf-488, 10 μg/ml, Thermo Fisher Scientific) for 15 min. Transferrin uptake was stopped by washing cells in ice-cold acidic buffer (150 mM NaCl, 20 mM citric acid and pH: 5). Cells were resuspended in FACS buffer and analyzed by flow cytometry. Samples were acquired using a BD AccuriC6 flow cytometer. Data were analyzed with Flowjo®_V10.5 (Tree Star, USA).

    HPG Incorporation Assay

    [0310] NK cells were seeded in 96-well plates (2×10.sup.5 cells/well). Cells were stimulated in R10AB containing IL-12 (10 ng/ml), IL-15 (1 ng/ml) and IL-18 (50 ng/ml) for 4.5h and afterwards incubated for 1.5h in methionine-free RPMI-1640 medium containing 10% dialyzed FBS and IL-12 (10 ng/ml), IL-15 (1 ng/ml) and IL-18 (50 ng/ml). Click-IT®HPG (50 μM, Life Technologies) was added for the last 30 min of the incubation. HPG incorporation into NK cells was stained with Click-iT® reaction cocktail (Thermo Fisher Scientific) and detected by flow cytometry. Samples were acquired using a BD AccuriC6 flow. Data were analyzed with Flowjo®_V10.5 (Tree Star, USA).

    Immunoblot Analysis

    [0311] Protein concentrations were determined by BCA protein assay kit (Thermo Fisher Scientific). Total cell lysates were separated using 4%-15% Mini Protean TGX Gel (Bio-Rad, Hercules Calif., USA), and transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer (Bio-Rad, Hercules Calif., USA). Membranes were probed with the following antibodies: anti-human CD71 mAb (13113), anti-human IRP1 mAb (20272), anti-human IRP2 mAb (37135), anti-human FTH1 mAb (4393), anti-human eIF4E mAb (2067), anti-human c-Myc mAb (5605) and anti-human β-actin mAb (3700) (all from Cell Signaling, USA). Blots were stained with appropriate secondary antibodies and the odyssey imaging system (LICOR, Lincoln Nebr., USA) was used for visualization, and the ImageJ software (1.48v) for quantification.

    RNA Sequencing

    [0312] RNA-seq was performed by Admera Health (USA). In brief, samples were isolated using ethanol precipitation. Quality check was performed using Tapestation RNA HS Assay (Agilent Technologies, USA) and quantified by Qubit RNA HS assay (Thermo Fisher Scientific). Ribosomal RNA depletion was performed with Ribo-zero Magnetic Gold Kit (MRZG12324, Illumina Inc., USA). Samples were randomly primed and fragmented based on manufacturer's recommendation (NEBNext® Ultra™ RNA Library Prep Kit for Illumina®).

    [0313] First strand was synthesized using Protoscript II Reverse Transcriptase with a longer extension period (40 min for 42° C.). All remaining steps for library construction were used according to the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. Illumina 8-nt dual-indices were used. Samples were pooled and sequencing on a HiSeq with a read length configuration of 150 paired-end.

    [0314] Reads were aligned to the human genome (UCSC version hg38AnalysisSet) with STAR (version 2.5.2.sup.4) using the multi-mapping settings ‘--outFilterMultimapNmax 10--outSAMmultNmax 1’. The output was sorted and indexed with samtools (version 1.7) and picard markDuplicates (version 2.9.2) was used to collapse samples run on different sequencing lanes. The qCount function of QuasR (version 1.20.05) was used to count the number of read (5′ends) overlapping with the exons of each gene assuming an exon union model (RefSeq genes downloaded from UCSC on 2017-09-01). All subsequent gene expression data analysis was done within the R software (R Foundation for Statistical Computing, Vienna, Austria). The differentially expressed genes were identified using the edgeR package (version 3.22.5).

    Quantitative Real-Time PCR

    [0315] RNA was isolated from NK cells using Trizol (Thermo Fisher Scientific) and chloroform (Sigma-Aldrich) according to manufacturer's protocol, then purified with RNeasy RNA purification mini kit (QIAGEN, Germany). RNA concentration was determined using the NanoDrop 2000C (Thermo Fisher Scientific). From purified RNA, cDNA was synthesized using the reverse transcriptase kit GoScript™ Reverse Transcriptase (Promega). Quantitative PCR for IFNG, TFRC and 18S mRNA was done in triplicate using commercially designed primers from Life Technologies (Hs00989291_m1, Hs00951083_m1, Hs03003631_g1). PCR reactions were performed using Go Tag G2 DNA Polymerase (Promega) according to manufacturer's protocol.

