Anti-host cytotoxic T cells of bone marrow transplant recipients with or without Graft-versus-Host-Disease are equally sensitive for cyclosporin A.

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Bone Marrow Transplantation, (1996] 18 73-78

Anti-host cytotoxic Τ cells of bone marrow transplant recipients with

or without graft-versus-host disease are equally sensitive to

cyclosporin Α

LM Liem, JT van Noort and Ε Goulmy

Department of Immunohematology and Blood Bank Leiden Univenity Hospital The Netherlands

Summary:

It is generally accepted that cytotoxic Τ cells (CTLs) play an important role in the pathogenesis of graft-ver-sus-host disease (GVHD). Nonetheless, anti-host CTLs can be observed in the peripheral blood of patients both with and without clinical signs of GVHD following HLA genotypically identical bone marrow transplantation. Thus we questioned whether a qualitative difference, such as a differential in vitro sensitivity to cyclosporin Α (CsA), may exist between the anti-host CTLs, generated from these groups of patients post-bone marrow trans-plantation (BMT). We analyzed anti-host CTL precur-sors (CTLp) of patients without clinical signs of GVHD, of patients suffering from acute GVHD (grade II-IV) and of patients suffering from acute GVHD followed by chronic GVHD for their in vitro sensitivity to the inhibitory effects of CsA. The results obtained in a total of 20 patients revealed anti-host CTLp frequencies (CTLpf) post-BMT in all three groups of patients and addition of CsA in the tests resulted in approximately 60% Inhibition of the CTLpf independent of the GVHD Status of the patients. Thus, in view of the in vitro CsA sensitivity, no difference exists between the anti-host CTLs in patients with or without GVHD.

Keywords: CTL, CsA, LDA, GVHD

The lesults of clinical bone manow transplantation (BMT) show that the selection of MHC identical siblmg bone mar iow donors does not guarantee avoidance of giaft-versus host disease (GVHD) graft failuie or disease-free survival ' It is beheved that dispanties of mmor histocompatibihty antigens (mHag) bt-tween donoi and lecipient constitute a potential nsk factoi for GVHD oi graft failure ^ Ovei the last few yeais, f vidence has accumulated that mHag spec lfic helper Τ cells (Th) could be rele ^ant to the pathogenesis of GVHD In vitro studies reporting on host directed Th cells have been descnbed in patients with GVHD s~1 Van Eis et alk »eported on the long-teim kinetics of Th cells in tesponse to host mHag in 16 patients and dcmonstrated that

sigmficant Th cell activity in vitro correlated with clinical acute GVHD Studies on the phenotype and function of Τ lymphocytes lnfiltrating the skin dunng GVHD following allogeneic HLA-identical BMT revealed CD8 cells,9 but predominantly CD4 cells 1 0' ' Most recent observations support the notion that mHag specific, IL-2 producmg Th cells are hkely to play a role m the pathogenesis of acute G V H D1 2 n

Several reports demonstrated the presence of anti host mHag-specific cytotoxic Τ cells (CTLs) in patients suffering from GVHD aftei HLA genotypically identical BMT I 4 1 9 However, the lole of anti-host CTLs in the development of GVHD is still controversial In our eaiher studies, we demonstiated the presence of anti host mHag-specific CTLs in the blood of patients undergoing GVHD Although patients with chronic GVHD tended to develop highei and more persistent levcls of anti-host CTL activity than thosc without GVHD, this finding was not statistically sigmficant16 Subsequent analyses at the quantitative level, le determination of the anti-host specific CTL precursoi fre-quency (CTLpf) levealed high frequencies of mHag spec-ific CTLs eaily after BMT irrespective of the GVHD Status of the patient 9° In vitro data in suppoit of a role of CTLs in GVHD in man were provided by Kaminski et aVx The fiequency of recipient-reactive CTLp piesent in donor PBL befoie BMT was found to predict the seventy of GVHD after BMT Howevei, the donoi-iecipient pairs in the latter study weie umelated HLA-matched, MLC-nonreactive individuals

