T-cell and natural killer cell development in thymectomized Xenopus

John D. HortonTrudy L HortonRobin DzialoIan GravenorRalph MinterPamela RitchieLanier GartlandMartin D, WatsonMax D, Cooper

T-ceU and natural killer celldevelopmentin thymectomized Xenopus

Autfiors' addresses

Jo!in Horton'.Trudy Horton', Robin Dzio!o', !on Gravenor̂

Ra!p!i Minter', Pamela Ritchti;', Lanier Gari!and^

Martin IVutson'. Max Cooper̂

'Department of Biological Sciences. University

of Durham. Durham, UK.

'Division of Developmental and Clinieal

Immunology and the Howard Hughes Medical

Institute at the University of Alabama at

Birmingham. Birmingham. Alabama, USA.

Correspondence to:

Jolin D. Horton

Department of Bio!ogieal Sciences

University of Durham

South Road

Durham DHl 3LE

UK

Fax: 44 1913 742417

e-mail: j,[email protected],uk

Acknowledgements

This research was supported by project grants

from the Leverhulme Trust (JDH) and the

University of Durham (JDH and MDW) and

dirough BBSRC studentsliips (IG and RM). MDC

is a Howard Hughes Medica! institute

investigator. We are indebted to

Dr. Louis Du Pasquier for his generosity in

supplying molectilar probes for identifying

Xenopus T ceUs and for the Xenoptis tumour eel! !ines

used in our NK stndies.

Immunohgical Reviews 1998

Vol. 166; 245-258Primed in Denmark. All rights reserved

Copyriglit © Mtinksgaard 1998

Immunological ReviewsISSNOIOS-2896

Stjmmary: The Xenopus ear!y-thymectomy model system is used to investi-gate the extent to which the thymus controls T-cell development and toprobe the evolution of natural killer (NK) ceils. Loss of T-cell function fol-lowing thymectomy, together with the paucity of cells expressing mono-clonal antibody-defined T-cel! surface markers, and greatly reducedexpression of T-cell receptor p transcripts in spleen, Hver and intestine,indicate that T-cell development is minimal in the absence ofthe thymus.Our findings therefore mitigate against the idea tbat a substantial extrathy-mic pathway of T-cell development exists in early vertebrate evolution.Rather, they suggest that in this amphibian representative T cells are pre-dominately thymus dependent. In vitro studies with control and thymecto-mized Xenopus splenocytes reveal that a non-T/non-B population and alsotwo T-cell subsets all display natural cytotoxicity towards aUogeneic thy-mus lymphoid tumour cells (which are deficient in MHC antigen expres-sion). Since Xenopus thymectomized early in larval development are perma-nently deficient in T cells, tbey may provide a useful phylogenetic modelfor the study of NK cells.

Introduction

The immune system ofthe clawed frog, Xenopus, has been char-acterised in depth (1). These and other phylogenetic studieshave helped confirm a high degree of similarity hetweenImmune systems of all jawed vertehrates and have heen usefulin identifying novel immune system components of likelyimportance in mammals. One such example of this is the recentdiscovery of a developmentally regulated type-I transmem-brane protein, called CTX, on cortical thymocytes in Xenopus(2). Gene homologues for CTX, a receptor believed to play animportant role in thymocyte development, have since beenidentified in birds (3), mice and humans (L. Du Pasquier, per-sonal communication). likewise, studies on fish have indicatedevolutionary conservation of natural killer (NK)-cell receptors:thus catfish non-specific cytotoxic cells (NCC) and human NK-cell killing are hlocked by monoclonal antibodies (mAbs)against a vimentin-like molecule on catfish NCC (4).

Xenopus offers an advantage over mammalian model systemsfor investigating the role of the thymus in immune systemdevelopment in that it is possihle to remove the Xenopus thymus

245

Horton et al • T-cell and NK development in Tx Xenopus

at a rudimentary stage of T-cell development (5-7), The first

part of this review describes experiments using the Xenopus

early-thymectomy model to investigate the extent to which the

thymus controls T-cell development. We then focus attention

on candidate NK cells in control and thymectomised (Tx) Xeno-

pus in a search revealing that several populations of natural cyto-

toxic cells exist at this level of evolution.

Effect of thymectomy on T-cell development

Background

Although the thymus plays a special role in T-cell development,the possibility of a substantial extrathymic T-cell maturationalpathway is still a topical, controversial issue. Considerable evi-dence exists for some degree of extrathymic T-cell develop-ment, in which the intestine of euthymic rodents is thought toplay a major role (8-12), Thus in mice it has been suggestedthat gut endoderm attracts lymphoid precursors and inducesthese to undergo dt novo T-cell development within the intestinalepithelium (the so called intraepithelial lymphocytes (IELs))(10, 12), Evidence for such extrathymic development includesthe finding of recombination-activating gene-1 (Rag-1) tran-scripts and DNA deletion circles (containing T-cell receptor(TCR) VDJ genes) within IELs, the Utter frequently expressingthe phenotype TCRY5/CD8aa or TCRap/CD8aa (8, 10, 13).However, other evidence indicates that most IELs in eutbymicmice are in fact derived from the thymus, or are dependent onthymus-derived factors (14, 15). Evidence that the liver isanother site of extratbymic T-ceil development emanates fromstudies characterising TCR molecules on liver lymphocytes(16-18), especially during early ontogeny, prior to tbymusdevelopment (19, 20). Involvement of tbe bone marrowmicroenvironment in supporting the differentiation of T-cellprecursors into CD4'̂ and CD8^ T cells has also recently beenreported (21). Altbough tbe emergence of some lympbocytesexpressing TCR's in congenitally athymic rodents is cited as evi-dence for extratbymic T-cell maturation (22), sucb T-celldevelopment could well be promoted by vestigial tbymus tissuefound in nude mice (23, 24). Significant extrathymic develop-ment of IELs appears unlikely in sheep, since thymectomy intitero is followed by persistent depletion of intestinal lympho-cytes (25). In birds, a comprehensive search has failed to pro-vide evidence for either TCRaP or TCRyS extratbymic T cells(26-29).

