7
V . Martell et al. IC sequences by 19. etic constitution 72. locyte cytotoxic-tanual of Tissue nbin-nylon wool. ILA Press, 1980, In Bodmer WF, patibility Testing Workshop Tech-ger-Verlag, 1989, membranes. :striction endonu-33.ling DNA restric-•n 137:266, 1984. 2 antigens. Immu-idley Β: Α DNA-led and ill-defined .40, 1988. polymorphism of 1989. on fragment length 'orkshop Southern t, Springer-Verlag, at the DNA level": um Genet 43:954, ntification of HLA-;. Tissue Antigens Watling DL, Tieber les at the jSIII locus ol 137:2627, 1986. DR and -DQ genes 39:751, 1986.
Induction of Minor Histocompatibility
Antigen-Spedfk T-Helper But Not T-Cytotoxic
Response Is Dependent on the Source of
Antigen-Presenting Cell
Cecile A. C. M. van Eis, Astrid Bakker, Jon J. van Rood,
and Eis Goulmy
ABSTRACT: We studied the accessory cell requirements for triggering in vivo primed human major histocompatibility complex class I— and class H—restricted Τ cells specific for minor histocompatibility antigens. We compared the antigen-presenting capacities of peripheral blood lymphocytes (PBL·) and Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL·), both derived from the same donor, to induce minor histocompatibility antigen—specific cytotoxic and proliferative Τ cells. PBL· and EBV-LCL· were equally effective as antigen-presenting cells to trigger cytotoxic-T-cell responses specific for minor histocompatibility antigens, some of which were reactive with Β cells only. In contrast, α clear difference was found between the capacities of the two antigen-presenting cell types to induce minor histocompatibility antigen—specific T-helper-cell responses. PBL· as antigen-presenting T-helper-cells could induce T-helper-T-helper-cell lines reactive against minor histocompatibility antigens presented on PBL·, on EBV-LCL·, or on both cell types as sttmulator cells. Unexpectedly, however, EBV-LCL· as antigen-presenting cells in all instances failed to induce T-helper-cell responses specific for minor histocompatibility antigens presented on
PBL· or on both PBL· and EBV-LCL· as sttmulator cells and could only trigger T-helper cells directed against B-cell-specific minor histocompatibility antigens. Our findings indicate α dichotomy in the capacity of EBV-LCL· to present minor histocompatibility antigens in the induction versus the effector phase of the in vitro T-helper-cell response. Furthermore, the results show different in vitro accessory cell requtrements for major histocompatibility complex cUss I— and class ll—restricted T-cell responses specific for human minor histocompatibility antigens. ABBREVIATIONS APC BMT CML EBV-LCL GvHD antigen-presenting cell bone marrow transplantation cell-mediated lympholysis Epstein-Barr virus— transformed lympho-blastoid cell line graft-versus-host disease mH antigen MHC PBL PLT minor histocompatibility antigen major histocompatibility complex peripheral blood lymphocytes primed lymphocyte test
From the Department of Immunohematology and Blood Bank, University Hospital Leiden, The Nether-lands.
Address reprint requests toC.A. C. M. van Eis, Leiden Unive'sity Hospital, Department of Immunohema-tology and Blood Bank, Building 1, E3-Q, PO Box 9600, 2300 RC Leiden, The Netherlands.
Receivedjuly 17, 1989; accepted November 22, 1989.
Human Immunology 28, 39-50 (1990)
© American Society for Histocompatibility and Immunogenetics, 1990
40 C. Α. C Μ. van Eis et al. Tc cell cytotoxic Τ cell Th cell prohferative/helper
Τ cell
INTRODUCTION
In humans, T-cell immunity to minor histocompatibility (mH) antigens is of particular interest for its presumed role in the development of graft-versus-host disease (GvHD) and graft rejection after bone marrow transplantation (BMT) from an HLA-identical sibling [1,2]. mH antigens are able to induce responses of both cytotoxic (Tc) and proiiferative/helper (Th) T-lymphocyte subsets [3-9]. In accordance with the general model [10,11], presentation of mH antigens to Tc cells was shown to occur in the presence of major histocompatibility complex (MHC) class I molecules [3—6], whereas Th-cell responses against mH antigens were MHC class II-restricted [7-9]. In the last few years, a series of studies on the principles of MHC class I— and class II—restricted antigen presentation to Τ cells has emphasized the central role of the antigen-presenting cell (APC) and the necessity for antigen processing [12,13]- Although the intracellular events involved in antigen presentation are still not fully understood, it has been proposed that distinct metabolic pathways of antigen degradation may be connected with the ultimate presentation of antigen in the context of either class I or class II molecules to Tc and Th cells, respectively [14]. Cell types that can function as APCs to Tc and Th cells include macrophages, dendritic cells, Β cells, and EBV-LCLs
[15-19]-Unlike presentation of nominal antigens such as tetanus toxoid or viral proteins, the particular APC requirements for mH antigen—specific T-cell activation have received little or no attention. Several reasons may account for this. First, the identity of these molecules, which are thought to be of endogenous origin, is still largely unknown. Second, mH antigens evoke mainly [20,21], although not exclusively [22], MHC-restricted T-cell-mediated immune responses that are not useful for informative protein biochemistry. Finally, in vivo priming prior to in vitro sensitization is generally required to detect mH antigen-specific responses. This means that in humans, functional studies are almost totally dependent on cells from patients who have undergone blood transfusion or kidney or bone marrow grafting.
