Minimal Recruitment and Activation of Dendritic Cells Within Renal Cell Carcinoma

1998;4:585-593. Published online March 1, 1998.Clin Cancer Res A J Troy, K L Summers, P J Davidson, et al. renal cell carcinoma.Minimal recruitment and activation of dendritic cells within

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Vol. 4, 585-593, March 1998 Clinical Cancer Research 585

Minimal Recruitment and Activation of Dendritic Cells within Renal

Cell Carcinoma’

Andrew J. Troy, Kelly L. Summers,

Peter J. T. Davidson, Christopher H. Atkinson,

and Derek N. J. Hart2

Hematology/immunology Research Group [A. J. T., K. L. S.,D. N. J. H.], Department of Urology [A. J. I., P. J. I. D.], OncologyService [C. H. A.], Christchurch Hospital, Christchurch, New Zealand

ABSTRACT

Dendritic cells (DCs) are predicted to participate innatural tumor immunity by migrating into tumors, where

they acquire antigen, undergo activation, and migrate tolymph nodes to initiate a T-lymphocyte response against

tumor-associated antigens. The presence of DCs using de-fined lineage markers and their function in human tumorshas not been assessed previously. The monocbonal antibodiesagainst CMRF-44 and CD83, which are differentiation/ac-tivation antigens on DCs, were used in immunohistobogicaland flow cytometry studies to analyze the DC subtypesinfiltrating 14 cases of human renal cell carcinoma (RCC).The functional immunocompetence of the DCs isolated fromRCC was assessed by testing their ability to stimulate an

allogeneic mixed leukocyte reaction. The majority of leuko-

cytes present within the RCC were macrophages (62% ±

14.7) or T lymphocytes (19% ± 9.5), with CD45” HLA-DR�

lineage-negative putative DCs accounting for less than 10%of the leukocytes present. Of these, a subset, comprising lessthan 1 % of total leukocytes, had an activated CMRF-44” orCD83� DC phenotype. Activated CMRF-44� and CD83�DCs were more evident outside the tumor in association withT-lymphocyte clusters. The number of CMRF-44� DCs cor-related closely with the number of 5-100-positive DCs. Iso-lation of DCs from eight RCCs was achieved, and flowcytometry studies confirmed the small proportion of acti-vated CMRF-44� DCs. The CMRF-44� DCs stimulated anallogeneic mixed leukocyte reaction, but the CMRF-44

DCs (normal tissue DC precursors and other cells) failed todo so. These results suggest that RCCs recruit few DCs intothe tumor substance, and the tumor environment fails toinitiate the expected protective activation of DCs. These two

Received 8/4/97; revised I 1/18/97; accepted 12/1/97.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.I A. J. I. was supported by a Schering Plow Fellowship from the Uro-logical Society of Australasia. Running costs for the project were

provided by the generosity of Urology Associates and the CancerSociety of New Zealand.2 To whom requests for reprints should be addressed, at HematologyDepartment, Christchurch Hospital, P. 0. Box 151, Christchurch, NewZealand. Phone: 64 3 3640 300; Fax 64 3 3640 750.

mechanisms, amongst others, may contribute to tumor es-

cape from immunosurveillance. In vitro loading of DCs with

tumor-associated antigens may be a useful therapeutic ma-neuver.

INTRODUCTION

DCs3 are specialist antigen-presenting cells that are found

in the interstitium of tissues and within epithelial surfaces (1, 2).

If DCs are to play a part in natural tumor immunity, DCs would

be expected to migrate into tumors, derive antigen, undergo

activation, and migrate to central lymphoid tissue to initiate any

T-lymphocyte response against TAAs. The failure of DCs to

migrate from the blood into malignant tissue or to be activated

and migrate out into draining lymph nodes represents a potential

tumor escape mechanism. Little is known about the migration of

DCs into human tumors, their state of activation and their

interactions, if any, with responding T lymphocytes. The immu-

nohistobogical studies performed to date using S-bOO staining

have shown that an increased number of DCs located within

tumors correlate with a better prognosis in coborectal adenocar-

cinoma (3); adenocarcinoma of the lung (4, 5); gastric (6),

esophageal (7), and nasopharyngeal (8) carcinomas; and papil-

lary carcinoma of the thyroid (9). These studies have not been

correlated with more detailed immunohistobogical analyses us-

ing mAbs. Furthermore, the widespread interest in using blood

DCs as “nature’s adjuvant” for tumor immunotherapy (10)

makes an understanding of how DCs function within tumors

vital for evolving strategies to use DCs in effective tumor

vaccines.

RCC is the most common malignancy of the kidney and

accounts for approximately 2-3% of all adult neoplasms (1 1).

The observation that large numbers of lymphocytes are often

found in RCC has encouraged investigators to develop immu-

notherapeutic treatments but with limited success (12, 13). The

presence or absence of DCs in RCC is unknown, despite the fact

that interstitial DCs were first defined in normal kidney some

time ago (14, 15). A recent study (16) described infiltration of

S-bOO-positive cells into RCC but not into benign adenomas.

