enrichment in tumor-reactive cd8+ t-lymphocytes by ... · pbl and lnl isolation. pbls were obtained...

[CANCER RESEARCH 51. 3891-3899. August 1. I99I|

Enrichment in Tumor-reactive CD8+ T-Lymphocytes by Positive Selection from theBlood and Lymph Nodes of Patients with Head and Neck Cancer1

Eric M. Letessier, Dae S. Heo, Thomas Okarma, Jonas T. Johnson, Ronald B. Herberman,and Theresa L. Whiteside2

Departments of Pathology [E. M. L., D. S. H., R. B. H., T. L. W.]. Otolaryngology [J. T. J.J, and Medicine [R. B. H.], University of Pittsburgh School of Medicine, andPittsburgh Cancer Institute [E. M. L., D. S. H., J. T. J., R. B. H., T. L. W.], Pittsburgh, Pennsylvania 15213, and Applied Immune Sciences, Mento Park, California94025 [T. O.I

ABSTRACTTo study antitumor functions of T-lymphocyte subpopulations in the

blood [peripheral blood lymphocytes (PBLs)l and tumor-draining lymphnodes (LNs) of patients (n = 16) with squamous cell carcinoma of thehead and neck (SCCHN), antibody-coated devices were used to positivelyselect CDS* or CD4* cells. The mean percentage of CDS* cells captured

on antibody-coated flasks from PBLs was 92% and that captured fromlymph node lymphocytes (LNLs) was 98%. The initial enrichment inCD4+ T-cells was comparable. CDS* T-lymphocytes captured from PBLs

proliferated as well as unseparated lymphocytes in both patients withSCCHN and normal donors, while captured CD4* PBLs of the patients

showed significantly lower expansion than those of normal volunteers.Unseparated LNLs proliferated as well as PBLs, but captured CD4+ or(1)8* LNLs failed to proliferate in the presence of interleukin 2 (100

units/ml) and phytohemagglutinin (5 Â¿ig/ml).The addition to capturedLNL cultures of irradiated autologous or allogeneic feeder cells significantly improved expansion of CDS* LNLs but not CD4+ LNLs. During15-day culture of captured CDS* PBLs or CD8+ LNLs in the presenceof feeder cells, a significant (P < 0.05) enrichment in CDS* T-cells was

maintained |94 Â±5% (mean Â±SEM) or 99.5 Â±0.1%, respectively, onday 15|. Capture of CDS* LNLs and their expansion resulted in theoutgrowth of CD8*CDllb" effectors which had no or little cytotoxicity

against Daudi, low cytotoxicity against K562, and very high levels ofcytotoxicity against 4 different natural killer cell-resistant SCCHN targets, as measured in 4-h â€¢'( r release assays. Such significant enrichment

in SCCHN-restricted cytotoxicity could be obtained with LNLs fromtumor-uninvolved LNs but not from tumor-involved LNs. Captured andcultured CD4* LNLs had no preferential anti-SCCHN cytotoxicity. Theaddition of irradiated autologous tumor cells to captured CDS* PBLs did

not result in improved proliferation or antitumor function of the effectorcells. Positive selection on antibody-coated flasks of CDS* T-lymphocytesfrom tumor-uninvolved LNs of patients with SCCHN led to the enrichment in SCCHN-restricted but the major histocompatibility complex-unrestricted effector cells in 15-day cultures. Thus, CDS* lymphocytes

separated from tumor-draining LNs in patients with head and neck cancercontained cytolytic T-cell precursors capable of developing into effectorswith preferential activity against SCCHN targets.

INTRODUCTIONCellular adoptive immunotherapy with LAK3 cells has re

cently been used as a novel treatment modality in some human

Received 2/26/91; accepted 5/15/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by NIH Grant 1PO 1-CÃ•4744501A2 and American Cancer

Society Grant IM588A (to T. L. W.). E. M. L. was supported by Associationpour le Recherche sur le Cancer. 94801. Villejuif. France.

2To whom requests for reprints should be addressed, at Pittsburgh CancerInstitute, Room W104I, Biomedicai Science Tower, DeSoto at O'Hara Street,

Pittsburgh, PA 15213.'The abbreviations used are: LAK, lymphokine-activated killer; IL. interleu

kin: rIL, recombinant IL; NK, natural killer; LN, lymph node; PBL, peripheralblood lymphocyte: LNL, lymph node lymphocyte; SCCHN. squamous cell carcinomas of the head and neck: I-LN, tumor-involved LN; NI-LN. tumor-uninvolved LN; TCM, tissue culture medium; PBS. phosphate-buffered saline; AIS,Applied Immune Sciences; LU, lytic unit; MoAb, monoclonal antibody; PHA,phytohemagglutinin antigen; PHA-p, PHA-purified; MHC, major histocompatibility complex; AuTu, autologous tumor; FITC. fluorescein isothiocyanate. PE,phycoerythrin.

solid tumors refractory to conventional therapies (1, 2). However, a considerable degree of toxicity associated with cellularadoptive immunotherapy (3) and very large numbers (e.g., 10")

of autologous effector cells required (1) have both been considered to be serious drawbacks. In addition, issues regardingoptimal delivery and distribution of transferred cells in tissuesand in the tumor, their in vivo autotumor cytotoxicity, andrequirements for in vitro activation of these cells have all beenintensely debated (4). In view of this controversy, it appearsreasonable to consider alternative forms of cellular therapywhich, at least theoretically, might eliminate some of the problems associated with LAK cell administration. For example,more homogeneous populations of IL2-activated NK cells (5)or of purified, IL2-activated CD4+ or CD8+ T-lymphocytes (6),

administered with lower doses of IL2 and/or other cytokines(6), may be therapeutically more effective than LAK cells.Alternative sources of cells, such as LNs draining the tumor ortumor-infiltrating lymphocytes, may provide better antitumoreffectors for therapy than those currently prepared from theperipheral blood (7, 8). Finally, in vitro sensitization of effectorswith irradiated autologous or allogeneic tumor cells or tumorantigens (9) could help in generating in vitro effector cells withcytotoxic reactivity against autologous tumor cells (10).

The future of cellular therapy depends to a large extent onimproved capabilities to select, activate in vitro, and expandsubpopulations of patients' PBLs, LNLs, or tumor-infiltrating

lymphocytes. Methodology has recently become available forseparating NK cells (11) and T-lymphocytes (12, 13) from thehuman peripheral blood in numbers sufficient for therapy. Theapplication of these developing technologies to obtaining selected subpopulations of cells which can be then used alone orin combination with other agents to treat cancer has been amost recent challenge.

