Parasite antigen specific human T cell lines and clones Major histocompatibility complex restriction and B cell helper function

Parasite antigen-specific human T cell lines and

clones. Major histocompatibility complex

restriction and B cell helper function.

T B Nutman, … , A S Fauci, D J Volkman

J Clin Invest.

1984;

73(6)

:1754-1762.

https://doi.org/10.1172/JCI111384

.

The development of T lymphocyte lines and clones of defined specificity has become an

important method for investigating both T cell recognition of foreign antigens as well as T

cell influence on B cells. In the present study, human antigen-specific T cell lines and

clones have been derived from a patient with a naturally acquired filarial infection. These T

cells are of the helper phenotype (Leu 1+, Leu 2-, Leu 3+) and are independent of

exogenous interleukin-2. Furthermore, these T cells have been shown to require both

antigen-presenting cells and antigen for optimal proliferation. Helper function mediated by

these T cells as manifested by the in vitro induction of parasite-specific antibody was

antigen-dose dependent, requiring much lower antigen concentrations than those

necessary to induce blastogenesis. More importantly, there is an absolute requirement of

the T cell line for HLA-DR histocompatible antigen-presenting cells; clones derived from this

T cell line show a more specific DR-related restriction--to only one of the two parental DR

haplotypes in antigen stimulated proliferative responses. Such parasite antigen specific

human helper T cell lines and clones should prove useful in exploring the fine control of the

host response to naturally acquired helminth infections. In addition, these long-term T cell

lines and clones can provide a potent tool for examining not only the events involved in

human T […]

Research Article

Parasite Antigen-specific

Human T Cell Lines and

Clones

Major Histocompatibility Complex

Restriction

and B

Cell

_Helper

Function

Thomas B. Nutman, Eric A. Ottesen, AnthonyS. Fauci, and David J. Volkman

Laboratory ofParasitic Diseasesand the Laboratory of

Immunoregulation, National Institute ofAllergy and

InfectiousDiseases, National Institutes of Health,

Bethesda, Maryland 2020S

A~bstract. The development of T lymphocyte

lines

and clones

of

defined specificity has become an

im-portant

method

for investigating

both T cell

recognition

of

foreign antigens

as

well as T cell

influence

on B cells.

In

the

present

study, human

antigen-specific

T cell lines

and

clones have been derived from

a

patient with

a

nat-urally acquired filarial infection.

These T cells are

of

the

helper phenotype

(Leu

1+,

Leu

2-,

Leu 3+) and are

independent of

exogenous interleukin-2.

Furthermore,

these

T

cells

have been

shown

to

require both

antigen-presenting

cells and

antigen for optimal proliferation.

Helper

function mediated

by these T cells as

mani-fested by the in vitro induction of

parasite-specific

an-tibody

was

antigen-dose dependent, requiring

much lower

antigen

concentrations

than those necessary to

induce

blastogenesis.

More

importantly,

there is

an

absolute

re-quirement of

the T

cell

line for

HLA-DR

histocompatible

antigen-presenting cells; clones derived from this

T cell

line show

a more

specific DR-related restriction-to only

one

of the

two

parental

DR

haplotypes in

antigen

stim-ulated

proliferative

responses.

Such

parasite antigen specific

human

helper

T

cell

lines and

clones

should

prove

useful

in exploring

the

fine

control of

the

host

response

to

naturally

acquired

helminth

infections.

In

addition,

these

long-term

T cell

lines and

clones can

provide

a

potent

tool for

examining

not

only

the events

involved in human T cell responses

to

parasite

antigens,

but

also

into

the

associated cellular

and

humoral

factors

necessary

for

the B cell responses

which

follow.

Receivedfor publication

18October1983 andin

revisedform

23January

1984.

The Journal of ClinicalInvestigation, Inc. Volume73, June 1984, 1754-1762

Introduction

The human filariases,

estimated to

affect

-250

million

people (1), are

chronic parasitic

helminth infections

that evoke host

immune

responseswhich have been

implicated

both

in

resistance to

infection (2, 3)

and

in the

pathogenesis ofthe various clinical

manifestations of

these

diseases (3). Though serological studies

reveal

that

infected individuals

develop

anti-filarial antibodies

ofdifferent

immunoglobulin

isotypes, the

manner

by which the

parasite antigens elicit

the

production of parasite-specific

an-tibodies

is not

well

characterized. More importantly, it has been

difficult

to

evaluate the

fine control

of the immune response to

parasite antigens

at

either the cellular

or

subcellular

level because

of the

lack

of

a

satisfactory in vitro model of

antigen-induced

T or B

lymphocyte function.

