High frequencies of cytotoxic T cell precursors against minor Histocompatibility antigens after HLA-identical BMT. Absence of corrleation with Graft-versus-Host-Disease.

Full text

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Bone Marrow Transplantation

1993, 11: 363-368

© Macmillan Press Ltd, 1993

High frequencies of cytotoxic Τ cell precursors against minor

histocompatibility antigens after HLA-identical BMT: absence of

correlation with GVHD

M. de Bueger, A. Bakker, H. Bontkes, J.J. van Rood & E. Goulmy

Department of Immunohaematology, University Hospital Leiden, Leiden The Netherlands

Summary:

Limiting dilution analysis was used to quantify the

frequency of cytotoxic Τ cell precursors (CTLp) against

minor histocompatibility (H) antigens induced by

HLA-identical BMT. The development of CTLp was

moni-tored serially in ten patients developing either acute

(n = 3), acute and chronic

(n

= 4) or no (n = 3) GVHD.

In blood samples of patients taken shortly after BMT

(<100 days) a high frequency of anti-recipient CTLp

was found (mean 1/3433). With time, this value

de-creased to hecome undetectable (< 1/500 000) beyond

400 days. This occurred also in patients still suffering

from chronic GVHD. In contrast, autologous BMT did

not induce any measurable recipient-reactive CTLp at

any time point after BMT, In the early phase of

reconstitution after BMT the frequency of CTLp against

allo HLA-antigens was measured in the same patients.

The absence of a consistent increase of allo-specific

CTLp indicates that the kinetics of CTLp against host

minor Η antigens does not merely reflect an

overaü

changed cytolytic potential shortly after BMT.

These results indicate that: (1) HLA-identical BMT

induces high frequencies of minor

Η antigen-specific

CTLps detectable in the blood during the early phase of

reconstitution, and (2) the frequency of

recipient-react-ive CTL measured in the peripheral blood is not an

adequate parameter for GVHD. These data therefore

challenge the clinical value of

in vitro

measurement of

recipient-reactive CTLs in the peripheral blood after

HLA-identical sibling BMT.

GVHD is still a major problem in allogeneic BMT,

occurring in 45% of recipients of HLA-genotypically

identical BM.

1

Despite much effort, the effector

raechanisms responsible for the pathogenesis of acute

and chronic GVHD in humans remain obscure. To

elucidate potential effector cells, several investigators

have sought to define

in vitro

parameters measurable in

the peripheral blood of recipients after BMT which

would correlate wilh the incidence and severity of

GVHD. Particularfy, phenotypic markers such as NK

cell number, CD4/CD8 Τ cell ratio

2

or percentage of

Correspondence: Dr E. Goulmy, Department of

Immuno-haematology, University Hospital Leiden, Rijnsburgerweg

10, 2333 AA Leiden, The Netherlands

Received 19 August 1992; accepted29 October 1992

MHC class II + cells,

3

as well as functional

immunolo-gical parameters such as NK function,

4

antibody

dependent cell-mediated cytotoxicity,

5

PHA-respons-iveness

6

·

7

and cytotoxic Τ cell responses to

allo-antigens

8

were found not to discriminate between

patients suffering from acute GVHD and those who did

not. As GVHD does not occur after BMT performed in

the absence of minor histocompatibility (H)

differ-ences

9

or when mature donor Τ cells have been

removed from the BM inoculum,

10

donor Τ cells

specific for recipient minor Η antigens have also been

considered as potential GVHD effector cells. Initial

studies demonstrated the presence of recipient-specific

CTLs in spleens of mice

11

·

12

or in peripheral blood of

individuals

13

·

14

suffering from GVHD after MHC

identical BMT, and uniformly implicated their role as

effector cells in GVHD. Subsequent murine studies

using BM inocula depleted of the Th or CTL

popula-tion confirmed the role of minor Η antigen specific

CTLs in the induction of GVHD, although in some

murine strains the Th cell population also appeared

essential.

15

'

16

By contrast, in a recent study in humans,

the presence of

recipient-specific CTLs in PBL, as opposed to recipient recipient-specific

Th cells,

17

proved to be insufficient and not required

for GVHD pathogenesis after HLA-identical BMT.

18

As in the latter study, host-antigen specific CTL lines

were generated by repeated

in vitro

restimulation, the

latter results only provided qualitative Information on

the presence of minor Η antigen-specific CTLs. In a

previous report we found that minor Η antigen-specific

CTLs could be quantified in PBL of a primed individual

using a primary 7 days limiting dilution (LD) assay.

19

We therefore have set out, using a LD assay, to

quantify the anti-minor CTL response at several time

points after minor H-disparate BMT in ten patients.

Our aim was to ascertain whether the frequency of

recipient-reactive cytotoxic Τ cell precursors measured

in the blood would be a sufficient parameter for the

incidence of GVHD after HLA-genotypically identical

BMT.

