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Much ado about minor hist0c0mpatibility antigens Elizabeth Simpson, Derry Roopenian and EIs Goulmy

ur knowledge of minor

histocompatibil i ty (H) anti-

gens has developed from

rodent studies and the large

body of clinical data on graft-versus-host

disease (GVHD), graf t -versus- leukaemia

(GVL) effect and host-versus-graft (HVG)

reaction in humans. The meeting provided

an exceptional opportuni ty to communicate

knowledge rapidly amassing in each area to

arrive at a better unders tanding of immune

responses to this hitherto enigmatic group

of transplantation antigens. The pioneering

work over the past decade, largely from the

efforts of Hans-Georg Rammensee, Kirsten

Fischer Lindahl and Thierry Boon, set the

paradigms that are now being strengthened

by the considerable data presented at this

meeting. The meeting was particularly timely

in that the genes encoding the pept ide

components of several minor H antigens

have been identified in rats, rnice and hu-

mans, and clinicians are seriously consider-

14 A t~, C i i i 9 9

Current knowledge of

histocompatibility (H) loci

located outside of the major

histocompatibility complex -

i.e. those encoding the so-called

minor H antigens - teas surveyed

at a recent meeting*.

ing strategies that employ minor H antigens

in the management of GVHD and GVL. A

summary, of advances and outstanding ques-

tions in this research area is given in Box I.

G e n e t i c s : o ld m e e t s n e w

The meeting was dedicated to the memory

of George Snell, who laid the immuno-

genetic foundations of the subject dur ing

his many years working at The Jackson

Laboratory (Bar Harbor, ME). It was fitting

that D. Bailey (Bar Harbor, ME), who was a

colleague of Snell in the 1960s and 1970s

and who created and analysed most of the

minor H antigen congenic strains studied

today, related the history of the way in

which the minor H loci in mice had been

inves t iga ted when it became apparent

that they segregated from the major histo-

compat ibi l i ty complex (MHC) and that

there were many of them. J. van Rood

(Leiden) provided a historical perspective

on the early discoveries of human H anti-

gens, both major and minor, and high-

lighted the communicat ion gaps that then

existed between the mouse experimentalists

and their clinically oriented counterparts.

He also questioned the assumption that only

loci that encode pept ide antigens contribute

to GVHD.

No such assumptions were made by the

retrospective genome screening approach

described by D. Ginsburg (Ann Arbor, MI).

He reported on a 10 cM scan using HLA-

identical donor- rec ip ient pairs in which

the recipient had developed severe GVHD,

in an a t tempt to localize genetic poly-

morphisms associated with the disease. The

I O~ VoJ I 9 M o o 3 *The 1st International Symposium on Minor Histocompatibility Antigens wa>

held at Bar Harbor, ME, USA, on 14-17 September I9u7

T R E N D S M N U N O L O G Y T O D A Y

250 pairs analysed, of which approximately

50% were HLA-A2, showed some sugges-

tive localizations that bordered on statistical

significance. However, the numbers ana-

lysed to date expressing any particular HLA

allele were too low to draw conclusions.

Nevertheless, W. Nichols from the same lab-

oratory demonstrated the ability of this ap-

proach to exclude the gene encoding CD31

as a candidate minor H antigen gene.

The still-unresolved issue of the number

of loci that encode minor H antigens is of

considerable conceptual and practical con-

cern, and was thus discussed in many con-

texts. Bailey recounted his estimates made

utilizing skin graft rejection as a criterion

for detecting minor H loci. For example,

considerations based on F2, backcross and

mutational analysis (which also resulted in

the isolation of the valuable H-2K bm series),

and the development of congenic and re-

combinant inbred strains, bring different

but frighteningly large (for those consid-

ering clinical issues) estimates, even in

those situations where the MHC is fixed.

D. Steinmuller (Bozeman, MT) discussed

the extraordinary sensitivity of skin to allo-

graft rejection, presumably because of the

rich cost imulatory context when minor

H antigens are presented to T cells by skin

Langerhans cells. Estimates made using the

skin allograft model may thus detect the

upper limit of possible minor H antigens.

Possibly consistent with these large esti-

mates, K. Fischer Lindahl (Dallas, TX) noted

that, while only 13 proteins are encoded by

the mitochondrial genome, there are now

four minor H epitopes, defined by class I-

restricted T cells, described from these

genes. Given the vastly greater number of

genes carried by nuclear DNA, she sug-

gested that there should be a very high

number of potential autosomal minor H loci

even considering the lower mutation rate of

nuclear DNA compared with mitochondrial

DNA.

The possibility that the number Of minor

H antigens may be functionally limited

was the subject of further discussions.