Epidemiological studies in

areas where

filariasis

is

endemic

have revealed

differential susceptibilities

to

infection

both

within

the

entire population

as

well

as

within

families studied (4).

Though the

cause

of this

difference in susceptibility has

not been

studied

directly in the filarial diseases, work done with

other

helminths (5-7) and

protozoa

(8) has

implicated

the

in-volvement

of

the

major histocompatibility

complex, MHC.'

Studies

done with

congenic

mouse

strains have shown that the

H-2

complex in

mice

may

affect

differences

of

antibody

titers

and

delayed type

hypersensitivity

to the

parasite Schistosoma

mansoni

(9). More

recently,

a

strong association between

HLA-D

specificity

and low

responsiveness

of

periperal

human

T

cells

to

antigen

extracted from

Schistosoma

japonicum

worms

has

beenreported

(10).

Though the

requirement of

MHC

recognition

of

antigen

by

T

cells

is well

established,

the events

controlling

this recognition

have

only lately begun

tobe

elucidated,

primarily

because of

1.Abbreviations used in this

paper:

AET,

2-aminoethylisothiouranium

bromide; BMA, filarialantigenderivedfromBrugia

malayi

adult

par-asites;

FACS,

fluorescence-activated cell

softer;

FCS,

fetalcalf

serum;

IL-2,interleukin2;MHC, major

histocompatibility

complex;

PBMC,

peripheralblood mononuclear

cell; PHA,

phytohemagglutin

A;

PWM,

theability to maintain antigen-specific Tlymphocytesinvitro (1 1-18). Initially, much of the workon Tcelllines(TCLs) and clones was carried outinthe murine system;however,the

meth-ods for developing long-term T cell populations specific for

particulate and soluble antigens have now been extended to

human cells(16, 17, 19-22). TCLs have been developedwhich

require histocompatible antigen-presenting cells at the

HLA-DRregion in order for antigen-specific proliferationto occur;

cloned cells from these and other lines have been developed andshown to

recognize

soluble and

particulate antigens

only in aDR-restricted fashion (16, 17, 21-25).

Similarly, when supernatants fromthese T cell cloneshave been

examined,

soluble factors have been shown to enhance

immunoglobulin production

(26).In

addition,

TCLs and clones of the

helper/inducer

phenotype have been showntobe capable ofhelpingBlymphocytes produce specific immunoglobulin (21, 24, 27).

In the present study we have

established

filarial

antigen-specific

human T cellsby the

technique of

repeated stimulation with a soluble parasite antigen and subsequent rest in the absence

of this antigen.

The T cells produced in this manner are

in-dependent of

exogenous

interleukin-2 (IL-2)

for their growth. Furthermore,this is the first

demonstration

thatantigen-specific,

IL-2-independent

TCLs and clones are able to be

developed

from

naturallyinfected subjects rather than

from

subjects

that wereartificially immunized. More importantly, work donewith these TCLs show that

parasite antigen recognition

is class II

(DR)restricted; these same cells provide help forBcellantibody production.

Methods

Antigens. Brugiamalayiadultantigen(BMA)waspreparedasasaline

extractof adultparasitesasdescribed and characterizedpreviously(28);

tetanustoxoidwasobtainedfrom the Massachusetts Public Health Service (agift of Dr. Thomas Folks).

Cell separation. Peripheral blood mononuclear cells(PBMC)were

obtained from heparinized blood. Leukocyteswere separated by low speedcentrifugation (200 g) for 8min andfurtherpurifiedon a Hypaque-Ficoll gradient ina standard fashion (29). PBMCwere cryopreserved

in RPMI-1640 (Biofluids, Rockville, MD) supplementedwith10%fetal calfserum(FCS), 25mmHepes (MABioproducts, Walkersville, MD), 80

_Ag/ml

gentamicin(Schering Corp., Kenilworth, NJ), 7.5% dimethyl

sulfoxideinarate-controlled cellfreezer (Cryo Med.,Mt.Clemens,MI), and stored in liquid nitrogen until use. Tcell-depleted mononuclear

cellswereobtained byrosettingwith 2-aminoethylisothiouranium

bro-mide (AET)asdescribed previously (30).

