Eur. J. Immunol. 1990. 20: 847-854

Alloreactive CD4+ CTL precursor

847

Stephen Manov, Robert I. LechlerO, J. Richard BatchelorO and Claire E. M. SharrockO

Individual variation in the frequency of HLA class II-specific cytotoxic T lymphocyte precursors

Department of Immunologyo, Royal Postgraduate Medical School, Hammersmith Hospital and Department of Immunologyo, St. Georges Hospital Medical School, London

The frequencies of HLA class II-specific cytotoxic T lymphocyte precursors (CTLp) were studied in number of unrelated individualsusing a limiting dilution analysis system optimized for the detection of CD4+ CTLp. Peripheral blood mononuclear cells (PBMC) were enriched for CD4+ T cells by immunomagnetic depletion of CD8+ T cells. In some allogeneic combinations high CTLp frequencies were obtained with no significant difference between PBMC and CD4-enriched PBMC populations. In other combinations CTLp frequencies in CD4-enriched PBMC were found to be at least twentyfold lower than in the starting, unfractionated PBMC, suggesting a predominance in these pairs of CD8+ CTLp. In addition there was variation in CTLp frequencies against the same set of HLA class I1 gene products between individuals, and variation in CTLp frequencies against different HLA class I1 gene products within individuals. The HLA class11 specificity of the assay system was demonstrated unequivocallywith detection of CTLp against HLA-DR1 expressed on a murine L cell transfectant.

1 Introduction

has been achieved using short-term in vitro cultures. The route of virus infection [14] and subsequent antigen CD4 and CD8 are cell surface markers which divide processing [15,16] may determine whether CD4 or CD8 T T lymphocytes into two main subpopulations, recognizing cells are activated in the virus-specific CTL response. antigen in the context of HLA class I and HLA class 11, respectively [l]. These CD antigens were also used initially The role of CD4+ CTL in allograft rejection is also unclear. to divide human T lymphocytes into functional subsets, CD4+ Tcells appear to make a minimal contribution to the CD8+ T cells comprising the cytotoxic/suppressor popula- primary in vitro CTL response against allogeneic MHC tion, and the CD4+ cells the helperhnducer population [2]. [17], but significant levels of HLA class II-specific cytotoxIt is becoming clear, however, that there is considerable icity can be obtained if CD8+ T cell activation is negated by functional heterogeneity within each of the two subsets. matching for HLA class I antigens [18-201 or by depletion For example, CD8+ Th cells have been described [3], and of the CD8+ T cell subset [11. there have been many reports of CD4+ T cell clonesnines with specific cytotoxic activity for viruses [4,5], autoanti- The detection of HLA class II-specific CTL after primary culture suggests that the precursor cells are present. There gens [6,7] and allogeneic HLA class I1 molecules [8,9]. have been few studies, however, on the frequency of CD4+ A potential role for CD4+ CTL in vitro has been suggested CTL precursors (CTLp) using LD analysis (LDA) and by the predominance of CD4+ T cells in the CTL response these have revealed CD4+ CTLp frequencies severalfold to certain herpes viruses [4,51. In addition, soluble anti- lower than those found in CD8+ subset [17,21]. These gen-specific CD4+ CTL may play a role in the antigen- studies suggested that the predominance of CD8+ T cells in specific suppression of the humoral response [101. Before the primary cytotoxic response against allogeneic MHC such data were available, however, the majority of virus- molecules was due to a low precursor frequency of CD4+ These studies used very few MLC combinations, and specific CTL were found to be of the CD8+ T cell subset a. which suggests that CD4+ CTL had no physiological role in used mitogen-activated Tcell blasts as targets, which have vivo, and were aberrant cells induced by in vitro culture been shown to be insensitive targets for measurement of conditions.This notion has been supported by the difficul- class II-specific CTL [18, 19, 221. ties in generating virus-specific CD4+ CTL in short-term cultures [ l l , 121 and the demonstration that non-cytotoxic The aim of the study reported here, was to determine Tcell clones can acquire cytotoxicity under certain culture whether there were CTLp frequency differences against conditions [13]. The generation of measles virus-specific HLA class I1antigens in a panel of unrelated individuals,as CD4+ CTL [5] and herpes virus-specificCTL [4], however, has previously been shown for HLA class1 antigens [23,24].This was achieved using an LDA system optimized for the detection of CD4+ CTL. Responder PBMC were immunomagnetically depleted of CD8+ Tcells, and B Iym[I 78771 phoblastoid cell line (BLCL) targets were used to increase the efficiency of detection of CD4+ CTL. In some MLC Supported by Nan Dimond trust. combinations vey high frequencies of CD4+ CTLp were obtained, comparable to those seen when unseparated Correspondence: Claire Sharrock, Department of Immunology, St. Georges Hospital, Medical School, Cranmer Terrace, London PBMC were used as responders, whereas in others, CD4+ CTLp frequencies were low or undetectable. These results SW17 ORE, GB suggest a variation in the cytotoxic T cell repertoire to Abbreviations: BLCL: B lymphoblastoid cell line CTLp: CTL allogeneic HLA class I1 molecules between unrelated individuals. precursor LDA: Limiting dilution analysis 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

