The Molecular Basis of Metal Recognition by T Cells

REVIEW

The Molecular Basis of Metal Recognition by T Cells Francesco Sinigaglia Roche Milano Ricerche, Milano, Italy

T lymphocytes play critical roles in a series of important immune responses, including delayed-type hypersensitivity and contact sensitivity. Metal haptens such as nickel are among the most common sensitizers. In general, T cells recognize fragments of proteins complexed to MHC molecules, Haptens, however, are too small to be antigenic by themselves. Recent experiments have sug-

gested that hapten-specific T cells recognize hapten-modified MHC-peptide complexes. Here I review the data supporting this hypothesis and speculate on the relevance of these findings for the understanding of chemical-induced

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blood mononuclear cells [8,11] and of T-cell lines, obtained from tissue biopsies of patients with nickel-contact dermatitis [12], Furthermore, Saltini et al isolated and characterized beryllium-specific T-cell lines from lung biopsies of patients with chronic beryllium disease [13], The beryllium-specific T cells were all CD4"*", Anticlass II, but not anti-class I antibodies inhibited beryllium-induced proliferation. Taken together, these results indicate that, in general, haptens are recognized by T cells in tbe context of self-MHC class II gene products, just as are peptide antigens,

he antigen-specific immune response is initiated by CD4"'" T lymphocytes that are activated by the antigen bound to a relevant major histocompatibility complex (MHC) class II protein on the surface of antigen-presenting cells (APCs) [1], Recognition of MHC proteins complexed with peptides derived from protein antigen stimulates DNA synthesis and cell division, and causes the release of lymphokines that mediate T-cell function [2], Recently, much progress has been made in understanding the processing of proteins and presentation of their fragments by MHC molecules on the surface of APCs [3], There is no evidence for the binding of nonpeptidic compounds to the "antigen-presenting groove" of MHC class I and class II molecules; however, in functional assays both class I and class II restricted T cells have been identified that recognized nonpeptidic compounds such as trinitrophenyl (TNP) [4,5], fluorescine [6], or metals (which may be referred to as haptens) [7-9], Most interestingly, metal-specific T cells have been implicated in several human diseases, in particular in patients with contact hypersensitivity [10], Contact hypersensitivity is a T-cell - dependent inflammatory response usually manifested as an eczematous reaction at the site of contact with the allergen. Metal haptens such as nickel are among the most common allergens. Because of their complexity and clinical importance, the molecular basis of recognition of haptens has intrigued basic and clinical immunologists for a long time. In this review I summarize experiments that suggest that T cells recognize metal-hapten-modified MHC-peptide complexes, METAL RECOGNITION BY T CELLS IS MHC RESTRICTED We studied T-cell responses to nickel or gold in patients with nickel or gold contact dermatitis, respectively. In both cases, nickel- or gold-specific CD4''" T-cell clones were isolated from patients' peripheral blood mononuclear cells [7,9], To determine whether MHC molecules are involved in metal recognition, we have studied the response of these clones using a panel of APCs with different HLA isotypes. The results were clear: the clones responded to the nickel in association with HLA-DRwl 1 [7] and to gold in association with DRl [9] or DNA (Romagnoli P, Sinigaglia F, unpublished). Using similar methods, other investigators also have shown MHC class II restriction of nickel-specific responses of peripheral Reprint request to: Dr, Francesco Sinigaglia, Roche Milano Ricerche, Via Olgettina 58,1-20132 Milano, Italy, "~ Abbreviations: APC, antigen-presenting cell; TNP, trinitrophenyl.

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hypersensitivities. Key words: hapten/mafor histocompatibility complex/peptide binding/delayed-type hypersensitivity. J Invest Dermatol 102:398-401, 1994

T CELLS RECOGNIZE HAPTEN-MODIFIED PEPTIDES The finding that metal recognition by T cells is MHC restricted suggests that the metal modifies the structure of the MHC molecules, the bound peptide, or botb, and that it is the modified structure that is recognized by the T cells. To distinguish between these possibilities we studied tbe molecular interaction of contact sensitivity-inducing Ni*'*' ions with the MHC peptide complex by analyzing, in a competition assay, the effect of nickel on peptide presentation to DRwl 1-restrictedT cells [14], The competition assay is based on the proliferative response of a "test T-cell clone" to a "test peptide" (Fig 1), It allows one to investigate whether nickel can inhibit the reaction by directly modifying the specific MHC molecule or by altering the antigenic peptide, either before or after its association witb the MHC protein [15], Initially we used two DRwl 1-restricted T-cell clones specific for different peptides. Nickel inhibited the response of one of the clones ; tested. There are two possible interpretations for this finding. By binding to MHC molecules nickel may prevent the subsequent • binding of the peptide, or by binding to the MHC-associated peptide it may modify the structure of the complex, rendering it no longer recognizable by the peptide-specific T cells. We showed that the latter was the case by treating APCs first with nickel and then with peptide, and vice versa, Pretreatment with nickel before pulsing with the peptide did not inhibit the subsequent stimulation of the peptide-specific T-cell clone. However, when the APCs were first pulsed with the peptide and then treated with nickel, we observed the same inhibition as witb APCs that were incubated with a mixture of the peptide and nickel. Thus, the interaction of nickel with the MHC-associated peptide modifies the peptide such that it is no longer recognized by the peptide-specific T-cell clone. Nickel appears not to interact with all peptides, because recognition of the other peptide tested was not inhibited by nickel treatment. Because histidine residues are known to interact with nickel we suspected that nickel interacted with histidine at position 6 of the peptide that

