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. 2017 Nov;152(3):425-438.
doi: 10.1111/imm.12779. Epub 2017 Aug 2.

Polyfunctional Response by ImmTAC (IMCgp100) Redirected CD8 + and CD4 + T Cells

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Free PMC article

Polyfunctional Response by ImmTAC (IMCgp100) Redirected CD8 + and CD4 + T Cells

Caroline Boudousquie et al. Immunology. .
Free PMC article

Abstract

The success of immune system-based cancer therapies depends on a broad immune response engaging a range of effector cells and mechanisms. Immune mobilizing monoclonal T cell receptors (TCRs) against cancer (ImmTAC™ molecules: fusion proteins consisting of a soluble, affinity enhanced TCR and an anti-CD3 scFv antibody) were previously shown to redirect CD8+ and CD4+ T cells against tumours. Here we present evidence that IMCgp100 (ImmTAC recognizing a peptide derived from the melanoma-specific protein, gp100, presented by HLA-A*0201) efficiently redirects and activates effector and memory cells from both CD8+ and CD4+ repertoires. Using isolated subpopulations of T cells, we find that both terminally differentiated and effector memory CD8+ T cells redirected by IMCgp100 are potent killers of melanoma cells. Furthermore, CD4+ effector memory T cells elicit potent cytotoxic activity leading to melanoma cell killing upon redirection by IMCgp100. The majority of T cell subsets belonging to both the CD8+ and CD4+ repertoires secrete key pro-inflammatory cytokines (tumour necrosis factor-α, interferon-γ, interleukin-6) and chemokines (macrophage inflammatory protein-1α-β, interferon-γ-inducible protein-10, monocyte chemoattractant protein-1). At an individual cell level, IMCgp100-redirected T cells display a polyfunctional phenotype, which is a hallmark of a potent anti-cancer response. This study demonstrates that IMCgp100 induces broad immune responses that extend beyond the induction of CD8+ T cell-mediated cytotoxicity. These findings are of particular importance because IMCgp100 is currently undergoing clinical trials as a single agent or in combination with check point inhibitors for patients with malignant melanoma.

Keywords: ImmTAC; T cell receptor; T cells; cancer; immunotherapy; melanoma.

