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Annu. Rev. Immunol. 1990. 8:501-30 Copyright © 1990 by Annual Reviews Inc. All rights reserved

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MOLECULES RELATED TO CLASS-I MAJOR Annu. Rev. Immunol. 1990.8:501-530. Downloaded from www.annualreviews.org Access provided by University of Central Florida on 02/22/15. For personal use only.

HISTOCOMP ATIBILITY COMPLEX ANTIGENS Iwona Stroynowski Division of Biology 147-75, California Institute of Technology, Pasadena, California 9 1 125 KEY WORDS:

major histocompatibility antigens, Qa, Tla.

INTRODUCTION Class-I transplantation antigens play an important role in immune recog­ nition. Their major known function involves antigen presentation during viral infections. It is now believed that the class-I antigens associate with small fragments derived from viral products and present these complexes to cytotoxic T cells (CTL) in a form recognizable by T cell receptors. Classical transplantation antigens are present on most cell types. Their level of expression is highest on adult lymphoid tissues, particularly in the B-cell compartment, and is barely detectable in brain, sperm, and early stages of embryogenesis ( 1 ). Most vertebrate species have two to three of these highly polymorphic class-I proteins. For example, in the mouse there are two (and sometimes three) classical transplantation antigens, H-2K, -D (and -L), with'" 1 00 alleles identified at the two major loci. The H-2K, -D and -L genes map to a region of chromosome 17 called the major histocompatibility complex (MHC).I The exon-intron organization of these genes reflects the structural organization of their products (2). The first exon encodes a leader peptide that is absent in the mature proteins; exons 2-4 encode the three external domains, 0: 1,0:2, and 0:3, each approxi1 For the purpose of this discussion, the H-2 (usually used to identify refer only to K, D, and L loci.

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502

STROYNOWSKI

mately 90 amino acids (aa) in length; exon 5 specifies a linker peptide, a transmembrane region, and a fragment of the intracellular domain, while exons 6, 7, and 8 encode the remaining portion of the 30-40 aa cytoplasmic tail. The class-I antigens are 44--48-kd glycoproteins and associate with a 1 2-kd 132 microglobulin (132m). Crystallographic analysis of the human transplantation antigen HLA-A2 suggests that (X3 domain of class-I mol­ ecules and /32m are structurally related to immunoglobulin domains and interact with each other (3). On top of them, the (X I and (X2 regions, folded into a flat [3 sheet and crisscrossed by two (X helixes, form th(: antigen binding site (3, 4). Recent advances in molecular biology have identified many genes similar to the classical transplantation antigen sequences. Some products of these class I-like genes were discovered previously by immunological approaches; others do not have known protein products, but their structure can be deduced from the DNA sequence. In contrast to the classical transplantation antigens, the class I-related molecules show little poly­ morphism, have a different tissue distribution and low levels of expression, and do not seem to participate in immune responses against virally infected cells. While no unambiguous definition of class I-related products can be made at this time, for the purpose of this review, the nonclassical, class 1like or related proteins are those that associate with 132m and are encoded by genes with an exon-intron organization similar to the classical H-2 genes. Class I-related genes and molecules have been described in many organisms, but the murine system is best characterized and contains the greatest number of potentially intact, H-2 homologous genes. This review focuses on the structure and expression of the mouse class I-related molecules. The status of research on soluble, phospholipid­ linked, and integral membrane molecules comprising this group is sum­ marized, structural motifs compatible with the HLA-A2 model are discussed, and common features are identified. A correlation is found between the variable positions in class I-related proteins and those identi­ fied as polymorphic in H-2 antigens. Similarities between class-I proteins from mouse and other species are referenced and the functional implica­ tions of these comparisons are considered. Among them is the possibility that at least some of the class I-related proteins have evolved as restriction elements for nonpolymorphic, species-specific antigens. ORGANIZATION OF CLASS-I GENES IN MOUSE

The class-I genes of the mouse are members of a multigene family con­ taining a minimum of 20-50 genes per haploid genome. They can be subdivided into seven different groups mapping to the H-2K, H-2D, Qa,

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QA AND TLA ANTIGENS

503

Tla, and Hmt subregions, plus the mCDI subfamily.2 The first six sub­ groups are found on chromosome 1 7; the location of the latter one is not known. The H-2K, H-2D, Qa, Tla, and some Hmt class-I genes share substantial DNA sequence homology with the classical H-2K, D, L genes as demonstrated by their cross-hybridization with the probes derived from the fourth exon of the H-2 genes. In contrast, the Mb-I and mCDI families originally identified by homology with human HLA and CDI gene frag­ ments, respectively, are less similar to H-2 genes. DNA sequencing of representative members from each of the subgroups reveals similar exon­ intron organization and the size of the predicted core polypeptides, as well as at least 25 homologous aa in the sequence of the a3 domains. Figure 1 shows the location of class I-related genes in the BALB/c (H2'1, C57BLjlO (H-2b) and C3H (H_2k)3 haplotypes. All these class I-like genes have, presumably, descended from a common genetic ancestor(s), but the precise evolutionary relationships cannot be reconstructed. The analysis is complicated by variation in the number of class I-like sequences in different mouse strains. Since either homologous or nonhomologous alignments of these genes can generate duplications/deletions, it is almost impossible to identify the true allelic relationships in different haplotypes. Nevertheless, attempts have been made to align homologous members from H-2b and H-2d haplotypes using as criteria restriction enzyme pattern similarities and hybridization with probes specific for individual genes or gene subsets (5, 6, 8, 9; Figure 1 ). Comparison of class-J gene organization in inbred haplotypes indicates that many can be deleted without obvious effect. Perhaps the most drastic is the extensive deletion of the QI-Q9 region and possibly of QIO gene in strains of the H-2fhaplotype ( 1 0, 1 1 ). Deletions involving the H-2D region genes D2, D3, and D4 have also been reported in laboratory-derived mutant strains such as dmi and dm2 ( 1 2). Other types of class-I gene rearrangements are also common. For example, alternating genes in the Qa region (Q4, Q6, Q8, and Q5, Q7, and Q9) may be more similar to one another than to adjacent genes (6, 1 3). Thus the locus may have evolved by duplication of an ancestral gene followed by duplication of the gene pairs. Tla has also been shown to contain duplicated regions (5, 6). The most extensive is a partial duplication of the Tie_TIDe BALBjc genes