These results uiged us to le-analyze the role of the anti host CTLs in the development ot GVHD Eaiher studies in kidney grafting showed that the in vitro resistance of lecipients' lymphocytes foi the immunosuppressive drug cyclosponn Α (CsA) was coirelated with a higher late of giaft loss " We theiefoic set out to investigate whethei we could find diffeiences in the in vitro CsA sensitivity of anti host CTLs in patients with and without GVHD Here we present the lesults obtained in 20 lecipients analyzed at the anti-host CTLp level at different times after HLA geno typically identical BMT

Conespondencc Di Ι Μ Liem Depaitmenl oi Immunohematology and Blood Bank Leiden Univeisity Hospital PO Box 9600 2300 RC Luden The Nethcilands

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Measurement of CsA sensitivity of CTL post-BMT LM Liem et al

74 Material and methods

Patients

Twenty HLA genotypically identical donor-recipient pairs

are included in this study. All except one patient (UPN

11) receivcd non Τ cell-depleted bone marrow. AU patients

received cyclophosphamide (60 mg/kg/day χ 2) and total

body irradiation (8 Gy) as pretransplant conditioning

regi-men, except UPN 9 who received 4 x 50 ng/kg

cyclophos-phamide, 5 x 1 mg/kg busulphan and five treatments with

Campath and UPN 11 who received 4 χ 150 mg

cyclophos-phamide, 4 χ 75 mg busulphan and five treatments with

Campath. Methotrexate (MTX) and/or CsA (and in one

case methylprednisone) was given as GVHD prophylaxis.

The relevant clinical information is summarized in Table

1. Patients are divided into groups according to their

GVHD status. The 'no GVHD' group consists of patients

without GVHD or with acute GVHD grade I (n = 7). The

'acute GVHD' group consists of patients with acute GVHD

grade II-IV (n = 7) and the 'chronic GVHD' group consists

of patients who suffered from chronic GVHD which was

prcceded by acute GVHD grade I or more (n - 6).

Blood samples

Heparinized blood samples were taken from the recipients

before and at regulär intervals after BMT and from their

bone marrow donors. Peripheral blood leukocytes (PBL)

were isolated by Ficoll-amidotrizoate density gradient

cen-trifugation and cryopreserved in liquid nitrogen. Α total of

61 samples post-BMT was used for analysis.

Cyclosporin Α

CsA was a kind gift from Sandoz, Basel, Switzerland. It

was dissolved at 1 mg/ml in ethanol. CsA was used in a

final dilution of 50 ng/ml culture medium, corresponding

to therapeutical levels, and was present during the whole

culture period.

Limiting dilution analysis

Responder cells (RC) were PBL from the donor and PBL

from the recipient at several dates after BMT. Seven

con-centrations of RC (ränge: 40000-625 cells per well) were

set up in round-bottom microtiter plates in the presence of

5000 irradiated (5 Gy) stimulator cells (SC) in a total

vol-ume of 200 μΐ RPMI 1640 medium supplemented with

10% pooled human serum and 20 U/ml rIL-2. Stimulator

cells were Epstein-Barr virus transformed Β cell lines

(EBV-BLCL) of the recipient before BMT or of an

unre-lated individual. EBV-BLCL were used as SC because of

the limited number of PBL of the recipients before BMT.