Patients and methods

Patients

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autologous BMT was also included As conditioning patients recerved Cy (60 mg/kg/day χ 2) and total body Irradiation (8 Gy) To prevent acute GVHD all patients were given MTX dunng the first 3 months except patient 9 who recerved CsA Acute GVHD was treared with prednisonc or methyl prednisone chronic GVHD was treated with prednisone and/or azathiopnne Relevant informdtion on the patients is summanzed in Table I

Blood samples

Hepannized blood samples were collected from recipi ents before and penodically after transplantation from their BM donors and from unrelated HLA typed individuals PBL were isolated by Ficoll-Isopaque denslly grddient centnfugation and stored in liquid nitrogen to be used as responder cells in hmiting dilution assays

Limiting dilution analysiv (LDA)

Seven concentranons of responder cells (RC) (lange 312-40000/well) were set up in round bottom microtiter plates in the presence of nradiatcd stimula tor cells (SC) in 200 μ\ of RPMI 1640 supplemented with 15% pooled human serum dnd 20 U/ml rIL 2 Responder cells were either PBL of the BM donors of the BM reupients at several dates after BMT and of unrelated HLA mismatched individuals Stimulator cells were Epstein-Barr Virus (EBV) transformed Β ceil lmes (5 χ 103/we)l 5000 cGy) of the BM recipient

before BMT or of an unrelated HLA typed mdividual Fiom cach dilution 24 wells were set up After 5 days 100 μ\ containing 20 U/ml rIL 2 was refieshed and at ddy 7 the wells were assayed for cytotoxicity towards ΙΟ4 ΡΗΑ Τ cell blasts of the original stimulator in a

Standard 5I Cr release assay EBV Β cell lmes were

nevor used as target cells to exelude as contribution of EBV antigen sptcific CTLp to the value measured

Cultures were considered positive when showmg lysis exceeding mean spontaneous release (24 wells contain ing no RC) plus 3 SDS LD welis never contamed any nonspecific cytotoxicity of autologous (= responder) PHA bla&ts

Statisticai analysis

Frequencies of responding CTLp 95% confidence intervals and goodness of fit were calculated using the mmimum chi squared Maximum likehhood and Jack mfe methods z( All expenments shown had a ρ > 0 05

for lineanty21 mdicating Single hit kinetics Frequencies

are referred to as high (>l/5000) low (1/100000-1/ 500000)orundetectable(<l/500000)

Results

Absence of correlation between frequencies of CTLp agmnst minor Η atitigens and GVHD after HLA identical BMT

We set out to quantify the anti recipient CTL response tnduced against minor Η antigens by HLA identical BMT and to monitor the kinetics of the anti minor CTLp frequency in time PBL of BM recipients at two to four dates in the first 2 years after transplantation were analysed from three patients suffermg from acute GVHD (Figure 1B) from four who developed addi tional chronic GVHD (Figure IC) and from three patients without any GVHD complications (Figure 1A) (see Table I) The frequency of CTLp in each of these blood samples against recipient specific minor Η anti gens was determined in a pnmary in vitro LD assay usjng EBV Β cells of the recipient before BMT as stimulator cells and testing the responding CTLp for minor Η antigen speafic lysis of the PHA Τ cells of the recipient before BMT All blood sampies of a Single

fable I Clinical Information concerninb the paticnts

No 1 2 3 4 5 6 7 β 9 10 11 At,e 33 42 24 17 36 28 26 39 30 19 /ahenü Sex in Μ Γ Μ F F Μ F F Γ F Μ Sex smatd No Ya Yes No Yts No Yes Ycs No Ycs No" Diagnom ANL· ANL ANL ALL ANL ANL ALL ANL ANL ANL ALL after BMT (llays) >7<Ό ^760 ^760 >760 >76O >76O >760 394 >760 -•760 760 Prophylaxe MTX MTX MTX MTX MTX MTX MTX CsA MTX MTX MTX GVHD Acute GVHDyade 0 0 0 I 11 II II II I I 0 „VHD Day1 33 54 23 18 28 17 21 Chromc GVHD 0 0 0 0 0 0 , 2 1 1 0

ANL acute non lymphoblasticieukemn 'Dayofonsetof acute GVHD

Gr-ideof chronic GVHD (0 nonc 1 hmited 2 extensive)

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OUANTITATION OF ΑΝΤΙ MINOR CTLp< AFTER BMT 365

h pt 1 . pt 2 • p t 3

donor 0 100 200 300 400-700 donor 0 100 200 300 400 700

Pt7 :

