Cell, Vol. 66, 1037-1049,
September
6, 1991, Copyright
0 1991 by Cell Press
Mutational Analysis of the Interaction between CD4 and Class II MHC: Class II Antigens Contact CD4 on a Surface Opposite the gpl20-Binding Site S. Fleury, ‘t D. Lamarre, l S. Meloche,’ S.-E. Ryu,* C. Cantin,’ W. A. Hendrickson,* and R.-P. Sekaly*t ‘Laboratoire d’lmmunologie lnstitut de Recherches Cliniques de Montreal Montreal, Quebec H2W lR7 Canada tD6partement de Microbiologic et d’lmmunologie Universite de Montreal Montreal, Quebec H3C 3J7 Canada *Department of Biochemistry and Molecular Biophysics Howard Hughes Medical Institute Columbia University New York, New York 10032
Summary Using functional and adhesion assays, we have studied the ability of 30 human CD4 mutants to interact with class II major histocompatibility complex (MHC) molecules and also with gp120 from human immunodeficiency virus. The mutants cover the four domains (Dl-D4) of CD4 and include several single-site substitutions. Analysis of the results, in the context of the CD4 crystal structure, shows that mutations that affect the interaction with class II MHC molecules are located on three exposed loops from CD4 domains 1 and 2. The specifically implicated residues, 19, 89, and 185, are separated from one another by 9 A, 24 A, and 24 A on one face of the CD4 molecule. Moreover, the class II binding site does not include residues 43 to 49 of the CD4 molecule, a region on an opposite face known to be involved in the binding of gp120. Introduction The CD4 molecule is a nonpolymorphic membrane glycoprotein composed of four extracellular domains (D, domain; Dl-D4) that share sequence homologies with immunoglobulin (lg) domains (Maddon et al., 1985, 1987; Williams and Barclay, 1988). The CD4 protein is expressed on a subpopulation of mature T cells (for review, see Littman, 1987). The T cell receptor (TcR) of these CD4+ cells recognizes antigen (Ag) only when presented by a class II molecule of the major histocompatibility complex (MHC) (Biddison et al., 1982, 1983; Swain, 1983). This absolute correlation between T cell specificity and CD4 phenotype is thought to result from a selective process in the thymus involving engagement of the TcR and CD4 with class II MHC molecules. The specific expression of the CD4 molecule on class II-restricted T cells has led to the hypothesis that CD4 interacts with a nonpolymorphic determinant of class II proteins on antigen-presenting celis (Marrack et al., 1983; Wilde et al., 1983; Swain et al., 1984). This association
has now been established through reconstitution of CD4class II-dependent T cell function and cell adhesion. Introduction of CD4 intoCD4-Tcell hybridomas greatly potentiates the response of these cells to Ag-specific stimulation when class II molecules are expressed on target cells (Sleckman et al., 1987; Gay et al., 1987; Lamarre et al., 1989a), and this response correlates with a physical association between the hybridoma and antigen-presenting cells (Lamarre et al., 1989a, 1989b). Other studies have also shown that class II MHC-positive cells can specifically aggregate with nonlymphoid cells expressing high levels of human CD4 (Doyle and Strominger, 1987). The CD4 protein serves as the primary cellular receptor for the human immunodeficiency virus (HIV) (Klatzmann et al., 1984a, 1984b; Dalgleish et al., 1984) through its specific binding to the gpl20 envelope glycoprotein of the virus (Maddon et al., 1986; McDougal et al., 1986). The gpl20-binding site on CD4 lies on the first N-terminal domain (Peterson and Seed, 1988; Landau et al., 1988; Arthos et al., 1988; Clayton et al., 1989; Brodsky et al., 1990; Ashkenazi et al., 1990). However, less is known about the regions of the CD4 molecule that interact with their physiological ligands, the class II MHC molecules. Previous mutational analysis (Clayton et al., 1989; Lamarre et al., 1989a) and antibody-blocking experiments (Lamarre et al., 1989b) have suggested that residues in Dl as well as D2 and D3 of the CD4 molecule are involved in the interaction with class II MHC molecules. However, nearly all of these mutations involved substitutions of several residues and included insertions or deletions. This makes it difficult to evaluate, in the case of impaired function, whether the effect is due to a direct impact of the mutated residues on the interaction of CD4 with class II MHC molecules or whether the impact is indirect and due to perturbation of the three-dimensional structure of CD4. An important point is that the three-dimensional structure of CD4 is now known. Crystal structures of soluble CD4 proteins constituting the two N-terminal domains, Dl and D2, were recently solved at atomic resolution (Wang et al., 1990; Ryu et al., 1990). Dl sharessignificant structural similarities with lg VK domains, as predicted from sequence homology comparisons (Maddon et al., 1987; Peterson and Seed, 1988; Clayton et al., 1988); however, the CC’ and CDRS-like loops from the lg dimer interface are shortened, and the CDR2-like loop is elongated. The folding of D2 resembles that of an lg constant domain, but differs significantly in the placement of a disulfide bond between strands in the same sheet and in the reduced length of the 6 strands. Dl and D2 share a common 8 strand (G strand in Dl, A strand in D2) corresponding to residues 88-103, which results in tight packing between these two domains, leading to a rod-shaped molecular fragment of approximate dimensions 25 x 25 x 60 A. In this article, we report the effect of a panel of mutants, which included several single residue substitutions, within the four domains of the human CD4 molecule on their interaction with class II MHC and gpl20 molecules. The
Cell
1038
structural integrity of the CDCmutated proteins was evaluated by a serological analysis using a panel of 36 anti-CD4 monoclonal antibodies (MAbs) and CD4 MAb clusters. Functional evaluation and adhesion property analysis of these CD4 mutated molecules were carried out following infection in a T cell hybridoma (3DT52.5.8). Effects that might be mediated indirectlythrough CD4-TcR interaction are excluded in the assay system that was used. In this report, we are also able to couple the functional assays with theatomicstructureof CD4, inordertospatiallydefine the residues on CD4 involved in binding to class II MHC molecules. Mutations that strongly affected recognition of class II MHC molecules in the functional and adhesion assays are clustered in three distinct segments. Two of these segments, which are located in Dl , are homologous to complementary determining regions (CD&) of lg. The third segment is located in D2 near the interface with Dl. Results Description, Expression, and Serological Analysis of CD4 Mutants Thirty CD4 mutant proteins (M9-M38) were expressed in the murine CD4- CD8- T cell hybridoma 3DT52.5.8 (see Table 1 for description). These mutations cover the four Ig-related domains of the CD4 molecule, Dl to D4. Nineteen mutations are located in Dl, 5 in D2, 2 in D3, and 4 in D4. These mutations introduced into CD4 have been described previously and consist of: epitope-loss mutations (M9-M20, M24-M28, M30, M31, and M35), which are random substitutions of 1 to 3 residues in Dl, D2, and D4 of the CD4 molecule (Peterson and Seed, 1988); linker-scanning mutations (M21-M23, M29, and M32), which are insertions of the dipeptide sequence serinelarginine (SR motif) at predicted hydrophilic sites in Dl and D2 (Mizukami et al., 1988); and homolog-scanning mutations (M33, M34, and M36-M38), which are substitutions of 3 to 8 residues of nonconsented sequences between the human and mouse CD4 molecule in D3 and D4 (Lamarre et al., 1989a). All the mutated proteins were stably expressed in the 3DT52.5.8 T cell hybridoma using a retroviral vector as previously described (Lamarre et al., 1989a). The cellular expression of the different CD4 mutants was determined by flow cytometry using appropriate anti-CD4 MAbs (see Table 1). A detailed serological analysis was undertaken to determine the structural impact of the mutations on the conformation of the CD4 protein. The results of this serological analysis are summarized in Table 1 as the total number of epitopes conserved (C), affected (A), and lost(L) for each mutant. The number of epitopes conserved for each mutant varies between 5 and 35, indicating that some mutations are more disruptive than others. Sixteen of the mutants are recognized by at least half of the antibodies, and all except M23 are detected by at least 30% of the antibodies. The M23 mutation (SR insertion after residue 57) interrupts the D 8 strand in Dl, which likely explains its global effect on most antibodies that map to the closely associated Dl and D2.
