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The major histocompatibility complex of the rat: A partial review
The Major Histoeompatibility Complex of the Rat: A Partial Review Jona',~han C. Howard The major histocompetibility complex of the rat is n o w called RT1, and this name is becoming widely accepted, In the past five years many recombinsnts have been reported withtn RT1 that enable distinct functional regions to be identified and located relative t o each other. RT1 does not at present look particularly like its closest k n o w n relative, tf-2. No doubt the genetic relationship will become apparent a t ' t h e I~NA level. The spontaneous diabetes mellitus of the BB r a t line is associated w i t h RTI. The data so far suggest that RTI "supplies a dominant susceptibility that becomes apparent only if protection conferred by a dominant gone mapping outside the MHC is w i t h d r a w n . It seems likely that the BB rat carries a recessive mUtation at this " p r o t e c t i v e " locus.
A
SURVEY of immunogenetic studies among vertebrate species reveals a situation reminiscent of certain popular spectator sports, with the mouse and human in the role of the likely season finalists. But there is also a group of contenders in the semifinals, including rat, guinea pig, and chicken, and I believe that the rat is the top contender (though this could be interpreted as "rooting for the home team"). The Syrian hamster is the unchallenged occupant of the cellar position in this league, a position it occupies by virtue of displaying a major histocompatibility complex (MHC) so defective in conventional properties that it scarcely qualifies for the designation at all. It is widely believed; quite wrongly, that almost nothing is known about the rat MHC. What is true, however, is that until recently rat immunogeneticists had not combined their efforts into a common pool, and th:re was no common agreement on elementary nomenelatural problems. It is possible,for example, to find publications from distinguished scientists still using the obsolete Ag-B nomenclature for t h e rat MHC. We must hope that the founding of Rat News Letter by Michael Fasting in 1977J and the inauguration of the series of international workshops on rat histocompatibility systems by Tom Gill in the same year will finally provide the necessary solid center of agreement and up-to-date pooled information. From an immunogenetic point of view, the rat will never be the league champion, no matter how h a r d w e work, because the animals are so expensive to maintain. This is, obviously, quite apart from the clear preeminence that the mouse now enjoys from the extraordinary distinction Of its immunogenetic investigators over the past 60 years. But we certainly have only ourselves to blame if our colleagues remain ignorant of the: real state of affairs in rat immunogenetics. The BB rai provides an/inimal modelfor juvenile onset diabetes of quite exceptional interest, and f o r once there isno established rivalmodel in the mouse.lt will b e interesting t o : s e e Whether ~the ~utoimmune features Of the r a t disease andits association with the major ::hist~mpatibility::cgmp!ex ~ ! ! .bring: a new rgency [6 :rat: immunogenetiCS!:in!!gene~l. 'In a n y Met.sboF.c,m.Vet.32, N6.7.SuppL 1 (Jub/), 1983
event, the major concentrationof research interest that the BB rat is bound to generate must force rat immunogeneticists to put their house in good order. The major histocompatibility complex of the rat is now called R T I 3 (The italics conform with standard practice for genetic symbols in the mouse). This name replaces H-I ~ and Ag-B. 4 The chromosomal environment of R T I is not well known. Although the linkage group now contains the glyoxalase marker Glo-! s and otlter enzyme loci? a n apparent homology with the mouse 11-2 and human HLA linkage groups, the chromosome carrying R T I is not known. Fu;thermore, the position of the eentromere in relation to R T I is not known, so the orientation of the linkage group on the printed page is arbitrary. It is curious that the chromosome bearing R T I should still be unknown. The rat karyotype is well-differentiated, 7 unlike that of the mouse, and rat cells have be~n used extensively in somatic cell hybridization, particularly recently. THE FIRST RECOMBINANTS: A AND B REGIONS DEFINED
For a long time, breeding experiments calculated to reveal genetic fine structure in the rat M H C produced no recombinantsbetween recognized MHC functions. In I977 the first two recombinants within the rat M H C were described from two different laboratories.S~9.In each case a "conventional '~ serologic marker was recombined from markers determining other typical MHC-specified functions, such as mixed lymphocyte response (MLR). ~(The term Conventional in this context always refers to antigens of the class ltype, which are carriedon all lymphooytes and probably on all tissue cells. In mice and rats they are expressed on From the Agrtcuhural Research-Council :lr~tttute of Animal Physiology. Babraham.':Cambridge..UK. Supported in part~by:)VIH gront~Al 13162 and by MRC grants 978/120~C and 979/4.~6/C. Reprint ~,eque~lxthouldbe'addresxed to Dr J,C: Howard. A.R.C
znsmute of Ailimai e~yaOto~i:'~iibmham.:!Ca~b~dge;CRZ ~a Z UK © 1983 by Grune K Straiten. Inc. 0026:.0495/320;92000951.00/0 41
42
JONATHAN HOWARD
erythrocytes, and this provides a quick and convenient means of typing for those antigens). The MLRassociated region was also shown to specify histocompatibility antigens causing skin graft rejection, R immune response genes, ~°and, more usefully, serologic naarkers carried on a lymphocyte subpopulation and B cells,s and absent from T cells, platelets, and erythrocytes." in other words, single recombination events were apparently separating all the conventional serologic alloantigen-specifying regions from regions specifying in-like antigens. It was obvious that this genetic structure was unlike that of the mouse (Fig 1). Because mouse la antigens are specified by genetic regions lying between regions coding for the conventional K and D ailoantigens, no single recombination event can separate all la antigens from all conventional ailoantigens. The rat MHC, on the other hand, looked in 1978 much more like the HLA region of humans, where H LA-DR representing In-like antigens, clearly lies to one side of the region specifying conventional HLA-A,B,C antigens and can be separated from it by single recombination events (Fig 1).