2The subfamilies an: defined by their genetic location (Figure I ) or, in the absence of this data, as for mCDl genes, by their unique DNA sequence composition. 'The nomenclature for BALB(c and C57BL/IO Qa and Tla genes is as proposed in Refs 5 and 6. For other genes the nomenclature used in initial reports is preserved. The superscripts for Qa gene identify the H-2 haplotype from which Qa gene was cloned. The superscripts for Tla genes refer to Tla haplotype (7).

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Organization of class I-related genes in H-2" (BALB/c), H_2h (C57B L/1O) and H_2k (C3H or AKR) mice, The number of genes corresponds to the lowest estimate predicted for each strain (5, 6, 1 2, 45 and 8, 82, 9 1 , 94, 95), The H-2K, H-2D, Qa, Tla and Hmt regions are defined by recombinant mice genetics and cloning data, The Mb-J maps to the Hmt region ( 1 42). The meDl maps outside of chromosome 1 7. The orientation and relative position of the gene clusters within the regions is not known, The BALBlc Tll- "37" cluster is shown proximal to the TI- TJO cluster in relation to the centromere, in accordance with Ref. 138. The gene designations are listed above the cloned class I sequences; the unlabeled class I genes are predicted by Southern hybridizations with family-specific probes. Fragments of genes (homologous to exons 1-3 but not 4-6 of H-2) are marked as 5'. The brackets show proposed deletions_ The genes are aligned to indicate putative allelic relationships (exceptions are listed in the text). Figure J

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QA AND TLA ANTIGENS

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(TIC --+ TIi", T2e --+ TI2e, T3c --+ T13c, T6c --+ TI4c, T7c --+ TI5", T8c --+ TI6", Tge --+ T17e, Tloe --+ "37'). Given the DNA sequence homology between H-2K, D, L, and the nonclassical genes and the evidence for contractions and expansions of class-I gene families (15),it could be proposed that the nonclassical genes were derived by a fairly recent series of duplications from the H-2 genes. Studies of evolutionary relationships among H-2D, L and the nonclassical genes mapping to the H-2K, Qa and Tla regions argue against this hypothesis (14). Thus the organization of the class I-related loci may be relatively new and frequently changing, but their origin may predate the origin of the H-2D/H-2K divergence.

CLASS I-RELATED MOLECULES IN MOUSE

Qa

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The Qa-2 antigens were discovered by Flaherty (16) during the serological analysis of two recom­ binant mouse strains, which differ at the Qa locus. Qa-2 antigens are ,,-,40-kd polypeptides associated with f32m (17). They are present on lymph nodes,spleen,thymus,bone marrow,and liver but are not detectable by serology on other tissues expressing H-2 antigens (16). They are abundant on primitive hemopoietic progenitors but are lost progressively upon differentiation (18). In adult thymus the Qa-2 determinants are present on some,but not all mature CD4+/CD8- and CD4- /CD8+ cells,and absent on CD4+ /CD8+ thymocytes (19,20). All Qa-2 positive cells (5-6% of all thymocytes) express CD3, but it is not known whether they include r/b as well as alP T cell receptor expressors (20). The Qa-2 antigens are structurally similar (21) and have three known alleles (Qa_2high, Qa_210W, and null) which correspond to the quan­ titative differences in expression (17, 22). T cells from Qa_2high strains express ",4--5 x 104 Qa-2 molecules/cell,Qa_2low strains express no more than'" 6 x 103 molecules/cell; B lymphocytes from the same strains have '" lO-fold lower levels of Qa-2 antigens (23). Recent expression analyses using sensitive techniques (cDNA cloning, Sl nuclease or RNase protec­ tion, polymerase chain reaction [PCR] assays) have detected Qa-2 tran­ scription in nonlymphoid tissues as well: lung,liver,kidney (24,25),heart, testes, and brain (N. Dlker and I. Stroynowski, unpublished). Whether this transcription pattern is accompanied by Qa-2 translation remains to be shown. Expression of Qa-2 mRNA can be also detected as early as day 8.5 of development (24). It reaches a transient peak on day lO­ I I postcoitum,and then declines, which contrasts sharply to the steady increases in H-2 RNA levels throughout embryogenesis (26). Protein data QA-2 ANTIGENS AND QA-2 SECRETED MOLECULES