D. Roopenian (Bar Harbor, ME) had previ-

ously found that, for a number of chromo-

somal regions, each 'locus' defined by skin

grafting within a congenic segment in-

cludes at least one CD8+-T-cell-recognized

epitope restricted by class I molecules and

one CD4+-T-cell-recognized epitope re-

stricted by class II. He discussed his esti-

mates of minor H epitopes presented on

lymphoid cells by class I molecules: these

were in the range of 10 to 20 in the context

of a given MHC allele. He attributed these

unexpectedly low numbers to the lowered

immunogenicity of lymphocytes compared

with skin allografts. A mathematical ap-

proach was used by P. Martin (Seattle, WA)

to deduce from retrospective bone marrow

transplant data a relatively small number

(seven) of minor H antigens important for

GVHD. He calculated that matching at a

smaller number (three) could significantly

decrease the risks of GVHD in MHC-

mismatched donor-recipient pairs. The

analysis was based on looking at disparities

recognized by the donor in the recipient and

assumed a normal frequency of bi-allelic

alleles in the population. These estimates

agreed well with those from E. Goulmy

(Leiden), who analysed HLA-A2-matched

bone marrow recipients and found a strong

correlation of acute GVHD with mismatch-

ing of one or more of five minor H antigens

(HA-I-HA-5), and that even mismatching

of one (HA-l) was associated with the high

grades of GVHD. HA-1 is immunodomi-

nant and acts as a 'major minor ' . E. Roosnek

(Geneva) reported the failure to detect

minor H antigen-specific cytotoxic T lym-

phocytes (CTLs) in HLA class I- or II-

mismatched pat ient-donor combinations,

but in his HLA-matched pairs, HA-1 CTLs

dominated the response. The clinical poten-

tial of such biochemically identified minor

H antigens should extend to bone marrow

donor selection, prediction of development

of GVHD and immunomodula t ion of

GVHD by T-cell receptor (TCR) peptide an-

tagonists. Interestingly, a high proportion

(10/12) of patients with acute GVHD also

had mismatches at CD4+-T-cell-recognized

epitopes, again emphasizing the impor-

tance of the synergistic effects of CD4 ~

T-cell and CD8 + T-cell collaboration.

I m m u n o d o m i n a n c e

The importance of immunodominance as

a mechanism that functionally limits

the number of clinically relevant minor H

M A R C H I 9 9 8 V o l . I 9 N o . 3 1 0 9

T R E N D S

antigens was a topic of considerable interest.

Thus, when multiple minor H differences

are present, as found between different

strains of MHC-matched mice or humans, a

small number of the total dominate the

response. C. Perreault (Montreal) compared

dominant with dominated H-2Db-binding

minor H peptides. Through experiments

comparing the amount of dominant pep-

tides recovered per cell with their ability to

stabilize class I proteins and to trigger

a T-cell response, he suggested a novel

mechanism to explain immunodominance.

Dominated peptides are at low peptide

density and thus only trigger TCRs with

high-affinity interactions, while dominant

peptides are found at a considerably higher

density and thus promote TCRs with lower-

affinity interactions. T cells directed at the

dominant pept ide are at a competi t ive

advantage in rive when they encounter anti-

gen-presenting cells because they can en-

gage and then release the number of pep-

tide ligands required for activation more

efficiently than TCRs recognizing rarer,

dominated peptides at a higher affinity. As

discussed below, now that several H-2D b-

restricted minor H antigens, both dominant

and dominated, have been defined at the

peptide level, this intriguing model can be

addressed more comprehensively.

Both P. Wettstein (Rochester, MN) and

R. Korngold (Philadelphia, PA) described

their studies analysing immunodominant

antigens detected in C57BL/6 mice in re-

sponse to BALB.B minor H antigens.

Wettstein showed results where the re-

sponse measured by cell-mediated cytolysis

assays after in vitro culture showed the ex-

pected immunodominance of the CTT2

(H4b), while secondary allograft rejection

showed no such discrimination between

dominant and immunodominated antigens.

These studies questioned the physiological

significance of dominant antigens defined

by i~t vitro CTL assays, at least as measured

by allograft rejection. He also described a

detailed analysis of TCR usage in response

to the H4 t' minor H antigen by comparing

V~ and V6 usage and complementarity-

determining region 3 (CDR3) sequences of

H4b-specific CTL clones with TCR spectre-

types of CD8 + T cells infiltrating primary,

secondary and tertiary H4b-disparate allo-

f'/I A t~, C H i 9 9 8

grafts. The relatively simple pattern of

Vc~- and VIB-gene usage and negative charges

in the TCR contact residues of the carboxy

region of CDR3 were all consistent with

the CTLs responding to the same minor H

antigen peptide. Korngold described stud-

ies investigating CD4 + and CD8 ~ T cells in

GVHD in irradiated BALB.B mice that re-

ceived C57BL/6 bone marrow. He analysed

TCR usage of T cells from the thoracic

duct of recipient mice and suggested two

predominant antigen specificities in the

B6--*BALB combination, but this number

was reduced to one in the case of B6 *CXBE.

