a characterization of human tonsillar lymphocytes after

A Characterization of Human TonsillarLymphocytes After Separation From OtherTonsillar Cells in an Isokinetic Gradient ofFicoll in Tissue Culture Medium

James K. V. Willson, Jr., BA, Jack L. Zaremba, BA, Annette M. Pitts,and Thomas G. Pretlow 11, MD

Purified human tonsillar lymphocytes responded less to mitogenic stimulation than didunseparated tonsillar cells. Their response to mitogens was restored when they werecombined with cells from all other gradient fractions. We interpret our data as evidencethat the majoritv of tonsillar lymphocytes require the presence of more rapidly sedi-menting cells for a maximum response to the tested mitogens. The purified tonsillarlymphoyctes were 47.0% lymphocytes that have detectable surface immunoglobulinand 29.9% lymphocytes that form rosettes with sheep red blood cells. The predominantcell surface immunoglobulin was IgM. Digestion of the tonsil with trpsin yieldedtenfold more plasma cells, more viable cells, and a larger proportion of blasts, his-tiocytes, and binucleated cells than were obtained by mechanical dissociation of thetissue. (Am J Pathol 83:341-358, 1976)

THE MIAMIMALIAN LYNIPHOID SYiSTE\m has been conceptualized as

consisting of t-o separate but interacting systems: the peripheral lym-phoid system and the central l-mphoid system.' Most in vitro studies ofthe human l-mphoid system have been conducted with lymphocytes ob-tained from peripheral blood; however, interest has increasingly beenfocused on the characteristics of the central lymphoid system. In vitrostudies of cells obtained from the palatine tonsil,2 '3 the thvmus 14.5 theappendix,'6 and other lImphoid tissue associated wvith the alimentarvcanal 17 have broadened our understanding of the human lI-mphoidsv stem.

WNe have recently reported a method for the separation of lymphocytesfrom the human palatine tonsil by velocity sedimentation in an isokineticgradient of Ficoll in tissue culture medium.'8 In the present communica-tion, w e have characterized the purified tonsillar lymphocytes obtained bythis method. Specifically, tonsillar lymphocytes wvere stimulated wvith the

From the Departments of Pathologp and Engineering Bioph\sicss. Lniversitx of Alabama MedicalCenter. Birmingham. Alabama

Supported b\ Grants CA-1.50S9 from the National Cancer Institute. CA-16764 from the NationalCancer Institute through the National Large Bowel Cancer Project. CA-164s30 from the NationalCancer Institute through the National Prostatic Cancer Project. DE-2670 from the National Instituteof Dental Research. and bh Grant PDT-9B from the American Cancer Society: Dr Pretlowu issupported by Research Career Development Awxard K4-CA-705S4-0:3

Accepted for publication January 26. 1976Address reprint requests to Dr Thomas G Pretlo%- II. Department of Pathologp. UIniversitv of

Alabama in Birmingham. Box 1S9. University Station. Birmingham. AL :352994341

342 WILLSON ET AL American Journalof Pathology

polvclonal mitogens phytohemagglutinin (PHA-P), pokeweed mitogen(PWM), and concanavalin A (Con A). The modal population of tonsillarlymphocytes from the isokinetic gradient responded maximally to mito-gens only when stimulated in the presence of more rapidly sedimentingcells. The separated modal population of tonsillar lymphocytes includedboth thymus-dependent (T) lymphocytes and bursa-equivalent (B) lvm-phocytes.

Materials and MethodsCdWs

Palatine tonsils were obtained in the operating room and placed immediately in sterilecold tissue culture medium. Sections were taken for microscopic examination. The tissuewas weighed, minced, and divided into three equal portions. Three different methods wereused in parallel to obtain the cells from tonsils in suspension. Enzymatic digestion wasperforned according to the procedure described in detail previouslv "' either with 0.25%trvpsin (Microbiological Associates, Bethesda, Md.) in Joklik's modification of minimumessential medium (Grand Island Biological Co., Grand Island, N.Y.) or with 0.05% SigmaTy-pe II collagenase (Sigma Chemical Co., St. Louis, Mo.) in Joklik's modification ofminimum essential medium with 10% fetal calf serum (Grand Island Biological Co.).Briefly, enzymatic digestion was accomplished in seven successive 20-minute periodsduring which the tissue fragments were maintained in suspension in 40 ml of enzymesolution at 22 C (room temperature) with a magnetic stirrer. At the end of each period, thesuspension of cells in enzvme solution was decanted from the fragments of tissue, and thefragments were resuspended in fresh enzyme solution. Cells were centrifuged out of thesolutions containing enzvmes with a centrifugal force of 90g for 8 minutes and resus-pended in cold joklik's tissue culture medium with 10% fetal calf serum. Cells from thesecond through seventh digestion periods were combined and filtered through a singlelayer of Nitex (TETKO Inc. Elmsford, N.Y.) with a pore diameter of 48 p. Cells from thefirst 20-minute digestion period contained many red blood cells and dead cells; these werediscarded. In preliminary experiments, we found that the cell suspensions contained thesame relative frequencies of each cell type after seven digestions as those observed when13 digestions were carried out as described previously.The third method for obtaining cells in suspension consisted of gently teasing the tissue