+ " - · + •

200 300 400 700 donor

Days after BMT 0 100 200 300 400-700

pient CTLp frequcncy ifler BMT is not affected by GVHD CTLp frcquencies dgdinst recipicnt mmor Η antigens were measured in PBL of unpnmcd donors ind of recipibiit at sever<ii dates ifter B\iT Results are shown of ten patients aftcr HLA ldcntical dllogeneic BMT suffenng from (A) no (B) acute or (C) acute + chronic GVHiD ind (D) one patienl after lutologous BMT Reciprocal values of the frequencics and the 95% confidence intcrvals are indicitcd pt = pitient no

for patient 1Γ whose PBL coniained only 1/35000

CTLp in the first sample available at 85 days (Figure

IC) Between 100 and 200 days after BMT, anti-minor

Η CTLp rould still be detected in PBL of all BM

recipient*

1

However, values found in distmct indi

viduals vaned strongly

(ränge 1/3086-1/110000) and

seemeJ mdependent of the State of GVHD in a given

patie.it (Figure 1A versus

Β versus C) All CTLp

frequencies had umformly declmed to low values

(1/111000-1/200000) between 200 and 400 days Be

yond 400 days CTLp frequencies weie undetectable

(<l/500000) in all patients, including those three

patients still suffenng from mild chionic GVHD at that

point in time (Figuie IC) Only in patient 9, who had

received presensitized BM, was a frequency of 1/80000

found Thus, a high frequency of anti-recipient CTLp

was found shortly after BMT declimng in time to

become low or undetectable beyond 200 days after

Figure 1 Kinetics of the anti recip were measured in PBL

eicBM e frequi

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ΒΜΤ in all ten patients analy&ed nrespective of their

GVHD Status

Although nonspecific cytolytic activity was never

observed in any of the CTLp hmiting dilution cultures

(see Materials and methods) we mcluded an additional

control to exclude the induction of recipient reactive

CTLs being merely due to the process of BMT

irrespective of minor histocompatibiiity differcnces

between donor and recipient As shown in Figure 1D,

histocompdhbihty differences between donor and reci

pient are indeed essential for the development of CTLp

after BMT because no anti recipient CTL precursors

(> 1/500 000) could be detected at any time point in

PBL of a recipient of autologous BMT (patient 1.1)

Frequencies of CTLp against allo HLA antigens are

not affccted by BMT

In 21 blood samples of 7 patients the frequency of allo

HLA antfgen reaciive CTLp was also determined This

was done for two reasons Firstly, the recurrence of

allo reactive CTL activity after BMT as measured in

bulk CTL cultures remains a controversial issue

fi ig 2Z

Sccondly, to exclude the possibihty that the observed

kinetics of minor Η anirgen specific CTLp could rcflect

an overall altcred cytolytic potential after BMT The

CTLp frequencies against allo HLA antigens werc

measured against EBV Β cells of a healthy unrelated

individual who had been selected on the basis of dt

icast four HLA differences with all recipients In

addition in each expenment the CTLp value between

the same two HLA disparate mdividuals was

measured This value served as indicator value for

inter expenmental Variation, known to be high in LD

assays and which in our hands ranged between 1/3500

and 1/9090 in ten expenments (data not shown) The

levels of allo CTLp frequencies vaned between the

distinct donor/recipient pairs, as was expected on the

basis of distinct genetic backgrounds, lmmumzation

history and HLA (Frgure 2) After BMT, m two

patients a considerable reduction of the allo CTLp

frequency was observed in the first blood sample,

whereas dt subsequent dates values resembled those of

the donors before BMT (patients 1 and 8) In one

patient a slight merease in the allo CTLp value was

observed after BMT (patient 2) In the remaining four

donor/rccipient pairs tested, BMT did not significantly

change the frequency of CTLb against allo HLA

antigens (Figure 2 patients 4 6,10 and 11)

Discussion

In PBL of recipients of HLA identical BMT a high

frequency of host reactive CTLs was observed dunng

the first 100 days which dechned in time to become

undetectable beyond 1 year after BMT The kinetics of

anti minor CTLp followed the same trend in all ten

recipients analysed, whether they suffered from moder

ate acute and/or chronic GVHD or not No patients

were analysed who developed severe acute or chronic

*

ü

i.

