[CANCER RESEARCH 37, 1159-1164, April 1977)

SUMMARY

Regional lymph node lymphocytes from patients withsquamous cancer of the head and neck were tested in vitrofor their ability to proliferate in response to phytohemagglutinin, concanavalin A, and allogeneic stimuli in one-waymixed lymphocyte culture. Their ability to act as cytotoxiceffectons in phytohemagglutinmn-dependent cellular cytotoxicity was also evaluated, and all results were comparedto normal lymph node on blood lymphocytes. The regionallymph node lymphocytes retained proliferative capabilitiesequal to those in control lymph nodes on blood, whereasthey were unable to mediate phytohemagglutinin-dependent cellular cytotoxicity. However, this was not a tumorrelated effect because normal lymph node lymphocyteswere also ineffective in this assay. The failure of the regionalimmune response to control early tumor growth could notbe accounted for by generalizednonspecificimmunosuppression in regional lymph node lymphocytes, inasmuch as these cells demonstrated normal in vitro activity.

INTRODUCTION

The regional immune response, as represented by draining RLN,3 will be the 1st level of interaction between adeveloping neoplasm and the host's immune capabilities.This occurs because tumor cells and/or tumor-related products will be drained into regional lymphatics prior to vasculanization of the tumor. The continued growth of the neoplasm might be related to an absolute lack of stimulation ofthe host's immune response (nonimmunogenicity). However, previous work showing changes in lymphoid subpopulations (13) as well as evidence of histological alterations(1, 2) in human RLN suggests that RLN are being influencedby the presence of the tumor.

An antigenic tumor might overcome the regional immuneresponse by causing specific immune suppression of reactivity toward itself on by generating nonspecific immunosuppression of the entire range of immune capabilities ofRLN. Thislatterpossibilitycouldbe relatedtothefactthat

I Research supported by NIH Grant CA 12800 and NIH Training Grant Al00431.

2 To whom requests for reprints should be addressed.3 The abbreviations used are: RLN, regional lymph node(s); PHA, phytohe

magglutinin; Con A, concanavalin A; MLC, mixed lymphocyte culture; DNL,distal lymph node(s); NLN, normal lymph node(s); PBL, peripheral bloodlymphocytes; PDCC, PHA-dependent cellular cytotoxicity; RPMI, RoswellPark Memorial Institute; LNL, lymph node lymphocyte(s); T-, thymus derived;B-, bursa equivalent; LN, lymph node.

Received September 27, 1976; accepted December 29, 1976.

RLN will be exposed to high concentrations of tumor products from a very early stage in tumor development. Bloodlymphocytes from patients with cancers have been reportedto demonstrate such nonspecific immunosuppression bothin vivo and in vitro (6, 10).

To test the hypothesis that the regional immune responsemay show generalized immunosuppression of reactivity,RLN lymphocytes from patients undergoing primary tumorsurgery with RLN dissection were tested in PHA, Con A, and1-way MLC-proliferative assays. The responsiveness ofthese RLN lymphocytes was compared to that for autologous DLN, NLN, and PBL. These same lymphocyte populations were also tested for their ability to function in shortterm (2- to 3-hr) PDCC. Those studies demonstrated noevidence of generalized immunosuppression in RLN, because RLN lymphocytes retained in vitro capabilities similarto those of their normal counterparts.

MATERIALS AND METHODS

Patients, Preparation and Separation of Lymphold Suspensions, and Analysis of Surface Receptors. These procodunes and results are described in detail in the precedingpaper(13).

Mitogen Stimulation. For mitogen stimulation studies,lymphocytes were suspended at a concentration of 1 x 106/ml in RPMI 1640 media supplemented with streptomycin,penicillin, 10% human AB serum (heat inactivated), and Lglutamine. One-tenth ml of this suspension (1 x 10@lymphocytes) was added to the wells of Cooke microtiten plates(Grand Island Biological Co., Grand Island, N. Y.), and 0.1ml of supplemented RPMI 1640 media with or without PHAon Con A was added. All tests were nun in triplicate. Theplates were incubated with 5% CO2 in a 37°humidifiedincubator. Twenty-four hr prior to harvesting, each well waspulsed with 2 @.tCiof [3H]thymidine (New England Nuclear,Boston, Mass.) in 20 @lof RPMI 1640 media. The plateswere harvested, and incorporated [3H]thymidine wascounted using a liquid scintillation system.