with scalpels in cold Joklik's tissue culture medium with 10%7c fetal calf serum. The tissuefragments were then stirred in joklik's tissue culture medium with 10% fetal serum for 20minutes; the fragments were allowed to settle; and the cell suspension was decanted. Cellcounts were performred using hemocvtometer chambers. Viability was assessed by observ-ing the ability of the cells to exclude trvpan blue."' Slides were prepared with thecytocentrifuge (Shandon Southern Instruments, Inc., Sewicklev, Pa.) and stained withWNright's stain. At least 500 cells were counted per slide.

CGl SIabIIonVelocity cell separation experiments were performed in the previously described isoki-

netic gradient." The theory and a detailed description of the technique and its appli-cations have been reviewed recently 21 and will, therefore, be omitted from this report.Tonsillar lymphocytes were separated from the other cells in the tonsil in the isokineticgradient exactly as described previously by centrifugation at 4 C for 16 minutes with acentrifugal force of 97g at the sample-gradient interface 13.7 cm from the center ofrevolution." The isokinetic gradient was collected in 4-im fractions, except for the first

Vol. 83, No. 2 HUMAN TONSILLAR LYMPHOCYTES 343May 1976

fraction which consisted of the 7-ml starting sample suspension. Fractions 7-12 from theisokinetic gradient were combined. These fractions contained the modal population oftonsillar lymphocytes. 1We used two methods to separate lymphocytes from human peripheral blood. Lvm-

phocytes were separated in the isokinetic gradient from leukocyte-rich plasma as reportedby Pretlow and Luberoff.22 Lymphocytes were also separated by the method described byBoyum as modified by Thorsby and Brathie.24 This separation was accomplished bvlayering blood anticoagulated with 10 units/ml of heparin over an equal volume of aseparating fluid of 69.6 parts of 8% Ficoll (Pharmacia Fine Chemicals, Piscatawav, N.J.)and 39.4 parts of 32.8% sodium metrazoate (Nyegaard, Oslo, Norway). Lymphocytes andmonocytes were isolated at the sample-gradient interface by centrifugation for 30 minutesat 20 C with a centrifugal force of 400g at the sample-medium interface.

Ctre systemCells cultured with mitogens were adjusted to a concentration of 5 X 10' viable

nucleated cells/ml in R.P.M.I. 1640 medium (Grand Island Biological Co.) with 10%fetal calf serum and 292 ug/ml glutamine (Grand Island Biological Co.). Peripheral bloodlymphocytes were cultured with 100 units/ml of penicillin and 100 ig/ml of streptomycin(both from Grand Island Biological Co.). These antibiotics were not sufficient for culturesof tonsillar cells; therefore, 50 jig/ml of gentamicin (Schering Pharmaceutical Corp.,Kenilworth, N.J.) was substituted for penicillin and streptomycin in cultures of tonsillarcells. Our preliminary experiments were in agreement with previous reports that gen-tamicin had no effect on the stimulation of lymphocyte cultures by mitogens. '2 A volumeof 0.2 ml of the suspension of cells was distributed with a Hamilton repeating dispenser(Hamilton Co., Whittier, Calif.) into each well of a microplate (Model No. IS-FB-96,Limbro Scientific Co., New Haven, Conn.). A final concentration of 1 x 10' viablenucleated cells per well was obtained. The following doses of mitogen were shown to giveoptimal stimulation of human peripheral blood lymphocytes and were added to triplicatecell cultures in 0.02 ml of RP.M.I. 1640 medium: 0.5 il of the 5-ml reconstituted stocksolution of PHA-P (Difco, Inc., Detroit, Mich.); 2 jig of Con A (INC Pharmaceuticals,Cleveland, Ohio); and 5.0 j1i of the 5-ml reconstituted stock solution of PWM (GrandIsland Biological Co.). Triplicate cultures were incubated without mitogens as controls.Cultures were incubated for 72 hours in a humid atmosphere of 5% C02 and 95% air at 37C.

A PocdweThe assay procedure was a modification of the microculture system originally described

by Hartzman et al.2 Six hours prior to termination of the culture, 0.02 ml of R.P.M.I. 1640medium containing 1 jiCi of 'H-thymidine [specific activity 2 Ci/mmole (New EnglandNuclear, Boston, Mass.)] was added to each culture. The cultures were terminated bvharvesting the cells with an automated harvesting apparatus (Otto Hiller Co., Madison,Wisc.) following the procedure of Harrison et al.2' The cultures were counted in a liquidscintillation counter (Beckman Instruments, Palo Alto, Calif.) for 1 minute. The results areexpressed as the mean of the counts per minute per culture.