HLAtyping. TissuetypingforHLA-Aand HLA-Bwasperformed

onPBMC ofvarious donors ofantigen-presentingcellsbyusing standard microcytotoxicityassays. BcellsweretypedforDRantigen inasimilar fashion. Alltyping wasperformedin the HLATyping Laboratoryof

the BloodBank atthe NationalInstitutesof Health.

Culture and cloning procedure. The method for preparing

antigen-specificTcelllines isthatofVolkman et al.(31),amodification ofthe

cyclic stimulation and rest method first described by Kimoto and

Fathman (32, 33).Fresh PBMC wereinitially cultured at a density of 5X

106

cells per wellin24-well cluster dishes (3524 Costar, Cambridge,

MA) inRPMI-1640 supplemented with 80lg/ml gentamicin, 25 mm

Hepes, 10% FCS (complete RPMI),and 25

Ag/ml

of BMA. The cells were incubated at370C in a humidified atmosphere in 5% CO2 for 5 d. Viable cells were separated on a Hypaque-Ficoll gradient and cultured (rested)for 10-12 d with freshly thawed autologous PBMC, whichwere

irradiated with 3,500 rads by exposuretoa calibrated 37Cs-source (Iso-medix, Parsippany,NJ)at aratio of 106viable cellsto4X106 irradiated PBMC. After the resting cycle, the procedurewasrepeated by separating viable cells, as indicated above, andcombining them with freshly prepared PBMCas a sourceofantigen-presenting cells along with fresh BMA (25

Ag/ml).

Four cycles of5-dstimulation with BMA and 9-11 drestculture in theabsence of antigen were repeated before cloning.

Cloningwasaccomplished by limiting dilution. Viable T cells were seeded at 0, 0.3, 1, 3, and 10 cells per well in U-bottomed 96-well microtiter dishes (Linbro, FlowLaboratories, McLean, VA) in 200 tl of mediumcontaining both BMA (25

Ag/ml)

and exogenous IL-2 (15% final concentration). Each well also contained 5X 104 autologous ir-radiatedantigen-presenting cells. Positive wellswerescoredby viewing inaninverted microscope after 12-14 d inculture; positive cellswere removed andrestarted on repeatedstimulation and restcycles in the absence of exogenous IL-2.

IL-2.Supernatantscontaining IL-2wereobtained from fresh PBMC of four donors whose cellswerepooled and incubatedat adensity of 2 X 106 cells per ml in RPMI 1640 and 0.5% FCS with 2

Ag/ml

of purified phytohemagglutininA(PHA;Burroughs WellcomeLaboratories, Research Triangle Park, NC)for 36 h at370C(34). The conditioned supernatant fromthis culturewasfiltered througha

0.45-,sm

filter (Mil-lipore Corp.; Bedford, MA) and storedat-20'C.

Proliferativeassays. Unless statedotherwise,assays forantigen-specific responses wereperformed by culturing 5X 103-1I X 104Tcellswith 1 and5 x 105irradiated(3,500 rads)antigen-presentingcellsand antigen ineither round-bottomed (1 x 105cells) orflat-bottomed (5X 105cells) 96-well microtiter dishes. Cultures were incubated for 3 d andthen

pulsedwith 1

ACi

of[3Hjthymidine for 16h.Cellswerecollectedon

glassfilters with a Titertek cell harvester (Flow Laboratories, Inc., Rock-ville, MD) and incorporation of[3H]thymidinewas measured byliquid scintillation spectroscopy. Dataareexpressedas mean countsper minute oftriplicatecultures.

Invitroantibody production. Unfractionated mononuclear cells or

aTcell-depleted fraction (AET negative) cells were placed inflat bottomed wellsofmicrotiter plates at a concentration of either 250,000 cells/ml

(unfractionated)or125,000 cells/ml (AETnegative) in complete RPMI.

Tothesecells were addedvarying numbersofTcellsderivedfrom the TCL in the presence or absence of varying concentrations of BMA

antigen.Thevolumewasnormalized at 220

AI,

and the cells were in-cubatedat37°C, 5%C02,and100%humidity for 10 d. The supernatants were harvested and immediately assayed.

