The impact of naturally occurring DR3 microvariants, DRw17 and DRw18, on T-cell allorecognition

The Impact of Naturally Occurring DR3 Microvariants, DRwl7 and DRwl8, on T-Cell Allorecognition Armead H. Johnson, Ting Fang Tang, Vernon CoweR, and Carolyn Katovich Hurley

ABSTRACT: The limited amino acid sequence differences between the DR3 microvariants, DRwl7 and DRw18, are found in the second variable region of the DR ~ chain (residues 26 and 28) as well as in framework residues 47 and 86. Using selected responder/stimulator combinations, alloprolfferative T-lymphocyte clones (TICI were generated which recognize either a supertypie DR3-related determinant(s) or only those T-cell recognition determinants created by the four amino acids which differ between DRwl7 and DRwI8. Results indicate that the microvariation creates potent T-cell recognition determinants while leaving the DR3-related determinant(s) unaffected. Several TIC were generated ABBREVIATIONS B-LCL B-lymphoblastoidcell line [HWS Inmr~ationalHistocompatibilityWorkshop mAb monoclonalantibody

which recognize the DRwl8 molecule strongly and the Dgw52c molecule weakly reflecting the sequence similarity between these molecules. In addition, one TIC was generated which recognizes DRwl8 and DRwI4,Dw9 but not Dgw14,Dw16 mo!eanles, a result not predicted by linear amino acid sequence comparisons. The intricate and sometimes unpredictable allorecogaition patterns observed demonstrate that the molecular context of a specificamino acid sequence is as important as the actual sequence in forming a T-cell recognition site and, thus, in shaping the immune response profile of a given allele. Human Immunohg~ 32. 4 6 - 5 5 (1991)

PBL TCGF TIC

peripheral blood lymphocyte T-cell growth factor T-lymphocymclone

INTRODUCTION DR, the predominant class II molecule on the cell surface, contains a highly polymorphic ,~ chain complexed with an invariant a chain. Within the 3 chain, the majorit-/ of the amino acids which differ among DR allelic variants are concentrated in the first domain (amino acids 1-94) and are clustered in three variable regions. These variable regions, in the extrapolated structural model of the class II molecule, line the antigen-binding cleft and control the interaction of the class II molecule with an antigen and/or a T-cell receptor [1-3]. Allorecogultion is thought to result from T-cell roeFrom the Departments of Pediatrics and Microbiology, G~rgetown UniverJity Medical School, Washington DC. Addres~reprint requeststo Dr. Armead H. Johnson, GeorgaownHospital, HLA laboratory, 3800 Reservoir Road N.W., Washington DC 20007. Received August 20, 1990; acceptedApril 11, 1991.

46 0198-88591911$3.S0

ognition of a foreign class II molecule complexed with endogenous peptide, a molecular interaction similar to that initiating an antigen-specific response [4-9]. In contrast to an antigen-specific response, however, an unusually large percen: of peripheral T cells bearing a structurally diverse repertoire of T-cell receptors recognize a class I1 alloantigen, a phenomenon which may result from the binding of multiple endogenous peptides to the class II molecules [4, 10]. Since a large percent of antigen-specific T-cell receptors can also function in allorecogeition [I1, 12], it is likely that the diverse population o f T cells utilized in an alloresponse may represent a cross section of the antigen-specific Tcell repertoire of an individual. Thus, while complex, the allogeneic response can be used to survey the effect of DR structural variation on multiple antigen-specific responses. HumanImmunology32, 46-55 (1991} © AmericanSocietyfor Histocompafibilityand Immunogenetics,1991

Recognition of DRwl7 and DRwI8 Microvariams

DR3 microvariants, D R w l 7 and DRwl8, share the DR3 serologic determinant(s) yet differ from each other by four amino acids at positions 26, 28, 47, and 86 in the/8 chain [13]. This study examines the effect of the amino acid substitutions found in the DRw17 and DRw18 microvariants on T
Reference stimulator lymphocyte panel. Panel peripheral blood lyraphocytes (PBL) were typed for HI.A-A, B, C, DR, and D Q using alloantisera from the Ninth and/or Tenth International Histocotepatibility Workshops (IHWS) as well as local typing trays using standard techniques [14]. In addition, selected cells expressing DRwlT, DRwI8, DRw13, DRw14, and D R w l l were used from the 10th IHWS reference B-lymphoblastoid cell line (B-LCL) panel [15]. HLA-D and -DP types were determined using a collection of local and IHWS typing reagents [16-18]. DRw52 subtypes for the stimulator PBL panel were determined by hybtidizadon with allele-specific oligonocleotide probes, D N A restriction fragment length polyraorphisra analysis, and/or T-lymphocyte clone (TIC) typing [13, 19].

