Lymphocyte activation gene 3: a novel therapeutic target in chronic lymphocytic leukemia

Abstract

A novel therapeutic approach in cancer, attempting to stimulate host anti-tumor immunity, involves blocking of immune checkpoints. Lymphocyte activation gene 3 (LAG3) is an immune checkpoint receptor expressed on activated/exhausted T cells. When engaged by the major histocompatibility complex (MHC) class II molecules, LAG3 negatively regulates T-cell function, thereby contributing to tumor escape. Intriguingly, a soluble LAG3 variant activates both immune and malignant MHC class II-presenting cells. In the study herein, we examined the role of LAG3 in the pathogenesis of chronic lymphocytic leukemia, an MHC class II-presenting malignancy, and show that chronic lymphocytic leukemia cells express and secrete LAG3. High levels of surface and soluble LAG3 were associated with the unmutated immunoglobulin variable heavy chain leukemic subtype and a shorter median time from diagnosis to first treatment. Utilizing a mechanism mediated through MHC class II engagement, recombinant soluble LAG3-Ig fusion protein, LAG3-Fc, activated chronic lymphocytic leukemia cells, induced anti-apoptotic pathways and protected the cells from spontaneous apoptosis, effects mediated by SYK, BTK and MAPK signaling. Moreover, LAG3 blocking antibody enhanced in vitro T-cell activation. Our data suggest that soluble LAG3 promotes leukemic cell activation and anti-apoptotic effects through its engagement with MHC class II. Furthermore, MHC class II-presenting chronic lymphocytic leukemia cells may affect LAG3-presenting T cells and impose immune exhaustion on their microenvironment; hence, blocking LAG3-MHC class II interactions is a potential therapeutic target in chronic lymphocytic leukemia.

Figure 1.

Full-length LAG3 expression in CLL patients. (A-C) Full-length LAG3 mRNA levels in CD19⁺ selected normal B cells and CLL cells quantitated by qPCR, and normalized to GAPDH. (A) LAG3 mRNA expression in normal B cells (n=7) vs. CLL cells (n=28). (B) LAG3 mRNA expression in M-IGHV cells (n=15) compared to UM-IGHV CLL cells (n=13). (C) Kaplan-Meier analysis of time from diagnosis to first treatment in CLL patients with “low” and “high” LAG3 mRNA levels (n=28), using the median value as cutoff level. (D-E) Detection of LAG3 protein levels by Western blot assay in CD19⁺ purified CLL cells, M-IGHV cells compared to UM-IGHV CLL cells. (D) Representative blot analyzing CLL cells from 8 patients, 4 M-IGHV and 4 UM-IGHV CLL (patients’ characteristics are presented in the Online Supplementary Table S1, according to the designated numbers). Actin was used to verify equal loading. (E) Statistic analysis of CLL cells from 16 patients with CLL, 8 M-IGHV compared to 8 UM-IGHV CLL cells. (F-I) LAG3 expression in peripheral blood samples, evaluated by flow cytometry. (F) Representative dot plots showing surface and intracellular expression of LAG3 in CLL cells. (G) Summary of surface LAG3 mean fluorescence intensity (MFI) normalized to isotype control, in normal B cells (n=8) compared to CLL cells (n=22), isolated from peripheral blood of healthy controls and patients with CLL, respectively. (H) Surface LAG3 MFI normalized to isotype control (MFIR) as detected in peripheral blood of M-IGHV (n=11) and UM-IGHV (n=11) CLL cells. (I) Summary of surface LAG3 MFIR in CLL, CD4⁺ and CD8⁺ cells isolated from peripheral blood of patients with CLL (n=22). CLL: chronic lymphocytic leukemia; LAG3: lymphocyte activation gene 3; ns: not significant.

Figure 2.

Soluble (s)LAG3 is associated with UM-IGHV status and progressive disease. (A-B) LAG3V3 mRNA levels encoding sLAG3 were quantitated in CD19⁺ selected normal B and CLL cells by qPCR, and normalized to GAPDH. (A) LAG3V3 mRNA levels in normal B cells (n=7), M-IGHV (n=12) and UM-IGHV (n=11) CLL cells. (B) Kaplan-Meier analysis of time from diagnosis to first treatment in patients with CLL (n=23), expressing “low” and “high” LAG3V3 mRNA levels, using the median value as cutoff level. (C-D) Serum sLAG3 levels in CLL patients and healthy individuals, determined by ELISA. (C) Serum sLAG3 levels in healthy controls (n=8) and patients with M-IGHV (n=16) and UM-IGHV (n=17) CLL. (D) Comparison between serum sLAG3 levels in CLL patients with either stable (n=17) or progressive (n=18) disease. (E) Measurement of sLAG3 levels in the medium of cultured CLL cells; negatively selected CLL cells were cultured and medium sLAG3 levels in the culture medium were determined by ELISA after 24, 48 and 72 hours (n=5). mRNA (RQ): messenger RNA (relative quantification).

Figure 3.