20

Target cells were PBL stimulated with 1 % PHA for 3 days

and restimulaled with irradiated (3 Gy) feeder cells once

every week for 1 or 2 weeks. In this way a Τ cell line of

each target was made to ensure sufficient target cells. Of

each dilution, 18 wclls were set up in two senes: one series

with and one without CsA in the culture medium. All

responder-stimulator combinations of each patient were set

up simultaneously and tested on the same day in the same

experiment. After 5 days, 100 μ.1 culture medium

contain-ing 20 U/ml rIL-2 was refreshed. At day 7, each well was

tested for cytotoxicity against 5000 Europium-labelled

23

Table 1 Relevant clinical data of the patients included in this study UPN 2 3 4 5 18 16 23 6 20 7 8 9 17 22 10 11 12 13 14 15 Sex (P/D) F/M M/M M/F M/F F/F F/M F/F F/M M/F F/F F/M M/M M/M M/M M/M M/F F/M F/M F/M F/F AML-M4 AML-Ml AML-M2 AML-M3 ALL AML-M4 ALL AML-M4 AA AML-M2 AML-M3 NHL/AML-M4 AML-Ml AML-M4 AML-M2 CML CML AML-M2 AML-M4 AML-M4 Conditioning Cy/TBI Cy/TBI Cy/TBI Cy/TBi Cy/TBI Cy/TBI Cy/TBI Cy/TBI Cy/TBI Cy/TBI Cy/TBI CBE Cy/TBI Cy/TBI Cy/TBI CBC Cy/TBI Cy/TBI Cy/TBI Cy/TBI Piophylüxis MTX MTX MTX MTX MTX CsA CsA + MTX CsA MTX CsA MTX CsA CsA MTX MP CsA CsA MTX MTX CsA aGVHD {day)" 0 0 0 0 0 I (+42) K+8) II (+14) II (+34) II—III (fl2) III (+54) III (+17) III (+11) III (+47) II (+27) II (+37) I—II (+11) IV (+28) II (+35) I (+17) cGVHD 0 0 0 0 0 0 0 0 0 0 0 0 0 0 severe severe mild seveie mild severe Survival (day.\)h ahve ahve ahve ahve ahve ahve ahve +175 +147 ahve ahve +107 +137 +158 ahve +243 ahve +397 alive ahve

UPN = uniquc patient number, P/D = sex oi patient (P) and donor (D), SAA = severe aplastic anemia, ALL = acute lymphoblastic leukemia, AML = acute mycloid leukemia with FAB classification, CML = chronic myeloid leukemia (chronic phase); NHL = non-Hodgkin lymphoma Cy/TBI = cyclophos-phamide and total body Irradiation, CBC = cyclophoscyclophos-phamide, busulphan, Campath and Τ cell depletion, CBE = cyclophoscyclophos-phamide, busulphan and Campath, CsA = cyclosporin A, MTX = methotrexate, MP = methylprednisone.

aDay of onset of acute GVHD

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Measurement of CsA sensitivity of CTL post BMT

LM üem et a/

target cells in the cell-mediated lympholysis (CML) test

EBV BLCL were nevei used as target cells to exclude the

contnbution of EBV antigen specific CTLp to the value

measured20 24 Wells were considered positive, when fluor

escence exceeded the mean spontaneous lelease (18 wells

containing no RC) plus 3 Standard deviations (s d ) 21

Statistical analysis

Frequencies of responding CTLp, their 95% confidence

mteival and the goodness of fit were calculated using the

Jackknifed maximum likehhood method25 The

Mann-Whitney U test was used to compare the effect of CsA in

the different patient groups

Results

Twenty patients were divided into thiee gioups aecording

to then GVHD Status PBLs were isolated before and at

different dates after HLA genotypically identical BMT and

used to determine host directed CTLpt of each patient in

hmiting dilution assays, with and without the addition of

CsA to the test

Figuie 1 demonstrates three repiesentative expenments

of the CTLpf with or without the addition of CsA measuied

in a patient without GVHD (UPN 4), in a patient with acute

GVHD (UPN 8) and in a patient with acute GVHD

fol-lowed by chrome GVHD (UPN 13) Independent of the

oeeunence of GVHD, the post-BMT anti-host CTLpf

inercase from day 30 post BMT onwards, reach maximum

levels around day 100 and decrease gradually thereafter

Figure 1 IS also representative for our gcncral findmg that

the CTLp are equally susceptible foi the addition of CsA in

the eulture medium in the three groups of patients studied

Figuie 2 demonstrates 61 anti host CTLpf measured,

with and without CsA in vitro, in a total of 20 patients

post BMT The mean CTLpf of the 'no GVHD' patients

without addition of CsA in the medium is 17 8/106 PBL

(ränge 0-62/106 PBL) The addition of CsA leads to strong

Inhibition of most CTLpf (% Inhibition = 56%, Table 2),

although two out of seven patients still show a significant

CTLpf in the presence of CsA (>25/106 PBL) The mean

CTLpf in the piesence of CsA is 7 8/106 PBL (lange 0

-68/106 PBL) Statistical analysis shows a significant

Inhi-bition of the CTLr/f by CsA piesent in the medium (P =

0 0138, Table 2)