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506

STROYNOWSKJ

are generally consistent with these observations. Qa-2 antigens do not reach significant levels of expression until 3 weeks after birth (28). Two genes in the H-2d haplotype and four genes in the H-2b haplotype encode cytoplasmic Qa-2 molecules in transfected L cells (29, 30). These include a single pair of genes Q6d, Q7d in the BALB/c strain (Qa_2Iow strain) and two gene pairs Q6b, Q7b and Q8b, Q9b in the C57BI/10 strain (Qa_2hi9h strain). They are all closely related to one another, with the Q6 more similar to Q8, and Q7 more similar to Q9 (13). In transfl�cted hepatomas (29, 31) and thymomas (32, 33, 34) only Q7 and Q9 encode membrane­ bound Qa-2. This is consistent with protein sequencing analysis of spleenic Qa-2 molecules which revealed two components (35). The sequence of the minor Qa-2 component is identical to the predicted Q7b or Q9b product but not the Q8b product. The sequence of the major component is two aa longer than the minor component and has been attributed to an alternative cleavage of the leader sequence from Q7b and/or Q9b (Figure 2). The Q7 and Q9 polypeptides are attached to the membranes of spleno­ cytes and transfected cells by a phospholipid anchor (29" 31, 33, 36) and, like many phosphatidylinositol (PI)-linked molecules, participate in vitro in T-cell activation (37). Interestingly the Qa-2 antigens on resting spleen cells have different sensitivity to PI-phospholipase C (PI-PLC) than do those antigens on activated T cells (38). Furthermore, the PI-PLC-sensitive Qa-2 is expressed on CD4 + /CD8- cells while the resistant form is found on CD4 -/CD8 + cells. During T-cell activation, the changes in the structural properties of Qa-2 are accompanied by a decrease in cell surface expression (19) and simultaneous secretion of "-' 39-kd soluble Qa-2 (39). L cells and hepatomas transfeeted with the Q7 or Q9 genes also secrete soluble "-' 39kd Qa-2 (29), smaller than the "-'40-kd molecules cleaved off the mem­ branes with the exogenous phospholipase C (29, 38). The mechanism responsible for the synthesis of membrane bound and secreted Qa-2 mol­ ecules from the same gene is unknown, but recent data show that activated T cells, L cells, and hepatomas process Q7/Q9 mRNA into two forms (N. Ulker and I. Stroynowski, unpublished). One mRNA lacks exon 5 and is predicted to encode a soluble "-' 39-kd protein. The secreted Qa-2 may be translated from this alternatively spliced mRNA. The challenge now is to determine the biological significance of the Qa-2 "switching" in activated T cells, reminiscent of "switching" from membrane-bound to secreted IgM in activated B cells. Minor constituents of the spleen Qa-2 soluble fractions may also include products of the Q6 and Q8 genes (predicted to be secreted; Figure 2), although the bulk of the Qa-2 molecules secreted from activated spleno­ cytes have an NH2-terminal sequence of the Q7, Q9 products (35). Further

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QA AND TLA ANTIGENS

S07

studies are necessary to determine the contribution of Q6 and Q8 genes to the in vivo Qa-2 phenotype. Similarly, the BALB/c Q8/9d gene, proposed to be a fusion of ancestral Q8d and Q9d sequences (30), needs to be examined for its potential to encode a Qa-2 like protein. Qa-2 antigens can serve as weak alloreactive targets for CTL in vivo and in vitro (40). Recognition of Qa-2 positive targets is H-2 independent and requires in vivo priming and in vitro restimulation. The identity of all the Qa-2 region products recognized by anti-Qa-2 CTL has not been established (41). Thus far transfection studies have demonstrated that BALB/c Q6d and Q7d genes encode IY.) and 1Y.2 epitopes recognized by bulk and cloned Qa-2 specific CTL (4 1 , 42), but no comparable data exist for the H-2b haplotype.

{Q4) SOLUBLE MOLECULES Qb-l is a ",41 -kd soluble glycoprotein detected by Robinson (43) in spleen lysates with anti f32m antibodies. Its synthesis has been also demonstrated in B and T cells from bone marrow and lymph nodes. Qb-l has three known alleles: Two differ in their iso­ electric points and the third is a null allele (43). L-cell transfection experi­ ments with C57BL/ IO Q4b and AKR Q4k genes showed that Q4 genes encode a soluble 4 1 -kd product indistinguishable from the two known polymorphic forms of Qb- l (44). The DNA sequences of Q4 genes (44, 45) contain a single nucleotide deletion in exon 5 which leads to a shortened carboxyl end because of a frameshift and a premature termination codon. The same deletion and deduced carboxyl end is found in the Q8b gene (13). An independent study (46) has revealed that a gene, tentatively identified as Q4P, is widely transcribed in adult mice. It remains to be shown whether the transcription of Q4P in spleen, thymus, lymph nodes, liver, lung, heart, kidney, testes, muscles, and brain is paralleled by a secretion of a Qb- l product into serum.