Lectin-dependent Cell-mediated Cytotoxicity. Severalmunine tumor lines were used as target cells to determinethe lectin-dependent cellular cytotoxicity of lymphoid cells:D6 (Gross virus-induced lymphoma) syngeneic in C3H mice;EL-4 leukemia syngeneic.in C57BL/6 mice; and G-35 (Grossvirus-induced lymphoma) syngeneic in BALB mice. D6 wasgrown in suspension in Tissue Culture Medium MJLA (Microbiological Associates, Bethesda, Md.) supplementedwith 10% heat-inactivated fetal calf serum. G-35 and EL-4were maintained in ascites form. Immediately before test

APRIL 1977 1159

Preservation of in Vitro Biological Functions in RegionalLymph Node Lymphocytes in Squamous Head and Neck Cancer1

Andrew Saxon2 and Joe Portaro

Department of Microbiology and Immunology, Immunobiology Group, UCLA School of Medicine, Los Angeles, California 90024

on June 29, 2018. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

A. Saxon and J. Portaro

ing, the target was labeled with 51Cnusing the technique ofWigzell (18). Sodium chromate, 0.4 pCi, was added to 10@tumor cells in 1 ml of media supplemented with 10% fetalcalf serum. After incubation at 37°for 90 mm, the targetswere washed 4 times with media and added to the effectorlymphocytes with an effector:targot ratio of 20:1 in a finalvolume of 0.2 ml. For D6, 1 x 10@targets were used pertube; for EL-4 and G-35, 2.5 x 10@targets were used pertube. PHA was suspended in media to the appropriate concentration (pi/ml) and added in 0.1 ml. The tubes werecentrifuged at 200 x g for 5 mm and incubated for 2 hr at 37°in a 5% CO2-humidified atmosphere. The reaction wasstopped by addition of 1 ml of cold media to each tube. Aftercentnifugation at 800 x g for 10 mm, the supennatants wereremoved, and radioactivity in the supennatants was determined in a gamma counter. Specific lysis was calculatedaccording to the formula:

% “Crreleasedinexperiment— % “Cr in minimum essential medium

% “Crreleasedby detergent —% “Cr. released in minimum essential medium

The percentage of radioactivity released by detergent reprosents the total lysable count (80% average). All tests weresetup intriplicate.

MLC. One-way MLC was performed in triplicate in Cookemicrotiten plates. Two x 10@responder lymphocytes wereadded to each well in 0.1 ml of RPMI 1640 media supplemented with L-glutamine, N-2-hydnoxyethylpiperazine-N ‘-2-ethanesulfonic acid buffer, gentamicin, and 20% human ABserum (complete RPMI). Stimulator cells were treated withmitomycin C (Sigma Chemical Co. , St. Louis, Mo.). Lymphocytes (15 x 106)were suspended in 1.0 ml of RPMI 1640containing 50 @Lgof mitomycin C, incubated for 37°withstirringfor30 mm, and washed 3 timeswith0.15N phosphate-buffered saline. After resuspension in completeRPMI, 2 x i0@lymphocytes were added to the test wells intriplicate in 0.1 ml of media. The MLC plates were pulsedwith [3H]thymidine and harvested in a manner identical withthe mitogen stimulation plates. Controls consisted of mesponder cells alone, mitomycin-treatod stimulator cellsalone, and allogeneic mitomycin-treated cells added together.

Statistical Analysis. Statistical analysisof PHAand Con Aproliferation was performed using Student's t test (2-way).

