A defect in KCa3.1 channel activity limits the ability of ... · Julianne Qualtieri,2 Trisha Wise-Draper,3 Laura Conforti1‡ The limited ability of cytotoxic T cells to infiltrate

SC I ENCE S I GNAL ING | R E S EARCH ART I C L E

CELL B IOLOGY

1Division of Nephrology, Department of Internal Medicine, University of Cincinnati,Cincinnati, OH 45267, USA. 2Department of Pathology, University of Cincinnati,Cincinnati, OH 45267, USA. 3Division of Hematology Oncology, Department of Inter-nal Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.*Present address: Department of Pediatrics, Faculty ofMedicine, University of Debrecen,Egyetem tér 1, Debrecen 4032, Hungary.†Present address: Department of Biophysics and Cell Biology, Faculty of Dentistry,University of Debrecen, Egyetem tér 1, Debrecen 4032, Hungary.‡Corresponding author. Email: [email protected]

Chimote et al., Sci. Signal. 11, eaaq1616 (2018) 24 April 2018

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A defect in KCa3.1 channel activity limits the ability ofCD8+ T cells from cancer patients to infiltrate anadenosine-rich microenvironmentAmeet A. Chimote,1 Andras Balajthy,1* Michael J. Arnold,1 Hannah S. Newton,1 Peter Hajdu,1†

Julianne Qualtieri,2 Trisha Wise-Draper,3 Laura Conforti1‡

The limited ability of cytotoxic T cells to infiltrate solid tumors hampers immune surveillance and the efficacy of im-munotherapies in cancer. Adenosine accumulates in solid tumors and inhibits tumor-specific T cells. Adenosine inhibitsT cell motility through the A2A receptor (A2AR) and suppression of KCa3.1 channels. We conducted three-dimensionalchemotaxis experiments to elucidate the effect of adenosine on the migration of peripheral blood CD8+ T cells fromhead and neck squamous cell carcinoma (HNSCC) patients. The chemotaxis of HNSCC CD8+ T cells was reduced in thepresence of adenosine, and the effect was greater on HNSCC CD8+ T cells than on healthy donor (HD) CD8+ T cells. Thisresponse correlated with the inability of CD8+ T cells to infiltrate tumors. The effect of adenosine was mimicked by anA2AR agonist and prevented by an A2AR antagonist. We found no differences in A2AR expression, 3′,5′-cyclic adenosinemonophosphate abundance, or protein kinase A type 1 activity between HNSCC and HD CD8+ T cells. We instead de-tected a decrease in KCa3.1 channel activity, but not expression, in HNSCCCD8+ T cells. Activation of KCa3.1 channels by1-EBIO restored the ability of HNSCC CD8+ T cells to chemotax in the presence of adenosine. Our data highlight themechanism underlying the increased sensitivity of HNSCC CD8+ T cells to adenosine and the potential therapeutic ben-efit of KCa3.1 channel activators, which could increase infiltration of these T cells into tumors.

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INTRODUCTIONThe immune system plays an important role in cancer. In many solidmalignancies, including head and neck squamous cell carcinoma(HNSCC), an increased infiltration of cytotoxic CD8+ T cells into thetumor mass is often associated with good prognosis and response totherapy (1–3). This knowledge is indeed at the foundation of immunetherapies that increase the number and functionality of cytotoxictumor-infiltrating lymphocytes (TILs). Adoptive T cell (ATC) transfer,chimeric antigen receptor (CAR)T cells, and checkpoint inhibitors haveshown promising results in many forms of cancer. Although thesetherapies are very effective in increasing the functional capabilities ofT cells, the modified T cells still maintain a limited ability to infiltratethe tumor mass and resist the immunosuppressive tumor micro-environment (TME) (4–7). The inability of tumor-specific T cells totraffic to a solid tumor represents a great challenge for effective immu-notherapy. The unique features of the TME contribute to the reducedinfiltration and functionality of TILs (8). Thus, understanding how theTME limits T cell infiltration is necessary for improving immune sur-veillance in cancer and developing effective immunotherapies.

The purine nucleoside adenosine accumulates in the TME and hasbeen associated with tumor progression, enhanced metastatic poten-tial, and poor prognosis (9–11). In vivo studies provide conclusive ev-idence of the importance of adenosine in cancer (12–15). Abrogationof the adenosine signaling pathway, either through knockdown of theadenosine A2A receptor (A2AR), a G protein (heterotrimeric guaninenucleotide–binding protein)–coupled receptor (GPCR) expressed in

immune cells, or by A2AR antagonists, reduces tumor burden in tumor-bearing mice, increases survival, and increases the efficacy of immuno-therapies (5, 6, 9, 16–18). Furthermore, knockdown of CD73, an enzymenecessary for adenosine production, completely restores the efficacy ofATC therapies and leads to long-term tumor-free survival of tumor-bearing mice (19, 20). Adenosine is thus emerging as an importantcheckpoint inhibitor of the antitumor T cell response (21). In addition,we have shown that adenosine limits cytokine release and motility inhuman peripheral blood T lymphocytes through calcium-activatedKCa3.1 potassium (K+) channels (22).

Ion channels regulatemultiple functions of T lymphocytes includingcytokine, granzyme B production, and motility (23–26). Two K+ chan-nels, the voltage-dependent Kv1.3 and the Ca2+-activated KCa3.1, reg-ulate the electrochemical driving force for Ca2+ influx that is necessaryfor nuclear factor of activated T cells nuclear translocation, gene expres-sion, and effector functions (26). These two channels also mediate theresponse to two key immune suppressive elements of the TME: hypoxia(Kv1.3) and adenosine (KCa3.1) (22, 27–29). Defects in Kv1.3 channelshave been reported in TILs and are associated with their diminishedcytotoxicity (30). The importance of K+ channels of T lymphocytes incancer was confirmed in mice where overexpression of the Kv1.3channel increased interferon-g (IFN-g) production, reduced tumor bur-den, and increased survival (31, 32). We have shown that in human Tlymphocytes, KCa3.1 channels reside at the uropod of polarizedmobileT cells and mediate the inhibitory effect of adenosine (22, 24). Adeno-sine, through A2AR, stimulates 3′,5′-cyclic adenosine monophosphate(cAMP) production and protein kinase A type 1 (PKAI) activation, in-hibits KCa3.1 channels, and suppresses T cell motility (22). We specu-lated that this mechanism could have important implications in theability of effector T cells to infiltrate the tumor mass. Furthermore, itmaybeparticularly important inHNSCC,where effectorT cells aremoresensitive to adenosine than are their healthy counterparts; that is, aden-osine inhibits proliferation and cytokine releasemore in HNSCC effectorT cells than in healthy donor (HD) cells (33). This enhanced sensitivity