Micro-enzyme-linkedimmunosorbent assayfortotal and

parasite-specificIgGand IgM. IgM and IgG in culture supernatants were measured byusingenzyme-linked immunosorbent assays. Flat-bottomed microtiter

plates(Dynatech Laboratories, Inc., Alexandria, VA) were coated with 0.1 ml of carbonate buffer, pH 9.6, containing either 10

Ag/ml

Fab fragment goat anti-human IgG or IgM (Fab fragment, Cappel Labo-ratories, Inc., Cochranville, PA) or 5

Ag/ml

of parasite-specific antigen (as described above)and allowed to incubate overnight at 4°C. The plates were then washed in PBScontaining Tween 20 (Sigma Chemical Co., St. Louis, MO). Samples were appropriately diluted, added to

chain-specific goat anti-IgG or anti-IgM conjugated to horseradish per-oxidase (Kirkegaard and Perry, Rockville, MD) was added to each well. The plates were incubated at370Cfor 2 h, washed again and allowed toreactwith o-phenylenediamine in a potassium phosphate buffer, pH 7.0,containing 0.006% H202. The reaction was stopped with 2 NHCL.

Color development was measured by using a multiplate reader. For total IgG and IgM the color development was related to a reference pool of human sera with a known amount of IgG orIgM(Meloy

Lab-oratories,Inc.,

Springfield,

VA). Forantigen-specificIgGand IgM, ar-bitrary units were defined on the basis of a reference serum to which all other samples were compared.

Fluorescence-activatedcell sorter (FACS)analysis. FACSanalysis was performed on an FACS II cell sorter (Becton-Dickinson, Sunnyvale, CA) in the laboratoryofDr. T. Chused. Direct staining was performed

using106 cells per determination. Mouse monoclonal antibodies to Leu 1, Leu 2, and Leu 3 conjugated to fluorescein isothiocynate (Becton-Dickinson) were incubated with the cells at40Cfor 30min.Cells were then washed and placed in the cell sorter. Cells stained with an unrelated,

fluoresceinated monoclonal antibody were used as a control. The Leu 1, Leu 2, and Leu 3 monoclonal antibodies recognize the total Tcell,cytotoxic/suppressor T cell,and helper/inducer T cell subsets,

respectively(35).

Statistical methods. Analysis of precursor frequencyinlimiting

di-lutionexperimentswasperformed by assuming random and independent

distribution ofthe T cells inmicrotiterwells andby applying the Poisson

analysis(36).Theremainder of the statistical analyses were performed

byusingatwo-tailedttest.

Donor ofthecellsusedinpropagatingtheTCL.Thepatient,a 29-yr-old white female, was diagnosed as havingloiasisin February, 1982,

afterresidingfor3 yr inGabon, WestAfrica,andsubsequentlydeveloping

thecharacteristic angioedematous"calabarswellings"on herextremities alongwithhighgradeeosinophilia. She hadhigh levelsofanti-filarial

IgG antibody,and theclinicaldiagnosis of loiasiswasconfirmedwhen anexcisionalbiopsy ofanodule thatappearedonher armaftertreatment

withdiethylcarbamazinerevealed themicrofilarial form ofLoaloa.The

patientrequired multiplecoursesofdiethylcarbamazine toprevent re-currentswelling(andpresumablyto kill all ofherparasites).Atthe time ofleukopheresis(when theoriginalcells wereobtained)thepatientwas

without clinical manifestations of disease and had adecreasing

anti-filarialantibody titer. Thesefindings are illustrated inFig. 1.

Results

Influence of

antigen

concentration on T

cell

proliferation.

The response

of unfractionated PBMC

to

both the

BMA

and

tetanus

toxoid (TT)

was

explored

initially.

AsseeninTable

I,

maximal

T

cell

proliferation

was

noted with

a

parasite antigen

concen-tration of

25

_gg/ml.

This level ofBMAthus became the

estab-lished concentration for further studies. The

responsetoTT is

also

shown in Table

I;

again,

a

dose-dependent

responsiveness

was seen.

BMA-specific

TCL and

clone

preparation.

PBMC fromthe

patient

were

stimulated with

BMA

(25

Atg/ml)

in vitro for 5d

andthen rested

in

the

absence

of

BMA. After four

cycles

of

stimulation and

rest,a

fraction of the

viable cellswasremoved

and cloned

by

limiting

dilution.

Fig.