Difficulties with the specificity of S- 100 expression and the

solubility of S-bOO has limited its use for characterizing DCs

further.

DCs have been identified in blood (17) and normal tissues

(14, 15) by demonstrating their high density expression of MHC

class II antigens and their lack of other mature hematopoietic

lineage markers (lin) using immunocytobogical techniques.

The recent availability of the relatively more DC-specific, mAbs

3 The abbreviations used are: DC, dendritic cell: TAA, tumor-associatedantigen; mAb, monocbonal antibody; RCC, renal cell carcinoma: NK,natural killer; HLA, human leukocyte antigen: FACS. fluorescence-activated cell sorter; MLR, mixed leukocyte reaction.




586 Dendritic Cells in Renal Cell Carcinoma

CMRF-44 (18) and HBlSa (19), combined with double-labeling

techniques. allows more accurate identification of DCs, as well

as providing information on their state of differentiation/activa-

tion. The CMRF-44 antibody recognizes an early activation

antigen, which is expressed on blood DCs, only after a short

period of culture ( 18, 20). HB1Sa detects the CD83 antigen, a

member of the immunogbobulin superfamily with an as yet

unknown function (21), which is expressed by activated DCs

and monocyte-derived DCs generated in culture with cytokines

(Mo-DC: Ref. 22).

In this study on DCs in RCC, we identified the CD45�/

HLA-DR�/lin� DC population in RCC and adjacent normal

kidney. and used the CMRF-44 and HBl5a mAbs to define the

activated DC subsets. Expression of the CDla antigen (23), a

marker of the Langerhans cell subset of DCs as well as the

up-regulation of the critical costimulator molecules, CD8O and

CD86 (24), were also assessed. These results and the functional

data on the DCs isolated from RCC suggest that deficient DC

activation and migration may contribute to RCC escape from

immune surveillance.

MATERIALS AND METHODS

Tissue Samples. Fresh tumor tissue was collected, fol-

lowing informed consent, from 14 radical nephrectomy speci-

mens, all removed for RCC. All patients were well at the time

of surgery with no evidence of metastases, and only one case (2)

had invasion of the resection margin of Gerotas fascia. There

were nine clear cell tumors, three papillary tumors, and two

granular tumors. After initial assessment by a histopathobogist,

samples were processed for routine histopathobogy. Samples for

immunohistobogy were mounted in OCT compound (Tissue

Tek), snap-frozen in liquid nitrogen. and stored at -80#{176}Cuntil

required for sectioning. Additional fresh tumor tissue was pro-

cessed immediately when available for further studies on the

isolated cell populations. For 5-100 staining, tissues obtained

from the same specimens were embedded in paraffin.

mAbs. The anti-CD3 (OKT3, IgG2a), anti-CD19

(FMC63, IgG2a), and anti-HLA-DR (L243, IgG2a) antibodies

were produced from hybridomas obtained from the American

Type Culture Collection (Rockville, MD). The antibodies

CMRF-l2 (CD45, IgGl), CMRF-lS (negative control, 1gM),

CMRF-3l (CD14, IgG2a), and CMRF-44 (activation antigen.

1gM: Ref. 18) were produced and characterized in this labora-

tory. The anti-CD83 mAb (Hbl5a, IgG2a; Ref. 19) was a gift

from Prof. T. F. Tedder (Durham, NC), and the anti p55 (K-2,

IgG I ) mAb was provided by Dr. E. Langhoff (Boston, MA; Ref.

25). The anti-CD16 mAb (HuNK-2, IgG2a) was a gift from

Prof. I. F. C. McKenzie (Melbourne, Australia), and the Na 1/34

mAb (CDIa, IgG2a) was a gift from Prof. A. J. McMichael

(Oxford, United Kingdom). The anti-CD8O (IgGla) mAb was

purchased from Becton Dickinson (Australia), and the anti-

CD86 (IgG2a) mAb was purchased from Serotek (United King-

dom). The anti-S-lOO polyclonal antibody was purchased from

Dako Corp. (Carpinteria, CA). The negative control mAb SalS

(IgG2a) and X63 (IgGI) were gifts from Prof. H. Zola (Ade-

laide, Australia). Phycoerythrin-conjugated CD14, CD19, and

negative control mAbs (IgG 1 , IgG2a) were purchased from

Becton Dickinson (Australia).

Immunoperoxidase (Single-Label) Staining. Cryostat-

cut sections (-20#{176}C; 4-6 p.m) were placed on gelatin-coated

slides and air-dried overnight, prior to fixing for 10 mm in cold

acetone (4#{176}C).Sections were prebbocked with 10% human AB

serum for 30 mm, followed by the application of the primary

mAb for 30 mm at room temperature. Sections were then

washed three times with PBS and then incubated with peroxi-

dase-conjugated goat anti-mouse IgG (Dako) for an additional

30 mm. After washing twice in PBS and once in TrisfHCl buffer

(pH 7.6), the sections were developed with 0.1% 3,3’-diamino-

benzidine for 3-10 mm before counterstaining with hematoxy-

bin.