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has been attributed to a reduction in adenosine deaminase activity andincreased A2AR signaling. To date, the chemotactic abilities of HNSCCCD8+ T cells and their response to adenosine have not been studied.Here, we investigated the effect of adenosine on the chemotaxis ofcirculating CD8+ T cells of HNSCC patients and the mechanismsthat mediate their heightened response to adenosine. We provide evi-dence of a role for KCa3.1 channels in the adenosine-mediated suppres-sion of the chemotaxis ofHNSCCCD8+T cells and suggest that KCa3.1channels may have therapeutic potential to increase the ability ofHNSCC CD8+ T cells to infiltrate an adenosine-rich TME.

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RESULTSThe chemotaxis of circulating CD8+ T cells of HNSCC patientsis impaired by adenosineThe TME is characterized by rapidly dividing tumor cells in a fibrousmatrix with a variable degree of immune cell infiltrate. Adenosineaccumulates in the hypoxic TME, and the increased adenosine concen-tration contributes to the inhibition of the antitumor immune responseby cytotoxic CD8+ T cells (10, 34). Experiments were performed toassess whether adenosine had any effect on the chemotaxis of HNSCCCD8+ T cells (for the demographic and clinicopathologic features of thepatients, see Table 1 and table S1, respectively). We studied the effect ofadenosine on the chemotaxis of activated HNSCC CD8+ T cells in atumor-like experimental setting and compared it to that of CD8+ T cellsfromHDs. To mimic the TME, we used the m-Slide Chemotaxis cham-ber, which enables the generation of a stable, three-dimensional (3D)collagenous matrix in which CD8+ T cells migrate in response to a che-mokine gradient (35, 36). All chemotaxis experiments were conductedwith CXCL12 (37). Also, all experiments were conducted on CD8+ Tcells activated in vitro with anti-CD3 and anti-CD28 antibodies for 3to 4 days, unless otherwise specified. In the absence of CXCL12, cellsexhibited random migration within the collagen matrix, whereas inthe presence of a CXCL12 gradient, the cells migrated toward the high-est CXCL12 concentration (Fig. 1, A and B, fig. S1, and table S2). Weobserved no significant differences in the baseline chemotactic responseof HD and HNSCC CD8+ T cells as indicated by similar values for Y-COM (y coordinate of the center of mass, that is, the averaged posi-tion the cells achieved at the end of the experiment; red triangles inFig. 1 and fig. S1), FMIy (y coordinate of the forward migrationindex), velocity, accumulated distance, directness, and Euclidean dis-tance (table S2). Hereafter, Y-COM is used to quantify a chemotacticresponse. To evaluate what effect adenosine had on the chemotaxis ofHD and HNSCC CD8+ T cells, we measured CXCL12-driven chemo-taxis in the presence or absence of a concomitant adenosine gradient.Adenosine inhibited chemotaxis and this effect was significantly morepronounced in HNSCC CD8+ T cells than in HD T cells (Fig. 1, A andB). Adenosine decreased Y-COM in cells from five of seven HDs(Fig. 1C). In contrast, HNSCC CD8+ T cells displayed greater sensitiv-ity to adenosine than did their healthy counterparts (Fig. 1D). ActivatedT cells fromHDs exhibited 26%overall reduction in the Y-COMvalues,whereas adenosine inhibited the Y-COM values of HNSCC donor cellsby 80% (Fig. 1E). Overall, HNSCC cells lost their chemotactic ability inthe presence of adenosine, as indicated by loss of significant differencesbetween Y-COM and X-COM values (the COM y and x coordinates),and between FMIy and FMIx (FMI in the x and y directions; Fig. 1B andTable 2). Note that adenosine did not induce any significant changes inother parameters that define T cell migration, such as cell velocity, di-rectness, or accumulated distance in either HDorHNSCCCD8+T cells


Table 1. Demographics of HNSCC patients enrolled in the study.Patients matching the inclusion criteria (n = 39) were enrolled in thestudy. ECOG (Eastern Cooperative Oncology Group) performance statusdescribes how the disease affects the daily living abilities of the patient. Forevaluating smoking status, pack years are calculated by multiplying thenumber of packs of cigarettes smoked per day by the number of years theperson has smoked. Tumor stage from T1 to T4 refers to the size and extentof the tumors. The involvement of regional lymph nodes is referred by N1toN3depending on the number and location of the lymphnodes involved. N0denotes absence of cancer in the regional lymph nodes.

Age (at the time of sample collection)
Years
Range
34 to 77
Mean
56
Variable
Number (%)
Gender

Male
31 (79)
Female
8 (21)
Site

Oral cavity
12 (31)
Oropharynx
17 (44)
Larynx
8 (21)
Hypopharynx
1 (2)
Nasopharynx
1 (2)
Tumor stage

T1
7 (18)
T2
10 (26)
T3
9 (23)
T4
11 (28)
Unknown
2 (5)
Nodal status

N0
8 (20)
N1
4 (10)
N2
24 (62)
N3
1 (3)
Unknown
2 (5)
ECOG performance status

0
25 (64)
1
9 (23)
2
3 (8)
Unknown
2 (5)
Smoking

No (<10 pack years)
15 (38)
Yes (>10 pack years)
24 (62)
Alcohol

No
28 (72)
Yes (>5 drinks/week)
11 (28)
p16 status

Positive
16 (41)
Negative
13 (33)
Unknown
10 (26)
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(Table 2). Overall, these data show that adenosine inhibits the chemo-taxis of HNSCCCD8+ T cells. To test whether adenosine accumulationmight explain the inability of CD8+ T cells to infiltrate the adenosine-rich TME,we performed immunohistochemical staining ofHNSCC tu-mors for CD8 and CD73 (ecto-5′-nucleotidase), an enzyme responsiblefor adenosine production that is used as a marker of adenosine abun-dance in solid tumors (19). We found a mixed degree of CD8+ infiltra-tion andCD73 expression inHNSCC tumors (Fig. 2, A and B, and tableS1). However, in the small cohort of patients with high intratumoralCD73 expression (n = 9), there was a negative correlation betweenthe effect of adenosine on chemotaxis in vitro and CD8+ T cell tumorinfiltration, that is, the patients whose CD8+ T cells chemotaxis wasmost inhibited by adenosine were also those that had the lowest CD8+

T cell infiltration into the tumor (Fig. 2C and table S1).