0014-2980/90/0404-0847$02SOJO

848

Eur. J. Immunol. 1990. 20: 847-854

S. Man, R. 1. Lechler, J. R. Batchelor and C. E. M. Sharrock

2 Materials and methods 2.1 Media The growth medium used (GM) consisted of RPMI 1640 (Flow, Irvine, Scotland) buffered with sodium hydrogen carbonate (Flow) and supplemented with penicillin-streptomycin (Flow), L-glutamine (Flow), and either 10% FCS (Sera-Lab., Crawleydown, GB, for BLCL) or 10% pooled heat-inactivated human AB serum (for LDA cultures). Hepes-buffered medium (HM) was used for immunomagnetic depletions, and was of similar composition to GM except that HM was buffered with a mixture of sodium hydrogen carbonate and Hepes (Flow). 2.2 Cell donors and targets cells Responder and stimulator PBMC for LDA were obtained from a panel of healthy individuals. The panel was HLA typed using serological reagents from the Xth Histocompatibility Workshop. In some cases DQ, DP and Dw types were assigned by RFLP analysis. BLCL for use as CTL targets were generated by EBV transformation of PBMC from all the individuals. BLCL were generated by infecting PBMC with virus using SN from the EBV-producing marmoset cell line B9-58. Homozygous BLCL from the Xth Histocompatibility Workshop were used to determine the specificity of CTL. In addition the NK-sensitive target cell line K-562 and the LAK-sensitive target cell line Daudi were used in some experiments. All cell lines were cultured in GM supplemented with Tylocine (anti-mycro-plasma, Flow Labs).The DR1-expressing murine L cell (5-3.1) was kindly provided by Drs. R. F! Sekaly and E. 0. Long, and the derivation of this line has been described in detail elsewhere [25]. Both 5-3.1 and the untransfected parental L cell line (DAP.3) were cultured in DMEM supplemented with L-glutamine, penicillin-streptomycin and 10% FCS.

2.3 Immunomagnetic depletion of CD8+ and NKH-l+ cells from PBMC PBMC were obtained by Ficoll-Hypaque (Pharmacia, Milton Keynes, GB) gradient centrifugation of heparinized peripheral blood. CD8+ T cells were depleted using antiCD8 mAb-coated magnetic beads (HLA class I Dynabeads, Dynal, Oslo, Norway). PBMC at a concentration of 2 x 107/mlin HM were dispensed into a 10-ml test tube and cooled in an ice bath. Anti-CD8-coated magnetic beads were mixed with PBMC at a ratio of 100 p1 of beads to lo7 PBMC. The tube containing the bead/PBMC mixture was then left on a Rock-N-Roller for 30 min at 4°C to allow rosetting of beads with CD8+ T cells. The contents of the tube were resuspended gently in 5 ml of ice-cold HM, and a magnet was applied to the side of the tube. Non-rosetted cells were removed using a Pasteur pipette and dispensed into a fresh tube.The rosetted population was washed twice more using the magnet and cold HM to free any remaining non-rosetted cells. The non-rosetted cells (CD8 depleted) were pooled, washed, resuspended in HM and subjected to two more cycles of immunomagnetic depletion using fresh antibody-coated beads. The simultaneous removal of CD8+ and NKH-1+ cells was achieved by immunomagnetic depletion using an indirect

method. PBMC were resuspended in HM and saturating amounts of anti-CD8 (Dakopatts, High Wycombe, GB) and anti-NKH-1 (Leu-19, Becton Dickinson, Cowley, GB) antibodies and left for 30 min on ice. After one wash with cold HM, immunomagnetic depletion was carried out as before, except that magnetic beads coated with sheep anti-mouse Ig antibody (M450 Dynabeads, Dynal) instead of anti-CD8 were used.