Representative monoclonal antibodies used for staining were grouped in eight different clusters (Figure l), based on the mapping of these antibodies to our CD4 mutants (Fleury et al., unpublished data) and to other previously published CD4 mutants (Sattentau et al., 1989; Brodsky et al., 1990) and based on their capacity to compete the binding of CDCspecific MAbs (Sattentau et al., 1986, 1989; Healey et al., 1990). This cluster analysis allows us to define better the impact of the different mutations and region(s) of the CD4 molecule affected. The majority of the 36 MAb epitopes are found in stretches of residues that are exposed to solvent and are homologous to CDR loops, as shown by the CD4 crystal structure (see Table 1). The first three clusters define regions closely related but spatially distinct. Clusters 1 (OKT4A), 2 (OKT4D), and 3 (Leu3a) delineate different regions between the CDRl-, CDR2-, and CDR3-like loops. Cluster 4 (L-93 and L-206) is specific for the CDRBlike loop, while cluster 5 (101.69 and 866.6.1) delineates the CDRl- and CDR2-like loops in Dl, and cluster 6 (L-68 and L-92) defines the CDRland CDRS-like loops in Dl. In D2, cluster 7 (OKT4B and MT-151) corresponds to the FG loop region. Cluster 8 (L-120 and MT-429) defines D3 and D4. Of the 27 CD4 mutants in Figure 1,18 mutants have lost only one cluster, 6 mutants have lost two clusters, and only 3 mutants have lost three clusters. These results clearly show that only local conformation changes are induced by the mutations, as reflected by the alteration of one, two, and in rare cases, three loops; the other regions are still intact and able to interact with other molecules, such as gp120 and class II MHC molecules. Functional Analysis of the CD4-Class II MHC Interaction Efficient stimulation of CD4+ T cells is dependent on the specific association of CD4 with class II MHC molecules on the antigen-presenting cells. However, CD4 also appears to associate with the TcR during the effector phase of the immune response (Saizawa et al., 1987; Kupfer et al., 1987). To isolate the CD4-class II component of this interaction, we have utilized the 3DT52.5 T cell hybridoma whose TcR is specific for the murine class I MHC protein H-2Dd. The interaction between the TcR on this hybridoma and the murine H-2Dd on the target cell is required for an IL-2 response (Greenstein et al., 1984). However, expression of human CD4 on the hybridoma and the human class II HLA-DP on the target cell (Table 2, WT and Figure 2) increases the IL-2 responss20- to 30-fold (Lamarre et al., 1989a, 1989b). The T cell hybridoma cell lines expressing the different CD4 mutants were tested for their IL-2 response in the presence of target cells display&g the murine H-2Dd class I protein alone or together with the human HLA-DP class II protein. To minimize cell line variation, hybridoma populations for each mutant were selected that gave similar responses when stimulated with an anti-TcR MAb. The results of these assays are summarized in Table 2. All of the CD4 hybridomas produced low, levels of IL-2 in the presence of DAP-3Dd stim,ulaJor&tls (see Table 2). Sev-
CD4Class 1039
II Recognition
Table 1. Description,
ExpressIon,
and Serological
Analysis of CD4 Mutants Epitopes
Mutants
Mutations
C
WT Dl
A
c
M. F. V.
36
0
0
18
M27
S19Y Q20K Q20H/H27R S23G1124VIH27L G38E Q40P Deletion F43S49 F43L T45P K46NIG47V G47R P48S insertion 48[SR] Insertion 55]SR] Insertion 57[SR] S60R E87L Q89L Q94K
26 19 25 4 18 23 12 ND 29 17 14 12 27 11 5 13 ND 21 21
7 14 6 4 15 9 18 ND 5 12 18 24 8 21 23 21 ND 10 14
3 3 5 2 3 4 6 ND 2 7 4 0 1 4 8 2 ND 5 1
15 11 26 13 9 23 8 17 23 12 10 10 7 9 11 10 33 16 26
M28 M29 M30 M31 M32
P122H Insertion 133[SR] Ql65E Q165P InsertIon 165[SR]
18 28 13 17 13
18 7 19 17 21
0 1 4 2 2
11 10 12 21 14
M33
277-280
10
14
9
M34
293-300
33
0
10
M35 M36
V326l 328-330
35 23
0 3
22 12
M37
349-356
18
6
14
M38
363-369
26
3
9
r M9 Ml0 CDRl Ml1 t Ml2 Ml3 Ml4 Ml5 Ml6 CDR2 Ml7 Ml8 Ml9 M20 M21 M22 M23 M24
D2
D3 h m h M
EAKT D--K R ATQLQKN KVAQLNNT
h m h m h m
KRE EEQ DSGQVLLE EGDKVKMD PTWSTPV -SRGVNQ
D4
The CD4 mutants M9-M20, M24-M28, M30, M31, and M35 consist of single or multiple point mutations with impaired antibody recognition. Mutants M21-M23. M29, and M32 are insertion mutants in which codons for serine-arginine residues were inserted after the indicated amino acid. Mutants M33, M34, and M36-M38 are clustered substitution mutants in which three to eight amino acids of human CD4 (T4) were replaced with the corresponding residues of the murine CD4 (L3T4). The numbering of CD4 amino acids is according to Maddon et al. (1985) and the alignment with L3T4 is according to Littman and Gettner (1987). The mutant CD4 cDNAs were subcloned into the retroviral vector pMNC-stuffer and introduced into the murine T cell hybridoma 3DT52.5.8. The cellular levels of the CD4 wild-type and CD4 mutants were determined by flow cytometry using OKT4 MAb (L-68 in the case of M20, M28, and M32) and are expressed as the mean fluorescence value (M. F. V.). The CD4 mutants were then stained with 36 murine anti-CD4 MAbs and analyzed on a FACScan flow cytometer. C, epitope conserved; A, epitope affected; and L, epitope lost. Based on the CD4 crystal structure (Wang et al., 1990; Ryu et al., 1990) three CDR-like loops are identified on Dl: CDRl-like (residues 19-24) CDR2-like (residues 41-59) and CDRS-like (residues 87-88). In D2, three loops are also characterized: loop BC (residues 122-126), loop CC’ (residues 133-135). and loop FG (residues 163-165). ND, not determined. B Ml2 was stained with only 10 MAbs.