The suggestion that extra thymic T cells occupy an inter-mediate pbylogenetic position between NK ceUs and thymus-derived T cells (30) and might reflect a more primitive patb-way of T-ceil development (3!) led us to search for thymus-

independent T-cell development at the amphibian level of ver-

tebrate evolution. In Xenopus, tbe tbymic anlage can be easily and

completely removed between 5 and 7 days of development (6),

At 5 days of age (stage 47), the tbymus comprises <500 cells

(32), including epithelial cells of tbe tbymic anlage and a few

lymphoid precursors, which began to enter the anlage during

the fourth day of larval development (33). Even by 7 days (stage

48), when thymocyte nimibers have reached approximately

4,000, fewer than 20% of these cells express tbe 120 kDa Xenopus

T-lympbocyte antigen (XTLA-1) (34, 35) and CD8 expression

is not yet identifiable (36). Tbe earliness of tbe thymectomy

operation, coupled with the fact that thymectoniized Xenoptis are

free of both tbe complex genetic defects inherent in nude

(athymic) rodents and direct maternal influence on tbe devel-

oping embryo, provide an unambiguous test model for assess-

ing tbe role of tbe thymus in supplying T cells to the rest of the

body. The question of whether or not T cells can be generated

in athymic Xmopus can now be more directly assessed witb the

acquisition of reliable markers for Xenopus T cells. Monoclonal

antibodies (mAbs) have been generated against Xenopus CD5

(37) and CD8 (38, 39) homologues, and other mAbs identify

less well characterised T-cell epitopes (34, 38). Additionally,

tbe Xenopus TCRp chain gene has recently been cloned (40).

Initial characterisation of lymphocyte development

in thymectomised Xenopus

Tbe effect of early (5-7 day) thymectomy on lymphocytedevelopment was initially examined in tbe spleen, a majorperipberal lymphoid tissue in Xenopus, which lacks lymphnodes. Altbough die overall growth of control and Tx Xenopuslaevis siblings over the 14-montb experimental period was com-parable (Fig. I), splenocyte numbers were reduced in Tx ani-mals to approximately 3 0-5 0% of control levels in all four agegroups examined (Table 1). Tbe athymic frogs routinely remainin excellent bealth at least until 9 months of age. However, wehave noted incidents of wasting or subcutaneous bydrops insome batches of older frogs. Both of these conditions, whichare of unknown aetiology, were more frequent in Tx animalstban in control siblings. Early tbymectomy carried out inanotber laboratory (41) indicated tbat most Tx Xenopus frogletsbecame infected by nematodes (Capillaria), and the 'flaky skin'disease caused by tbis parasite was corrected by tbymusimplantation. Tbus while most athymic Xenopus survive remark-ably well, lack of a thymus can lead to serious immunologicdeficiencies and susceptibility to certain pathogens.

When splenocytes were analysed for T and B-cell surfacemarkers by flow cytometry (Fig, 2), the mean percentage ofcells expressing the pan-T-celi marker CD5 was found to grad-

246 Immimoiogicol Reviews 166/ J 998


Fig. 1. The Xenopus thymectomy model system.A. At S weeks of age the paired, piginentedthymus gland (T) is readily visible through thetransparent skin of the dorsal pharyngealregion of a control larva, lying jusl posterior tothe eyes. B. Note absence of thymus in the 5-week-old larva thai had been thymectomized(Tx) ai 5 days of age. prior to T-lymphocytedevelopment. Blood vessels (red/black incolour) and nerves (white) are seen traversingthe pharyngeal region, the black abdominalregions can be seen posteriorly. Followingmetamorphosis al 7-8 weeks, conirol (1C) andTx (ID) siblings (here 6 months old) continueto display comparable growth rates, despiteabsence of bonu fide T cells. Absence ofthymus inTx frogs is confirmed by dissection andhisiological analysis of the ihymic regions.Magnification: x4 (A, B); xO.7 (CD).

ually increase with age in normal (control) X. laevjs. from 28%

in one-month-old larvae (age group I) to 62% at 10-14months of age (group IV). (Xenopus B cells do not constitutivelyexpress CD5 (37).) The percentage of CD8* splenocytes like-wise increased from 11% in larvae to 27% in 10—14-month-old adults. Approximately 20% larval and 35% adult spleencells from controls expressed the XTLA-1 antigen. In the Txanimals, however, XTLA-1*, CD5^ and CD8-̂ (F17mAb) sple-nocytes were virtually undetectable at all ages (Fig. 2; data notshown), in keeping with an earlier study revealing loss ofXTLA-1 + cells from diverse lymphoid tissue sites of early-TxXenopus (35). In contrast, the B-cell percentages were consis-tently higher in Tx animals, as expected from the initial char-

acterisation of B cells and Ig levels in such frogs (42). Typical

staining patterns for splenocytes from 10-14-month control

and Tx frogs are shown in Fig, 3.

