T-Cell-Independent and T-Cell-Dependent B-Cell Responses to

INFECTION ANDIMMUNITY,

JUlY

1990, p.2337-2342 Vol. 58, No.7 0019-9567/90/072337-06$02.00/0

CopyrightC 1990,American Society forMicrobiology

T-Cell-Independent

and

T-Cell-Dependent

B-Cell

Responses

to

Exposed Variant Surface Glycoprotein Epitopes

in

Trypanosome-Infected

Mice

DAVID M. REINITZAND JOHN M. MANSFIELD* DepartmentofVeterinary Science, University of Wisconsin-Madison,

1655Linden

Drive, Madison,

Wisconsin 53706 Received 12 December1989/Accepted10April1990

TheT-cell dependency of B-cell responsestovariant surface glycoprotein (VSG) epitopes exposedin their native surface conformation on Trypanosoma brucei rhodesiense clone LouTat 1 was investigated. T-cell requirementswereexamined by analyses of gammaglobulinpreparationsderived fromtrypanosome-infected BALB/cnude(nulnu)andthymus-intact(nu/+)mice. Aradioimmunoassaywasusedtoselectivelyquantitate antibody binding tonative VSG 1 epitopespresenton the surface of viable trypanosomes. Suchanalyses of VSG-specificantibody in infected mice demonstrated that in the absence of T cells therewas asignificant B-cell response toexposedVSG epitopes; however, inthepresenceof T cells these surfaceepitope-specificresponses were greatly enhanced. Incontrasttoinfection, immunization of mice withpurifiedVSG 1 or paraformalde-hyde-fixed parasites elicitedsignificantVSG surfaceepitope-specific responsesonly in the presence ofTcells (i.e., in nul+ miceonly). VSG-specific antibodyresponses in mice infected with three other clonal T. brucei rhodesiense populations (LouTat 1.2, 1.5, and 1.9) were found to be similar in this pattern, although not identical, to the anti-LouTat 1 responses. Animportant exception was that mice infected with LouTat 1.8 required T cells to produce VSG surface-specific antibody. Thus, the VSG surface epitope-specific B-cell responses in trypanosome-infected mice represent composite T-cell-independent and T-cell-dependent pro-cesses, andasignificantly stronger response is made in the presence of T cells. However, immunization with VSG in the absence of infection elicited only T-cell-dependent responses. Since therelative contribution of T-cell-independent and T-cell-dependent processes to the total VSG-specific antibody produced during infection was variable (as seen with the absence ofa T-cell-independent response to LouTat 1.8), this may reflect differencesintheprimary structure ordisplay of VSGmoleculesonthetrypanosome membraneormay represent activeparasite interference with someepitope-specificB-cellresponses.

African sleeping sickness is caused by infection with the protozoanparasitesTrypanosoma brucei rhodesienseandT. bruceigambiense.Approximately 50million Africans living in sub-Saharan regionsareatriskof being infected (7). The blood-borne trypanosomes evade temporallyprotective im-muneresponsesby transcriptional switching ofgenes encod-ingthe variantsurface glycoprotein(VSG), which covers the plasma membrane of the parasite (5). Studies of VSG gene expression have revealedthatthe regulation of these genes is amongthe mostunusualandcomplexofeucaryotic systems studiedtodate (8). Theinteraction oftheparasitewith cells and products oftheimmune systemalsois extremely com-plex.Tobetter understandbasic immunological characteris-tics of this infectious disease, it is one of the long-term goals of thislaboratorytopreciselymapB- andT-cellepitopes on the VSG molecule that are involved in temporally protective immune responses.

Previous studies from this laboratory (19) demonstrated that independent regulation ofB-cell responses to surface andsubsurfaceepitopes on the VSG molecule occurs during infection. However, the T-cell requirements of these B-cell responses arepoorly understood. Rigorous determination of T-cellinvolvementin these responses should clearly precede detailed attempts to map potential T-cell epitopes. Earlier studies have shown that athymic nude mice can control T. brucei rhodesiense (1, 9, 14), T. brucei brucei (3), and T. congolense(17) infections butnot infections with unrelated

*Correspondingauthor.

trypanosomes suchas T. musculi(18). Such findings dem-onstrated that thymus-independent (TI) B-cell responses artificially isolated from thymus-dependent (TD) mecha-nisms are sufficient tocontrol relapsing parasitemias; how-ever, theircontributiontotheoverall B-cellresponsein the normal thymus-intact hostis unclear. A quantitative com-parison ofVSG-specific B-cellresponsesin infectedathymic (nulnu) and thymus-intact

(nul+)

mice may directly deter-minetherelative contributions ofTIand TD processestothe temporally

protective

immuneresponse.