eral of the mutants showed moderate to marked reduction in IL-2 production at saturating concentration of effector or target cells. These results, normalized to the IL-2 production in the absence of class II protein, are shown in Figures 3A.and 38. Eleven mutants significantly affected the IL-2 response, yielding only a 1.5- to 6-fold stimulation, in contrast to the 20- to 30-fold stimulation observed with the native CD4 protein. For each of these mutants, this ineffi-
cient stimulation of IL-2 was reproduced in a minimum of five different populations of the hybridomas derived from two independent retroviral vector infections. The remaining CD4 mutants gave at least a g-fold stimulation of IL-2 production and were therefore considered not to have a deleterious effect on CD4 recognition of class II protein. In Dl, 8 out of 19 CD4 mutants showed only a 1.5- to B-fold increase in IL-2 production in the presence of target
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7:4;-Class
II Recognition
Table 2. Ability of CD4 Mutants to Maintain a Class II MHC-Dependent CD4-Class MHC Interaction (IL-2 U/ml)
IL-2 Production
and to Bind rgp120
II
Anti-CD4 MAb (IL-2 U/ml)
Anti-T&t Stimulation (IL-2 U/ml)
Rosette Formation
rgp120 Binding
Mutants
Mutations
Dd
DdDP
w-r
-
330
7,040
930
35,000
+
11.5
S19Y Q20K Q20HIH27R S23GA24VIH27L G38E Q40P Deletion F43S49 F43L T45P K46NIG47V G47R P48S Insertion 48[SR] Insertion 55[SR] Insertion 57[SR] S60R E67L Q89L Q94H
340 220 350 200 445 500 210 200 230 270 230 500 320 170 380 750 200 185 530
1,000 4,000 500 4,200 4,000 3,880 4,330 7,570 350 400 370 4,400 7,040 1,010 660 8,600 7,570 550 4,780
ND ND 700 ND 640 370 640 ND ND ND 350 770 920 580 640 1,200 ND 450 ND
30,000 32,000 24,800 31,100 17,400 30,720 21,760 33,700 31,120 32,000 26,670 35,000 32,000 24,240 35,000 32,000 28,900 21,760 32,000
+ + + + + + -
+
19.2 25.8 46.9 7.5 62.9 306.7 NSB NSB NSB NSB NSB 9.9 NSB NSB NSB 15.4 13.9 7.0 13.0
D2 M28 M29 M30 M31 M32
Pl22H Insertion 133[SR] Q165E Q165P Insertion 165[SR]
600 210 580 940 330
5,830 4,000 5,400 5,400 900
ND 350 ND 900 380
19.100 17.280 32,000 32,000 32,000
+ + + -
13.5 10.1 11.1 14.7 7.5
D3 M33
277-280
200
4,000
580
21,600
+
M34
293-300
200
2,980
ND
24,600
+
8.0
D4 M35 M36
V326l 328-330
530 200
4.880 1,700
620 ND
32,000 32,000
+ +
9.6 15.1
M37
349-356
200
630
ND
14.700
+I-
14.5
M36
363-369
190
2,050
ND
35,000
+
19.7
Kd (nM)
Dl M9 Ml0 Ml1 Ml2 Ml3 Ml4 Ml5 Ml6 Ml7 Ml6 Ml9 M20 M21 M22 M23 M24 M25 M26 M27
h EA KT mD--K hRATQLQKN mKVAQLNNT
h KRE mEEQ hDSGQVLLE mEGDKVKMD h P T WS T P V m-SRGVNQ
+ + + +
NSB
The ability of the CD4 wild-type (WT) and CD4 mutants M9 to M38 to interact with class II MHC molecules was evaluated in a functional and an adhesion assay. Shown are IL-2 production (U/ml) and rosette formation (f, presence of conjugates; -, absence of conjugates) obtained by the 3DT52.5.8 cells expressing the various CD4 mutants following a 20 hr coculture period with the murine DAP-3 cells expressing Dd or DdDP. Results of independent functional assays did not show more than a 15% variation. Each coculture was also carried out in the presence of 20 nM of the CDCspecific MAbs OKT4B or L-68. The 3DT52.5.8 effector cells were also stimulated with coated anti-TcR MAb F23.1 to evaluate their intrinsic ability to produce IL-2. The rgpl20-binding properties of the CD4 mutants were determined by incubating the 3DT52.5.8 transfectants with increasing concentrations of ‘2SI-labeled rgpl20 for 4 hr at 25OC in the presence or absence of a lOO-fold excess of rgpl20. The saturation binding curves were then analyzed by nonlinear least-squares regression analysis according to mass action law (DeLBan et al., 1982). The results are reported as the dissociation constants (Kds) in nM. The Kd for each CD4 mutant was determined by two separate rgpl20-binding assays, and only 10% of variation was observed. NSB, no specific binding; ND, not determined.
tants were tested for their capacity to associate with p561Ck and for the autophosphorylation of the tyrosine kinase upon cross-linking of CD4 with CDCspecific MAbs; indeed, our results (S. Fleury, unpublished data) show that all mutated CD4 molecules were efficiently associated with p!Wk. Furthermore, significant phosphorylation of the
tyrosine kinase was observed 60 s after cross-linking of the mutated CD4 molecules. In D3 and D4, mutant M37, which was made by substituting human residues with corresponding murine residues in the region spanning amino acids 349-356, shows only a 3-fold increase in IL-2 production (Table 2 and Figure
Cdl 1042
38) in the presence of the appropriate target cells. Mutants M33, M34, M35, M36, and M38 displayed significant class II MHC binding, as demonstrated by a 9- to 20-fold increase in IL-2 production in the presence of class II MHC molecules on target cells (Figure 38). From these res.ults, it appears that in D3 and D4, of those CD4 mutants tested, only residues 349-356 could be involved in the CD4-class II MHC interaction. Figure 2. Schematic Description of the Cellular System Used to Evaluate the CD4-Class II Interaction The TcR of the T cell hybridoma 3DT52.5 is specific for the mouse class I antigen (H-2Dd). An L3T4- variant of 3DT52.5, called 3DT52.5.8, was infected with a retrovirus vector containing a cDNA encoding the human wild-type CD4 (I1 B-3 hybridoma). Mutated CD4 moleculeswere also introduced in the 3DT52.5.8 hybridoma. (A) shows the interaction between effector cells expressing only the TcR and target cells expressing H-2Dd molecules. (6) shows that the introduction of the human CD4 molecule in the effector cells does not lead to a significant increase in IL-2 production, when cocultured with target cells expressing only class I molecules. (C)shows that when CD4 and its ligand (HtADP molecule) are expressed on effector and target cells, respectively, a significant increase (greater than 20-fold) in IL-2 production is obSeNed, compared with (B), where target cells expressed only the TcR ligand (H-2Dd).
Analysis of the Adhesion Properties of the CD4 Mutants to Class II MHC-Positive Cells A qualitative binding assay was performed concurrently to determine the effect of each mutation on the physical interaction between CD4 and class II MHC molecules. Hybridoma T cells expressing TcR and wild-type CD4 molecules will form conjugates with target cells expressing both TcR and CD4 ligands, i.e., murine class I (H-2Dd) and human class II (HLA-DP), respectively (Figures 4a and 4b). Conjugate formation is specifically inhibited by a MAb specific for CD4 (Figure 4~). In contrast to other assays used to study the adhesion function of CD4 (Doyle and Strominger, 1987; Clayton et al., 1989; Rosenstein et al., 1990), conjugate formation with the 3DT52.5.8 hybridoma does
~
21
01
D2
D3
D4
B
C
CD4 mutants Figure 3. Quantitative Affinity
Analysis of a Panel of CD4 Mutants for Their Capacity to Interact with Class II MHC Molecules and for Their rgpl20Sinding
The CD4 wild type (WT) and CD4 mutants M9-M38 were analyzed for their ability to produce IL-2 ([A] and [B]). Results are reported as the ratio of IL-2 production obtained in the presence of class II in their absence(DAP Dd). The CD4 mutants were also analyzed for their capacity to bind ‘ZWabeled ([Cl and [D]). The results are reported as the ratio of the equilibrium binding constants (Ka:l/Kd) of CD4 wild-type molecule.
with DAP-3 cells expressing either Dd or DdDP molecules (DAP DdDP) over the value obtained rgp120 in whole-cell saturationbinding assays the vario@ CD$m&ts overthe value of the
CDCClass 1043
II Recognition
Figure 4. Ability of CD4
Mutants to Form Rosetteswith Target DAP DdDPCells
The 3DT52.5.9 cells expressing the CD4 wild-type and CD4 mutants were,analyzedfor their ability to form rosetteswhen coculturedwith murine fibroblastic DAP cells expressing Dd or DaDP. Following coculture, the cells were observed by phase-contrast microscopy and photographed under 100 x magnification. (a) CD4 wild-type (I1 B-3 hybridoma) and DAP Dd cells. (b) CD4 wild-type and DAP DdDP cells. (c) CD4 wild-type and DAP DdDP cells in the presence of OKT4B MAb (20 nM). (d) Mutant M9 (S19Y) and DAP DdDP cells. (e) Mutant Ml5 (deletion in F43-549) and DAP DdDP cells. (9 Mutant Ml5 and DAP DdDP cells in the presence of OKT4S MAb (20 nM).
not require centrifugation or expression of unusually high levels of the CD4 molecule. However, CD4 mutants that did not show an increase in IL-2 production of greater than 1.5-to B-fold (M9, Mll, M17, M16, M19, M22, M23, M26, M31, M32, and M37) upon interaction with class II MHC molecules (Table 2 and Figures 3A and 38) also failed to form rosettes with class II-positive cells (Table 2 and Figure 4d). M15, which corresponds to a partial deletion of the CDR2 loop, still allows rosette formation (Figure 4e), and binding is inhibited by CDCspecific MAb (Table 2 and Figure 4f). Other mutants in the four domains of the CD4 molecule, which all showed significant increases in IL-2 production (greater than g-fold) in the presence of class II MHC-positive cells, also physically interact with cells expressing DdDP, as demonstrated by the formation of conjugates in the adhesion assay (Table 2). Furthermore, rosette formation (Figure 4c) was inhibited by MAbs specific to the CD4 molecule (OKT4B or L-68), thus confirming the specificity of this assay. Taken together, these experiments suggest that residues on CD4 that affect the interaction with class II MHC molecules in the functional assay also affect the adhesion assay. These critical residues for class II MHC binding are located on protruding loops on Dl and D2 of CD4, while residues 43 to 49 are excluded from the class II MHCbinding site.