2shows the

limiting

dilution

analysis of plates seeded

at

3,

1,

0.3,

and 0 cells per well. A

linear plot consistent with

an

independent

Poisson

distribution

5,000

-e

.Q

-1,000

C, 0t

.1

im

DEC DEC

DEC DEC

Cells Obtained forCloning

I I

NovJan Mar May JulySept Nov Jan Mar MayJuly Sept Nov

Ciaber+ + + + +

Absolute

Eiosinoophl 5,4204,% 610 13 14 18 1per mmtl

Figure 1.Timecourseofpatient's clinicalcondition and anti-filarial

IgGantibodytiters. Arrowsindicate pointsat whichthe patient re-ceiveddiethylcarbamazine (DEC).

ofasingle cell typewasobtained. We have designatedasclones the T cell populations derived from limiting dilution plates seededat oneT cell per well or less (> a 95%

probability

of true clonality); however given the low clonal efficiency, even

TableL

Proliferative

ResponsesofUnfractionated PBMCs*

as aFunctionofIn VitroAntigen Concentration

Antigen Antigenconcentrationt Proliferativeresponses§ cpm

None 0 226

BMA 0.01 582

0.1 3,214

1 6,882

10 7,094

25 8,625

50 5,685

100 6,516

TT 1/2,000 7,415

1/5,000 9,281

1/10,000 9,321

1/20,000 2,341

* I X

101

cells cultured in round-bottomed wells and

assayed

on

day 5.

100, 90 Qa,

i

a) c, a-85 80

A

0

0 1 2 3 4 5 6 7 8 9 10 TCells per Well

z z 4 I U

c.

u-z a.

Figure2. Limiting dilution analysis ofBMA-specific T cell clones. Tcells were seededat anaverage of3,

1,0.3, or 0 cells perwell andthepositive and nega-tive wellswerescored after

14d. Thenumberof nega-tive wellswere48/60, 52/ 60,58/60, and60/60,

re-spectively.

the cells

seeded

at 3 or 10cells per well are

likely

tobe true

clones.

Subsequently,

wehave beenable toimprovethe clonal

efficiency significantly whereby

the precursor

frequency

(ob-tained

by

extrapolating

the

point

atwhich 37% ofthewellswere

negative) of clonable T cells has approached oneper 1.5 cells

compared with one per 13 cells in the present

study (data

not

shown).

Antigen

specificity of the TCL. In order to study the TCL

for

antigen specificity,

a

proliferative

assaywas

performed

with

autologousantigen-presenting cells alone or with the

addition

of

Tcells

(from

the

TCL),

antigen,

orboth T cells and

antigen

(Fig. 3).

Proliferation

wasnoted only in the presence

of

Tcells and

parasite

antigen. Stimulation with

an

antigen (TT)

towhich the

patient's unfractionated PBMC

werecapable

of responding

(Table I) was unable toinduce theTcell

line derived

from

this

same

patient

to

proliferate.

Cytofluorometric

analysis

of

the

TCL.

BMA-specific

Tcells werestainedwith anti-Leu 1, -Leu 2, and -Leu 3, and examined in the cell sorter. As seen in Fig. 4, after two cycles ofstimulation andrest(panel A), 90%

of

the cells were Leu I +and Leu 3+;

I

a. Figure 3.Analysisof

anti-gen-specificproliferative

re-sponses ofthehumanT

cellline in the presenceor

absenceof specific antigen andantigen-presenting cells

(APC). Thedata are ex-pressedasmean

[3Hjthymidine

uptakein

cpm±SD. 1.00 0.80 0.60 0.40 0.20 z z 1.00 z 0.80 u r 0.60 a.

C_

0.40 z w_ 0.20 i 0.00 a. Z 1.00 z I 0.80 sr 0.60 u CL 0.40 z Z 0.20 c 0.0c Ma

I

0 0 . Control

Leu 1+

10 100 CHANNEL NUMBER Control Leu2+ 10 100 CHANNEL NUMBER Lt Control

Leu3 +

0 10 100 CHANNEL NUMBER

B

Z 1.00n

z

< 0.80 M ) 0.60 X. 0.40 G 0.20 LU z 0.00 uj 0 z 1.00 z 0.801 0.60

it

0.40 z 0.20 Q 0.00 4r1.00w1

l_o:o

L.0

u 0.60H

z l 0.40 0.20 0.00 CLa n Control

Leu 1+

10 100

CHANNEL NUMBER

l-- Control

Leu2+

10 100

CHANNEL NUMBER

Control

'I

n

Leu3+

10 100 CHANNEL NUMBER

Figure4.FACSanalysisof the parasite-specificTcellline. Viable cells were separatedfrom dead cells by Hypaque-Ficoll gradient sedi-mentation and stained with the designated fluoresceinated

mono-clonalantibody (Leu 1, Leu 2, Leu 3) or withafluoresceinated con-trolmonoclonalantibody. Cells were then analyzed on a FACS-I1 (Beckton-Dickinson). A represents the FACS analysisafter two

coursesof antigen presentation and an intervening rest.Brepresents theFACS analysis just before cloning.

after the

fourth

cycle (panel B), all cells in thisparticular TCL were of the helper/inducer phenotype (35) as determined by

FACS analysis.