QB-l

Soluble Q l O molecules in mice were first pre­ dicted from class I-like cDNA clones derived from liver (47). These cDNAs and the QI0 gene (45, 47, 48) carry a 13-bp deletion in exon 5 and encode a truncated class I-like molecule. Immunoprecipitations with antibodies against a synthetic peptide from the predicted Q l O carboxyl terminus and with xenoantiserum against H-2 antigens showed that the Q IO is secreted in vivo into serum and in vitro into cell culture media ( 1 1 , 49, 50, 5 1 , 52) . It has a molecular weight of ",40 kd and is associated with f32m (50, 51). In serum, Q I 0 is a part of a high molecular weight complex of 200-300 kd and is probably identical to class-I complexes detected earlier with rabbit anti H-2 antisera (53). Its serum concentrations range from un­ detectable to '" 60 ,ug/ml in different strains ( 1 1 ). QIO SOLUBLE MOLECULES

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Annu. Rev. Immunol. 1990.8:501-530. Downloaded from www.annualreviews.org Access provided by University of Central Florida on 02/22/15. For personal use only.

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H-2 polymorphic

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HLA polymorphic

1

class

variable

183

190

230

a3 H-2 consensus

DSPKAHVTHHPASEGKVTl

01' 02k 04b 05'

.P .

07d OBb OJOb 02d T30 T13' Tla"-l ·

37 "

"

·

2 3

Thy 19.4

Mb ·l

meOl.l

.2

. • . . . . . . . .

.....

VYHEGLPEPLTLAW

�.

q

280

190

lINKEA

i" �

300

310

310

330

340

350

CYTOPLASMIC

HyDROPHOBIC

RRRNKGGOGEOCALAPSRAAPRAlI SIS

S • . • . . . • • • • • • . . • . • • I . . • • • AFV R6RNTGGKVRONP.DPGROS. Q.SOISLLOCKVMVHOSLSLl s . . ...ANV . • • • • • . . WPSlOlWWll .... . P......VIl....AFV KRRRNTGGOGGOYAPAPA • . . SS.K1 • • • • • • • • • .AT • • • V .0 • • • • AI.GAV .AFV AA • • • • S • • . . . ANV. 1. ...V. WPSlElWWIl • • . •

·

. . .

.tr

"

I

07d OB� OJOb 02d nb

�

S.D. I. SHI.D.lWPSlKlWWYl • .. . F.... NAl. • • • • • F • • • AI.V ...MV • • . .

T13c

.L. OTSMP. • • . QSSHP.

T1aa-l

• • .

·2 ·3

-37Thy 19.4

.

@j

ALGFYPAOITLTWOLNGEELTOOMEL VETAPAGOGTFOKWASVVVPLGKEONY

a.o............................................................................

EPPPYTVSN VIIA LVVlGAVIVIGAVV II GVHVSFV

as'

"0

... .

.P..1 • • • . • • • • • . Y • • • • . . • • • • • • • • • • • • . • . • • • . . I • • T • • • . . • . . • • . • • • . • • Al . . • 5,.E • . K • • • 0 " .K . • • • . . . • • • .P . • T • • . . • • • P • • Y • • • • • • • • • • . • • H . • • • • • • . • • • . I • • • • • • • • • • • • • . • • • . • • A • • • • S.E • . K . • • • • . • • • • • • • • • • • • .P . • • • • • • • . • • • OE . • . • • • • • • • • • • • • . • • • . . . • • • • . • . • • • . • • • • • . • . • • • • • • A • . . • . . • • • Y • • • • • . . • • • . • • . . • • • EP • • . Y • • • . . . P • • 0 . . . • • • . • . • • . S • • IHI • . RD.. DO • . . . DVI . . • • • . • • • • • • . VA• . . . S . • • . • . . • • • A • • • • . • • . . . • • • T • • I .H • • KI.POR. T . • . • • . fH • • • PE • • • • • • RD. SHO • • . . • HI • • . • 5 . • • • • • • • • A • • • Sl.E.HI • . • • • N • • • . S • • I • • TKH A EK.V. WlSSV.S 'AH. HRO. V.HVS . • • • KPVWVHJ)HR. DOEO.GTHRGOfL.NA. E. WYlO.no.EAGEEAGlA.R.K.SS.GGO I.Y • EK.V. WLSSV.S.H.HLO.V.HVS • • • • KPVWVM.! lItR. OOEO.GTHRGOFl.NA. E. WYLO.TLO.EA6EEAGLA.R.K.SS.66001.Y •

Z7�

Mb ·1

160

50

1

. .....

