Incidence of anti-host cytotoxic and proliferative T cell responses after HLA-identical bone marrow transplantation (BMT).

Sncidence of Anti-Host Cytotoxic and Proliferative Τ Cell Responses After

HLA-Identicai Bone Marrow Transplantation (BMT)

Cecile van Eis, Astrid Bakker, Ferry Zwaan,* Jon J van Rood, and Eis Goulmy

T

been described in patients after HLA genotypically identical BMT '2 Almost certainly, differences for minor

histocompatibihty (H) antigens account for the tnggenng of such an anti-host immune response It has been hypothesized that anti-host cytotoxic (CTL) as well as proliferative (PLT) Τ cell reactivities might be relevant for the deveiopment of GVHD We previously showed an association between the presence of circulating anti-host CTLs and the occurrence of GVHD in 27 patients1 On the other hand anti-host PLTs

have been isolated from skin biopsies at the Sites of GVHD 4

This report descnbes a case study concerning both kinetic and functional aspects of the anti-host CTL- and PLT activities occurnng after BMT and discusses the possible impact for the clinical outcome

MATERIALS AND METHODS Patient

Α 38 year old femaie patient with acute myeloid leukemia in 1° remission received bone marrow from her HLA Α Β Cw DR identical MLC nonreactive sibhng after conditiomng with cyclo phosphamide (60 mg/kg/day χ 2) and total body Irradiation (8 Gy) As prophylaxis for acute GVHD cyclosporme (CyA) was given for the first 100 days At about two months after BMT CyA was temporanly interrupted for about one week Acute GVHD grade III IV diagnosed at day 12 post BMT was treated with predniso lone (20 mg/kg/12hr for twodays then tapered off) Thereafter, the patient developed severe chronic GVHD and died 267 days post BMT

Blood Samples and Τ Cell Lines

Penpheral Blood Leukocytes (PBL) were obtained by density gra dient centnfugation of hepannized blood samples from the patient before and at 27 90 154 and 180 days after BMT from the donor and from healthy unrelated HLA typed volunteers Patient's post BMT PBL and donor s PBL were cocultured with 30 Gy irradiated patient's pre BMT PBL for six days Hereafter, responder cells were maintained as Τ cell lines by weekly exposure to host specific cells and IL 2 (Biotest) which allowed testing for host-specific CTL and PLT activity as well as blocking of the latter activity in the presence ofdifferent 1 300 diluted monoclonal anübodies

hereafter (day 90, 54%, day 154, 100% and day 190, 92% specific lysis) On the other hand, significant anti-host PLT activity was detected only at day 90 (22 000 cpm) No in vitro anti-host CTL- or PLT activity was generated from donor's PBL

Longitudinal Τ Cell Subset Analysis

No unequivocal correlation was found between the CD4/ CD8 subset distnbutions and the anti-host Τ cell reactivities of patient's post-BMT Τ cell lines All cultures were 100% CD3+ (not shown) and were predommantly of the CD4+ Τ

helper cell phenotype (Fig lc and ld) Nevertheless some Τ cell lines exhibited strong anti-host CTL activity (day 90, day 154, day 180) or failed to display anti-host PLT activity (day 27, day 154 and day 180)

CTL and PLT Specificity Analyses at Day 90 Post-BMT Since the Τ cell line obtained 90 days after BMT contained both anti-host CTL and PLT activities, we tested its speci-ficity on extended panels of HLA types target- and stimula-tor cells (Fig 2a and °b) The CTLs might recognize rather frequently occurnng host specific antigens, most probably restncted via the HLA-B7 seif HLA molecule (Fig 2a) The PLT analysis showed strong reactivity towards a part of the DR2+ and DR3+ stimulator cell panel In addition several

non DR matched stimulator cells were recognized The host-specific PLT activity was significantly blocked in the presence of moAbs OKT3 (CD3, 99%), OKT4 (CD4, 80%), B8 11 2 (HLA-DR, 73%) and IB6 (HLA-DR, 42%) but not in the presence of moAbs FK18 (CD8,13%), W6/32 (HLA-A, B, C, 15%), Bl 1 G6 (beta2M, 16%), SPV-L3 (HLA-DQ, 12%) and B7 21 2 (HLA-DP, 12%)