RESULTS

Proliferative Response of RLN Lymphocytes followingStimulation with PHA. The ability of RLN and NLN lymphocytes to proliferate in response to PHA was examined usingfinal PHA dilutions of 1:10, 1:100, 1:1000, and 1:2500. SampIes were examined on Days 2 through 6 (Chart 1). Themaximal response was seen at a 1:100 dilution for each of 2NLN and 2 RLN. A total of 3 NLN and 3 RLN were tested for

their kinetic response to PHA, (1:100) and all demonstrateda maximum responsebetweenDays3 and4 witha gradualdecline in [3Hjthymidine incorporation thereafter (Chart 2).

To test the relative reactivity of LNL and PBL to PHA, a

Normal Lymph Node Regional Lymph Node

80

c.@600

x

@200

V@ 3 4 5 6 0 2 3 4 5 6

DAYS DAYS

Chart 1. Proliferative response as measured by [3H]thymidine incorporation of RLN and NLN lymphocytes to serial dilutions of PHA over a 6-day timecourse. PHA final dilutions were 1:10 (U), 1:100 (•),and 1:1000 (A).

% specificlysis=

80

60

C.)0

x

IC', 40

0.0

20

0 2 3 4 5 6

DAYS

Chart 2. Proliferative response of 3 NLN ( 0) and 3 RLN (•)to PHA, finaldilution 1:100, over a 6-day time course.

total of 8 NLN and 12 RLN were tested on Day 3 for proliferation after stimulation with PHA (1:100) (Chart 3). Each sampie was tested simultaneously with a sample of normal PBLfrom an age-matched control. Both groups demonstrated alevel of proliferation similar to that for normal PBL (although RLN and NLN lymphocytes were not tested simultaneously). No RLN on NLN demonstrated a markedly dopressed response to PHA. However, RLN from the 2 patientswith squamous cancer of the head and neck who receivedpreoperative radiation showed a decreased PHA response(Chart 3). The average background reactivitios for RLN,NLN, and PBL as assayed on Day 3 without the addition ofPHA were 1463, 1239, and 1051 cpm, respectively.

Con A Response. The ability of NLN and RLN lymphocytes to incorporate [3H]thymidine in response to Con A wasevaluated on Day 3 using 10 @gof Con A per ml, conditionspreviously found to be optimal for stimulation of PBL (12).

1160 CANCERRESEARCHVOL. 37

on June 29, 2018. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

RLN Reactivity in Cancer

the same patients were capable of lysing the targets in thepresence of PHA (Chart 5). These same LN cells were shownto be able to proliferate normally at 3 days in response toPHA stimulation. T- and B-cell fractions from several of thesame LN preparations were also tested for their ability tomediate PDCC, and both fractions remained relatively mactive (Chart 5). The ability to mediate PDCC can be found inboth the T- and B-cell fractions from normal PBL separatedin this manner (B. Bonavida, personal observation). For removal of any lymphocyte membrane-bound material thatmight block the PDCC activity of these LNL, LNL werewashed 12 times in media after incubation at 37°for up to 1hr. This procedure did not increase PDCC by LN cells, nordid incubation of the LN lymphocytes at 37°for up to 24 hrprior to testing.

LNL Dose Response and Kinetics in 1-Way MLC. Oneway MLC was established using stimulator:nesponder cellratios of 1:2, 1:1, and 2:1 . NLN and RLN lymphocytes mesponded equally well at the 1:2 and 1:1 ratios with only aminor decrease in [3H]thymidmne incorporation at the 2:1ratio. Normal PBL showed a clearly superior response at the1:1 ratio; this ratio was therefore used for subsequent studies. The proliferative response by LNL was relatively constant on Days 3 through 6 with some decline thereafter (datanot shown). For further studies, cultures were tested on Day5 inasmuch as this has been shown to be the time ofmaximal PBL response in the MLC (19).

Comparison of Reactivity of NLN and RLN Lymphocytesto Allogeneic Stimuli in MLC. NLN and RLN lymphocyteswere tested fresh against 2 allogeneic sets of mitomycin C-treated PBL. The response of a nonrelated normal PBLagainst each stimulator cell was taken as base line, and theratio between the LNL response and PBL response to theallogeneic stimulus was calculated.

‘ cpm (LNLA) ,Responseindex = ______— vs. PBL (mitomycinC-treated)cpm (r-DL,,

@80

160

140

12(

80

60

4'

.