Ibdni 1ction of Human T and B LynyhocytesGoat antisera to human IgM, IgG, and IgA were kindly provided by Dr. Max D. Cooper

(University of Alabama Medical Center, Birmingham, Ala.) after purification with solidphase immunoabsorbent columns." Antibodies to each immunoglobulin class were con-jugated with fluorescein isothiocyanate.' Purified lvmphocytes from tonsils were stainedwith the fluorescein-labeled, class-specific antibodies in the presence of 0.1 % sodium azide


at 4 C. WNet-mount preparations w ere examined by light and fluorescence microscopy witha Leitz Orthoplan microscope. WVe determined the percentage of positive cells by countingoil immersion fields alternateiv by light and fluorescence microscopy. At least 200 l%-m-phocytes Xwere counted per slide.

Sheep red cells, stored in Alsever's solution (Colorado Serum Co. Laboratories. Denser.Colo.) at 4 C for not more than a month. were used for the E-rosette test.31 Sheep red cellswere washed once with phosphate-buffered saline and then four times with Hanksbalanced salt solution (HBSS) and 5It fetal calf serum. A 1% suspension 0.2 ml) of thesesheep red cells was incubated with 1.0 to 1.3 X 10( tonsillar lymphocytes in 0.2 ml of HBSSand 5% fetal calf serum for 20 minutes at 22 C. The suspension was then centrifuged for 5minutes at 26g at 4 C. The pellet was undisturbed and incubated at 4 C overnight. Thecells were then gently resuspended and the E rosettes counted in the presence of trypanblue. Viable nucleated cells binding three or more red cells were considered rosettes Atleast :300 viable nucleated cells w-ere counted per slide.

Results

Mitogenic Stimulation of Peripheral Blood Lymphocytes

We initially tested the culture system with ly-mphocytes separated fromhuman peripheral blood either by velocity sedimentation in the isokineticgradient or by the Bovum method. Lymphocytes separated in the isoki-netic gradient were obtained as >99.0% of the nucleated cells. Thelymphocytes isolated by the Bovum method contained 10 to 14% mono-cvtes. The relative incorporations of 3H-thymidine by cultures of l-m-phocytes separated by these two methods are compared in Table 1. Thecultures of lymphocytes containing monocytes had twofold greater in-corporation of 3H-thvmidine than cultures of purified lymphocytes whenstimulated with PHA-P, threefold greater when stimulated wvith Con A.and fivefold greater when stimulated with PWM. Others have obtainedsimilar results.32Cel Suspensions From Tonsils

The total number of cells and the proportions of individual kinds ofcells obtained by the three methods used to disaggregate tonsil %verecompared (Table 2). Disaggregation of tonsil wvith 0.23% trypsin gave thegreatest number of tonsillar cells of all types, except neutrophils. The totalnumber of cells per gram of tonsil obtained from 13 exchanges of 0.25%trypsin was greater for this series of experiments than reported pre-Viouslv.18 WN'e have found enormous differences in the relative efficienciesof different lots of tr-psin used to disaggregate the human tonsil. Thesedifferences occurred even among lots from the same supplier and did notcorrelate with the specific activity of the trypsin. The reduction in thenumber of exchanges of enzyme (0.25%c trypsin or 0.05% collagenase)from 1:3 to 7 resulted in a 32%E decrease in the total number of cells pergram obtained with 0.25% trvpsin and a 52% decrease in the total number

HUMAN TONSILLAR LYMPHOCYTES

CO.'. CO

-HO

C\0s)C'I) LOo-CD

CO)

C')

-H

coJN-

- N-

C\D

c;

C')

CD _

CV)t

ON-

CD _

N- C

CDs

_' _

CDt

CD 0

_00

C')0

,.

C00GsoCD C')LOCDcli

r- 0CDO)CM..

co 0qC o~

N-

c'0

C'J)

-ft

o0)

--4'

C C

N-

" 0)

CDC0(6co

'

OD 'D0Q: - E

o a, 0 (, 0 Eav

>%

Vol. 83, No. 2May 1976

345

cC0)

-C

a.C

00

4-C

Ec '-

o _

0

00

4-C

-E

< _-

00

4-c

2&E

00

aa

0ICVJN

C)0a)

CLaCDa.

02V020

0-

m

0.

0

E0

CL

E

z7E

0U-

Q

0.aC,,aa

E-J

'BC

0

E

a

I-

atax

CD

E

E

E

a

0.

=EE E

31

VCD

0

0Da.E~

's3C Do CD)> C

coQ

C C

C) CD0~

<CDCLOC

nC E.-

-o

a~E

_0>n '

Co.0o

a. co0a0a

I0E0. *

a

Go0a

0

a

C.)o0-E-J

-o,ZO

CD

a.!