The presentstudy addressestwobasic questions concern-ing the regulation of VSG surface epitope-specific B-cell responses.First,whatarethe T-cellrequirementsfor B-cell responsesmountedtoVSGepitopes exposed in their native conformation on viabletrypanosomes duringinfection, and arethey similarafterimmunizationwithpurifiedVSG prep-arations? Knowledge of such basic characteristics of these temporally protective immuneresponsesis of interestin its own

right

and is relevant in the contextof future investiga-tions (above). Second, are the patterns ofTI or TD VSG-specific B-cell responses observed in clonal trypanosome populationsimmutable? That is, arethe generalpatterns of such responses thesamefor each variantpopulationtoarise during infection,or aretheremarkeddifferences that may be attributable to differences in VSG structure or in parasite immunodulatory effects? Such a broader picture of TD B-cell function is relevant to the disease process, because the host must mount successive VSG-specific B-cell re-sponsesto many variantpopulations.

MATERIALS ANDMETHODS

Mice. Athymic nude (nulnu) BALB/c mice and their thymus-intact (nul+) littermates were obtained from the University of Wisconsin Gnotobiotic Laboratory. Micewere age matched (8 to 10 weeks) and weighed 20to 25 gat the onsetof all experiments. Outbred mice, used for expanding cryopreserved trypanosome clonalpopulations (stabilates), were obtained from the University of Wisconsin Charmany Farms mouse colony. All mice were housed in American Association for Accreditation of Laboratory Animal Care-approvedfacilities and were handled according toNational Institutes of Health guidelines.

Infection withtrypanosomes. Frozen stabilates ofT.brucei rhodesiense clones LouTat 1, 1.2, 1.5, 1.8, and 1.9 were thawed and expanded in cyclophosphamide (Cytoxan, 300 mg/kg of body weight; Mead Johnson and Co., Evansville, Ind.)-immunosuppressed outbred mice. When these mice exhibited systemic parasitemias of 2 x 108 to 5 x 108

trypanosomes per ml (typically in 3 to 5 days), they were exsanguinated, and parasiteswerepurifiedfromheparinized blood by DEAE-cellulose chromatography (10). Infections

were subsequently initiated in nude and thymus-intact mice by intraperitoneal injection of 106 washed viable

trypano-somes in 300 ,u of PBS (10 mM P04 [pH 8.0], 150 mM NaCl)-1% glucose.

Immunization of mice. Paraformaldehyde-fixed

trypano-somes used for immunization were prepared by incubation ofpurified live parasites in 1% paraformaldehyde-150 mM NaCl (pH 7.4) for 1 h at 20°C, followed by washing four times in PBS(10 mM P04, 150mMNaCl)atpH7.4. BALB/c nulnuandnul+ mice wereinjected intraperitoneallywithup

to 1010 fixedtrypanosomes. In addition, BALB/c micewere immunized with up to 500 jig of VSG isolated from clone LouTat1, 1.2, 1.5, 1.8, or 1.9 as previouslydescribed (20). Briefly, mice were injected intraperitoneally with 500 ,ug of purified VSG (see below) inPBS andexsanguinated 10days later(see Results). VSG used for immunizationwaspurified from trypanosome clones after distilled water lysis in the

presence of ZnCl2 and phenylmethylsulfonyl fluoride (4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealedasingle Coomassieblue-stained band(Mr, 61,000to 65,000, depending uponthe variant population)when 30 ,ug of protein was loaded per lane. The purified proteins in

approximately 3 ml were dialyzed against two changes of PBS (pH 7.4) (2 liters per 24 h), and theirA280 was deter-minedby using thePBSontheoutside of thedialysis bagas a spectrophotometer blank. Exactly 3,000

RI

of protein solution (1 mg/ml) and the PBS blanks were placed in separate preweighed vessels, lyophilized, and weighed; the difference was assumed to be the weight of the VSG. Extinctioncoefficientswerethencalculatedas

E2nm

(i.e.,

the A280

ofa 10-mg/ml solution)for the LouTat 1 (E= 6.71)and 1.5

(E = 4.67) VSG molecules. These values were used to determinethequantityof VSG forimmunization of BALB/c nude mice.