The A2AR mediates the suppressive effect of adenosine onthe chemotaxis of HNSCC CD8+ T cellsWe conducted experiments to define whether the A2AR mediates theinhibitory effect of adenosine on HNSCC CD8+ T cells. The selectiveA2AR agonist CGS21680 suppressed the chemotaxis of HNSCC CD8+

T cells in a way comparable to adenosine (Fig. 3, A and B) (22, 38).We observed that the Y-COM of HNSCC CD8+ T cells was reducedby CGS21680 (Fig. 3A). In four of these six patients, we simultaneouslytested the effect of adenosine, and the Y-COM was not significantly dif-ferent from that in the presence of CGS21680 (Fig. 3A). Overall, the de-gree of Y-COM reduction was similar in the presence of CGS21680 andadenosine (Fig. 3B). The involvement of the A2ARwas further confirmedwith SCH58261, a selective A2AR competitive antagonist (22). The inhib-


itory effect of adenosine on HNSCC CD8+ T cells was abrogated whenthe cells were pretreated with SCH58261 (Fig. 3C). The cells pretreatedwith SCH58261 still migrated toward CXCL12 even in the presence ofadenosine, as shown by the increased Y-COM values when comparedto cells not treated with SCH58621 (Fig. 3C). The adenosine-induced re-duction in the Y-COM value of HNSCC CD8+ T cell chemotaxis wasblocked by SCH58261 (Fig. 3D). Overall, we showed that the adenosine-mediated inhibition of chemotaxis of HNSCC CD8+ T cells occursthrough the A2AR. These data suggest that blockade of the A2AR couldincrease the ability of circulating CD8+ T cells of HNSCC patients tomigrate toward a chemokine.

A2AR expression and cAMP-PKA signaling are not altered inHNSCC CD8+ T cellsThe A2AR signals through the activation of adenylate cyclase and, inturn, induces an increase in cAMP, PKAI activation, and inhibitionof KCa3.1 channels (11, 22). Experiments were performed to assesswhether alterations in components of the adenosine signaling pathwaymediate the inhibitory effect of adenosine on the chemotaxis ofHNSCCCD8+ T cells. Increased A2AR abundance, increased cAMP-PKAIsignaling, or decreased KCa3.1 activity in HNSCC CD8+ T cells couldexplain their enhanced sensitivity to adenosine. We measured ADORA2Aexpression (Fig. 4A) as well as A2AR protein abundance (Fig. 4, B to D)in CD8+ T cells in HDs and HNSCC patients and found no significantdifferences in either mRNA or protein expression. We further investi-gated whether there were any differences in the signaling pathwaydownstream of the A2AR.We did not observe any significant differencein the intracellular cAMP concentrations in activated HD and HNSCC

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Y-C

OM

(μμm

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CXCL12 CXCL12 + ADO

Y-C

OM

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)

P < 0.001D

HD HNSCC

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P = 0.012

ADOCXCL12

ADOCXCL12

HD HNSCC

E

CO

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Y

Fig. 1. HNSCC CD8+ T cells exhibit reduced chemotaxis in the presence of adenosine. (A andB) Trajectories of CD8+ T cellsmigrating along either a CXCL12 gradient (greentriangles) 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 areshown 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 cellsmigrating along either a CXCL12 gradient or a combination gradient of CXCL12with adenosine in HDs (n = 7 donors) (C) and HNSCC patients (n = 16 patients) (D). (E) Percentageinhibition 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 linerepresents 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.

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CD8+ T cells (Fig. 4E). Similarly, we could not detect statistically signif-icant differences in PKA activity after A2AR stimulation between HDand HNSCC CD8+ T cells (Fig. 4F). Overall, our results show that de-


creasedmigration of HNSCCCD8+ T cells in the presence of adenosinewas not due to any differences in A2AR abundance or proximalsignaling.

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 agradient of either CXCL12 or CXCL12 and adenosine (ADO), and the indicated values were measured. Results are presented as means ± SEM for all measuredvalues. 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, forwardmigration index in the direction of the x axis (represents the efficiency of migration perpendicular to the chemokine gradient respectively).

Parameter
CXCL12 CXCL12 + ADO P value (CXCL12 versusCXCL12 + ADO)
HD (n = 5)

X-COM (mm)
5.660 ± 4.739 1.586 ± 5.282 0.610
Y-COM (mm)
43.279 ± 9.221* 33.366 ± 10.424† 0.134
FMIx
0.016 ± 0.019 −0.001 ± 0.015 0.523
FMIy
0.141 ± 0.031‡ 0.109 ± 0.029§ 0.001
Directness
0.203 ± 0.024 0.202 ± 0.017 0.925
Velocity (mm/s)
0.210 ± 0.021 0.182 ± 0.021 0.478
Accumulated distance (mm)
15.462 ± 31.352 272.336 ± 30.909 0.475
Euclidean distance (mm)
62.122 ± 6.277 56.081 ± 10.104 0.526
HNSCC (n = 6)

X-COM (mm)
−7.125 ± 9.596 3.943 ± 11.983 0.629
Y-COM (mm)
46.773 ± 18.178ǁ¶ 4.281 ± 4.829# 0.029
FMIx
−0.030 ± 0.023 0.024 ± 0.036 0.404
FMIy
0.191 ± 0.039** 0.005 ± 0.020†† <0.001
Directness
0.256 ± 0.040 0.188 ± 0.017 0.080
Velocity (mm/s)
0.146 ± 0.042 0.139 ± 0.040 0.495
Accumulated distance (mm)
11.297 ± 62.163 206.839 ± 59.620 0.725
Euclidean distance (mm)
38.126 ± 8.524 38.145 ± 10.953 0.438¶
*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 = 0.031versus 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).