Search for T cells in diverse lymphoid tissues following

th/mectomy

The 10—14-month-old control and early-Tx X. luevis were alsoanalysed for T-cell development in the liver and intestine - sitesof putative extra thymic T-cell development in mammals. Lym-phocyte numbers obtained from Xenopus liver and gut variedappreciably from one preparation to another, making reliablecomparison of absolute numbers of these cells in control andTx frogs impractical. Mean percentages of mAb-stained lym-

Immunologicdl Reviews 166/1998 247

HHorton et a! • T-cell and NK development in Tx Xenopus

CD5*

III IV I II III IV

Age groups

III IV

Fig, 2. Ontogeny of T and B cells in the spleens of control andthymectomised Xenopus. Mean percentages of sp!enocytes (±SD) stainingfor CD5, CDS, or IgM are shown in control (•) and thymectomized ( o)XenopQs kevis. Exp!anation of age groups I-JV, the number of experimentsperformed oti each group, and the mean absolute splenoeyte numbers inthese animals are shown in Table I, Splenocytes, prepared as describedelsewhere (5!), were washed in amphibian strength PBS (APBScontaining 0.1% NaN; and 0,1% BSA) before incubation with either F17(anti-CD8) (38) or D8 (anti-IgM) (38) primary mAb, After washing, thecells were incubated with FITC-labelled, rabbit anti-mouse

immunoglobuiin, adsorbed with 5% Xenopus serum. Cells were thenwashed with AFBS contait:iing 1% normal mouse sertim and counter-stained with PE-labelled, 2B1 mAb (anti-CD5) (37), The control mouseantibody for indirect FITC stainings was CT3, an anti-chicken CD3 (87);the phycoerythrin control was mouse IgG conjugated to phycoerythrin.Five to ten thousand lymphocytes were analysed using Becton DickinsonFACScan or Coulter XL flow cytometers. Gates were set by forward andside scatter to exclude erythrocytes, granulocytes and dead cells fromanalysis, the latter being confirmed by propidium iodide staining.

phocytes in spleen, liver and gut (intestine) of control and Tx

froglets are given in Table 2, Representative staining patterns are

shown in Fig, 4,

Mean percentages of CD5+ lymphocytes recorded in

spleens of control frogs (70%) were similar to those found in

gut (75%) and liver (59%). AbouE half of these Tcells co-

expressed CD8, although the proportion of intestinal Tcells

expressing the CD8 epitope recognised by mAb AM22 tended

to be somewhat higher. The levels of splenic and hepatic lym-

phocytes staining with the D4 mAb, which identifies an 80 kDa

homodimer on Xenopus Tcells (38), were consistently just

below the total percentage of CD5+ T cells. In contrast, dual

Table 1. Effect of thymectomy on splenoeyte development Table 2. Percentages of mAh-stained lymphocytes in spleen, liver andgut of control and thymectomised Xenopus

Splenoeyte numbers' at different ages

IV

(1 month) {2-5 months) (fr-9 months) (10-14 months)

Control

Tx0.3

0.1

5.4±3.5

2,4±1.5

13,7±5.8

5.7±4.0

22.4±15,6

12.6±10.8

'Values represent mean cell numbers+1 standard deviation (xiO'), At 1 montb(age group I), 4-8 larval spleens were pooled; thereafter 5-7 individual spleenswere examined at each time point. Thymectomy was performed at 5-7 days of ageand animals reared at 23°C,

Control

Tx

{±S.D.) of mAb-stained cells

Organ

Spleen

Liver

Gut

Spleen

Liver

Gut

2B1

70±13

59 ±375+3

6±3

21+11

32+10

AM22

33±9

31 ±4

49±8

5±3

20±8

27+7

F17

33±7

3O±3

35+8

2±0

4±2

5±5

D4

68±13

56±3

17±11

2±0

5±4

4+3

l^arkers were set to exclude from positive analyses 98% of cells stained witb con-trol mouse antibody. Mean percentages of lymphocytes (± standard deviation)expressing CD5 (mAb 2B1), CD8 (mAbs AM22 & F17), and the 80 kDa epitoperecognised by mAb D4 (specificity unknown} are given, Spleen, liver and gut wereexamined from 5 animals (10-14 months old) in each experimental group. Thymec-tomy performed at 5-7 days of age,

248 Immtmological Reviews !66 / !998


CONTROL

35% 29% 52%

: ,1

''••'W.. . , n.Tif

40%

Fig. 3. Representative flow cytometricanalysis of mAb stained splenocytes fromcontrol and thymectomised adult Xenopus,Splenocytes from 1 -year-old X. laevis werestained sequentially with anti-CD8 (FITC) andthen anti-CD5 (PE) mAhs or anti-IgM (FITC)followed by anti-CD5 (PE) and surfacefluorescence evaluated by flow cytometry. as inFig. 2. Percentages of CD5'CDS"lymphocytes(putative helper T cells), CD5*CD8+lymphocytes (cytotoxic T cells), and CD5-IgM+ B cells are indicated. Quadrants set toexclude 98% cells stained with control reagentsfrom positive analysis.

THYMECTOMIZED

:

<1%

' 1 •

(1%

' ' 'J ' • ' • • " •

84%

CD8

colour flow cytometry revealed that a significant population of

CD 5+ T cells in the gut were D4".

In Tx Xenopus, the epitopes recognised hy the Fl 7 anti-CD8

mAb and the D4 mAh were not found on splenocytes, and were

often undetectable in liver and gut. However, splenocytes

expressing low levels of CD5 and the AM22 anti-CD8 epitopes

were relatively abundant in the Tx frogs. (The AM22 mAb may

detect a CD8a epitope and the F17 mAb may detect a CDBp

epitope. but this has not yet been proven.) Approximately 30%

of the lymphocytes from liver and intestine of these Tx frogs

were CDS'" and AM22i", but none expressed the distinctly

higher fluorescence intensities of these markers typical of con-

trol T cells (Fig. 4). Compared with controls, elevated propor-

tions of IgM'̂ B cells were found in the spleen, liver and gut of

these Tx frogs. The XT-1 mAb was not used in these studies,

because we have recently found the XTLA-1 antigen is not iden-

tifiable in some batches of control X. lams (43).