2.4 LD cultures The LDA used was that described by Sharrock et al. [23]. The level of reproducibility of this system has been established in previous studies from this laboratory [23,26]. To check that the reproducibility in the current experiments was similar to that reported in the previous studies, two responder : stimulator pairs were assayed on two or more occasions, and the reproducibility levels were similar to those obtained previously (see footnote to Table 1). LD cultures were set up in 96-well V-bottom plates (Sterilin, Teddington, Middlesex, GB). Graded numbers of PBMC or CD8-depleted PBMC (10 000312/well) were co-cultured with 5 x 104 X-irradiated (3000 rad) stimulator PBMC. Thirty replicates were set up for each responder cell concentration. On days 3 and 6 the LD cultures were fed with GM containing rIL2 (Boehringer Mannheim, Mannheim, FRG) to give a final concentration of 5 U/ml in each well. CTL assays were performed on day 10 [20].

2.5 Bulk MLC In some experiments LD conditions were used to assess the effect of immunomagnetic depletion on bulk lymphocyte populations. In these experiments, responders at lO'Vwe11 were mixed with 5 x leirradiated stimulators.The cultures were fed with GM containing rIL 2 as for the LDA cultures and assayed for CTL activity on day 10.

2.6 CTL assay Target BLCL were labeled with W r (200 pCi = 7.4 MBq per lo7 targets) for 1 h at 37°C. They were washed three times before resuspending in GM at a concentration of l@/ml. Before adding 100 p1 of the target cell suspension, 150 p1 of SN was removed from each LDA culture well. Controls for the CTL assay consisted of 6 wells for total release (targets incubated with 1% Triton-X) and 24-36 wells for spontaneous release (targets in absence of responders). Cultures using BLCL targets were incubated for 4 h; when L cell targets were used the incubation time was extended to 7 h. At the end of the incubation period, the assay plates were centrifuged and 100 pl of SN was removed from each well. The amount of 51Cr released into the SN was determined using a gamma spectrometer (LKB, Bromma, Sweden). Specific lysis was determined using the following formula: % Specific lysis =

(Experimental counts - spontaneous release) x loo (Total release - spontaneous release)

Eur. J. Immunol. 1990. 20: 847-854

2.7 Statistical analysis Experimental wells were scored as positive if the counts exceeded the mean plus 3 SD of the control wells (spontaneous release). The frequency of CTLp was determined using maximum likelihood analysis [27] of the experimental data. A computer program [23] was used to estimate CTLp frequency, to provide 95% confidence limits, to determine points for the best straight line, and to provide the probability that the experimental data fitted the single hit model. Standard LD graphs were obtained by plotting the lines determined by the computer program, together with the actual experimental data points. CTLp frequencies were considered significantly different from each other if their 95% confidence limits were non-overlapping. Linearity of the experimental data was assessed using regression analysis and non-linear data rejected.

2.8 Indirect immunofluorescence and FCM The following mAb were used to assess the efficiency of immunomagnetic depletions; anti-CD3 (OKT3, American Type Culture Collection, Rockville, MD), anti-CD4 (Leu3a, Becton Dickinson), anti-CD8 (Dako-T8, Dakopatts), and anti-NKH-1 (Leu-19, Becton Dickinson). Cell populations were stained by indirect immunofluorescence. Cells were incubated on ice with saturating amounts of mAb for 30min. After two washes with cold PBS supplemented with 5% FCS, the cells were incubated with FITCconjugated sheep anti-mouse IgG (Amersham Int., Amersham, GB) for a further 30 min at 4 "C in the dark.The cells were washed three times before being fixed with 1%

Alloreactive CD4+ CTL precursor

849

paraformaldehyde in PBS and stored in the dark at 4°C. Negative controls for each cell population studied consisted of cells stained with the FITC-conjugated second antibody alone. The percentage of positive cells stained with each antibody was determined using FCM (Epics Profile analyzer, Coulter Electronics, Luton, GB).

3 Results 3.1 Immunomagnetic depletion of CD8+ T cells from PBMC PBMC were depleted of CD8+ T cells using anti-CD8coated magnetic beads. PBMC and CD8-depleted PBMC were subjected to phenotypic analysis for every LDA experiment. The results from a representative experiment are shown in Fig. 1.This procedure was seen to be efficient with between 0.26% to 3% CD8+ Tcells remaining after immunomagnetic depletion in twelve experiments, furthermore no increase in the proportion of CD8+ T cells was seen in the CD8-depleted population after 10days of culture. There was an increase in the proportion of cells expressing NK cell markers (detected by Leu-19 antibody) in both the PBMC and the CD8-depleted PBMC populations after 10 days of culture. The residual CD8+ cells left after immunomagnetic depletion expressed low levels of CD8, and double staining demonstrated that these cells also expressed the NKH-1 marker (data not shown).