.I • .: •1I

.1 i S.

..

0

. 0

C-)0

‘CI

C-)

0.

0

-@, Normal Normal Regional

PBL LN LN

Chart 3. Proliferative response by PBL, NLN lymphocytes, and RLN lymphocytes on Day 3 to PHA at a final dilution of 1:100. 0, radiated LN.

@ Normal Normal Regional

PBL LN LNChart 4. PDCC by normal PBL, NLN lymphocytes, and RLN lymphocytes.

Results are expressed as percentage of specific lysis (see text) in a 3-hr “Crrelease assay with EL-4 as the target cell.

The mean [3H]thymidine uptake for 3 NLN was 135,932 ±16,855(5.D.)for3 RLN was 143,469±15,063,and for4normal PBL it was 140,954 ±20,860. Again no clear differonce between the various lymphoid populations was demonstrated with the concentration of Con A used.

LNL Ability to Mediate PDCC. All RLN and NLN lymphocytes demonstrated either an absent on markedly diminished ability to mediate PDCC against the D6 target ascompared to PBL from age-matched controls (Chart 4).Therefore, the ability of PBL from the patients was compared to their LNL with regard to their ability to act aseffectons in PDCC. For these studies, EL-4 and G-35 targetswere used. Again both NLN and RLN cells did not demonstrate the ability to act as offectors in PDCC, while PBL from

.1.

I.

S

S

I

S@ I;

V

S

,121

10

8

U,Cl)>@@.1

.@ 4

C)6)0.

0

Cl)Cl)

3'U

C,)

0

Patients Regional Regional LNPBL LN TandBcells

Chart 5. PDCC by patients' PBL, unseparated RLN lymphocytes, and RLNlymphocytes separated into T- (A) and B- (U) lymphocyte fractions. 0, A.lysis without PHA; U, A, lysis in the presence of PHA. Results are expressedas percentage of specific lysis (see text) in a 3-hr “Crrelease assay using D6and G-35 as target cells.

1161APRIL 1977

on June 29, 2018. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

ComparisonbetweenRLN and DLN responsein 1-wayMLCreactivity to a pair of allogeneic mitomycin C-treatedPBLResponse

indexStimulator

A StimulatorBRLNI

1.0 0.6DLNI 3.31.8RLN,

0.55 0.82DLN2 1.01.9RLN3

0.52 0.94DLN3 0.641.2RLN4

1.1 1.2DLN4 1.51.6RLN5

2.5 1.9DLN5 2.21.4RLN,

1.3 1.7DLN, 1.31.8Radiated

RLN7 0.18 0.12DLN7 0.250.21Radiated

RLNN 0.26 0.17DLN, 0.28 0.18

A. Saxon and J. Portaro

Table 1The strength of the allogeneic stimulus will vary betweenpairs of stimulator and responder cells, making misleadingindividual comparisons between reactivity by NLN lymphocytes from noncancer patients and RLN lymphocytes fromcancer patients toward one given allogeneic stimulus.Thus, the general reactivity between the 2 groups was compared (Chart 6). RLN and NLN lymphocytes responded in afashion similar to that of the allogeneic PBL stimulus. NLNor nonradiated RLN cells all demonstrated at least a 10-foldincrease in [3Hjthymidine incorporation as compared to thesame unstimulated lymphocytes. However, RLN obtainedfrom 2 patients who had received local radiation therapyshowed a clearly decreased responsiveness (Chart 6).

Comparison between RLN and DLN Lymphocyte Reactlvfty In MLC. RLN and DLN lymphocytes from the samecancer patient were compared for their ability to respond in1-way MLC. Since both LN were from the same individual,comparisons are valid between RLN and DLN response to agiven allogeneic stimulus. In 4 of 6 pairs tested, the RLNlymphocytes showed a decreased proliferation in the MLCwhen compared to DLN lymphocytes (Table 1). This differential reactivity was always present for the pair of allogeneicstimuli. In 1 pair of RLN and DLN tested no difference inreactivity was found, while in the other pair RLN cells meacted somewhat better than did DLN cells. Again no LNLfrom any untreated patient were found that did not respondwell to allogeneic stimuli. We did examine RLN and DLNfrom 2 patients with squamous cancer of the head and neckwho had undergone radiation therapy. In each case the radiated RLN and DLN lymphocytes demonstrated a marked

depression in alloreactivity (Table 1).

