Targeted knockdown of the adenosine A2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells

Abstract

In solid malignancies, including head and neck squamous cell carcinoma (HNSCC), the immunosuppressive molecule adenosine, which accumulates in the tumor, suppresses cytotoxic CD8⁺ T cell functions including chemotaxis and tumor infiltration. Adenosine functions through binding to the adenosine A_2A receptor (A_2AR) present on T cells. In order to increase T cell migration into the tumor, the negative effect of adenosine must be abrogated. Systemic drug treatments targeting A_2AR are available; however, they could lead to negative toxicities due to the broad expression of this receptor. Herein, we developed a lipid nanoparticle (NP)-based targeted delivery approach to knock down A_2AR in T cells in order to increase their chemotaxis in the presence of adenosine. By using flow cytometry, immunofluorescence, qRT-PCR, and 3D-chemotaxis, we demonstrated that CD45RO-labeled nanoparticles delivering ADORA2A gene-silencing-RNAs decreased ADORA2A mRNA expression and rescued the chemotaxis of HNSCC CD8⁺ memory T cells. Overall, the data indicate that targeting the adenosine signaling pathway with lipid NPs is successful at suppressing the inhibitory effect of adenosine on the chemotaxis of HNSCC memory T cells, which could ultimately help increase T cell infiltration into the tumor.

Graphical abstract

Figure 1

ADORA2A siRNAs decrease ADORA2A mRNA and A_2AR protein expression (A) Representation of the structure of the lipid NPs labeled with fluorescent streptavidin and biotinylated targeting antibody and loaded with siRNAs. PE: phosphoethanolamine; PEG: polyethylene glycol (B) Relative quantity (RQ) of ADORA2A mRNA expression in activated HD CD3⁺ or CD8⁺ T cells 24 h post-transfection with 10 nM scr or ADORA2A siRNAs (n = 5; 3 CD3⁺ T cell samples, 2 CD8⁺ T cell samples). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as the reference gene. Data are shown as mean ± standard deviation and significance is determined by a paired Student’s t test. (C) (Left) Fold change of normalized A_2AR protein expression in activated HD CD3⁺ or CD8⁺ T cells 24 h (n = 5; 3 CD3⁺ T cell samples, 2 CD8⁺ T cell samples), 48 h (n = 5; 3 CD3⁺ T cell samples, 2 CD8⁺ T cell samples), or 72 h (n = 4; 2 CD3⁺ T cell samples, 2 CD8⁺ T cell samples) post-transfection with 10 nM ADORA2A siRNAs as compared to scr-RNAs. (Right) The normalized MFI of A_2AR expression showing the highest degree of A_2AR protein knockdown post-transfection with 10 nM ADORA2A siRNAs as compared to corresponding scr-RNAs for each individual sample regardless of time point at which knockdown occurred. Data are shown as mean ± standard error of the mean (n = 5). Data are compared using a paired Student’s t test. (D) Flow cytometry gating strategy for determining A_2AR expression in transfected cells. The cells were first gated on T lymphocytes and then on GFP fluorescence (indicating that the cells were successfully transfected with siRNAs) to determine A_2AR expression. The unstained sample is indicated in orange in the histogram while the A_2AR expression of cells treated with scr-RNAs is indicated in blue and the A_2AR expression of cells treated with ADORA2A siRNAs is indicated in red. (E) Representative histogram showing A_2AR antibody specificity where CD3⁺ T cells were stained with A_2AR antibody ± peptide followed by secondary antibody. Unstained cells are indicated in gray, A_2AR antibody + peptide is indicated in red and A_2AR antibody alone is indicated in blue. Shown here is a representative experiment from two identical experiments performed in HDs.