3.2 Immunomagnetic depletion of NKH-l+ cells abrogates lysis of Daudi targets but does not affect lysis of allogeneic targets The number of NKH-1+ cells (detected by Leu-19 antibody) expanded in both the CD8-depleted PBMC and untreated PBMC populations after culture. The role of NKH-1 cells in mediating cytotoxicity of BLCL was investigated by subjecting PBMC already depleted of CD8+ cells to an additional immunomagnetic depletion using an antibody against NKH-1 (Leu-19, see Sect. 2.3). The cytotoxic activity of depleted and undepleted PBMC populations after LDA culture against the same stimulator is shown in Fig. 2. The untreated PBMC were highly cytotoxic against allogeneic stimulator targets, K-562, and Daudi targets but not against autologous BLCL. CD8-depleted PBMC exhibited significant lysis of the stimulator target, but at a markedly reduced level, indicating that the bulk of the cytotoxic activity in untreated PBMC was mediated by CD8+ T cells. The weak, but significant lysis mediated by CD8-depleted PBMC was consistent, and similar results (data not shown) were obtained in three further experiments using different responders and targets from those of Fig. 2. Reduced lysis of K-562 targets was also seen, although there was no significant change in lysis against Daudi targets.

Figure I. Phenotypic analysis of LDA cultufes. PBMC (a) or CD8-depleted PBMC (b) were stained with anti-CD4, anti-CD8 and anti-NKH-1 (Leu-19) antibodies o n days 0 (W) and 10 ( 0 )of culture.The percentage of positive cells stained with each antibody was determined by FCM. Negative controls for PBMC and CD8-depleted PBMC were always < 1.5%.

When PBMC were depleted of both CD8+ and NKH-l+ cells, the cytotoxicity against Daudi targets was lost, while lytic activity was retained against both K-562 and allogeneic stimulator targets. There was no significant difference between specific lysis of allogeneic BLCL mediated by either CD8-depleted (Fig. 2b) or CD8 plus NKH-1-

850

Eur. J. Immunol. 1990.20: 847-854

S. Man, R. I. Lechler, J. R. Batchelor and C. E. M. Sharrock

HLA class 11-matchedtargets when the contents of 30 wells were subjected to split-well analysis. There was no correlation in specific lysis of the two HLA classI1-matched targets when PBMC (containing both CD4+ and CD8+ cells) were used as responder cells (see Fig. 3a). PBMC r2

4.284.p0.05

1

0

2

4 6 8 1 rIL2 added (u/ml)

0

1

2

i i p 7 , U ‘

0

20

40

60

80

%Specific Lysis WTIOO-81sB-LCL

10

CDE-depleted PBMC

0 0

60

2

4 6 8 1 rIL2 added (u/rnl)

0

1

2

801 r 2 4. 81 4, pc0.0001

-

50 -

20 0

20

40

60

80

%Specific Lysis WT100-BIS B-LCL

l0

o 0

2

4 6 8 1 rlL2 added (ulml)

b 0

1

2

Figure 2. Depletion of NKH-l+ (Leu-19) cells abolishes lysis of Daudi target cells but does not affect specific lysis of allogeneic BLCL targets. PBMC (A), CDS-depleted PBMC (B) and CDVNKH-l+-depleted PBMC (C) from MB (HLAAl, B8 DR3 DQ2 DRw52) were cultured with irradiated allogeneic JRB PBMC (HLA A2,3 B44,62 DR4 DQ3 DRw53). The cultures were fed with GM and varying concentrations of rIL2 on days 3 and 6, before being assayed for cytotoxic activity on day 10. Specificlysiswas calculated from the mean of six replicate cultures. ( 0 )JRB (0)auto; (W) Daudi; (A)K-562.

Figure 3. Split-well analysis of PBMC and CD8-depleted PBMC cultures against allogeneic targets matched for HLA class 11. PBMC (a) and CDS-depleted PBMC (b) from SF (HLAA1,26B7,12DR3DQ2DRw52)were set up in primary MLC against NF PBMC (HLA A32 B14,15 DR1 DQl). The cultures were incubated for 10days under LDA conditions (see Sect. 2.4) before the contents of individual wells were split and tested for CTL activity against NF BLCL (stimulator) and WTlOOBIS BLCL (HLA A l l B35 DR1 DQ1). The points plotted represent specific lysis results from individual wells. Significant correlation occurs with a probability of < 0.05.

depleted PBMC (Fig. 2c). These results demonstrate that removal of NKH-l+ cells removes cells able to lyse Daudi targets, but not T cells able to lyse allogeneic targets. In addition these results indicate that the killing of allogeneic BLCL targets under these conditions was specific, as no significant lysis of either autologous BLCL (Fig. 2a, b and c) or third-party BLCL (data not shown) was ever seen despite the presence of cells able to lyse K-562 and Daudi targets. Targets