T-lymphocyte cloning and expansion. Human alloreacrive, proliferative T I C were generated by stimulating responder PBL with gamma-irradiated (3000 Lad) stiteulatur PBL in primary mixed lymphocyte culture for 4 days. Viable primed cells were isolated using Ficoll-Hypaque gradients and cloned at 0.3 cells/well by limiting dilution in RPMI 1640 medium containing 5 raM HEPE$ buffer and 50 tzg/ral gearamycin (complete medium), 10% human A + plasma, and 20% T-cell growth factor (TCGF) with 104 irradiated stimulator PBL/well in 60-well microtest plates (Robbius Scientific, Mountain View, CA). T C G F was obtained by stimulation of PBL with 1% PHA-P (Difco, Detroit, MI). After 7 days, growing cells were transferred to 96-well U-bottom microtiter trays containing 10' irradiated stimulator cells/well in 0.1 ml of complete medium containing 10% A + plasma. After 3 days of incubation, 0.1 ral of complete medium containing 10% A + plasma and 20% TCGF was added to each well and, on day 7, growing cells were transferred into 24-well culture trays. T I C generated in this manner were maintained by the weekly addition of 20% TCGF on day 3 followed by the addition o f irradiated feeder cells plus 20% TCGF on day 7. Irradiated feeder cells consisted of the original stimulator PBL up to the 24-well stage and then the original stimulator mixed 1 : 5 with a pool of PBL from unrelated individuals. All T I C that were nonreactive

47

with the original responder and responded by proliferation to the original stimulator were expanded for chatacterization using the above schedule of feeding. The TLC were maintained at approximately 2 to 4 x 10~ cells/ml during expansion.

Assay for proliferative T-lymphocyte activity. T I C were tested for allospecitic function in a proliferation assay in which an individual T I C (104 cells) was added to cultures of 5 x 104 irradiated stimulator cells per well. PBL used as stimulators were irradiated at 3000 rad and assayed in 96-well U-bottom plates; B-LCL used as stimulator cells were irradiated at 10,000 sad and assayed in 96-well flat-bottote plates. The cultures, set up in triplicate, were incubated for 72 hr. During the final 18 to 20 hr of incubation, L0 ~Ci of 3H-thymidiue (5 Ci/mM) (Amersham, Chicago, IL) was added to each well. Cultures were harvested unto glass fiber filters and radiolabal incorporation was raeasured [19]. Results were clustered into positive and negative by fitting mixtures of two or three normal distributions with common variance to the log-transformed proliferation responses using maximum likelihood methods. The cutpoint that minimizes the misclassification rate between high and low valued distributions was calculated as indicated by Namboodiri and colleagues [20]. The means and outpoints reported are expressed on the original scale. Linear contrasts [21] were constructed to test for a difference in mean proliferation levels among DR3, DRw52c, and other D R types. The dichotiteizad values were correlated to class II specificities and a correlation coefficient calculated using the Milwaukee serum analysis program obtained from the Milwaukee Blood Center (Milwaukee, WI).

Characterization of TLC by monoclonMantibody (mAb) blocking. Six raurine raAbs specific for HLA class II molecules were used in blocking assays: raAbs GSP4.1 and 1.243, which react with raonoteorphic determinants on D R and DRw52 molecules; teAb 7.3.19.1, which reacts with a DRw52 determinant found on DR3-associated D R and DRw52 molecules [22]; mAb 33.1, which reacts with a determinant on D Q w l , DQw3, and DQw4 molecules; raAb SFR16-PI.2, which reacts with a determinant on DQw2, DQw3, and DQw4 molecules; mAb B7/21, which reacts with a teonoteorphie determinant on D P molecules [23]; and mAb BBM.1, which reacts with/32-microglobulin [24]. Optimal concentrations of each mAb for blocking of proliferation were predetermined using a range of mAb concentrations known to fall on the saturation plateau as well as the exponential portion of the raAb binding curve as determined by flow cytoraetry using the stimulator cell. For blocking assays, 0.05 ml o f a m A b diluted at three predetermined

48

A.H. Johnson et ~.

concentrations in complete medium containing 10% A + plasma was added to 0.1 ml aliquots containing 5 x 104 stimulator cells in complete median plus 10% A + plasma in U-bottom microtiter trays. Stimulator cells and mAbs, set up in triplicate, were incubated 30 rain before the addition of 1 x 104 T I C in 0.05 ml/wen complete medium containing 10% A + plasma. The cells were incubated for 72 hr, pulsed overnight with 1.0 mCi 3H-thymidine, and harvested onto glass fiber filters for measurement of radiolabel incorporation. A mAb was considered m block a response if the level of stimulation as measured by 3H-thymidine incorporation was decreased by at least 50% in the presence of the mAb compared to the negative control using mAb BBM. 1 at one of the concentrations tested.