RT1 RAT
A
¢I~ ,=
The discovery of intra-MHC recombinants led to a new seriousness in trying to deal with nomenclatura| problems. In particular, the two regions defined by recombination were named A (conventional, class l, K,D-like, HLA-A,B,C-Iike) and B (B lymphocytes, class II, MLC-stimulating, la-likc, HLA-DR-Iike), 1° and a numerical series of defined laboratory recombinants was inaugurated, to which all rat immunogeneticists now subscribe. THE SECOND PHASE: t-I-6 AND THE C REGION
To the right of the B region there exists another histocompatibility locus. Originally named 1t-6 by Stark and colleagues, t2 this locus was noted to determine relatively slow first-set skin graft rejection and to be closely linked to the conventionally defined MHC, but to be recombinable from it. Two apparently reciprocal recombinants, r3 and r4, between the a and u haplotypes, were recovered from 67 F2 progeny. The curious genetic region defined by r3 and r4 has been named C, and has been further characterized by recombinants r5, r14 and r15. '3a' Immunization
'0
a
E
¢
glo grc
J H-2
HLA
MAN
K1 K2
A
~
S
B .......
J m
.....
O
C
A
mi
am
,
U
Os2Qa3
11. Q a l
Fig. 1, A scflemati~ comparison of the MHCs of rat, mouse, and man. The centromare is drawn to the left for mouse and man. The rat MHC is aligned relative to the glyoxaLsse-1 locus glo-l, Regions specifying class I polypeptides are indicated by squares, and class II polypept|des by circles. T h e Imnail circles in 14-2 called LIMP represent the low molecular weight pclypeptldoa recently described,s~Shaded areas show the presumed location o f class ll| products, complement components C4. C2, Factor B in mouse and man. Polymerphlc complement componer~ts |ir~ked to MHC have not been described In the rat. References to features of the rat MHC can bs found in the text. Squares denoting class I-determinlng loci are represented in three ways. Serial squares indicate the presence of a locus specifying -; known major hlst0competibilRy antigen capable of inducl,~g • plimary allogenelc cytotoxic T cell response in vitro and capable o f forming restricting assoc~Jtor~ with minor histocompat|bi|tW antigens or virus antigens. Stipphxd squares Indicate the presence of probable presence of other ¢~Jalm! I o ~ known am'y from sero~gt¢ or structural properties. Open squares :,ndlcate t h e location o f know n "'medial'" histo¢ompstibittty afltigeaS,tO capsble of evoking secondary unrestricted 8|logene|c cytntox|c T cellS,but Probably incapable of f ~ l r d n g restricted associations with minor histocompatibllity antigens or viruses.
MHC OF THE RAT
between C region-incompatible recombinants generates a potent cytotoxie T lymphocyte (CTL) response specific for C-region productsJ r~5 The properties of this response are most unusual and need emphasis. A typical MHC incompatibility will evoke a primary CTL response in vitro. C-region incompatibility induces no in vitro primary response: It is, in this sense, a weak incompatibility. The typical C-region CTL response is seen only after in vivo printing and in vitro boosting with C-incompatible cells. Unlike a response against a typical minor histocompatibility antigen, however, CTL against C-region products are not restricted in their specificity to the products o f any other MHC region. The quality of C-region antigens is therefore equivocal: They are weak in the sense of requiring prior immunization to evoke an in vitro CTL response, while the response has the typical specificity of major alloantigens in not being MHC-restricted. Ailoantigens determined by the C-region are probably responsible at least in part for the remarkable data of Marshak et al, who found a weak but complex alloantigenic system closely linked to the MHC, requiring priming and boosting to evoke an unrestricted CTL r e s p o n s e . 16.t7
Serologic investigations of C-region products have recently demonstrated lymphocyte alloantigens apparently structurally related to those specified by the A region. =~ The constellation of properties displayed by the C region are so strongly reminiscent of those of the Q a / T l a region of the mouse tt-2 complex ~9''° that the possibility of homology cannot be excluded. There is no known comparable MHC region in HLA. HETEROGENEITY OF la ANTIGENS: DISTINCTION BETV'¢EEN B AND D REGIONS BY RECOMBINATION
One ~.'xpects an MHC to specify more than one la glycoprotein. In the mouse the l-Aand I-E molecules are now well-defined, with four loci specifying their constituent a and B chains. 2~The original definition of the rat t~ region was identical to that of the whole mouse ! region in the sense that it included lr-gene function, strong MLC-stimulating activity, and serologic antigens expressed predominantly by B lymphocytes.9"2ZIt was therefore reasonable that, as defined, the B region might prove complex, and specify more than one+Ia-like glycoprotein. :This has provedto be the case. Evidence that the rat M H C as a whole specified giycoproteins corresponding to I-A and I-E came from immunoprecipitation studies using mouse alloantisera specific for mouse I-A or I-E products. These reagents cross-reacted extensively onrat cells(without marked polymorphicspecificity) and wereshown+t0 precipitate two independent la-like glycoproteins discriminableby two-dimensional p01yaerylamide gel eleetroph0resis. 2'
43
Partial N-terminal sequences of a and/~ polypeptides precipitated by rat alloantisera indicated that both I-A and I-E homologues were coded in R T I . 24Rat alloantisera also discriminated two molecules using sequential precipitation in appropriate cross-reactive strain combinationsY Finally, mouse monoclonal antibodies have been raised+ against rat niembrane glycoproteins and shown clearly to be specific for two independent la-like molecules. :+,27 The loci specifying these two serologieally defined la-likemolecules have now been separated by the r12 recombination. 2s The two la loci defined by the r12 recombinant haplotype have been named R T I B and R T I D . Combined serologic and immunochemicai studies have shown that the molecule apparently homologous with mouse I-A is specified by R T I B , and that homologous with mouse I-E by R T I D . 