Q-0

346 WILLSON ET AL

CO

0cCIO

EI

0

0

-0

0

0c

0

0

0

0

I-

00

0

o

I0D0

0

American Journalof Pathology

Co,

0

0

0Q

3C3

0

x

4-

0

-C0

x

.v0

IL)

V

0-

n~00D 0

0 =

CDz

0

0-

-0°Cc

0

t0

In

0 -

0

0L -E

E0_

.0 0

0

0 0> CNcoC .00 0CV, Xv- 0

0 Q

>% 0N

r- 0

C0

-

o

_00 0

r -

occ

6 6+ +

6 6

6 O

O 6LAI C\D6 6CM U)

CD

O 6

CO) C\J

.H .

CM cO

C; c-m. .

-C- C~)

CII) 0)

6 CD

o r-

+ -H

c;c

r-

r- CDi

-H +

0)

LA

CD

CD

00

0

0

-H4co

co0

CD6

CD0

tD6

c;)-H

cocLco

cDco

ur-

(0

Pl, '+ + te0C CD O CD Pr-

0)a 0)- CK-

cq) Ce) CMcl)LAr -C-

-H +IVCD0l C, Le) Ur_

0V) cl 4~C-

U.C)Co C-)

c0CC

U CI- co

LA LAOC\ 0 0e

00 D00 -CD

c 00 aciss 0-C 0

0C0 0x X- 00> 0

EE =_E 00 0

E- 0E 0

_0 0

CCD

0

a 6.

x _5

EE E00 0C0 0

E 0>

0 0--0B

o- 0 >X 0

-05 >0a>. 4-+CO)


of cells obtained w ith 0.05%-c collagenase. We obtained a greater numberof cells using mechanical methods than reported previously;18 however,the viability of these cells wvas significantly- less (Table 2) than the viabilityof cells obtained in suspension by digestion with enzymes.Isokin,eti Separation of Tonsillar Cells

Followving sedimentation in the isokinetic gradient for 16 minutes usinga centrifugal force of 97g at 4 C, the modal population of lymphocytes waslocated in Fractions 7-12 (Text-figure 1). A detailed frequency distribu-tion of all types of tonsillar cells in the isokinetic gradient has beenreported previously.18 The modal population of lymphocytes contained9:3.2 ± 3.6c lymphocytes when suspensions of cells wvere obtained wvithtrvpsin (Figure 1), 94.4 ± 1.4%c lymphocytes when suspensions of cellswere obtained w-ith 0.05%c collagenase, and 94.4 ± 0.9c lymphocytes-hen suspensions of cells wvere obtained by mechanical methods.Response to Mitogens

The modal populations of lymphocytes separated from tonsillar cells,were cultured for 72 hours wvith PHA-P, Con A. or PWAM. The 3H-thymidine incorporation by these cultures (Table :3) stimulated with PHA-

300 -

01.100

LaI 1.020-

1,000LYMPHOCYTES

4.0-J-J

U. 3.00

z0 2.0-J

1.0

5 10 15 20FRACTION NUMBER

TEXT-FIGTRE 1-The frequenc!- distribution of tonsillar cells after separation of cells from thehuiman palatine tonsil in the isokinetic gradient of Ficoll in tissue culture medium. Centrifugation iscarried out at 97,g measured at the sample-gradient interface for 16 minutes at 4 C. 4n arrou marksthe sample-gradient interface on the densit' plot.


Table 3-3H-Thymidine Uptake of Separated Tonsillar Lymphocytes*

Mitogens

Method of Control PHA-P Con A PWMdisaggregation (counts/min) (counts/min) (counts/min) (counts/min)

0.25% Trypsin 278 ± 216 2,926 2,013 959 ± 1,409 356 ± 259(63-753) (74-5,606) (36-4,104) (59-667)

0.05%Collagenase 342 r 187 3,067 -2,384 1,390 ±2,030 321 ±225(94-575) (400-7,920) (182-5,838) (52-576)

Mechanical 240 ± 242 1,770 r 1,988 840 1,681 254 ± 254(38-723) (141-6,138) (74-4,633) (60-795)

Data from seven tonsils; values are mean counts per minute of triplicate cultures ± 1 SD.Minimum-maximum values are given in parentheses.

P was tenfold greater than that of the unstimulated, control cultures.Cultures stimulated N-ith Con A incorporated fourfold more 3H-thymi-dine than unstimulated, control cultures. Similar responses were observedfrom lymphocytes obtained with either trvpsin or collagenase. Tonsillarlxmphocytes obtained in suspension by mechanical methods respondedslightly- less vigorously than lymphocytes obtained with tn-psin or collage-nase. The response to PWVM1, after 72 hours, was not significantly greaterthan controls for any separated lymphocytes.

There wvas a large variability in the responses of lymphocytes among thedifferent tonsils wve examined. This variability was evident in the largestandard deviations and in the wide ranges of values reported for eachexperimental condition (Table 3). This variability could not be correlatedwith either the method of disaggregation or with the clinical parametersof age, sex, or hypertrophy of the tonsil.