Figures

Figure 1
Figure 1
IMCgp100 induced killing capacity of different CD8+ T cell subpopulations. (a) Isolated CD8+ T cells (here obtained from Donor 8) were FACS sorted, according to cell surface labelling of CD45RA and CCR7 markers, into four subpopulations: naive, effector memory (Tem), CD45RA effector memory (Temra) and central memory (Tcm). Melanoma target cells (Mel526 cell line: HLA‐A2+ve, gp100+ve) were incubated with each of the CD8+ T cell subpopulations (Naive, Tem, Temra and Tcm) as well as the total pool of CD8+ T cells with or without addition of IMCgp100 at 82 pm. The A375 cell line (HLA‐A2+ve, gp100−ve) was used as a negative control. The number of apoptotic cells/area (in mm2) was determined every 2 hr by the IncuCyte imaging system. (b) The killing curves produced for the individual CD8+ T cell subsets from seven donors using non‐linear logistic growth mixed effects model. (c) Fitted values and standard errors for the model fitted to each cell population. Asym representing Asymptote (the maximum level of the curve), Xmid (the time taken to reach half the level of the Asymptote) and Scale (controls the angle of the curve) (d) Representative images obtained from Donor 8 at the end point (44 hr) of the IcuCyte assay. Red crosses and blue dots depict apoptotic cancer cells and apoptotic T cells, respectively.
Figure 2
Figure 2
IMCgp100‐induced killing capacity of different CD4+ T cell subpopulations. (a) Magnetically enriched CD4+ T cells (here obtained from Donor 16) were FACS sorted, according to cell surface labelling of CD45RA and CCR7 markers, into four subpopulations, including: naive, effector memory (Tem) and central memory (Tcm). Melanoma target cells (Mel526 cell line: HLA‐A2+ve, gp100+ve) were incubated with each of the CD8+ T cell subpopulations (naive, Tem and Tcm) as well as the total pool of CD4+ T cells with or without addition of IMCgp100 at 82 pm. The A375 cell line (HLA‐A2+ve, gp100−ve) was used as a negative control. The number of apoptotic cells/area (in mm2) was determined every 2 hr by the Incucyte imaging system. (b) The killing curves produced for the individual CD4+ T cell subsets from eight donors using non‐linear logistic growth mixed effects model, except naive subpopulation, which was fixed with a linear mixed effects model due to the lack of killing; the intercept: −0·51, standard error: 1·96; slope: 0·165, standard error: 0·1. (c) Fitted values and standard errors for the model fitted to each cell population. Asym representing asymptote (the maximum level of the curve), Xmid (the time taken to reach half the level of the asymptote) and Scale (controls the angle of the curve). (d) Representative images obtained from Donor 16 at the end point (44 hr) of the IcuCyte assay. Red crosses and blue dots depict apoptotic cancer cells and apoptotic T cells, respectively.
Figure 3
Figure 3
Cytotoxic T cell killing is not impacted by regulatory T (Treg) cells. (a) The killing curves for total CD8+ T cell population and a mixture of CD8+ T cells and Treg cells at 1 : 1 ratio obtained from four donors were generated using a non‐linear mixed effects model. (b) Fitted values and standard errors for the model are presented in the table. Asym representing Asymptote (the maximum level of the curve), Xmid (the time taken to reach half the level of the Asymptote) and Scale (controls the angle of the curve).
Figure 4
Figure 4
Activation status of the individual CD8+ and CD4+ T cell subsets upon IMCgp100 redirection. (a) Mel526 cells were incubated with naive, effector memory (Tem), CD45RA effector memory (Temra) and central memory (Tcm). CD8+ T cells along with IMCgp100 at decreasing concentrations (131, 82, 31, 12 and 1 pm). Interferon‐γ (IFNγ) release was measured using ELISpot and represented as a number of spots. Means obtained from four donors are presented. (b) The same assay was carried out using the individual CD4+ T cell subpopulations (naive, Tcm and Tem). SEM; standard error of the mean.
Figure 5
Figure 5
IMCgp100‐induced cytokine and chemokine secretion by CD8+ (a) and CD4+ (b) T cell subsets. Mel526 cells were incubated with specific T cell subpopulations and supplemented with IMCgp100 at 82 pm for 44 hr (CD8+ culture) or 48 hr (CD4+ culture). Selected cytokines [tumour necrosis factor‐α (TNFα), interferon‐γ (IFNγ), interleukin‐2 (IL‐2), IL‐15, IL‐10, IL‐6, IL‐4] and chemokines [macrophage inflammatory protein‐1α/β (MIP‐1α/β), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), monocyte induced by IFN‐γ (MIG), IFN‐γ‐inducible protein‐10 (IP‐10), monocyte chemoattractant protein 1 (MCP‐1)] were measured in the culture medium using Luminex assay. Interleukin‐4 was only measured in the culture medium from CD4+ cells as CD8+ T cells do not produce it. Cytokines and chemokines were measured in duplicates from three (a) or two (b) donors.
Figure 6
Figure 6
Polyfunctionality of different CD8+ T cell subpopulations induced by IMCgp100 stimulation. Peripheral blood mononuclear cells (PBMC) were incubated with the Mel526 cell line in the presence of increasing concentrations of IMCgp100 (31, 82 and 131 pm) for 16 hr. PBMC incubated with melanoma cells pulsed with gp100 peptide at 1 μm served as a positive control, whereas PBMC incubated with Mel526 cells in the absence of IMCgp100 served as a negative control. T cell subpopulations were identified using CCR7 and CD45RA markers and the amounts of selected proteins [CD107, interferon‐γ (IFNγ), interleukin‐2 (IL‐2), macrophage inflammatory protein 1β (MIP1β) and tumour necrosis factor‐α (TNFα)] were measured in each subpopulation using FACS. The pie charts show the proportion of cells expressing between one and five of the markers tested (mean values from three donors). Blue, light blue, yellow, orange, brown and red slices refer to a proportion of cells expressing none, one, two, three, four or all five of the tested markers, respectively. The pie chart arcs indicate the specific proteins being produced.
Figure 7
Figure 7
Polyfunctionality of different CD4+ T cell subpopulations induced by IMCgp100 treatment. Peripheral blood mononuclear cells (PBMC) were incubated with the Mel526 cell line in the presence of increasing concentrations of IMCgp100 (31, 82 and 131 pm) for 16 hr. PBMC incubated with melanoma cells pulsed with gp100 peptide at 1 μm served as a positive control, whereas PBMC incubated with Mel526 cells in the absence of IMCgp100 served as a negative control. T cell subpopulations were identified using CCR7 and CD45RA markers and the amounts of selected proteins (CD107, interferon‐γ (IFNγ), interleukin‐2 (IL‐2), macrophage inflammatory protein 1β (MIP1β) and tumour necrosis factor‐α (TNFα)] were measured in each subpopulation using FACS. The pie charts show the proportion of cells expressing between one and five of the markers tested (mean values from three donors). Blue, light blue, yellow, orange, brown and red slices refer to a proportion of cells expressing none, one, two, three, four or all five of the tested markers respectively. The pie chart arcs indicate the specific proteins being produced.

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