29The order of loci is R T I A - B - D . It should be noted that the present use of R T I B refers only to the locus specifying the I-A homologue, while the former use of the same term referred to the homologue of the whole mouse /-region. It would seem reasonable to introduce the term "/-region" into the rat MHC to cover both RTI B- and RTI D-specifying loci. In this case the loci can be referred to as I-B (I-A homologue) and I-D (I-E homologue), and their products similarly. The only known recombinant within the rat/-region is rl 2, and it separates the ot and/~ chain loci of I-B and the ~ and B chain loci of 1-D. in the mouse nearly all intra-l-region recombinations fall between I-E/3 and I-E c~. One might hazard to guess that the highfrequency recombination site recently postulated within 1-E in the mouse3° may not be present in the rat MHC. The arrangement of the la loci in the rat appears therefore to be more symmeti'ical than those of the mouse, where three of the four la polypeptides are specified in the region conventionally termed 1-,4, while only the c~ chain of the I-E product is actually coded in 1-E. In the mouse a number of defects in the transcription and expression of the I-E molecule have been recorded. As a result, many 1-1-2 haplotypes have no detectable I-E expression. There is no evidence for this anomaly in the rat. Every haplotype tested with the /-D-specific xenogeneic monoelonal antibody MRC OX 17 has proved positive.27'3~ Formal evidence that/-B and I-D both lie between R T I , 4 and R T I C i s not available at the moment. From the indirect evidence available, however, there seems tobe little doubt that this isthe ease. R T I E A N D THE:GROVVTH AND REPRODUCTION COMPLEX
The growth and repr0duction ~ m p l e x ( G R C ) was named t0 describe a mutantphenotype-irlvolving low
44
JONATHAN HOWARD
male fertility and reduced body size) "~'3~The trait was shown to map dose to the MHC, and the recombinant r l l showed the gene order to be A-(B-D).GRC) 4 From the low recombination frequency between RTIA and GRC ~2it is tempting to locate GRC to the:left of RTIC. Recently, the rl I recombinant has been used to demonstrate the close linkage of a new cell-surface ~dloantigen, RTI E, with the GRC. ~ Overall, the properties of the R T I E product resenable those of a class I molecule. Evidence has been presented for the expression of R T I E on erythrocyte membranes, and immunoprecipitation studies have shown heavy and light polypeptides corresponding tolerably well in apparent molecular weight to those of conventional class ! glycoproteins) ~ R T I E products seem also to excite cellular immunity, as judged by the development of cytotoxic efl'eetor cells:'6 RT1C, RTIE AND THE MOUSE PARADIGM
On the strength of all the evidence presently available it is reasonable to describe the order of loci in RT! as A-B-D-E(GRC)-C (Fig 1). Because R T I E has class I-like properties and lies to the opposite side of the typical /-region from RTIA, Gill and his colleagues ~s'-~7have been tempted to draw a formal analogy between the K-I-Dstructure of 1t-2 and the A-I-E structure of RTI. in both cases, loci specifying class 11 molecules are flanked by loci specifying class ! molecules. The main objection to this simplifying proposition is that there is no evidence that R T I E specifies a major transplantation antigen rather than another antigen of the R T I C type. it may be significant that experiments demonstrating the generation of cytotoxic cells specific for R T I E products were conducted using the protocol required for R T I C antigens, namely in vivo priming and in vitro boost. Furthermore, exten~sive experiments in the author'slaboratory have failed to provide any unequivocal evidence for either primary CTL or MHC restriction of secondary CTL induced by the products of any locus other than R T I A (A.M. Livingstone, unpublished results). Similar results are reported from Gunther and colleagues) s For the moment it is rather important that R T I E and R T I C be compared formally. Although the recombination frequencies separating these two loci from R T I A seem fairly different at present (about 3% for R T I A - R T I C "compared with about 0.5% for R T I A - R T I E ) , similar variations in recombination frequency have been reported between homol0gous pairs of loci in !t-2)~ The expression of RTI E products on erythrocytes~ is a further difference from the reported properties of R T I C products. Curiously, however, R T I E products are not detectable on erythrocytes of certain haplotypes (although present in an
crythrocyte lysate)) ~ It remains possible, therefore, that R T I C products are also present but have not yet been detected. My own strong preference is to consider R F I E to be more closely related to R T I C than to RTIA. If the homology between R T I C and the Q a / T l a region of mouse !1-2 is correct, then we should expect to find several linked loci with similar properties. It should be emphasized, though, that until laboratories carrying the informative R T I E and R T I C recombinants compare their results formally, it remains perfectly possible that R T I E and R T I C are literally one and the same thing. It is natural to attempt a formal comparison between RT1 and H-2. The main source of omfusion lies in the lack of a well-defined sec,~nd locus determining strong class I products in the rat. Allth~ classic class i alloantigenie activity.is concentrated in the products of the A region, while R T I E and R T I C determine weak and equivocal antigens of uncertain status. There is some evidence for more than one distinct antigenic:product from RTIA. Microsequeneing of the 45Kpolypeptides precipitated by alloantisera from normal iymphocytes suggested a limited heterogeneity: ° The data were compatible with two polypeptides present in equivalent amounts and presenting strikingly similar but not identical N-terminal sequences within each haplotype. Between haplotypes the sequences were more distinct. Such limited sequence heterogeneity between products of distinct genes on.a single chromosome is typical of very closely linked members of a multigene family. It is more reminiscent, for example, of the relationship within the tt-2D family, ~* than between K and D:" A similar conclusion has been reached recently on serologic grounds using a panel of monoclonal alloantibodies. The results suggest the expression by RTI of a series of at least three very similar molecules, all of which share one common specificity, two of which share a second, and one of which carries a unique specificity: 3 Again, this situation is reminiscent of the H-2K or H-2D families, where members of each closely linked series share most but not all the specificities characteristic of the particular haplotype: ~ Finally, two conventional immunopreeipitation studies have demonstrated structural heterogeneity among class I alloantigens in the rat. zS"4s'~ Unfortunately, none of these studes has combined serologic, immunochemical, and immunogenetic techniques to demonstrate formally what the genetic basis may be for the observed heterogeaeity or which structures correspond to the class 1 major histocompatibility antigens defined a s such on the basts of T cell-mediated responses. Until these comparisons a r e made the organization of the rat M H C and its formal relationship with tl-2 remains sub judice.