Since highly purified lymphocytes from peripheral blood responded lessvigorously to mitogens than lymphocytes adulterated with monocvtes(Table 1), wve wondered if there could be a similar helper cell populationthat had been separated from the modal population of tonsillar lvm-phocytes. WNe did not have all types of tonsillar cells in sufficient purity toallow- demonstration of a specific cell type, the presence of wvhich mightenhance the response of the purified tonsillar lymphocytes to mitogens;therefore, -e combined the tonsillar cells from all 22 fractions of theisokinetic gradient (Text-figure 1). Thus, the modal population of lym-phocvtes (Fractions 7-12) w-as supplemented by the more rapidly sedi-menting cells found in Fractions 13-23. These more rapidly sedimentingtonsillar cells are illustrated in Figure 2; they include lymphocytes, imma-ture lymphoid cells, histiocytes, plasma cells, and an unidentified binu-cleated cell. Cells obtained from the combination of all gradient fractions


responded more vigorously to mitogens than purified tonsillar l\-m-phocvtes (Table 4). Unseparated tonsillar cells (Table 3) also respondedmore vigorously to mitogens than purified lymphocytes.T and B Populations of Tonsillar Lymphocytes

The purified tonsillar lymphocytes were charcterized as either B (bursa-equivalent) lymphocytes by the demonstration of surface immunoglobu-lin or T (th\-mus-dependent) lymphocytes by formation of nonimmune Erosettes (Table 6). The presence of surface immunoglobulin on lym-phoyctes after exposure to either 0.25%/-c trypsin or 0.05%c collagenase at 22C was not significantly different from the surface immunoglobulin de-tected on lymphocytes disaggregated by mechanical methods. Lym-phocytes from mechanically disaggregated tonsils were 47.0 ± 2.6-c Blymphocytes, and Ig\I was the predominant immunoglobulin class. Me-chanically disaggregated tonsils contained 29.9 ± 7. Txc T lymphocytes.Disaggregation with 0.25%cc trypsin decreased the E-rosette formation by-lymphocytes to 46-c of the E-rosette formation by lymphocytes disagre-gated mechanically. The capacity to form E rosettes was restored byincubation of the trypsin-disaggregated lv-mphoc\-tes for 20 hours inR.P.M.I. medium supplemented with 10Vc% fetal calf serum at 37 C.Disaggregation with 0.05%c collagenase did not alter the formation of Erosettes.Discussion

In this paper wve have compared various methods for the disaggregationof human tonsil into single cells. As in our initial study of the humantonsil,18 we found that disaggregation with trypsin gave the largest num-ber of all types of tonsillar cells and the greatest proportion of plasmacells. histioc\ tes, blasts, and binucleated cells. In discussing their disag-gregation of mouse lymphoid tissue. Steinman and Cohn 3335emphasized

Table 4-3H-Thymidine Uptake of Combined Tonsillar Cells After Separation*

MitogenMethod of Control PHA-P Con A PWMdisaggregation (counts/min) (counts/min) (counts/min) (counts/min)0.25% Trypsin 138 = 82 5,583 z 2,304 5,145 z 3,258 496 z 310

(64-226) (3,245-7,849) (1,559-7,924) (241-841)

0.05%Collagenase 91 -66 7,681 -4,472 10,781 = 12,359 683 z830(45-166) (2,573-10,893) (1,426-24,791) (60-1,625)

Mechanical 90 - 25 3,936 2,524 5,698 _8,283 257 - 119(73-119) (1,643-6,641) (357-15,242) (181-394)

Data from three tonsils; values are mean counts per minute of triplicate cultures , 1 SD.Minimum-maximum values are given in parentheses.

350 WILLSON ETAL American Journalof Pathology

Table 5-3H-Thymidine Uptake of Unseparated Tonsillar Cells'

Mitogens

Method of Control PHA-P Con A PWMdisaggregation (counts/min) (counts/min) (counts/min) (counts/min)0.25% Trypsin 77 = 32 9,849 = 4,310 12,490 = 5,693 1,298 t 416

(44-107) (5,929-14,465) (6,060-16,914) (817-1,616)0.05% 138 149 4,626 + 1,008 5,469 = 3,292 540 r 234

Collagenase (46-310) (3,770-5,737) (1,798-8,160) (297-737)Mechanical 84 =49 4,693 -3,232 3,059 -2,983 224 = 161

(63-141) (1,508-7,971) (784-6,438) (41-341)Data from three tonsils; values are mean counts per minute of triplicate cultures T 1 SD;

minimum-maximum values are given in parentheses.