BA C

CD73High

CD8LowCD8High

CD73Low

P = 0.0301ρ = –0.700

CD

8+T

cel

ls (c

ells

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2 )

CD

8+T

cel

ls (c

ells

/mm

2 )

Y-COM (% inhibition)

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 mm. (B) Bargraph showing thenumber of CD8+ T cells (cells/mm2) within the tumor region in 16HNSCC tumors. Please note that donor HNC-52 has ameanCD8+ T cell infiltration value of5 cells/mm2. Thebroken red line represents themedian value for the 16HNSCCpatients. The tumorswithCD8+ T cell infiltration above themedian valuewere considered to be“well infiltrated” (referred to as high in table S1), whereas the tumors with CD8+ T cell infiltration below themedian valuewere considered to be “poorly infiltrated” (referred aslow in table S1). The bars representmeans± SEM. (C) Correlationbetween CD8+ T cell infiltration andpercentage reduction of the Y-COMvalues in the presence of CXCL12 andadenosine (values shown in Fig. 1E) in nine HNSCC patients thatwere scored as CD73High (see table S1). Correlationwasmeasured by Spearman rank-order correlation test (P=0.0301; correlation coefficient, r = −0.700).

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KCa3.1 channel activity is reduced in HNSCC CD8+ T cellsKCa3.1 channels are downstream of PKA in the adenosine signalingpathway, and we have previously shown that adenosine reduces T cellmotility by inhibiting KCa3.1 channels (22). Hence, we conductedexperiments to measure whether alterations in KCa3.1 channels inHNSCC CD8+ T cells could explain the adenosine-dependent inhibi-tion of chemotaxis. KCa3.1 channels are present at the uropod of mi-grating T cells and mediate their migration (24). KCa3.1 channels arealso involved in the chemotaxis of HD and HNSCC CD8+ T cells be-cause the KCa3.1-specific blocker TRAM-34 inhibited their migration(fig. S2) (39). We thus evaluated the expression and function of KCa3.1channels in resting and activated HNSCC CD8+ T cells and comparedthem to those of HD CD8+ T cells (Fig. 5). KCa3.1 channel expressionin T lymphocytes increases with activation (40). Patch-clamp exper-iments showed that the KCa3.1 activity (defined by the whole-cellKCa3.1 conductance normalized to the cell membrane capacitance)was significantly lower in activated HNSCC CD8+ T cells as comparedto their HD counterparts (Fig. 5, A and B, and table S3). No differenceswere observed in resting HD or HNSCC CD8+ T cells (table S3). There


were no changes in the Kv1.3 activity (re-ported here as current density: peakcurrent/capacitance) in either resting oractivated HD or HNSCC CD8+ T cells(Fig. 5C and table S3). The data were nor-malized for the capacitance because therewas a significant difference in cell capaci-tance between activated, but not resting,HD and HNSCC CD8+ T cells (tableS3). Because activation increased KCa3.1channel expression and capacitance,which is a measure of the cell size, thesedata raise the possibility that HNSCCCD8+ T cells are less activated than HDCD8+ T cells. We thus measured KCa3.1expression by flow cytometry in restingand activated CD8+ T cells in HD andHNSCC patients and observed that uponactivation, KCa3.1 channel abundancewas similarly increased in both HD andHNSCC CD8+ T cells (Fig. 5, D and E).In both HD and HNSCC CD8+ T cells,there were comparable increases in theMFI of KCa3.1 after activation (Fig. 5F).We also measured CD69 abundance as amarker of activation and observed no sta-tistically significant difference betweenHNSCC and HD CD8+ T cells (fig. S3).This suggests that the decreased KCa3.1channel activity in HNSCC CD8+ T cellsis not likely due to a decrease in channelsurface expression or defective T cell acti-vation. Overall, these data show that thereis reducedKCa3.1 activity, but not expres-sion, in HNSCC CD8+ T cells.

Activation of KCa3.1 restores thechemotaxis of HNSCC CD8+ T cellsin the presence of adenosineExperimentswere conducted to determine

whether activation of KCa3.1 channels by 1-ethyl-2-benzimidazolinone(1-EBIO), a selective positive modulator of KCa3.1 channels, abolishedthe inhibitory effect of adenosine on the chemotaxis of HNSCC CD8+

T cells (41). We evaluated whether increasing the activity of KCa3.1channelswith 1-EBIOwould enable theHNSCCCD8+Tcells tomigratetoward a chemokine even in the presence of adenosine. 1-EBIO in-creased the activity ofKCa3.1 channels in activatedHNSCCCD8+Tcellsto a level comparable to that of the baseline conductance in HDCD8+

T cells (without 1-EBIO) (Fig. 6A). Consistent with our earlier findings(Fig. 5, A and B), KCa3.1 conductance in HNSCC CD8+ T cells in theabsence of 1-EBIO was also significantly lower than that in HD CD8+

T cells. The Y-COM values of HNSCC CD8+ T cells that underwentchemotaxis toward CXCL12 were significantly reduced in the presenceof adenosine (Fig. 6B), similar to our earlier finding (Fig. 1). However,whenHNSCCCD8+Tcells from the same individualswere preincubatedwith1-EBIO, theY-COMvalues in thepresenceof adenosinewere almostthreefold greater than those of cells in the presence of adenosinewithout1-EBIO. Thus, the inhibitory effect of adenosine on the chemotaxis ofHNSCCCD8+ T cells was blocked by 1-EBIO (Fig. 6C). A similar effect

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+

+

ADO –+ –

Fig. 3. A2AR mediates the suppressive effect of adenosine on the chemotaxis of HNSCC CD8+ T cells. (A) Y-COM valuesfor HNSCC CD8+ T cells migrating along either a CXCL12 gradient (n = 6 patients), a combination gradient of CXCL12 withCGS21680 (n = 6 patients), or CXCL12 with adenosine (n = 4 patients). (B) Percentage inhibition in the Y-COM values for eachindividual experiment shown in (A) after incubationwithCGS21680or adenosine.Horizontal red lines representmeanvalues foreach group. (C) Y-COM values for HNSCC CD8+ T cells pretreated with or without 1 mMSCH58261migrating toward CXCL12 inthe presence of adenosine. Untreated CD8+ T cells in a CXCL12 gradient were used as controls (n = 5 patients). (D) Percentageinhibition 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 measuresanalysis 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.

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was caused by NS309, a more potent activator of KCa3.1 channels (42).Preincubation of CD8+ T cells from two HNSCC patients with NS309reversed the inhibitory effect of adenosine on chemotaxis to CXCL12(fig. S4). These findings suggest that enhancing KCa3.1 function inHNSCC CD8+ T cells restores their ability to chemotax in the presenceof adenosine.