3.3 CDSdepleted PBMC mediate HLA class 11-specific lysis The specificity of the CD8-depleted PBMC populations was investigated using split-well analysis of LDA cultures. The results shown in Fig. 3b show that the CD8-depleted PBMC population mediates specific lysis of allogeneic target sharing HLA class I1 antigens only. This is shown by the highly significant correlation between lysis of allogeneic

Figure 4. CD8-depleted PBMC mediate specific lysis of murine transfectants expressing HLA-DR1. PBMC (0)or CD8-depleted PBMC (W) fromTD (HLA A1,2 B35 DR6 DQ1 DRw52) were set up in primary MLC against GW PBMC (HLAA1,31 B8,51 DR1,3 DQ1,2 DRw52). These cultures were tested for cytotoxic activity after 10 days of culture (see Sect. 2.6).The target cells used were stimulator BLCL (GW), DR1-expressing murine transfectant (5-3.1) and HLA-negative parent L cell line (DAP.3). Specificlysis was calculated from the mean of six replicate cultures after a 7-h Cr-release assay.

Eur. J. Immunol. 1990. 20: 847-854

Alloreactive CD4+ CTL precursor

A more stringent test for the presence of HLA class IIspecific CTL was to assay CD8-depleted PBMC cultures against targets expressing only a single HLA class I1 gene product. In Fig. 4, it can be seen that PBMC and CD8depleted populations can both lyse stimulator BLCL targets; however, only the CDS-depleted population was able to lyse 5-3.1, a murine L cell transfectant expressing HLA-DR1. This lysis was D R l specific as no lysis of the untransfected fibroblast, DAP.3, was seen. These results confirm the data of Fig. 2 in that the CTL generated after MLC were predominantly HLA class I specific, whereas CDS depletion of PBMC prior to MLC results in the development of CTL which are HLA class I1 specific.

851

3.4 Variation in CD4+ CTLp frequency depending on the allogeneic responder :stimulator combination used Having shown that the depletion of CD8+ T cells from PBMC allows development of HLA class 11-specific CTL, LDA was used to estimate the frequency of CTLp after CD8 depletion of PBMC. As shown inTable 1, a large range of CTLp frequencies was seen among the different responder : stimulator MLC combinations used, and very high CTLp frequencies were obtained for some combinations. In contrast, the CTLp frequencies obtained with untreated PBMC had a much smaller range (Table 2).

Table 1. Allogeneic MHC-reactive CD4+ C I Z p frequencies in unrelated individuals Responder and H L A typea) NP" NF'

Stimulator and HLA type

DRl DQ1 DR1 DQ1 DR1 DQl DRl DQ1 DR1 DQ1

TD JRB SF MK

NF

DR6 DQl DRw52 DR4 DQ3 DRw53 DR3 DQ2 DRw52 DR7 DQ2 DRw53 DRl DQ1 (autologous)

MB MB MB MB

DR3 DQ2 DRw52 DR3 DQ2 DRw52 DR3 DQ2 DRw52 DR3 DQ2 DRw52

JRB MK SF SM

DR4 DQ3 DRw53 DR7 DQ2 DRw53 DR3 DQ2 DRw52 DR2 DQ1

SF SFI, SFf) SFf) SP) SF SFf)

DR3 DQ2 DRw52 DR3 DQ2 DRw52 DR3 DQ2 DRw52 DR3 DQ2 DRw52 DR3 DQ2 DRw52 DR3 DQ2 DRw52 DR3 DQ2 DRw52

NF BR JRB JM MK SM SF

DR1 DQ1 DR2 DQ1 DR4 DQ3 DQ3 DRw53 DR3 DR5 DQ2 DRw52 DR7 DQw2 DRw53 DR2 DQ 1 DR3 DQ2 DRw52 (autologous)

SD JM

DR3,4 DQ2.3 DRw52.53 DR5 DQ3 DRw52

m

W'

NF) NF)

NF

DR1 DQ1 DR2 DQ1

Frequencyh) (range) 1 : 1016 1 : 3635 1: 3902 1 : 34 837 1 : 75OO8

1327) 4544) (3031- 5023) (20003- 60588) (33865-166 132)

1: 1479) 1 : 4951 1 : 26 778* 1: 38987*

(1 124- 1936) (3828- 6402) (1638')- 43754) (23 038- 65 976)

1: 1: 1: 1:

8340 1 : 12 174 1 :39 587* 1 : 93 702

(1481- 2420) (2379- 3945) ( 4 6 0 - 7789) (6 198- 11 221) (8805- 16833) (19659- 79715) (42 037-208 867)

1: 2182* 1 :38 949*

(1716- 2774) (23 035- 65 976)