RESULTS

Alloprolifirative TLC were derived from priming combinations with a subtle or with a substantial DR difference. In priming TC1 (Table 1), the difference between the mismatched D R alleles expressed by the responder (DRI) and the stimulator (DRwl8) is substantial; the D R 13 chains are expected to differ by 14 amino acids in the first domain. In contrast, the second (TC2) and third (TC4) primings utilized responder and stimulator who express DRw17 and DRwl8, microvariants of DR3 which differ by four amino acids. After cloning and expansion, functional alloproliferarive T I C were characterized with a selected stimulator panel of PBL from 51 individuals, including the family of each original stimulator, and with D g w l 7 , DRw18, DRwl4, DRw13, or DRw11 class II homozygous BLCI.s to determine which HLA molecule stimulated the proliferation. The panel of stimulators shown in Table 2 contains primarily DR3- and DRw52c-posirive cells.

Although 34 non-DR3-posirive stimulator cel/s were tested, only 21 stimulator cells which include all nonDR3 PBL recognized by any of the T I C and which represent most non-DR3 D R specificitier are shown. The stimulator panel was designed m include cells with different DR/DQ, DR/DRw52, and D R / D P associations in order to facilitate selection of T I C that recognize the DR3, DRw17, or D R w l 8 molecules. In order to confirm the identity of the class II gone products recognized by T I C reactive with cells expressing DRw17 and/or DRwI8, representative T I C , TC1.18 (specific for cells expressing DR3), TC4.26 (specific for cells expressing DRwl 7), TC2.zi0 (specific for cells expressing DRwlS), and TC2.27 (specific for cells expressing D R w I 8 or DRw52c) were tested for proliferation in the presence of four different concentrations of DR-, DQ-, and DP-specific mAbs (Fig. I). mAbs GSP4.1 and 1.243, directed against nonpolymorphic DR- and DRw52-associated determinants, blocked the reactivity of all of the TIC. Similarly, all T I C were blocked by mAb 7.3.19.1 which is directed against a DRw52-related determinant localized on D R and DRw52 molecules. None of the TLC were blocked by DQ-, DP-, or class I-specific mAbs (mAbs 33.1, SFRI6-PI.2, B7/21, and BBM.1). The blocking data support the PBL panel specificity analysis identifying DRw17 and/or DRw18 as the molecule recognized by the alloreactive T I C .

Alloprolifirative TLC define DR3 subtypes, DRwI7 and DRwlS. The majority o f TLC in each priming recognize the subtypic specificities, DRw17 or DRwlS. TLC detecting a DRwl8-related specificity were derived in both the TC1 and the TC2 primings even though the D R difference between the responder and stimulator used for the TC1 priming was not as subtle as in the TC2 combination (Table 1). All T I C specific for srimu-

TABLE 1 Priming combinations for generation of aUoproliferative T-lymphocyte clones HLA type" Priming

Donor ID.

D

DR

TC1

R~ 1579 S6 1559

D-~,w8.1 D-,w8.t

DRI,w8 DRwt8,w8

TC2

R 1077

Dw3,w5

DRwl7,wl I

TC4

S 1563 R 1563

D-,D-,-

DRwt8,wl 1 DRwl8,wl 1

S 1077

Dw3,w5

DRwl7,wl I

DRw52

DQ

DP

DRw52a

DQwS,w4 DQw4,w4

DPwl,w3 DPwt,w3

DRw52a,w52b

DQw2,w7

DPw4,w4

DRw52a,w52b DRw52a,w52b

DQw4,w7 DQw4,w7

DPwl DPwI

DRw52a,w52b

DQw2,w7

DPw4,w4

• Poten~dclassn alieficdifferencesrecognizedbythe respondermeunderlined.TheDRwlt Lllelesexpressedby 1563~d 1077differ(Leeel M..submitted).The DRand DQ ~ll©lesexpressedbycell I~63havebeencharacterizedby oligonucleoudeWpir~and cDNAsequencesanalysis. ~R, responder,S, st~nub~or. cHLA-Dspe¢ificiw undefinedbyour typingre~en~.