In the 'acute GVHD' gioup the mean CTLpf without

CsA present is 96/106 PBL (lange 0-992/106 PBL) The

CTLpf found m the picscncc of CsA are mostly very low

or absent (rrcan CTLpf = 31 4/106 PBL, ränge 0-403/10f

PBL), but ilso here two out of seven patients still show a

significant CTLpf Thiee out of seven 'acute GVHD' pati

ents have a higher CTLpl (M00/106 PBL) than 'no

GVHD' patients at at Ieast one time point post-BMT,

whercas the others do not diffei in then CTLpf Statistical

analysis shows that CsA in the medium causes a significant

Inhibition of the CTLpf (% Inhibition = 67%, Ρ = 0 0187)

The mean CTLpf of chrome GVHD patients is the high

est of the thiee groups (mean = 161/10

6

PBL, ränge

Ο-Ι 801/10

f1

PBL) Thiee out ol six 'chrome GVHD' patients

UPN 4

No GVHD

50 r

> ο c ω er α> LL ο. 1-υ 40 30 20 10 η J donor 50 100 150 200 250

Days after BMT

UPN 8

Acute GVHD 300 350 ο donor 50 100 150 200 250 Days after BMT UPN 13 Chrome GVHD 0 300 350 250 > 200 ο a> 150 er £ 100 LL °r 50 I 0 donor 50 4 3 Β Π3 +-* (/) 2 Q 0 100 150 200 250 Days after BMT 300 350

Figure 1 Kmetics of CTLpf after BMT of a patient without GVHD (UPN 4) ot a patient sutfermg from acute GVHD (UPN 8) and of τ patient suftenng from acute GVHD followed by chrome GVHD (UPN 13) GVHD Status indicated with -in astensk is lepiesented as giade I (1 = very mild) to IV (4 = seveie) CTLpf without CsA ate represented with a stiaight linc CTLpt in the piesence of CsA aie lepiesentcd with a dashed line

have highei CTLpf than 'no GVHD patients at at Ieast one

time point post BMT Α majonty ol chrome GVHD

pati-ents (four out of six) has also a lelatively high CTLpf in

the piesence of CsA The mean CTLpf in the presence of

CsA is 69 5/10

6

PBL (lange 0-891/10

6

PBL) The

Mann-Whitney U test for companson shows that CTLpf in the

absence 01 piesence of CsA aie not significantly diffeicnt

(% Inhibition = 57%, Ρ = 0 1066) Summaii7ing, our lesults

suggest that CsA leads to a significant deciease in CTLpf

in patients without GVHD and patients with acute GVHD,

but not in patients with chrome GVHD Α large vanation

is found in post-BMT CTLpf between patients with GVHD

The data are summaiized m Table 2 The mean CTLpf

obseived in the thiee groups of patients without oi with

CsA added in the eulture medium are indicated and is the

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Measurement of CsA sensitivity of CTL post BMT LM Uem ei al 76 Acute GVHD 100 80 No GVHD 20 >-ο c ω σ CD Lp h r υ 60 40 — Μ 1600 1100 600 100 80 60 40 20 ChromcGVHD \ \ \ NoCsA - Η - U P N 2 Ar UPN4 - • - UPN 16 k. UPN 23 CsA — 1 — UPN 3 * UPN 5 β UPN 18 NoCsA H B - U P N 6 * UPN 8 • UPN 17 -b. UPN 22 . CsA * - UPN 7 -8— UPN 9 -•- UPN 20 No CsA CsA — 1 — UPN 10 - • - UPN 12 β UPN 14 - • - UPN 11 -Ar UPN 13 • UPN 15 -Mean

Figure 2 Each hnc represents the CTLpf with and without CsA of onc sample post BMT All samples of onc patient arc represenled with thc same Ime and symbol

Table 2 CsA sensitivity ol C FLpf in difierent patient groups

Paticnts No GVHD η = 7 Acute GVHD η = 1 Chiomc GVHD η = 6 No samples 20 23 18 CsA added No Yes No Yes No Yes Mean CTlpj (/10() 178 7 9 96 0 31 4 161 0 69 5 sd 184 16 7 228 2 89 8 421 2 208 6 % Inhibition 56 67 57 Mann-Whitney /> = 0 0Π8 Ρ = 00187 ^ = 0 1066