Histological analysis of the spleen from 1-year-old control

frogs indicated that CDS''T cells were concentrated in the zone

near the boundary region of the white and red pulp. CD5+ cells

were also scattered throughout the white pulp follicles

(Fig. 5A), which are rich in IgM+ B lymphocytes (44, 45). The

distrihution of the CD8+ (AM22+) subpopulation of cells was

similar to that of the total population of CD5+ T lymphocytes.

Splenic CDS+ and CD8+ cells were not identifiable in splenic

sections from Tx siblings (Fig. SB). CD5+and CD8+ (AM22+)

lymphocytes were found throughout the intestine of control

Xenopus (Figs SC & SD), both in the lamina propria, where nod-

ular lymphoid tissue and more diffuse lymphocyte collections

are seen, and in the intestinal epithelium (the IELs), near the

hasement memhrane. These CDS+ and CDS"'lymphocytes typ-

ically displayed a circumferential "rim" pattern of staining. In

contrast, the CD5'". CD8''' cells identified by FACS in the intes-

tine of Tx frogs were not histologically visihle, even with

hiotin-avidin enhancement of the mAb staining (Fig. SE).

Search for TCR transcripts following thymectomy

The most stringent evidence for the existence of T cells would

he the detection of TCR gene rearrangement and expression.

Anti-Xenoptis TCR antibodies are not yet available, but complete

TCRp cDNA clones have recently been isolated and sequenced

(40), This information was used to design consensus Vp and Cp

ImmunoJogico] Reviews 166/1998 249


CONTROL

SPLEEN

CD5

CD8(AM22)

77

38

D4

35

75

THYMECTOMIZED

SPLEEN

CD5

CDS(AM 22)

CDS(F17)

D4

LIVER GUT

LIVER

31

31

GUT

' - '''

1 A

42

25

1

"- -

6

Fig. 4. Representative flow cytometric analysisof mAb-stained lymphocytes from spleen,liver and gut of control and thymectomisedadnlt Xenopus. Lymphocytes were prepared asdescribed elsewhere from the spleen (51), hver(43) and intestine (88) of individual i-year-oldX, laevis and stained separately with each of thefour mAbs shown. Markers set to exclude 98%cells stained with control reagents from positiveanalysis. Traces representative of five separateexperiments, for which mean data is shown inTable 2,

region primers tbat would detect transcripts in cells tbat hadundergone VDJp rearrangement. When splenic and intestinallympbocytes from 1-year-old control frogs were analysed forthe presence of TCRp transcripts by a reverse transcription poly-merase chain reaction (RT-PCR) and subsequent Soutbern blotanalysis, fully rearranged TCRp transcripts were readilydetected. In contrast, our initial efforts to detect TCR|3 trans-cripts in splenic lympbocytes from tbree pairs of early-Tx sib-lings were unsuccessful (Fig, 6), altbough sucb transcripts weredetectable at relatively low levels in intestinal lympbocytes.Transcripts for IgM and elongation factor la were detectedequally in Tx and control frogs.

Subsequent titration of the number of cells used to isolatethe RNA from which tbe cDNA template was reverse trans-cribed revealed tbat approximately 10̂ lymphocytes from hverand intestine of control Xenopus were minimally required todetect TCRp transcripts; somewhat greater numbers (6x10^)were needed from the spleen. Tbis semi-quantitative PCRproved sufficiently sensitive to detect low levels of TCRp tran-scripts in intestine, liver and spleen from eight additional Txanimals studied. This PCR analysis indicated that more than twolog higher numbers of cells were required to detect TCRP tran-

scripts in all three lympboid tissues from Tx frogs (Fig, 7),

Given tbe prolonged opportunity for clonal expansion in 1-

year-old animals, tbis constitutes powerful evidence for mini-

ma! T-cell development in Xenopus thymectomised between 5

and 7 days of larval development.

Functional deficits in Tx Xenopus

Previous analyses bave shown that early-Tx Xenopus develop acrippled tbymus-dependent immune system, as judged byimpaired allo reactivity in vivo (S, 46) and in vitro (47), loss ofhelper activity for antibody responses to thymus-dependentantigens (48), and inability of lymphocytes to respond toT-cell mitogen stimulation by enhanced proliferation (47, 49,50) and release of lympbokines (51). Nevertheless, tbe possi-bihty that certain residual "T-cell-like" functions (e,g, chronicallograft rejection (52) and low level T-cell mitogen reactivity(53)) can develop in some early-Tx froglets bas also been noted

(7).Recent functional studies of 1 -year-old control and Tx X.

loevis fail to provide evidence for extra tbymic T-cell maturation.Tbese experiments (54) revealed that MF€C-disparate skingrafts from X, luevis/X. mudleri (LM3) clonal Xenopus were rejected