S-100 staining was performed on sections cut from forma-

bin-fixed, paraffin-embedded tissues. These were dewaxed and

rehydrated before staining as for fresh tissue, except that rabbit

anti-S-bOO and peroxidase-conjugated goat anti-rabbit IgG (Bio-

source International, CA) were used.

Immunoalkaline Phosphatase/Peroxidase (Double-Label)

Staining. Sections were treated as for single staining up to the

first primary mAb incubation. Following this, sections were

incubated with biotinylated goat anti-mouse IgG (Dako) diluted

1:200 for 30 mm, washed three times in PBS, and then incu-

bated for 30 mm in Extravidin#{174} alkaline phosphatase (Sigma

Chemical Co., St. Louis, MO) at 1 :200 dilution. After washing

two times in PBS and once in TrisIHCL buffer (pH 7.6), the

sections were developed with Fast Blue (Sigma) for 5-10 mm.

Sections were then washed with PBS before a 30-mm incuba-

tion in 10% mouse serum. Incubation with the second mAb was

carried out as for single immunoperoxidase staining, including

developing the color reaction with 3,3’-diaminobenzidine.

Immunohistobogical Assessment. T lymphocytes (CD3).B lymphocytes (CD19), NK cells (CD16), and monocyte/macro-

phages (CD14 and CD1 Ib) were identified and counted as cells

when identifiable nuclei were present. Potential DCs were identi-

fled as CD45�. lin (CD3, CDI9, CDI6, CD14, and CD1 lb)

using double-labeling and further confirmed to be HLA-DR posi-

tive. CMRF-44� DC and CDla� LC were identified by double-

labeling with CD14 to exclude macrophages, which may also

express these antigens. CMRF-44� and CD83� DCs were consid-

ered on the basis of data published previously (18, 19) to be

partially activated.

Sections were examined at X400 for cell types present in

large numbers and at X 100 for cell types present in low num-

bers through an eyepiece graticube, giving a field of view of

0.044 and 0.772 mm2, respectively. All counts were converted

to number per mm2 for comparison. Positively stained cells

were counted in 5-10 random, nonoverlapping fields of tumor

tissue, excluding areas containing dense stroma or large lymph-

oid aggregates. Cell numbers are expressed as a percentage of

the total leukocyte count (mean % ± SD), as determined by

CD4S staining. Separate counts were made within the area of

lymphoid aggregates when present.

Isolation of Tumor Containing Leukocyte Populations.

Tissues were minced into small pieces in sterile 10% FCS/RPMI

media (RPMI 1640 supplemented with 100 units/mb penicillin,

1 m�i glutamine, and 100 units/mI streptomycin) containing

colbagenase (0.5 mg/mI) and DNase (1 .0 pg/mb) and incubated

at 37#{176}Cfor 1-2 h with gentle agitation. The resulting cell

suspension was washed in PBS twice before filtering through a




Clinical Cancer Research 587

Table I DC response to each tumor

Cell counts are expressed as the number of cells per mm2 and are also expressed as a percentage of total CD4S leukocytes (boldface). S- 100counts were not converted to percentages, because these were obtained from different samples of the same tumor. Counts were averaged over S fields

for CD45 and CDI4 and 10 fields for CMRF-44 and CD83. Individual I-lymphocyte and NK cell counts have been omitted for clarity (theyrepresented 8-40% of leukocytes; see Fig. 3A).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Mean±SD

Histology” CC CC CC P CC CC CC CC CC” p CC P G G

Stage (TNM)’ T� T4 13b T3� T3� T35 Tib T� T2 T3� 1, 13b 12 T�b

Total leukocytes 531 603 349 413 363 640 1152 739 631 619 513 925 308 354 581 ± 239(CD45)

