Research ArticleCell Biology

A defect in KCa3.1 channel activity limits the ability of CD8+ T cells from cancer patients to infiltrate an adenosine-rich microenvironment

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Science Signaling  24 Apr 2018:
Vol. 11, Issue 527, eaaq1616
DOI: 10.1126/scisignal.aaq1616
  • Fig. 1 HNSCC CD8+ T cells exhibit reduced chemotaxis in the presence of adenosine.

    (A and B) Trajectories of CD8+ T cells migrating along either a CXCL12 gradient (green triangles) or a combination gradient of CXCL12 with adenosine (ADO, blue triangles) in a representative HD (A) and HNSCC patient (B). Trajectories of at least 15 to 20 cells are shown for each condition, and the starting point of each cell trajectory is artificially set to the same origin. The red triangles represent Y-COM. (C and D) Y-COM values for cells migrating along either a CXCL12 gradient or a combination gradient of CXCL12 with adenosine in HDs (n = 7 donors) (C) and HNSCC patients (n = 16 patients) (D). (E) Percentage inhibition in the Y-COM values in the presence of CXCL12 and adenosine [values shown in (C) and (D)] in HD (n = 7 donors) and HNSCC (n = 16 patients). Horizontal red line represents mean values for each group. Data in (C) and (D) were analyzed by paired Student’s t test and in (E) by Mann-Whitney rank sum test.

  • Fig. 2 Tumor infiltration is dependent on the sensitivity of circulating CD8+ T cells to adenosine.

    (A) Immunohistochemistry of CD8 (top) and CD73 (bottom) expression (brown signal) in representative HNSCC tumor tissues showing low and high infiltration by CD8+ T cells and low and high CD73 expression (table S1). Scale bars, 100 μm. (B) Bar graph showing the number of CD8+ T cells (cells/mm2) within the tumor region in 16 HNSCC tumors. Please note that donor HNC-52 has a mean CD8+ T cell infiltration value of 5 cells/mm2. The broken red line represents the median value for the 16 HNSCC patients. The tumors with CD8+ T cell infiltration above the median value were considered to be “well infiltrated” (referred to as high in table S1), whereas the tumors with CD8+ T cell infiltration below the median value were considered to be “poorly infiltrated” (referred as low in table S1). The bars represent means ± SEM. (C) Correlation between CD8+ T cell infiltration and percentage reduction of the Y-COM values in the presence of CXCL12 and adenosine (values shown in Fig. 1E) in nine HNSCC patients that were scored as CD73High (see table S1). Correlation was measured by Spearman rank-order correlation test (P = 0.0301; correlation coefficient, ρ = −0.700).

  • Fig. 3 A2AR mediates the suppressive effect of adenosine on the chemotaxis of HNSCC CD8+ T cells.

    (A) Y-COM values for HNSCC CD8+ T cells migrating along either a CXCL12 gradient (n = 6 patients), a combination gradient of CXCL12 with CGS21680 (n = 6 patients), or CXCL12 with adenosine (n = 4 patients). (B) Percentage inhibition in the Y-COM values for each individual experiment shown in (A) after incubation with CGS21680 or adenosine. Horizontal red lines represent mean values for each group. (C) Y-COM values for HNSCC CD8+ T cells pretreated with or without 1 μM SCH58261 migrating toward CXCL12 in the presence of adenosine. Untreated CD8+ T cells in a CXCL12 gradient were used as controls (n = 5 patients). (D) Percentage inhibition in the Y-COM values by adenosine for each of the donors shown in (C) with or without SCH58261 pretreatment. Horizontal red line represents mean values for each group. Data in (A) and (C) were analyzed by one-way repeated measures analysis of variance (ANOVA) [P = 0.010 for (A) and P = 0.001 for (C)]; data in (B) and (D) were analyzed by Student’s t test.

  • Fig. 4 A2AR expression and A2AR signaling are not altered in HNSCC CD8+ T cells.

    (A) ADORA2A expression in activated HD and HNSCC CD8+ T cells was quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Data are the fold change in ADORA2A expression relative to GAPDH expression. The data were normalized to the mean ADORA2A expression in HD. Data are means ± SEM for from four HD and five HNSCC patients. (B) Representative flow cytometry histograms showing A2AR expression in resting and activated CD8+ T cells from HD and HNSCC. (C and D) Mean fluorescence intensity (MFI) of A2AR measured in resting (C) and activated (D) CD8+ T cells from HD (n = 6 donors) and HNSCC patients (n = 7 patients). (E) cAMP concentration in CD8+ T cells from HD (n = 7 donors) and HNSCC patients (n = 7 patients). (F) Relative PKA activity in CD8+ T cells from HD (n = 3 donors) and HNSCC patients (n = 4 patients). Horizontal red line represents mean values for each group. Data in (C), (D), and (F) were analyzed by Mann-Whitney rank sum test; data in (A) and (E) were analyzed by Student’s t test.