gp120-Binding Properties of CD4 Mutants To determine the extent of the overlap between the gpl20-binding and class II MHC-binding domains, the affinity of the CD4 mutants for recombinant gp120 (rgp120) was evaluated in a quantitative whole-cell radioligandbinding assay. Results of these binding studies (Table 2 and Figures 3C and 30) revealed the presence of a single population of binding sites for all the CD4 mutants that specifically bound 1251-labeled rgp120, with dissociation constants (Kds) ranging from 7 to 306 nM. In accord with other studies (Peterson and Seed, 1988; Landau et al., 1988; Arthos et al., 1988; Clayton et al., 1989; Brodsky et al., 1990; Ashkenazi et al., 1990), the CDRBlike loop in Dl is critical for recognition of gp120 (Figures 3C and 3D). Seven of the 8 mutants in this region did not bind to gpl20, and the remaining mutant (Q4OP) had a 27-fold decreased affinity. Of particular interest is the mutant with deletion of residues 43-49, which does not bind to gp120 (Figure 3C) but still interacts with the class II protein (Figure 3A). The CD4 mutants M9, MlO, Ml 1, and M13, which bear mutations between residues 19 to 38, displayed a 2- to g-fold reduction in binding affinity for rgpl20. Interestingly, 4 mutants, Ml 2, M26, M32, and M34, which encompass three of the four domains of the CD4 molecule, bound lz51-labeled rgp120 with significantly higher affinities (1.5-fold) than the wild-type CD4 molecule (Table 2 and Figures
Cell 1044
Table 3. Characteristics
and Effects of CD4 Residues on Class II Binding Fractional Solvent Accessibility
Domain (Loop)
Mutation
Residue
Side Chain
S19Y Q20K G36E Q40P F43L T45P G47R P46S Deletion F43S49 55[SR] 57[SR]
0.93 0.66 0.31 0.72 0.70 0.62 0.43 1.05
1.03 0.66 -
Cluster(s) Lost
% IL-2 Ratio (MutanUWT)
Residue Involved in CD4-Class II Interaction
6 2 5, 6 4, 5 ND 5 2.5
14 66 43 41 177 7 6 43
+ TC -
4. 5, 6 1,2 2, 3, 5 1 ND
97 26 6 54 177
-
Dl CDRl
CDR2
I CDR3
0.65 0.90 0.69 1.06
-
-
E67L Q69L Q94K
0.17 0.16 0.66 0.63 0.72 0.40
0.00 0.00 0.94 0.53 0.69 0.61
7. 1
14 43
+ -
P122H Q165E Q165P
0.96 0.97 0.97
1 .oo 0.96 0.96
None 7 7
46 29 14
+ +
2, 6
D2 FG
-
Fractional accessibility values for all amino acids involved in single substitutions into the CD4 molecule are shown. Values close to 1 .OO indicate that residues or lateral side chains are highly exposed, while amino acids with values close to 0.00 correspond to buried residues. Clusters that were lost for each CD4 mutant are designated by their respective number, as described in Figure 1. To compare the IL-2 production of the 3DT52.5.6-expressing CD4 mutants with IL-2 production of those expressing CD4 wild type, the results were expressed as the percentage of the ratio of the IL-2 ratio of the various CD4 mutants over the values of the IL-2 ratio of the CD4 wild-type ([CD4 mutant IL-2 ratio/CD4 WT IL-2 ratio] x 100). CD4 residues that are involved in the interaction with class II MHC molecules are identified by a”+“, while”-” indicates that the CD4 residues are not implicated in the binding with class II MHC molecules. TC, to be confirmed by mutagenesis;‘ND, not determined.
3C and 3D). Surprisingly, a mutation in D3 of CD4 (M33) completely abolished the interaction with rgpl20. All other mutants in D2, 03, and D4 (except M33) exhibited binding affinities for 1z51-labeledrgpl20 that were comparable to that of the wild-type CD4 molecule. Discussion A functional and an adhesion assay were used to identify the residues on CD4 that are involved in the interaction with their physiological ligands, class II MHC molecules. Analysis of a large panel of mutant CD4 molecules spanning the four external domains of CD4 indicates that several residues in Dl, D2, and possibly D4 are involved. Mutations that affected class II MHC binding in both the functional and adhesion assays include amino acids S19 (M9), Q20and H27(Mll), T45(M17), K46andG47(M18), G47(M19),A55(M22),S57(M23),Q89(M26),Q165(M31, M32), and the cluster substitution 349-356 (M37). These CD4 mutants showed an increase in IL-2 production of only 1.5- to 6-fold when class II MHC molecules were expressed on target cells, compared with a 20- to 30-fold increase in IL-2 production when the wild-type CD4 molecule was expressed in the T cell hybridoma. Furthermore, they did not form conjugates with target cells expressing class II MHC molecules. The relevance of these results to the interaction between CD4 and class II MHC molecules has been further evaluated in light of the CD4 crystal structure, which is now available.
In Dl , substitution of the serine at position 19 (M9), which corresponds to the first amino acid of the CDRl-like loop, greatly affected class II MHC binding. Serological analysis (Table l), CD4 cluster analysis (Figure l), and gp120binding results (Table 2) suggest that this mutation did not & Fig. Slead to major conformational changes. Table 3 shows that this mutant has lost only one out of eight antibody clusters. The single substitution at position Q20K (MlO) had no effect on the interaction of CD4 with class II MHC molecules. However, the combination of mutations on Q20 from the CDRl-like loop and H27, which follows in 8 strand C, abolished the interaction of the CD4 molecule with class II MHC molecules, quite possibly by altering the orientation of the CDRl -like loop; this leads to serological and functional changes in the CDRP-like loop. This is shown by the loss of two specific MAb epitopes (Figure 1) of the CDRP-like loop (161.69 and 666.6.1) and by a substantial decrease (Cfold) in the capacity of this mutated molecule to bind gp120 (Table 2). Spatial localization of S19 (yellow residue) and Q20 (wen residue), as shown from the CD4 crystal structure,JFigures 5A and 5C) and from fractional solvent accessibility values (Table 3) indicates that these residues are pointing out from the CDRllike loop, while H27 is only partially exposed from a 8 strand. Moreo%er, the lateral side chain of the serine 19 is highly exposed (Table 3 and Figures 5A and 58) suggesting that residue 19 could constiWte a contact site with class II. The importance of-therCDRl-like loop has been suggested in previous studies showing that substitutions
CDCClass 1045
II Recognition
Figure 5. Crystal Structure Class II MHC Binding
Location of CD4 Residues Implicated
in
Drawings for (A) and (C) were produced with QUANTA (Polygen) using the atomic coordinates for the DlD2 fragment of human CD4 as reported with Brookhaven entry name lCD4 (Ryu et al., 1990). (6) in association with stereodrawing (A) provides a key to residue locations. Residues identified in this mutational analysis as having a direct involvement in binding to class II molecules are drawn in yellow (19, 89, 165); residues for which the results are conflicting or inconclusive are drawn in green (20, 45, 46, 47); and those residues that qualified as candidates by solvent exposure and serological analysis and, when included in tested mutants, continued to show substantial IL-2 production (greater than g-fold enhancement ratios) and rosette formation in the adhesion assay are drawn in pink (23, 24, 40, 41, 42, 43, 44, 46, 49, 60, 87, 94, 122, 133). Four residues (27, 38, 55. 57) for which mutant proteins ware defective in antibody interactions at noncontiguous epitopes and/or which have low side chain fractional accessibility (
September
6, 1991, Copyright
0 1991 by Cell Press
Mutational Analysis of the Interaction between CD4 and Class II MHC: Class II Antigens Contact CD4 on a Surface Opposite the gpl20-Binding Site S. Fleury, ‘t D. Lamarre, l S. Meloche,’ S.-E. Ryu,* C. Cantin,’ W. A. Hendrickson,* and R.-P. Sekaly*t ‘Laboratoire d’lmmunologie lnstitut de Recherches Cliniques de Montreal Montreal, Quebec H2W lR7 Canada tD6partement de Microbiologic et d’lmmunologie Universite de Montreal Montreal, Quebec H3C 3J7 Canada *Department of Biochemistry and Molecular Biophysics Howard Hughes Medical Institute Columbia University New York, New York 10032
