Immunotherapeutic targeting of LIGHT/LTβR/HVEM pathway fully recapitulates the reduced cytotoxic phenotype of LIGHT-deficient T cells

Abstract

Tumor necrosis factor (TNF)/TNF receptor (TNFR) superfamily members play essential roles in the development of the different phases of the immune response. Mouse LIGHT (TNFSF14) is a type II transmembrane protein with a C-terminus extracellular TNF homology domain (THD) that assembles in homotrimers and regulates the course of the immune responses by signaling through 2 receptors, the herpes virus entry mediator (HVEM, TNFRSF14) and the lymphotoxin β receptor (LTβR, TNFRSF3). LIGHT is a membrane-bound protein transiently expressed on activated T cells, natural killer (NK) cells and immature dendritic cells that can be proteolytically cleaved by a metalloprotease and released to the extracellular milieu. The immunotherapeutic potential of LIGHT blockade was evaluated in vivo. Administration of an antagonist of LIGHT interaction with its receptors attenuated the course of graft-versus-host reaction and recapitulated the reduced cytotoxic activity of LIGHT-deficient T cells adoptively transferred into non-irradiated semiallogeneic recipients. The lack of LIGHT expression on donor T cells or blockade of LIGHT interaction with its receptors slowed down the rate of T cell proliferation and decreased the frequency of precursor alloreactive T cells, retarding T cell differentiation toward effector T cells. The blockade of LIGHT/LTβR/HVEM pathway was associated with delayed downregulation of interleukin-7Rα and delayed upregulation of inducible costimulatory molecule expression on donor alloreactive CD8 T cells that are typical features of impaired T cell differentiation. These results expose the relevance of LIGHT/LTβR/HVEM interaction for the potential therapeutic control of the allogeneic immune responses mediated by alloreactive CD8 T cells that can contribute to prolong allograft survival.

Keywords: Alloreactivity; CD258); CD270); DcR3 (TNFRSF6b); HVEM (TNFRSF14; LIGHT (TNFSF14; LTβR (TNFRSF3); co-stimulation; cytotoxicity; graft rejection; graft-vs.-host disease; transplantation.

Figure 1.

Effective blockade of LIGHT/LTβR and LIGHT/HVEM interactions using a mouse anti-mouse LIGHT monoclonal antibody. 2.5 × 10⁵ NIH-3T3 cells transduced with GFP-tagged murine LIGHT (blue solid lines) or GFP-transduced NIH-3T3 cells (red solid lines) were incubated with a mouse anti-mouse LIGHT mAb 3D11. After an incubation step, antibody binding was revealed with biotinylated rat anti-mouse IgG_2b followed by allophycocyanin-coupled streptavidin (A, upper panel). To further demonstrate the specificity of the anti-LIGHT mAb, 1 μg/well of Hilyte 647-labeled anti-LIGHT mAb alone (blue solid line) or 1 μg/well Hilyte 647-labeled anti-LIGHT mAb preincubated with 2 μg/well FF-LIGHT fusion protein (red solid line) were added to LIGHT-GFP transduced NIH 3T3 cells (A, lower panel). (B) 2.5 × 10⁵ LIGHT-transduced NIH-3T3 cells were pre-incubated for 30 min at room temperature with anti-LIGHT mAb (clone 3D11, blue solid lines) or mouse IgG_2b isotype control (red dotted lines). Then, cells were stained with LTβR-Ig (upper panel) or HVEM-Ig (lower panel). Binding of LTβR-Ig was revealed with biotinylated anti-hIgG and binding of HVEM-Ig was revealed with biotinylated anti-mouse IgG_2a, both followed by allophycocyanin-coupled streptavidin. The baseline background staining is represented by hIgG₁ Fc fragment (upper panel, black solid lines) to LIGHT-transduced NIH-3T3 cells or the binding of the Fc fragment mIgG_2a (lower panel, black solid lines). The mean fluorescence intensity (MFI) is indicated in each plot. (C) Serial dilutions of anti-LIGHT mAb (clone 3D11) were preincubated with a fixed amount of 1 μg/well of soluble recombinant FF control (red solid line) or FF-LIGHT protein (black solid line). Then, 1 × 10⁵ LIGHT-transduced NIH-3T3 were added to the reaction. After a washing step, a biotinylated rat anti-mouse IgG_2b conjugate was further incubated and developed by SA-APC. The mean fluorescence intensity (MFI) binding of anti-LIGHT mAb to LIGHT transduced cells in the presence of FF control or FF-LIGHT is plotted. (D) The association and dissociation constant rates of 3D11 mAb binding to distinct immobilized mouse FcγR were calculated by surface plasmon resonance and from those values the equilibrium dissociation constant K_D for each of them was determined.

Figure 2.