    RNA-Mediated Interference

    [0316] NK92, NKL or Jurkat cells (2×10.sup.6) were transfected with pools of siRNA targeting ACO1, IREB2 or control-scrambled siRNA (each 10 pmoles) (QIAGEN) using the AMAXA cell line V nucleofection kit (Lonza). Afterwards cells were rested for 72 hours and phenotypically and functionally analyzed. Knockdown efficiency was assessed by immunoblot analyses of the respective proteins.

    CRISPR Editing

    [0317] A 24-well cell culture plate with 1 ml R10AB containing IL-2 (50 U/ml; NK92 cells) or R10FBS (Jurkat cells) was prepared and pre-warmed at 37° C. For CRISPR-Cas9 mediated IREB2 gene knockout following sgRNAs from IDT were used: Hs.Cas9.IREB2.1 AA (Ref no. 220257866) or Alt-R CRISPR-Cas9 Negative Control (Ref no. 224163224). Guide RNA complexes were formed by combining the crRNA and tracrRNA in equal molar amounts in IDT Duplex buffer (30 mM HEPES, pH 4.5, 100 mM potassium acetate) at 20 μM concentration by heating the oligos at 95° C. for 5 min and slowly cooling to room temperature. An equal volume of CAS9 nuclease (QB3 MacroLab, University of California, Berkeley) was added and incubated at room temperature for 15 min. NK92 or Jurkat cells (2 ×10.sup.6) were washed in PBS and resuspended in electroporation solution (AMAXA cell line V nucleofection kit, Lonza). RNP solution (3 μM final RNP concentration) was added and electroporated with the recommended program. Cells were transferred into pre-warmed media and rested cells for indicated time points. NK92 cells were rested for 5 days, followed by phenotypically and functionally analysis. Knockdown efficiency of IRP2 was assessed by immunoblot. Jurkat cells were rested for 2 days, followed by single cell sorting into 96-well plates. Clones were expanded and knockout efficiency of IRP2 was assessed by immunoblot analyses and positive clones were expanded for phenotypical and functional analysis and lentiviral transduction of IRP2.

    IRP2 CAR Construction

    [0318] The human IRP2 was synthesized as gene strings (GeneArt, Thermo Fischer Scientific). IRP2 (NM_004136.4) was then cloned into a third-generation self-inactivating lentiviral expression vector, pELNS, with expression driven by the elongation factor-1α (EF-1α) promoter, in frame with T2A and the second generation anti-PSMA CAR. The scfv for the anti-PSMA CAR derived from monoclonal antibody J591 used as the tumor-targeting moiety, while the intracellular domain consists of CD28 costimulatory domain and CD3zeta chain. In the control vector IRP2 has been substituted with the reporter gene eGFP.

    Recombinant Lentivirus Production

    [0319] 24 hours before transfection, HEK-293 cells were seeded (5×10.sup.6 cells/5 ml media). All plasmid DNA was purified using the Endotoxin-free Plasmid Maxiprep Kit (Sigma). HEK-293T cells were transfected with 1.3 pmoles psPAX2 (lentiviral packaging plasmid) and 0.72 pmoles pMD2G (VSV-G envelope expressing plasmid) and 1.64 pmoles of pLV-EFIA>mCherry(ns):P2A:EGF or PLV-EIFIA>hIREB2:P2A:EGFP (Vector Builder) using Lipofectamine 2000 (Invitrogen) and Optimem medium (Invitrogen, Life Technologies). The viral supernatant was collected 48 and 72 hours after transduction. Viral particles were concentrated using VIVASPIN 20 (Sartorius) and viral supernatants were stored at −80° C. Lentiviral particles of CAR constructs were produced as described by Giordano-Attianese et al., Nat Biotechnol, 2020, 38, 426-432).

    Lentiviral Transduction of Jurkat Cells

    [0320] Jurkat cells were seeded in U-bottom 96 well plates (5×10.sup.5 cells/well) using R10FBS. Viral supernatant was thawed and Jurkat cells were transduced with different virus dilutions ranging from 1:16 to 1:1′160′000. Plate was centrifuged at 400×g for 3 minutes and incubated for at 37° C. for 24 hours. Afterwards medium was changed and cells were rested for 2 more days. Transduction efficiency was assessed by analyzing GFP expression by flow cytometry. GFP.sup.+ cells were flow-sorted (frequency 10-30% positive cells) and expanded for phenotypical analysis. Lentiviral overexpression of IRP2 was assessed by immunoblot analyses.