This pattern of Leu reactivity (Leu 1+, Leu 2-, Leu

3+)

has

remained

stable on the cells in culture

for

>6 mo.

Ability of TCL

to

stimulate

antibody production. When

au-tologous

unfractionated

PBMC were cultured in the presence ofincreasing numbers ofTcellsfrom the TCL in the absence of

antigen,

nominal

amountsof total andantigen-specific IgG and IgM were produced. When parasite antigen was

added,

however, the ability to produce both total and parasite specific antibody was markedly enhanced (Fig. 5).

The dose-response curves of the titration of the TCL against the in vitro production of antibody by these unfractionated PBMC demonstrated that as few as 500 T cells could elicit polyclonal helper activity (Fig. 5 A, C), whereas 1,000 T cells were necessary to help in the production of parasite-specific

antibody

(Fig. 5 B, D). Maximum help, both polyclonal and

Uuu (J

n

rnlL

1O,0K

-8,0C

CD

6,X0

9

4,0C

2,0X

0

x

1c

0 500 1,000 2,500A Number ofTCells Added

#

x _:v

Figure 5. In vitro induction of BMA-specific and totalIgGandIgM

as afunction of increasing numbers ofTcells added. Open circlesor

bars indicate culturestowhichnoantigenwasadded. Dark circlesor barsindicate culturestowhich BMAwasadded. Dataareexpressed as meanvalues±SD of triplicate cultures. ResponsestoPWMare

specific,occurredat2,500 T cellsatallconcentrations of antigen tested (data not shown). T cells from the TCL added to un-fractionatedcells stimulated production ofatleastasmuch (and

usually greater) polyclonal immunoglobulin and antigen-specific immunoglobulin than did the addition of pokeweed mitogen (PWM)orPWM +BMAtounfractionated cells.

The requirements for the antigen-specific helpwerestudied further. When Tcells were depleted from the unfractionated PBMC

population,

thereby removing residualTcells (presum-ably with both helper andsuppressorfunction)thatwerecapable

ofrespondingtotheantigen,a morestrikinginvitroantibody response was seen(Fig. 6). Both antigen-specific and total IgM

(panel A) and IgG (panel B)were measured inculture

super-natantsafterthe addition ofvaryingconcentrations of BMAto

2,500 T cells and 125,000 AET-negative PBMC. Maximum

anti-BMA antibody responsesto BMA stimulation were seen

atthelowestantigen concentrationsused:astheconcentration

ofBMAwasincreased, amodestsuppressionof theanti-BMA antibody response wasseen. Such wasnotthecase, however,

500 1,000 2, Numberof T Cells Added

usedas apoint of reference. The ordinates of the bargraphsare

identicaltothoseof the corresponding linegraphs.Arepresentsthe totalIgGresponses;BrepresentstheBMA-specific IgGresponse;C

representsthe total IgMresponses;and Drepresentsthe

BMA-spe-cificIgMresponse.

for totalimmunoglobulin production, asall concentrationsof

BMA thatresulted in specific antibodyresponsesalso resulted

inmarkedincreases in the secretion oftotal IgG and IgM. Thus, BMAaddedtoaBMA-specific TCL in thepresenceofautologous

Bcells andmonocytesresults both inatruepolyclonalresponse

(perhaps more marked athigh antigen concentrations) and a

predominantly antigen-specific responseatlower antigen

con-centrations.

MHC restriction

of

antigen presentation to the TCLand clones. The capacity of allogeneic antigen-presenting cells to

substitute forautologous antigen-presentingcells in the

presen-tationof BMAtothese T cellswasnext explored.The donor

ofthe TCLwastypedas HLA-A 1, 3,-B 14, 39,and -DR 7, 8. Antigen-presenting cellswerethen obtained from five

indi-viduals who were heterozygous for the DR8 haplotype, four

individuals who were heterozygousfor DR7, and sixpatients who had neither the DR7 northe DR8 haplotype. As

dem-onstratedinTableII,the TCLwascapableofrecognizingBMA

inassociation witheitherDR7orDR8allogeneic antigen-pre-A

30

30

30

20

)o

40

mo~~~~~~~-1'

4~~~~~~~-a

e 4

0 0.01 0.1 1

IBMAI inpg/ml

-v

ID

0n T.