In earlier studies we analyzed the role of mH antigen-specific T-cell responses in patients after HLA-identical BMT. Here, we aimed at designing experimental conditions that would produce in vitro triggering of both cytotoxic and prolifera-tive T-cell subsets against mH antigens from patients' posttransplant cells. For this purpose, we compared two different types of APCs in the induction and the effector phase of the response. We discovered that APC conditions that were sufficient for the induction of Tc-cell responses appeared to be inadequate for Th-cell responses. This differential APC defect was exhibited by Epstein-Barr virus—transformed lymphoblastoid cell lines (EBV-LCLs).
MATERIALS AND METHODS
Patients. Sixteen patients (6 males and 10 females) received bone marrow from their HLA-A, -B, -Cw, and -DR-identical, mixed lymphocyte culture-nonreactive sibling donor. The patients' median age was 32 years (ränge 15—42). One patient
an Eis et al. lelper igens is of fersus-host on (BMT) responses sets [3-9}. mtigens to cy complex Η antigens studies on tation to Τ (APC) and jlar events η proposed iected wich or class II function as , and EBV-al proteins, Nation have . First, the s origin, is though not :hat are not prior to in responses. Dendent on ;y or bone 1 responses perimental d prolifera-t cells. For ion and the 5 that were lequate for pstein-Barr irrow from Tionreactive )ne patient hamide (50 0). Fifteen ol for these irradiation
Induction of Minor Η Antigen—Specifk Τ Cells 41
TABLE 1 Flow chart of the induction and effector phase of host-specifk Tc-and Th-cell lines
Responder cells 1 Post-BMT PBLs 2. Post-BMT PBLs Induction phase A P C added on days 0 and 6 Host* PBLs Host EBV-LCLs Feeder cells for maintenance of the culture Host EBV-LCLs and donor PBLs Host EBV-LCLs and donor PBLs Effector Target cells in the cell-mediated lympholysis assays Host PHA-blasts Host EBV-LCLs Donor PHA-blasts Donor EBV-LCLs Host PHA-blasts Host EBV-LCLs Donor PHA-blasts Donor EBV-LCLs phase Stimulator cells in pnmed lymphocyte tests Host PBLs Host EBV-LCLs Donor PBLs Donor EBV-LCLs Host PBLs Host EBV-LCLs Donor PBLs Donor EBV-LCLs
1 Antigen presenting cells
1 Denved from the patients before transplantanon.
(800 rad). As prophylaxis for acute GvHD, 11 patients received methotrexate (patients 1-8, 10, 12, 13) and 5 patients received cyclosporine Α (patients 9, 11, 14-16) during the first 100 days after grafting.
Blood samples. Heparinized blood samples were collected from recipients before
and periodically after transplantation and from the sibling donors. Posttransplant blood sampling was distributed equally over the first 2 years after BMT, and one blood sample was taken 5 years after grafting. Peripheral blood lymphocytes (PBLs) were isolated by Ficoll-Isopaque density gradient centrifugation, washed, and resuspended in RPMI-1640 dimethylsulfoxide (final concentration 10%) for cryopreservation in liquid nitrogen.
Tissue culture medium. All cultures were performed in RPMI-1640 supplemented
with antibiotics (gentamicin) and 15% human serum for T-cell cultures or 10% fetal calf serum for EBV-LCLs at 37°C in a humidified atmosphere of 5% CO2.