Copyright © 1994 by The Society for Investigative Dermatology, Inc,

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Proliferation

No proliferation F i g u r e 1. Schematic representation of the competition assay to evaluate the effect of nickel on peptide presentation. Solid circle, antigenic peptide; X, nickel; TH, helper T cell.

was susceptible to inhibition. To test this possibility we synthesized and tested a variant peptide in which the histidine was replaced by lysine. Interestingly, the variant peptide was still recognized by the peptide-specific T-cell clone but this recognition was no longer inhibitable by nickel. The crucial role of the histidine residue in the interaction of nickel with the peptide in solution was confirmed by nuclear magnetic resonance (NMR) spectroscopy. Interaction of nickel with the histidine-containing peptide was apparent from the dramatic change in the NMR spectrum. In contrast, no interaction w i t h nickel was evident in the case of the Iysine-variant peptide [14]. These results provided the first evidence for the physical interaction of a hapten with an MHC-bound peptide. Non-metal haptens appear to be recognized in a similar way by T cells. Nalefski and Rao have recently shown that the ligand recognized by the p-azobezenarsonate-specific T cell is a complex of a haptenated peptide bound to the mouse I-A'' molecule. They identified a peptide fragment, generated by enzymatic cleavage of arsonate-conjugated ovalbumin, which stimulates an arsonate-specific T-cell clone. The corresponding synthetic peptide, when conjugated with arsonate, was efficiently recognized by the arsonate-specific T cells [16]. Similarly, Weltzien et al have shown that TNPreactive cytotoxic T cells also recognize a hapten-modified peptide, in this case bound to a mouse class I MHC molecule. Several T'NPspecific, H-2K''-restricted mouse CTL clones were identified, which specifically lysed target cells in the presence of tryptic digests of TNP-modified bovine serum albumin [17]. ONE T CELL CAN RECOGNIZE A HAPTEN IN ASSOCIATION WITH DIFFERENT PEPTIDES In MHC-binding peptides that were isolated from class I or class II molecules one or few amino acids recur at certain positions with very high frequencies. Such residues, called "anchor" residues, are

Figure 2. A simplified view of the different mode of interaction of nickel and gold with MHC class II molecules. Class II molecules hold peptides of 12 to 25 residues by anchoring two or three peptide side chains into complementary pockets of the class II groove. The MHC-associated nickel peptides may be formed by cellular processing of nickel-modified proteins or by conjugation of nickel to peptides already bound to the binding groove of MHC molecules.

characteristic for each class I or class II allele examined and therefore define distinct sequence motifs for peptide binding to a given MHC molecule [18-21]. The side chains of the anchor residues interact with specific pockets in the peptide-binding groove (Fig 2) [ 2 2 24]. Whereas peptides interact with both the groove of the presenting M H C molecule and the T-cell receptor (TCR), haptens appear to interact mainly or exclusively with the TCR. Thus, Martin et al showed that several TNP-specific T-cell clones reacted with peptides containing the MHC class I K''-binding motif (octamers with phenylalanine or tyrosine and leucine in positions 5 and 8, respectively), but differing in their remaining amino acid sequences, provided that they contained a TNP-conjugated lysine in position 4 [25]. These results indicated that TNP may be attached to any MHC-binding, Iysine-containing peptide and that various peptideT N P / M H C complexes are recognized by the same T cell. A similar situation has also been reported for TNP-specific class Il-restricted T-cell lines.* TNP-reactive, I-A"--, I-A-"-, and I-E-f-restricted T-cell lines proliferated in response to tryptic peptides from TNP-bovine serum albumin and synthetic TNP peptides. Some of the T-cell clones were crossreactive to TNP peptides of different sequences. Nickel interacts with peptide-MHC class II complexes at the cell surface. The peptides are diverse; in cells not exposed to foreign antigens they are all derived from a potentially very large number of self proteins. It is conceivable that nickel interacts with several such self peptides and that at least some T cells recognize a diverse set of nickel-modified peptides. T cells that recognize haptens irrespective of the structure of the carrier peptide are likely to be confronted with TCR Iigands at higher densities than T cells that recognize the side chains of one particular peptide only. It has been speculated that CD4"*' T cells not * Kohler J, Martin S, Weltzien HU: Hapten-peptides as antigenir topes for class II MHC-restricted, TNP-specifie, CD4+ T cells. Imrr 189:57, 1993