CD

C) a

(A

3

_Figure

6. Total and

BMA-specific

immu-a

noglobulin

responsesin T

cell-depleted

mononuclearcellstowhichwereadded

increasingdoses ofantigen.Arepresents

theIgM responses.Brepresentsthe IgG

responses.

Open

bars indicated total

im-10 munoglobulin and hatched bars indicate

antigen-specific responses.

senting cells. Only in the absence of either of these DRtypes

wastherenoproliferationtothe antigen. Furthermore,as dem-onstrated inTable II,one of the clones derived from the TCL

was capable of recognizing BMA in association withDR7 an-tigen-presenting cellsandnotDR8allogeneic antigen-presenting

cells. Anotherclone recognized antigenwithonlyDR8 butnot

with DR7. Each clone we tested recognized antigen only in

association withtherestriction elementpresentonDR7orDR8

cells; noclone recognized antigenwithautologous antigen-pre-senting cells alone; this observation implies that none of the clones was restricted to a DR7, 8 hybrid Ia. In addition, no

clone testedproliferated toautologous antigen-presenting cells alone(without antigen present), afinding thatsuggeststhe

ab-sence ofautoreactive clones.

Similarities ordifferencesattheHLA-Aor-B locus played no role in antigen presentation tothe Tcell line.

Discussion

Thedevelopment of human T cell lines of defined specificity canprovideapowerfultool fordissectingtheregulatory

mech-anismsinvolvedin T-T and T-Bcellinteractions. Thepresent

studyconfirmstheearlierobservations (31)whichdemonstrate

thatantigen-specific humanTcelllines and clonescan be

ob-tained independent ofexogenousgrowth factors andprovides evidence that these TCLs can be derived not only from im-munized subjects but also from patients withnaturallyacquired parasiteinfections. Furthermore, theseT cellsexhibit fineMHC

restriction to either of the two parental DR haplotypes, and

functionally they have the capacityto provide helpto B cells fortheproduction of polyclonal immunoglobulin andspecific antibody.

The technique ofrepeatedcyclesof antigenstimulationand

rest selects fora subset ofTcells capable of supporting their

own

growth.

In

addition,

afterfour cycles of antigen stimulation,

the resulting TCL is comprised almost exclusively of

antigen-specific cells sothat cloning from this highly

antigen-specific

linecan yield, with reasonableassurance, clones of predefined specificity. These resultscontrastmarkedly with the

techniques

generally used in human T cell cloning in which cloning is performed after one initial in vitro antigen stimulation and where specificprecursorfrequency is often <1%.Furthermore,

since theseclonesand TCLsareindependentofexogenous IL-2, theycan be assayed for both polyclonalandantigen-specific lymphokine production inthe absenceofexogenousmaterials that might be contaminated with other lymphokines. Suchassays are in progressat thepresent time inour laboratory.

Cloned human T cells that mediate helper activity inthe formation of both specific and nonspecific antibodyhavealready been described (21, 24, 27). However, since these lines have hadanabsolute requirement ofexogenousIL-2 (usually in the

form ofPHA supernatants containingmanyfactors including PHA) for growth and maintenance, the helper activitymaybe

relatedtothe action of contaminatingexogenousfactorsonthe T cells and not directly to the function of the T cell being studied. Thus, the findingthatthis helper activityisindependent ofexogenousIL-2 in human TCLs and clones is of additional importance.

The induction of specific anti-parasite antibodyby cloned humanTcellswasclearly antigen-dose dependent. Stimulation ofthe T cells with lowconcentrations ofantigen induced optimal antigen-specific antibody production by autologousBcells. In

contrast, the concentration for optimal antigen-induced lym-phocyte proliferation was consistently 100-1,000-fold higher than thatseenfor in vitro antibody production(Table I). This worksupportssimilar observationsreportedin keyhole limpet

hemocyanin-induced (37, 38)orinfluenza A-induced(24)

spe-cific antibody formation wherepeakspecific antibodyproduction

5,000

B

'E

.0

0

1,o000[

-V

Qa)

CD

T.