that the frequencx- distributions of the types of cells which they obtainedwere highly dependent upon the techniques employed for disaggregationof the tissue. We know of no other comparison of techniques for thedisaggregation of human tonsil but would emphasize that the conven-tional, mechanical methods used to disaggregate tonsil give tenfold fe'werplasma cells. fewer v-iable cells. and a lower proportion of cells other thanlymphocytes than are obtained by digestion with trypsin. The differentmethods used to disaggregate human tonsil affected the responses of thetonsillar lymphocytes to mitogenic stimulation only- modestly. In contrastto our findings, it has been previously reported that prior treatment mvithtrypsin decreases the responses of lymphocytes to mitogens."The response of tonsillar cells to mitogens has been studied pre-

viouslX_2,8,9 Others found, as we have reported, significant response tostimulation with PHA-P and Con A; however, except for the unseparatedcells obtained with trypsin, our cultures of both unseparated and purifiedtonsillar cells did not respond to PWMN1 as vigorously- as those described byothers.2'8 The population of purified tonsillar lymphocytes wvhich we ob-tained bv v-elocit- sedimentation in the isokinetic gradient demonstrated adiminished response to stimulation with the polyclonal mitogens whencompared with unseparated tonsillar cells. WNe considered the possibilitythat this decrease in response Xwas caused by the processing in the isoki-netic gradient; how%vever, wve found that combination of the separated cellsfrom all gradient fractions in culture restored their responses to mitogens.The diminished response of the purified tonsillar lymphocyte was notcaused by processing in the gradient; rather, it appears likely that a helpercell population was among the more rapidly sedimenting cells and was.therefore, separated from the modal population of tonsillar lymphocytes.In cultures of purified blood lymphocytes. we showed a significant en-

351Vol. 83, No. 2 HUMAN TONSILLAR LYMPHOCYTESMay 1976

IN

0c'ooC')CV) -

C.' !Es

in

cr, C8

in CDI-HIpI-C', IN

ui CD+,JC.ic

'lt-"

IN U)06 C-i

l)

Cvi C',

Hcl~+o C")

a CO

r-. i

a) INCi CI

Cm CV

r--. o-C14 _

-H f

oa-^ *C^ )^q in 0C-H -H

ITaqC) LQCD*_ CIN 0 j -

O CI-

0 0'1- a;_o-

CI CM. .

*i aiCM-4-

CD

0,-0 0V--

'Nco~INo qC OD

C.4M-

0 0

-H4 CM

T-1

_OIN

-0

CM CM

a0C

c aD

1.- 0 C

CM 0 06 6 2

0

aci_ U0 -D

^

.

4-40 40O L-

w

a

0co

0

w

40.

E

<(Zo

4-

0

a

E0

coU-cDi

0

E-J-

a

0

m

a

a

a0

cn

00.O-

D

C..

0aa

C.IC

aaa-cCD

aa

FE

0.

Ea

cEE

.

Ecn,o

EE

C C

E

c_

E-00c o °3

* -I00

CDiss cj

C

o~~~~~~~~~~~~~~~~~~~~r )C

> . +-

C0o

0 0

-aLO C

0 0C na


hancement of mitogenic stimulation when lymphocytes were culturedwith monocvtes. The identity of and requirement for a helper cell popu-lation for the optimal response of the lvmphocvte to mitogenic stimulationis a subject of controversy. Greaves et al.2 found that optimal mitogenicstimulation of tonsillar lymphocytes was independent of phagocytic cells.In contrast, Geha and Merler8 found that the addition of adherent cells tocultures of tonsillar lymphocytes resulted in a modest enhancement oftheir responses to mitogens. Other reports have described potentiation ofthe mitogenic stimulation of lymphocytes from peripheral blood by mon-ocytes.7"'7' Oppenheim et al." found this effect only with suboptimaldoses of mitogen.The variability in the responses of tonsillar cells to mitogens among the

tonsils we studied could not be correlated with any pathological or clinicalparameter which we could identify. This degree of variability was notobserved when lymphocytes from the peripheral blood of different indi-viduals were stimulated with mitogens. In studying the production ofantibody to sheep red blood cells by human tonsillar cells in vitro, Hoff-man et al.3 and Watanabe et al.4 noted considerable variability among thedifferent tonsils they studied.

In our study, the proportions of tonsillar lymphocytes having surfacemarkers for either B lymphocytes or T lvmphocvtes varied slightly fromthose reported previously. Greaves et al. found 43.2% of the unseparatedtonsillar lymphocytes to be B lymphocytes and 54.8% to be T lm-phocytes.2 Thomas and Phillips'3 reported that tonsillar lymphocytes con-tained 40 to 60% B lvmphocvtes. Tabata et al. 2 found that 22.6% oftonsillar cells formed E rosettes and that 38.7% formed EAC rosettes. Wefound IgM to be the predominant surface immunoglobulin class presenton the tonsillar B lymphocyte. We are not aware of other data whichdefines the proportions of different immunoglobulin classes on tonsillar BIymphocvtes. It has been reported recently that the predominant B Iym-phocyte found in the human appendix was also of the IgM class.'6 Exceptfor the reduction of E-rosette-forming lymphocytes by digestion withtrypsin, enzymatic digestion of the tonsillar tissue had minimal effect onthe surface markers assayed. We were particularly surprised to find thatenzvmatic digestion of the tissue had minimal effect on the B lymphocytemarkers. Purified collagenase has been noted previously, not to affectdetermination of lymphocyte surface markers;'3 however, trvpsin hasbeen used to reduce the detectable surface immunoglobulin on lm-phocytes.40 The preservation of the surface markers on the lymphocyteswe studied may result from several differences between our technique fordigestion and that described by Preud'homme and Seligmann;40 alterna-


tivelv, there mav be differences in the sensitivities of our staining pro-cedure.References