In this manuscript, we describe our experience with a devicethat has been utilized to capture human T-lymphocyte subpop-ulations from LNs and the peripheral blood of patients withhead and neck cancer. This device has already facilitated theselection of CD8+ lymphocytes from the peripheral blood of

patients with acquired immunodeficiency syndrome, and thetherapeutic efficacy of these cells is being tested in an ongoingclinical trial (12). Evidence is provided for the enrichment intumor-reactive CD8+ T-lymphocyte subpopulations by positiveselection on antibody-coated surfaces from LNLs and PBLs ofpatients with SCCHN.

MATERIALS AND METHODS

Patients. Twenty-five patients with SCCHN (6 females and 19 males)and 1 male patient with glandular metastatic adenocarcinoma aged 48-83 years (59 Â±9 years, mean Â±SD) were included in this study (Table1). None of the patients was previously treated with radiation therapy.Five patients had recurrent disease. All patients underwent surgicalresections of primary tumors and/or modified radical neck dissections.The primary tumor sites were: larynx, 5; hypopharynx, 2; oral cavity,

3891

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CAPTURE AND GROWTH OF CDS* AND CD4* T-CELLS

Table 1 Clinical staging and histopathology of patients with SCCHN

CasePBL123456789101112LNL1314151617181920212223242526Age(yr)/M51/M83/M50/M56/M65/F64/M51/M54/M48/F65/M67/M56/M64/F61/M46/M59/M54/F56/M62/M59/M68/F73/M53/M47/M67/F65/MSiteTRANS"SPRMTCLOTFOMCLOTFOMTONGRMTFOMRMTTONGFOMTONSHYPORMTBOTBOT/FOMTRANSTRANSTONSSUBMANDRMTTONGFOMFOMPrimarystageT3NOT4N2T4N1T3NOT2NOT3NOT4NOT3NOT3NOMOTINOT4N2T3N1TINOT4NOT3NOT3NOT1N2T3N1T3NOT4NOT2NOT2N2T2NOT4N1T2N2PathologyModPoorPoorModModWellModModPoorModWellModAdenoid/cysticModModWellPoorModModModMod/CISMETWellPoorWellModILN1/30*3/424/380/210/10/11/261/310/772/252/420/173/280/400/325/262/210/340/591/131/242/230/153/642/60

* Trans, transglottis: BOT, base of tongue: FOM. floor of mouth: RMT.

retromandibular trigone: GLOT, glottis: TONS, tonsil; HYPO, hypopharynx;SP, sinus pyriform; SUBMAND, submandibular; Mod. moderately differentiated;Poor, poorly differentiated; Well, well differentiated; CIS, carcinoma in situ;MET: salivary mÃ©tastasesof glandular carcinoma.

* Number of involved LN/total examined by histopathology.

10; and oropharynx, 9. Of 26 patients, 16 had at least one metastaticnode, as determined by histopathology. Whenever possible, I-LNs andNI-LNs were obtained from the same patients (14 of 16 cases).

PBL and LNL Isolation. PBLs were obtained from venous blood ofpatients prior to surgery and isolated by Ficoll-Hypaque centrifugation.The recovered lymphocytes were washed twice and resuspended inTCM consisting of RPMI 1640 (Gibco, Grand Island, NY) supplemented with 2 Mm L-glutamine, gentamicin, streptomycin, penicillin,and 10% (v/v) human pooled AB serum.

Samples of cervical LNs were obtained from the resected specimensby a pathologist. The samples were placed in TCM and immediatelycarried by hand to the laboratory. The LNs were disaggregated by thepassage through a wire screen (10 Mm). Suspensions of I-LNs or NI-LNs were separated on differential Ficoll-Hypaque gradients as described earlier (14). The recovered cells (tumor cells from the upperand lymph node cells from the lower gradient interface) were washedtwice in TCM and checked for viability using a trypan blue dye.

Flow Cytometry. The phenotype of fresh or cultured PBL and LNLcells was determined by two-color flow cytometry. Cells were adjustedto a concentration of 5 x 105/ml in PBS-0.1% (v/v) sodium azide, and0.2 ml of this cell suspension was incubated with 5 n\ of fluorescein- orphycoerythrin-labeled monoclonal antibodies for 30 min at 4Â°C.The

cells were then washed three times in PBS-sodium azide and fixed in2% (w/v) paraformaldehyde solution in PBS. Two-color analysis wasperformed on FACScan (Becton-Dickinson FACS system. MountainView, CA). The monoclonal antibodies used included the followingspecificities: Leu4 (anti-CD3), Leu2a (anti-CD8), Leu3a (anti-CD4),Leul9(anti-CD56), Leu 15 (anti-CD 11b), anti-IL2R (anti-CD25), anti-HLA-DR, Leulla (anti-CD 16), anti-TCR a/0, and anti-TCR 1. Ascontrols, mouse isotypes IgGl and IgG2a were used in all experiments.Cells in the "lymphogate" were always stained with anti-CD45 (FITC)

and anti-CD 14 (PE) monoclonal antibodies to estimate the percentageof all leukocytes and monocytes, respectively. The lymphogate typicallycontained >90% lymphocytes. All antibodies were purchased fromBecton Dickinson, except for anti-TCR 1, which was obtained from TCell Sciences, Cambridge, MA.

Enrichment in CD4+ and CDS* of PBLs and LNLs. Enriched populations of CD4+ and CDS* cells were obtained from PBLs and LNLs

by positive selection with a panning technique (13). Briefly, T25 (25cm2) culture flasks covalently coated with anti-CD4 or anti-CD8 mono

clonal antibodies (proprietary process) were generously provided byAIS, Menlo Park, CA. To facilitate covalent attachment of monoclonalantibodies, the surface styryl groups of polystyrene T25 flasks (CorningGlassworks, Corning, NY) were substituted with bromoacetamidegroups. Antibodies were covalently coupled to the bromoacetamide-activated T25 flasks "bycoating with 5 ml of a 50 ng/ml solution of

anti-CD4 or anti-CD8 in PBS for 2 h at room temperature. After theexcess antibody solution was removed, the flasks were washed extensively with PBS and then sterilized by electron beam irradiation. Theflasks are commercially available from AIS.