Summary Using functional and adhesion assays, we have studied the ability of 30 human CD4 mutants to interact with class II major histocompatibility complex (MHC) molecules and also with gp120 from human immunodeficiency virus. The mutants cover the four domains (Dl-D4) of CD4 and include several single-site substitutions. Analysis of the results, in the context of the CD4 crystal structure, shows that mutations that affect the interaction with class II MHC molecules are located on three exposed loops from CD4 domains 1 and 2. The specifically implicated residues, 19, 89, and 185, are separated from one another by 9 A, 24 A, and 24 A on one face of the CD4 molecule. Moreover, the class II binding site does not include residues 43 to 49 of the CD4 molecule, a region on an opposite face known to be involved in the binding of gp120. Introduction The CD4 molecule is a nonpolymorphic membrane glycoprotein composed of four extracellular domains (D, domain; Dl-D4) that share sequence homologies with immunoglobulin (lg) domains (Maddon et al., 1985, 1987; Williams and Barclay, 1988). The CD4 protein is expressed on a subpopulation of mature T cells (for review, see Littman, 1987). The T cell receptor (TcR) of these CD4+ cells recognizes antigen (Ag) only when presented by a class II molecule of the major histocompatibility complex (MHC) (Biddison et al., 1982, 1983; Swain, 1983). This absolute correlation between T cell specificity and CD4 phenotype is thought to result from a selective process in the thymus involving engagement of the TcR and CD4 with class II MHC molecules. The specific expression of the CD4 molecule on class II-restricted T cells has led to the hypothesis that CD4 interacts with a nonpolymorphic determinant of class II proteins on antigen-presenting celis (Marrack et al., 1983; Wilde et al., 1983; Swain et al., 1984). This association
has now been established through reconstitution of CD4class II-dependent T cell function and cell adhesion. Introduction of CD4 intoCD4-Tcell hybridomas greatly potentiates the response of these cells to Ag-specific stimulation when class II molecules are expressed on target cells (Sleckman et al., 1987; Gay et al., 1987; Lamarre et al., 1989a), and this response correlates with a physical association between the hybridoma and antigen-presenting cells (Lamarre et al., 1989a, 1989b). Other studies have also shown that class II MHC-positive cells can specifically aggregate with nonlymphoid cells expressing high levels of human CD4 (Doyle and Strominger, 1987). The CD4 protein serves as the primary cellular receptor for the human immunodeficiency virus (HIV) (Klatzmann et al., 1984a, 1984b; Dalgleish et al., 1984) through its specific binding to the gpl20 envelope glycoprotein of the virus (Maddon et al., 1986; McDougal et al., 1986). The gpl20-binding site on CD4 lies on the first N-terminal domain (Peterson and Seed, 1988; Landau et al., 1988; Arthos et al., 1988; Clayton et al., 1989; Brodsky et al., 1990; Ashkenazi et al., 1990). However, less is known about the regions of the CD4 molecule that interact with their physiological ligands, the class II MHC molecules. Previous mutational analysis (Clayton et al., 1989; Lamarre et al., 1989a) and antibody-blocking experiments (Lamarre et al., 1989b) have suggested that residues in Dl as well as D2 and D3 of the CD4 molecule are involved in the interaction with class II MHC molecules. However, nearly all of these mutations involved substitutions of several residues and included insertions or deletions. This makes it difficult to evaluate, in the case of impaired function, whether the effect is due to a direct impact of the mutated residues on the interaction of CD4 with class II MHC molecules or whether the impact is indirect and due to perturbation of the three-dimensional structure of CD4. An important point is that the three-dimensional structure of CD4 is now known. Crystal structures of soluble CD4 proteins constituting the two N-terminal domains, Dl and D2, were recently solved at atomic resolution (Wang et al., 1990; Ryu et al., 1990). Dl sharessignificant structural similarities with lg VK domains, as predicted from sequence homology comparisons (Maddon et al., 1987; Peterson and Seed, 1988; Clayton et al., 1988); however, the CC’ and CDRS-like loops from the lg dimer interface are shortened, and the CDR2-like loop is elongated. The folding of D2 resembles that of an lg constant domain, but differs significantly in the placement of a disulfide bond between strands in the same sheet and in the reduced length of the 6 strands. Dl and D2 share a common 8 strand (G strand in Dl, A strand in D2) corresponding to residues 88-103, which results in tight packing between these two domains, leading to a rod-shaped molecular fragment of approximate dimensions 25 x 25 x 60 A. In this article, we report the effect of a panel of mutants, which included several single residue substitutions, within the four domains of the human CD4 molecule on their interaction with class II MHC and gpl20 molecules. The
Cell
1038
structural integrity of the CDCmutated proteins was evaluated by a serological analysis using a panel of 36 anti-CD4 monoclonal antibodies (MAbs) and CD4 MAb clusters. Functional evaluation and adhesion property analysis of these CD4 mutated molecules were carried out following infection in a T cell hybridoma (3DT52.5.8). Effects that might be mediated indirectlythrough CD4-TcR interaction are excluded in the assay system that was used. In this report, we are also able to couple the functional assays with theatomicstructureof CD4, inordertospatiallydefine the residues on CD4 involved in binding to class II MHC molecules. Mutations that strongly affected recognition of class II MHC molecules in the functional and adhesion assays are clustered in three distinct segments. Two of these segments, which are located in Dl , are homologous to complementary determining regions (CD&) of lg. The third segment is located in D2 near the interface with Dl. Results Description, Expression, and Serological Analysis of CD4 Mutants Thirty CD4 mutant proteins (M9-M38) were expressed in the murine CD4- CD8- T cell hybridoma 3DT52.5.8 (see Table 1 for description). These mutations cover the four Ig-related domains of the CD4 molecule, Dl to D4. Nineteen mutations are located in Dl, 5 in D2, 2 in D3, and 4 in D4. These mutations introduced into CD4 have been described previously and consist of: epitope-loss mutations (M9-M20, M24-M28, M30, M31, and M35), which are random substitutions of 1 to 3 residues in Dl, D2, and D4 of the CD4 molecule (Peterson and Seed, 1988); linker-scanning mutations (M21-M23, M29, and M32), which are insertions of the dipeptide sequence serinelarginine (SR motif) at predicted hydrophilic sites in Dl and D2 (Mizukami et al., 1988); and homolog-scanning mutations (M33, M34, and M36-M38), which are substitutions of 3 to 8 residues of nonconsented sequences between the human and mouse CD4 molecule in D3 and D4 (Lamarre et al., 1989a). All the mutated proteins were stably expressed in the 3DT52.5.8 T cell hybridoma using a retroviral vector as previously described (Lamarre et al., 1989a). The cellular expression of the different CD4 mutants was determined by flow cytometry using appropriate anti-CD4 MAbs (see Table 1). A detailed serological analysis was undertaken to determine the structural impact of the mutations on the conformation of the CD4 protein. The results of this serological analysis are summarized in Table 1 as the total number of epitopes conserved (C), affected (A), and lost(L) for each mutant. The number of epitopes conserved for each mutant varies between 5 and 35, indicating that some mutations are more disruptive than others. Sixteen of the mutants are recognized by at least half of the antibodies, and all except M23 are detected by at least 30% of the antibodies. The M23 mutation (SR insertion after residue 57) interrupts the D 8 strand in Dl, which likely explains its global effect on most antibodies that map to the closely associated Dl and D2.