Titration ofanti-yandanti-,u reagents.Sinceacomparison

ofnulnuand nul+ immune responses was thebasis of this

study,itwas imperative toensurethat the assayprocedure usedwas notsignificantly biased towarddetection of VSG-specific immunoglobulin M (IgM) (i.e., nulnu responses) versus immunoglobulin G (IgG) antibody binding to the surface ofany LouTat clonal parasite populations. To

ad-dressthis,theaffinity-purified125I-labeledrabbitanti-mouse reagent was acarefullytitrated mixture ofanti-,uandanti--y

preparations. This

125I-labeled

reagent was then tested in control

solid-phase

assayswith

affinity-purified IgG

and

IgM

antibodies. The rabbit

anti-y

reagent was made in our

laboratory

by

using

amousenormalgamma

globulin

agarose

affinity

column

(Affi-Gel

10)

and

accordingly

detected

pri-marily IgG; however,

it didpossess to alesserextentsome

anti-,u

activity.

To allow

equivalent

detection of ,uand -y

isotypes,

this reagent was

spiked

with

commercially

avail-able

affinity-purified

rabbit anti-mouse

IgM

(no. 0611-3181;

Cooper

Biomedical).

Various ratios of these two

affinity-purified

reagents

(1:100

to

100:1)

wereiodinated andused in

solid-phase

assays.

Dynatech

ImmulonIIwellswerecoated

separately

with

affinity-purified

mouse

IgG

or

IgM (no.

15381 or

M1520,

respectively;

Sigma

Chemical

Co.,

St.

Louis,

Mo.)

(50

jig/ml

in

PBS)

andwashedthree

times;

5 x 104

cpm

ofthe

rabbit

anti-mouse

reagent

(,u

and-yatvarious

ratios)

in PBS with 0.1% bovine serum albumin was

added,

and

preparations

were allowed to incubate for 2 h at

4°C.

The wellswerethen washedthreetimesin PBSwith0.1% bovine

serum

albumin,

and thebound

radioactivity

wasdetermined

by using

a gamma counter. When the

anti--y

globulin

and

anti-,u

preparations

wereiodinated and usedataratio of40:1

(vol/vol),

the mixturewould bind

18,500

+ 450 cpminwells coated with either

IgG

or

IgM. Furthermore,

inwells coated with 25

p,g

of each

isotype

per

ml,

the titrated

reagent

bound

32,300

+ 750 cpm. All

subsequent

iodinations in the

study

used a 40:1

(vol/vol)

mixture of these

anti--y

globulin

and

anti-,u

reagents.

Thus,

wellscoated with50 ,ugof

IgG

or

IgM

bound

equal

amounts of the titrated

1251I-labeled

reagent.

Furthermore,

Immulon II wellsarecoatedto

nonspecifically

bind all

proteins

equivalently.

These data indicate that

IgG

and

IgM

bind

similarly

ifnot

equally;

however,

the actual amounts bound areunknown.

Quantitation

of

surface-specific

B-cellresponses

by

RIA. A

radioimmunoassay

assay

(RIA)

was

developed

inthis labo-ratory

(19)

thatallowed

quantitation

of

antibody binding

to

exposed

VSG

epitopes

presented

on the surface of live

trypanosomes.

Briefly,

fresh whole serum

samples

from

trypanosome-infected

or immunized BALB/c mice were clarified of

lipoproteins by

dextransulfatetreatment, and the gamma

globulin

fractions were isolated

by

ammonium sul-fate

precipitation

followed

by

dialysis

against

PBS

(20).

These

purified

gamma

globulin

fractions exhibited much lower

background binding

than whole sera when live

try-panosomes were used as

binding

targets.

On the

day

of assay, fresh

parasites

of the relevant variant

type

were isolated from the blood of

immunosuppressed

mice

(10),

suspended

in PBS-1%

glucose

with 20%

heat-inactivated

fetal bovine serum, and

aliquoted

into 96-well

vinyl

assay

plates

(no. 2595; Costar,

Cambridge,

Mass.)

at 5 x 107

parasites

in 50 ,u per well. Then

50-,ul

samples

of various dilutions ofthe

purified

gamma

globulin

preparations

were addedandincubatedat

4°C

for1h with

gentle shaking.

The

plates

were then washed two times

(10

min,

1,500

x

g),

suspended

in 50

,ul

ofPBS-1%

glucose-20%

heat-inactivated fetal bovine serum

(per

well)

with

105

cpm

of

1251I-labeled

rabbit anti-mouse gamma

globulin

(specific

activity,

21

X

109

cpm/mg;

-y and ,u at

40:1),

incubated as

above,

and washed three times. Plateswere

subsequently

cut

apart

into individual

wells,

and bound

radioactivity

wasdetermined

by

using

a gamma counter. The

affinity-purified

rabbit

anti-mouse

antibody

was iodinated with

lodogen

(Pierce

Chem-ical

Co., Rockford, Ill.)

as

previously

described

(20).