188oe) 3063 5989

(77')(2906-

a) HLA phenotypes obtained by serological typing. b) Frequency of CTLp in CD8- and NKH-1-depleted PBMC (frequencies marked with * were obtained from PBMC depleted of CD8+ cells only), range represents 95% confidence limits for frequency estimate. c) Frequencies obtained in the same experiment. d) Frequency obtained from CD8-depleted PBMC vs. JRB in the same experimental = 1 : 1096 (856-1403). Results from two further experiments for MB vs. JRB were 1: 1262 (988-1612) and 1:2611 (2054-3319). e) Frequency obtained from CD8-depleted PBMC vs. NF in same experiment = 1:2273 (1676-2420). Results from two further experiments for SF vs. NF were 1:701 (539-920) and 1430 (1118-1829). f) Frequencies obtained in the same experiment.

Table 2. Frequency of allogeneic MHC-reactive CTLp in unrelated individuals Responder and HLA typea) MB MB MB MB MB SF SF SF JM

A1 B8 DR3 DQ2 DRw52

A1 B8 DR3 DQ2 DRw52 A1 B8 DR3 DQ2 DRw52 A1 B8 DR3 DQ2 DRw52 A 1 B8 DR3 DQ2 DRw52 Al,2Y B7,12 DR3 DQ2 DRw52 A1 2 9 B7.12 DR3 DQ2 DRw52 A1.29 B7.12 DR3 DQ2 DRw52 Al,2 B8 DR5 DQ3 DRw52

Stimulator and HLA type SM SF GW JRB SD NF MK MB MR

A11.24B13,15DR2 A1,29B7,12DR3DQ2DRw52 A1,31 B8,51 DR1,3 DQ1.3DRw52 A2.3 B12.15 DR4 DQ3 DRw53 Al,11 B8,35 DR3,4DRw52DRw53 A32Bl4.15DRlDQ1 A1.Y B12,17DR7DQ7DRw53 A1 B8 DR3 DQ2 DRw53 A325 B7,8 DR2 DQ1

a) HLA phenotypes determined by serological typing. b) Frequency of CTLp in PBMC with 95% confidence limits shown in parentheses.

Frequencyh) (range) 1 :2006 1 :2098 1 :2623 1 :2672 1 :6296 1 : 2133 1 :2611 1 : 5141 1 :4815

(1562-2577) ( 1650-2667)

(2059-3342) (2009-3401) (4742-8360) ( 1638-2779) (2054-3319) (3984-6834) (3736-6205)

852

S. Man, R. I. Lechler, J. R. Batchelor and C. E. M. Sharrock

Split-well analysis confirmed that the high CTLp frequencies detected were due to the presence of stimulator specific CTLp, as there was a negligible contribution from either autologous or third-party-reactive CTLp (see Fig. 5). Previous results (Figs. 3,4) demonstrated that CDS-depleted PBMC mediated HLA class 11-specific lysis. There was no significant difference between the CTLp frequencies obtained with CD8-depleted PBMC or with PBMC depleted of both CD8+ and NKH-l+ cells, suggesting that NKH-1 effectors did not contribute to the lysis of allogeneic BLCL targets (see footnotes d and e, Table 1). The high frequencies of CD4+ CTLp obtained were not significantly different from the CTLp frequencies obtained from the same allogeneic combinations using untreated cells/well (XIO

0.0

E

0.2

0.4

4,

0.6

0.8

1.0

e

u.l

J

Targets

Frequencies (95% confidence limits)

o DR4B-LCL

f=1: 4973 (3815-6482) A Third party B-LCL (DR7) f=1: 40535 (22387-73395) 0

Autologous B-LCL

Not detectable.

Figure 5. Specificity of LDA cultures primed against HLA-DR4 determined by split-well analysis. LDA cultures were set up using CD8-depleted PBMC from MB (HLA A1 B8 DR3 DQ2 DRw52) and stimulator PBMC from JRB (HLA A2,3 B44,62 DR4 DQ3 DRw53). At the end of a 10-day culture the contents of individual culture wells were split three ways and assayed against DR4 BLCL (PE117, HLAA24B60DR4DQ3 DRw53), autologous BLCL (MB), and third-party BLCL (LBUF, HLA A30B13 DR7 DQ2).