Total lin 82 82 41 45 23 122 91 127 45 102 73 127 36 68 76 ± 35

leukocytes 15 14 12 11 6 19 8 17 7 16 14 13 12 19 13.1±4.1%lin DCs 17.9

3.420.0

3.321.1

6.0

12.1

2.9

15.2

4.2

24.6

3.8

41.4

3.6

19.0

2.6

19.6

3.1

16.1

2.6

21.8

4.2

25.3

2.7

8.0

2.6

11.5

3.2

19.5 ± 8.0

3.4 ± 0.9%

CMRF44� DCs 0.500.9

0.300.5

1.20.35

0.700.17

0.28

0.081.6

0.250.70

0.061.0

0.140.0

0.00.69

0.112.50.49

0.97

0.100.35

0.110.28

0.080.79 ± 0.650.15 ± 0.13%

CD83 DCs 1.7

0.320.7

0.121.6

0.461.4

0.340.55

0.153.50.55

3.5

0.301.6

0.220.18

0.030.18

0.031.1

0.212.3

0.250

0

0.69

0.19

1.4 ± 1.1

0.23 ± 0.16%

CDIa LCs 0.35

0.070.20.04

2.20.43

7.21.7

0.280.08

0.820.13

1.80.16

0.55

0.076.51.03

3.20.52

0.14

0.03

1.2

0.13

0.0

0.0

1.9

0.54

1.8 ± 2.3

0.35 ± 0.48%CD86� DCs 0.0

00.0

00.0

0

0.0

0

0.0

0

0.0

0

0.14

0.010.0

00.0

00.0

00.0

00.0

00.0

00.0

00.01 ± 0.04

0.00%

S-l00 NA” NA NA 9.5 0.14 0 1.1 1.9 3.6 0.7 0.3 NA 0.7 0.8 1.87 ± 2.9

“ CC, clear cell: P. papillary clear cell; G. granular clear cell.h Cystic variety of clear cell.

C. ThM, Tumor-Node-Metastasis classification.d NA, not available.

70 �im nylon cell strainer (Falcon; Becton Dickinson, Australia)

to remove undigested material. This single-cell suspension was

layered over a Ficoll-Hypaque density gradient (density, 1.077;

Pharmacia, Uppsala, Sweden) and centrifuged at 400 X g for 20

mm to obtain mononuclear cells. This step excluded granubo-

cytes but retained some tumor cells (identified as CD45 cells).

Cell viability was always >80%, as confirmed by trypan blue

exclusion.

Flow Cytometry Analysis and Sorting. Mononuclearcells were labeled with primary mAb for 30 mm at 4#{176}C.Fob-

bowing one wash in PBS, labeled cells were detected by incu-

bation with FITC-conjugated sheep anti-mouse IgG (Silenus

Laboratories, Australia) for 30 mm at 4#{176}C.Following blocking

for 10 mm in 10% mouse serum, cells were double-labeled with

phycoerythrin-conjugated CD14 mAb. Appropriate gating and

flow cytometry analysis were performed using Lysis software

on a FACS (FACS Vantage; Becton Dickinson). Contaminating

CMRF-44�-activated B lymphocytes were not considered, be-

cause B lymphocytes were not present in the mononuclear cell

populations. For analysis of DC phenotype, mononuclear cells

were labeled with a mix of lineage markers (CD3, CDI lb,

CD14, CD16, and CD19), and the lin population was sorted

and then reanalyzed for the expression of DC activation anti-

gens.

For functional studies, the mononuclear cell suspension

was sorted after labeling, as described in the results. Single

labeling was used for three experiments. In an additional five

double-labeling studies, the four different populations consist-

ing of CMRF-44�/CDl4 (activated DCs), CMRF-44�/CD l4�

(activated monocytes), CMRF-44/CDl4� (resting mono-

cytes), and CMRF-441CD14 (other cells, including resting

DCs) were isolated.

MLR. Tumor cell suspension and the sorted leukocyte

populations were cocultured with 1 X l0� allogeneic normal

peripheral blood T lymphocytes. Cultures were maintained at

37#{176}Cand 5% CO2 in 96-microwell, round-bottomed plates for 6

days. Wells were pulsed with [3Hjthymidine (5 Ci/mmol; Am-

ersham) in the last 16-18 h of culture. [3HjThymidine incorpo-

ration was measured on a liquid scintillation analyzer to give a

direct readout of T-lymphocyte proliferation. Responses were

reported as the mean value ± SE of triplicate wells.

RESULTS

Immunohistological Analysis of the Leukocytes InfiltratingRCC

Identification of Leukocyte Types within Cryostat

Sections. A summary of the results of the immunostaining of

the 14 tumors analyzed are shown in Tables 1 and 2 and Figs.

1 and 2. The majority of the leukocytes infiltrating the RCCs

were either CDl4� macrophages or CD3� T lymphocytes,

accounting for 62% ± 14.7 and 19% ± 9.5, respectively, of the

leukocytes present. NK cells (CDl6� lymphoid cells) were

present in small numbers (1 1% ± 8. 1), whereas B lymphocytes

were virtually never detected. Total CD45�/lin cells, which

include the DCs, comprised 13. 1% ± 4. 1 of the total leukocyte

population in RCCs. These numbers were not significantly

different from the proportion of CD45�/lin leukocytes seen in

normal kidney (12.7% ± 3.0). Morphobogically, at least 25% of

these were large cells with dendritic processes, suggesting a

degree of activation (Fig. 1A). When only these cells were

counted, the potential DCs were found to comprise 3.4% ± 0.9

in RCCs and 4.4% ± 2. 1 in normal kidney. Double-labeling to

identify lin and HLA-DR� cells, although difficult to analyze

accurately due to HLA-DR expression on endothelium and the

tumor cells, gave cell counts similar to the CD45�fbin counts,

with a minimum estimate of 50% of these cells having a

CD45�fl-ILA-DR�/lin phenotype. CDla�/CDl4 LC ac-





Table 2 DC numbers in T-bymphocyte aggregates at tumor margins

All counts were made at X400 and converted to numbers per mm2 on matched lymphoid areas (three to five, depending on sample) for CMRF-44and CD83. Direct comparison of 5-100 counts was not possible because different samples of the tumor are required for processing.