CD

C_,

3

11

.0

E

f

500

loo0

50

IBMAJ inMg/ml

I,

/2

/

Table II. Filarial Antigen Presentation Is MHC Restricted

HLA Proliferativeresponse* Antigen-presenting

cells Donor A B DR Ag TCL Clone A Clone B

D.Z. A.B. B.A. D.W. C.T. K.S. H.T.

1,

3 28, 32 23, 23 1, 26 29, 30 2, 3 2, 32

I.R. 33, 33

T.B. 28, 32

A.P. 2, 25

Non-DR-related D.B. 3, 30 J.B. J.J. J.L. J.J. C.D. 26, 28 1, 23 30, 34 1,33 28, 30

14,

39 7,42 39, 52 7,40 18,54 7,60 35, 58 35,B18 7, 42 27, 35

45,

57 40, 41 49,53 42, 39 42, 49 45, 42 cpm

7, 8 - 6,084

+ 20,987t

3,7 - 5,508

+ 11,748t

5,7 -

3,482

+ 10,289t

1,

7 - 1,146

+

2,900t

6.3, 7 - 1,044

+ 8,006t

2, 8 - 2,642

+

_13,683t

6, 8 - 2,737

+

_6,503t

6.3, 8 - 450

+

_3,521t

3,8 - NT

+ NT

3, 8 - 779

+

6,044t

2,2 -5,6 -5,6 -1,3 -3,6 -3,5 -1,783 1,758 3,708

3,417

1,297 1,298 5,055 4,425 5,234 5,063 7,670

7,733

NT,nottested. *Meanoftriplicateculture. *Significantdifference(P<0.05)between itand itscorrespondingcontrol.

occurs inresponsetoantigen concentrationsseverallogslower than optimal blastogenic antigenconcentrations.

The number of helperT cells inoursystemsimilarlyaffects the formation ofspecific antibody,withhighnumberscapable

ofsuppressingthespecificresponse.Thisphenomenonhas been

seenwith clonedmousecells(39, 40)andantigen-specifichuman

helperTcells (26). In these IL-2-dependent systems, limiting quantities of IL-2orculture nutrientshave beenimplicated as

thecause for thesuppression seenat highcell numbers.

Alter-natively, andmorelikely in view of theIL-2 independence of

our T cells, an excess ofhelper factors could turn off

immu-noglobulin production by actingat theB cell level (41). Also, aclonemay besecreting morethan one _factor, one of which

helpswhile the othersuppresses(but onlyathigh concentrations). Theprocessofparasite antigen recognition bythese T cell

clones is noteworthy. An absolute requirement for

histocom-patible antigen-presentingcells isseen.Similar to other human

TCLandclones,these filarialparasite-specificT cellsrecognize antigen onlyin association with discreteimmuneresponsegene

separate clonesthat are able to recognize BMA do soonly in the context of either one or the otherparental Ia haplotypes. Thisfinding lends further credenceto theconceptthat thehuman T cellnetwork contains clones that

recognize

each of theparental immune response gene productsand that an individual Tcell recognizes soluble parasite antigen, at least,in thepresence of one, but not both, Iaalleles. Our study,

therefore,

extends to

parasite antigens a finding already noted for keyhole limpet hemocyanin

recognition by specific

humanTcell clones(31), namely thathybrid paternal-maternal Iarecognition (reported in the murine model) (32, 42)is notnecessary forantigen

pre-sentation by human Tcells.

Finally, this study

clearly

indicates the usefulnessof exog-enous

IL-2-independent, antigen-specific

TCLs ininvestigating the nature of T cell

recognition

of

antigen

andthe B cellresponses thatoccur as a

result

of this

recognition.

The

ability

toextend

theseobservationstothe

immunology

of

parasite

infection has

already provided important

insights

into theimmunoregulatory

mechanisms involved.

Itshould also

help

pinpoint

the important

antigens

necessary for

inducing

cellular and

thereby

humoral

responses;

this

then should enable us to

begin

to manipulate these

immune

responses in

hopes

of

establishing protective

im-munity

to

organisms

causing

these diseases of worldwide

im-portance.

Acknowledgments

Theauthors wishtothank Dr.DavidAllingfor hisexpertassistance in thestatisticalanalyses utilized in these studies and the National Institute ofAllergyandInfectious Diseases/LaboratoryofParasitic Diseases ed-itorial staff for help in preparing this manuscript.

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