  • Fig. 5 KCa3.1 channel activity is reduced in HNSCC CD8+ T cells.

    (A) Representative KCa3.1 currents in CD8+ T cells recorded in whole-cell voltage clamp configuration from an HD and HNSCC patient. Currents were normalized for the maximum current at +40 mV to ease comparison of the KCa3.1 conductance at hyperpolarizing voltages. (B) KCa3.1 conductance (normalized to cell capacitance, G/C) measured in activated CD8+ T cells from HD (n = 30 cells, six donors) and HNSCC patients (n = 21 cells, four patients). (C) Kv1.3 channel current density measured in activated CD8+ T cells from HD (n = 25 cells, five donors) and HNSCC patients (n = 21 cells, four patients). For (B) and (C), the data are normalized to values measured in activated CD8+ T cells from HD, and the bars represent mean ± SEM. (D) Representative flow cytometry histograms showing KCa3.1 expression in resting and activated CD8+ T cells from HD and HNSCC patients. (E) MFI of KCa3.1 measured in resting and activated CD8+ T cells from HD (n = 6 donors) and HNSCC patients (n = 7 patients). (F) KCa3.1 MFI in activated CD8+ T cells from HD (n = 6 donors) and HNSCC (n = 7 patients). Horizontal red line represents mean values for each group. Data in (B), (C), and (F) were analyzed by Mann-Whitney rank sum test; data in (E) were analyzed by paired Student’s t test.

  • Fig. 6 Activation of KCa3.1 channels restores the chemotaxis of HNSCC CD8+ T cells in the presence of adenosine.

    (A) KCa3.1 channel conductance in the presence or absence of 100 μM 1-EBIO was measured in activated CD8+ T cells from HD (n = 17 cells, four donors) and HNSCC patients (n = 24 cells, five patients). The data were normalized to untreated (no 1-EBIO) activated cells from HD. The data are means ± SEM. (B) Y-COM values calculated for HNSCC CD8+ T cells migrating along either a CXCL12 gradient or a combination gradient of CXCL12 with adenosine with or without preincubation with 20 μM 1-EBIO (n = 5 patients). (C) Percentage inhibition in the Y-COM values (B) of the cells pretreated with 1-EBIO. Horizontal red line represents mean values for each group. Data in (A) were analyzed by two-way ANOVA, whereas data in (B) were analyzed with one-way repeated measures ANOVA (P = 0.009) and (C) with paired Student’s t test.

  • Table 1 Demographics of HNSCC patients enrolled in the study.

    Patients matching the inclusion criteria (n = 39) were enrolled in the study. ECOG (Eastern Cooperative Oncology Group) performance status describes how the disease affects the daily living abilities of the patient. For evaluating smoking status, pack years are calculated by multiplying the number of packs of cigarettes smoked per day by the number of years the person has smoked. Tumor stage from T1 to T4 refers to the size and extent of the tumors. The involvement of regional lymph nodes is referred by N1 to N3 depending on the number and location of the lymph nodes involved. N0 denotes absence of cancer in the regional lymph nodes.

    Age (at the time of sample collection)Years
      Range34 to 77
      Mean56
    VariableNumber (%)
    Gender
      Male31 (79)
      Female8 (21)
    Site
      Oral cavity12 (31)
      Oropharynx17 (44)
      Larynx8 (21)
      Hypopharynx1 (2)
      Nasopharynx1 (2)
    Tumor stage
      T17 (18)
      T210 (26)
      T39 (23)
      T411 (28)
      Unknown2 (5)
    Nodal status
      N08 (20)
      N14 (10)
      N224 (62)
      N31 (3)
      Unknown2 (5)
    ECOG performance status
      025 (64)
      19 (23)
      23 (8)
      Unknown2 (5)
    Smoking
      No (<10 pack years)15 (38)
      Yes (>10 pack years)24 (62)
    Alcohol
      No28 (72)
      Yes (>5 drinks/week)11 (28)
    p16 status
      Positive16 (41)
      Negative13 (33)
      Unknown10 (26)
  • Table 2 Effect of adenosine on the chemotaxis of HD and HNSCC CD8+ T cells.