Case number

DC phenotype 2 5 6 7 8 9 10 1 1 14

CMRF-44� CDI4 136 556 447 415 513 386 284 358 449

CD83� CD14 113 426 295 243 333 147 159 352 277

5-100 NA” NA 95 177 150 132 95 NA 159

a NA, not available.

counted for 0.35% ± 0.48 of leukocytes in RCCs and 0.02% ±

0.03 of leukocytes in normal kidney and was elevated in RCCs

due to the increased counts in the cases with papillary and cystic

RCCs.

Identification of 5-100 Cells in Paraffin Sections. The

results for the 5- 100 antigen staining in paraffin sections cannot

be compared directly with the frozen sections because the for-

mer are necessarily cut from a slightly different area of the

tumor. Nonetheless, staining for S-bOO antigen identified only

small numbers of positive cells in 9 of 10 cases (Fig. 1D; Table

1 ). They were present in greater numbers where T lymphocytes

had accumulated. In over one-half the cases, weak S- 100 stain-

ing of tumor cells was noted.

Identification of Activated DCs. CD14 beukocytes,

which expressed the CMRF-44 antigen, i.e., activated DCs,

accounted for only 0.15% ± 0.13 of the total leukocytes infib-

trating the tumors. The CD83 antigen, which is induced after the

CMRF-44 antigen (20) and may undergo some subsequent

down-regulation (26), was expressed by a similar number

(0.23% ± 0. 16) of tumor leukocytes (Fig. 1C). These data

suggested that only a small proportion (5-10%) of the lin/

HLA-DR� putative DCs identified within the RCCs are acti-

vated. The number of activated DCs in the adjacent normal

kidney was much less, with only 0.04% ± 0.04 CMRF-44�/

CD14 cells and 0.04% ± 0.04 CD83� cells present. No

specific localization of DCs was found within the tumors with

respect to necrotic or inflammatory areas.

Staining for the p55 antigen (25) was also performed, but

accurate assessment was difficult because of tumor cell staining

in close to one-half of the cases. In those cases that could be

assessed, the number of p55-positive cells was similar to the

number of CMRF-44- and CD83-staining cells.

The expression of the CD8O and CD86 costimulator mob-

ecules was examined. No CD80� cells were detected in either

normal or RCC tissue. Small numbers of CD86� cells were

present in many of the cases, but when double staining with

CD14 was performed to exclude CD86� macrophages, only one

case was found to have CD86�/CDl4 cells present.

Although the number of activated DCs within the RCCs

was limited, CMRF-44 � or CD83 � DCs were more evident just

outside the tumor in the pseudocapsule. Here, relatively large

numbers of activated DCs were seen in the T-lymphocyte clus-

ters that develop. In nine tumors, in which sufficiently well-

defined T-cell aggregates had formed, it was possible to count

the number of DCs in the clusters (Fig. 1, E and F; Table 2).

Perhaps consistent with the earlier induction of the CMRF-44

antigen on blood DCs in vitro (20), more CMRF-44�/CDl4

cells than CD83�/CD14 cells were present in the bymphoid

areas.

FACS Analysis of Leukocytes in Single-Cell TumorSuspensions

Flow cytometric analyses on the mononuclear cell sus-

pensions isolated from digested tumor tissue were performed

on eight tumors (Table 3). Side scatter and forward scatter

were used to define typical “bymphoid” and “monocytic-

macrophage” electronic gates. The bymphoid gate contained

>95% CD4S’ leukocytes and consisted predominantly of

CD3� cells. The monocytic-macrophage gate, containing

>85% CD4S’ leukocytes, was analyzed more extensively to

identify potential DC populations. CD14� cells accounted for

over 50% of the monocytic-macrophage gate. A variable

number of these macrophages expressed the CMRF-44 anti-

gen and accounted for the bulk of the CMRF-44�-positive

cells within the tumors (Fig. 3A). Activated CMRF-44’/

CD14 DC comprised only 1.5% ± 1.02 of the total mono-

cyte-macrophage gated cell population. The contaminating

CD45 tumor cells did not express the CMRF-44 or CD14

antigens. The remaining population of CD14/CMRF-44

cells was presumed to contain unactivated DCs, contaminat-

ing tumor cells, and occasional CD14 lymphoid cells. Over-

all, these leukocyte populations occurred in similar propor-

tions to that observed in direct tissue staining.

To facilitate a more direct comparison of the flow cytom-

etry data with the immunohistobogical results, lin cells were

sorted, and the CD4S’ cells were analyzed in five cases (Fig.