Recognition of DRwl7 and DRwl8 Microvarian¢s

49

TABLE 2 DR3-related alloproliferative T-cell clones Stimulators

T < e U dories-

H L A type

•

D

DR

DQ

DP

4.26

4,52

03 nz

00

181S

,I.4

1.0 1.3

12

is.s

S2a S2a

4.6

0.s o4

104

S2a

4.7

1.4

I I

00

1568

0.0

181

52a

4.5

10

06

01

12;

37

z7 ?

52alS3

2

1.2

G t077b 958 373 1o64

3 35 3 t4 30 3o

17 I~ 17 4 17 l:.e

521 52a,52b 52a.53 52b $2b

2 2.7 2.3 2 2.7

1.4 4.4 1.0

~0S3

~.r

~: :

S2~lS3

~,2

13 13.1 a 134 1311~ 134 13 9 131 I1.11 .... 3 14.10 14.13 13,2 12.8 i1 ~ 11~ 8,1 81 8.1 41

$2b,S2¢ $2c.52b $2¢,$3 52c,52b 52c,53 52¢.53 52¢ 52¢,52b S2".S~" 526 S2b 52b 526 12~ 52b

2,6 6.7 6,4 6,6 6.3 5,2 6.5 6,7 S.S S.$ S,S 6.6 5,7 7,S 7,6 4,6 4,5 7.5 3.5

2041 lsSgb •

156D lOl 634 258 1000 1082 1233 2011 ~00e ZG2 1426 1145 1066 1129 260 1571 h 956 2040 1096

3.0 19.5 19.0 19.0 0.4 0,O 0.0 0,0 0.0 9,0 918 0.2 0,0 o,I 0,0 8,0 0,1 0,0 0,O

1116

at

;099 0,2 1406 nt TCGF C~uc4 M ~ u m Cootie4

7,4

I0,2 92

53

.to

1,6

22.8 16.4 2.t

4.2 4,0 4,5 1.0

1

1.18

I

2.40

2.3L

2.85

2.95

, 04 13 01 03

7 01 0~ 04 02

02 0O 02 n~

3,01

ot 0~ 01 nt

00 nt Ol ol o o nt ~ O2 n~

04 01 o5 1o 01

0 S 09 03 10 01

03 O0 01 02 03

0 I 01 os 04 8.1

oo

os

09

01

02

o2

.I

02

o2

00 00 0S 01 07 ot

03 0~ 03 00 01 ot

01 01 00 02 05 07

0.1 010 ] 011 1o

02 00 09 00

00 16 00

07 nl 02 01

00 01 nt 00 nt oo oo nl 00 01

] S 0 ~

0 3 00

0 0 0 2

0 0 0 O

oo 33 00

09 o i 23 03

O3 04 18 02

03 03 18 oo

110 4 .~

53

S.S 2,6

0.0 ~ 1

2.27

2.144

!iiiii, iiii!iii

01 01 . . . . O0 0S 06 02 18 0~ 04 03 oi 02 OO 02 nt nt 02 01 io 06

2,3

2.124

t

2,3 4.0 3.4 4 0,O I,O 4.0 2.4 1.3 4.0 0.0

53.53

2.65

01

02 "-ql 0.0 09

~

Ol

0.2 ~ 02 o~ 02 n 01 0 1 02 ~

00 01

0 0 0O

03 0 I

0.1 00

24 0 ~ ~23 oo

o; 0 I 04 oo

oo nt 4 1 02

os 0.2 3.9 oo

02 O0 2-'IF~'] 0 5 t 13.21 02 ~ 1 I 1.8J 00 04 07 05 02 00 0 4 ~ 0 ]

09 03 31 ol

I 6 07 10 10

05 10

22 0:3 22 15 07

13

0.S 01 07 ol 0.1 12

1.1 00 .00 Ol 100 .o0

01 00 00 01

04 00 02 01

01 G3 03 0S

O0 O0

0.1 O1~

O~ 01

O4 02 7? Ol

09 nt 47 oo

17 02 62 ol

• Tridla~ed Ihyrnidine incorporation expressed ~ × 10 "s. A s o r e of 0,0 = <,14 cpm. Positive responses de~n'nincd by maximum likelihood ~ 0 in bold and boxed. Data shown represent means of triplicate cultures. *O~iginai respond©r/stimulator.

See Table t for details.