DISCUSSION

This report shows that anti-host CTLs and -PLTs do not necessanly emerge with similar kmetics after HLA identical BMT Furthermore, in the course of acute GVHD grade 1II-IV, Τ cell reactivities specific for patient's pre-BMT cells became apparent only after a penod of complete immune unresponsiveness The stnking coincidence of the induction

RESULTS

Longitudinal Anti Host CTL and PLT Pattern

Different patterns of anti-host CTL- and PLT activities were detected in patient's post BMT Τ cell lines (27, 90, 154, 190 days after BMT), as IS shown in Fig la and lb Anti-host CTL activity was absent at day 27, but strongly developed

From the Departments of Immunohaematology, Haematology,* Umversity Hospital Leiden, Leiden, The Netherlands

Address repnnt requests to Depts of Immunohaematology and Blood bank Umversity Hospital Leiden, Ρ Ο Box 9600, 2300 RC Leiden The Netherlands

© 1989 by Appleton & Lange Ine 0041 1345/89/$3 001Η Ο

2988 _{VAN ELS, BAKKER, ZWAAN ET AL} % lVMS CTLs 0 0 -ι—ι 0 • 0 -( | 1 20 10 ;PM χ ί ο - ; PLTs

π

1

|—

ΙΠΠΠΠ

Fig 1. The antl-host CTL (a) and -PLT (b) reactivities and Τ cell subset distribution (c,d) of donor's (D, black bars) and

patient's post-BMT (day 27, 90, 154 and 180, open bars) Τ cell lines. Target and stimulator cells were 5 χ 103 51Cr labeled

PHA-Induced Τ cell blasts (E:T ratlo 40:1) and 1 χ 10" irradiated PBL (R:S ratio 1:10) from the patient pre-BMT. No CTL- and PLT actlvitles were found against donor-derived target and stimulator cells (not showr}.

Α, Β antl-host CTL restriotion analysis _{* lysis}

Fig 2. CTL- (a) and PLT (b) specificity analyses of patient's host speciftc 90 days post-BMT Τ cell line. The upper two bars of each analysis represent the activity against patient's (i.e., pre-BMT) ard donor's cells respectively. Unre-lated HLA-A, -B and -DR typed cells were used as target or stimulator cells (see legende Fig 1).

of antl-host Τ cell reactivities with the time of CyA with-drawal, also found in two other patients5 led to the

assump-üon that both the in vivo ongoing host specific CTL and PLT responses, in contrast to normal alloreactive CTL and PLT responses (not shown), were susceptible to the activity of

CyA Nevertheless, the different time patterns of the

post-BMT antl-host CTL and -PLT activities suggested that these

two functional subsets could also be subject to different regulatory mechanisms Further, the unique presence of antl-host CTLs in the absence of PLT activity dunng the course of chronic GVHD might point at a functional mvolve-ment of the former Τ cell subset Patient's anti-host CTLs at

90 days post-BMT seemed to recognize (a) minor Η gen(s) in the context of HLA-B7 Simultaneously, the anti-host PLT activity was, according to the panel- and blocking analyses, most probably restricted via HLA-DR The ques-tion remains unanswered whether the same minor Η antigens can be recognised in diverse restnction contexts The con-trasting longitudinal anti-host CTL and -PLT activity pat-terns might favour the involvement of different minor Η antigens in the activation of both anti-host Τ cell subsets This lssue IS currently investigated at the clonal level

ACKNOWLEDGMENTS

Supported by the Dutch Foundation for Medical and Health Research (MEDIGON 900-509-001), the J Α Cohen Institute for Radiopathology and Radiation Protection (IRS) and Biotest Α G , Frankfurt, FRG

We would like to thank D van Gorp and Τ Westerop for secretanal help and the Leiden Umversity Fund and the Sandoz Research Foundation for financially supporting the presentation of this paper

REFERENCES

1 Goulmy E, Gratama JW, Blokland E, et al Nature 302 159 1983

2 Irle C, Chapuis B, Jeannet M, et al Transpl Proc 1 2674 1987

3 Goulmy Ε In Morris P, Tilney Ν (eds) Transplant Revue Philadelphia, Saunders, vol 2, ρ 29, 1988

4 Reinsmoen NL, Kersey JH and Bach FH Human Immunol 11 249, 1984