250 Immunologica! Reviews 166/1998

Horton et al • T<ell and NK development in Tx Xenopus

-^•^•'•'if i " w i i * ^* ' •*-• A - ; • ? : ' • ^

Fig. 5. Imraunohistochemical analysis ofspleen and gut sections from control andthymectomised Xenopus adults. Four to six-micromelrc cryostat sections from spleen andintestine of 1-year-old X. loevis were fixed inacetotie and incubated with anti-CDS (A-C) oranti-CD8 (AM22) mAbs (D-E).ImmunosUining employed a biotin-avidin-peroxida.se detection technique (36). A. Spleenfrom control showing CD.S+ cells in red pulp(RP) margitial zone, jusl outside white pulp(WP), which also conuins a few scatteredT cells. B. Thymectomised spleen showingabsence of CDS*" cells. Arrows in A and B pointto bntmdary layer which separates white 2ndred pulp, and whicb frequently displaysperoxidase reaction product even in controlCT3-sUined sections. C . D. Intestine fromcontrol Xenopus stained for CDS* (C) and CD8*(D) intraepithelial lymphocytes (IEL). Nodularcollections (arrowed) of lymphocytes in laminapropria also express these antigens. E. Intestinefrom thymectomised animal showing absenceof identifiable CDS* lymphocytes. Ceils of theintestine are stained heavily with methyl greencounterstain, but no specific peroxidasereaction product is found in the thymectomisedanimal. L = ltnnen of gut. Data representativefrom five separate experiments. Magnification:X200.

by control recipients in <3 weeks, whereas grafts persisted in

perfect condition for >100 days on all Tx recipients. Immuno-histochemical analysis illustrated that LM3 grafts on controlsbecame heavily infiltrated by CD5"̂ and CD8+ lymphocytes,whereas MHC-disparate grafts on Tx animals remained devoidof such cells. In vitro-cultured splenocytes from Tx siblings ofthe grafted animals proved unreactive to the T-cell mitogenconcanavalin A (ConA), whereas in keeping with the higherproportion of B cells, splenocytes from Tx frogs responded bet-ter (higher incorporation of ^H-thymidine (TdR)) to Escherichiacoli lipopolysaccharide (LPS) than did splenocytes from con-trols. Mitogen reactivity of lymphocytes from 1 -year-old Xenopusliver and intestine have also been analysed. CeUs were culturedfor 3 days with ConA, or che pan-lymphocyte stimulator, phor-bol myristate acetate (PMA). and examined for mitogen-induced 'H-TdR uptake (Fig. 8). ConA was highly stimulatoryfor lymphocytes extracted from spleen, liver and gut from con-

trols, whereas cells from all lymphoid tissues from Tx frogs

failed to respond. The viability of cultured lymphocytes from

spleen, hver and intestine of Tx frogs is demonstrated by their

ability to proliferate in response to PMA stimtilation.

Effect of thymectomy; summary

This analysis of the Immune system in Xenopus thymectomizedas 5-7 day larvae indicates the virtual ahsence of identifiableT cells, greatly reduced expression of TCRp transcripts, and lossof detectable T-cell function. Our study thus provides evidencethat T-cell development is minimal in the absence of the thy-mus in this amphibian representative. On the other hand, theappearance of CD5'". AM22'" cells (which may representCD8aK+ cells) in the liver and gut, together with the low levelsof TCR|3 transcripts in lymphoid cells of Tx frogs, indicates avery limited developmeni of cells with "T-like" feattires inthese athymic animals. It seems likely that these latter cells have

Immunologitfll Reviews i 66/1998 251


Spleen Intestine

normal Tx normal Tx

Fij. 6. Analysis of TCRp, IgM and elongation factor la transcripts insplenic and intestinal lymphocytes from control and thymectomizedXenopus adults. Total RNA was obtained from splenic and intestinallymphocytes from one pair of thymectomised (Tx) X. loevis (I -year-old)and one pair of normal (control) siblings. First strand cDNA template wassynthesised using oligo dT. PCR amplification followed witholigonucleotide primers corresponding to TCRp chain framework region2, position 79-99, 5' AAC ATG TTC TGG TAC CAA CAG 3' and a reverseprimer complementing constant region position 742—762, S' AGC CGTGAC AAT GAG CCC ATA 3', Primers amphfying portions of CM at position569-589, 5' TAT AAA CCT GTG ATG CAA TCT 3' in exon 1 and reverseposition 740-760, 5' GGATGG AGG GTG AAT AGA CAC 3' in exon 2, Thesequence of a 5' primer in exon 4, position 675—695 of elongation factor1 a is CCA TGG TTT AAG GGA TGG AAA and its 3' partner in exon 5,position 890-91 0 is AGT TGT TAC ATT AAC AGG GGC. Cyclingparameters were as follows: 1 minat 94°C, 1 minat S8°C, 30 minat 72°Cfor 30 cycles followed by a 10 mine extension at 72°, For Southern blotof the PCR products, I Ofil of a SOpl PCR volume were electrophoresed ona 4% Nusieve GTG agarose gel. The resultant gel was Southern blotted tonylon aiid UV cross-linked. Oligonucleotide probes corresponding toposition 670-690 of TCR Cp, 690-710 of C|j, and 795-815 of EF la wereS' end labelled with ^'P 7ATP and hybridized to the membrane at 42° in50% formamide. The membrane was washed to a stringency of 45° inO.IXSSC, 0,1% SDS,

emerged extra tbymicalty, but it is conceivable that they devel-oped from cells influenced by the tbymus for the very briefperiod prior to larval thymectomy The question of whethersome yS T-hneage cells can develop in Tx Xenopus also remainsunanswered. The Y§ T-cell receptor genes also arose early in ver-tebrate evolution, baving been described togetber with aP TCRgenes in cartilaginous fish (55), birds and mammals, althoughnot yet defined in Xenoptjs, Tbe severe impairment of T-celldevelopment demonstrated in Tx Xenopus nevertheless mitigatesagainst a substantial extrathymic pathway of T-cell develop-ment eariy in vertebrate evolution.

