Checkpoint inhibition enhances cell contacts between CD4+ T cells and Hodgkin-Reed-Sternberg cells of classic Hodgkin lymphoma

Abstract

Although checkpoint molecules like CTLA-4 and PD1 have been described several years ago, checkpoint inhibitors such as nivolumab (an anti-PD-1 antibody) have only recently been used to treat classic Hodgkin lymphoma (cHL). Several studies have shown convincing therapeutic effects of nivolumab in cHL. However, the mechanism of action of nivolumab in cHL is not fully understood. The aim of this study was to monitor changes in cell motility and cell contacts after administration of nivolumab to an in vitro model of cHL as well as to native hyperplastic lymphoid tissue and native human tissue from cHL. In both tissue and in vitro, CD4+, CD8+, CD30+ and CD20+ cell velocities were unchanged after nivolumab incubation. In contrast, in primary cHL tissue, the duration of cell contacts between CD4+ T cells and Hodgkin-Reed-Sternberg cells was significantly increased after 5 hours of nivolumab treatment, and the number of contacts with HRS cells was also slightly increased for CD4+ T cells (not significant), suggesting that CD4+ T cells in particular contribute to the cytotoxicity observed as a result of nivolumab therapy. There was no change in the duration of cell contacts in the hyperplastic lymphoid tissue after nivolumab incubation. In conclusion, we show here for the first time by imaging of native lymphoma tissue an enhanced interaction of CD4+ T cells and Hodgkin-Reed-Sternberg cells in cHL after nivolumab administration.

Figure 1.

Effects of nivolumab on T-cell motility in microchannels in the presence of Hodgkin-Reed-Sternberg cells. (A) Schematic overview of experimental setup with 4x10 μm microchannels connecting 2 reservoirs containing either CD4⁺ or CD8⁺ T cells (left side) and classic Hodgkin lymphoma (cHL) cell lines (right side). (B) PD-L1 expression in the cHL cell lines L-428 and L-1236 (black) compared with isotype control (grey). (C) Velocity of resting CD4⁺ and CD8⁺ T cells in microchannels with and without nivolumab or with IgG4k isotype control in presence of cHL cell lines. (D) Velocity of activated CD4⁺ and CD8⁺ T cells in micro-channels with and without nivolumab or with IgG4k isotype control in presence of cHL cell lines. (E) Duration of cell-cell contacts between Hodgkin-Reed-Sternberg (HRS) cells of cell line L-1236 and resting CD4⁺ or CD8⁺ T cells with or without nivolumab or with IgG4k isotype control. (F) Duration of cell-cell contacts between HRS cells of cell line L-428 and resting CD4⁺ or CD8⁺ T cells with or without nivolumab or with IgG4k isotype control. (G) Example of several short duration contacts between a resting CD4⁺ T cell and a HRS cell without nivolumab (HRS cell green, CD4⁺ T cell red). (H) Example of a long duration contact between a resting CD4⁺ T cell and a HRS cell with nivolumab (HRS cell green, CD4⁺ T cell red).

Figure 2.

Cell velocity and duration of cell-cell contacts in hyperplastic lymphoid tissue. Each dot represents the mean of a movie. Typically, 3 movies per condition were obtained. (A) Velocity of CD4⁺ T cells in hyperplastic lymphoid tissue (from 13 donors) with and without nivolumab or IgG4k isotype control antibody; *P<0.05, Kruskal-Wallis test with Dunn´s post hoc test for multiple testing. (B) Velocity of CD8⁺ T cells in hyperplastic lymphoid tissue (from 13 donors) with and without nivolumab or IgG4k isotype control antibody. (C) Velocity of CD20⁺ B cells in hyperplastic lymphoid tissue (from 13 donors) with and without nivolumab or IgG4k isotype control antibody. (D) Duration of cell-cell contacts between CD4⁺ T cells and CD20⁺ B cells in hyperplastic lymphoid tissue (from 13 donors) with and without nivolumab or IgG4k isotype control antibody; *P<0.05, Kruskal-Wallis test with Dunn´s post hoc test for multiple testing. (E) Duration of cell-cell contacts between CD8⁺ T cells and CD20⁺ B cells in hyperplastic lymphoid tissue (from 13 donors) with and without nivolumab or IgG4k isotype control antibody. (F) Examples for a short cell contact (left) between a CD8⁺ T cell (arrow head) and a CD20⁺ B cell (arrow, green cell) and longer duration cell contact (right) between a CD8⁺ T cell (arrow head, red cell) and a CD20⁺ B cell (arrow, green cell); CD8⁺ T cells labeled in red, CD20⁺ B cells labeled in green.

Figure 3.

Nivolumab incubation extends cell-cell contacts between CD4⁺ T cells and Hodgkin-Reed-Sternberg cells in classic Hodgkin lymphoma. The study analyzed 6 cases of classic Hodgkin lymphoma (cHL), including 5 cases in both untreated and nivolumab-treated conditions, and 1 case in untreated condition only. Each dot represents the mean of a movie. Typically, 3 movies per condition were obtained. (A) Velocity of CD4⁺ and CD8⁺ T cells as well as CD30⁺ Hodgkin-Reed-Sternberg (HRS) cells in cHL in untreated condition (0 hours [h]) and after incubation with nivolumab for 3 h or 5 h. (B) Number of cell-cell contacts between individual CD4⁺ T cells/movie and CD30⁺ HRS cells, individual CD8⁺ T cells/movie and CD30⁺ HRS cells as well as CD4⁺ and CD8⁺ T cells in cHL in untreated condition (0 h) and after incubation with nivolumab (3 h and 5 h). (C) Duration of cell-cell contacts between CD4⁺ T cells and CD30⁺ HRS cells, CD8⁺ T cells and CD30⁺ HRS cells as well as CD4⁺ and CD8⁺ T cells in cHL in untreated condition (0 h) and after incubation with nivolumab (3 h and 5 h). *P<0.05, Kruskal-Wallis test with Dunn´s post hoc test for multiple testing. (D) Duration of cell-cell contacts between CD4⁺ T cells and CD30⁺ HRS cells as well as CD8⁺ T cells and CD30⁺ HRS cells after 5 h nivolumab incubation. Cases were stratified based on major histocompatibility complex (MHC) I and II expression in the HRS cells. *P<0.05, Kruskal-Wallis test with Dunn´s post hoc test for multiple testing. (E) Examples for a short cell contact (left) between 3 CD4⁺ T cells (arrow heads) and a CD30⁺ HRS cell (#) in untreated condition and long duration cell contact (right) between a CD4⁺ T cell (arrow head) and a CD30⁺ HRS cell (#); CD4⁺ T cells labeled in pink, CD30⁺ HRS cells labeled in green; confocal micrsopscopy images 40x magnification.

Figure 4.

Cell tracks in classic Hodgkin lymphoma without and with nivolumab. Top row shows images (merged, CD4 pink, CD8 red, CD30 green) and the respective tracks of the cells in a 15-minute (min) movie of untreated classic Hodgkin lymphoma (cHL) tissue. Bottom row shows images (merged, CD4 pink, CD8 red, CD30 green) and the respective tracks of the cells in a 15-minute movie of cHL tissue from the same case after incubation with nivolumab for 5 hours. Scale bars are in ym/sec, with each individual scale bar 10 μM.