' Not tested.

lator cells expressing D R w I 8 respond to the 10th IHWS D g w l 8 reference B-LCL (cell 9021) hut not to DRwl 7 reference B-LCL (cells 9019, 9022, 9023) (data not shown). TLC TC1.6, derived from priming T C I , responds to all five DRwlS-positive cells tested. All (11/11) DRwl7-positive cells tested did not stimulate clone TC1.6. One non-DR3-pusitive stimulator [cell 260 (DR2,wl 1)] also induced positive responses from clone TC1.6. Thirty-three T I C reactive with cells expressing D R w l 8 were derived from the TC2 priming, Nine of these respond to all DRwlS-positive stimulators and do not respond to any of the 34 non-DR3positive stimulators. T I C TC2.40 in Table 2 is representative of this group o f TLC. An additional 10 T I C reactive with cells expressing

DRw18 derived from priming TC2 respond t o Dgw18 cells and from one to five of the 45 non-DRwl8-positire cells. TMs latter group is represented in Table 2 by T I C TC2.31, TC2.85, TC2.95, and TC2.65. Most of the noo-DR3-positive cells that stimulate DRwl8-specific T I C express one of the D R alleles in the DRw52 family [DR3, DR5 (DRw11,DRw12), DRw6 (DRwlS,DRwI4), DRw8] although the non-DR3 cells recognized by each TLC differ. These non-DR3 recngnition patterns are reproducible and do not correlate to any known I-ILA-DR, DQ, or D P specificity nor with unusually high responses of the respoader to TCGF. Similar to the alloresponse to the mouse bm 12molecule [10], this heterogeneous response to the limited difference between the DR3 microvariants may result from

50

A.H. Johnson et al.

A

~

8

I:~D

,"

,:,~00

,:4h,

u~b o a ~ l . . . 0

ZCgaZ

. .o

i:Io~

U°Ab ~ . . . ° . .

the utilization of multiple T-cell receptors with varying specificity for D R and peptide. The remaining 14 clones recognize ceils expressing DRw18 plus cells expressing a specific, second DR/DRw52 molecule as discussed

bdow. T I C specific for a DRwl7-relared specificity were derived from the TC4 priming and are represented by T I C TC4.26 and TC4.52. T I C TC4.52 responds to 10 of the 11 DRw17-positive cells tested but does not respond m the DRw18-positive cells or to any of the 34 non-DR3-positive cells. The one DRw17-positive cell (cell 1064) characterized as nonstimulatory using statistical analysis stimulates proliferarion at a level of 1.4 x 103 cpm which is just below the cut point of 1.5 x 103 cpm required to be classified as a positive response. T I C TC4.26 responds to all 11 of the DRw17-positive cells and none of the DRw18-positive cells but does respond to two of the non-DR3-positive cells [cells 1233 (DRI,wl3) and 1099 (DR2,wl0)]. These T I C respond to 10th IHWS DRwI7 reference B-LCLs (cells 9019, 9022, 9023) but not to the DRw18 reference BLCL (cell 9021) (data not shown).

An alloproliferative TLC defines a supertypic DR3-rdated specificity. One T I C from the T C I priming, TCI.18,

I:,ooo

.,.oDo

FIGURE 1 Monoclonal antibody inhibition of the alloproliferative response of T-lymphocyte clones. The following mAbs were used: DR specific (A GSP4.1, • 12.43, or [] 7.3.19.11; DQwl,w3,w4 specific (~' 33.1); DQw2,w3,w4 specific (X SFR16-PI.2); DP specific (ll B7/21); and •z--microglobulin (class I) specific (O BBM.I). Stimulation of the TIC in counts per minute are shown at four dilutions of mAh. TIC: (A) TCI.18 (DR3), (B) TC4.26 (DRwI7), (C) TC2.40 (DRwl8), and (D) TC2.27 (DRw18+DRw52c).

proliferates exclusively to both DRwI8- and DRwl7positive stimulators. This T I C was derived from a priming utilizing a DRwlS-positive stimulator and a nonDR3-positive responder. The substantial D R mismatch between responder and stimulator resulted in recognition of a supertypic, DR3-relared determinant although the priming also produced a T I C (TC1.6) which recognizes cells expressing DRw18 as described above.