Search for candidate NK cells in thymectomized Xenopus

Baci<ground

Mammahan NK cells are large granular lymphocytes distrib-uted in diverse lympboid tissues. Tbese cells, wbich can be dis-tinguished from T cells by their lack of rearranging TCRs,exhibit innate, non-antigen-specific defence against certaintumour targets and virally infected cells, especially those tbatare deficient in MHC class I protein expression (5 6). Such aber-rant targets cannot be dealt with by cytotoxic T lymphocytes(CTL), whose clonally distributed antigen receptors are MHICrestricted. Tbe reahsation that NK cells provide crucial immunedefence, together witb increasing awareness that these cells areimportant regulators of adaptive immunity (57), has lead to awealth of research attempting to defme the molecular basis ofantigen receptors and target ligands involved in the activationand inhibition of mamtnahan NK cells (58-62), NK-cell inhib-itory receptors in mouse and man are beginning to be charac-terised in considerable deptb and it is now realised that the twodifferent structural types of inhibitory receptor existed beforethe divergence of primates and rodents (61).

Since NK cells are believed to be the evolutionary forerun-ners of CTL (63), studies on lower vertebrates may reveal novelanimal models to explore crucial issues concerning NK-cellbiology For example, the identification of cytoplasmic CD3proteins and botb surface CDS and interleukin (IL)-2 receptorson cbicken NK cells (64) support the notion (65) that T cellsand NK cells derive from a common progenitor. As mentionedin the introduction, studies on catfish indicate the conservationof NK-cell antigen receptors (4). Heterogeneous populationsof non-specific cytotoxic cells (NCC) bave recently beendescribed in catfisb, where separable peripberal blood leuco-cyte populations from unimmunised animals are spontane-ously cytotoxic to eitber allogeneic or virally infected targets(66), Cytotoxicity mediated by these two populations is notinhibited by an anti-NCC mAb (4, 66), vv̂ hereas tbe xenoreac-tive NCC cells extracted from catfisb kidney are inhibited bytbis mAb, Distinct populations of alloreactive and xenoreactivenon-specific cytotoxic cells have also been observed in tumour-bearing damselfisb (67), Altbougb kiUing by fisb cytotoxiccells frequently displays lack of antigen specificity, the unavail-ability of either T-cell-depleted fish or appropriate anti-fishT-cel! antibodies (68) precludes documentation as to whethersuch killing represents diverse NK-like populations rather thanT cells.

Amongst amphibians, NK activity against xenogeneictumour targets and viruses is suspected in several anuran spe-cies and its role in the very effective tumour immunity at thisevolutionary level has been discussed (69). Our ongoing

252 Immunological Reviews 166/1998


10-'

10

Thymectomised

Normal

Fig. 7. Approximate mean (± SD) numbers ofcells needed to detect TCRp transcripts indiverse lymphoid tissues of control andthymectomized adult Xenopus. Numbers oflymphocytes from spleen, liver and intesdne(from 1 -year-old X. lams) used lo extract RNAwere titrated [o allow a semi-quantitacive RT-PCR, based on methodology given in Fig. 6.Data from eight separate experiments, each onindividual normal (control) and 5-6-day-chymectomised Xenopos.

Spleen Liver

50,000

•

•

Medium

ConA

PMA

Spleen Liver Gut Spleen Liver Gut

fig. 8. Mitogen responsiveness of lymphocytesfrom control and thymectomised adnltXenopus. Graph shows mean DPM ^H-TdRincorporation for lymphocytes from spleen, liverand gut of individual control and chymectomizedX. laais aged 12 months, stimulated in vitro with2.5 fig ml-1 concanavalin A (ConA) or lOngmh'phorbol myristace acetate (PMA). or cultured inmedium alone. Lymphocytes were cultured at27°C for 48 h, before being pulsed with ^H-TdR18-20 h prior to harvesting and liquidscintillation counting. Data are representative ofthree separate experiments.

Control Thymectomised

research is attempting to identify candidate NK cells in Tx Xeno-

pus.

Xenopus splenocytes exhibit NK-like activity

The remarkable capacity of early-Tx Xenopus to survive in the

absence of bona fide T cells (see above) attests to the importance

of otber elements of host defence in these frogs. Altbough

powerful antibiotic magainins in Xenopus skin (70) may be

essential in this respect, it seems likely that NK cells play an

important role in tbe survival of Tx Xenopus. Our initial experi-

ments (71, 71) revealed that splenoeyte effectors taken directly

from Tx (1-year-old) X. laevis, but not from control sibhngs.

showed natural lytic activity towards allogeneic thymus

tumour-derived lymphoid cells (6-h ^'Cr release assay). (Thy-

mus tumour-derived lymphoid cell lines, such as the B3B7 cells

from homozygous Xeiiopus laevis family ff, are appropriate targets

for candidate NK cells, since they are deficient in class I

(class Ia) and II MHC antigen expression (73),) In contrast,

some Tx batches of Xenopus tested directly ex vivo fail to lyse allo-

tumour targets (71). Such variation in natural cytotoxicity may

well relate to differences in natural antigenic challenge in vivo

and associated cytokine modulation. In this respect lytic capac-

ity towards allogeneic tumour cells can be readily induced in

control Xenopus and elevated in Tx frogs by a single injection of

Immunological Reviews 166/1998 253


tumour cells, with maximal splenocyte cytotoxicity occurring3 days post-injection (71). These investigations also revealedthat splenocytes, even those from tumour-injected frogs, wereunable to lyse allogeneic splenic lymphohlasts or erythrocytetargets, even when the latter are coated with IgY (the XenopusIgG equivalent); moreover, we were unable to demonstrate anysplenocyte-induced lysis of the hmnan NK-cell target K562.