    Lentiviral Transduction of Primary T Cells

    [0321] Blood samples were obtained from healthy donors after written informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated by standard density-gradient centrifugation protocols (Lymphoprep; Fresenius Kabi). CD4+ and CD8.sup.+ T cells were positively selected using magnetic CD4.sup.+ and CD8+ beads (Miltenyi Biotec). Purified CD4.sup.+ and CD8.sup.+ T cells were cultured in R10AB. CD4.sup.+ and CD8.sup.+ T cells were plated into a 24-well cell culture plate and stimulated with anti-CD3 and anti-CD28 monoclonal antibody-coated beads (Invitrogen, Life Technologies) in a ratio of 1:1 in R10AB containing IL-2 (150 U/ml). T cells were transduced with lentiviral particles at 18-22 hours after activation in cell culture plates coated with Retronectin (Takara Bio). Every 24 hours medium was replaced with fresh IL-2 (150 U/ml). 5 days after transduction cells were analyzed for CD71 expression by flow cytometry. Samples were acquired using CytoFLEX flow cytometer (Beckman Coulter). Data were analyzed with Flowjo® V10.5 (Tree Star, USA). Lentiviral transduction of primary T cells with CAR constructs were conducted as described.sup.7.

    Activation and Proliferation of Transduced Primary CAR T Cells

    [0322] Transduced T cells expressing the CAR or CAR_IREB2 were adjusted for equivalent CAR expression. To stimulate the CAR a polyclonal anti-Fab antibody (Jackson Immuno Research) was used. Briefly, a 96-well plate was coated with 20 μg/ml anti-Fab in PBS for 4 hours at 37° C. Primary T cells were loaded prior to activation with the cell-proliferation dye Cell Trace violet (CTV; 1 μM, Thermo Fisher Scientific). Plate was washed twice and stained T cells were seeded (1×10.sup.5/well) and stimulated for 5 days. CTV dilution and CD71 expression was analyzed by flow cytometry. Samples were acquired using BD FACS LSR II flow cytometer (BD Bioscience). Data were analyzed with Flowjo®_V10.5 (Tree Star, USA).

    Statistical Analysis

    [0323] Data are presented as mean±SEM. Statistical significance was determined by either using unpaired two-tailed Student's t test or paired two-tailed Student's t test using GraphPad Prism 8.00 (GraphPad Software). For comparison of increases (before versus after) in paired samples, a simple linear-regression model was used. P values of less than 0.05 were considered statistically significant.

    Example 9: The IRP/IRE Regulatory System Orchestrates CD71 Expression in NK Cells

    [0324] Thus far, the experiments established (i) iron uptake via CD71 as a critical metabolic checkpoint controlling NK cell proliferation, and (ii) preferential upregulation of CD71 on activated CE as compared to NV NK cells. Next, the inventors asked how CD71 per se was regulated in NV and CE NK cells. To address this question, the inventors first assessed whether induction of CD71 relied on NK cell transcriptional activity. As in previous experiments, CD71 was induced to a greater extent in cytokine-stimulated CE than NV NK cells (FIG. 5A, upper panel). In both subsets, inhibition of transcription—using actinomycin—prevented stimulation-induced upregulation of CD71, as did blocking of translation with cycloheximide (FIG. 5A lower panel). Thus, transcription and translation were similarly required in both cell subsets. Next, we examined the possibility that transcription of TFRC might be differentially regulated between NV and CE NK cells. To do so, we analyzed transcript abundance of TFRC. Indeed, TFRC mRNA levels were higher in activated CE than NV NK cells, yet further induced in both subsets upon stimulation (FIG. 7A). c-Myc is a key transcription factor regulating TFRC in various immune cells. Given the increased abundance of TFRC mRNA among activated CE over NV NK cells, preferential c-Myc induction in CE NK cells could thus explain differential regulation of CD71 between activated CE and NV NK cells. However, c-Myc was equally induced in both NK cell subsets (FIG. 5F). Together these data established a symmetric need in activated NV and CE NK cells for continuous transcription and translation to support expression of CD71.

    [0325] Many genes involved in cellular iron homeostasis contain iron responsive elements (IREs) in the 5′ or 3′ UTR of their mRNA. Iron regulatory proteins 1 and 2 (IRP1 and IRP2) bind IREs, thereby controlling mRNA stability and translation. TFRC mRNA contains 5 IREs in the 3′UTR, where binding of IRPs stabilizes the mRNA and facilitates translation. As per text book, this occurs under iron-deficient conditions. The inventors reasoned that, irrespective of cellular iron abundance, increased expression of IRPs selectively in CE NK cells would explain higher TFRC transcript abundance and enhanced activation-dependent CD71 expression in these cells. Supporting this idea, protein abundance of both IRP1 and IRP2 were higher in quiescent and activated CE NK cells (FIG. 6A middle and lower panels). These data were compatible with IRPs generating a pseudo iron deficient state in a cell subset-specific manner, namely in CE NK cells, thus selectively controlling abundance of a distinct set of proteins at the post-transcriptional level. To further probe this notion, we analyzed transcript and protein abundance of FTH1 (encoding the ferritin heavy chain), which contains an IRE in the 5′UTR—where binding of IRPs inhibits translation. FTH1 mRNA was increased in activated CE NK cells (FIG. 7B)—yet despite these higher transcript levels, protein abundance of ferritin heavy chain was, if anything, lower in both unstimulated and activated CE NK cells (FIG. 6E). This finding was highly suggestive of IRPs, with their CE vs. NV NK cell specific abundance, being involved in regulating translation of IRE containing mRNAs in these cells.