Control

wells in each assay included normal gamma

globulin

and immune mouse

variant-specific antibody

preparations.

All

TI AND TD VSG-SPECIFIC B-CELL RESPONSES 2339 values represent the means of triplicate or quadruplicate

values.

RESULTS

Dose responses to paraformaldehyde-fixed trypanosomes and purified VSG. Before comparative studies of nulnu or nul+ mice immunized with purified VSG or paraformalde-hyde-fixedparasites, various control experiments were con-ducted to validate the assay procedures. Initially, a dose-response experiment was done to determine the optimal amounts ofthe immunogens used for maximal stimulation and to ensure that the same dose was equally effective in nulnu and nul+ animals. Three mice (nulnu or nul+) were immunized with 10, 100, or 500 ,ug of purified VSG or with 106, 108, or

1010

paraformaldehyde-fixed trypanosomes, and gamma globulin was prepared 10 days later. Day 10 was chosen based on previous kinetic analyses of surface-spe-cific anti-VSG responses in B1O.BR and C3HeB/FeJ mice (19; see below). These preparations were assayed at a 1:10 dilution (see Materials and Methods) for binding to native surface-exposed VSG epitopes displayed on live LouTat parasites. Immunization with

1010

fixedparasites or with 500 ,ug of purified LouTat 1 VSG consistently elicited maximal activity against surface-specific VSG. Typical results after immunization of nu/+ mice with 10, 100, or 500 pLg of purified LouTat 1 VSG were 1,000 + 250, 1,150 + 210, and

3,250 + 350 cpm bound, respectively. In addition, the binding activity of gamma globulin (diluted 1:10) obtained after immunizationwith

106,

108,or

1010

fixedparasites was 550 + 430, 440+ 375, and 3,410± 330cpm, with background countsofapproximately 1,000 cpm(Fig. 1 and 2). Thus, 500

jig

of purified LouTat 1 VSG and a dose of 1010 fixed trypanosomeswere used to generate the data in Fig. 1. The nulnu mice didnotproduce detectable anti-VSG activity on day 10 after immunization with any of these doses (Fig. 1). To examine further, mu globulin (prepared as above) was harvested after 7 and 14 days and assayed as above; how-ever, as on day 10 no anti-VSG activity was detectable at these times afterimmunization.

Kinetics ofantibodyproduction ininfectednu/nuand nu/+ mice. Before comparative studies of the immune responses tovarious LouTat parasite clones (1, 1.2, 1.5, 1.8, and 1.9), a brief examination of the kinetics of these responses in nulnuversus nul+ mice was conducted. Three mice (nulnu ornul+) wereinfected with 106live LouTat 1 parasites and gamma globulin was prepared 7, 10, or 14 days later and assayed forantibody binding to VSG epitopes exposed on live trypanosomes, as previously described (19) (see Mate-rials and Methods). Binding data revealed similar but not identical kinetics ofantibody production in nulnuandnul+ animals. Although both substrains produced maximal spe-cificantibody concentrations on day 10 and lower levels on day14, thenulnu miceproduced slightlymore VSGsurface epitope-specific antibody on day 7 than did nul+ animals. Typical results from the binding assay for nulnu and

nul+

gamma globulin samples were as follows: day 7, 2,820 ± 475 and2,355 ± 475 cpm; day 10, 3,210 ± 390and 3,370 ± 440 cpm; and day 14, 2,130 ± 475 and 2,270 ± 275 cpm. These bindingvalues andkinetics were similar to thosepreviously measured in B1O.BR and C3HeB/FeJmiceinfected with T. rhodesiense (LouTat 1 or 1.5) and C57BL/6 nulnu mice infected with T. congolense (17, 19). Since both the

nulnu

andnul+ miceproduced peak serumconcentrationsofVSG surfaceepitope-specificantibody 10 dayspostinfection,this time pointwaschosen for all comparative studies (Fig. 1 and

o 3.0-z 0 CL2.0-0 1.0 -12 3 4 1 2 3 4 1 2 3 4 1 2 3 4

-Log [SeraDilution]