Some alloproliferative TLC recognize cells expressing DRwlSorcdlsexpressingDRw52c, Although the majori v / o f T L C specific for cells expressing DRw18 do not respond to any DRw52c-positive cells tested, two T I C (TC2.27 and TC2.144) were observed to weakly recognize many cells expressing DRw52c on repeated testing (Table 2). A log likelihood ratios test favored three distributions for stimulator cells tested with T I C TC2.27 and TC2.144 in contrast to the two distributions favored for all other T I C , The first cluster included all DRwl8-positive cells (cut point = >3.5 for TC2.27 and >4.3 for TC2.144). The second cluster included all DRw52c-positive cells plus several non-DRw18, nunDRw52c positive cells (cut point = >0.2 for TC2.27 and >0.3 for TC2.144). The DRw52c-positive stimulator cells express DRw52c in conjunction with DRwl 3

Recognition of DRwI7 and DRwI8 Microvariants

51

TABLE 3 Linear contrast of mean proliferation levels among DRwI8, DRw52c, and other DR alleles T-lymphocyteclones Class II alleles Dl~.wl8 Dgw52¢ O t h er DR D R w I 8 vs. DRw52c ~ D Kwl8 vs. other DKw52c vs. other

TC2.27

TC2.144

TC2.31

TC2.40

7125 -* 3350 J 1230 ± 333 277 ± 266

18,697 ± 4427 1756 ± 1087 355 -* 404

4446 ± 2446 76 ± 95 105 ± 149

22,724 ± 16,416 182 ± 165 233 ± 530

p < 0.0001 p < 0.0001 p < 0.0001

p < 0.0001 p < 0.0001 p < 0.B001

p < O.000t p < 0.0001 p = 0.7012

p < 0.0001 ? < 0.0001 p < 0.6164

• Mealt counts per minute ± standazd deviation. *Linear contrasts ninon8 g~oups with one degree of freedom.

as well as one ceil which expresses DRw52c in conjunction with D R w l I (cell 2015). The third cluster included only non-DRwl8 or DRw52copositive cells. The mean counts per minute generated in the proliferation assays as determined for the DRw18, the DRw52c, and the negative control stimulators for T I C TC2.27 and TC2.1zl4 are summarized in Table 3. Two T I C (TC2.31 and TC2.40) that do not recognize DRw52c are included for comparison. To test for a difference in mean proliferation levels among DR.3, DRw52c, and other D R types, the means of the three groups were compared by linear contrast among groups. The mean responses o f TC2.27 and TC2.144 to the nine DRw52c-positive cells were significantly greater than the background responses to 18 cells expressing non-DR3, non-DRw52c alleles (p < 0.0001) yet not as strong as the positive response to the five cells expressing DRw18 (p < 0.0001). For the T I C that do not recognize DRw52c, the mean responses to cells expressing DRw52c were nor different from the group of negative control stimulators.

One alloproliferative TLC recognizes cells that ex#ress DRwI8 or DRwl4,Dw9 but not cells that express DRwI4,Dwl6. One T I C from the TC2 priming, TC2.124, recognizes stimulators expressing DRw18 or DRw14,Dw9 with equal levels of proliferation (Table 2) but does not recognize stimulator cells expressing DRw14,Dw16 (data not shown). In addition to reference stimulator PBL, IHWS B-LCL 9054 and 9057, cells expressing DRwI4,Dwg, reproducibly stimulated prolifetudon ofTC2.124, while IHWS B-LCL 9064 and 9099, cells expressing DRw14,Dw16, reproducibly failed to stimulate proliferation by T I C TC2.124. TC2.124 also recognizes stimulator cells from an individual (cell 2008) expressing a third DRw14 haplotype ( D R w l 4 , D - ) . By c D N A sequence analysis, this haplotype expresses the same D R allele as DRw14,Dwg, but

differs in the DRw52 allele expressed [Hurley, in preporation]. DISCUSSION Among DR alleles, the DR3 microvariants are unusual in that they differ from each other in the second variable region (residues 26 and 28) while the first and the third variable regions remain unchanged [13]. In the Brown model of class lI structure [1], residues 26 (DRwl7/tyr; DRw18/phe) arm 28 (DRw17/asp; DRw18/giu) are located on a ,8 pleated sheet on the floor of the antigenbinding groove and are predicted to interact with antigen (Fig. 2A). Indeed, it has been shown that, although a more drastic substitution, the interchange of Glu/Val at a position in the mouse l-E molecule analogous to position 28 in D R ~ chain controls the efficiency of binding of pigeon cytochrome c to I-E molecules, and, hence, the availability of the antigen-class II complex for T-cell receptor stimulation [25]. Thus, we expect that one or both substitutions at positions 26 and 28 will affect the binding of some peptides to these microvariants. Two additional sites of variation between DRw17 and DRw18 are found at ~8-chain residues 47 and 86, both located in the more conserved regions of the D R molecule. These residues have one of the two alternative amino acids found at these positions in all D R molecules. The substitution at position 86 is the only difference found between many microvariants (e.g., alleles of DR4, DRw 11, DRw8, an d DRw 13 ) and has beer* shown to affect allorecngnitioa [26]. The majority of alloprollferative T I C derived in this study are specific for either D R w l 7 or DRw18 molecules indicating that the four amino acids which differ between the ~ chains of DRw17 and DRw18 are not trivial and create a potent T-cell recognition determinant(s). Although the majority of the TLC were derived in priming combinations between individuals expressing

52

A

A.H. Johnson et ai.