1 u1

e

-

c

a

E C

.ti

PBMC (see SF vs. NF, MB vs. JRB,Table 2). Low precursor frequencies were obtained for some HLA class IImismatched combinations and all autologous combinations, with frequencies close to the limits of detectability (Table 1). No correlation of CD4+ CTLp frequency with serological typing could be made, although when HLA class I1 antigens were matched (see MB vs. SF in 'hble 1) low CTLp frequencies were obtained. There was considerable variation in the CD4+ CTLp frequencies obtained between different individuals against the same allo-MHC class I1 antigens, as can be seen inTable 1.Three individuals (Ml3,SF and NF)produced different CD4+ CTLp frequencies against MK. This was not due to variation between experiments, as such significant frequency differences could be obtained in the same experiment (see frequencies obtained using NF or MB in Table 1) and the frequencies within one responder : stimulator pair were reproducible over a number of experiments (see footnotesd and e, Table 1).These results suggest that differences exist in the cytotoxic T cell repertoire to HLA class I1 molecules between individuals. 3.5 Frequency of CTLp against HLA-DR1 expressed on a murine L cell transfectant

.

.ti

Eur. J. Immunol. 1990.20: 847-854

The interpretation of CTLp frequency data between class 11-mismatched MLC combinations is difficult as there is the possibility of multiple class I1 target antigens being recognized on a BLCL target, e.g. DR, DQ, DP and DRw52 or DRw53 molecules could serve as target antigens. This problem can be overcome by the use of murine or human transfectants expressing single HLA class 11 gene products as CTL targets, allowing detection of CTLp specific for single HLA class I1 molecules. The results in Fig. 6 demonstrate the LDA system used was sensitive enough to detect CTLp specific for HLA-DR1 expressed on a murine L cell transfectant. There was no specificity for xenogeneic determinants as no CTLp could be detected using untransfected mouse fibroblasts as targets. The frequency of HLA class I1 CTLp detected using a BLCL target was significantly higher than when the DR1+ transfectant was used.The specificity of the CTLp detected against the DR1-expressing BLCL was demonstrated by the extremely low CTLp frequency detected against a third party (DR7) BLCL.

4 Discussion .

e

U

rrequency 195% confidence limits)

f = l : 7 0 5 1 (5364-9268) f = l : 1 9 7 9 5 (13386-29272)

f-1: 1 4 2 5 9 5 (53808-377885) Not detectable

Figure6. Frequency of CD4+ CTLp specific for HLA-DRl expressed on a murine L cell transfectant. PBMC from SF (HLA A1,29 B7,12 DR3 DQ2 DRw52) were depleted of CD8+ and NKH-l+ cells, before setting up in LDA cultures against EM PBMC (HLAA2,ll B35,40 DR1,3 DQ1,2 DRw52). CTLp frequencies were determined against DR1+ BLCL (NF A32 B14,15 DR1 DQl), DR1+ murine L cell transfectant (5-3.1), HLA-negative parent murine L cell line (DAP.3) and third-party BLCL (MK HLAA1,9 B12,17 DR7 DQ2 DRw53).

In this study high frequencies of HLA class 11-specific CTLp were found using an LDA system optimized for the detection of CD4+ CTL. Successful detection of CD4+ CTLp was achieved as a result of two modifications to previous LDA protocols: first, the conditions for functional maturation of CD4+ CTL were optimized by immunomagnetic depletion of CD8+ T cells and second, the sensitivity of CTL detection was optimized using BLCL as targets for the CTL assay. BLCL constitutively express high levels of HLA gene products (HLA class I and 11) and accessory molecules (LFA-1, LFA-3, ICAM-l), which contribute to their sensitivity as targets for detecting allo-MHC-reactive P T T

LIL.

The high frequencies obtained using this protocol are in contrast to previous studies [17, 211 in which frequencies of

Eur. J. Immunol. 1990. 20: 847-854

allo-MHC-reactive CD4+ CTLp were reported to be severalfold lower than allo-MHC-reactive CTLp frequencies in unseparated T cell populations. The discrepancy between these results and the current study may be explained by the different protocols used. Both the previous LDA studies utilized mitogen-activated T cell blasts as CTL targets which have been shown to be relatively insensitive to HLA class 11-specific CTL [18, 19, 221. Furthermore, only a small number of allogeneic combinations were used in these studies, and the current study demonstrates that there is variation of CTLp depending on the allogeneic combination used. The specificity of the CTLp detected in the current assay was shown unequivocally to be for allogeneic HLA class I1 gene products by split-well analysis and by successful specific lysis of a DR1-expressing murine L cell transfectant. The use of allogeneic BLCL as targets to detect HLA class 11-specificCTL could result in lysis of BLCL by either HLA-restricted EBV-specific CTL or by NK-like populations generated as a result of MLC [28]. NKH-1 is a marker expressed on NK cells and a subset of Tcells [29]; however, depletion of NKH-11+ cells from CD4-enriched PBMC, abolished the lysis of Daudi but not K-562 targets. These results might be explained by the heterogeneity of K-562 killers in terms of cell surface markers [30] and improved results may be obtained by depletion using a cocktail of antibodies against NK markers. The depletion of NKH-l+ cells also had no effect on the specific lysis of allogeneic BLCL targets and no significant changes in CD4+ CTLp frequencies were seen in comparison to undepleted populations. These results clearly demonstrate that the lysis of K-562 cells (NK-sensitive target) and Daudi cells (LAKsensitive target) was distinct from the lysis of allogeneic BLCL targets. No significant lysis of third party or autologous BLCL was observed confirming the specificity of the CTLp detected for allogeneic HLA class 11.The existence of distinct effector populations has been suggested by studies utilizing cold target competition experiments which have demonstrated that K-562 effectors are distinct from allo-MHC-specificeffectors [31]. It is also likely that K-562 and Daudi effectors are utilizing different lytic mechanisms to allo-MHC-specificCTL generated after primary culture ~321.