MHC OF THE RAT
IMMUNE RESPONSE GENES IN THE RAT
Some of the earliest studie~ on Ir gene control were performed using synthetic polypeptides in congenic rat strains..High and low antibody responsiveness to HGAL were shown to be linked to the MHC, 47 and immunization of recombinant animals showed that differential responsiveness mapped to the I (BID) region, t° Both I-B and I-D subregions probably control differential responsiveness. The rl 2 (BID) recombination was, in fact, discovered by a discordance between RTIA and immune responsiveness to GLT. :a In this case differential responsiveness mapped to I-D. More recently the in vitro proliferative responses to bovine and pork insulins have been shown to be under MHClinked genetic control, 4"'49 and differential responsiveness apparently maps to I-B (D.V. Cramer, personal communication). It was, of course, to be expected by analogy with mouse data that both the la molecules of the rat should be involved in antigen presentation. COMPLEMENTATION IN Ir GENE SYSTEMS
There is no molecular or genetic evidence yet that I-B or I-D region products display novel specificities in FI hybrid cells because of formation of hybrid a/3 dimers. It would, however, be extraordinary if this effect did not occur, as it is well known in the mouse.-'° Furthermore, Gunther and colleagues s~ observed anomalous high responsiveness to TGAL in Ft hybrids between R T ! congenic rats carrying low and intermediate response alleles. Although the molecular basis for this effect was not demonstrated, it seems likely from mouse studies that hybrid c~/~/-region dimers, causing a novel specificity of antigen presentation, were responsible. A start has been made in defining the association between la polypeptide structure and immune responsiveness in the rat. Massey and Wilson 5' have shown that in vitro proliferative responses to GT are under R T l - t i n k e d control. Unexpectedly, the two highresponder haplotypes, R T I = and R T I ¢, appear to be equivalent in the sense that cells bearing either of these haplotypes are able to present GT indiscriminately to each other. The implication that these two haplotypes might share identical class 11 polypeptides has apparently been confirmed by studies on the structures recognized by an RT! De-specific monoclonal antibody.at This antibody precipitated RTI D molecules carrying an apparently common B chain from the a. c, o, and f haplotypes. Subsequent work has shown that R T I f also belongs to the G T reciprocal presentation group (G. Massey,' persOnal communication); The RTI D ~8 chain involved in this cross, presentation group is also assc,e!ated with the/r,gene:colttrolled
45
immune response against the class l M H C molecule. RTIA=. s3.s4 Inthis case, however, the association is with low responsiveness. Positive correlation between an la polypeptide and unresponsiveness is suggestive that the immune response in question may be regulated in part by suppression. It is tempting to relate these findings in the rat to the 1-E-suppressed immune response to LDH isozymes and other antigens recently illuminated by Baxevanis et al. ~5 EXPERIMENTAL AUTOIMMUNE DISEASE
The classic experimental investigation of autoimmune disease in rats is the experimental allergic encephalomyelitis (EAE) induced in LEW (formerly known as Lewis) rats by footpad injection of guinea pig spinal cord or\myelin basic protein in Freund's complete adjuvant. -~ As with most autoimmune diseases, EAE has resisted a simple immunogenetic analysis, while still presenting tantalizing evidence that genetic factors in general, and the MHC i'n particular, play an important predeterminative role. There is no doubt that the LEW rat is pecu|iarly~susceptible to the induction of EAE, but it has been difficult to apportion responsibility betweea IVlHC and background genes. 57-5q The study of EAE in rats has been particularly effective: although a similar condition can be induced in mice, no mouse strain is as susceptible to the severe disease as the LEW rat. Nevertheless, some ~mmunogenetic studies have confirmed an association between H-2 and EAE, ~° and, most imp¢,rtantly, an indication that the disease process entails l~ecognition of antigen in association with I-A products has been obtained by McDevitt and colleagues.6~Theit" investigation showed that the induction of EAE could'be prevented by passive transfer of high-titred anti-l-A monoclonal antibodies into mice at risk. This important and rational protocol seems likely to be of considerable value in the analysis of experimenial fiutoimmune disease, and may even contain the germ Of a valid clinical procedure. Perhaps the most important theoretical implication to develop from these:studies on EAE in mice and rats is that the disease state contains a t least two components at the immunologic level: First, from the evidence for a positive association between particular M H C alleles and disease, even v,;~,en the al/ele is in the heterozygous condition, sTJs from the'direct evidence for MHC-restrieted presentation of the sensitizing antigen u and from the protection experiments using in vivo administration of anti-l-A antibodies, 6t it is clear that t h e development of t h e disease S t a t e involves a conventional form of MHC-restrieted T c e l l induction on antigen-presenting cells.It is likely that this process
46
JONATHAN HOWARD
is subject t o / r gone control such that presentation of the encephalitogenic peptides in ass~)ciat~on with some class !1 antigens will not lead to an immune response. Susceptible animals are therefore high responders, and the healthy or resistant animals are the low responders. Arguably, the resistant animals are those that have what is sometimes called an "It-gone defect." (When the problems of autoimmunity are put into this context, one can readily see the merit in an explanation for lr genes in terms of cross-reactive self-tolerance). 6~'~ Secondly, there is convincing evidence that under certain conditions a state of active immunosuppression can be established in susceptible animals? ~ In general this state follows immunization with nonencephalitogenie forms of the antigen, and can be ab~gated by treatment with cyclophosphamide.~ The immunogenetics of illduction of suppression are not known but are an obvious avenue of investigation, it would be of great interest to know whether a "resistant" genotype may not only represent one in which the MHC (or other factors) does not predispose to induction of a pathogenic response, but also one that predisposes to the induction of suppression. IMMUNOGENETICS OF BB RATS