B

':~'~'~~J"~/~. '!. e~,~.,.~.~ ¢

D

the two microvariants D R w l 7 and DRwl8, these subtypic determinants also are recognized in priming combinations where *he difference between DR alleles expressed by responder and stimulator is substantial as shown here for DRw18 (priming TC 1, TC 1.6) and elsewhere for DRw17 [27, 28]. The recognition patterns of these alloreactive T I C were correlated to the protein sequences of the DR .8 chains in an attempt to understand the structure of the T-cell al/orecngnition epitope(s). Inspection of amino acid sequences of all published D R .8 chains could identify no linear sequence unique to either DRw17 or DRw18. Furthermore, it appears that several of the DRw17/DRw18 differences, notably variable region residues 26 and 28, which are located on the floor of the antigen binding groove, are presumably hidden from the T-cell receptor. Therefore, one explanation of the reactivity is that the DRw17- or DRw18-related determinant recognized by the T I C may result, in part, from a differential binding o f endogenous peptides. This hypothesis is supported by studies which show that T I C TC4.26, which recognizes stimulator cells expressing DRw17, does not recognize a DRwl7-positive B-LCL with an antigen processing defect [29]. The limited differences between DRw17 and DRw18 do not appear to alter a supertypic, DR3-reluted recognition determinant(s) since one T I C recognizes both DRw17 and DRw18. Since DRw17 and

FIGURE 2 A model of the antigenbinding site of HI.A-DR [1] showing the amino acid differences between two class II molecules. (A) DRwl8 and DRw17, fl chains differ at positions 26, 28, 47, 86; (B) DRwI8 and DRw52c, ,8 chains differ at pasitions 10,11, 12, 37, 57, 60, 7,t, 86; (C) DRw18 and DRwl4.01 (Dw9), chains differ at positions 28, 37, 57, 60, 71, 73, 74, 77, 86; (D) DRwI8 and DRw14.02 (Dw16), 3 chains differ at positions 71, 73, 74, 77. Amino acids that differ a~ represented by closed circles (0) and she amino acid substitutions are indicated by their single letter code. The first amino acid listed is the residue found in DRw18.

D R w l 8 are indistinguishable to some T I C , the effect of microvariation of DRw17 and DRw18 on specific immune responses may vary. Consequently, in some instances these subtle differences may not affect presentation of some antigenic peptides or the thymic selection of some T-cell receptors. Inspection of all reported D R ,8-chain sequences to define the shared DR3-related determinant(s) reveals that there are no sequences unique only to these two DR3 microvariants. The third variable region located in an a helix, a prime T-cell recognition site [1, 10, 30-34], is shared by DRw17 and DRw18 but also shared with DRw52a. Among DR alleles, gene conversion appears to have been a potent force which has shuffled a limited number of nucleotide segments among genes creating new alleles. It has been hypothesized that a gene conversion involving .8-chain genes encoding DRw6 and DRw52a alleles gave rise to the DR3 fl-chaln precursor. This precursor shared the third variable region with the DRw52a 3 chain while retaining the remainder of the DRw6 .8 chain. Additional gene conversion events resulted in DRw17 and DRw18,8 chains which share the amino-terminal portion of the second variable region (amino acids 26.-36) with the DRw52a and DRw52c fl chains, respectively [13, 35] (Fig. 2). The large number (i.e., 19) of T I C that recognize the DRw18 molecule allowed us to investigate the possibility that the gene conversion generated shared T-cell recognition deter-