Representative monoclonal antibodies used for staining were grouped in eight different clusters (Figure l), based on the mapping of these antibodies to our CD4 mutants (Fleury et al., unpublished data) and to other previously published CD4 mutants (Sattentau et al., 1989; Brodsky et al., 1990) and based on their capacity to compete the binding of CDCspecific MAbs (Sattentau et al., 1986, 1989; Healey et al., 1990). This cluster analysis allows us to define better the impact of the different mutations and region(s) of the CD4 molecule affected. The majority of the 36 MAb epitopes are found in stretches of residues that are exposed to solvent and are homologous to CDR loops, as shown by the CD4 crystal structure (see Table 1). The first three clusters define regions closely related but spatially distinct. Clusters 1 (OKT4A), 2 (OKT4D), and 3 (Leu3a) delineate different regions between the CDRl-, CDR2-, and CDR3-like loops. Cluster 4 (L-93 and L-206) is specific for the CDRBlike loop, while cluster 5 (101.69 and 866.6.1) delineates the CDRl- and CDR2-like loops in Dl, and cluster 6 (L-68 and L-92) defines the CDRland CDRS-like loops in Dl. In D2, cluster 7 (OKT4B and MT-151) corresponds to the FG loop region. Cluster 8 (L-120 and MT-429) defines D3 and D4. Of the 27 CD4 mutants in Figure 1,18 mutants have lost only one cluster, 6 mutants have lost two clusters, and only 3 mutants have lost three clusters. These results clearly show that only local conformation changes are induced by the mutations, as reflected by the alteration of one, two, and in rare cases, three loops; the other regions are still intact and able to interact with other molecules, such as gp120 and class II MHC molecules. Functional Analysis of the CD4-Class II MHC Interaction Efficient stimulation of CD4+ T cells is dependent on the specific association of CD4 with class II MHC molecules on the antigen-presenting cells. However, CD4 also appears to associate with the TcR during the effector phase of the immune response (Saizawa et al., 1987; Kupfer et al., 1987). To isolate the CD4-class II component of this interaction, we have utilized the 3DT52.5 T cell hybridoma whose TcR is specific for the murine class I MHC protein H-2Dd. The interaction between the TcR on this hybridoma and the murine H-2Dd on the target cell is required for an IL-2 response (Greenstein et al., 1984). However, expression of human CD4 on the hybridoma and the human class II HLA-DP on the target cell (Table 2, WT and Figure 2) increases the IL-2 responss20- to 30-fold (Lamarre et al., 1989a, 1989b). The T cell hybridoma cell lines expressing the different CD4 mutants were tested for their IL-2 response in the presence of target cells display&g the murine H-2Dd class I protein alone or together with the human HLA-DP class II protein. To minimize cell line variation, hybridoma populations for each mutant were selected that gave similar responses when stimulated with an anti-TcR MAb. The results of these assays are summarized in Table 2. All of the CD4 hybridomas produced low, levels of IL-2 in the presence of DAP-3Dd stim,ulaJor&tls (see Table 2). Sev-
CD4Class 1039
II Recognition
Table 1. Description,
ExpressIon,
and Serological
Analysis of CD4 Mutants Epitopes
Mutants
Mutations
C
WT Dl
A
c
M. F. V.
36
0
0
18
M27
S19Y Q20K Q20H/H27R S23G1124VIH27L G38E Q40P Deletion F43S49 F43L T45P K46NIG47V G47R P48S insertion 48[SR] Insertion 55]SR] Insertion 57[SR] S60R E87L Q89L Q94K
26 19 25 4 18 23 12 ND 29 17 14 12 27 11 5 13 ND 21 21
7 14 6 4 15 9 18 ND 5 12 18 24 8 21 23 21 ND 10 14
3 3 5 2 3 4 6 ND 2 7 4 0 1 4 8 2 ND 5 1
15 11 26 13 9 23 8 17 23 12 10 10 7 9 11 10 33 16 26
M28 M29 M30 M31 M32
P122H Insertion 133[SR] Ql65E Q165P InsertIon 165[SR]
18 28 13 17 13
18 7 19 17 21
0 1 4 2 2
11 10 12 21 14
M33
277-280
10
14
9
M34
293-300
33
0
10
M35 M36
V326l 328-330
35 23
0 3
22 12
M37
349-356
18
6
14
M38
363-369
26
3
9
r M9 Ml0 CDRl Ml1 t Ml2 Ml3 Ml4 Ml5 Ml6 CDR2 Ml7 Ml8 Ml9 M20 M21 M22 M23 M24
D2
D3 h m h M
EAKT D--K R ATQLQKN KVAQLNNT
h m h m h m
KRE EEQ DSGQVLLE EGDKVKMD PTWSTPV -SRGVNQ
D4
The CD4 mutants M9-M20, M24-M28, M30, M31, and M35 consist of single or multiple point mutations with impaired antibody recognition. Mutants M21-M23. M29, and M32 are insertion mutants in which codons for serine-arginine residues were inserted after the indicated amino acid. Mutants M33, M34, and M36-M38 are clustered substitution mutants in which three to eight amino acids of human CD4 (T4) were replaced with the corresponding residues of the murine CD4 (L3T4). The numbering of CD4 amino acids is according to Maddon et al. (1985) and the alignment with L3T4 is according to Littman and Gettner (1987). The mutant CD4 cDNAs were subcloned into the retroviral vector pMNC-stuffer and introduced into the murine T cell hybridoma 3DT52.5.8. The cellular levels of the CD4 wild-type and CD4 mutants were determined by flow cytometry using OKT4 MAb (L-68 in the case of M20, M28, and M32) and are expressed as the mean fluorescence value (M. F. V.). The CD4 mutants were then stained with 36 murine anti-CD4 MAbs and analyzed on a FACScan flow cytometer. C, epitope conserved; A, epitope affected; and L, epitope lost. Based on the CD4 crystal structure (Wang et al., 1990; Ryu et al., 1990) three CDR-like loops are identified on Dl: CDRl-like (residues 19-24) CDR2-like (residues 41-59) and CDRS-like (residues 87-88). In D2, three loops are also characterized: loop BC (residues 122-126), loop CC’ (residues 133-135). and loop FG (residues 163-165). ND, not determined. B Ml2 was stained with only 10 MAbs.
eral of the mutants showed moderate to marked reduction in IL-2 production at saturating concentration of effector or target cells. These results, normalized to the IL-2 production in the absence of class II protein, are shown in Figures 3A.and 38. Eleven mutants significantly affected the IL-2 response, yielding only a 1.5- to 6-fold stimulation, in contrast to the 20- to 30-fold stimulation observed with the native CD4 protein. For each of these mutants, this ineffi-
cient stimulation of IL-2 was reproduced in a minimum of five different populations of the hybridomas derived from two independent retroviral vector infections. The remaining CD4 mutants gave at least a g-fold stimulation of IL-2 production and were therefore considered not to have a deleterious effect on CD4 recognition of class II protein. In Dl, 8 out of 19 CD4 mutants showed only a 1.5- to B-fold increase in IL-2 production in the presence of target
8 2.
g!k F a pi53 LYQ-
3 0,s 505
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5
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=,,,z
“a222
n n 0 n n n n c n
n E n n n n 0 c!
n n c n n n n n
n n n n n n E n
n 4 n n n n n n
0
0
at.4
n
nmm~m~7~~mmmm rmmm00m~00m~mm n m0mcn~nmmmrmm
at4
at.4
at.4
at4
aN
nmm5r7rcmmmmmmm~~~~~mmm mmmmmc7Emmmmm 0mm00nca~n~mm mm0mm~00mnm0m m~0mm0on~mnum n n n q n n 0
mm~mmrmmmrrrm
n
at4
n n 0 n n n n 0
n n n n n n n L!