DISCUSSION

$0

.

0

@ I0

.

0 •8 .

0 •

. .4

The cells that proliferate in response to stimulation withPHA and Con A have been demonstrated to be overlappingsubpopulations of T-cells (15). Animal LN cells and PBLhave been shown to respond to these mitogens equally well(16). Our data demonstrate that human NLN lymphocytesalso show a degree of lectin-induced proliferation comparable to that of PBL. RLN lymphocytes from patients with earlymelanoma or squamous head and neck cancer did not differin maximal [3H]thymidmne incorporation from either NLNlymphocytes or normal PBL. RLN that had been radiated,however, showed clearly a diminished PHA response. Theequality in RLN and NLN response was in spite of the factthat RLN had an average of 21% less T-cells by sheep ABCrosette testing. Indeed, if the cpm [3H]thymidmnepen numberof T-cells are calculated for our data, then RLN lymphocyteswere more reactive on this basis than either normal LN cellsor PBL. The suggestion that RLN T-cells may be activated invivo and therefore show increased reactivity is supported bythe observation that RLN had elevated levels of lymphocytesthat form spontaneous rosettes with human ABC (A. Saxon,personal observation). This surface marker has been meported to characterize activated T-cells (14).

However, our data can be manipulated to show a docreased response to mitogens in RLN by calculating a stimulation index (cpm with PHA/cpm without PHA). Since thebackground [3H]thymidino incorporation (cpm withoutPHA) in RLN lymphocytes was 1.4 times greater than innormal PBL and 1.2 times greater than in NLN lymphocytes,

4

3

2

0

Cs'a:

C

0

CO

Normal RegionalLN LN

Chart 6. Proliferative response by NLN (0) and RLN (•)lymphocytes toallogeneic stimuli in 1-way MLC. A, radiated RLN lymphocytes. Proliferationwas measured by [3H]thymidine incorporation and expressed as a stimulationratio (see text).

CANCERRESEARCHVOL. 371162

on June 29, 2018. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

RLN Reactivity in Cancer

the stimulation indices would reflect these differences. Thesignificance of the increased background in RLN is notclean. It may represent increased spontaneous DNA synthesis, which has been shown in RLN in animal models (7), onincreased trapping of [3H]thymidine within the cytoplasmicpool of RLN lymphocytes. Because of these various possibilities, we have presented our data as maximum cpm andgiven separately the percentage of cells in the various populations and the background cpm.

The lymphocytes that respond to allogeneic stimulation inMLC are a small population of approximately 2 to 6% of theT-lymphocytes (8). Both NLN lymphocytes from normalsand RLN lymphocytes from cancer patients reacted to allogeneic stimuli equally well. Two-thirds of DLN demonstrated an increased reactivity in MLC when compared toRLN from the same individual. Since DLN contained anaverage of 10% more T-cells, this increased reactivity maysimply relate to the presence of an increased number ofreactive cells. While NLN contained 20% more T-cells thanRLN, differential reactivity may not have been demonstrablebecause direct comparisons could not be made. Certainlyall NLN, RLN, and DLN showed the ability to respond toallogeneic stimuli. RLN and DLN from patients who hadreceived local radiation therapy exhibited MLC reactivityfan below that seen with any other lymphoid suspensiontested. This is in spite of the fact that these radiated LN hadT-cell percentages similar to that seen in nonnadiated DLN.Either the subpopulation of T-cells responsible for alloreactivity was specifically diminished by the radiation or, whatis more likely, the reactivity of lymphocytes in general wascompromised by radiation damage. The latter interpretationis supported by the fact that the PHA response by theseradiated RLN was also markedly depressed. An alternativeexplanation that cannot be excluded is that those patientswho received radiation therapy had mono extensive tumorsand this may have played an undefined role in the observedresults.

