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.
1Despite 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
2or 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,
3as well as functional
immunolo-gical parameters such as NK function,
4antibody
dependent cell-mediated cytotoxicity,
5PHA-respons-iveness
6·
7and cytotoxic Τ cell responses to
allo-antigens
8were 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
9or when mature donor Τ cells have been
removed from the BM inoculum,
10donor 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·
12or in peripheral blood of
individuals
13·
14suffering 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'
16By contrast, in a recent study in humans,
the presence of
recipient-specific CTLs in PBL, as opposed to recipient recipient-specific
Th cells,
17proved to be insufficient and not required
for GVHD pathogenesis after HLA-identical BMT.
18As 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.
19We 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
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)
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 donorDays 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*
1However, 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
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 2ZSccondly, 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
23Secondly, 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 DFurthermore, the anti recipient
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)
References
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8 TILKIN AF, BAGOT M, DREYFUS F et al Recon stitution of cytotoxic Τ cell activity following allogeneic bone marrow transplantaiton m man Γ/αηιρΙαΜαΙιοη 1987,44 303-307
9 BEATTY PG, HERVE Ρ Immunogcnetic faclors lele vant to acute GVHD In Grajt versus Host Disease Immunology, Paihophysiology and Treatmutt Buiakoft SJ, Deeg HJ Ferrara J Atkmson Κ (eds) Dekker, New York, 1989, pp 415-423
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