FIG. 1. Quantitation of TI andTD B-cell responses specificfor native VSG surface-exposed epitopes. BALB/c athymic (nulnu) or thymus-intact (nul+) littermate mice were infected or immunized (three mice pertreatment) with T. bruceirhodesiensecloneLouTat 1, and gamma globulin fractions were derived from day 10pooled serumsamples. Dilutions of thesepreparationswereincubated with freshly isolated viable trypanosomes, which were subsequently washed and incubated with an affinity-purified '25I-labeled rabbit anti-mouseimmunoglobulinreagent(,u and-yspecific). The parasites were thenwashed, and bound radioactivitywasdeterminedwith a gamma counter (see Materials and Methods). (A) Background binding by normal gamma globulinbeforeinfection; (B)aggregateTI and TD binding activity of day 10 gamma globulin derived from BALB/c nulnu (TI) or nul+ (TI and TD) mice infected with 106 LouTat 1 trypanosomes; (C) exclusive TD binding displayed by gamma globulin derived from mice immunized with 500 p.g of purifiedVSG; (D) exclusive TD binding by gamma globulinderived from mice immunized with 1010paraformaldehyde-fixed LouTat 1 parasites.

2). In addition, at day 10, both the nulnu and nul+ animals have cleared the first systemic parasitemia and the number of trypanosomes per milliliter of blood is relatively low (<105 cells per ml) (5, 6, 9). Thus day 10 postinfection represented a valid time point to harvest VSG surface epitope-specific antibody; both nu/nu and nu/+ mice pro-duced peakconcentrationsinserum, and parasitemias were at an equivalentand low level.

Nude mouseB-cell responsesto trypanosome VSG. BALB/c nude (nulnu) mice or their thymus-intact (nul+)littermates were infected with106liveparasites or were immunized with purified VSG (500 ,ug) or paraformaldehyde-fixed trypano-somes

(1010

cells). Ten days after immunization or infection, sera were collected and then analyzed for the presence of antibody binding to native exposed VSG epitopes displayed on the surface of the live parasites. The kinetics of the BALB/c nul+ response after immunization or infection was examined, and day 10 was found to represent the maximal concentrations in serum, whereas the nulnu mice did not respond to immunization (see above). An RIA employing freshly isolatedlive trypanosomes as binding targets, which selectively detects only surface specific B-cell responses (and not buried VSG epitope specific responses), was used to assaygamma globulin preparations derived from the nude andthymus-intactmice(19). Figure 1 shows the RIA results

7.0 z

06.0-:L

5.0

D

0 4.0-3.0 2.0 1.0 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

-Log[Sera Dilution]

FIG. 2. Differential VSG surface epitope-specific B-cell

re-sponseselicitedduring activeinfection with clonal populations of

LouTat trypanosomes. Groups of BALB/c athymic (nulnu) or

thymus-intact (nul+) littermate mice (three mice per treatment)

wereinfected (106parasites) withoneof fourclonalpopulations of

LouTat parasites (1.2, 1.5, 1.8, 1.9), which had been isolated

previouslyatvarious times(10to50days)frommicewith relapsing

parasitemias afteraLouTat1-initiatedinfection. Thegamma

glob-ulin preparations derived from the nulnu andnul+ mice infected

with LouTat 1.2, 1.5, or1.9trypanosomes alldisplayed significant

bindingtotherespective VSGsurface-exposedepitopes. However,

infection with LouTat 1.8 elicited specific binding activity exclu-sivelyinnul+ animals. Thus,aggregateB-cellresponsestoLouTat

1.2, 1.5, and 1.9 surface VSG epitopes resulted from significant

contributions by TI and TD mechanisms, whereas LouTat 1.8

infection selectively elicitedTDresponses. The RIA allowing

sep-aratequantitationofTI(nulnu) andTDplus TI(nul+) VSG surface epitope-specific B-cell responses during trypanosome infection is described in the legend to Fig. 1. The dashed lines represent the bindingby pooledgammaglobulin preparationsobtained from each

treatmentgroupbeforetrypanosome infection.

from LouTat 1-infected or immunized mice. The athymic

and thymus-intact mice were able to mount VSG-specific B-cell responses to exposed epitopes after infection with LouTat 1(Fig.1B).Thenul+ mice madesomewhat stronger anti-VSG responses when infected, as indicated by the higher binding at the 10-2 serum dilution. Whether this

represents a higher concentration of antibody or higher affinity is not clear from these data, although the similar slopes of theselineswould indicatethat theseVSG-specific antibodypopulations have similar overallaffinities but differ in concentration.