Cell-mediated lysis of target cells in the presence of PHA(PDCC) occurs in 2 hr and is not dependent on proliferation(3). The lymphocyte subpopulation(s) that mediate this activity remain ill-defined. In normal PBL, both the T-ennichedand B-enriched fractions derived by our separation technique can effect PDCC, although the B-enriched fractionis more active. PBL from normals and the cancer patientswere effective in the PDCC assay, while LN cells from thesame normals and cancer patients were ineffective. Thesesame LN lymphocytes were simultaneously shown to proliforate normally oven 3 days in response to PHA. Furthermore, neither the purified T- on B-populations of LN, RLN,or NLN were capable of lysing targets in the presence ofPHA. These data suggest that the cell population(s) nesponsible for PDCC are either absent on not functional in NLNand RLN.

The proliferative response to PHA, Con A, and allogeneicstimuli is a property of T-cell subpopulations (9). The reactivity of NLN in all 3 assays supports the concept that LN Tcells share proliferative capabilities similar to their PBLcounterparts. While RLN demonstrated a decreased proportion of T-cells, these RLN retained intact T-cell respondermechanisms as measured by proliferative response to thevarious stimuli. There may be some decrease in reactivity in

MLC related to diminished T-cell numbers, but certainly no

generalized immunosuppression of T-coll reactivity wasdemonstrated in RLN. This agrees with the reports of Bunket a!. (4), who reported that LN cells from animals bearingsmall and large tumors responded to mitogens with intactT-cell proliferation. One must be aware of the fact, howoven, that microcultume systems may give different resultsthan do macmoculture systems (10). Lymphocytes from radiated RLN showed decreased PHA and MLC reactivity fan outof proportion to any change in the percentage of T-cellspresent. This correlates with evidence that lymphocytesshow diminished reactivity after in vivo and in vitro madiation exposure (5, 17).

Both NLN and RLN lymphocytes were unable to mediatelysis of target cells in the presence of PHA, while PBL fromthe same donors retained this activity. The lymphocytesresponsible for PDCC appear to be compartmentalized outside both NLN and RLN, being found in blood and spleen. Aparallel situation occurs in that the cells responsible forantibody-dependent cell-mediated cytotoxicity are notfound in NLN or RLN, while they are found in peripheralbloodorspleen(11,12).

Overall, the methods used in this study indicate that RLNfrom patients with early localized tumors do not demonstrate depressed immune reactive capabilities when compared to NLN. We would propose that inability of RLN tocontrol early growth and local spread of a tumor does notrelate to generalized suppression of RLN reactivity but mayrelate to tumor-specific suppression of LN activity. The tumommay effect this by modulation of the immune responseto its own advantage through the generation of host suppressom cells on other blocking agencies that prevent thedevelopment on expression of an effective specific immuneresponse.

ACKNOWLEDGMENTS

The authors would like to express their thanks to Dr. John L. Fahey for hisencouragement and helpful suggestions in preparing this manuscript, to Dr.Ben Bonavida for performing some of the cytotoxicity assays and for reviewing this manuscript, and to Joanne Bihr for her excellent technical assistance.

REFERENCES

1. Berlinger, N. T. , Tsaknaklides, V., Pollak, K., Adams, G. L., Yang, M.,and Good, R. A. Immunologic Assessment of Regional Lymph NodeHistology in Relation to Survival in Head and Neck Carcinoma. Cancer,37:697-705, 1976.

2. Black, M., and Speer, F. D. Sinus Histiocytosis of Lymph Nodes inCancer. Surg. Gynecol. Obstet., 106:163-175, 1958.

3, Bonavida, B. , and Bradley, T. P. Studies on the Induction and Expression of T Cell-mediated Immunity. v. Lectin-Induced Non-specific Cellmediated Cytotoxicity by Alloimmune Lymphocytes. Transplantation,21:94-102,1976.

4, Burk, M. W. , Vu, S., Ristow, 5. 5., and McKhawn, C. F. Refractoriness ofLymph Node Cells from Tumor Bearing Animals. Intern. J. Cancer,15:99-108,1975.