BB rats are Wistar-derived albino rats carrying the R T ! ~ allele typical of this provenance. There is no
reported evidence that the line is heterogeneous at the MHC. When spontaneous diabetes mellitus (IDDM) was first observed in the colony maintained at BioBreeding Laboratories of Canada Ltd., theL:olony had been closed to new stock for 6 years (Chappell, personal communication). In view of the high standard of maintenance and observation at BioBreeding Laboratories, it seems unlikely that the diabetic condition was in fact present for 6 years before it was noticed. Fur=hermore, IDD M has not been reported from the many other laboratories holding Wistar-derived albino colonies. The single reasonable explanation for the sudden onset of IDDM in t h e BB colony is that a mutation occurred that, in association with the BB genetic background, predisposed strongly to the disease. That some single event occurred that spread through only a part of the colony is suggested by the fact that lines of rats from the 'BB colony with no IDDM are widely used as forms of control:for the typical diabetic BB rat. Such are, for example the W and V lines segregated from the colony by Butler at the University'of Massachusetts (L. Butler, personaLc~mmunication). A similar nondiabetie line of BB rat is maintained by Thibert in Ottawa. Both susceptible and control lines of BB rat are apparently homozygous for the R T ! = haplotypc. One would, therefore, be strongly inclined to belicve that the mutation that predisposes
to IDDM occurred at another locus. Before assuming this, however, one should remember that the H , 2 K b mutation H-2 t"'~ predisposes the mouse carrying it to the induction of autoimmune thyroiditis, 67 a susceptibility characteristic of the 11-2 k and 11-2 d haplotypes a:~d not of the H-2 b. o n ~ with the use of a panel of monoclonal antibodies hiis-it been possible to show convincingly that the H - 2 K pr~uct of H-2 b"l is serologically distinct from that of tl-2"b. ~ One wonders, therefore, whether a mutation at R T I " would have been noticed yet. An argument against the relevant mutation of the BB rat having been at the MHC concerns the susceptibility of segregat;ng populations to IDDM. In the studies of Colic and her colleagues69 it is clear that IDDM is associated with R T I " , whether it is in the homozygous or heterozygous condition. It is also the case, however, that F~ hybrids between IDDM-suseeptible and nonsusceptible control BB rats are invariably nondiabetic, arguing strongly that the relevant differonce between these two rat lines is not at the MHC, which would, of course, be heterozygous for the putative dominant R T l - l i n k e d susceptibility marker in the Ft hybrids. It seems likely that the principal differences between diabetic land nondiabetic BB rats lies at a single locus unrelated to the MHC. The outstanding characteristic of the normal allele at this locus seems to be that it protects ~,the BB rat from a susceptibility to IDDM that it possesses by virtue of other genetic characteristics. Of these characteristics only one is seriously recognized, namely, the dominant susceptibility associated with R T I ~ . This susceptibility is only apparent if the rat is also homozygous for the mutant allele at the "protective" locus. Striking evidence that the difference between a vulnerable and invulnerable subline of BB ratsmay be due to the activity of ff single locus emerges from the genetic studies of Butler "at the University of. Massachusetts (personal communication). This investig~/t0r has developed inbred lines of IDDl~l-susceptible arid control BB rats. The most interesting case is that of the W control line, which was extracted from the fifth generation of brother x sister mating of the susceptible BA line. Presumably, at the 5th B x S generation the BA line was still segregating for the "protective" allele. However, at the 12th B x S generation the susceptible allele had evidently been fixed in the BA line, and the protective allele fixed in the W line. At this time the incidence'of IDDM in the BA line was about 6.0%, a figure typical of all BB lines from which segregating genetic factors have been eliminated b y inbreeding:The incidence of IDDM in the W line was zero. All F~ progeny in the BA x W cross were normal,
MHC OF THE RAT
47 Table 1, R T f Haplotypos and RT1 Congentc Strains
Typical I.trsins
RT1 a b c d e f g h
ACI, DA, AVN BUF, A L B AUG, PVG BD'V BD"V AS2 KGH HW
i
BI
k
SHR, WKA LEW
I
m n
o p t
u x y z
MNR BN MR RP TO WF, AO nes~ved for wilds Reserved for unknowns Reserved for unknowns
LEW congdsnic LEW.1A(AVN) LEW. 1a LEW. 1C (AUG) LEW. 1D(BDEt LEW.1E(BDVII) LEW. 1F(AS2)
PVG ¢ongenic
PVG-RTf"(DA) PVG
BN congenic
Othol's
BN.B4(DA) BN. 1B(BUR BN. I C(AUG|
BN, 1G(KGH) LEW. 1H(HW) DA. 1l(Bi) PVG. 1K(VVKA) PVG-RTI~{AGUS) PVG-RT;*(F344) PVG~RTtt(LEW)
BN. IK(WKA) BN. 1L(LEW)
LEW.1N(BN)
PVG-RTf"(BN) PVG-RT I*(IC1)
BN
LEW. 1U(WP)
PVG.RTt"(AO)
BN. 1U(WF)
LEW
AUG.1N(BN)
Inforrne, tion concerning the origins and availability of the rats listed on this table can be obtained as follows. LEW strains: Dr O. Stark, Department of Biology. Facult! of General Medicine, Albertov 4, Prague 2. Czechoslovakia; and Dr E. GiJnther0 Mex-Planckqnstitut f~r Immunbiologie, 78 Freiburg-Zahringen, St~beweg 51, W. Ge~'many. PVG strains: Dr G.W. Butcher, Department of Immunology, Agricultural Research Council, Institute of AnimrJl Physio~'3y, Bsbrahem, Cambridge CB2 4AT, UK; BN stratus: Dr T.J. Gill IlL Department'of Pathology, Unwersity of Pittsburgh School of Me.d~cine. Pittsburgh, PA 15261. Dr Gill also maintains the largest collection of inbred rat strains in the world. His colony provides an invaluable and uni~m resource.