DISCUSSIONThe unequivocal prognostic and therapeutic significance of CD8+ T cellinfiltration in most solid tumors and the known immunosuppressive


properties of adenosine have driven thestudies reported herein. The data that wepresent suggest that the chemotaxis of cir-culating HNSCC CD8+ T cells may becompromised in an adenosine-rich mi-croenvironment because these cells, con-trary to their healthy counterparts, havereduced KCa3.1 channel activity. Further-more, these data highlight the therapeuticpotential of KCa3.1 activators to increasepenetration of CD8+ T cells into tumorsby abrogating the inhibitory effect of aden-osine on the chemotaxis of CD8+ T cells.

The failure of immune surveillance incancer has been attributed to the lack ofeffective major histocompatibility com-plex presentation by cancer cells and theimmunosuppressive properties of the TME(8). New immunotherapies have been de-signed to increase the functionality of Tcells and their ability to resist the TME(6, 43, 44). A prerequisite for cytotoxicfunctionality is direct contact with tumorcells. To this effect, a high intratumoralCD8+/Treg (regulatory T cell) ratio is asso-ciated with good prognosis and responseto therapy in multiple solid malignancies,including HNSCC (45). Thus, infiltrationof CD8+ T cells is a limiting step in the ef-ficacy of immune surveillance and immu-notherapies in cancer. The TME is rich inthe immunosuppressant adenosine (10).We showed that adenosine inhibitedCD8+ T cell chemotaxis, and this effectwas enhanced in cells from HNSCC pa-tients. This is consistent with immuno-histochemical data of various solid tumorsshowing an inverse correlation betweenCD73 in the tumor and the infiltration ofCD8+ TILs (46–50). The advantage of thestudies that we have performed here overthe correlative studies in tissue samples isthatwe have used a collagen-rich 3Dmicro-environment where we have full controlover the experimental conditions used,whereas in vivo, the TME is a complexmix-ture of metabolic and waste products andtumor cells. This 3D system enables us to

exclusively study the effect of adenosine on chemotaxis. We found thatadenosine inhibited chemotaxis of HNSCCCD8+ T cells. The chemotaxisexperiments reported herein showed that HNSCCCD8+ T cells lost di-rectionality in the presence of adenosine but maintained their velocityand overall distance traveled. The effect of adenosine in 3D chemotaxisexperiments does not fully recapitulate what we have previously ob-served in a 2D migration assay on intercellular adhesion molecule–1surfaces, where adenosine inhibited integrin-mediated random migra-tion of HD CD3+ T cells by reducing their velocity (22). The differentexperimental conditions and signaling pathways triggered by the differ-ent migratory stimuli may explain these discrepancies.

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Fig. 4. A2AR expression and A2AR signaling are not altered in HNSCC CD8+ T cells. (A) ADORA2A expression in acti-vated 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 tothemean ADORA2A expression in HD. Data aremeans ± SEM for from four HD and five HNSCC patients. (B) Representativeflow cytometry histograms showing A2AR expression in resting and activated CD8+ T cells fromHD and HNSCC. (C andD) Mean fluorescence intensity (MFI) of A2ARmeasured in resting (C) and activated (D) CD8+ T cells fromHD (n=6donors)and HNSCC patients (n = 7 patients). (E) cAMP concentration in CD8+ T cells fromHD (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 representsmean values for each group. Data in (C), (D), and (F) were analyzed byMann-Whitney ranksum test; data in (A) and (E) were analyzed by Student’s t test.

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We found that adenosine had a more substantial inhibitory effecton the migration of HNSCC CD8+ T cells than HDCD8+ T cells. Thedegree of chemotaxis inhibition by adenosine inHNSCCCD8+ T cellsnegatively correlated with the number of CD8+ TILs in CD73-positivetumors underscoring the importance of this effect on immune surveil-lance in cancer. This also raises the possibility that the chemotacticsensitivity to adenosine of circulating CD8+ T cells could be used asaminimally invasive biomarker ofCD8+TIL infiltration and, possibly,


prognosis. Larger population studies are necessary to confirm thispossibility.

The increased sensitivity to adenosine that we observed in HNSCCCD8+ T cells is in agreement with the heightened suppression of cyto-kine production andproliferation ofHNSCCCD8+T cells by adenosinereported previously by Mandapathil et al. (33). They attributed thisincreased sensitivity to the reduced ability of HNSCC effector T cells(Teff) to degrade and internalize adenosine as well as an amplified

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ls)

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Fig. 5. KCa3.1 channel activity is reduced inHNSCCCD8+ T cells. (A) Representative KCa3.1 currents in CD8+ T cells recorded inwhole-cell voltage clamp configuration froman HD and HNSCC patient. Currents were normalized for themaximum current at +40mV to ease comparison of the KCa3.1 conductance at hyperpolarizing voltages. (B) KCa3.1conductance (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) Kv1.3channel 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 arenormalized to valuesmeasured in activated CD8+ T cells fromHD, and the bars representmean ± SEM. (D) Representative flow cytometry histograms showing KCa3.1 expressionin resting andactivatedCD8+ T cells fromHDandHNSCCpatients. (E)MFI of KCa3.1measured in resting and activatedCD8+ T cells fromHD (n=6donors) andHNSCCpatients (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.

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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 orabsence of 100 mM1-EBIOwasmeasured in activated CD8+ T cells fromHD (n = 17 cells, four donors) and HNSCC patients (n = 24 cells, five patients). The data were normalized tountreated (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 acombination gradient of CXCL12 with adenosine with or without preincubation with 20 mM 1-EBIO (n = 5 patients). (C) Percentage inhibition in the Y-COM values (B) of the cellspretreatedwith 1-EBIO. Horizontal red line representsmean values for each group. Data in (A) were analyzed by two-way ANOVA,whereas data in (B) were analyzedwith one-wayrepeated measures ANOVA (P = 0.009) and (C) with paired Student’s t test.