1. Good RA, Peterson, RDA, Gabrielsen, AE: The thymus: Current concepts. Post-grad Med 36:505-315, 1964

2. Greaves NI, Janossy G, Doenhoff NI: Selective triggering of human T and Blymphocytes in vitro by polvclonal mitogens. J Exp Med 140:1-18. 1974

3. Hoffrnann M1K, Schmidt D, Oettgen HF: Production of antibody to sheep redblood cells by human tonsil cells in vitro. Nature 24:3:408-410, 1973

4. Watanabe T, Yoshizaki K, Yagura T, Yamamura Y: In vitro antibody formation byhuman tonsil lymphocytes. J Immunol 113:608-616, 1974

3. Slover JL, \eltri RWN, Sprinkle PM: In vitro IgNM antibody synthesis by humantonsil derived lymphocytes. J Immunol 111: 1&3-188. 197:3

6. Smith RS, Sherman NA, Newcomb RW: Synthesis and secretion of immunoglobu-lin, including IgA, by human tonsil and adenoid tissue cultured in vitro. Int ArchAllergy Appl Immunol 46:785-801, 1974

7. Wiig JN, Thunold S: Electrophoresis of lymphoid cells: Characterization of humanB and T cells in peripheral lymphoid tissues. Clin Exp Immunol 13:497-506. 1973

8. Geha RS, \Nerler E: Response of human th-mus-derived (T) and non-thy-mus-de-rived (B) lymphocytes to mitogenic stimulation in vitro. Eur J Immunol 4:19:3-199.1974

9. Oettgen HF, Silber R. Nliescher PA, Hirschhorn K: Stimulation of human tonsillarlymphocytes in vitro. Clin Exp Immunol 1:77-84. 1966

10. Siegel I, Grieco NIH, Gupta S: Subpopulations of B lymphocytes in human tonsilsand peripheral blood. Lancet 2:213-216, 1974

11. Hurtado RC, Rola-Pleszczvnski M, Merida MA, Hensen SA, V'incent MM, ThongYH, Bellanti JA: The immunologic role of tonsillar tissues in local cell-mediatedimmune responses. Pediatr Res 8:414, 1974 (Abstr)

12. Tabata T, Enomoto T, Fujimura N, Hiramatsu K: Immunological function ofhuman tonsils, with special reference to E- and EAC-binding lymphocytes. ActaOtolaryngol 77:150-134, 1974

13. Thomas DB, Phillips B: The separation of human B lymphocytes on a digestibleimmunoadsorbent column. Eur J Immunol 3:740-742, 1973

14. Yu DTY, Peter JB, Paulus HE, Nies KM: Human lymphocyte subpopulationsstudy- of T and B cells and their density distribution. Clin Immunol Immunopathol2:333-342, 1974

13. Carr MC, Stites DP, Fudenberg HH: Dissociation of responses to phytohaemag-glutinin and adult allogeneic lymphocytes in human foetal lymphoid tissues. Nature[New Biol] 241:279-281, 197-3

16. Jubert AX', \cBride CM\t, M\avligit G, Gutterman JU, Hersh EM\: Immunoglobulinson the surface of human appendix lymphocytes. Immunol Comm 3:1-9. 1974

17. \Mavligit GM1, Jubert A, Gutterman JU, Reed RC. Hersh EM1: Subpopulations ofthymus dependent and thymus independent lymphocytes in human gut associatedlymphoid tissues. Surg Gynecol Obstet 140:397-400, 19735

18. Willson JKV, Luberoff DE, Pitts A, Pretlow TG II: A method for the separation oflymphocytes and plasma cells from the human palatine tonsil using sedimentation inan isokinetic gradient of Ficoll in tissue culture medium. Immunology 28:161-170.1973

19. Boyse EA, Old LJ, Thomas G: A report on some observations with a simplifiedcv-totoxic test. Transplant Bull 29:63-67, 1962

20. Pretlowv TG: Estimation of experimental conditions that permit cell separations by-

354 WILLSON ET AL American Joumalof Pathology

velocitv sedimentation on isokinetic gradients of Ficoll in tissue culture medium.Anal Biochem 41:248-255, 1971

21. Pretlow TG II, Weir EE, Zettergren JG: Problems connected with the separation ofdifferent kinds of cells. Int Rev Exp Pathol 14:91-204, 1975

22. Pretlow TG II, Luberoff DE: A new method for separating lymphocvtes andgranulocytes from human peripheral blood using programmed gradient sedimenta-tion in an isokinetic gradient. Immunology 24:85-92, 1973