7

i /

Pt 1

- K P t 2

* Pt4

• -Pt6

X Pt8

••· Pt 10

-TA- Pt V 4

X

no GvHD 1 noGvHD aGvHD aGvHD CGvHD CGvHD

ABMT

Donor BMT 100 200 300 400 Days after BMT

tigurei CTLp frequency against allogeneic HLA antigens is not

systematically influcnccd by BMT CTLp frequencics against an

unrelated HLA disparalc individual were medsured using LDA in

the donor before md the recipients after BMT CfLp vahits are

Kivcn

GVHD Measurement of high frequencies in patients

lacking any signs of acute GVHD and undetectable

values in patients suffermg from chronic GVHD late

after BMT, most clearly indicated that the quantity of

host reactive CTLp in the penpheral blood is not a

predictive parameter for the oecurrence of GVHD

after HLA-identical BMT

This observation can be interpreted in three ways

Firstly, the parameter measured may not mdi^ate a

specific change in the anti-minor CTL population after

BMT but may merely reflect the changing lymphocyte

composition dunng the hematopoietic reconstitution

after BMT

23

Secondly, CTLs recogmzing

recipient-specific mtnor

Η antjgens may not be relevant for the

induction or pathogenesis of CVHD Thirdly, minor

specific CTLs may be involved in GVHD, but the

incorrect in vitro parameter was measured The first

explanation that the high frequency of anti minor

CTLp shortly after BMT fs mereiy due to the rapid

recurrence of CD8

+

Τ cells, is contradicted by the fact

that frequencies of CTLp to other anugens such as

allogeneic HLA antigens as well as to mitogens are

unaffected or even decreased in the first 3 months after

BMT (Figure 2)

6 2 D

Furthermore, the anti recipient

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QTJ ΑΝΤΓΤΑΤΙΟΝ OF ΑΝΊI MINOR CTLps AFTER BMT 367

reinjection of some, but not all, TNFct-producing, noncytolytic Τ cell clones reactive to minor Η antigens resulted in G V H D ,2 4 2e> whereas injection of cytolytic Τ

cell clones specific for other minor Η antigens did not u However, these studies, in fact, do not excJude a

contnbution of CTLs to G V H D pathogenesis, but rather stress that involvement of anti-minor Τ cells in G V H D does not solely depend on their CTL/Th phenotype, but may also depend on their lymphokine production and the minor Η antjgen recognuea

Α third Interpretation is that anti-mmor CTLs can be relevant in G V H D in vivo, however, that the in vitro measurement performed is not a relevant one The blood may not be the appropriate Site for momtonng those CTLs potentialJy relevant in G V H D The ob-served kinetics of recipient-reactive CTLp in PBL could well be attnbuted to an initial penpheral expan-sion as a result of activation by residual host A P C , followed by redistnbution and migration out of the blood into the target tissues Whereas this lymphocyte migration was demonstrated in the rat,2 6 thus far no

systematic functional analysis of lymphocytes tnfiltrat-mg GVHD-damaged oigans has been performed in humans Another argument for measurement of an inappropnate in Vttro parameter may be that those CTLs with antigen specificities relevant in G V H D were not measured The CTL population induced by B M T over a multiple minor Η barrier followed by in vitro boosting using lymphoid cells may, as in the mouse,2 7

recognize only a hmited number of 'dominant' minor Η antigens In a recent m u n n e study lt was questioned whether these minor Η antjgens, dominant in the induction of an in vitro CTL response, are indeed the ones relevant for the mduction of G V H D in vivo 2H In

addition, using a LD protocol with EBV-BLCL as stimulator cells, CTLs reactive with recipient minor Η antigens expressed only on lymphoid cells or on both lymphoid and parenchymal tissues can be detected 2 9

However, CTLs reactive to tissue-specific minor anti-gens expressed only by the parenchymal ceils of G V H D target organs are not detected using this assay, whereas they might strongly contnbutc to the local G V H D phenomena The latter hypothesis would be compatible with the finding that the only two in vitro assays currently available with predictive value for the deve-lopment of anti-minor G V H D , use skin cells as stimulator cells 1 0 3 1 Also, CTLs specifii. for the skin

specific m u n n e minor EPA-1 have been shown to infhct GVHD-hke tissue destruction when mjeeted in vivo λ 2 Should the latter assumption be correct, ι e

should the CTLs recognizing relevant G V H D mmors not have been detected using this assay, then the question anses as to what the in vivo function of the high frequent host-reaetwe CTLs detected in PBL shortly after B M T could be One possibihty would be that these CTLs define minor Η antigens restneted to the hematopoietic Imeage2 9 and therefore could be

selectively involved in graft-versus-leukerma reactiv-ity 1 3 3 4 Whereas no patients suffenng from leukemic

relapse were mcluded in this study, van Eis et al found that within a panel of 16, only the two patients

developing a relapse did not have measurable anti-reci-pient CTLs 1 8

In conclusion, a hmiting dilution method is desenbed for quantitation of minor Η antigen-specific CTL precursors induced by HLA-identical B M T The fre-quency of CTLp against recipient antigens in the penpheral blood is shown not to coirelate with the development of G V H D in vivo These resuits clial-lenge the chnical value of in vitro assays measurmg potential G V H D effector cell populations m the penpheral blood Furthermore, it raises the question of what the host-reactive CTLs present in high frequen-cies shortly after B M T might do in vivo

Acknowledgements

The authors thank Dr F Claas and Dr F Kontng far cnncal reading of the manusenpt This work was supported by the Dutch Health Insurance Society and by the J Α Cohen Institute for radiopathology and radiation protection (1RS)

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