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only recognize peptide-MHC complexes but somehow are also able to "sense" the ciensity of TCR ligands on the surface of APCs i,e,, to respond differently to high- and low-density TCR ligands [26,27], High-density ligands favor the development of so-called T h l cells, which secrete IFN-y and tumor necrosis factor (TNF), whereas low-density ligands favor the development of so-called Th2 cells, which secrete IL-4 and IL-5 [28,29], T h l responses that are responsible for contact bypersensitivity [30] may be preferentially induced by haptens such as T N P and Ni, which generate ligands at high density on the surface of APCs,

GOLD INTERACTION WITH MHC CLASS II MOLECULES Gold salts have long been used to slow or stop the progression of rheumatoid arthritis [31,32], Unfortunately, in up to one-third of the patients the treatment with gold has to be stopped because of toxicity [33], The most common toxic effects are delayed-type hypersensitivity reactions involving the skin and mucous membranes. Although different mechanisms have been proposed, based upon various effects of gold compounds observed in a number of different model systems [34], an adequate explanation for the therapeutic effect and/or the toxic reactions has remained elusive. Recently, we have shown that gold salts, as nickel, inhibited peptide-induced responses of peptide-specific T-cell clones [9], Several lines of evidence, however, suggested that, unlike nickel, gold binds directly to the MHC class II molecule (Fig 2), First, we have shown that in a competition assay, pretreatment of the antigen-presenting cells with gold before pulsing with the antigenic peptide results in complete inhibition of the peptide-induced proliferation, t Second, by incubating radioactive gold with a panel of class Il-positive and -negative cell lines, we could demonstrate the direct binding of gold to class II molecules,t Further studies will be needed to determine the structural basis for gold binding to MHC class II molecules and whether other molecules, distinct from MHC but contributing to T-cell activation, might also be involved in interaction with gold. In any case, the available data provide an explanation for both the therapeutic and toxic effects of gold treatment. Modification of the putative "autoimmune peptide-MHC complexes" by gold may prevent the stimulation of autoreactive T cells in RA, and may, at the same time, elicit gold-specific T cells responsible for the delayed-type hypersensitivity reactions that are observed in some patients.

T H E ROLE OF MHC GENE PRODUCTS IN HAPTENSPECIFIC T CELL RESPONSES T-cell clones with specificity for haptens have been shown, in general, to recognize hapten-modified peptides in association with M H C class II molecules on the surface of the antigen-presenting cell. Genes of the MHC class II (HLA-DR, -DQ, and -DP) that are associated with susceptibility to autoimmune disorders [35,36] are likely candidates as susceptibility genes in metal allergies. Indeed, Richeldi et al [37], examining the MHC class II genes in 33 cases with chronic beryllium disease and 44 controls, have shown a negative association with HLA-DPBl*0401 and a positive association with HLA-DPBl*0201 alleles. Interestingly, the two DP alleles differ in only a few residues of the MHC-binding site. More controversial findings have been reported for nickel allergy. The initial association between nickel allergy and HLA-DQA genes [38] could not be confirmed [39], Thus, the involvement of MHC molecules in susceptibility to metal allergies seems to be variable, depending on which metal one examines. As discussed, metals, via their chemically reactive groups, could attach themselves to peptides bound to molecules and induce MHC-restricted responses to the con-

t Romagnoli P, Takacs B, Sinigaglia F: Immunologically relevant binding of gold to HLA-DR molecules (in preparation)

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jugates. Variations in the family of peptides, influenced by MHC polymorphism, could account, at least in part, for differences in ease of sensitization to such agents.

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ESDR ANNUAL MEETING The 24th Annual Meeting of the European Society for Dermatological Research (ESDR) will be held September 24-27, 1994 at the Vienna General Hospital, Vienna, Austria. As a leading forum for research in dermatology and skin biology, the ESDR provides a broad platform for the presentation of the latest scientific results in the form of oral or poster communications. The program will be highlighted by guest lectures from renowned scientists on new and exciting developments in molecular and cellular biology and their impact on dennatology. The closing date for submission of abstracts is May 20, 1994. Abstract forms and meeting registration information can be obtained by writing to: Prof. Dr. Wolfram Sterry, ESDR Secretary, Department of Dermatology, University of Ulm Medical School, Oberer Eselsberg 40, D-89081 ULM, Germany. Fax: 49-731-502-3772.