Prior to panning, antibody-coated flasks were washed three timeswith Dulbecco's phosphate-buffered saline-calcium-/magnesium-free

medium (Gibco, Grand Island, NY)- Fresh or fresh-cryopreserved PBLsor LNLs were diluted in TCM to a concentration of 20 x IO6cells/mland added to the flask in a volume of 2 ml. In some cases, 40 x IO6

cells were not available, and fewer cells had to be used for capture.Flasks were then centrifuged at 1000 rpm for 5 min and incubated lyingflat for l h at room temperature. Following incubation, nonadherentcells were collected, counted, and used for flow cytometry or as autol-ogous feeder cells for growth of separated LNLs. Sometimes, cells notcaptured on anti-CD8 antibody-coated flasks were used for a newpositive selection to capture CD4* cells. After cell capture, flasks werewashed 5 times with Dulbecco's phosphate-buffered saline-calcium-/

magnesium-free medium to completely remove nonadherent cells.TCM (2 ml) containing rIL2 (100 units/ml; Cetus, Emeryville, CA)and PHA-p (5 Â¿ig/ml;Sigma Chemical Co., St. Louis, MO) were thenadded, and the flasks were incubated in a humidified atmosphere of 5%CO2 in air at 37Â°Cfor 72 h. During this incubation, most CDS* cells

spontaneously separated from the antibody-coated surfaces. To recoverall cells, gentle pipeting was used. The harvested cells were countedand transferred to standard tissue culture flasks for further expansion.For satisfactory culture of captured LNL populations nonadherent cells(3 x IO6) were irradiated (5000 rads) and added back to autologouscaptured cells on day 1. The cultures were sampled for cell counts at 2-to 3-day intervals and maintained at a cell density of 2 x 106/rnl by

adding fresh TCM containing 1L2. The cells were allowed to proliferateuntil sufficient numbers were obtained for phenotypic and functionalassays, and in some cases, cultures were maintained for 40 days. Foldexpansion was calculated by dividing the final number of cells in cultureby the initial cell number determined on day 3, by which time allcaptured cells had detached.

The same procedure was followed for the capture of CD4* cells

except that AIS flasks coated with anti-CD4 monoclonal antibody wereused. To recover CD4* T-cells, vigorous pipeting was necessary.

The proportion of CDS* or CD4* cells captured on antibody-coated

flasks was determined using the following formula:

% subset captured =

1 - % positive cells in noncaptured population% positive cells in unseparated population

x 100

The percentages of CDS* or CD4* cells were determined by flow

cytometry as described above.Tumor Cell Lines. The SCCHN lines used in these studies were

established from tumor biopsies as described by us earlier (15). Thelines were maintained in minimal essential medium (Gibco) supplemented with 15% (v/v) fetal calf serum, split, and passaged as needed.All other tumor cell lines were obtained from American Type CultureCollection and cultured in RPMI 1640 supplemented with 10% fetalcalf serum. The lines were tested for Mycoplasma using the Gene-Probeassay at monthly intervals.

Culture of PBLs with Tumor Cells. Positively selected CDS* or CD4*

cells from the peripheral blood obtained from patients with SCCHNwere cultured, with or without tumor cells, in TCM as described above.Irradiated (5000 rads) autologous tumor cells (SCCHN lines) were

3892




added to these cultures as stimulators on day 1, following spontaneousrelease of captured cells from antibody-coated surfaces, at a lympho-cyte:tumor cell ratio of 20:1. The cultures were restimulated weekly byaddition of irradiated tumor cells at the same ratio. Control cultureswere established with unseparated PBLs from the same patients.

Cytotoxicity Assays. A 4-h 5'Cr release cytotoxicity miniassay wasperformed as described earlier (16). Briefly, target cells (NK-sensitiveK562 or NK-resistant Daudi, allogeneic or autologous SCCHN orother solid tumor cell lines) were incubated with 150 nG of Na"enrÃ³mate (5 mCi/ml; NEN, Boston, MA) for l h at 37Â°C.The cellswere washed three times, and 1 x IO3 5'Cr-labeled target cells wereplated in each well of 96-well plates and mixed with effector cells ateffectontarget cell ratios ranging from 20:1 to 1.2:1. Cells were centri-fuged at 65 x g and incubated for 4 h at 37Â°Cin an atmosphere of 5%CO2 in air. Supernatants were harvested and MCr released was measured

in a beta counter (17). Spontaneous release, always <10% of maximalrelease for K562 and Daudi and never >25% for solid tumor cell lines,was measured after incubation of target cells with medium only. Maximal release was determined in wells containing target cells only, afterthe addition of Triton X-100. The percentage of specific lysis wasdetermined using the following formula:

Experimental mean cpm - Spontaneous release mean cpmMaximal mean cpm - Spontaneous release mean cpm x 100

Results were expressed in LU, calculated according to the method ofPross et al. (18). One LU was defined as the number of effector cellsrequired for 20% lysis of 1 x IO'1target cells, and the number of LUpresent in IO7 effector cells was calculated. All cytotoxicity data areexpressed as LU20/107 effector cells.

Statistical Analysis. The significance of differences between functional and phenotypic characteristics of positively selected and noncap-tured cells was calculated using Student's t test. When a paired test was

not appropriate, the one-way analysis of variance was used (Tables 3and 4).

RESULTSCapture of CD8+ or CD4+ T-Cells. PBLs and LNLs obtained

from patients with SCCHN were used to determine the efficacyof antibody-coated T25 flasks in capturing CD8+ or CD4+ T-cells. Only cell preparations containing at least 40 x IO6 cells

were used in these experiments, to better control consistencyof the capturing process. In comparison to freshly isolatedLNLs or PBLs phenotyped before the capture, cells not captured on flasks coated with anti-CD8 MoAb were significantly(P < 0.001) depleted of CD8+ cells (Table 2). In PBLs,CD3~CD56+ cells were also significantly depleted (P < 0.02),

an indication that a proportion (about 30%) of NK cells, thosewhich express the CD8+CD56+ phenotype, also reacted withthis antibody. As shown in Fig. 1, a mean of 92% of CD8+ cellsin PBLs and of 90% in LNLs were captured on antibody-coatedflasks during the process.