3B). These represented 8.1% ± 2.4 of the total leukocytes

(lymphoid and monocyte-macrophage gates). Approximately

one-half these cells were HLA-DR�, i.e., approaching the pro-

portion of total DCs identified by tissue staining. The CMRF-44

antigen was expressed on only a subpopulation of lin cells

(15.6% ± 9.0), i.e., - 1% of total leukocytes, a figure that

relates closely to the bow percentage of CMRF-44� DCs esti-

mated by direct staining. The CD83 antigen was not detected by

this method, possibly because of its sensitivity to proteases.

Functional Studies

To examine the functional capacity of these tumor leuko-

cyte populations as antigen-presenting cells, various cell popu-

lations were tested as stimubators in an albogeneic MLR. Limited

cells were available in each case; therefore, only certain sorted




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)i� �Fig. I Immunohistobogical analysis identifies a CD45�fHLA-DR�ilin population of putative DCs, a subset of which express the CMRF-44 CD83

differentiation/activation antigens within RCC. A, a putative DC (arrow) stained for CD45 (golden brown) but not CD3, CD1 lb. CDI4, CDI6, or

CDI9 (lin. blue black) compared with the CD14-positive macrophage (arrowhead, X400). B, single CMRF-44�/CDl4 DC (arrow) surroundedby many CDl4� macrophages (blue, X400). C. CD83� DCs were seen among RCC cells (single-label immunoperoxidase). D, S-lO0- DC (white

arrow) in RCC (single-label immunoperoxidase). Note weak S-l00� tumor cells. DCs are found in lymphoid aggregates at the margins of RCCs. E.

CMRF-44�/CDl4 DC (yellow) and CDl4� macrophages (blue) are identified (no counterstain). F, CD8Y DCs are seen among the lymphocytes

(counterstained) in a lymphoid aggregate.

populations could be tested. The first two analyses established 4447CD14 DCs were found to be at least three to five times

that the RCC leukocyte population was albostimulatory (data not more potent stimulators of the abbogeneic T-lymphocyte re-

shown) and that the lin population contained the albostimula- sponse than the CD14� macrophages (Fig. 4B). The CMRF-44

tory cells (data not shown). An additional case was analyzed cells, i.e. , the population that included the presumed resting or

after sorting the tumor mononuclear cell suspension into unactivated tumor-associated DCs (and some contaminating tu-

CMRF-44� and CMRF-44 populations. The CMRF-44� pop- mor cells), failed to stimulate T lymphocytes in all five cases

ubation was clearly the most effective at stimulating the albo- tested. Blood DCs, sorted from two additional patients, were

MLR (Fig. 4A). Five additional cases were sorted using double- found to fully stimulate T lymphocytes in an albogeneic MLR

labeling to identify the CMRF-44�/CDl4 DCs. The CMRF- (Fig. 4D).




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CDI4 CD3 CDI9 CDI6 TotalS, IinDC 44DC 83�DC CDIa

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DC subtypes in normal kidney and RCC

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Fig. 2 Leukocyte subsets and DC subsets in 14 cases of RCCs. The

percentages were calculated as a proportion of total leukocyte (CD45 �)

numbers averaged over 5-10 fields (see “Materials and Methods”), and

each symbol represents a separate case. For absolute numbers, see Table

2. In A. CD14 macrophages. CD3 T lymphocytes, CD19 B lympho-cytes, CDI6 NK cells, lin DCs, CMRF-44�/CDI4 DCs. and CD83�

DCs are shown. The latter three DC populations are presented on anexpanded scale in B and are compared with the percentages of the same

cell types in the adjacent normal renal tissue.

DISCUSSION

These studies suggest that there is minimal recruitment and

activation of DCs within RCCs and that the CMRF-4�F DCs

present may not function normally. To date, no marker for

resting human DCs, or less differentiated members of the DC

lineage, has been discovered; therefore, unactivated putative

DCs were defined as CD4S’ cells backing mature lineage-

specific markers and the activated DC subset defined by

CMRF-44 and CD83 staining. This is the first study to use these

techniques to describe the DC response in human cancer. Rel-

ativeby few putative DCs compared with macrophages were

present in the tumors, and most of these were present in a resting

state with a normal tissue DC phenotype, i.e., CMRF-44/

CD83 - . Only a minority of the DCs showed signs of activation,

phenotyped as CMRF-44�/CD83�. Conversely, the majority of

DCs located in the T-cell aggregates within the normal tissue

surrounding the tumor had an activated CMRF-44�/CD83�

phenotype consistent with that documented for interdigitating

DCs in normal lymph nodes (18, 19).

Tumors are known to produce a modified inflammatory

response, and it was expected that the majority of leukocytes

present would be either macrophages or T lymphocytes. We

established that 13.1% ± 4.1 of the leukocytes present did not

express the lineage markers of macrophages, T lymphocytes, B

lymphocytes, or NK cells and have a phenotype consistent with

DCs. Admittedly, not all of these lin cells will be DCs,

particularly the proportion with lymphoid features, and these

may represent cells similar to the lin lymphoid cells noted in

blood DC preparations (20). Nonetheless, the CD45�fHLA-

DR� lin phenotype of the majority of these cells and the fact

that -25% of them had a dendritic morphology raise the prob-

ability that a significant proportion are DCs. When morphology

was taken into consideration and only large 45�/lin cells were

counted, these cells were found to constitute 3.4% ± 0.9 of total

beukocyte numbers. The number of these lin� cells in RCCs,

when compared with numbers within normal kidney, showed no

significant difference.