Soluble (s)LAG3 binds and activates MHC class II molecules on CLL cells. (A-B) Detection of sLAG binding to CLL cells: peripheral blood CLL cells were incubated for 15 min with either LAG3-Fc, LAG3-Fc after pre-incubation with anti-LAG3 blocking antibody, or Ig-Fc that served as control. LAG3 binding to CLL cells was detected by flow cytometry, using a fluorophore-conjugated secondary antibody (anti-human Fc). (A) Representative dot plot analysis showing that LAG3-Fc binding to CLL cells (middle box) was completely abolished by anti-LAG3 blocking antibody (right box). (B) The mean fluorescence intensity (MFI) of LAG3-binding CLL cells after incubation with LAG3-Fc (middle bar) decreases to control levels after pre-incubation with anti-LAG3 antibodies (right bar); cumulative results of 11 experiments. (C) Measurement of CD86 surface expression on CLL cells in response to sLAG3 activation: CLL cells were incubated with either LAG3-Fc or Ig-Fc that served as control for 24 hours, and surface CD86 expression was analyzed on CD5⁺/CD23⁺ gated CLL cells by flow cytometry. C-Left: representative dot plots showing an increase in surface CD86 expression on CLL cells in the presence of LAG3-Fc. C-middle: surface CD86 MFI levels on LAG3-Fc-activated CLL cells are presented as fold change increase over control (Ig-Fc) levels, n=11. C-Right: comparison of CD86 MFI expression on CLL cells incubated with either Ig-Fc (control, left bar), LAG3-Fc (middle bar) or LAG3-Fc pre-incubated with anti-LAG3 blocking antibody [right bar, (n=7)]. (D) Changes in the mean fluorescence levels (normalized to baseline) of pERK and pAKT in CLL cells, measured by flow cytometry at 0, 5, 15, 45, 60 and 120 min after activation by LAG3-Fc (n=5). CLL: chronic lymphocytic leukemia; LAG3: lymphocyte activation gene 3.

Figure 4.

Soluble (s)LAG3 protects CLL cells from spontaneous apoptosis. (A-F) Peripheral blood CLL cells were incubated for 48 or 72 hours with either Ig-Fc (control), LAG3-Fc, or with LAG3-Fc pre-incubated with anti-LAG3 blocking antibody aimed at the MHCII molecules binding site. Cell viability was determined by flow cytometry, using an Annexin V/PI apoptosis detection kit. The levels of anti-apoptotic proteins and cleaved PARP were determined by Western blot analysis and quantified. (A) Representative dot plots showing the percentage of apoptotic cells in the presence of Ig-Fc (left), LAG3-Fc (middle) and LAG3-Fc with anti-LAG3 blocking antibody (right). (B-C) Percentage of live CLL cells in the presence of either Ig-Fc (control) or LAG3-Fc, as seen in 10 independent experiments after 48 (B) and 72 hours (C) incubation. (D) The percentage of live CLL cells in the presence of Ig-Fc control [marked as (−)], or LAG3-Fc [marked as (+)], with or without 1 hour pre-incubation with PD98059, wortmannin (left graph), ibrutinib, R406 or idelalisib (right graph). After being cultured for 48 hours, cell viability was determined by flow cytometry, using an Annexin V/PI apoptosis detection kit (n=7). (E) The percentage of live CLL cells cultured with either Ig-Fc (left bar), LAG3-Fc (middle bar), or LAG3-Fc pre-incubated with anti-LAG3 (right bar), for 1 hour, washed and incubated for an additional 72 hours before being analyzed by flow cytometry (n=6). (F) Representative Western blot analysis showing the levels of cleaved PARP, MCL-1, Bcl-XL and Bcl-2 in CLL cells after 72 hours incubation with Ig-Fc as a control [marked (−)] or LAG3-Fc [marked (+)]. Actin was used to verify equal loading. (G) Cumulative results from 8 independent experiments, performed as described in Figure 4F. Shown are quantified levels of cleaved PARP, MCL-1, Bcl-XL and Bcl-2 in LAG3-Fc activated CLL cells, normalized to control (incubation with Ig-Fc). (H) The percentage of apoptotic CLL cells increased following LAG3 blockade. The levels of apoptotic CLL cells were determined after 72 hours incubation with anti-LAG3 blocking antibody and normalized to control levels in 15 independent experiments. LAG3: lymphocyte activation gene 3; CLL: chronic lymphocytic leukemia; ns: not significant.

Figure 5.

Increased surface LAG3 expression on CD8⁺ tumor infiltrating lymphocytes and blocking LAG3 enhance in vitro T-cell activation. (A) The expression of surface LAG3 on CD4⁺ and CD8⁺ cells (analyzed by flow cytometry), in 10 paired samples of peripheral blood (PB) and secondary lymphoid tissues [sec. LN; lymph node (n=3) or spleen cells (n=7)] from patients with CLL. (B) A representative dot plot analysis, showing co-expression of surface LAG3 and PD1 on lymph node-derived CD8⁺ lymphocytes isolated from a patient with CLL (n=5). (C) CD69 expression on activated T cells, with or without LAG3 and PD1 blockade. Cells from 7 patients with CLL were incubated for 48 hours in the presence or absence of the indicated blocking antibodies or with IgG1 isotype control. T cells were then activated by CD3/CD28 beads for 6 hours and CD69 expression was analyzed in CD4⁺ (left) and CD8⁺ cells (right) by flow cytometry. LAG3: lymphocyte activation gene 3; MFI: mean fluorescence intensity; ns: not significant.