To evaluate the capture process on T25 flasks coated withanti-CD4 antibody, PBLs or LNLs previously depleted of CD8+

cells as described above (i.e., noncaptured cells) were transferredto and incubated in these flasks. This sequence allowed forcapture and growth of separated populations of both CD8+ andCD4+ lymphocytes. The second capture procedure for CD4+

lymphocytes was very effective, as shown in Fig. 1: MoAb-captured cells contained 98% of CD4+ T-lymphocytes. Sincethe previously panned cells contained few, if any, CD56+ lymphocytes, these were not captured on flasks coated with anti-CD4 monoclonal antibody (Fig. 1).

Growth of Captured Populations. Positively selected CD8+ orCD4+ lymphocytes were cultured in the presence of IL2 for up

to 15 days. Proliferation of lymphocyte subpopulations cap-

Table 2 Phenotype of peripheral blood or lymph node lymphocytes obtained frompatients with SCCHN before and after capture on anti-CD8 or anti-CD4

antibody-coated flasks

cor CDS* CD4* CD3-CD56*

PBL capture on anti-CDSMoAb-coated flasks"

Fresh cellsNoncaptured cells

LNL first capture on anti-CD8-coated flasks'

Fresh cellsNoncaptured cells

LNL second capture onanti-CD4-coated flask/

Before captureNoncaptured cells

63 Â±2*

44 Â±10

68 Â±658 Â±10

76 Â±530 Â±8*

22 Â±52 Â±\c'd

10Â±11 Â±Ie

3Â± l6Â±2

41 Â±847 Â±10

59 Â±653 Â±10

67 Â±54Â±2*

26 Â±316Â±3C'"

1.6 Â±0.41.0 Â±0.4

1.3 Â±0.14Â± 1

" PBLs obtained from 8 patients with SCCHN were phenotyped prior to

capture (fresh cells) and at the end of the capturing procedure (noncaptured cells).4 % positive cells, mean Â±SEM.c CDS* cells were significantly depleted (P < 0.001) from noncaptured cells.d CD56* cells were significantly depleted (P < 0.02) from noncaptured cells.' LNL were obtained from 6 patients with SCCHN and were phenotyped prior

to and after capture.^LNL were initially depleted of CD8* lymphocytes on flasks containing anti-

CDS antibody and were then transferred to flasks coated with anti-CD4 antibody.Flow cytometry was performed using CD8*-depleted LNL prior to capture andCD4-depleted, noncaptured LNLs.

*CD4* cells were significantly (P < 0.001) depleted from noncaptured LNLas were CD3* cells (P< 0.001).

B

PBL LNL

100

80 -

V)

IliUQLUOC

60 -

OvP

40 -

20 -

CD8 + CD3-CD56+ CD3-CD56 + CD4-I

Fig. l. The percentage of cell subsets captured sequentially on anti-CD8 MoAb-coated flasks (-4) and anti-CD4 MoAb-coated flasks (B). The data (columns) aremean percentages (bars, Â±SEM)of captured cells calculated from flow cytometrymeasurements of fresh and noncaptured cells in PBLs of 4 patients and LNLs of6 patients with SCCHN. Suspensions (40 x IO6 cells/flask) of PBLs or LNLsobtained from patients with SCCHN were first placed in flasks (T25) coated withanti-CDS MoAb. Following capture, the noncaptured LNLs were transferred toflasks coated with anti-CD4 MoAb.

tured from the peripheral blood of normal volunteers or patientswith SCCHN was compared with that of unseparated cells inbulk cultures (Table 3). Captured CD8+ T-lymphocytes prolif

erated as well as unseparated lymphocytes in both patients andnormal donors, and their growth was comparable to that ofCD8+ cells captured from the blood of normal individuals(Table 3). In contrast, captured CD4+ T-lymphocytes of pa

tients with SCCHN proliferated poorly, while those obtainedfrom normal donors showed excellent proliferation in culture(Table 3).

3893



CAPTURE AND GROWTH OF CDSÂ»AND CD4Â»T-CELLS

Table 3 Proliferation of CDS* and CD4* T-lymphocytes captured from the

peripheral blood of patients with SCCHN or normal volunteersUnseparated PBLs or captured CDS* or CD4* T-lymphocytes obtained from

patients with SCCHN as well as from normal donors were cultured in the presenceof rIL2 (100 units/ml) and PHA (5 Â»jg/mladded at day 0) for 15 days withoutthe addition of irradiated autologous feeder cells.

SCCHN patientsUnseparatedCDS*CD4*

Normal donorsUnseparatedCDS*

CD4No.85

37

45Fold

expansion48

Â±1639 Â±10

5Â±0.532

Â±4.821Â±7.8

98 Â±37.0Â°Mean Â±SEM.

Table 4 Proliferation of Unseparated or captured CDS* or CD4* lymph node

lymphocytes obtained from patients with SCCHN and cultured in the presence orabsence of feeder cells

Unseparated or captured CDS* or CD4* LNLs were cultured in the presence

of 100 units/ml of rlL2 and PHA (5 Â¿ig/mladded on day 0) for 15 days. Toobtain feeder cells, noncaptured autologous LNLs were collected, washed, irradiated at 5000 rads, and then added back to captured CDS* or CD4* LNLs at aconcentration of 1.5 x 10'cells/ml on dayO. Allogeneic feeder cells were Epstein-Barr virus-infected B-cell lines irradiated at 20.000 rads and added to capturedLNLs at a concentration of 0.2 x 106/ml on day 0 of culture. The difference inproliferation between CDS* and CD4* LNLs in culture was significant at P <

0.05.

No feeder cells+autologous feeders+allogeneic feedersUnseparated31

Â±12(7)Â°ND*

NDCDS*0.4

Â±0.5 (4)34Â± 10(11)15 Â±6(4)CD4*0(2)

2 Â±0.8 (7)7 Â±2 (4)

Â°Fold expansion, mean Â±SEM. Numbers in parentheses, numbers of cultures

studied.* ND. not determined.

When unseparated LNLs were cultured in the presence ofIL2 for 15 days, they generally proliferated as well as PBLs.However, captured CD4* or CDS* LNLs failed to proliferate

under the same culture conditions (Table 4). To improve expansion of captured LNLs subpopulations, autologous irradiated noncaptured cells were added back to the captured CDS*or CD4+ cells at a concentration of 1.5 x 106/rnl on day 0 of

culture. The addition of irradiated autologous or allogeneicfeeder cells considerably improved the fold expansion of separated CDS* LNLs (Table 4). However, only a small increase in

proliferation was observed after the addition of autologousfeeder cells to cultures of CD4* LNLs in patients with SCCHN(Table 4). It appeared that captured CD4+ LNLs of patients

with SCCHN responded poorly by proliferation to IL2 plusPHA even in the presence of feeder cells. In additional experiments, proliferation of CD4+ LNLs in response to IL2 was not

enhanced by stimulation with concanavalin A (10 Mg/ml) and/or phorbol ester (1 ng/ml) (data not shown).