.P . . • • . . • • • . . . • • • . • . . • • • . • • • • • • • . . • • . • • • . . • • • • . • • . • • • . • • . • • • • . • A • • . • . • • • . . • • • • • H • • • • . • . • • • • • .P . . • • • . C.H • . 0.0 • • • • . • • • . . • • • H.I . . . • . • • • . . • . • • • • • . • • . 5 • • . . . • • • • • • . • . . • • • • • . • • • • H . • • • . • . • . • • • .P.E.K • • • • H • . 0.0 • • • K . • • • • . . • • • • • • • . • . • • • . . . . • • • • • • • • • • EN • • . • • • • • . • • • • • • • • K . • • • • E • • • • • • • • . . • • .P • • • • • • • • • • • Y.A • • • • . . • • • • . • • • • • • • . • . . • • • . . . . T • • • • • • • • • • • . . • • . . . • • • . • • • • • • • . • • • N . • • • . • • • • • • • GRW .P . • . . • • . • • • • Y.A • • • • • • • • • • • • . • . • • • • • . . . • • • • • . • • • • • , . • • . . • • • • • • . • • • • . • • . • • • • • • • • N . • • • . • • . . • • • .P.. T. • • • • • G • • • O • • • • • • • • • • • . . • • • • • • • • . . . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • . .P • • • • • • • • . 1 . • • A . . . . • • • • • • • • • • • . . . • . . • • . • • • • • • • . . • • • • • • . • • • . . • • • • • H.I 95% (for the Qa region products) to 64% (for Mb- l ) . The most divergent antigens are the murine equivalents o f the CDI antigens, mCD l . 1 and mCD 1 .2, with no discernible homology to the H2 proteins in their a l and 0(2 domains and only � 27% homology in the a 3 domain. This is comparable t o the similarities found between the class­ I H-2 antigens and the class-II antigens. Because of these pronounced differences between the mCD 1 proteins and other class I-like proteins, the two groups are discussed here separately. The aa sequences (Figure 2) fail to reveal any features incompatible with their expression (with a possible exception ofmCD 1 .2; see Ref. 95 below). In searching these sequences for structural features shared with the H-2 antigens two criteria were considered: (a) the pattern of variability at each position, and (b) the potential to fold into the HLA-A2-like structure (3). Of the 53 positions conserved in the a l and a 2 of HLA and H-2 antigens ( 1 1 8), 28 are conserved in the MHC-coded class I-like proteins (Figure 2). Only a couple of the nonconserved residues appear significantly more variable than others (positions 44 and 1 65). The substitutions in the con­ served residues occur at different positions in different proteins and do not fall into specific patterns; thus the identification of these class-I proteins as Qa or TL products is not possible on this basis.

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518

STROYNOWSKI

The analysis of variable residues among the class I-related proteins shows a striking correspondence with the residues identified as poly­ morphic in H-2 antigens. The variable residues in the nonclassical MHC proteins are those that show more than five substitutions (Figure 2). Many of these variable positions correspond to the aa pointing into the antigen­ binding cleft in the model of HLA-A2 (positions 9, 24, 63, 66, 73, 97, 1 1 4, 1 1 6, 1 52, 1 55, 1 56) or to those proposed to point towards the T cell receptor (positions 69, 1 50, I SS, 1 63). Some of the variable positions (for example 1 56) have substitutions not found in the mouse or human transplantation antigens (C, S, H, V). Hence MHC-coded class I-like proteins may fold into structures similar to the HLA-A2 and serve as restriction elements for antigens that are not presented by the classical H-2 molecules. The very limited polymorphism of the class I-related proteins suggests that these proteins may complex with antigens which are themselves non­ polymorphic. Consistent with this, the allelic substitutions correspond to aa located outside of the antigen binding cleft. Thus Q4k protein differs from Q4b in the 1X3 domain (A236 --+ S236; Y262 --+ H2d and Q7b differs from its Q9b pseudo allele in the 1X2, at a position outside of the cleft (Q173 --+ EI73). The major regions of the variability between TL related proteins (T3b , T l 3C, Tlaa-I , -2, -3) fal l into the regions outside of the major groove (positions 34-43, 48-49, 86, 1 06-1 08, 1 82). There are also examples to the contrary; Q7d differs from Q7b in 1X3 domain (T228 --+ M228) but also in the IX I residue that corresponds to a polymorphic position in H-2 antigens and points into the cleft (M66 --+ T66). Q l Ok differs from Q l OQ in a residue of a f3 strand that points into the binding site (Ms --+ Ts) ; Q I 0k differs from Q l Ob in the highly conserved residue that faces the cleft (Y84 --+ H84). These examples may indicate that the class I-like proteins evolved to accommodate a low level of poly­ morphism of the presented antigen(s). Several other lines of evidence are consistent with the proposition that class I-related proteins have a structural similarity to the HLA-A2. Cross­ reactivity of the Qa and H-2 products at the CTL level (60, 6 1 , 1 1 9) and at the serological level ( 1 20, 1 2 1) indicates that they share similar epitopes. Substitution of exon 3 of the H-2Dd with the homologous region from the Q7d gene leads to the expression of hybrid molecules with serological epitopes of H-2Dd and Q7d ( 1 22). Many of the critical aa, which in HLA­ A2 are involved in the formation of the 3-D structure, are co nserved in class I-like proteins. These include proline PIS which forms the turn between f3 strands and P47 and Pso (one or both) found before first IX helix of the IX I domain. Residues that contact f32m (Q96, Q I 1 5 and G120) are present in all the molecules (except Mb- l where Q l l5 --+ K l l 5 and G120 --+ LI20). Many of the downward pointing residues on the IX helixes that contact the f3 pleated

QA AND TLA ANTIGENS

519

(W60 , L160 , L 168 , L 179, A 1 53) are identical or are conservatively sub­ stituted (exceptions in Q2k: L I 60 � M 1 60 ; in TL -s: L1 79 � Q I 79; in TL-s and Q4k: A I 53 � T I 53) . The intradomain disulfide bridges between cysteines

sheet

CIOI

�

C I 64 and C203

�

C259 are conserved and so are the salt bridges

between H3-D29 and RI I I -D I 29 (exception in Q l k: D I29

�

S I29).