The data also show that both TI and TD processes

contribute toresponses madeto the native conformational form ofthe VSG surface coatduring active infection. This

canbe seenbycomparison of thecurves withinFig. 1B. At the higher dilutions (e.g., 10-2) the TI sera showed no significant surface specific binding, whereas the infected nul+ mouse sera displayed maximal binding. This binding

representsthe TD enhancementof the nativeVSG surface-specific B-cellresponses, since the contribution byTI

anti-body binding has been diluted to background levels. Thus theTIandTD compartmentsof thisresponseeach

indepen-dently display maximal

binding

in the RIA

used,

and there-fore both

probably

represent

significant

componentsofthe total native VSG surface-specificresponseininfectedmice. Further dissection and quantitation of the TI and TD re-sponsescould notbe madefromthese

data;

however,

sera

obtained from LouTat 1-infected nul+ mice

(Fig. 1B)

con-sistently displayed VSG surface

epitope-specific

binding

activity equivalent to that ofnulnu mouse sera

assayed

at

10-foldhigher concentrations.Thus, the enhancementofthe native VSG surface

epitope-specific

response in LouTat 1-infected mice by the presenceofTcells may accountfor the production of the

majority

of this

specific

antibody

population.

Finally,

it should be noted that the

higher

titer of

antibody

in LouTat 1-infectednul+ mice

(relative

tothatof nulnu

mice)

was

consistently

observed

during

infection with other LouTat1 serodeme variant

antigenic

types

(Fig. 2).

When the

athymic

nulnu and

thymus-intact

nul+ animals were immunizedwithpurified VSG (500

,ug)

or

paraformal-dehyde-fixedtrypanosomes

(1010

cells),

ananti-VSG surface epitope-specificresponsewasdetected

only

in thenul+mice (Fig. 1C and D). The

day

10 gamma

globulin

preparations

from the immunized nude mice showed no

binding activity

greater thanthatofthe normalserum controls

(Fig.

2A)

to the surface oflive LouTat 1 trypanosomes. Immunization with various doses ofthese

antigens

also failed to elicit a

surface

epitope-specific

immuneresponsetoviableLouTat 1

parasites

in nudemice. In contrast,

purified

VSG 1 prepara-tions or fixed LouTat 1

parasites

elicited VSG

surface-specific

responsesin the

thymus-intact

nul+littermatemice. These nul+ TD B-cell responses were weaker than those triggered

by

LouTat 1

infection,

as shown

by

the lack of

significant

binding

in the

higher

immunizedserum dilutions

(>10-2;

Fig.

1Cand

D)

as

compared

with the infectedserum

binding profiles

(Fig. 1B).

Thus,

purified

VSG

(500

,ug per mouse) or fixed trypanosome

preparations

(1010

cells per mouse) elicited TD native VSG surface

epitope-specific

B-cellresponses,albeitless

strongly

than

composite

(TI

plus

TD) responses elicited

during

infection. It wouldtherefore seemthat thesemolecules presentatleastasubsetofthose VSG

surface-specific

epitopes

displayed

by

thelive LouTat 1

parasites.

Differential B-cell responses to LouTat 1 serodeme variant antigenictypes in nude mice. To further examinethe TI and TD components of the aggregate VSG surface

epitope-specific

B-cell responses, BALB/c nulnu and nul+ mice were infected with four additional cloned variant

antigenic

types(VATs)of the LouTat 1serodeme.

Figure

2shows the VSG

surface-specific

antibody

binding

resultsobtainedfrom RIA

analyses

of

day

10 serum

samples

from mice infected with clone LouTat

1.2,

1.5, 1.8,

or 1.9. These additional T. brucei rhodesiense clones

displayed

immune response kinet-ics identical to that of LouTat 1

(see Discussion).

As observed with LouTat 1

(Fig. 1),

infection with these addi-tional VATs

triggered

more

vigorous

VSG

surface-specific

B-cell responses in nul+ mice than in the

athymic

nulnu mice. A

single

exception

was observed with LouTat 1.9-infected mice at alow serum dilution

(i.e., 10-1).

Analyses

of sera obtained from LouTat 1.8-infected nulnu mice re-vealed that this T. brucei rhodesiense clone didnotelicitaTI VSG surface

epitope-specific

B-cell response in nude mice. However, the infected

thymus-intact

nul+ littermate mice did

produce

antibody

toVSG surface

epitopes displayed

on liveLouTat1.8

parasites.