The number of activated DCs present in RCCs was readily

assessed using the CMRF-44 and HB 1Sa antibodies. These

mAbs detect activation/differentiation antigens induced on

blood DCs and found on DCs in the T-lymphoid areas of lymph

nodes (18, 19). Activated CMRF-44� or CD83� DCs were

present in comparatively small numbers in RCCs, representing

0. 13 and 0.23% of tumor leukocytes, respectively. The numbers

using either antibody are very similar, showing a strong corre-

lation reminiscent of the dual staining CMRF-44’7CD83�

blood DC population (20). These activated DC counts represent

5-10% of the total (bin) population of DCs. We speculate that

these DCs are likely to have been activated either by inflamma-

tory stimuli from the tumor or as a result of an interaction with

T lymphocytes. The proportion of activated DCs, although

small, was several times larger than that seen within the normal

kidney. No expression of CD8O on DCs was detected, and with

the exception of one case, no CD86�/l4 cells could be iden-

rifled.

Similar numbers of CDla� cells were identified in the

tumors. Whereas p55 staining was difficult to assess, it also

appeared to identify similar numbers of activated DCs. The

number of CMRF-44� and CD83� DCs identified in frozen

sections correlated with the number of 5-100 staining DCs in

paraffin sections. It has been unclear at what stage 5-100 is

expressed in normal DC differentiation/activation, but this first

attempt at a comparison suggests that this may be a relatively

late event. Another study, which used the 5-100 antibody to

define DCs within RCCs, demonstrated three or less S-bOO-

positive cells per X 100 field in 6 of 10 tumors (16). We found

this to be the case in 8 of 10 tumors. It is noteworthy that the

numbers of S-l0O� cells are also considerably fewer in RCCs

than in tumors of epitheliab organs (3. 5-9).

The ability to isolate viable beukocytes from RCCs was of

major benefit to the study. Using flow cytometry, it was possible

to confirm our immunohistobogy findings by demonstrating a

significant bin�fHLA-DR�/CD45� population of cells with the

scatter characteristics of DCs. The sensitivity of flow cytometry




A

ri�

B

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c)

CONTROL

5b’5� (bit)) ‘4’)lo (I 175) 15% (236)


Table 3 FACS a nalysis of tumor mon onuclear cell suspensi on (myeloi d gate)”

DC phenotype

Case number

Mean ± SD2 3 4 6 8 10 1 1 13

CMRF-44�/CDl4 DCs 0.0 1.0 1.4 1.3 3.5 2.3 1.2 1.2 1.5 ± 1.02

CMRF-44�/CDl4� macrophages 19.6 25.9 31.5 23.8 41.7 3.3 5.3 41.0 24.0 ± 14.4

CMRF-44/CDI4� macrophages 65.1 42.7 24.4 21.5 39.8 9.8 42.6 41.0 35.9 ± 16.9

CMRF-44�/CD14 cellsb 15.9 30.3 42.7 52.6 14.7 78.9 46.4 17.0 37.3 ± 22.4

a Results are expressed as percentages of each cell type in the myeloid gate. The myeloid gate included between S and 30% of total cells, and

when analyzed, >85% of CD45� cells. The nonleukocytes fall in the CMRF-44 CDI4 population.b Heterogeneous population including DCs (-50% HLA-DR� cells).

FSC

7.06% .� . ,�

�:

.- .

� � .....,.rt...

. . ). . 1.59%

CMRF44

CD3,llb,14,16,19 CD45 HLA-DR CMRF44

Fig. 3 FACS analysis of tumor leukocyte suspension. In A, the analysis was performed on the gated mononuclear population. Within the

mononuclear gate, the majority of the cells are CDl4� macrophages. Only 1.59% of this sample are CDl4/CMRF-44� DC (one of eightrepresentative analyses; see Table 3). B, analysis of sorted lin cells sorted from the mononuclear gate. In this sample, lin cells account for 7.06%.

The majority are CD45 leukocytes, with close to 50% of these being HLA-DR�, probable DCs. Only a small proportion (15%) of lin cells areactivated DCs expressing CMRF-44. Solid line, the test mAb: broken line, the control mAb labeling. Parentheses, mean channel fluorescence.

excludes the possibility that the lin cells included low density

CDl4� or CD3� cells, which were not detected by immuno-

histological techniques. Flow cytometry, likewise, confirmed

that CMRF�44tactivated DCs comprise a very small portion of

the tumor leukocyte infiltrate. We also compared the stimulatory

abilities of DCs and macrophages, albeit with difficulty, because

only limited numbers of cells were obtained from finite tissue

samples.