Generation of bost-specific T-cell lines. The generation of host-specific T-cell lines
is essentially based on the protocol described earlier [3]: 4 Χ 106 posttransplant
PBLs from 16 patients were stimulated with different host-specific lymphocytes as APCs; i.e., either with 4 X 106 3000-rad-irradiated PBLs or with 0.8 x 106
5000-rad-irradiated EBV-LCLs, both derived from the patient before transplant-ation (see Table 1). After 6 days 0.1 x 106 responder cells were specifically
42 C. Α. C. Μ. van Eis et al. cells might be generated when using EBV-LCLs as APCs, especially since all 16 recipients and all but three donors (i.e., patients 2, 4, 12) had an EBV-positive Status prior to transplantation. To identify the latter possibilities, the Tc- and Th-cell activities were assessed against different target Th-cells and stimulator Th-cells including EBV-LCLs of both host and donor origin.
Cell-mediated lympholysis assay (CML). Cytotoxic activity of the host-sensitized T-cell lines was assessed against host T-T-cell blasts, host EBV-LCLs, and, as negative controls, donor T-cell blasts and donor EBV-LCLs in the chromium release assay (see Table 1) [25]. Briefly, 5 Χ 103 51CR-labeled target cells [T-cell blasts,
generated by treatment of PBLs with 1% phytohemagglutinin mitogen (Difco) for 3 days, or EBV-LCLs)} were incubated together with 2 x 105 effector cells
in round-bottom microtker plates so that the effector cell-to-target cell (E: T) ratio was 4 0 : 1 (other Ε: Τ ratios were also tested, but data are not shown). After 4 hr at 37°C the supernatants were harvested for counting in a gamma counter (Packard Instruments, Bruxelles). Percentages of specific 5 1Cr release were
calcu-lated according to the following formula: (ER - SR/MR- SR) X 100%, in which expenmental, spontaneous, and maximal release (ER, SR, and MR) were the 5 1Cr
release by target cells measured in the presence of, respectively, effector cells, culture medium alone, and culture medium with the detergent zaponine. Based on values obtained with different Ε: Τ ratios and on repeated results, 0—20% lysis was considered background activity; responses above 20% lysis were considered positive.
Proliferation assay. Proliferative activity of the host-sensitized T-cell lines was assessed against host PBLs, host EBV-LCLs, and, as negative controls, donor PBLs and donor EBV-LCLs in a primed lymphocyte test (PLT) (see Table 1). Minimally 7 days after the last Stimulation and 5 days after the last addition of IL-2, 1 Χ 104
Τ cells were seeded in flat-bottomed microtiter plates and were stimulated with 1 X 105 3000-rad-irradiated PBLs or with 0.25 Χ 105 5000-rad-irradiated
EBV-LCLs in a total volume of 150 μΐ. After 48 hr of incubation, 1 μ θ of tritiated thymidine was added for the last 18 hr. The cultures were collected with a multiple-sample harvester, and isotope incorporation was measured in a liquid scintillation counter. The results were expressed as the mean counts per minute (cpm) of triplicate cultures. Standard deviations were always below 20% or me-dium background. Based on spontaneous background proliferation of the T-cell lines, responses below 4000 cpm, which corresponded with a Stimulation index (SI) of < 4 [SI = cpm in the presence of stimulator cells/(cpm of responder cells alone + cpm of stimulator cells alone)], were considered negative. Responses of 4000 cpm or higher were considered positive.
RESULTS
Host-Specific Cytotoxic Activity of Posttransplant T-Cell Lines Induced with
Host PBLs as APCs
Μ van Eis et al
ially since all 16 in EBV-posmve the Tc- and Th-stimulator cells ost-sensitized T-and, as negative um release assay s [T-cell blasts, mitogen (Difco) 05 effector cells arget cell (Ε Τ) Dt shown) After gamma counter ease were calcu-100%, in which R.) were the 51Cr y, effector cells, zaponine Based lts, 0-20% lysis were considered
T-cell hnes was -ols, donorPBLs >le 1) Minimally ofIL-2, 1 x 104 stimulated wtth irradiated EBV-μ,Ο of tntiated ollected with a ured in a liquid unts per rrunute ow 20% or me-ιοη of the T-cell miulation index * responder cells 'e Responsesof luced with ne intervai after The induced T-id EBV-LCLs of 1-16} Fourteen Dwed significant ainst host T-cell patients 1, 3—7, activity against 43 % lysis ι ι ι ι ϊ ι host EBV-LCL host Τ blasts donor EBV-LCL donor Τ blasts 1 2 3 4 5 β 7 θ β 10 11 12 13 14 15 1β
patient
% lysis 100 80 -Τ host EBV-LCL host Τ blasts donor EBV-LCL donor Τ blasts 8 9 10 11 12 13 14 15 16patient
44 C. Α. C. Μ. van Eis et al. host T-cell blasts. Furthermore, all T-cell lines cytotoxic for host T-cell blasts (except 15) also lysed host EBV-LCLs, whereas T-cell line 8 showed weak cytolysis only against host EBV-LCLs as target cells. The presence of EBV antigen-specific background Tc-cell activity as revealed by donor EBV-LCL lysis was found only in cultures 2 and 4.