Lymphokine-activated natural c/totoxicity

Lymphokine-activated killing (LAK) in Xenopus is suggested (71)by the ohservation that when splenocytes from 1-year-old Txfrogs were cultured for 48 h in growth factor-rich medium(GFM) from ConA-stimulated cells {probably containing IL-2-like material (74)), their capacity to kill allogeneic tumour tar-get cells was significantly enhanced (Fig, 9). Although thiscytokine-indnced cytotoxicity is reminiscent of LAK activity,the question of whether culture in GFM induces splenocytesfrom Tx frogs to kill a wide array of target types was not exam-ined. It would be interesting to observe whether prolongedincubation of Xenopus splenocytes in GFM (>48 hours) results inthe increased promiscuity of target cell killing that is typical ofmammalian LAK cells (75). LAK activity was first suspected inXenopus splenocytes from frogs injected with the iridovirus FV3,which, after restimulation in vilro in the presence of T-cellgrowth factor-rich supernatant, exhibited non-specific cyto-toxicity (76). The potential of GFM to stimulate NK-like activ-ity in Tx splenocytes is consistent with previous studies, wheresupernatants from activated T cells were shown to induceenhanced tritiated TdR incorporation by Ig" splenocytes fromTx Xenopus (51). In hnmans, a NK-cell prohferation-inducingfactor distinct from IL-2 has recently heen identified (7 7),

Splenocytes from control Xenopus are also induced to displayanti-tumour cytotoxicity following culture in tumour medium,but this was not dependent on addition of GFM (Fig. 9). Itseems likely (see below) that T lymphocytes in the controlspleen, hy responding to elements in the culture medium,achieve effective activation of NK-like cells, without the needfor addition of exogenous T-cell-derived cytokines. In theabsence of splenic T cells, such exogenous cytokines are crucialfor in vitro activation of NK-like cells.

Cellular basis of NK-like activityMagnetic cell sorting of mAb-labelled T and B cells has recentlyheen used to examine the cellular basis of Xenopus splenocytecytotoxicity towards allogeneic thymns tumour-derived lym-phoid cells, flow cytometry being employed to confirm thepurity of spleen subpopulations (78). Splenocytes from controlfrogs, depleted of T cells and then cultured for 48 hours.

behaved hke unsorted Tx splenocytes, in that they became allo-cytotoxic effectors, but only after culture in GFM. Sorting withanti-Ig mAbs revealed that purified B cells from hoth controland Tx Xenopus, even after culture in GFM, failed to kill allotu-mour targets, whereas B-cell-depleted splenocytes from thesesame frogs hecame effective killers (Fig, 10). These experimentstherefore indicate that non-T/non-B splenic leucocytes inunimmunised control and Tx Xenopus can display natural lyticactivity towards allogeneic tumour cell targets and representNK candidates, (The viahility of purified B cells produced hyimmunomagnetic sorting was shown hy their ability torespond to the B-cell mitogen, LPS (72).)

Experiments employing magnetic cell sorting have alsoindicated that cytotoxicity against tumour cells (but not alloge-neic lymphoblast targets) is mediated by CD5+ T cells fromXenopus spleen following culture in GPM (Fig. 10), This findingthat T cells from non-immunised, outbred X. laevis can lyse allo-geneic B3B7 tumour cells is of interest, since in vitro cytotoxicityof allogeneic splenic lymphoblasts by CTL requires prior Jn vivoimmunisation with allogeneic lymphocytes or skin grafts, andrestimulation of splenocytes in vitro for 5 days (79). Onr obser-vation that cultured T cells can kill allogeneic tumour cells isconsistent with recent findings from early thymectomy exper-iments (80), indicating that T cells play an important role inthe rejection of ff strain thymus tumour cells implanted intominor histocompatibility antigen-disparate ff Xenopus hosts.

The question of which T-cell subsets in unimmunisedcontrol Xenopus are capable of lysing allothymus tumour targetsfollowing cell cnlture has been investigated through sequentialmagnetic sorting (78); this procedure obtained cytotoxicT cells (CD8+CD5+), putative helper T cells (CD8-CD5+), anda non-T-cell subset (CD8- CD5-). The CD8- lymphocytesbecame effective killers after a 48-h culture in GFM (Fig, 11), afinding that was not entirely unexpected in view of the knownabihty of mammahan GTL to become NK-like after IL-2 treat-ment. More surprising was the allocytotoxicity exhibited bythe CD8- CD5+ T-cell population cultured in GFM. (There isno anti-CD4 reagent available to identify the Xenopus CD4+T cells,) This population achieved higher levels of tumour celllysis than the enriched CD8+T-cell (and T-cell-depleted) sub-populations. In mammals, CD4-̂ CTL clones exist that can killtargets relatively quickly, probably through binding to thetumour necrosis factor receptor family memher Fas (81, 82).Interestingly, the presence of Fas has recently been demon-strated on apoptosing Xenopus lymphocytes (L, N. Ruben, per-sonal communication).