as were all backeross progeny in the direction (BA x W) × W. in the backeross (BA x W) x BA, however, the incidence of IDDM was about 30%, compatible with a single segregating recessive factor in th~ BA:line with a penetrance of about 60%. If the BB rat is to develop its full potential as a model for ht man juvenile onset diabetes 'mellitus, it is essential that inbred lines, preferably congenie for relevant genetic markers, be used and become widely available. The situation at present is unsatisfactory, with no general agreement to use only manifestly inbred lines and congenicr,: The situation is; of course, complicated by the lx~r health of IDDM rats; particularly in conjunction with the IDDM-associated immunodefieiency, and inbreeding depression is a major obstacle. Nevertheless, if genetically well-defined animals are not made available quickly, the emphasis of research interest is likely to move rapidly and irretrievably over to the N O D mouse2°'~z and pressure will grow to make this interesting but little known model for IDDM generally available. APPENDIX 1::INBRED STRAINS OF RATS Contrary to popular belief, there arc large numbers
of inbred rat strains, :Indeed, a *:list containing more
than ! 50 strains has recently been compiled and published by Festing72in Rat News Letter 10.* APPENDIX 2: R T I CONGENIC SERIES There are three major congenic series of rats available at pr,;sent, based on the LEW, PVG, and BN
backgrounds (Table 1). The LEW series was established by Stark and his colleagues in Prague and is also maintained by Gunther at Freiburg. The PVG series was established by Butcher and Howard in Cambridge, England. The BN series was begun by Joy Palm in Philadelphia and has been maintained and extended by Gill and his colleagues in Pittsburgh. This last laboratory has an extensive collection of other congenics. APPENDIX 3 : R T 1 RECOMBINANTS
T~bl¢ 2 ~ s all the laboratory recombinant haplof type, o:..-~TI presently recorded. Thts hst is constantly ~:,lg extended.
*(Ra'~:NewsLetter isavailable from Dr. M,F.W~ Fcsting, Medical Research CoUnCil Laboraiory Animals Centre,. Woodmanstcrne Road, C.arshalton, Srai-rcy~SM5 4EF, UK)
48
JONATHAN HOWARD
Table2. Recombinant RT1Hlplotypes
rI
r2 r3 r4 t5 r6
rT" rB r9 t rlO
Suam
A
B
FVG.R 1 LEW.1AR1 LEW.1WR ! LEW. 1AR2
a n u a n u a a
c u
a c u
n
I
LEW, 1WR2 PVG.R7 PVG.R8
r11
--
I
r ~2(p3)
V~'RC
n u I
r14 r15
D
E
C
a u I
n u I
Refe+enCe 8 9 11 t1 13 80 81 81 82 32
i
34
!
28 14 14
a u
• The PVG,R7 line is about to become extinct. Liver DNA and RT f I--cont~tining ted! hybrids h~tve been p~esetved. tAn r9 recombinant line was never established and the recombinant chrt~--.,c~ne is extinct (T. Nero
APPENDIX
4:
MONOCLONAL
ANTIBODIES
AGAINST
RT ! PRODUCTS
The first use of antibody-secreting plasma cell hybrids to prepare monoclonal antibodies against
MHC alloantigens in any species, was in the rat. ~3 Since then, it has become apparent that ailoimmtmizations in the rat provide a particularly favorable system for generating anti-MHC monoclonals in large num~ r s : *s5 Xenoimntunization of mice with rat cells or cell products has also been a useful method of generating monocional antibodies specific for RTI products, usually but not always without ailelie specificity. Xenoantibodies against class ! ~7and class 1126'27products have been described. A compilation of monoclonal allo- and xenoantibodies specific for RTI products is presently in preparation by Butcher and Howard for publication in lmmut~ogenetics. ACKNOWLEDGMENT
i should like to record a particular debt of gratitude to Dr. Errol Marliss and to the Juvenile Diabetes Foundation for the invitation to attend the workshop on the BB rat, and for the intensiveintroduction to the pressing problems of juvenile onset diabetes. I am also grateful to Dr. G.W. Butcher for helpful conversations and for reading the manuscript, and to Jenny Richards for typing the manuscript.