Phenotypic Analysis of Captured and Cultured (1)8* andCD4+ Subpopulations. The phenotype of the cells in cultures ofcaptured CDS* or CD4* T-cells was tested serially by two-colorflow cytometry. The phenotypic markers of cultured CDS* cells

captured from the PBLs of normal volunteers were the same asthose in cultures of captured CDS* cells from the PBLs ofpatients with SCCHN (data not shown). In cultures of CDS*

cells obtained from the PBLs of patients, a mean percentage Â±SEM of CD3*CD8* cells was 94 Â±5% and of CD3*CD4* cells

10 Â±6% between days 10 and 15 of growth (n = 6).Capture and expansion in culture of CDS* T-lymphocytes

from LNs of patients with SCCHN were of particular interest,because we hoped to study antitumor cytotoxicity of these cells,which usually represent a relatively small proportion of LNLs

(i.e., 10 Â±1% in Table 2). Phenotypic markers of proliferatingCDS* LNLs are compared to those of unseparated LNLs

(paired cultures) in Table 5. In early (day 7) and later (day 15)cultures, the percentages of CDS* T-cells were similar, and they

were significantly (P < 0.05) higher in antibody-captured thanin unseparated cell cultures. Conversely, the percentages ofCD4* T-cells were significantly (P < 0.05) lower. In unsepa

rated cultures, the CD4:CD8 ratio was 3.1 in early cultures,and it increased to 8.0 in 15-day cultures. In cultures of capturedCDS* LNLs, the CD4:CD8 ratio remained stable at 0.2 (Table

5). The great majority of proliferating LNLs wereCD8*CDllb~, i.e., expressed phenotypic markers associated

with cytotoxic cells.The capture and expansion in vitro of the populations en

riched in CD4*LNLs were investigated next. As might bepredicted, based on the relative proportions of CD4* and CDS*T-cells in LNLs (Table 2), early cultures (day 3) of positivelyselected CD4* LNLs contained nearly pure CD4* cells (datanot shown). These cultures of CD4* LNLs did not proliferate

well, however, even in the presence of irradiated autologousfeeder cells (Table 4). In comparison to unseparated LNLscultures (Table 6), those of captured CD4* LNLs containedsignificantly fewer CDS* T-cells. By day 15, however, thephenotype of the positively selected CD4* LNLs and unsepa-

Table 5 Phenotypic analysis of captured CD/t* and unseparated lymph node

lymphocytes obtained from patients with SCCHN and cultured in the presence ofr!L2

CDS* captured cells and unseparated LNLs were cultured in the presence of100 units/ml of rIL2 and irradiated autologous feeder cells. Two-color flowcytometry was performed on cultured cells at different times Â¡nculture

Captured CDS* Unseparated

MarkersDay 8 Day 15 Day 7 Day 15

CD3*CDS*CD4*CD3*CD56*CD3-CD56*CD8*CDllb*HLA

DR*CD25*TCRa/Ãi**TCR>/o*f98

0.6Â°91720.189661.8*4*70.151610990.7100Â±0.196Â±3*7Â±3*6+

1.5015Â±

551+946+

14NDÂ¿0.293

Â±524+1375Â±1118

+252+18Â±869

Â±2676Â±25890.593

+410Â±580

Â±35Â±34Â±

26Â±540

Â±1343+43NDND

" % positive cells, means Â±SEM.* Significant differences (P < 0.05) between percentages of CDS* and CD4*

cells in captured CDS* versus unseparated LNL cultures.' One experiment.d ND. not determined.

Table 6 Phenotypic analysis of captured CD4* and unseparated lymph node

lymphocytes obtained from patients with SCCHN and cultured in the presence ofrIL2

CD4* T-cells captured on antibody-coated flasks and unseparated cultures of

LNLs were expanded Â¡nthe presence of 100 units/ml of 1L2 and irradiatedautologous feeder cells. Two-color flow cytometry was performed at differenttimes in culture.

Captured CD4* Unseparated

MarkersCD3*CD4*CDS*CD3*CD56*CD3-CD56*CD8*CDllb*HLA-DR*CD25*Day7(1

=5)97Â±0.5Â°87+43Â±

1.7*0.7

0.20.20.10.30.2462139

11Dav

15(1 =5)96

.489.63.6*4

.73

.220.726

Â±4.532Â±11.8Day

7(1=7)93

4751124131813218

8692676

25Day

15(1 =4)93

Â±580+310Â±55Â±34Â±26

+540Â±1343Â±40

" % positive cells, mean + SEM.* Differences in the percentages of CDS* cells between captured CD4* and

unseparated cells Â»eresignificant at P < 0.05.

3894




rated LNLs was quite similar (Table 6).Overall, the phenotypic data indicated that a significant en

richment in CDS* LNLs could be obtained following their

capture on the antibody-coated surfaces and expansion in thepresence of IL2 and autologous feeder cells. On the other hand,selecting for CD4* T-cells from LNLs did not offer substantial

advantages over unseparated LNLs cultures: growth of capturedCD4* LNLs was never satisfactory, and enrichment in CD4*

cells in 15-day cultures was not better than that with unseparated LNLs.