5. Byfield, P. E., Stratton, J. A., and Cole, D. E. The Effect of RadiationTherapy on the Kinetics of In Vitro Lymphocyte Response to Mitogens.Clin. Immunol. Immunopathol., 6:22-30, 1976.

6. Catalona, W. J., Sample, F. W., and Chretien, P. B. Lymphocyte Reactivity in Cancer Patients: Correlation with Tumor Histology and ClinicalStage. Cancer, 31:65-71 . 1973.

7, Edwards, A. J., Rowland, G. F., Sumner, M. R., and Hurd, C. M. Changesin Lymphoreticular Tissues during Growth of a Murine Adenocarcinoma.II. Nucleic Acid Content and Synthesis in Lymph Nodes, Spleen andThymus. J. NatI. Cancer Inst., 42:313—320,1971.

1163APRIL 1977

on June 29, 2018. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

A. Saxon and J. Portaro

8. Ford, W. L., Simmonds, S. J., and Atkins, R. C. Early Cellular Events in aSystemic Graft vs. Host Reaction. II. Autoradiographic Estimates of theFrequency of Donor Lymphocytes Which Respond to Each Ag-B Determined Antigenic Complex. J. Exptl. Med., 141:681-696, 1975.

9. Greaves, M., and Janossy, G. Elucidation of Selective T and B Lymphocyte Responses by Cell Surface Ligands. Transplant. Rev., 11:87-130,1972.

10. Harris, J., Stewart, T., and Sengar, D. P. Quantitation of T and B Lymphocytes in Peripheral Blood of Patients with Solid Tumors. I. Relation toOther Parameters of In Vivo and In Vitro Immune Competence. Can.Med. Assoc.J.,112:940-952,1975.

11. O'Toole, C., Saxon, A., and Bohrer, R. Human Lymph Node Lymphocytes Fail to Effect Lysis of Antibody Coated Target Cells. Clin. Exptl.Immunol., 27:165-171, 1977.

12. Samarat, C., Brochier, J., and Revillard, J. P. Distribution of Cells Binding Erythrocyte-Antibody (EA) Complexes in Human Lymphoid Populations. Scand. J. Immunol., 5:221-231, 1976.

13. Saxon, A., and Portis, J. Lymphoid Subpopulation Changes in Regional

Lymph Nodes in Squamous Head and Neck Cancer. Cancer Res.,37:1154-1158,1977.

14. Sheldon, P. J., and Holborow, E. J. Human Erythrocyte Formation withMitogen Stimulated Human Lymphocytes—AMarker for the Demonstration of Activated T cells. J. Immunol. Methods, 7:379—386,1975.

15. Shortman, K., von Boehmer, H., Lipp, J., and Hopper, K. Subpopulations of T Lymphocytes. Transplant. Rev., 25:163-210, 1975.

16. Strobo, J. D. Phytohemagglutinin and Concanavalin A: Probes for Murine “T―Cell Activation and Differentiation. Transplant. Rev., 11:60-82,1972.

17. Tarpley, J. L., Potvin, C., and Chretien, P. B. Prolonged Depression ofCellular Immunity in Cured Laryngopharyngeal Cancer Patients Treatedwith Radiation Therapy. Cancer, 35:638-644, 1975.

18. Wigzell, H. Quantitative Titrations of Mouse H-2 Antibodies Using Cr―-Labelled Target Cells. Transplantation, 3:423-429, 1965.

19. Wilson, D. D., Blyth, J. L. , and Nowell, P. C. Quantitative Studies on theMixed Lymphocyte Interaction in Rats. III. Kinetics of the Response. J.Exptl. Med., 128:1157-1181, 1968.

1164 CANCERRESEARCHVOL. 37

on June 29, 2018. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1977;37:1159-1164. Cancer Res   Andrew Saxon and Joe Portaro  Node Lymphocytes in Squamous Head and Neck Cancer

Biological Functions in Regional Lymphin VitroPreservation of

  Updated version

  http://cancerres.aacrjournals.org/content/37/4/1159

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/37/4/1159To request permission to re-use all or part of this article, use this link

on June 29, 2018. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from