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A2AR signaling as compared to HDs’ Teff (33). We observed a compa-rable effect of adenosine and the A2AR agonist CGS21680 on the chemo-taxis ofHNSCCCD8+Tcells, suggesting that differences in the adenosinesignaling pathway and not adenosine degradation are responsible for theheightened effect of adenosine on chemotaxis. It is well established thatA2ARmediates the effect of adenosine in human T cells (5, 16, 17, 51, 52).We have previously shown that adenosine inhibits themotility of humanCD3+ T cells and cytokine release through A2AR stimulation, activationof adenylyl cyclase and PKAI, and ultimately inhibition of KCa3.1 chan-nels (22). Therefore, it is anticipated that an increase in A2AR, cAMP, andPKAI in HNSCC CD8+ T cells could amplify the response to adenosine.Similarly, a reduction in functional KCa3.1 channels could reduce thefunctionality of the cells once they are exposed to adenosine, but it couldalso contribute to the reduced ability of HNSCC T cells to produceinterleukin-2 and IFN-g as compared to HDs’ cells, which was previ-ously reported (33). Our data indicated no significant differences inA2AR expression, cAMP abundance, or A2AR-stimulated PKAI activa-tion in HNSCC T cells. We instead detected a decrease in KCa3.1 ac-tivity. This was not due to reduced KCa3.1 expression. Activated HDand HNSCC CD8+ T cells had similar amounts of KCa3.1 membraneproteins as determined by flow cytometry. Despite the low KCa3.1whole-cell conductance, we observed no difference in baseline chemotax-is (in the absence of adenosine) betweenHNSCCandHDCD8+Tcells. Itappears that only a fraction of KCa3.1 channel activity may be sufficientto guarantee chemotaxis. Further single-channel electrophysiologicalstudies as well as evaluation of the downstream signaling pathwaysare necessary to understand the mechanism by which KCa3.1 channelsregulate chemotaxis in T cells.

The findings reported in this article indicate that restoring KCa3.1activity in HNSCC CD8+ T cells by 1-EBIO and NS309 abrogated theinhibitory effect of adenosine. KCa3.1 are Ca2+-sensitive K+ channelsthat open upon an increase in intracellular Ca2+ concentration. 1-EBIOis a positive modulator of KCa3.1 channels, which increases their Ca2+

sensitivity (41). Eil et al. (31) showed that pharmacological activation ofKCa3.1 channels by positive modulators such as 1-EBIO improvedCD8+ T cell function in vitro and may potentially be of therapeuticuse in cancer. The data that we presented suggest that KCa3.1 activatorscould have therapeutic benefit by restoring the chemotactic capacity ofCD8+ T cells in an adenosine-rich TME, whichwould favor tumor pen-etration. The effect of KCa3.1 activation was comparable to that ob-tained by A2AR blockade, which has already shown to be of clearbenefit in combination with anti-programmed death protein 1 (anti-PD1) antibody orCARTcells in preclinicalmodels of solidmalignancies(5, 6, 17). A therapy based on KCa3.1 activation would have advantagesoverA2AR inhibition. KCa3.1 channels are downstreamof otherGPCRs.Prostaglandin E2, which is also present in the TME, inhibits KCa3.1channels in mast cells (53). Thus, activation of KCa3.1 channels couldbe amore effective approach for improving immune surveillance and theresponse to immune therapies in cancer because it could simultaneouslycounteract multiple immune suppressive components of the TME.

MATERIALS AND METHODSHuman subjectsStudies were conducted on peripheral blood obtained from 39 deiden-tifiedHNSCCpatients in the age range of 34 to 77. The eligibility criteriafor patient inclusion in the study were a positive diagnosis for HNSCCconfirmed by tissue biopsy and no administration of radiation orchemotherapy before the time of drawing the blood (see Table 1 for pa-


tient demographics and table S1 for clinical information). The data onthe study subjectswere collected andmanagedusingResearchElectronicData Capture (REDCap) tools hosted at the University of Cincinnati.Peripheral blood was also drawn from 20 age-matched (±5 years)HDs (8 female and 12male) in the age range of 24 to 67 years.Discardedblood units from Hoxworth Blood Center (University of Cincinnati)were used for preliminary chemotaxis validation experiments as wellas for the ADORA2A RT-qPCR and the 1-EBIO electrophysiologicalexperiments. Informed consent was obtained from all HNSCC pa-tients and HDs. The study and informed consent forms were ap-proved by the University of Cincinnati Institutional Review Board(IRB no. 2014-4755).

Reagents and chemicalsHuman serum, L-glutamine, adenosine, SCH58261, 1-EBIO, and sodi-umhydroxidewere purchased fromSigma-Aldrich.Hepes, RPMI 1640,fetal bovine serum, penicillin, streptomycin, and phosphate-bufferedsaline (PBS) were obtained from Gibco. Rat tail collagen I was ob-tained from Corning Inc. CGS21680 hydrochloride and NS309 werepurchased fromTocris Bioscience, whereas CXCL12was obtained fromR&D Systems. TRAM-34 was a gift from H. Wulff (Department ofPharmacology, University of California Davis). Stock solutions ofSCH58261, CGS21680, TRAM-34, 1-EBIO, andNS309were preparedin dimethyl sulfoxide and used at 0.1% dilution. Stock solution ofCXCL12 was prepared in sterile PBS containing 0.1% bovine serumalbumin.

Cell isolation and activationPeripheral bloodmononuclear cells (PBMCs) were isolated fromwholeblood by Ficoll-Paque density gradient centrifugation (GE HealthcareBio-Sciences), as described previously (54). CD8+ T cells were sub-sequently isolated from PBMCs by negative selection using the EasySepHuman CD8+ T Cell Enrichment Kit (STEMCELL Technologies Inc.)according to the manufacturer’s instructions. The CD8+ T cells weremaintained in RPMI 1640 medium supplemented with 10% humanserum, penicillin (200 U/ml), streptomycin (200 mg/ml), 1 mM L-glutamine, and 10 mM Hepes (54). Cells were activated for 72 to96 hours in a cell culture dish coatedwithmouse anti-humanCD3 anti-body (10 mg/ml) (BioLegend) and mouse anti-human CD28 antibody(10 mg/ml) (BioLegend).

Reverse transcription quantitative polymerase chain reactionTotal RNA was isolated from activated HD and HNSCC CD8+ T cellsusing the E.Z.N.A. total RNA isolation Kit (Omega Bio-tek) as per themanufacturer’s instructions. Six hundred and fifty nanograms of RNAwas used to synthesize complementary DNA (cDNA) using theMaximaFirst Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scien-tific) as per the manufacturer’s instructions. Predesigned primers forRT-qPCR were obtained using TaqMan Gene Expression Assays (Ap-plied Biosystems, Thermo Fisher Scientific) to detect the expression ofADORA2A (assay ID: Hs001169123_m1) and GAPDH (assay ID:HS03929097_g1). The RT-qPCRwas set up in a 96-well plate by adding30 ng of cDNA, 1× TaqMan Gene Expression Master Mix (AppliedBiosystems), and 1 ml of TaqMan Gene Expression Assay primers. Allsamples were run in quadruplicate. GAPDH was used as an internalcontrol. RT-qPCR was cycled in Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems). CT values were measuredusing StepOne software version 2.1 (Applied Biosystems). CT valuesfor ADORA2A were normalized against measured CT values for

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GAPDH, and the DDCT values were calculated as described previ-ously (55). Relative quantity values, representing the fold change inADORA2A gene expression in HNSCC CD8+ T cells compared toHD CD8+ T cells, were calculated as the 2−DDCT values.