0ummmmmmm
n n n n n n n n 0
n n n n n n n
mm
y n n
n
n n IL! E n n n
n n n n
rmmmmmmmmmm
n n q 0 n n n 3
rrmrrmcrrr:
n n n c 0 z p 0 n n n n n 0 7 q n 4 0 0 PIN
pa3
UoIIJasuI
-1zw
-0lW - 6W
,461s
-1lW nozo
-ZlW tlLZH/HOZO
-EIW
38EE)
-9lW -PlW
wwlaa dOP0
lLZHlA~Zl10EZS
6s-~td
-LlW
-8lW d9*1
-6lW
-0zw MLtD
S8i’d
hLVD/NSVM
[MS]8V
-zzw UO!iJasUl
[klS]SS
-9ZW -tzw
-LZW
-8ZW
-6ZW
-0EW
-1EW
-ZEW
-EZW
XV60
d99lO
UO!IJOSUI
IMSILO UO~IJ=UI
iusIg~
69c-c9c ~114~IJJ~YS-96fd 9~6-6~6~14~IJJ~US-LEW occ-ezc WII(~IJJ~US-SEW 19zcn -SEW OOE-EBB ~~4~IJJ~YS-VEW OBZ-LLZ U/UWJJWS-GCW
odr(l
7:4;-Class
II Recognition
Table 2. Ability of CD4 Mutants to Maintain a Class II MHC-Dependent CD4-Class MHC Interaction (IL-2 U/ml)
IL-2 Production
and to Bind rgp120
II
Anti-CD4 MAb (IL-2 U/ml)
Anti-T&t Stimulation (IL-2 U/ml)
Rosette Formation
rgp120 Binding
Mutants
Mutations
Dd
DdDP
w-r
-
330
7,040
930
35,000
+
11.5
S19Y Q20K Q20HIH27R S23GA24VIH27L G38E Q40P Deletion F43S49 F43L T45P K46NIG47V G47R P48S Insertion 48[SR] Insertion 55[SR] Insertion 57[SR] S60R E67L Q89L Q94H
340 220 350 200 445 500 210 200 230 270 230 500 320 170 380 750 200 185 530
1,000 4,000 500 4,200 4,000 3,880 4,330 7,570 350 400 370 4,400 7,040 1,010 660 8,600 7,570 550 4,780
ND ND 700 ND 640 370 640 ND ND ND 350 770 920 580 640 1,200 ND 450 ND
30,000 32,000 24,800 31,100 17,400 30,720 21,760 33,700 31,120 32,000 26,670 35,000 32,000 24,240 35,000 32,000 28,900 21,760 32,000
+ + + + + + -
+
19.2 25.8 46.9 7.5 62.9 306.7 NSB NSB NSB NSB NSB 9.9 NSB NSB NSB 15.4 13.9 7.0 13.0
D2 M28 M29 M30 M31 M32
Pl22H Insertion 133[SR] Q165E Q165P Insertion 165[SR]
600 210 580 940 330
5,830 4,000 5,400 5,400 900
ND 350 ND 900 380
19.100 17.280 32,000 32,000 32,000
+ + + -
13.5 10.1 11.1 14.7 7.5
D3 M33
277-280
200
4,000
580
21,600
+
M34
293-300
200
2,980
ND
24,600
+
8.0
D4 M35 M36
V326l 328-330
530 200
4.880 1,700
620 ND
32,000 32,000
+ +
9.6 15.1
M37
349-356
200
630
ND
14.700
+I-
14.5
M36
363-369
190
2,050
ND
35,000
+
19.7
Kd (nM)
Dl M9 Ml0 Ml1 Ml2 Ml3 Ml4 Ml5 Ml6 Ml7 Ml6 Ml9 M20 M21 M22 M23 M24 M25 M26 M27
h EA KT mD--K hRATQLQKN mKVAQLNNT
h KRE mEEQ hDSGQVLLE mEGDKVKMD h P T WS T P V m-SRGVNQ
+ + + +
NSB
The ability of the CD4 wild-type (WT) and CD4 mutants M9 to M38 to interact with class II MHC molecules was evaluated in a functional and an adhesion assay. Shown are IL-2 production (U/ml) and rosette formation (f, presence of conjugates; -, absence of conjugates) obtained by the 3DT52.5.8 cells expressing the various CD4 mutants following a 20 hr coculture period with the murine DAP-3 cells expressing Dd or DdDP. Results of independent functional assays did not show more than a 15% variation. Each coculture was also carried out in the presence of 20 nM of the CDCspecific MAbs OKT4B or L-68. The 3DT52.5.8 effector cells were also stimulated with coated anti-TcR MAb F23.1 to evaluate their intrinsic ability to produce IL-2. The rgpl20-binding properties of the CD4 mutants were determined by incubating the 3DT52.5.8 transfectants with increasing concentrations of ‘2SI-labeled rgpl20 for 4 hr at 25OC in the presence or absence of a lOO-fold excess of rgpl20. The saturation binding curves were then analyzed by nonlinear least-squares regression analysis according to mass action law (DeLBan et al., 1982). The results are reported as the dissociation constants (Kds) in nM. The Kd for each CD4 mutant was determined by two separate rgpl20-binding assays, and only 10% of variation was observed. NSB, no specific binding; ND, not determined.
tants were tested for their capacity to associate with p561Ck and for the autophosphorylation of the tyrosine kinase upon cross-linking of CD4 with CDCspecific MAbs; indeed, our results (S. Fleury, unpublished data) show that all mutated CD4 molecules were efficiently associated with p!Wk. Furthermore, significant phosphorylation of the
tyrosine kinase was observed 60 s after cross-linking of the mutated CD4 molecules. In D3 and D4, mutant M37, which was made by substituting human residues with corresponding murine residues in the region spanning amino acids 349-356, shows only a 3-fold increase in IL-2 production (Table 2 and Figure
Cdl 1042
38) in the presence of the appropriate target cells. Mutants M33, M34, M35, M36, and M38 displayed significant class II MHC binding, as demonstrated by a 9- to 20-fold increase in IL-2 production in the presence of class II MHC molecules on target cells (Figure 38). From these res.ults, it appears that in D3 and D4, of those CD4 mutants tested, only residues 349-356 could be involved in the CD4-class II MHC interaction. Figure 2. Schematic Description of the Cellular System Used to Evaluate the CD4-Class II Interaction The TcR of the T cell hybridoma 3DT52.5 is specific for the mouse class I antigen (H-2Dd). An L3T4- variant of 3DT52.5, called 3DT52.5.8, was infected with a retrovirus vector containing a cDNA encoding the human wild-type CD4 (I1 B-3 hybridoma). Mutated CD4 moleculeswere also introduced in the 3DT52.5.8 hybridoma. (A) shows the interaction between effector cells expressing only the TcR and target cells expressing H-2Dd molecules. (6) shows that the introduction of the human CD4 molecule in the effector cells does not lead to a significant increase in IL-2 production, when cocultured with target cells expressing only class I molecules. (C)shows that when CD4 and its ligand (HtADP molecule) are expressed on effector and target cells, respectively, a significant increase (greater than 20-fold) in IL-2 production is obSeNed, compared with (B), where target cells expressed only the TcR ligand (H-2Dd).
Analysis of the Adhesion Properties of the CD4 Mutants to Class II MHC-Positive Cells A qualitative binding assay was performed concurrently to determine the effect of each mutation on the physical interaction between CD4 and class II MHC molecules. Hybridoma T cells expressing TcR and wild-type CD4 molecules will form conjugates with target cells expressing both TcR and CD4 ligands, i.e., murine class I (H-2Dd) and human class II (HLA-DP), respectively (Figures 4a and 4b). Conjugate formation is specifically inhibited by a MAb specific for CD4 (Figure 4~). In contrast to other assays used to study the adhesion function of CD4 (Doyle and Strominger, 1987; Clayton et al., 1989; Rosenstein et al., 1990), conjugate formation with the 3DT52.5.8 hybridoma does
~
21
01
D2
D3
D4
B
C
CD4 mutants Figure 3. Quantitative Affinity
Analysis of a Panel of CD4 Mutants for Their Capacity to Interact with Class II MHC Molecules and for Their rgpl20Sinding
The CD4 wild type (WT) and CD4 mutants M9-M38 were analyzed for their ability to produce IL-2 ([A] and [B]). Results are reported as the ratio of IL-2 production obtained in the presence of class II in their absence(DAP Dd). The CD4 mutants were also analyzed for their capacity to bind ‘ZWabeled ([Cl and [D]). The results are reported as the ratio of the equilibrium binding constants (Ka:l/Kd) of CD4 wild-type molecule.
with DAP-3 cells expressing either Dd or DdDP molecules (DAP DdDP) over the value obtained rgp120 in whole-cell saturationbinding assays the vario@ CD$m&ts overthe value of the
CDCClass 1043
II Recognition
Figure 4. Ability of CD4
Mutants to Form Rosetteswith Target DAP DdDPCells
The 3DT52.5.9 cells expressing the CD4 wild-type and CD4 mutants were,analyzedfor their ability to form rosetteswhen coculturedwith murine fibroblastic DAP cells expressing Dd or DaDP. Following coculture, the cells were observed by phase-contrast microscopy and photographed under 100 x magnification. (a) CD4 wild-type (I1 B-3 hybridoma) and DAP Dd cells. (b) CD4 wild-type and DAP DdDP cells. (c) CD4 wild-type and DAP DdDP cells in the presence of OKT4B MAb (20 nM). (d) Mutant M9 (S19Y) and DAP DdDP cells. (e) Mutant Ml5 (deletion in F43-549) and DAP DdDP cells. (9 Mutant Ml5 and DAP DdDP cells in the presence of OKT4S MAb (20 nM).