Host-Specific Cytotoxic Activity of Posttransplant T-Cell Lines Induced with Host EBV-LCLs as APCs
Likewise, it was possible by using host EBV-LCLs as APCs to induce host-specific cytotoxic T-cell lines firom patients 1, 3-7, and 9-16 [Figure 1(B)]. In general, the magnitude of the lysis against host EBV-LCLs as target cells was more pro-nounced than against host T-cell blasts in the EBV-LCL-induced cultures. In one case host-specific cytotoxic activity against T-cell blasts was only detected after Stimulation with host EBV-LCLs as APCs [Figure 1(B), patient 8]. Like host PBLs, host EBV-LCLs as APCs were not able to elicit host-specific Tc cells from patient 2. Furthermore, the presence of EB V antigen-spedfic background Tc-cell activity as revealed by donor EBV-LCL lysis was found in a considerable number of T-cell lines [Figure 1(B), patients 2, 3, 5, 6, 9-11, 13, and 16].
Host-Specific Helper-Cell Activity of Posttransplant T-Cell Lines Induced with Host PBLs as APCs
Subsequently, the T-cell lines sensitized with the two different host APCs (see Table 1) were tested for proliferative activity against PBLs and EBV-LCLs of host and donor origin as stimulator cells [Figure 2(A) and (B), 1—16]. As illustrated in Figure 2(A), 10 of the 16 T-cell lines induced with host PBLs as APCs showed weak (4-10 x 103 cpm, T-cell lines 1, 5, and 8) or strong (>10 Χ 103 cpm,
T-cell lines 7, 9-11, 14-16) specific proliferative activity against host PBLs as stimulator cells. None of these cultures showed proliferative activity against donor PBLs, except T-cell line 9, which also responded against donor PBLs, although to a four times lesser degree than against host PBLs, possibly reflecting a back-ground of "autoreactive Th cells." The 10 host PBL-reactive T-cell lines were to a variable extent also proliferative against host EBV-LCLs: Three cultures showed EBV antigen-specific background Th-cell activity against donor EBV-LCL, i.e., T-cell lines 4, 11, and 16. Of the cultures that did not proliferate against host PBLs, only one (i.e., 12) showed significant proliferation against host EBV-LCLs, which, however, could not be interpreted as host-specific because donor EBV-LCLs were not available for evaluation.
Host-Specific Helper-Cell Activity of Posttransplant T-Cell Lines Induced with Host EBV-LCLs as APCs
van Eis et al T-cell blasts veak cytolysis igen—specific is found only :ed with ; host-specific )] In general, /as more pro-iltures In one detected after ike host PBLs, s from patient Fc-cell activity number of T-duced with ost APCs (see /-LCLs of host ..s lllustrated in APCs showed x 103 cpm, T-hast PBLs as y against donor 5BLs, although Meeting a back-11 hnes were to ultures showed EBV-LCL, ι e , ite against host ost EBV-LCLs, se donor EBV-lduced with ne of the T-cell erative activity , in 13 eultures, ainst host EBV-se found against ) and thus must ity However, 2 ferative activity h was therefore 12) which also 45 CPM χ 10-3 70 60 -50 40 30 20 10 0 Α host EBV-LCL host PBL donor EBV-LCL donor PBL 1 2 3 4 5 β 7 8 9 10 11 12 13 14 15 16
patient
CPM χ 10-3 70 60 -host EBV-LCL host PBL donor EBV-LCL donor PBL \ '\ I I f\ 7 8 9 10 11 12 13 14 15 16patient
46 C. A C M. van Eis et al. TABLE 2 Capacity of host PBLs and host EBV-LCLs to induce mH
antigen—specific Tc cells Effectot phase Host-specific Tc-cell reactivities against target cells" Induction phase APC* (« = 44)» PBLs EBV-LCLs T-cell blasts + EBV-LCLs + 24 25 T-cell blasts EBV-LCLs T-cell blasts EBV-LCLs 9 T-ceii blasts EBV-LCLs 10 10
" Denved from pauencs before transplantacion.