23. Boyum A: Separation of leucocvtes from blood and bone marrow: Isolation ofleucocytes from human blood. A two-phase system for removal of red cells withmethylcellulose as erythrocyte-aggregating agent. Scand J Clin Lab Invest 21(Suppl 97):9-29, 1968

24. Thorsby E, Bratlie A: Histocompatibilitv Testing. Edited by PI Terasaki. Copenha-gen, Munksgaard, 1970, p 655

25. Levin DM, Rosenstreich DL, Wahl SM, Reynolds HY: Peyer's patch lymphocytes:Demonstration of the integrity of afferent and efferent T-ell functions in the guineapig and rat. J Immunol 113:1935-1941, 1974

26. Kreth HW, Herzenberg LA: Fluorescence-activated cell sorting of human T and Blvmphocytes. I. Direct evidence that lymphocytes with a high density of membrane-bound immunoglobulin are precursors of plasmacytes. Cell Immunol 12:396-406,1974

27. Hartzman RJ, Segall M, Bach ML, Bach FH: Histocompatibility matching. VI.Miniaturization of the mixed leukocyte culture test: A preliminary report. Trans-plantation 11:268-273, 1971

28. Harrison M R, Thurman GB, Thomas GM: A simple and versatile harvesting devicefor processing radioactive label incorporated into and/or released from cells inmicroculture. J Immunol Meth4:11-16, 1974

29. Cooper NMD, Lawton AR, Bockman DE: Aggammaglobulinaemia with B lvm-phocvtes: Specific defect of plasma-cell differentiation. Lancet 2:791-795, 1971

,30. Kincade PW, Cooper MD: Development and distribution of immunoglobulin-containing cells in the chicken: An immunofluorescent analysis using purified anti-bodies to M,Y and light chains. J Immunol 106:371-382, 1971

31. Aiuti F, Cerottini JC, Coombs RRA, Cooper M, Dickler HB, Frsiland SS, FudenbergHH, Greaves M F, Grey HM, Kunkel HG, Natvig JB, Preud'homme JL, Rabellino E,Ritts RE, Rowe DS, Seligmann M, Siegal FP, Stjernsward J, Terry WD, WybranJ: Identification, enumeration, and isolation of B and T lymphocytes from humanperipheral blood: Report of a WHO/IARC-sponsored workshop on human B and Tcells, London, 1974. Scand J Immunol 3:521-532, 1974

32. Alter Bj, Bach FH: Lymphocyte reactivitv in vitro. I. Cellular reconstitution ofpurified lymphocyte response. Cell Immunol 1:207-218, 1970

:3.3. Steinman RM, Cohn ZA: Identification of a novel cell type in peripheral lymphoidorgans of mice. I. Morphology, quantitation, tissue distribution. J Exp Med137:1142-1162, 1973

.34. Steinman RM, Cohn ZA: Identification of a novel cell type in peripheral lymphoidorgans of mice. II. Functional properties in vitro. J Exp Med 139:380-397, 1974

35. Steinman RM, Lustig DS, Cohn ZA. Identification of a novel cell type in peripherallvmphoid organs of mice. III. Functional properties in vivo. j Exp Med139:1431-1445, 1974

36. Lindahl-Kiessling K, Peterson RDA: The mechanism of phytohemagglutinin(PHA) action. III. Stimulation of lymphocvtes by allogeneic lymphocytes and phy-tohemagglutinin. Exp Cell Res 55:85-87, 1969

37. Lohrmann HP, Novikovs L, Graw RG Jr: Cellular interactions in the proliferativeresponse of human T and B lymphocytes to phytomitogens and allogeneic lym-phocytes. J. Exp Med 139:1553-1567, 1974


:S. Epstein LB. Kreth HWV. Herzenberg LA: Fluorescence-activated cell sorting ofhuman T and B lymphocytes. II. Identification of the cell ty-pe responsible forinterferon production and cell proliferation in response to mitogens. Cell Immunol12:407-421. 1974

39. Oppenheim JJ. Leventhal BG. Hersh EM: The transformation of column-purifiedlymphocytes wvith nonspecific and specific antigenic stimuli. J Immunol 101:262-270.1968

40. Preud'homme JL. Seligmann NI: Surface bound immunoglobulins as a cell markerin human lymphoproliferative diseases. Blood 40:777-794. 1972


[Illustrations follow]

_V

fe

_

II

w

I010

A

Figure 1-Tonsillar lymphocytes obtained by combining Fractions 7-12 of the isoki-netic gradient following separation of tonsillar cells obtained in suspension withtrypsin. These represent the modal population of lymphocytes which are 93.2 - 3.6%of the nucleated cells.

.. .. .... .. .

~~~ad

4

I

:4

qw

_- I_ ..

MD

a:

.4

*w;

Fgu 2-Tonsillar cells from Fraction 20 of the isokinetic gradient. These morerapidly sedimenting tonsillar cells include lymphocytes, immature lymphoid cells,histiocytes, plasma cells, and an unidentified binucleated cell.

i,4..

a characterization of human tonsillar lymphocytes after

Documents