Repeated

analyses

of nulnumouse

demon-TI AND TD VSG-SPECIFIC B-CELL RESPONSES 2341 strateVSG surface epitope-specific responses. Thus,

infec-tion with this particular cloneofLouTat parasitesselectively elicited only TD VSG surface-specific responses. Overall these data indicate that during the course of trypanosome infection TI and TD VSG surface epitope-specific B-cell responses occur to different trypanosomes (i.e., different VATs of the same serodeme: LouTat 1, 1.2, 1.5, and 1.9). However,there may beexceptions when onlyTDresponses are made to exposed VSG epitopes (i.e., as with LouTat 1.8). The frequency of this phenomenon was one of five VATs in this study. However, only five VATs were exam-ined.

DISCUSSION

The purpose ofthese studies was to examine the T-cell dependency of B-cell responses, specific for exposed epitopes of the VSG, that occur during active T. brucei rhodesiense infection. In addition, in these studies we ex-aminedB-cell responsestoVSG epitopes after presentation of murine hosts with alternate forms of VSG (i.e., purified solubleVSG and VSGdisplayedonparaformaldehyde-fixed parasites). An RIA capable ofselectively quantitating VSG surface epitope-specific antibody responses was used to analyzegamma globulinpreparations derived from trypano-some-infected orVSG-immunized BALB/c nulnu and nul+ mice. Bothsubstrainsof mice wereinfected; gamma globulin was prepared after 7, 10, or 14 days and subjected to RIA analysis. Acomparison of the kinetics and maximal specific antibody concentrations in sera (see Results) revealed that these responses in nulnu andnul+ mice were similar, with both substrains reaching maximal concentrations in serum onday10. Inaddition, theaffinity-purified125I-labeled rabbit anti-mouse gammaglobulin reagent was a carefully titrated mixture of anti--y and anti-,u antibodies (40:1), which de-tected IgGandIgMatapproximately similarconcentrations (basedon IgG and IgM standards; see Materials and Meth-ods). Finally, the lownumbers oftrypanosomes in infected nulnu andnul+ mice werealsoequivalentatday 10; thefirst systemic parasitemiawascleared by this timepostinfection. This experimental design allowed an examination of the

biologically

relevant protective VSG-specific B-cell re-sponses that occur in vivo during active infection. Results obtained were thus not complicated by binding of B-cell products to buried or sequestered VSG epitopes not nor-mally displayed on the VSG surface coat of live

parasites;

the role ofthesenonprotective responses is not well under-stood, and they may be associated primarily with immuno-pathological reactions observed during infection (11, 22).

Overall,theresultsdemonstratefor the first time thatVSG surface epitope-specific B-cell responses in infected hosts represent aggregate TI and TD responses, whereas the immunization procedures selectively triggered TD VSG sur-face-specific responses (see below). The infected athymic nulnu mice clearly made only TI surface VSG epitope-specific responses, whereas nul+ mice produced much stronger responses in the presence of T cells (Fig. 1; see Results). This difference in binding may be due to the production of higher concentrations of nul+ specific anti-bodiesratherthanhigher-affinity antibodiesasevidencedby the similar slope of the lines in Fig.

1B.

Similar patterns (within error) of B-cellresponseswere also observedduring infection with other VATs of the LouTat 1 serodeme (Fig. 2). Thus, in the presence of T cells a significantly stronger VSG surface epitope-specific responsewas mounted during active infection with LouTat 1, 1.2, 1.5, 1.8, and 1.9.

Whether this T-cell augmentation has as its basis classical MHCrestricted and

antigen-processing

cell-dependent

stim-ulation of VSG-specific T-helper cells is clearly not ad-dressed by data presented here and is

currently

under investigation.

The T-cell-independent component of the VSG surface epitope-specific B-cellresponse was influenced bythe mode of VSGpresentation (ortheparticular

infecting

trypanosome clone, see below) to the host. In contrast, the TD anti-surface responses were invariably observedregardless ofthe mode ofpresentation; immunization with purified VSG or paraformaldehyde-fixed trypanosomes triggered surface epitope-specific B-cell responses only in the thymus-intact nul+ mice. The athymic nulnu littermates did not develop surface-specific responses underthesecircumstances. Since the athymic mice did not respond, these particular immuni-zation procedures must selectively stimulate TD responses. Such results probably indicate that specific responses in nul+ immunized animals result from classical major histo-compatibility complex (MHC) restrictedprocessing and pre-sentation of monomeric VSG molecule determinants to VSG-specific TH cells. These responses would not be ex-pected to occur in the nulnu animals. In addition, the immunized nul+ mouse anti-surface VSG responses were significantly weaker than those elicited during infection (as compared with responses ofnul+ infected mice; Fig. 1B),as evidenced by the lack of binding at the higher serum dilu-tions. Thus, purified VSG molecules or paraformaldehyde-fixed trypanosomes must expose or present only certain of the surface epitopes and trigger only TD VSG surface epitope-specific B-cell responses. Indeed, the weak nature of the anti-surface B-cell responses elicited by the fixed para-sites may indicate that this immunogen poorly presents native surface epitopes or presents only a small fraction of all epitopes, displayed on the live trypanosome surface VSG. However, the distinction between infection and im-munizationis not trivial; parasite-derived factors may influ-enceimmune responsiveness in many ways.