Since previous analyses by skin grafting

and CTL generation have found each of

these combinations to differ at a number of

loci, it might be concluded that the number

of antigens involved in GVHD is substan-

tially lower than the number involved in

skin allograft rejection.

H o w m i n o r H pept ides get into the groove and c o m e to be recognized Biochemical questions discussed at the

meeting included antigen processing, which

quantitatively and qualitatively controls the

loading of peptides into MHC molecules,

and the structural relationship between the

pept ide-MHC and the TCR, which is crucial

for an understanding of the structural re-

quirements for T-cell triggering. In addition

to relating his earlier studies into the genetics

and biochemical basis of minor H antigens

and class I-binding motifs, H-G. Rammensee

(T/ibingen) discussed the role of the proteo-

some and its regulator, P28, in processing

short peptides from cytoplasmic proteins,

and the rules for cutting and editing that are

emerging. He also described the role of the

chaperone molecules protein disulphide iso-

merase (PDI) and GP96, which appear to

bind to peptides carried by the transporter

associated with antigen processing (TAP)

that do not readily bind to class I proteins,

possibly providing the opportunity for

'nibbling' to trim peptides to the right length.

TAP proteins are crucial for the transport

of most peptides into the endoplasmic

reticulum. G. Butcher (Cambridge) described

a 13mer rat mitochondrial peptide derived

from ATPase 6, with two alleles differing at a

single internal amino acid substitution that

i I 0 V o ~- ! 9 M o . 3

are both recognizable by MHC class la-

restricted T cells. This single amino acid

change is important for processing by the A

allelic form of TAP, while it is crucial for TAP-

dependent transport of antigenic peptides.

J. Taurog (Dallas, TX) presented evidence

for a novel locus in the mouse MHC pro-

ducing a 'cim'-like effect on peptide load-

ing. He named it Cim2 (Ciml being Tap), and

mapped it between K and the Tap/Lmp gene

complex, and thus distinct from known

antigen-processing genes. It affects the load-

ing of male-specific peptides into HLA-B27

expressed from a transgene, and leads to

quantitative differences in target cell lysis

by B27-restricted cytotoxic effector cells,

and differences in the high-performance

liquid chromatography profiles of peptides

eluted from endogenous as well as B27-

transgenic class I molecules.

The close, reciprocal relationship be-

tween amino acid residues of the pept ide-

MHC complex and the corresponding TCR

was examined by S. Nathenson (Bronx,

NY). He used a novel approach involving

mice transgenic for the TCR Vc~ receptor of

an H-2Kb-binding vesicular stomatitis virus

(VSV) peptide antigen to probe the CDR3

sequences of the TCR V[3 that are required

for antigen recognition. Most VSV-specific

T cells used V613, with position 98 assum-

ing prominence primarily through contact

with the opposingly charged 'point up'

residue at position 6 of the peptide.

Ident i f icat ion of m i n o r !-I genes There has been an explosion of information

on the molecular identification of the pep-

tide components of minor H antigens recog-

nized by T cells. Fischer Lindahl described

her continuing investigation into mitochon-

drially encoded minor H antigens, in which

she took advantage of the small size (16 kb)

of the mitochondrial genome to compare

the DNA sequences of those carried by in-

formative mouse strains. Allelic forms of

perfectly functional mitochondrial proteins

(ND1, CO1, ATPase6) gave rise to minor H

antigen peptides showing sometimes very

conservative single amino acid changes.

Several of these are presented by the mono-

morphic H-2M3 class Ib protein, while two

(one in rat and one in mouse) are presented

T R E N D S I M M U N O L O G Y T O D A Y

by conventional class Ia proteins. She dis-

cussed results suggesting that the mito-

chondrial proteins may not follow the

normal cytoplasmic processing route. More-

over, it was particularly intriguing that con-

servative amino acid changes required for

allelic discrimination of the NDl-der ived

minor H pepide pointed 'down' into the

F pocket of the class I H-2M3 groove. Thus,

in contrast to the results discussed by

Nathenson and Wettstein, the polymorphic

residues of this peptide do not seem to point

up and thus cannot easily interact directly

with the TCR.