REFERENCES
I. Festing MFW: Rat News Letter. Medical Research Council Labora!ory Anima|s ('entre. ISSN 0309-1848 2. Gill T J. et al: Report of the First International Workshop on Alloantigcnic Systems ;~ the Rat. Pittsburgh. June 6-8, 1977. Transpla+,t Proc i0:271-285, 1978 3, Stark O, Kre.n V: Erythrocytc and transplantation antigens in inbred.strains of raL~. VI. The histocompatibility-! system and its alleles. Folia Biol (Praha) ! 3:356--360. 196"1 4. Elkins WL, Palm J: Identification of a single strong histocompatibility locus in the rat by normal spleen-cell transfer. Ann NY Acad Sci :2~:~,73--580, 1966 5. Stoic V+ Kunz HW, Gill TJ 11|: The linkage ofglyoxalase-I to the major histocompatibility complex in the rat. J Immunol 125:! 167-1 ! 70, 1980 6. VandeBerg JL, Bittner GN, Meyer GS, et ah Linkage of neuraminida~- and a-mnnnosidase to the major histocompatibility complex in the rat. J lmmur=ogenct 8:239-242, 198 ! 7. tlungcrford DA, Nowel! PC: J Morphol ! 13:275, 1963 8. Butcher GW, Howard JC: A recombinant in the major histocompatibility complex of the rat. Nature 226;362-364, 1977 9. Stark O, Gtlnther E, Kohoutova M, ct ah Genetic recombination in the major histocompatibility complex. (H-I, An-B) of the rat. immunogenetics 5:1993-1997, 1977 !0. GIlnthcr E, Stark O, Koch C: Genetic definition of I regiondetermined antigens of the rat major histocompatibility complex. Eur J lmmunot 8:206-2|2, 1978 t I. Davies liftS, Butcher GW: Kidney alloantigens determined by two regions Of the rat major histocompatibility complex, lmmunogenetics 6:!71-18! 1":)78 12. Kohoutova ',~ ";unther E, Stark O, Vojcik L; Further two rccombinants at t : -ajor histocompabitility complex of tbo rat. Folia Biol (Praha) ,",+. ~ - 4 0 7 , 1978 13. Lynch DH, IP,.~ ,. CW:Genetie identificztlon of a locus
linked to the rat M H C that codes for a membrane antigen detectable
with cytotoxic lymphocytcs. J Immunol 121:2367-2375, 197g 14. Wonigcit K, Hanisch L, Steiniger B. et ah Analysis of the two new R T I C region recombinants" r14 and r15 and a LEW subline lacking expression of C-region antigens. (unpublished o~¢rvations) t5. Gilnther E, Wurst W. Stock W: Specificity of anti-RTlC alloantisera and cytotoxic T lymphocytes. (unpublished observations) 16. Marshak A, Doherty PC, Wilson DB: The control of spocificity of cytotoxic T !ymphocytes by the major histocompatibility complex (An-B) in rats and identification of a new alloantigen ~ystem showing no Ag-B restriction. J Exp Med 146:1773-1790, 1977 17. Sieck T, Wilson DB: Studies of the MHC-linked "CT" alloantigenicsystem in rats. II. Evidence for more than one locus and several different determinants. Immunogen©tics 14:293-302, 198 i I 8. Haustein D, Stock W, Gflnther E: Rat major histocompatibility RTI.C antigens of restricted tissue distributionconsist of two polypcptide chains with molecular weights of about 42000 and 12500. Immunogenet[cs 15:27l=278, 1982 19. Fischer Lindahl X, Langhornc J: Medial histocompatibility antigens, Scand J immunol 14:643--654, 1981 20. Flaherty L: The " T t a , region antigens, in Doff M (¢d): The Role of the Major Histocompatibility Complex in Immunobiology. New" York, Garland Prccs, 1981, pp 33-57 21. Klein J, Juretic A, Baxcvanis CN, et ah The tradhional and a new version of the mouse 11-2 complex. Nature 291:455-460, 198 ! 22. Howard JC: Recombinants tn the major histocompatibility comp|¢X. Rat News Letter 1:8-9,1977 23: Blankenhorn EPo Cecka .!M, Fre|inger J, et ah Structure of la antigens from the rat, Mouse :alloantiscra dcmonstrat© at least two
di~eaentmoleculars~es, Eur:J Immunol I0i145:!5 i~ !980 24. Cecka JM, Blankenhorn EP, GtStze Diet al::Microsequence
MHC OF TttE RAT
analysis of ia antigens from three strains of rats. Eur J |mmunol 10:140-145, 1980 25. Sporer R, Black G, Rigiero C, et al: The major histocompatibility complex of the rat, RTI. !1. Biochemical evidence for a complex genetic organization, lmmunogenetics 7:50725 ! 4, 1978 26. McMastcr WR, Williams AFt Identification of la glycoproreins in rat thymus and purification from rat spleen. Eur J lmmunol 9:426-433, 1979 27. Fukumoto T, McMastcr WR, Williams AFt Mouse mono~ clonal antibodies. Two la antigens and expression of la and class ! antigens in rat thymus. EurJ lmm,nol 12:237-243, 1982 28. Lobel SA, Cramer DV: Demonstration of a new genetic locus in the major histocompatibility system of the rat. Immunogenetics 13:465-473, 1981 29. Blankenhorn EP, Symington FW, Cramcr DV: (submitted for publication) 30..Stcinmetz M, Minard K, Horvath S, et al: A molecular map of the immune response i-gion from the major histocompatibility complex of the mouse. Nature 300:35--42, 1982 31. Butcher GW, Diamond AG, Clark M, ct ah Similarity of RTI D/~ chains from independent rat haplotypes. Transplant Proc (in press) 32. Kunz HW, Gill TJ !I!, Dixon BD, et ah The growth and reproduction complex in the rat: Genes linkod to the major histocompatibility complex that affect development. J Exp MOd 152:15061518, 1981 33. Gill TJ i11, Kunz HW: Gene complex controlling genetic and fertility linked to the major histocompatibility complex in the rat. Am J Patho196:!85-202, 1979 34. Kunz HW, Gill TJ Iil, Liebert M. et ah Gene order in the major histocompatibility complex of the ral. Immunogenetics 13:371-379, 1981 35. Kunz HW, Gill TJ III, Mura DN: The identification and mapping of a s~0nd class I locus in the major