Figure 2

CD8-NPs are specific toward CD8⁺ T cells (A) Flow cytometry gating strategy for determining specificity of CD8-NPs for CD8⁺ T cells. Cells were first gated based on the CD3⁺ lymphocyte population (left). The unstained sample was then used to determine the quadrant gate placement for the CD4 stain and NP fluorescence (middle). A representative plot for the distribution of CD8-NPs in CD4^– and CD4⁺ T cells is shown on the right. Quadrant 1 represents the CD4^– T cells that are positive for CD8-NPs. Quadrant 2 represents the CD4⁺ T cells that are positive for CD8-NPs. Quadrant 3 represents CD4⁺ T cells that are negative for CD8-NPs. Quadrant 4 represents CD4^– T cells that are negative for CD8-NPs. Representative plots for n = 4 independent experiments. (B) Distribution of CD8-NPs in HD CD4^– and CD4⁺ T cells within a CD3⁺ T cell population (the percent of CD8-NPs bound to CD4^– T cells versus the percent of CD8-NPs bound to the CD4⁺ T cells; n = 4). (C) Percentage of HD CD4^– and CD4⁺ T cells within a CD3⁺ T cell population that are positive for CD8-NPs (n = 4). (B and C) Data are bar graphs representing the mean ± standard deviation. Comparisons are made using paired Student’s t test. (D) Flow cytometry gating strategy for determining non-specific binding of NPs (labeled with IgG antibody) for CD8⁺ T cells. Cells were first gated based on the CD3⁺ lymphocyte population (left). The unstained sample was then used to determine the quadrant gate placement for the CD4 stain and NP fluorescence (middle). A representative plot for the distribution of IgG-NPs in CD4^– and CD4⁺ T cells is shown on the right. Quadrant 1 represents the CD4^– T cells that are positive for IgG-NPs. Quadrant 2 represents the CD4⁺ T cells that are positive for IgG-NPs. Quadrant 3 represents CD4⁺ T cells that are negative for IgG-NPs. Quadrant 4 represents CD4^– T cells that are negative for IgG-NPs. Representative plots for n = 3 independent experiments.

Figure 3

ADORA2A siRNAs delivered via CD45RO-NPs knock down ADORA2A mRNA expression in CD8⁺ memory T cells (A) RQ of ADORA2A mRNA expression in activated CD8⁺ T cells 48–72 h post-incubation with CD8-NPs loaded with 200 pmol scr or ADORA2A siRNAs (n = 2 for both time points). (B) RQ of ADORA2A mRNA expression in activated CD8 memory T cells 72 h post-incubation with CD45RO-NPs loaded with 200 pmol scramble or ADORA2A siRNAs (n = 2). (A and B) 18S rRNA was used as reference gene. Data are represented as mean ± standard deviation. Significance determined by paired Student’s t test.

Figure 4

CD8-NPs co-localize with the lysosome (A) Representative confocal images of an activated CD3⁺ T cell incubated with CD8-NPs (top panel) and CD45RO-NPs (bottom panel) for 24 h. NPs visualized in green. Lysosomal marker LAMP-1 is visualized in red. Merge of NPs with LAMP-1 is shown with co-localization indicated by the presence of yellow. All images shown are shown at the same scale. (B) Single cell percentage of NP raw integrated density (NP intensity) in the lysosome as compared to total NP intensity in the cell. Approximately 20–45 cells were visualized from a total of two donors. Data represented as boxplots: line indicates the median; lower box is the 25^th percentile; upper box is the 75^th percentile; and whiskers represent the 10^th and 90^th percentiles. Each dot represents an individual cell. Significance determined by Mann-Whitney rank sum test.

Figure 5

CD45RO(A_2AR)-NPs rescue the chemotactic ability of HNSCC CD8⁺ memory T cells in the presence of adenosine (A and B) Representative two point trajectories of T cells migrating along the CXCL10 gradient (green) or CXCL10 + adenosine (ADO) gradient (blue) in HNSCC CD8⁺ memory T cells treated with (A) CD45RO(scr)-NPs or (B) CD45RO(A_2AR)-NPs. Trajectories artificially set to start at the origin with the red triangle representing the Y-COM. (C) Y-COM of HNSCC CD8⁺ memory T cells treated with (left) CD45RO(scr)-NPs (n = 6) or (right) CD45RO(A_2AR)-NPs (n = 6) in the presence of CXCL10 or CXCL10 + adenosine. Significance was determined using (left) a Wilcoxon signed rank test or (right) paired Student’s t test. (D) Percent inhibition of Y-COM of HNSCC CD8⁺ memory T cells in the presence of CXCL10 + adenosine compared to CXCL10 alone when treated with CD45RO(scr)-NPs (n = 6) compared to CD45RO(A_2AR)-NPs (n = 6). Data are represented single dot plots (colored dots; wherein each dot represents a single donor) and as mean ± standard deviation (shown in black). Significance was determined using Student’s t test.