Antitumor Cytotoxicity of Cultures Enriched in CD8+ or CD4+Cells. Antitumor cytotoxicity of captured CDS* orCD4* LNLs

cultured in the presence of IL2 was tested on several tumor celltargets including allogeneic SCCHN targets. In the initial experiments (n = 4), these effectors appeared to have relativelylow cytotoxicity (i.e., 0-50 LU/107 cells) against NK-resistantbut LAK-sensitive Daudi targets and relatively high levels ofcytotoxicity (i.e., 160-884 LU/107 cells) against two SCCHN

cell lines (PCI-1 and -13), which were NK resistant. Antitumorcytotoxicities of captured CDS* PBLs (n = 6) of patients with

SCCHN were not significantly different from those obtainedfor CDS* LNLs (data not shown). In additional experiments,

it was possible to compare antitumor cytotoxicity of LNLsseparated into CDS* cells and CD4* cells with that of unsepa

rated LNLs following their expansion in culture (Fig. 2). Unseparated LNLs showed high levels of NK activity, intermediatelevels of activity against PCI-1, and low activity against Dauditargets (Fig. 2). Captured and cultured CD4+ LNLs showed a

different pattern of cytotoxicity, with low or almost no cytotoxicity against all four tumor cell targets used (Fig. 2). In contrast,captured and expanded CD8+ LNLs showed little, if any, cy

totoxicity against Daudi, had relatively low cytotoxicity againstK562, and exhibited substantial levels of activity against fourdifferent NK-resistant SCCHN targets (only data for two targets, PCI-1 and PCI-13, are shown in Fig. 2). Also indicated inFig. 2 is an excellent purity of captured and cultured CDS* andCD4* populations used in the cytotoxicity experiments. These

(A

UJO

600

500-

400-

300 -

200

100-

K562

PCI 1

PCI 13

Q DAUDI

UNSEPARATED CD8 +( 9 4 % â€¢2 )

CD4 +(90%-2)

Fig. 2. Antitumor cytotoxicity of unseparated (n = 5) and positively selectedCD8* (n = 9) and CD4* (n = 8) cells obtained from LNs of patients with SCCHN.

The cells were cultured in the presence of 100 units/ml of rIL2 and PHA (5 fig/ml added on day 2) for 15 days, and their cytotoxicity was determined in 4-h "Crrelease assays against a panel of targets consisting of K562, Daudi. and two NK-resistant SCCHN cell lines (PCI-1 and PCI-13). Numbers in parentheses, purityof cultures. The data (columns) are between LU/107 cells Â±SEM(bars).

results suggested that capture of CDS* LNLs and their subsequent growth might lead to an outgrowth of CDS* antitumor

effectors with a more restricted cytotoxic activity againstSCCHN cell lines than that seen in unseparated cultures ofLNLs.

Since the observed enrichment in cytotoxicity againstSCCHN targets was variable in cultures of captured CD8+LNLs, it was important to examine T-lymphocytes from I-LNsas opposed to NI-LNs for antitumor activity. As shown in Fig.3 for representative paired cultures, enrichment in cytotoxicityagainst PCI-1 or PCI-13 was greater in CD8*-captured LNLsfrom NI-LNs than I-LN. It can also be seen, in the sameexperiment, that separated CDS* LNLs had a significantly

higher (P < 0.05) antitumor cytotoxicity against SCCHN targets than unseparated LNLs only in cultures established withcells isolated from NI-LNs. These findings were consistentlyreproduced in 3 paired experiments, and they suggested thatthe presence of tumor cells in the lymph node might havecontributed to poor enrichment in antitumor effector cells evenin captured CDS* subpopulations.

Cytotoxicity against SCCHN Targets. The cytotoxicity experiments performed with captured CDS* LNLs as effectors indi

cated that they mediated lysis of SCCHN targets better thanthat of Daudi targets. To more closely examine this preferentiallysis of SCCHN, we compared cytotoxicity of several capturedCDS* and CD4* LNL preparations against a broader panel of

tumor cell targets. A panel of human cultured tumor cell lines,including several SCCHN lines (15), was assembled (Fig. 4)and tested in 4-h 5'Cr release assays with different preparations

of effector cells. The ability of each effector cell to lyse all thetargets in the panel was determined in the same experiment.The spontaneous release for different solid tumor targets rangedfrom 7 to 25%. Because cytotoxicity of CDS* or CD4* effectorcells against Daudi was consistently <20-30 LU/107 cells (see

Fig. 2) and that against other tumor cell targets in the panelwas very high, the percentage increase in lysis relative to Daudiwas calculated for CDS* and CD4* effectors. As shown in Fig.

4, for a paired experiment with effectors from the same LN,the percentage increase in cytotoxicity against SCCHN targetswas very substantial for CDS* LNLs, while that for CD4*

LNLs was minimal. The results of additional cytotoxicity experiments in which the ability of captured CDS* LNLs to lyseSCCHN targets was compared to that of CD4* LNLs aresummarized in Table 7. These data indicated that CDS* LNLs

captured and expanded in the presence of IL2 and irradiatedautologous feeder cells exhibited preferential cytotoxicityagainst SCCHN cell lines.

Additions of AuTu Cells to Captured Lymphocytes. To evaluate possible effects of AuTu cells on proliferation and antitu-mor cytotoxicity of captured CDS* lymphocytes, we studiedCDS* PBLs in those patients with SCCHN for whom AuTu

cell lines were available. Irradiated AuTu cells were added tocaptured CDS* cells on day 1 of growth at the 20:1 lympho-cyte:tumor cell ratio. These CDS* PBLs proliferated significantly less well (P< 0.05) than CDS* not stimulated with AuTu

cells (e.g., fold expansion was 3 Â±0.7 (mean Â±SEM; n = 7)versus 39 Â±10 (n = 5), respectively).

Next, the phenotype in culture was compared between CDS*

PBLs obtained from patients with SCCHN and cultured in thepresence or absence of AuTu cell lines. The data shown in Fig.5 indicated that no phenotypic differences existed between thesecultures, except for somewhat increased percentages of activated (CD25*, HLA-DR*) cells and CD3~CD56* cells in the

3895




Fig. 3. Antitumor cytotoxicity of LNLs obtained from tumor-involved and tumor non-involved lymph nodes of one patient withSCCHN. Unseparated and positively selectedCDS* LNLs were cultured in the presence of

100 units/ml of rlL2 and PHA (5 Mg/ml) andirradiated feeder cells for 7 days. Cytotoxicityof CDS* LNLs was determined in 4-h "Crrelease assays against Daudi and two alloge-neic SCCHN cell lines. The data (points) arepercentages of specific lysis at different effector: target cell ratios. They are representativeof experiments performed with paired LNLsfrom tumor-involved and -uninvolved LNs ofthree patients with SCCHN.

to>â€¢

-UNSEPARATED N-I-LNL(C08Â«:35%)

-CDS* N.|-LNL(CD8 +: 95%)

PCI 1

OAUDI

10:1 5:1

EFFECTOR:TARGET RATIO

2.5:1 20:1

UNSEPARATED I-LNL (CD8+: 38%)CD8+ I-LNL (CDS* :98%)