Mouse LIGHT is rapidly upregulated on activated T cells after polyclonal stimulation. (A) Naïve C57BL/6 splenocytes (2 × 10⁵) were left untreated or were stimulated with PMA (100 ng/ml) and ionomycin (500 ng/ml) for 5 h. 1 μg/well Hilyte-647-labeled anti mouse LIGHT (3D11) or Hylite-647-labeled isotype control mouse IgG_2b were added to cultures during incubation in a final volume of 250 μl. A lineage cocktail to gate out CD19⁺ CD11c⁺ Ly6G⁺ cells was included in the staining. The expression of mouse LIGHT was then analyzed on live resting and activated CD4 and CD8 T cells. (B) The expression of mouse LIGHT on the surface of resting or PMA plus ionomycin activated CD4⁺ and CD8⁺ T cells was also determined with the anti-LIGHT antibody pre-incubated with Flag-Foldon LIGHT (FF-LIGHT) (2 μg FF-LIGHT per well). The percentage of each population is represented in each quadrant. PMA: Phorbol Myristate Acetate. Iono: Ionomycin.

Figure 3.

LIGHT blockade attenuated the course of graft-versus-host reaction in a mouse model of alloreactivity. Semiallogeneic splenocytes from C57BL/6 (70 × 10⁶) were intravenously transferred into non irradiated CB6F1 recipients, which were treated with 1 mg of isotype mouse IgG_2b control (red squares) or anti-LIGHT mAb (3D11, green triangles) at day 0. In a third experimental group, semiallogeneic splenocytes from LIGHT-deficient mice (70 × 10⁶) were injected into CB6F1 mice (orange diamonds). The fourth group represents the syngeneic control group, in which 70 × 10⁶ F1 splenocytes was injected into F1 recipients (blue circles). The absolute number of host bone marrow (A), thymocytes (B), double positive thymocytes (C) and splenocytes (D) was determined 19 days after the adoptive transfer. Data represent a pool of 3 independent experiments. Statistical significance is indicated as follows: *p < 0.05, **p < 0.005, ***p < 0.0005, and ns, non-significant.

Figure 4.

The proliferation index and frequency of donor CD4⁺ and CD8⁺ alloreactive T cells is altered after LIGHT blockade or in LIGHT-deficient T cells. (A-B) 70×10⁶ of CFSE (carboxyfluorescein diacetate succinmidyl ester)-labeled B6 WT or CFSE-labeled B6 LIGHT-deficient splenocytes were adoptively transferred into non-irradiated F1 recipients and treated with 1 mg of isotype-control (mouse IgG_2b) or mouse anti-mouse LIGHT (3D11) mAb at day 0. Three days later, the Proliferative Index (PI) and percentage of Precursor Frequency (PF) of donor alloreactive CD4⁺ and CD8⁺ T cells were determined using the ModFit LT software. Black line profile deconvoluted into cells that had divided once (green), twice (light violet), 3x (light blue), 4x (yellow), 5x (red), 6x (dark violet) and 7x or more (light green). X-axis represents CFSE fluorescence on a log scale and Y axis indicates cell counts (number of events). Data are representative of 2 independent experiments with 3 mice per group. Bars indicate mean ± SEM, and unpaired t test was used to compare differences between groups. Statistical significance was indicated as follows: *p < 0.05, **p < 0.005, ***p < 0.0005, and ns, non-significant.

Figure 5.

Altered expression of differentiation (IL-7Rα and KLRG-1) and costimulatory (ICOS) surface markers is associated with delayed donor CD8 T cell differentiation in the absence of LIGHT. The pattern of expression of costimulatory and differentiation markers was assessed at days 5 and 10 after adoptive transfer of 70 × 10⁶ of donor syngeneic or allogeneic B6 WT or B6 LIGHT-deficient splenocytes into F1 recipients. Recipients were treated on the day of the adoptive transfer with 1 mg of isotype control mouse IgG_2b or anti-LIGHT mAb, clone 3D11. The expression of differentiation markers and costimulatory molecules was assessed by flow cytometry on host (blue lines) and donor (red solid lines) CD4⁺ and CD8⁺ T cells. The percentage of donor alloreactive CD8⁺ B6 WT or B6 LIGHT-deficient splenocytes that express IL7Rα or KLRG-1 (A-B) was evaluated 5 and 10 days after the adoptive transfer into F1 recipients. The delayed maturation of effector CD8⁺ T cells (ICOS negative) in the absence of LIGHT was also depicted (C-D). (E) The expression of the costimulatory markers BTLA and HVEM was also assessed at days 5 and 10. Bars indicate mean ± SEM. Statistical significance and p value was calculated using unpaired Student´s t test. The following criterion of significance was used: *p < 0.05, **p < 0.005, ***p < 0.0005, and ns, non-significant. Data are representative of 2 independent experiments with 3 mice per group.