* In total, 44 cultures were generated from lymphocytes obtained from 13 patients at different time mtervals after transplaatation and were tested for hosr-speciiV Tc activity
responded against EBV-LCLs but not against PBLs of host origin, we could not conclude whether this reactivity was host- or EBV antigen—specinc because EBV-LCLs of donor origin were not available for evaluation.
Induction of Host-Specific Tc- and Th-Cell Lines Longitudinally after Transplantation
As shown above, PBLs and EBV-LCLs from the same individuals elicited discrep-ancy in their induction capacities of mH antigen-specinc Th-cell responses; both cell types, however, functioned equally well as APCs for the induction of mH antigen-specinc Tc-cell responses. In order to assess whether these observations were limited to a certain in vivo responder Status or reflected a general feature we tested the in vivo primed lymphocytes from 13 patients (i.e., 2 - 6 , 8—11, 13—16) for their accessory cell requirements more than once after transplantation. It appeared that in 44 cultures induced with host PBLs and simultaneously with host EBV-LCLs as APCs the trends for Tc- (Table 2) and Th-cell (Table 3) responses were similar to the single observations. The longirudinal observations definitely showed that in no instances were EBV-LCLs as APCs capable of inducing Th cells recognizing mH antigens present on PBLs alone (Table 3, 0 versus 6 cases when using PBLs as APCs) or on both PBLs and EBV-LCLs (0 versus 15 cases) but were as able as PBLs to reveal Th cells lspecific for mH antigens uniquely present on EBV-LCLs (9 versus 10 cases).
DISCUSSION
• - ( * • « * - ,
Μ. van Eis et al. ce mH hase )* EBV-LCLs 25 10
;nt time intervals after
n, we could not c because EBV-elicited discrep-responses; both duction of mH se observations general feature e., 2-6, 8-11, transplan tation. kaneously with i-cell (Table 3) lal observations ible of inducing e 3, 0 versus 6 Ls (0 versus 15 r mH antigens >r the detection itized respond-between PBLs
Induction of Minor Η Antigen—Specific Τ Cells 47 TABLE 3 Capacity of host PBLs and host EBV-LCLs to induce mH
antigen—specific Th cells
Effector phase Induction phase
Host-specific Th-cell reactivines againsc second
stimultor cells* APCs* (« = 44)* PBLs EBV-LCLs PBLs + EBV-LCLs + PBLs + EBV-LCLs PBLs EBV-LCLs + PBLs EBV-LCLs 15 10 13 35
" Denved from patients befbre transplantauon
1 In total, 44 culrures were generated from lymphocytes obtained from 13 patients at different time intervals after
transplantation and were tested for host-specific Th-cell activity
and EBV-LCLs. Our study mainly concerns a technical finding, but, in addition, it addresses for the first time the question of whether in humans cytotoxic and proliferative T-cell responses to mH antigens have equal accessory cell require-ments for their in vitro induction.
Our results indicate that for the induction of mH antigen Tc-cell responses, both types of APC behave equally efficiendy. In contrast, the induction of mH antigen—specific Th-cell responses appeared to be strongly dependent on the host APC cell type. Typical host-specific Th-cell responses which were observed after induction with host PBLs as APCs could be classified into three types of activities; either against PBLs or EBV-LCLs of one individuai or against both stimulator cell types at the same time. The later activity indicates sharing of certain stimulating mH antigen epitopes on both stimulator cells. In contrast, host EBV-LCLs as APCs in all instances failed to elicit mH antigen—specific Th-cell responses di-rected against PBLs alone or against both PBLs and EBV-LCLs as stimulator cells. On the other hand, in some cases they were able, as were PBLs, to elicit host-specific Th cells recognizing EBV-LCLs alone. These trends were consistent in multiple observations per patient made over a period of 1 month to 2 years after transplant, herewith excluding an impact of the immunocompromised Status of the patients early after transplantation. Moreover, the patients appeared to have normal allogeneic anti-HLA Tc- and Th-cell responses in all, including the earliest, posttransplant blood samples (data not shown).