    Activated CD8+ T cells from HD and HNSCC patients were exposed to a gradient of either CXCL12 or CXCL12 and adenosine (ADO), and the indicated values were measured. Results are presented as means ± SEM for all measured values. Y-COM, center of mass along the y axis, along the chemokine gradient; X-COM, center of mass along the x axis, perpendicular to the chemokine gradient; FMIy, forward migration index in the direction of the y axis (represents the efficiency of forward migration toward the chemokine gradient); FMIx, forward migration index in the direction of the x axis (represents the efficiency of migration perpendicular to the chemokine gradient respectively).

    ParameterCXCL12CXCL12 + ADOP value (CXCL12 versus
    CXCL12 + ADO)
    HD (n = 5)
    X-COM (μm)5.660 ± 4.7391.586 ± 5.2820.610
    Y-COM (μm)43.279 ± 9.221*33.366 ± 10.4240.134
    FMIx0.016 ± 0.019−0.001 ± 0.0150.523
    FMIy0.141 ± 0.0310.109 ± 0.029§0.001
    Directness0.203 ± 0.0240.202 ± 0.0170.925
    Velocity (μm/s)0.210 ± 0.0210.182 ± 0.0210.478
    Accumulated distance (μm)315.462 ± 31.352272.336 ± 30.9090.475
    Euclidean distance (μm)62.122 ± 6.27756.081 ± 10.1040.526
    HNSCC (n = 6)
    X-COM (μm)−7.125 ± 9.5963.943 ± 11.9830.629
    Y-COM (μm)46.773 ± 18.178ǁ¶4.281 ± 4.829#0.029
    FMIx−0.030 ± 0.0230.024 ± 0.0360.404
    FMIy0.191 ± 0.039**0.005 ± 0.020††<0.001
    Directness0.256 ± 0.0400.188 ± 0.0170.080
    Velocity (μm/s)0.146 ± 0.0420.139 ± 0.0400.495
    Accumulated distance (μm)211.297 ± 62.163206.839 ± 59.6200.725
    Euclidean distance (μm)38.126 ± 8.52438.145 ± 10.9530.438

    *P = 0.002 versus X-COM (in HD).

    P = 0.0876 versus X-COM (in HD).

    P = 0.008 versus FMIx (in HD).

    §P = 0.046 versus FMIx (in HD).

    ǁP = 0.031 versus X-COM (in HNSCC).

    ¶Statistical significance is measured by Wilcoxon signed-rank test; all other P values are measured by paired Student’s t test.

    #P = 0.970 versus X-COM (in HNSCC).

    **P = 0.011 versus FMIx (in HNSCC).

    ††P = 0.610 versus FMIx (in HNSCC).

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/11/527/eaaq1616/DC1

      Fig. S1. HD and HNSCC CD8+ T cells chemotax toward CXCL12.

      Fig. S2. KCa3.1 channel blockade inhibits the chemotaxis of HD and HNSCC CD8+ T cells.

      Fig. S3. Activation of CD8+ T cells from HD and HNSCC patients.

      Fig. S4. Activation of KCa3.1 channels by NS309 restores the chemotaxis of HNSCC CD8+ T cells in the presence of adenosine.

      Table S1. Clinicopathologic characteristics of individual HNSCC patients.

      Table S2. CD8+ T cells from HDs and HNSCC patients chemotax toward CXCL12 similarly.

      Table S3. Electrophysiological parameters of resting and activated CD8+ T cells isolated from HD and HNSCC patients.

    • Supplementary Materials for:

      A defect in KCa3.1 channel activity limits the ability of CD8+ T cells from cancer patients to infiltrate an adenosine-rich microenvironment

      Ameet A. Chimote, Andras Balajthy, Michael J. Arnold, Hannah S. Newton, Peter Hajdu, Julianne Qualtieri, Trisha Wise-Draper, Laura Conforti*

      *Corresponding author. Email: laura.conforti{at}uc.edu

      This PDF file includes:

      • Fig. S1. HD and HNSCC CD8+ T cells chemotax toward CXCL12.
      • Fig. S2. KCa3.1 channel blockade inhibits the chemotaxis of HD and HNSCC CD8+ T cells.
      • Fig. S3. Activation of CD8+ T cells from HD and HNSCC patients.
      • Fig. S4. Activation of KCa3.1 channels by NS309 restores the chemotaxis of HNSCC CD8+ T cells in the presence of adenosine.
      • Table S1. Clinicopathologic characteristics of individual HNSCC patients.
      • Table S2. CD8+ T cells from HDs and HNSCC patients chemotax toward CXCL12 similarly.
      • Table S3. Electrophysiological parameters of resting and activated CD8+ T cells isolated from HD and HNSCC patients.

      [Download PDF]


      © 2018 American Association for the Advancement of Science

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