PCI 1

PCI 13

DAUDI

10:1 5:1

EFFECTOR:TARGET RATIO

2.5:1

Q-D

OCLU

LUV)<LUceCJ

00

co>_l0Â°

5000 n

4000 -

3000 -

2000 -

1000 -

K562 FEMXFig. 4. Antitumor cytotoxicity of positively selected CDS* and CD4* LNLs

obtained from a patient with SCCHN. The cells were cultured in the presence of100 units/ml of rIL2, PHA (5 ng/ml added on day 0). and irradiated autologousfeeder cells for 15 days. Their cytotoxicity was determined in 4-h "Cr releaseassays against a panel of targets consisting of K562. Daudi, three NK-resistantallogeneic SCCHN cell lines (PCI-1. PCI-13, PC1-4A). and a melanoma cell line(FEMX). The percentage increase in cytotoxicity over Daudi was determinedusing the following formula:

Cytotoxicity (LU/107 cells) against a targetCytotoxicity (LU/107 cells) against Daudi

The data shown are for a paired experiment. Other cytotoxicity experimentsperformed are summarized in Table 7.

cultures stimulated with AuTu (no significant difference). Also,in two cases, paired samples of CD8+ PBLs growing in the

presence or absence of AuTu were compared, and no significantdifferences in the percentages of CD3+CD8+ T-lymphocytes

were observed. It is important to note that, although a substantial proportion of CD3~CD56+ cells were initially captured on

anti-CD8 MoAb-coated flasks, few of them proliferated relativeto the CD3+CD56~ population. In both types of cultures,CD3+CD8+ T-lymphocytes were the major proliferating cell

type (mean, 95 or 99%). Nearly all of these T-cells expressedT-cell receptor a//3, except that, in one culture with AuTu, T-cells expressing T-cell receptor y/o were 15%. In general, theaddition of AuTu cells to captured CD8+ T-lymphocytes of

patients with SCCHN did not result in improved growth orenrichment in activated T-cells as compared to cultures grownwith feeder cells and IL2 alone.

Antitumor function of lymphocytes cultured in the presenceor absence of AuTu was also tested using CD8+ cells captured

from the peripheral blood of patients with SCCHN. No signif-

Table 7 Enrichment in antitumor cytotoxicity against SCCHN cell linesmediated by positively selected CDS* lymph node lymphocytes obtained from

patients with SCCHN and tested against various tumor cell targets

Tumorcelltarget"K562

PCI-1PCI- i 3PC1-4AFEMXCaptured

CDS*(n =10)880

Â±490"4420Â±2100C3980 Â±2120'3100Â± 1500C

350 Â±310Captured

CD4*(n =6)630

Â±230760 Â±520

1070 Â±72080 Â±40

100 Â±90Â°Cytotoxicity was determined in 4-h "Cr release assays performed with a

panel of tumor cell targets including Daudi and the targets listed. PCI cell linesare NK cell-resistant SCCHN cell lines established in our laboratory (15). FEMXis an NK-resistant melanoma cell line. Effector cells were CDS* or CD4* LNLscaptured on antibody-coated surfaces and expanded in the presence of IL2 for 15days as described in "Materials and Methods."

* The percentage increase in cytotoxicity over Daudi (mean Â±SD) was deter

mined as detailed in the legend to Fig. 4. The data, were obtained from paired aswell as nonpaired experiments with CDS* and CD4* LNLs from the same LN

or LNs from different patients, respectively.c Significant (P < 0.05) differences between cytotoxicity mediated by CDS*

versus CD4* effectors.

3896




100 -i

O

LU

>

inOCL

CD3 + CDS CD4+ CD3-CD56+ DR* CD25Â«Fig. 5. Phenotypic markers on positively selected CDS* lymphocytes obtained

from the peripheral blood of 10 patients with SCCHN and cultured in the presenceof 100 units/ml of rIL2. PHA (5 /ig/nil). and irradiated autologous feeder cells,with (n â€”6) or without)Â« = 6) the addition of irradiated AuTu cells as stimulatorsat a PBLiAuTu ratio of 20:1. The cultures were sampled for flow cytometrybetween days 10 and 20 of growth. The data (columns) are mean percentages ofpositive cells Â±SEM (bars). No significant differences between cultures with orwithout AuTu were detected.

icant differences in levels of cytotoxicity against K562, Dandi.or PCI-1 were observed in any of the six cultures studied (datanot shown), which contained up to 98% of CD3+CD8+ T-

lymphocytes. Cytotoxicity against AuTu was also measured incultures primed with AuTu; however, it was never higher thanthat against K562 targets (190 Â±20 LU/107 cells, mean Â±SEM). Thus, in vitro stimulation with AuTu of captured CD8+

PBLs of patients with SCCHN did not lead to preferentialexpansion of AuTu-reactive effectors under conditions used forthese cultures.

DISCUSSION

The ability to successfully separate lymphocyte subsets fromthe blood and tissues of normal individuals and patients withvarious diseases, including cancer, is essential for definition offunctional characteristics of these cells. Often, however, freshlyseparated lymphocyte subpopulations are not available in numbers sufficient for functional assays. It is particularly difficultto obtain purified or enriched subpopulations of tissue-infiltrating lymphocytes, which are frequently recovered in limitedquantities from surgical biopsies and have to be cultured in thepresence of rIL2 to increase their numbers (19). These lympho-kine-activated cells acquire, in vitro, an ability to kill variouscell targets via non-MHC-restricted mechanisms (20). However, LAK cells grown in long-term cultures with IL2 arephenotypically and functionally heterogeneous (21). Differentlymphocyte populations present in these cultures might influence each other's functions (22). For example, CD3~CD56+and CD3+CD56+ cells separated by sorting and tested forantitumor cytotoxicity in 5lCr release assays had a much higher

effector cell function than that measured in the unseparatedparent culture (22). This observation indicated that inhibitoryor regulatory activities in unseparated cultures may modifyeffector function and proliferation of the cultured cells. Sorted,highly purified lymphocyte subpopulations are generally difficult to culture without additions of conditioned media, irradiated feeder cells, or both (23-26). Thus, both effector cellfunction and proliferation are strongly dependent on the purityof culture and growth factors available at the time of expansion.

Since both parameters obviously play an important role ingrowth of therapeutically useful lymphoid cells, recent attentionhas focused on a better understanding of culture requirementsfor highly purified subpopulations of human lymphocytes.