254 Immimologica! Reviews 166/1998


Control

10:1 20:1 40:1

E:T ratio

10:1 20:1

E:T ratio

40:1

Fig. 9. Elevated cytotoxicity towards BjB?lymphoid tumour targets by splenocytes fromcontrol and thymectomised adult XenopusfoUowing in vitro culture. Splenocytes from one-year-old control and thymectomised (Tx) X. laeviswere tested directly ex vivo (0 hrs) and then againafter 48 h in tumour culture medium alone (med)or in culture medium supplemented with 25%growth factor-rich medium (GFM) generatedfrom ConA-stimulated cells. Data show meanpercentage specific '•'Cr release (±SE) from 5x10*B3B7 targets at various effector: target (E: T) ratiosfollowing 6-h assay. (All cell culture andcytotoxicily assays carried oul at 27°C.) Nineseparate experiments were carried out withspleens from individual control frogs, five withindividual Tx frogs.

u

40

30

20

10

0

-10

40

30 -

20

10

0 +

-10

CD5-ve

5:1 10:1

Ig-ve

20:1

5:1 10:1 20:1E:T ratio

40

30

20 4

10

0

-10

40 -r

30 -

20 -

10

0

-10

CD5+ve

5:1 10:1

Ig+ve

20:1

5:1 10:1

E:T ratio20:1

Fig. 10. Cytotoxicity towards allogeneic B3B7lymphoid tumour targets mediated bysplenocyte suhpopulations from control andthymectomised adult Xenopus. The subsets wereobtained by magnetic bead separation (78) usinganti-CD5 mAb 2B1 to deplete/enrich for T cellsor a 50:50 mix of anti-IgM mAb 8E4 (89) and acocktail of three anti-Xenopus Ig light chain mAbs(90) to deplete/enrich for B cells. The mAb-coated splenocytes were then incubated withMACS anti-mouse Ig paramagnetic microbeadsand populations enriched (>95%) or depleted(<2%) for T or B cells were collected by magneticcell sorting. The sorted cells were cultured for 48hours in medium alone or in GFM and thenincubated with BjB, targets at various effector:target (E:T) ratios for a 6-h ^'Cr release assay. Topgraphs sbow mean data for T-cell-depletedsplenocytes (CD5-) and purified T cells (CD5+)from four control animals. Mean data for B-cell-depleted splenocytes (Ig-) and purified B cells(Ig+) from three 1 -year-old control frogs areshown in tbe middle grapbs, and from threesibhng Tx frogs in the bottom graphs. Meanspecific 5'Cr release ±SE from 5x10* targets isshown.

U

_ Tx Ig-ve

50 -

40

30

20

10 +

0

-10

5:1 10:1

E:T ratio20:1

60

50

40

30

20

10

0

-10

Tx Ig+ve

Medium

GFM

5:1 10:1 20:1

E:T ratio



CD8 +ve (CD5+ve) CDS -ve (CD5+ve) CD8 -ve (CD5-ve)

20:1 20:1 20:1

Fig. 11, Cytotoxicity towards allogeneic BjB, lymphoid tumour cellsmediated by two T-cell subsets and a non-T-splenocyte popnlationfrom adult Xenopus. Splenocytes from a control, 1 -year-old frog wereinitially incubated with mAb AM2 2 to label CD8+cells and a subset highlyenriched (98%) for CD8 collected by magnetic separation (78). The CD8-cells were then incubated with mAb 2B1 (anti-CD5) and again

magnetically .sorted to produce highly enriched (99%) CD5+ (CD8") andalso CD5" CDS populations. Each of these three populations was culturedfor 48 h in medium alone or in growth factor-rich medium (GFM — seetext) and then tested for tytotoxicity against B1B7 targets al variouseffector: target (E:T) ratios in 6-h ^'Cr release assay. Mean data from threeanimals show specific '̂ Cr release ±SE from 5 x i 0̂ targets.

Ontogeny of NK-like activity

Our initial ontogenetic studies of NK-like activity revealed that

splenocytes from 6—7-week-old (stage 56/57) X. laevis larvae

were unable to display cytotoxicity towards B3B7 cells, even

after 2 days in vitro culture in GFM (78), These in vitro findings

are consistent with in vivo experiments in which Xenopus larvae,

in contrast to young adults, were unable to destroy minor H

antigen-disparate tymphoid tumour cells (73, 80, 83). Even

though in vivo reactivity towards MHC-disparate tumour cells by

tbe larval immune system has been described (73), our in vitro

studies indicate chac components of such larval aiiocytotoxicity

remain defective.

The lack of NK-like activity in Xenopus larvae is not unex-

pected, given tbat MHC class I antigen expression is essential

for inhibition of NK ceils (59, 60) and tbat MHG class Ia anti-

gens are not detectable on Xenopus cells prior to metamorphosis

(84). If NK cells were functional in the MHC class I-negative

Stage, larvae might risk auto immtrne destruction!

Xenopus NK cells: summary and future directions

The above experiments employing adult Xenopus splenocyte

populations indicate tbat a non-T/non-B population and two

T-cell subsets all display natural cytotoxicity towards allogeneic

lymphoid tumour cells, following a 48-h culture in growth fac-

tor-rich medium; such natural cytotoxicity is not evident when

larval splenocytes were similarly tested. Early-Tx Xenopus, with

their dearth of T cells, may provide a useful phylogenetic

model for generating and screening of mAbs against the non-

T-cytotoxic population, that is likely to include candidate NK

cells. Tbe generation of mAbs that identify Xmopus candidate NK

cells, using both hybridoma and phage display (85. 86) tech-

nologies, is a major current focus. Such mAbs will allow us to

explore lymphoid tissue sites where NK-cell development is

initiated, wbich will include the gnt, since this site is a major

reservoir of NK-Hke cells in birds (64). If novel receptors are

identified on Xenopus NK candidates witb the anti-Xenoptis mAbs

generated, conservation of tbese receptors on mammalian NK

cells will be sought.

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T-cell and natural killer cell development in thymectomized Xenopus