Patients were divided into three groups aotording to GVHD Status Twenty post BMT samples of seven patients without GVHD or acute GVHD giade I 23 samples of seven patients with acute GVHD II—IV and 18 samples of patienls with chrome GVHD following acute GVHD grade I or more weie tested The percentage Inhibition = (1 (mean CILpf with CsA/mcm CTLpf without CsA)) χ 100% s d = Standard deviation Ρ values companng CTI pf without CsA with CTLpf with CsA are calculated using the Mann-Whitney U test

highest in the 'chrome GVHD' group The effect of CsA presented as the percentage Inhibition of the mean CTLpf IS, although comparable, the strongest in the acute GVHD group

Discussion

More than a decade ago, lt was estabhshed by in vitro expenments that CsA mediates suppression of pnmary CTL responses 2 6 Studies by Hess et αΓΊ subsequently dem onstrated the resistance to CsA of pnmed CTL Similarly, on the CTLp level, the frequency of CTL grown out of pnmed mixed lymphocyte eulture cells is not influenced by CsA Yet CsA has a profound mfluence on thc pnmary activation of alloreactive CTLp induced in MLC bulk

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Measurement of CsA sensitivity of CTL post-BMT LM Liem ei al

agdinst HLA antigens toward which no antibodies were present (acceptable mismatches) were sensitive to CsA in vitro " Additional observations supporting the notion that a decreased in vitro CsA sensitivity of organ donor reactive CTLp is correlated with a higher nsk of graft rejection was substantiated by studies in corneal grafting34 and heart transplantation v

These results prompted us to investigate whether a decrease m CsA sensitivity of CTLp would correlate with an mcreased nsk for GVHD after HLA genotypically ldent-lcal BMT To lest this assumption, we analyzed the in vitro CsA sensitivity of the anti host CTLpf in a 7-day limitmg dilution assay Our results differ from those descnbed for organ transplantation, but are in lme with previous results wheie no differences in mhibitory effects of IL-2 pro-duction mediated by CsA were observed between patients with or without acute GVHD 1 6 We observed no clear dif-ference between patients suffenng from either acute or chronic GVHD and patients with no GVHD Neither was there a difference in CsA sensitivity of patients who used MTX or CsA as GVHD prophylaxis, nor did we notice an mfiuence of dispanties for the known minor histocompat-ibihty antigens between bone marrow donor and lecipient4 CsA is able to mhibil the CTLpf in vitro in the thrce groups of patients, although an mterindividual vanabihty in the in vitro CsA sensitivity was noted, a phenomenon descnbed before by o t h e r sT O 1 7 The appaient tendency of enhanced resistance to CsA of the 'chronic GVHD' gioup (Mann-Whitney U test, Ρ = 0 1066) must therefore be interpieted with care in view of the mterindividual CTLpf and CsA vanabihty previously mentioned

Yet the increment of CsA-resistant CTL observed in the chronic GVHD patients may be mdicative of a higher nsk of devclopment ol chronic GVHD The hmited patients' matenal diü not allow us to evaluate whether higher

con-centrations of CsA would break this lesistance The pres-ence of this relatively resistant anti-host CTL population may also reflect the composiüon of a diffeient CTL popu-lation as opposed to acute GVHD, related to the chronic form of GVHD

In chronic GVHD the conlmuous Stimulation of GVHD related Τ cells might give nse to an mciease m CD45R0'~

memory Τ cells Recent data suggests that CD45R01" Τ cells are less susceptible to the mlv'oitoiy effects of CsA than CD45RA1 naive Τ cells1 8 An inercase in CsA resistant CD45R0^ Τ cells will then decrease the overall CsA sensi-tivity of the total PBL pool

The aim of this study was to find a raison d'etre for the anti-host CTL observed in patients without any chmcal signs of GVHD Heieto we investigated whether there may exist a differential sus^eptibihty for CsA between anti host CTL observed in patients with or without GVHD Our data show however that in an HLA genotypically identical Situ-ation no corrclSitu-ation is found post-BMT between the in vitto sensitivity for CsA of anti-host CTLs and the oecurrence of chmcal GVHD

Acknowledgements

The authors would like to thank Dr FHJ Claas and Μ Oudshoorn for cntically leviewing this manusenpt and Piofessoi JT van Rood

for helpful discussions This work was supported by the JA Cohen Institute for Radiopathology and Radiation Protection (IRS), by EC grant BIO2 CT92 0300 and by a grant from the MACROPA Foundation

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