Recognition of DRwI7 and DRwl8 Microvariants

53

minants. Two TLC specific for a DRwl8-related specimight also inhibit T-cell recognition. Since TLC do recficity from the TC2 priming did recognize stimulators ogeize DRwl4.01, the molecular interactions involved that express DRw52c, although weakly. Although resiin the recognition of DRwl4,Owg-positive cells cannot dues 26 and 28 are shared, D R w l 8 and DRw52 differ be explained on the basis of sequence similarities. dramatically at position 74, a basic acginine (DRwlS) The D R molecule is only one of the antigen-bieding compared to an uncharged glutamine (DRw52c). Since molecules used by the immune system to present antiresidue 74, in the D R B chain a helix, is in a position to gen to T-cell receptors. This repertoire of antigen-bindinteract with T-cell receptors [1, 3, 3ll-32], it is surprising molecules is thought to provide an individual with ing that any of the T I C can recognize both DRwl8 and the capability to recognize a multitude of diverse forDRw52c molecules. This amino acid difference may be eign antigens, in fact, each antigen-binding molecule the cause of the significantly weaker response observed may differ in the antigens bound as shown for the both at the level of proliferation and in the frequency of mouse homolognes of the D R and D Q molecules [37, TLC exhibiting this dual recognition pattern. Similar 38]. Since, in the human, D R and D Q aUeles are not differential levels of recognition by alloreactive T I C randomly associated on most haplotypes, it is likely that have been observed in studies of the effect of amino D I U D Q associations may have evolved to maximize the acid substitutions in the a helical region of HLA-A2 antigen-binding potential for an individual. This may be microvariants [31,321. true in the case of the DR3 microvariants which are associated with different D Q alleles (DRwI7,DQw2 One T I C unexpectantly also recognized some and DRwl 8,DQwZi). These differential D Q associations DRw14-positive stimulators as well as DRwl8.positive may compensate for the differences in the immune restimulators. In contrast to the different levels of proliferation observed with the T I C , which detect cells exsponse profiles of D R w l 7 and DRwl8. Thus, selective advantage could be one of the forces driving the linkage pressing DRw18/Dgw52c, the level of proliferative redisequilibrium which is so characteristic of some HLA sponse to both D R w l 8 and D R w I 4 was equally as haplotypes. strong. Two sequences for D R w I 4 corresponding to The intricate and sometimes unpredictable allo~ the HLA-D types Dw9 (Dgwl4.01) and Dw16 recognition patterns observed in this study support the (DR14.02) have been reported [36]. The ~-chain allele view that a D R molecule must be viewed as a dynamic encoding DRwl4.02 has been hypothesized to have whole. Structural similarities or differences between arisen by a gene conversion event between DRw18 and DR allelic products often cannot be predicted by priD R I B-chain genes resulting in a sharing of amino acids mary amino acid sequence comparisons and must be 1-70 between DRw18 and DRwI4.02 B chains, a secdefined in functiunal assays using T-cell reagents or antition including the first and the second variable regions bodies. This is of great significance in the field of transand the majority of the DRw17/DRw18 differences plantation in which matching protocols based on seg(Fig. 2D). In contrast, the DRwI4.01 ~8 chain differs ments of shared amino acid sequences have been from DRw18 by multiple substitutions (10) in the secproposed [391. Clearly, the molecular context of a speond and third variable regions as well as in the framecific sequence is just as important as the sequence itself work regions (Fig. 2C). While sequence similarities in forming a T-cell or antibody recognition site. in addimight predict T-cell recognition of D R w l 8 and tion, D R alleUc variation and, specifically, microvariaDgwl4.02, T I C TC2.124 dearly recognizes stimuladun as found between DRw17 and DRw18 alleles, may tors expressing the DRwI4.01 allele but not the not produce a single effect but may or may not alter the DRw14.02 allele. In fact, none of the T I C recognize ability of the molecule to present a specific antigen or stimulators expressing DRwlZt.02. Thus, it is likely that select a specific T-cell receptor. Thus, microvaciatiun the differences between D R w I 8 and DRw14.02 in the appears to create distinct but overlapping immune rechain a helix (residues 71, 73, 74, 77) strongly inhibit sponse profiles for these DR3 allefic products. T-cell recognition o f both allelic products. Since three of these residues (71, 73, and 77) are shared by DRwI4.01 and DRwI4.02 alleles, one would predict that residue 74 may be key in preventing the recognition of DKw14.02 by TC2.124. The difference at resiACKNOWLEDGME1wrs due 74 between DKw18 and DRwlZL02 is substantial, a This work was supported by Grant AI 23371 from the Nabasic arginine for a nonpolar, smaller alanine. Interesttional Institutes of Health. We would like m thank Dr. Chris ingly, DRwI4.01 also has a substantial difference at coAmos for the statistical analysis of the TLC responses and the members of our laboratories for their assistance in the prodon 74 compared to DRwlS, an acidic glutamic acid curement of cells and the serologic and cellular typing. compared to the basic atginine, an interchange which

54

A.H. Johnson et al.

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