Alloreactive CD4+ CTL precursor

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frequencies within individuals against different HLA class I1 molecules. The variation in observed CTLp frequencies may result from variation in other parameters such as clone size of the cultures and the sensitivity of targets to lysis. However, clone size is a measure of Tcell proliferation and it has been shown that only a proportion of proliferating cells are cytotoxic [8]. Differential sensitivity of target cells was not investigated in the current study, but it should be noted that the same BLCL target cell could result in low and high CTLp frequencies (MK and SF BLCL in Table 1). The variation in CTLp frequencies observed suggests that there is variation in the cytotoxicTcell repertoire to HLA class I1 antigens between individuals. In order to examine the T cell repertoire to HLA class I1 antigens further the specificity of the CTLp must be determined.This is difficult with BLCL as potentially the products of nine isotypematched class I1 molecules (DR, DQ, DP, DRw52 or DRw53) may be expressed in a heterozygous cell line, in addition to possible isotype-mismatched pairs [36]. Murine or human transfectants expressing HLA class I1 molecules may allow a better means of determining Tcell specificity, as responses to a single HLA-DR gene product can be examined separately. Murine L cell transfectants have been used successfully to define the interaction between antigenicpeptide and MHC [37], however, success has been limited with allo-MHC-reactiveTcell clones [38]. This may be due to the importance of species-specific accessory molecule interactions in allo-MHC recognition [39]or to the association of cell lineage-specificendogenous peptides with allogeneic MHC [25,40]. Such proposals may explain the decreased CTLp frequencies obtained with a DR1-expressing murine transfectant compared to a homozygous DR1-expressing BLCL. Alternatively the higher frequencies obtained with BLCL may represent the cumulative CTLp frequencies against multiple class I1 gene products (isotype-matchedand mismatched). Studies are in progress to distinguish between these possibilities by determining CTL responses to DR1 expressed in murine and human transfectants.

The demonstration of a highly specific, sensitive LDA for detection of HLA class 11-specificCTL allows study of the role of CD4+ CTL in allograft rejection and evaluation of Specific lysis of allogeneic BLCL targets in this study and current models of allo-MHC recognition [41]. The imporothers [l, 331 was seen to be lower in CD4+-enriched tance of matching for class I1 antigens in clinical transplanPBMC than for CD8-enriched or untreated PBMC. This tation is well established [42]; however, it has been shown in was the case even in allogeneic combinations where CD4+ this study that there is extensive variation in the frequency CTLp frequencies were high. These results suggest that of CTLp between different HLA class 11-incompatible CD4+ CTL may have different activation requirements combinations. The structural basis for such allo-MHCfrom those of CD8+ CTL.The predominance of CD8+ CTL reactive CTLp frequency differences will now be investiafter primary MLC may reflect this difference, with a gated using a panel of transfectants expressing HLA-DR selective growth advantage over CD4+ CTL; alternatively, molecules. CD8+ Tcells may play an immunoregulatoryrole for CD4+ CTL. The demonstration of functional markers for CTL in We are grateful to the volunteers who donated blood for the work in study. The authors also wish to thank Ms. Maria Daley for help the CD8+ Tcell subset [34] and the CD4+ Tcell subset [35] this with flow cytometry, and the Tissue Typing laboratory (RPMS) for may provide the means to investigate activation require- HLA typing and phlebotomy services. ments for CD8+ CTL and CD4+ CTL. Received August 10, 1989; in revised form November 28, 1989.

Our primary aim in this study was to study the cytotoxic T cell repertoire to HLA class antigens in unrelated individuals. There was clear variation between individuals in CTLp frequencies against the same allogeneicHLA class I1 molecules. In addition, there was variation in CTLp

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Individual variation in the frequency of HLA class II-specific cytotoxic T lymphocyte precursors.

The frequencies of HLA class II-specific cytotoxic T lymphocyte precursors (CTLp) were studied in number of unrelated individuals using a limiting dil...
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