In contrast, the iX , and iX2 domains of mCD l protein do not share these structural similarities with the H-2 antigens. However, they have retained some of the features of the class-I molecules: disulfide bridge between

CIOI -+ CI64 (in mCD l . l but not in m C D 1 . 2) and the conserved residues at

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positions 96, 1 00, 1 60, 1 72, and 1 79.

In the Ci3 domain all the class I-related molecules share sequence motifs

characteristic of immunoglobulin-type domains: a patch of alternating hydrophobic amino acids starting at position 245, and 1 2 residues (posi­ tions 1 8 5, 20 1 , 203, 208, 209, 2 1 0, 2 1 7, 240, 259, 26 1 , 263) that are also conserved in the class-II MHC molecules. Two of these residues cor­ respond to cysteines (C 20rC259) that form a disulfide bridge, while a tryp­

tophan at position 2 1 7 is found in all immunoglobulin domains. In addition

there are four more residues shared by all the class I-like proteins (positions

22 1 , 226, 235, 266). There is a heterogeneity in the length and sequence of predicted carboxyl

termini of class I-like proteins. Since the carb oxyl terminus often encodes signals that target the polypeptide to specific intracellular compartments,

it may also control points of intersection between the class I-like proteins

and the peptide antigens. Hence the COOH end of class-I polypeptides may be of primary importance for their biological function. Proteins that

lack cytoplasmic domains (Q4, Q8, and Q l O) are likely to be secreted. All other class I-like proteins in Figure 2 may be membrane bound. It is difficult to predict which of them will be integral membrane proteins and which will be attached by a phospholipid anchor, as is the case for the Q7 antigen. The molecular features of the protein precursor destined for cleavage and subsequent PI attachment are not well defined, but they include a hydrophobic region similar to transmembrane domains of the classical H-2 antigens and a shortened cytoplasmic tail (N. Ulker, I. Stroy­ nowski, unpublished).

Inspection of the cytoplasmic regions of several class I-like molecules

reveals the presence of cysteine residues. By analogy with the intracellular

cysteines of CD I -s that are thought to mediate association with CD8, it is possible that Ql\ Q2\ Tlaa and "37" have a potential to form disulfide

bridges with other proteins. Unpaired cysteines in the extracellular

domains of Q5, TL-s, "37" and mCD I-s may serve the same purpose.

Possibly interactions of class-I proteins with other polypeptides may affect

their ability to transduce activation signal(s) and/or alter the responses

Annu. Rev. Immunol. 1990.8:501-530. Downloaded from www.annualreviews.org Access provided by University of Central Florida on 02/22/15. For personal use only.

520

STROYNOWSKI

of cells engaged in interaction with the class-I complexes via the T cell receptors. It is important to stress that these structural interpretations are critically dependent on several hypotheses. Among them is the assumption that class I-related transcripts are processed in a way similar to H-2 RNAs. This is not always true as the alternative splicing has been described for a number of Qa genes. For example, the Q7 and Q9 RNAs lacking exons 4--7 (24) or exon 5 (N. Ulker and I. Stroynowski, unpublished) were identified. Some Q 1 0 gene transcripts lack exon 3 (25). It is not known whether these truncated mRNAs direct synthesis of stable proteins. If they do, a new family of class I-related proteins should be considered.

FUNCTIONAL IMPLICATIONS AND CONCLUSIONS Despite extensive data and major efforts, no function has been assigned to class I-related products. Initially it was suggested that class I-like sequences are pseudogenes created by recent H-2 gene duplications, but this now appears unlikely ( 1 4). Because deletions in the Qa and Tla loci are frequent (as expected for a family of closely related genes) it has been proposed that they have no biological significance Of, at best, play a minor role as donors in H-2 gene conversion events. Although the importance of gene conversion as the major mechanism driving H-2 polymorphism is still controversial the role of the class I-like loci in generation of new H2 alleles has been established ( 1 23). While the mouse genome carries a number of class-I pseudogenes (Table 2), the majority of the sequenced class I-like genes appear intact and likely to encode proteins. When considering potential functions of these polypeptides it is important to recognize the diversity in their structures and homology to H-2 molecules. On this basis alone, one can expect different categories of potential functions. It is especially difficult to guess the functions of the class-I proteins that differ substantially from H-2K, -D, L ; for example, the mCD ! products. Out of � 1 00 invariant positions in a " a2, and a3 of classical trans­ plantation antigens ( 1 1 8), only 2 1 are conserved in mCD 1 . This is less than half the number of positions conserved in the rat Fc receptor which was recently shown to be structurally related to MHC c1ass-I antigens ( 1 24). The p53 Fc receptor from baby rats binds IgG from mothers' milk and represents a fi r st case of nonclassical protein for which a function has been defined. A role as a receptor for virus entry has been suggested for another, highly divergent, class I-like protein encoded by a human cytomegalovirus ( 1 25). At present there is no way of knowing how many more class I-like genes with homologies undetectable by Southern blot hybridizations are encoded in the mouse genome and what their functions may be. -

521

QA AND TLA ANTIGENS Table 2

Current status of sequenced (or partially sequenced) class I-like genes and t he i r

products in mouse Demonstrated expression Gene k

C3H

Qi' 4h Q 4k Q b Q5 5k Q 7d Q 7b Q

C3H

Ql

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Strain of origin

b

Q8 h Q9

0b Ql 0 Ql Q 0k Ql D2d k K2

Tl' T2"