It is of some significance that the CMRF-44 DCs ob-

tamed from RCCs were relatively poor stimubators in our ex-

periments, although an additional effect from contaminating

tumor cells in the MLR cannot be readily excluded. Interstitial

DCs isolated from mouse and rat require a period of in vitro

culture to induce allostimubatory (costimubatory) activity (27).

However, in humans, functional albostimulatory activity in the

DC lineage arises early with the CD34� precursor (28) and

blood DCs (including the CMRF-44, freshly isolated DCs;

Ref. 29) stimulate a strong MLR. Isolated human Langerhans

cells also act as potent albostimubatory cells (30). Thus, the

failure of RCC-associated interstitial CMRF-44 DCs to stim-

ulate an albogeneic MLR is remarkable. Indeed, these results

have parallels with the situation in rheumatoid arthritis, where it

appears that despite an inflammatory environment, inhibitory

factors prevent full DC activation and expression of a costimu-

bator phenotype (31). Any effect on the DCs within RCCs

appears to be localized to the tumor because fresh blood DCs

from RCC patients were able to stimulate T lymphocytes max-

imally. Both interleukin 10 (32) and transforming growth factor

1� (33) have negative effects on DCs, and both have been shown

to be produced by RCCs (34).

The CDl4/CMRF-44� DCs were considerably more po-

tent stimubators of albogeneic T-bymphocyte proliferation than

were isolated CD l4� tumor macrophages, and this suggests that

at least some DCs avoid active tumor suppression of their

function. A predominantly activated CMRF-44� and CD83�

DC population was noted in the presence of T-lymphoid aggre-

gates. This population may represent the DCs induced to mi-

grate out of the tumor. We speculate that the interaction between

these CMRF-44� DCs and the corresponding T lymphocytes

may involve recognition of TAA. It is possible that these ag-

gregates represent a peripheral version of DC-T-lymphocyte

interactions normally seen in the lymph node. Equally, these T

lymphocytes (specific or nonspecific) may represent an efferent

response reacting to the DCs that have not yet reached regional

lymph nodes. Whether the relatively few CMRF-44� DC or T

lymphocytes noted in these clusters are capable of trafficking




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10 100 1000 10000

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10 100 1000 10000 100000 100

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Fig. 4 Analysis of tumor-infiltrating DC abbostimulatory potential. Individual tumor cell mononuclear cell suspensions were prepared, and the

electronically gated lymphoid and monocyte-macrophage cell populations were sorted after labeling. A, CMRF-44� cells but not CMRF-44 cells

are allostimulatory (one experiment). B, CMRF-447CD14 DCs are potent allostimulatory cells. CMRF-44� CD14’ cells stimulated minimally inthis experiment only. CMRF-44 cells were not allostimulatory (one of two experiments). Results are presented as [3H]thymidine uptake (mean cpm)

using 2 x l0� responders and the absolute stimulator cell numbers as indicated. C. CMRF-44� tumor DCs stimulate as potently as blood DCs, whereas

CMRF-44 tumor DCs stimulate poorly. In D, fresh blood DCs from two patients with stage T, tumors were able to stimulate T lymphocytesmaximally (as determined by phorbol myristate acetate and ionomycin stimulation). A-D, bars, SE.

normally is another important question. It has to be conceded

that little is known about the reaction of DCs in pathological

circumstances and to what extent these results reflect normal or

abnormal DC responses to tissue damage.

It seems that RCCs do not recruit DCs into the tumor above

the numbers seen in normal tissue: (a) these first clinical data

reinforce a similar concept proposed as a result of rat studies in

vivo (27), and recent experiments demonstrate that a tumor cell

line secreting vascubar-endothelial growth factor suppressed in

vitro DC production (35); (b) our functional studies on the

CMRF-44 tumor DC population also raise the possibility that

there is a component of the tumor environment that suppresses

their normal costimubatory function. This is supported by the

finding of the lack of CD8O or CD86 up-regulation; and (c) the

paucity of CMRF-44� DCs in RCCs also prompts us to spec-

ulate that RCCs lack the inflammatory/noxious signals, such as

lipopolysaccharide, tumor necrosis factor-a, or chemokines,

required to initiate significant DC activation/migration (36). If

DC function is compromised at these three levels, tumor escape

from immunosurveibbance is likely to result.

These data establish that the new reagents CMRF-44 and

CDS3 can be exploited to study DCs in tumor biology, and

extended studies on breast cancer are now in progress. Further-

more, it encourages the concept that in vitro loading of autobo-

gous DCs with TAA and subsequent vaccination of patients

with these DCs may reinforce one phase of the immune re-

sponse to RCCs sufficiently to initiate a therapeutic immune

response.

ACKNOWLEDGMENTS

We thank the urobogists and pathologists at Christchurch Hospitaland Medlab South for help in obtaining nephrectomy specimens.

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Minimal Recruitment and Activation of Dendritic Cells Within Renal Cell Carcinoma

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