The infected host surface VSG epitope-specific B-cell responsesclearlyrepresent aggregate TI and TD responses, and both of these mechanisms are responsible for the pro-duction of a significant fraction of the specific antibody produced. An exception to this general trend was noted during RIA analysis of gammaglobulin obtained from Lou-Tat1.8-infectedmice. Infection with this particular trypano-some clone resulted in the production of VSG surface epitope-specific antibody exclusively by TD mechanisms. This was shown by theinability of the LouTat 1.8-infected nulnu mice to produce any anti-VSG 1.8 antibodies that reacted with exposedepitopes (Fig. 2C). These data collec-tively demonstrate that TI and TD VSG surface epitope-specific B-cell responses are made to most (four of five) trypanosome variants that arise during relapsing para-sitemias initiated by LouTat 1; however, growth of certain clones (i.e., LouTat 1.8) may result in the production of VSG-specificantibodies by TDmechanisms alone. Whether this phenomenon results from some significant conforma-tional alteration in the surface coat, lack of an appropriate second signal to B cells, parasite-specific immunosuppres-sion, or "holes" in the TIB-cell repertoire currently is under investigation. Finally, differences in the course ofinfection in thenulnuand

nu/+

mice may beimportant also.

Other studies involving trypanosome infection of nude mice (6, 14, 21) orB-cellresponses to surface VSG epitopes (2, 6, 13, 15, 16, 22) have not directly examined the T-cell dependencyoftheseimmune responses.Various experimen-VOL.58,1990

tal systems and species of trypanosomes have been used, making comparisons difficult or inconclusive; however, the data presented here are in general agreement with previous studies. For example, athymic (nulnu) and thymus-intact (normal) C57BL/6 mice infected with T. congolense pro-duced similar peak surface VSG epitope-specific anti-bodytiters when examined in a complement-mediated lysis assay (17). Further, the kinetics of these responses were significantly different; thymus-intact mice made peak re-sponses earlier (day 6) and maintained these levels longer (through day 14). These careful studies also showed that the assay of Pinder et al. was much more sensitive to the mu immunoglobulinisotype (the only isotype produced by nulnu mice), owing to its highly efficient rate of complement fixation. Although this assay is difficult to directly compare with the present study, and since species differences may exist (T. brucei rhodesiense versus T. congolense), it was clear the T. congolense-specific B-cell responses to surface VSG epitopes were modulated by the presence of T cells in the thymus-intact mice. Thus, these immune responses probably also represent aggregate TI and TD responses. Finally,studies from this laboratory (19; C. M. Theodos and J. M. Mansfield, J. Immunol., in press; C. M. Theodos, D. M. Reinitz, and J. M. Mansfield, J. Immunol., in press) and others (2, 15, 16) have examined monoclonal or poly-clonal antibody binding to parasite VSG. In general, these experiments were not intended to examine the role of T cells, yet they have indirectly shown such involvementby induction of hybridoma VSG-specific IgG antibodies. Thus, B-cellresponses specific forVSG surface-exposed epitopes on the LouTat serodeme of T. brucei rhodesiense, and probablyother trypanosome species and subspecies, repre-sentaggregate TI and TD processes. Inasmuch as there was little datadirectly addressing such T-cell dependency,more basicinformationwasneededbeforeT-cellepitope mapping studies ofthe VSG molecule. The present study provides some of thisnecessary information, whichserves as abase for our current investigations of B- and T-cell epitopes detectable on the VSG molecule and the means by which immune responses to them are regulated (Theodos and Mansfield, in press; Theodos et al., inpress).

ACKNOWLEDGMENTS

This workwassupported by Public Health ServicegrantAI-22441 from theNational Institutes of HealthtoJohnM.Mansfield andby Public Health Service postdoctoral award AI-07876 from the Na-tional Institutes of HealthtoDavid M.Reinitz.

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