Flow cytometryCD8+ T cells (~1 × 106 cells per condition) were fixed with 4% para-formaldehyde (Affymetrix) and stained with ATTO 488–conjugatedmouse anti-human KCa3.1 antibody (clone 6C1, Alomone Labs). Cellswere then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) andstained with Alexa Fluor 405–conjugated mouse anti-human A2ARantibody (clone 2D1, Novus Biologicals). To test T cell activation,CD8+ T cells were stained live with Alexa Flour 488–conjugated anti-CD69 antibody (Clone FN50, BioLegend) and fixed with 1% para-formaldehyde. Data were collected on an LSR II flow cytometer (BDBiosciences) and analyzed with FlowJo software (FlowJo LLC).

ChemotaxisThree-dimensional chemotaxis was performed using the m-SlideChemotaxis assay (ibidi GmbH) according to the manufacturer’s in-structions. Briefly, ~1 × 106 activated CD8+ T cells were incorporatedin a type I rat tail collagen gel (Corning) and added to the central ob-servation chamber of the m-Slide Chemotaxis. In all experiments,CXCL12 (8 mg/ml) was added to themigrationmedium in the reservoirto the left of the observation chamber, whilemigrationmediumwithoutchemokine was added to the reservoir to the right of the observationchamber, thus generating a chemokine gradient tomeasure the baselinechemotaxis. To assess the effects of adenosine or CGS21680 on chemo-taxis, we established simultaneous adenosine (or CGS21680) and che-mokine gradients in a separate chamber by injecting adenosine (10 mM)or CGS21680 (10 mM) and CXCL12 (8 mg/ml) into the left reservoir.The slide was mounted on the stage of an inverted Zeiss LSM 710 mi-croscope (Carl ZeissMicroscopyGmbH) equippedwith a 37°C incuba-tor. Cell migration was recorded by time-lapse video microscopy, withbright-field images acquired every 3 s for up to 3 hours. Cell tracking onthe time-lapse imageswas performed using the “Manual Tracking plug-in” on ImageJ software (National Institutes of Health), and the datawere analyzed using theChemotaxis andMigrationTool (ibidi GmbH).On average, 15 to 20 cells were tracked per condition. The followingchemotactic parameters were derived: (i) COM (the average positionalong the relevant axis that the cells reached by the end of the exper-iment), (ii) Euclidean distance (the linear distance between thestarting point and ending point of a cell), (iii) accumulated distance(the total distance traveled by the cell during the course of the entiremicroscopy recording), (iv) FMI (the ratio between the net distancetraveled along the relevant axis and the accumulated distance), (v) di-rectness (the ratio between the Euclidean distance and the accumu-lated distance, denotes the tendency of the cells to migrate along astraight line), and (vi) velocity (36). We defined a positive chemotaxiseffect if the cellsmigrated along the chemokine gradient (y axis) that is,if the Y-COM was significantly greater than X-COM and FMIy wassignificantly greater than FMIx.

cAMP determinationActivated CD8+ T cells were lysed using 0.1 M HCl at a final concen-tration of 1 × 106 cells/ml. Intracellular cAMP concentrations weremeasured in T cell lysates using the acetylated procedure of the DirectcAMP ELISA Kit (Enzo Life Sciences) according to the manufacturer’sinstructions.


PKA kinase activity assayActivated CD8+ T cells were stimulated with 1 mM CGS21680 for30 min. Cell lysates were prepared as previously described, and totalprotein content was measured using BCA Protein Assay (55). The celllysates were diluted to equal protein concentrations using kinase ac-tivity assay buffer. The PKA kinase activity was measured using a PKAkinase activity kit (Enzo Life Sciences) as described by the manufacturer,with a reaction time of 90 min at 30°C and a development time of 60 min.The relative kinase activity was determined by the absorbance of the sam-ple at 450 nm divided by the amount (micrograms) of crude protein.

ImmunohistochemistrySlides prepared from formalin-fixed paraffin-embedded (FFPE) tumorbiopsy specimens from 16 HNSCC cases were deparaffinized andstained with a monoclonal rabbit anti-human CD8 antibody (cloneSP57, VentanaMedical Systems) in a Ventana BenchMark ULTRA auto-mated IHC slide staining system (Ventana Medical Systems). For CD73staining, FFPE sections were stained with a mouse monoclonal anti-human CD73 antibody (clone 1D7, Abcam). The ultraViewUniversalDAB Detection Kit (Ventana Medical Systems) containing a horse-radish peroxide multimer and 3,3′-diaminobenzidine tetrahy-drochloride (DAB) chromogen was used for indirect detection of theprimary antibody. The slides were counterstained with hematoxylin, andimages were obtained at ×10 magnification on an Olympus BX53 lightmicroscope (Olympus Corporation) or on a Leica DMi8 inverted micro-scope with Leica Application Suite X software (Leica Microsystems Inc.).Tumor regions in the stained slides were identified by a pathologist, and atleast 4 to 10 fields per slide were imaged. CD8+ T cell infiltration in thetumor area was digitally quantitated by drawing a region of interest(ROI) around the tumor region and counting the number of cells (brownsignal) within the ROI using NIS-Elements Viewer software (NikonInstruments Inc.). Data were expressed as cells counted per square milli-meter, and the median value of CD8+ infiltration in all of the measuredROIs was determined. Tumors with CD8+ infiltration values (cells/mm2)above and below the median were considered as “high” and “low” infil-trated, respectively. Slides stained for CD73 were visually assessed forCD73 staining (brown signal) in the tumor and stromal regions by a pa-thologist and characterized as having high or low CD73 staining in theseregions. To eliminate a subjective bias in reporting the CD8+ infiltration aswell as CD73 staining, microscopy and the image analysis were performedblinded.