    [0326] To genetically interrogate the IRPs' role in regulating CD71 of NK cells—and thus their proliferation—the inventors went on to utilize the NK cell line, NK92. In these cells, IRP1 and IRP2 levels were each selectively reduced using an siRNA approach (FIG. 7C-D). Notably, no consistent change in CD71 protein expression was observed upon silencing of IRP1, whereas CD71 abundance significantly dropped in IRP2-silenced cells (FIG. 7E). Accordingly, increased ferritin heavy chain expression was also found when silencing IRP2, but not of IRP1 (FIG. 7F). To ascertain robustness of this finding, the inventors repeated these experiments using a second NK cell line (NKL cells). Similar to NK92 cells, silencing of IRP2 significantly lowered CD71 and increased ferritin heavy chain expression also in this cell line (FIG. 7 G-K). Lastly, knocking out IRP2 using CRISPR/Cas9 technology (FIG. 7L), also reduced CD71 expression among NK92 cells, which directly translated into reduced proliferation rates (FIG. 7M). These data thus recapitulated the findings made in CE vs. NV NK cells through genetic manipulation, and established the IRE/IRP regulatory axis—and specifically IRE/IRP2—is an important system regulating proliferation in NK cells/NK cell lines through governing expression of CD71.

    Example 10: Enforced IRP Expression is a Molecular Module Also Supporting T Cell Proliferation

    [0327] Adoptive cell therapy using engineered chimeric antigen receptor (CAR) T cells is a promising approach for control of various malignancies, in particular the treatment of hematologic malignancies. However, not all patients respond to the CAR T cell therapy, some relapse—and treatment of solid cancer remains uniquely challenging. Building on the results from the NK cell studies, the inventors reasoned that genetically enforcing pseudo iron deficiency may specifically improve activation-driven (i.e. context dependent) proliferation—and thus the therapeutic potential—also of (CAR) T cells. To begin to examine the role of IRPs in regulating expression of CD71 and interlinked proliferation in T cells, the inventors first suppressed abundance of IRP1 and IRP2 in Jurkat T cells, using siRNA technology (FIG. 8A-B). Similar to NK cell lines, reduction of IRP2 was the dominant factor in reducing expression of CD71 and increasing protein abundance of the ferritin heavy chain (FIG. 8 C-D). Inversely, lentiviral overexpression of IRP2 (LV-IREB2) in IRP2 knockout (ko) Jurkat cells increased CD71 expression as compared to cells transduced with a control vector coding for mCherry (LV-mCherry) (FIG. 8 E-F, top and lower left panels). LV-IREB2 dependent increased cell surface expression of CD71 was associated with more rapid Jurkat T cell proliferation (FIG. 8F, lower right panel). Importantly, expression of CD71 was regulated by lentiviral transduction of LV-IREB2 also in human primary CD4.sup.+ and CD8.sup.+ T cells (FIG. 8G-H).

    [0328] Encouraged by these observations, the inventors went on to test how pseudo iron deficiency—enforced through the expression of IREB2—affected regulation of CD71 and interlinked proliferation in primary human T cells expressing a CAR. For these proof of concept experiments, a CAR T cell model targeting the human prostate-specific membrane antigen (hPSMA) was used. CD4.sup.+ T cells were either lentivirally transduced with a vector encoding for the CAR (CAR), or both the CAR and IRP2 (CAR IREB2). Overexpression of IRP2 in CAR_IREB2 T cells was confirmed by Western blot analyses (FIG. 81). When assessed under non-activating conditions, overexpression of IRP2 did not affect cell-surface expression of CD71 (FIG. 8J, upper panels). However, expression of CD71 was consistently higher on CAR_IREB2 when compared to CAR T cells upon crosslinking CARs with an α-Fab antibody (FIG. 8J, lower panels). CAR_IREB2 T cells did not spontaneously proliferate (FIG. 8K, upper panel), yet increased IRP2-driven, and hence strictly activation-dependent expression of CD71 was sufficient to drive superior proliferation (FIG. 8K, lower panel). Taken together, these data demonstrate that IRP2 is regulating expression of CD71 and interlinked cell proliferation also in T cells, and specifically CAR T cells. Importantly, inducing pseudo iron deficiency through overexpression of IRP2 in CAR T cells enhanced proliferation in a strictly activation, i.e. context dependent manner.