E. Simpson (London) provided back-

ground information about the immuno-

genetics of H-Y antigens, and the localiz-

ation of their genes onto a region of the

mouse Y chromosome lying between Zfy-1

and Zfy-2. D. Scott (London) then described

the identification by expression cloning of

H-Y peptides restricted by three different

class I alleles - K k , D k and D b - derived from

two genes: Uty and Smcy. V. Engelhard

(Charlottesville, VA) and Goulmy have also

shown that the human homologue SMCY encodes two H-Y epitopes, restricted by

HLA-A*0201 and B7, respectively, identified

initially by the peptide elution approach.

Engelhard reported on his finding that

natural SMCY peptides preferentially rec-

ognized by CTLs can be post-translationally

modified by covalent cystine dimers, making

the link between gene and peptide more

opaque in the cases where such modification

occurs. The function of neither Uty nor Smcy is known, but their genes are ubiquitously

transcribed, and their sequences suggest

they may be DNA- or protein-binding

factors localized in the nucleus.

A substantial effort is now being made to

map genes encoding autosomal minor H

peptide epitopes in humans. There was a

strong feeling that this could be speeded up

by international collaboration. Discussions

on the best way of conducting this con-

cluded that initially the central provision of

lymphoblast cell line (LCL) target cells from

large families that are well-characterized at

the DNA level and carry the most common

HLA restriction molecules would be the

best way forward. This would facilitate the

identification of T-cell clones from different

laboratories with the same specificity.

P. Beatty (Salt Lake City, UT) illustrated

the use of family panels to search for the

chromosomal location of genes: he localized

genes encoding two different B7-restricted

epitopes identified by T-cell clones isolated

from a patient with GVHD to chromosome

11 and chromosome 22, following transfec-

tion of B7 into LCL cells of families in which

the minor H epitope gene was segregating.

Progress towards unders tanding the

molecular basis of minor H antigens

encoded by the autosomes was described

by several laboratories. In humans, J. den

Haan (Leiden) described the identification,

in collaboration with Engelhard, of an

HLA-A*0201-restricted HA-1 peptide. As

described by M. Wilke (Leiden), the peptide

appears to be the product of a novel cDNA

of which the allelic form shows a single

amino acid difference. E. van den Weil-

Kemenade (Nijmegen) reported on the

identification of cDNA presumably derived

from a human minor H gene whose antigen

is found only on lymphoid cells.

A. Zuberi (Bar Harbor, ME), in collabo-

ration with the Shastri laboratory (Berkeley,

CA), reported on the use of a 'positional

cloning' approach to proceed from genetic

position to cloned DNA fragments. He used

YAC transfectants to narrow down the site

of the H-2Db-restricted component of the

classical minor H locus H3 (located on

mouse chromosome 2), and then used im-

munoselection by minor H allele-specific

CTLs to clone the gene positionally. This has

led to the identification of a single peptide

differing from the reciprocal allele by two

amino acid substitutions, the product of a

novel 8.5 kb transcript whose sequence

shows hallmarks of a transcription factor. N.

Shastri, in collaboration with the Roopenian

laboratory, used T cells modified to express a

reporter gene to clone a cDNA encoding an

allele of the classically defined H13 antigen

encoded on mouse chromosome 2; the recip-

rocally antigenic cognate Db-binding pep-

tide, which surprisingly did not exhibit the

allele-specific motif, was also identified. The

gene appears to be a novel one, and the anti-

genicity resides in a remarkably conservative

single amino acid substitution in comparison

with the reciprocal allele. Clearly, with these

molecular definitions, minor H antigens are

no longer mysterious.

Both M. Strausbach (Rochester, MN) and

S. Joyce (Hershey, PA) utilized an expres-

sion library and a synthetic peptide screen-

ing approach to identify a number of bio-

chemical mimics of the H4 b antigen. Such

synthetic mimotopes of minor H antigens

could be of value to induce immunomodu-

lation and to increase understanding of

the basic biochemistry of minor H peptide

recognition. Immunomodulat ion of GVHD

with minor H antigen-specific TCR antagon-

ists was reported by den Haan. P. Schlegel

(T/ibingen) took a more generic approach

using promiscuous synthetic gut-associated

lymphoid tissue (GALT) peptides that bind

both mouse and human class II proteins,

and was able to ameliorate murine GVHD.

The potential clinical applicat ion of

minor H peptides to treat the GVL effect

was addressed. E. Warren (Seattle, WA)

reported specific inhibition of acute

myeloblastic leukaemia (AML) engraftment

when minor H antigen-specific CTL clones

reactive with haematopoietic cells were co-

cultured with AML cells before injection

into nonobese diabetic-severe combined

immunodeficiency mice. T. Mutis (Leiden)

showed that CTLs generated from normal

individuals against a known minor H

antigen-specific pept ide presented in

dendritic cells could lyse leukaemic cells

expressing the naturally processed HLA-

A2-pept ide ligand.