not require centrifugation or expression of unusually high levels of the CD4 molecule. However, CD4 mutants that did not show an increase in IL-2 production of greater than 1.5-to B-fold (M9, Mll, M17, M16, M19, M22, M23, M26, M31, M32, and M37) upon interaction with class II MHC molecules (Table 2 and Figures 3A and 38) also failed to form rosettes with class II-positive cells (Table 2 and Figure 4d). M15, which corresponds to a partial deletion of the CDR2 loop, still allows rosette formation (Figure 4e), and binding is inhibited by CDCspecific MAb (Table 2 and Figure 4f). Other mutants in the four domains of the CD4 molecule, which all showed significant increases in IL-2 production (greater than g-fold) in the presence of class II MHC-positive cells, also physically interact with cells expressing DdDP, as demonstrated by the formation of conjugates in the adhesion assay (Table 2). Furthermore, rosette formation (Figure 4c) was inhibited by MAbs specific to the CD4 molecule (OKT4B or L-68), thus confirming the specificity of this assay. Taken together, these experiments suggest that residues on CD4 that affect the interaction with class II MHC molecules in the functional assay also affect the adhesion assay. These critical residues for class II MHC binding are located on protruding loops on Dl and D2 of CD4, while residues 43 to 49 are excluded from the class II MHCbinding site.
gp120-Binding Properties of CD4 Mutants To determine the extent of the overlap between the gpl20-binding and class II MHC-binding domains, the affinity of the CD4 mutants for recombinant gp120 (rgp120) was evaluated in a quantitative whole-cell radioligandbinding assay. Results of these binding studies (Table 2 and Figures 3C and 30) revealed the presence of a single population of binding sites for all the CD4 mutants that specifically bound 1251-labeled rgp120, with dissociation constants (Kds) ranging from 7 to 306 nM. In accord with other studies (Peterson and Seed, 1988; Landau et al., 1988; Arthos et al., 1988; Clayton et al., 1989; Brodsky et al., 1990; Ashkenazi et al., 1990), the CDRBlike loop in Dl is critical for recognition of gp120 (Figures 3C and 3D). Seven of the 8 mutants in this region did not bind to gpl20, and the remaining mutant (Q4OP) had a 27-fold decreased affinity. Of particular interest is the mutant with deletion of residues 43-49, which does not bind to gp120 (Figure 3C) but still interacts with the class II protein (Figure 3A). The CD4 mutants M9, MlO, Ml 1, and M13, which bear mutations between residues 19 to 38, displayed a 2- to g-fold reduction in binding affinity for rgpl20. Interestingly, 4 mutants, Ml 2, M26, M32, and M34, which encompass three of the four domains of the CD4 molecule, bound lz51-labeled rgp120 with significantly higher affinities (1.5-fold) than the wild-type CD4 molecule (Table 2 and Figures
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Table 3. Characteristics
and Effects of CD4 Residues on Class II Binding Fractional Solvent Accessibility
Domain (Loop)
Mutation
Residue
Side Chain
S19Y Q20K G36E Q40P F43L T45P G47R P46S Deletion F43S49 55[SR] 57[SR]
0.93 0.66 0.31 0.72 0.70 0.62 0.43 1.05
1.03 0.66 -
Cluster(s) Lost
% IL-2 Ratio (MutanUWT)
Residue Involved in CD4-Class II Interaction
6 2 5, 6 4, 5 ND 5 2.5
14 66 43 41 177 7 6 43
+ TC -
4. 5, 6 1,2 2, 3, 5 1 ND
97 26 6 54 177
-
Dl CDRl
CDR2
I CDR3
0.65 0.90 0.69 1.06
-
-
E67L Q69L Q94K
0.17 0.16 0.66 0.63 0.72 0.40
0.00 0.00 0.94 0.53 0.69 0.61
7. 1
14 43
+ -
P122H Q165E Q165P
0.96 0.97 0.97
1 .oo 0.96 0.96
None 7 7
46 29 14
+ +
2, 6
D2 FG
-
Fractional accessibility values for all amino acids involved in single substitutions into the CD4 molecule are shown. Values close to 1 .OO indicate that residues or lateral side chains are highly exposed, while amino acids with values close to 0.00 correspond to buried residues. Clusters that were lost for each CD4 mutant are designated by their respective number, as described in Figure 1. To compare the IL-2 production of the 3DT52.5.6-expressing CD4 mutants with IL-2 production of those expressing CD4 wild type, the results were expressed as the percentage of the ratio of the IL-2 ratio of the various CD4 mutants over the values of the IL-2 ratio of the CD4 wild-type ([CD4 mutant IL-2 ratio/CD4 WT IL-2 ratio] x 100). CD4 residues that are involved in the interaction with class II MHC molecules are identified by a”+“, while”-” indicates that the CD4 residues are not implicated in the binding with class II MHC molecules. TC, to be confirmed by mutagenesis;‘ND, not determined.
3C and 3D). Surprisingly, a mutation in D3 of CD4 (M33) completely abolished the interaction with rgpl20. All other mutants in D2, 03, and D4 (except M33) exhibited binding affinities for 1z51-labeledrgpl20 that were comparable to that of the wild-type CD4 molecule. Discussion A functional and an adhesion assay were used to identify the residues on CD4 that are involved in the interaction with their physiological ligands, class II MHC molecules. Analysis of a large panel of mutant CD4 molecules spanning the four external domains of CD4 indicates that several residues in Dl, D2, and possibly D4 are involved. Mutations that affected class II MHC binding in both the functional and adhesion assays include amino acids S19 (M9), Q20and H27(Mll), T45(M17), K46andG47(M18), G47(M19),A55(M22),S57(M23),Q89(M26),Q165(M31, M32), and the cluster substitution 349-356 (M37). These CD4 mutants showed an increase in IL-2 production of only 1.5- to 6-fold when class II MHC molecules were expressed on target cells, compared with a 20- to 30-fold increase in IL-2 production when the wild-type CD4 molecule was expressed in the T cell hybridoma. Furthermore, they did not form conjugates with target cells expressing class II MHC molecules. The relevance of these results to the interaction between CD4 and class II MHC molecules has been further evaluated in light of the CD4 crystal structure, which is now available.
In Dl , substitution of the serine at position 19 (M9), which corresponds to the first amino acid of the CDRl-like loop, greatly affected class II MHC binding. Serological analysis (Table l), CD4 cluster analysis (Figure l), and gp120binding results (Table 2) suggest that this mutation did not & Fig. Slead to major conformational changes. Table 3 shows that this mutant has lost only one out of eight antibody clusters. The single substitution at position Q20K (MlO) had no effect on the interaction of CD4 with class II MHC molecules. However, the combination of mutations on Q20 from the CDRl-like loop and H27, which follows in 8 strand C, abolished the interaction of the CD4 molecule with class II MHC molecules, quite possibly by altering the orientation of the CDRl -like loop; this leads to serological and functional changes in the CDRP-like loop. This is shown by the loss of two specific MAb epitopes (Figure 1) of the CDRP-like loop (161.69 and 666.6.1) and by a substantial decrease (Cfold) in the capacity of this mutated molecule to bind gp120 (Table 2). Spatial localization of S19 (yellow residue) and Q20 (wen residue), as shown from the CD4 crystal structure,JFigures 5A and 5C) and from fractional solvent accessibility values (Table 3) indicates that these residues are pointing out from the CDRllike loop, while H27 is only partially exposed from a 8 strand. Moreo%er, the lateral side chain of the serine 19 is highly exposed (Table 3 and Figures 5A and 58) suggesting that residue 19 could constiWte a contact site with class II. The importance of-therCDRl-like loop has been suggested in previous studies showing that substitutions
CDCClass 1045
II Recognition
Figure 5. Crystal Structure Class II MHC Binding
Location of CD4 Residues Implicated
in
Drawings for (A) and (C) were produced with QUANTA (Polygen) using the atomic coordinates for the DlD2 fragment of human CD4 as reported with Brookhaven entry name lCD4 (Ryu et al., 1990). (6) in association with stereodrawing (A) provides a key to residue locations. Residues identified in this mutational analysis as having a direct involvement in binding to class II molecules are drawn in yellow (19, 89, 165); residues for which the results are conflicting or inconclusive are drawn in green (20, 45, 46, 47); and those residues that qualified as candidates by solvent exposure and serological analysis and, when included in tested mutants, continued to show substantial IL-2 production (greater than g-fold enhancement ratios) and rosette formation in the adhesion assay are drawn in pink (23, 24, 40, 41, 42, 43, 44, 46, 49, 60, 87, 94, 122, 133). Four residues (27, 38, 55. 57) for which mutant proteins ware defective in antibody interactions at noncontiguous epitopes and/or which have low side chain fractional accessibility (