48 C A C M. van Eis et al
antigen—specific Th cells in a PLT, but at the same time they are unable to function as APCs for the inductton of these Th cells.
Clearly, variabihty exists in the APC capacities between different cell types; for example, dendntic cells are more potent in presenting foreign antigens than other cell types [16]. As for a number of nominal antigens, EBV-LCLs function adequately as APCs for the induction of Th cells [18,19}· Further, it has recently been found that EBV-LCLs are just as active as dendritic cells in providing the cellular requirements for the induction of growth of individual CD4 + human Τ cells [27]. In our study, Stimulation with EBV-LCLs in some cases resulted in the induction of host-specific Th cells reactive against EBV-LCLs alone, as well as in the generation of EB V antigen-specific Th cells. So, the defect of EBV-LCLs to induce certain m H antigen-specific Th-cell responses is probably not due to a defective interaction with Th cells in general but rather to the APC requirements for mH antigen Th cells in particular.
Clearly, the setting of our study does not allow the delineation of the compo-nents necessary for mH antigen APC function. Yet, as to the question of why EBV-LCLs cannot induce mH antigen—specific Th-cell responses whereas PBLs from the same individual can, several options might be worth mentioning. First, EBV-LCLs could fail to express certain Th-cell-defined mH antigens which are present on cells within the PBL fraction. We do not favor this possibility since EBV-LCLs function as secondary stimulator cells for such responses in the PLT (Table 3) and since k is unlikely that the antigens involved in the induction and the effector phase of a Th-cell response would differ. In this regard it is worth mentioning that our study also revealed examples of cell-type-dependent presen-tation of host-specific antigens; in some instances host antigen-specific Tc and Th cells were elicited—regardless of the APC cell type used—which only responded to host EBV-LCLs in CML and PLT (Tables 2 and 3), pointing at a "B cell-ness" of their reactivity.
Second, a defective APC function of EBV-LCL may relate to accessory cell functions such as soluble or cell-bound auxiliary Signals. If so, then in vivo primed mH antigen—specific Th cells, but not Tc cells, may depend on those signals not provided by EBV-LCLs.
Third, the in vitro growth of mH-antigen—specific Th cells could be disadvan-taged or inhibited in the EBV-LCL-induced cultures. This possibility is favored by our finding that the latter cultures, in contrast to those induced by PBL, mainly expressed the cytotoxic/suppressor cell (CD8) phenotype (data not shown). This phenotype prevalence may indicate that the CD4 cells had been overgrown. Nevertheless, the EBV antigen-specific Th-cell responses were commonly de-tected in EBV-LCL-induced cultures, suggesting that CD8 predominance does not exclude Th-cell activity per se. In itself, the triggering of EBV antigen-specific Th-cell responses could be a complicating factor, if the frequency of cells respond-ing to EBV would be much higher than that of the mH antigen-respondrespond-ing cells. AU recipients and most donors in our study had a positive EBV Status pretransplantation. However, an EBV-positive donor Status was not always pre-dictive for the generation of EBV antigen-specific Th-cell activity. Estimation of Th-cell precursor frequencies for mH antigens as well as for EBV antigens will give insight into this matter.
. van Eis et al. ible to function renr cell types; ι antigens than -LCLs function it has recently > providing the 34 + human Τ resulted in the ie, as well as in f EBV-LCLs to y not due to a l requirements of the compo-lestion of why whereas PBLs ntioning. First, ;ens which are ossibility since ses in the PLT induction and ard it is worth ;ndent presen-cific Tc and Th >nly responded . "B cell—ness" accessory cell in vivo primed ose signals not d be disadvan-ility is favored >y PBL, mainly t shown). This :n overgrown. rommonly de-iminance does itigen—specific cells respond-n—responding 'e EBV Status ot always pre-Estimation of 7 antigens will . for presenta-lls are thought BMT [1,2]. Α •ts specific for s causing this
Induction of Minor Η Antigen-Specific Τ Cells 49
ACKNOWLEDGMENTS
The authors would like to thank Drs. C. Janeway, A. Lanzavecchia, P. Matzinger, and A. Termijtelen for critical reading of the manuscript, and Dr. F. Zwaan of the Hematology Department, University Hospital, Leiden, for his help in obtaining blood samples from the patients. This work was supported by the Dutch Foundation for Medical and Health Research (Medigon 900-509-001), the J. A. Cohen Institute for Radiopathology and Radiation Protection (IRS), and Biotest A. G., Frankfurt, FRG.
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