Various techniques have been used in the past to separatehuman lymphocytes, among them gradients of different separation media (27), resetting techniques (28), panning on solidsurfaces coated with antibodies (14, 29), complement-dependent lysis with monoclonal antibodies specific for surface antigens on lymphocyte subpopulations (30), and cell sorting byflow cytometry (30, 31). Negative selection has been consideredpreferable, because of a possibility that positively selected lymphocytes may become activated through interactions with theligands used for selection or may retain these ligands on theirsurface following the separation process. On the other hand, ithas been difficult to separate by negative selection those sub-populations that represent only small proportions of total lymphocytes, because of inadequate purity and poor recovery. Positive selection with MoAbs appears to perform better in thisregard. It is feasible to perform such positive selections withreasonably high efficiency and without a concern for retainingthe MoAb on cell surface when AIS antibody-coated flasks areused.4 Furthermore, serial panning of selected populations on

two or more flasks, each coated with different MoAbs, allowsfor an efficient use of human cell suspensions. For example, inthe case of human LNLs in patients with SCCHN, we wereable to capture a majority of CD8+ cells representing about

10% of the total LNLs and to expand these cells in culture withrelatively minor contamination from CD4* lymphocytes. Theenrichment in CDS* T-lymphocytes was excellent in these

cultures.In patients with SCCHN, cervical lymph nodes are often the

initial metastatic site (32). Tumor-draining lymph nodes havebeen reported to be a good source of autotumor effector cellsin patients with melanoma (33) and pancreatic cancer (34).However, in SCCHN, fresh LNLs had lower proliferative responses to nonspecific as well as specific (AuTu) activators andlower cytotoxicity than PBLs, as demonstrated by us earlier(35). We have also shown that LNLs obtained from I-LNs orNI-LNs were deficient in the ability to produce certain cyto-kines: tumor necrosis factor Â«,IL-1/3, or 7-interferon spontaneously or even after in vitro activation with PHA or lipopoly-saccharide (35). Exogenous IL2 alone did not appear to correctthis defect in LNLs (35). We, therefore, wished to determinewhether T-lymphocyte subpopulations separated by antibodycapture from LNLs could acquire improved functional andproliferative responses. However, captured CD4+ or CD8+

LNLs proliferated less well than unseparated LNLs in vitro.Although better proliferation of CD8+ LNLs was obtained inthe presence of feeder cells, captured CD4+ LNLs proliferatedsignificantly less well than CD8+ cells even when autologous or

allogeneic feeder cells were added to cultures. Such additionsof irradiated feeder cells were necessary for optimal in vitroexpansion of CD8+ LNLs.

Antitumor effector cell function of captured and culturedCD8+ LNLs against NK-resistant allogeneic SCCHN targets

was generally higher on the per cell basis than that of unseparated LNLs, especially in LNs not involved by the tumor. Also,captured and expanded CD8+ LNLs showed a more selective

pattern of cytotoxicity than unseparated LNLs, with little and,in some cultures, no cytotoxicity against Daudi targets (Fig. 2).CD4+ LNLs demonstrated little antitumor cytotoxicity in cul-

4 AIS. proprietary process. United States patent 4933410.

3897




ture. These observations indicated that a selective enrichmentin effectors with cytotoxicity restricted to SCCHN targets mightbe achieved by capture and expansion of the CD8+ LNL subsetin patients with SCCHN. Indeed, when CD8+ and CD4+ effec

tors were tested for cytotoxicity against a panel of tumor celllines, including SCCHN, only CDS* LNLs showed significantly

increased lysis of SCCHN targets in relation to Daudi targets(Fig. 4; Table 7). This type of MHC-unrestricted but SCCHN-restricted antitumor cytotoxicity mediated by CD3*CD8+ in

vitro activated effector cells was similar to that described byBarnd et al. (34) for T-cell lines obtained from a tumor-drainingLNs of a patient with pancreatic cancer. These T-cells recognized tumor-associated mucins via the T-cell receptor and selectively lysed tumor cells expressing large and regularly repeated mucin epitopes (34). Although various mucin moleculesare known to be produced by tumors of epithelial cell origin, ithas not been demonstrated that SCCHN cell lines used in ourexperiments express them. Nevertheless, it is possible thatCD3+CD8* effectors captured from LNLs of patients of

SCCHN and cultured in IL2 recognize an antigen complexcommon to various SCCHN tumor cells but not present ontumor cells of nonepithelial origin. Further studies are neededto confirm the presence and define the nature of such an antigenon SCCHN cell lines.

Captured and cultured CDS* LNLs from patients with

SCCHN did not respond to stimulation with irradiated AuTucells by increased proliferation or antitumor activity. This observation appeared to contradict the hypothesis advanced abovethat SCCHN-restricted cytotoxicity mediated by CD3+CD8+

LNLs following their culture in IL2 was a result of specificMHC-unrestricted recognition of a tumor-associated antigen.However, it is important to realize that the SCCHN cell linesused in these experiments produce prostaglandin E2,5 whichmay exert profound inhibitory effects on growth of CD8+ T-

lymphocytes (36). Such growth inhibitory effects of SCCHNwere indeed observed when captured CD8+ PBL-T lymphocytes

were incubated in the presence of AuTu. Also, the presence ofthe tumor in LNs adversely affected in vitro generation of anti-SCCHN CDS* effectors as shown in our experiments. Thus,NI-LNLs appeared to be a better source for capture of CD8+

cytotoxic lymphocytes with preferential reactivity againstSCCHN targets than I-LNLs. Overall, LNLs, and especiallyNI-LNLs, obtained from patients with head and neck cancercould be successfully used for enrichment in cytolytic CD8+SCCHN-restricted effector cells.

Capture of lymphocyte subpopulations on antibody-coatedsurfaces can be easily up-scaled for preparation of selected cellpopulations of human mononuclear cells for adoptive immu-notherapy. It can also be down-scaled to capture small numbersof effectors on antibody-coated microtiter plates. We haveutilized both of these approaches to obtain, in the formerinstance, highly enriched CDS* T-cells that are being used in

therapy of patients with acquired immunodeficiency syndrome(12) and, in the second instance, to separate and growCD3+CD8+ T-cells from human tumor-infiltrating lymphocytes

that are being evaluated for AuTu specificity in our laboratories.

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1991;51:3891-3899. Cancer Res Eric M. Letessier, Dae S. Heo, Thomas Okarma, et al. Head and Neck CancerSelection from the Blood and Lymph Nodes of Patients with

T-Lymphocytes by Positive+Enrichment in Tumor-reactive CD8

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