BIO C3H BIO C3H BALB/c BIO BIO BIO BIO

SWR C3H BALB/c C 3H

RALB/c A

3b

B I O, B6

T

Product k Ql k Q2 Q4b (Q b- I) k Q4

probably i/J k

Q5

Q7d (Qa-2) Q7b (Qa-2) Q8b

Q9b (Qa-2) h QJO Q l Oq k QlO

D2d probably i/J probably i/J probably i/J T3 b (TLb)

T/a"-l

A A

Tla I Tlaa-2

T1aa-2 ' T5

A

Tlaa-2

T1aa-2

TJO' T1 3'

b TJ 3 "

37

"

"-

BALB/c

probably i/J probably i/J T l 3' (TL')

B6

probably i/J

BALB/c BALB/c

BALB/c

"37"

Thy 19.4

BALB/c

Mb-l

BIO

Mb-I

BALB/c

mCD l . l

mCD1.2

C3H

mCD 1 .2

mCDl.l

a

Thy

1 9.4

RNA level

? widespread

?

? widespread widespread ? widespread liver liver liver ? ? ? ? leukemias ? ? ?

Protein level ? ? spleen ?

?

? lymphoid lymphoid ? lymphoid liver liver liver ?

References 45 45

44 45

44 45 141

and "

13 13 13 48

and '

47

45

63 64

?

1 39

leukemias

70, 7 1

140 73 73 73

?

?

9

widespread thymus, leukemias

thymus, leukemias

5

spleen ?

82

widespread

?

95

widespread

?

95

8

widespread widespread ?

92 94

D. Nickerson, personal communication.

It is perhaps more instructive to discuss MHC products that are highly homologous to the H-2 antigens. They have the potential to carry out some of the nonimmunological functions suggested for the H-2 proteins, including a role in adhesion ( 1 26) and contact inhibition ( 1 27), or a role in expression of hormone receptors (insulin, glucagon, interleukin 2, epidermal growth factor, y-endorphin, luteinizing hormone [reviewed in 1 28]). The structural diversity of different class-I products, their unique patterns

Annu. Rev. Immunol. 1990.8:501-530. Downloaded from www.annualreviews.org Access provided by University of Central Florida on 02/22/15. For personal use only.

522

STROYNOWSKI

of expression, and low polymorphism may represent adaptations for these types of function. It is also interesting to consider potential immunological functions of class I-like proteins, in particular their antigen-presenting potential. Although there is no evidence that they can act as restriction elements for conventional antigens (8 1 , 1 29), the structure of many class I-like polypeptides is compatible with the 3-D model for HLA-A2. Furthermore, several of them (Qa- l , Qa-2, Hmt, and others) can act as weak trans­ plantation antigens and/or alloantigens (80). The recent data suggest that allorecognition of the Hmt involves presentation of antigen in a peptide form ( 1 42). Like H-2 antigens, many of the class I-related proteins are inducible with interferons ( l , 73, and 1. Stroynowski, unpublished), a property thought to increase the efficiency of target cell recognition by C TL. If indeed the class I-related proteins interact with T cell receptors, do they guide cytotoxic reactions or stimulate proliferation? Both alter­ natives are possible: the Qa-2 antigen can be recognized by CTL (40) but can also trigger T-cell activation (37). During differentiation both these pathways may be used to amplify or eliminate specific cell lineages marked by individual class I-like antigens. It is intriguing that tht; developmental

pattern of expression and tissue distribution of some class I-like proteins are distinct from H-2 antigens. These properties served initially as a basis for identifying Qa and TL molecules as "differentiation antigens." Two functions were proposed for secreted class-I molecules: a role in the induction of tolerance to H-2 alloantigens (47), and hormone-like function in embryogenesis (55). The first of these propositions is not easily reconciled with the in vitro studies showing that animals with soluble class­ I proteins in their serum have CTL reactive with membrane-bound form of these proteins ( 1 30) . The role of the Qa-region genes in regulation of the rate of cleavage in preimplantation embryo has been suggested (27), but the mechanisms involved are not understood and may well include Qa-2 soluble molecules or antigens and their potential nonimmunological functions. If some of the class I-like molecules act as restriction elements what do they present? It seems reasonable to assume that the nonpolymorphic structures will bind invariant antigens. These may include endogenously coded "self" antigens, for example, embryonal or differentiation antigens, consistent with "differentiation" and "cell lineage selection" hypotheses. These types of antigens may also be present on some tumors. Two embry­ onal carcinomas negative for H-2, but expressing gene "37", "Q7," and another unidentified class I-like gene were recently described ( 1 3 1). It was proposed that rejection of these tumors is mediated by the class I-like

Annu. Rev. Immunol. 1990.8:501-530. Downloaded from www.annualreviews.org Access provided by University of Central Florida on 02/22/15. For personal use only.