ElectrophysiologyKCa3.1 andKv1.3 currents in CD8+T cells weremeasured inwhole-cellvoltage-clamp configuration using an AxoPatch 200B Amplifier (Mo-lecular Devices). The external solution contained either 160 mMNaCl,4.5 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, and 10 mM Hepes (pH7.4) (Fig. 5, A to C), or 145 mM Na-aspartate, 5 mM KCl, 2.5 mMCaCl2, 1.0 mM MgCl2, 5.5 mM glucose, and 10 mM Hepes (pH 7.4)(Fig. 6A), and the pipette solution contained 145 mM K-aspartate,10 mM K2EGTA, 8.5 mM CaCl2, 2 mM MgCl2, and 10 mM Hepes(pH 7.2), 290 to 310 mosmol (1 mM free Ca2+ concentration). Currentswere induced by 200-ms ramp depolarization from −120 to +40 mVfrom a holding potential of −70 mV every 10 s. The macroscopic con-ductance of KCa3.1 channels (GKCa3.1) was calculated as a ratio of thelinear fraction ofmacroscopic current slope to the slope of ramp voltage

stimulus after subtraction of the leak current GðpSÞ ¼ IslopepAmsð Þf g

VslopeVmsð Þf g. The

slope conductance was measured between −100 and −80 mV to avoid

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contamination by the Kv1.3 current. Kv1.3 currents were determinedfrom the same ramp protocol at +40 mV after subtraction of theKCa3.1 current extrapolated by linear regression.

Statistical analysisStatistical analyses were performed using Student’s t test (paired or un-paired), Mann-Whitney rank sum test, Wilcoxon signed-rank test (inexperiments where samples failed normality), and ANOVA as indi-cated. Post hoc testing onANOVAwas done bymultiple pairwise com-parison procedures using the Holm-Sidak method. Statistical analysiswas performed using SigmaPlot 13.0 (Systat Software Inc.). Outlierswere determined by Grubb’s test (GraphPad Software). P value of lessthan or equal to 0.05 was defined as statistically significant. The corre-lation between CD8+ T cell infiltration and inhibition of Y-COM wasanalyzed by Spearman’s rank-order correlation.

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SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/11/527/eaaq1616/DC1Fig. 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 cellsin 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 fromHD and HNSCC patients.

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Acknowledgments: We would like to acknowledge clinical coordinators from theUniversity of Cincinnati Cancer Institute’s clinical trials office (UCCI CTO) for their assistancein collection of patient samples. We also thank H. Duncan (Division of Nephrology andHypertension, Department of Internal Medicine, University of Cincinnati) for assistancewith IRB regulatory affairs. We would like to thank R. Jandarov (Department of Biostatisticsand Bioinformatics, University of Cincinnati) for valuable advice on statistical analysis.We would also like to thank the Center for Clinical and Translational Science and Training(CCTST), University of Cincinnati, for providing REDCap for effective patient datamanagement. We are grateful to H. Wulff (Department of Pharmacology, University ofCalifornia Davis) for providing TRAM-34. Confocal microscopy images were acquired atthe Live Microscopy Core, Department of Pharmacology and Systems Physiology,University of Cincinnati. Flow cytometry experiments were performed at Shriners Hospitalfor Children Flow Cytometry Core, Cincinnati, OH. Funding: This work was funded bygrant support from the NIH (grant R01CA95286), a Pilot grant from the UCCI, and aJust-In-Time Core Grant from CCTST, University of Cincinnati to L.C. T.W.-D. was supportedby a Clinical and Translational Science Award (CTSA)–awarded KL2 Mentored grant, aHematology Oncology Translational Science Award (HOTSA)/Hematology Oncology PilotGrant Award (HOPGA) from the Division of Hematology Oncology at the Universityof Cincinnati and a grant from CCTST (1UL1TR001425-01). A.B. was cofinanced byCampus Hungary Program (B2/1SZ/12351) and National Excellence Program(TÁMOP 4.2.4. A/2-11-1-2012-0001 B). P.H. was supported in part by the Bolyai JánosFellowship (Hungarian Academy of Sciences). Author contributions: Conception anddesign: A.A.C., A.B., and L.C. Development of methodology: A.A.C., L.C., A.B., M.J.A., andH.S.N. Acquisition of data: A.A.C., A.B., M.J.A., H.S.N., P.H., and J.Q. Provision and managementof patients: T.W.-D. Provision of patient data: T.W.-D. and J.Q. Analysis and interpretationof data: A.A.C., A.B., M.J.A., H.S.N., P.H., J.Q., T.W.-D., and L.C. Writing, review, and/or revision ofthe manuscript: A.A.C. and L.C. Study supervision: L.C. All authors discussed theresults and commented on the manuscript. Competing interests: The authors declarethat they have no competing interests. Data and materials availability: All dataneeded to evaluate the conclusions in the paper are present in the paper or theSupplementary Materials.

Submitted 9 October 2017Accepted 22 March 2018Published 24 April 201810.1126/scisignal.aaq1616

Citation: A. A. Chimote, A. Balajthy, M. J. Arnold, H. S. Newton, P. Hajdu, J. Qualtieri, T. Wise-Draper,L. Conforti, A defect in KCa3.1 channel activity limits the ability of CD8+ T cells from cancer patientsto infiltrate an adenosine-rich microenvironment. Sci. Signal. 11, eaaq1616 (2018).

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

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

DOI: 10.1126/scisignal.aaq1616 (527), eaaq1616.11Sci. Signal.

infiltration of adenosine-rich solid tumors. channel activators may help augment T cell+ T cell migration in the presence of adenosine, suggesting that K+CD8

activity, but not adenosine receptor expression or signaling. Treatment with a KCa3.1 channel agonist restored patient ) channel+ T cells to adenosine correlated with reduced KCa3.1 potassium (K+The increased sensitivity of patient CD8

and found that adenosine inhibited the chemotaxis of T cells from cancer patients more than T cells from healthy donors. T cells in a 3D chemotaxis assay+. analyzed the migration of CD8et alincluding the nucleoside adenosine. Chimote

T cell accumulation in solid tumors is limited by multiple factors found within the tumor microenvironment, channel activity curbs T cell migration+Reduced K

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A defect in KCa3.1 channel activity limits the ability of ... · Julianne Qualtieri,2 Trisha Wise-Draper,3 Laura Conforti1‡ The limited ability of cytotoxic T cells to infiltrate

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