F u n c t i o n of genes e n c o d i n g t u m o u r and m i n o r H an t igens P. van der Bruggen (Brussels) described in-

triguing examples of genes discovered from

analysis of MHC-restricted cells reactive to

endogenous peptides that encode tumour-

related antigens. One of these was a mutated

form of the 'cell-death gene' caspase 8 (in-

volving a single amino acid change) that gen-

erated a T-cell-recognized epitope expressed

by a carcinoma. The mutant form of caspase

8 was less effective at promoting apoptosis,

thus giving the tumour cell a growth advan-

tage. In another tumour, the peptide was de-

rived from a mutant CDK4; this molecule

normally binds cyclin but is inhibited by the

mutation, thus accounting for both the onco-

genic and antigenic effect. In a third example,

a mutation in ~-catenin affects proliferation.

M A R C H 1998 V o I . I 9 N o . 3 I I I

T R E N D S • ! :

The MAGE genes have also been identified

as tumour antigens in this way but their

physiological role is still unknown.

Similarly, with the exception of mito-

chondrial minor H genes, the functions of

the products of the minor H genes remain to

be identified. Given that several of them

possess features of transcriptional regulat-

ory molecules (e.g. Smcy, Uty and H3a), it

is unlikely that their natural function is im-

munological. Instead, and as hypothesized

by Townsend in 1985 (Ref. 1), the peptide

antigens are coincidental to genetic poly-

morphism in coding sequences. Thus, they

are immunological manifestations of slow

sequence divergence in evolutionarily con-

served genes that normally comprise self-

antigens. In any case, understanding how

these genes function biologically, in combi-

nation with a detailed understanding of how

their polymorphisms lead to self-nonself

discrimination, may suggest routes to

manipulate the immune response antigens

to advantage in transplantation situations.

Towards specific immunomodulation Nonspecific immunosuppression is cur-

rently the predominant treatment for

transplantation, but carries disadvantages,

with increased susceptibility to infections

and tumours. H. Waldmann presented

experimental data that strongly suggest that

certain combinations of minor histocompat-

ibility antigens, given under an 'umbrella'

of nondepleting antibodies to CD4 and

CD8, can induce a state of operational toler-

ance. This could be extended to additional

histocompatibility antigens if presented on

grafts also expressing the antigens to which

tolerance had been induced. The possibility

of substituting identified minor H peptides

during the induction phase may be an

improvement and prevent some compli-

cations. In that context, P. Chandler (London)

discussed results showing that adminis-

tration of an immunodominant H-Y/D b

peptide in vivo prolongs survival of male

skin grafts on syngeneic female mice. While

the mechanisms remain obscure, it could be

useful if such protocols can be applied to

a clinical setting.

Finally, one of the most striking exam-

ples of novel minor H antigen immuno-

biology comes from studies of the eye.

B. Kasander (Boston, MA) related his

studies showing that this site is immune-

privileged in that minor H antigens are con-

siderably more immunogenic than MHC

alloantigens. Minor H antigen-specific

CTLs can be generated and induce rejection

of corneal allografts because the antigens

can be presented indirectly, by host

antigen-presenting cells, while MHC

alloantigens generally are not. This serves

as an excellent model not only to examine

the mechanisms of 'indirect recognition' but

also to study responses to minor H antigens

in isolation. Thus, in the eye, the minor H

antigens are major H barriers and the MHC

antigens are minor barriers. So the distinc-

tion of who is minor and who is major lies

in the eye of the beholder. If only George

Snell were here!

This meeting was made possible with the gener- ous support from The National Heart, Lung and Blood Institute, the National Institute of Allergy and Infectious Diseases, and the Burroughs Wellcome fund.

Elizabeth Simpson (esimpson@rpms.ac.uk) is

at the Transplantation Biology Group, MRC

Clinical Sciences Centre, ICSM, Hammersmith

Hospital, Du Cane Road, London, UK W12

ONN; Derry Roopenian is at The Jackson

Laboratory, Bar Harbor, ME 04609, LISA:

Els Goulmy is at the Dept of hnmum>

haematology and Bloodbank, AZL, Building 1,

E3-Q, University Hospital, Postbus 9600, 2300

RC Leiden, The Netherlands.

R e f e r e n c e

1 Townsend, A.R.M., Gotch, R.M and Davey, I. (1985) Cell 42, 457-467

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