QA AND TLA ANTIGENS

523

structures complexed with tumor antigens, such as the embryonic antigens that are absent during the induction stage of T-cell tolerance. Stress pro­ teins marking damaged cells for possible destruction represent another class of potential endogenous antigens ( 1 32). Another category is carbo­ hydrates, or other unusual nonpeptide antigens derived from bacteria and fungi ( 1 33). What are the possible ligands for the class I-like/antigen complexes? Several investigators have suggested that y/) T cell receptors may be involved ( 1 32, 1 34). During ontogeny the y/) T cells are coordinately expressed with some of the known Qa, Tla genes. In adult mice only a small fraction of the y/) T cells home to peripheral lymph tissue; the great majority are found in skin, gut, and lung, hence the proposed function of yb cells and class I-like restriction elements in the immune defenses against pathogens in these tissues ( 1 33). Not all the yb receptors are specific for class I-like antigens, but a few cases with targets in Tla have been reported ( 1 34). Interestingly the majority of the y/) cells are CD4 - CDS - , and at least some Qa and Tla products are predicted to be CDS independent. If class I-related proteins serve as restriction elements, several paradoxes have to be explained: absence of highly homologous Qa and Tla sets of genes in other species, extensive deletions of Qa genes in some inbred mice, and low levels of expression of many class I-like products. Possibly the antigens presented by class I-like structures are species specific and impose structural constraints on their restriction elements. In other words, class I-like molecules may have coevolved with their antigens. Deletion of the Qa genes in some mice could have been preceded by the deletion of the genes encoding their respective "self" antigens. Alternatively, these mutant strains may carry a set of restriction elements with redundant functions or, if environmental pathogens are presented, the mutant mice may be less fit outside of the laboratory. It has often been said that nonclassical proteins can be distinguished from the H-2 antigens by their restricted tissue distribution. Recently, more sensitive assays have demonstrated that transcription of the class 1related genes is often widespread (Table 2), but overall much lower than the H-2 expression (at least l O-fold lower for Qa-2 in spleen; l OOO-fold or lower for other genes, like Thy 19.4). Thus the apparent absolute tissue tropisms of various class-I products may simply reflect the inability of some assays to detect low levels of expression in some cell types. Are such low levels of expression sufficient for antigen presenting functions? A fraction of a percent of the H-2 is enough to present influenza peptides ( 1 36, 1 37). This is comparable to the expression level of some of the class I-related molecules. The dichotomy in the quantity of the classical and

Annu. Rev. Immunol. 1990.8:501-530. Downloaded from www.annualreviews.org Access provided by University of Central Florida on 02/22/15. For personal use only.

524

STROYNOWSKI

nonclassical restriction elements may correlate with their affinities for the respective antigens: whereas H-2 evolved for maximum degeneracy to accommodate a whole spectrum of polymorphic peptide:s, the non-H-2 class I proteins may be dedicated to a limited set of invariant and "well fitting" antigens. The low expression of the class I-related proteins may help to evade self-reactivity and account for apparent inability of the Qa and TL molecules to present viral antigens. Biochemical studies of the class I-related proteins and putative antigens bound to them, as well as experiments with transgenic mice overproducing Qa and TL, may help to test some of these hypotheses. At present, class I-like molecules remain as enigmatic as when they were first discovered. Their number, variation in structure, and tissue distribution suggest a multitude of functions. To identify them all may require an element of chance but for the immediate future the obvious challenge is to establish their sites of expression during development and to determine the intracellular compartments where they may encounter potential ligands. ACKNOWLEDGMENTS I am pleased to acknowledge the help of many colleagues who have contributed their results prior to publication, especially Drs. M . Soloski, J. Forman, D. Singer, K. Fischer Lindahl, and C. Milstein. I also thank Drs. D. Nickerson, K. Brorson, P. Bjorkman, and L. Hood for critical reading of the manuscript, and N. King for typing. Supported by NIH grant AI 1 7565.

Literature Cited

I . Singer, D. S., Maguire, J. E. 1 989. Regulation of the expression of class I MHC genes. CRC Crit. Rev. Immun. In press 2. Hood, L., Steinmetz, M . , Malissen, 8. 1 983. Genes of the major histocom­ patibility complex of the mouse. Annu. Rev. Immunol. I: 529 3. Bjorkman, P. J., Saper, M. A., Samra­ oui, 8., Bennett, W. S . , Strominger, J . L., Wiley, D. C. 1 987. Structure o f the human class I histocompatibility anti­ gen, HLA-A2. Nature 329: 506 4. Bjorkman, P. J., Saper, M. A., Samra­ oui, 8., Bennett, W. S., Strominger, J . L . , Wiley, D. C. 1987. The foreign anti­ gen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329: 5 1 2 5. Fisher, D . A., Hunt, S . W . III, Hood,

L. 1 985. Structure of a gene encoding a murine thymus leukemia antigen, and organization of Tla genes in the BALB/c mouse. J. Exp. Med. 1 62 : 528 6. Weiss, E. H., Golden, L., Fahrner, K., Mellor, A. L., Devlin, J. J . , Bullman, H., Tiddens, H., Bud, H., Flavell, R. A. 1 984. Organization and evolution of the class I gene family in the major histocompatibility

complex

of

the

C57BL/1 O mouse. Nature 3 10: 650 7. Chen, Y.-T., Obata, Y, Stockert, E., Takahashi, T . , Old, L. Y. 1 987. Tla­ region genes and their products. Immu­ nolo Res. 6: 30 8. Brown, G. D., Ch oi Y., Egan, G., Meruelo, D. 1 988. Extension of the H2Tlb molecular map. Immunogenetics 27: 239 ,

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