histocompatibility complex of the rat. J lmmunol 128:402-408, 1982 36. Liebert M, Kunz HW. Gill TJ Ill, et ah CML characterization of a product of a second Class i locus in the rat MHC. lmmunogcnetics 16:!43-156, 1982 37. Gill T J, Kunz HW, Schaid DJ, e! ah Orientation of loci in the major histocompatibility complex of the rat and its comparison to man and the mouse. J lmmunogenet 9:281-293, 1~82 38. Gunther E, Wurst W: RTI.C gene products do not restrict cytotoxic T lymphocytes against minor histocompatibility antigens. Transplant Proc (in press) 39. Klein J: Biology of the Mouse Histocompadbility-2 Complex. Berlin, Heidelberg, New York, Springer-Voting,1975, p 213 40. Blankenhorn El), Cecka JM, Goctze D, et at: Partial Nterminal amino acid sequence of rat transplantation antigens. Nature 274:90-92, 1978 41. Scars DW, Wilson PH: Biochemical evidence for structurally distinct H-2Dd antigens differing in serological properties, lmmunogenetics t3:275-284, 1981 42. Maizels RM, Fre!inger JA. Hood L: Partial amino acid sequences of :mouse transplantation antigens, lmmunogeneties 7:425-444, !978 43. Misra DN, Nocman SA. Kunz HW, ¢t ah Identification of two c!ass I antigens codod by the major histocompatibility complex of the rat by using monoclonal antibodies, J lmmunol 128:16511658. 1982 44. Dcmant P, lvanyiD: Further molecular complexities of H-2K and D-rcgio n antigeus: Naturd 290:146-149.~1981 45. Sporer R, Manson LA. G6tze D: A biochemical analysis of themembrane-ass~iated 8ene products of the major histocompatibility complexof thelraL j |aimun01122:2162-2166; 1979 46. Natori T, Ohhashi T, KotaniT; et al:Tl~e molecular identifi-
49 cation of two scrologically defined gene products of the rat major
histocompatibility complex. J Immunol 122:19! 1-1915, 1979 47. Gtlnthcr E, Stark O: The major histocompatibility system of the rat (AgB or !f-2 system), in G0tzc D (¢d): the Organisation of the Major Histocompatibility System in Animals. Heidelberg, Sprlnger-Verlag, 1977, pp 207-253 48. Inomata T, Fujimoto Y, Natori T, et ah The genetic control of the immune response to i,sulins in the rat. Transplant Proc (in press) 49. Lobel SA, Shonnard JW, Miller J, et ai: Studies on the immune response to bovine insulin in inbred and wild rats. Transplant Proc 13:1394-1396, 1982 50. Kimoto M, Fathman CG: Antigen-reactive T cell clones. I. Transcomplementing hybrid I-A~reg;on gene products function effectively in antigen presentation. J Exp Meal 152:759-770, 1980 51. Gilnther E, Rude E: Geneticcomplementationofhistocompatibility-linkodIr genes in the rat. J lmmunoi 115:1387-I 393, 1975 52. Massey GR, Wilson DB: Presentation to T cells of antigens under Ir gene control in the rat. Transplant Proc 13:1397-1399, 1981 53. Butcher GW, Corvalan JRF, Licence DR, et ah Immune response (it) genes controlling responsiveness to major transplantstion antigens. Specific major histocompatibility complex-linked defect for antibody responses to class I atloantigens. J Exp Med 155:303-320, 1982 54. Howard JC, Butcher GW: The mechanism of graft rejection and thc concept of antigenic strength. Scand J lmmunol 14:687691, 1981 55. Baxcvanis CN, Nagy ZA, Klein J: A novel type of T-T cell interaction removes the requirement for I-B region in the tl-2 complex. Proo Natl Acad Sci USA 78:3809-3813, 1981 56. Paterson PY: Experimental allergic encephalomyelitis and autoimmune disease. Adv Immunol 5:13 !-208, 1966 57. Gasser DL, Newlin CM, Palm J, et ah Genetic control of susceptibility to experimental allergic encephalomyelitis in rats. Science 181:872-873, 1973 58. Williams RM, Moore MJ: E,inkageofsusceptibility toexpcrimental allergic encephalomyelitis t0xthe major histocompatibility locus in the rat. J EXp Mezl i 38:775-782, 1973 59. Gasser DL, Palm J, Gonatas NK:~;enetic control of suscepti. bdtty to experimenta!allergzc encephalomyeJ,tts and the Ag-B locus of rats. J lmmunol 115:431-433, 1975 60. Bernard CC,A: Experimental autoimmune encephalomyelitis in mice: Genetic control of susceptibility. J Immunogenet 3:263274,1976 61. Steinman L Rosenhaum JT, SriramS, et ah lnvivoeffectsof antibodies to immune response gent products: Preventiofi of experimental allergic encephalomyelitis. Proo Natl Acad Sci USA 78:71 ! 1-7114, 1981 62. Ben-Nun A, Wekerl¢ H, Cohen IR: The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur J lmmunol ! 1:195-199, 1981 63. Matzinger P: A one-receptor vie,.v of T-cell behaviour. Nature 292:497-501,1981 64. Schwartz RH: A clonal deletion model for ir gene control of the immune response. Scand J lmmunol 7.'3-10, I978 65. Swierkosz JE, Swanborg RH: Suppressor cell control of unresponsiveness to experimental allergic encephalomyelitis. J Immunol 115:631.633, 1975 66. Lando Z, Tcitelbaum D, Arnon R: Effect of cyclophosphamide on suppressor cell activity in m~ce unresponsive to EAE. J Immunol 123:2156--2160. 1979 67; Maron R , Klein J, Cohen IR: Mutations at tt-2K or tl-21) alter immune response phenotypo Ofautoiramune thyroiditis, lmmunogeaetics 15:625-627.1982
50 68. Shermat, LA. Randolph CP: Monoclonal anti.il-2K b antibodies detect serological differences between ll-2K ~' mutants. Immunogeneti~ 12:183-186, 1981 69+ Colic E, Guttmann RD, Sccmayer T, et al: Spontaneous diabetes mellitus syndrome in the rat. IV. Immunogenetic interactions of MHC and non-MHC components of the syndrome. This volume. 70. Tochino V, Kanaya T, Makino S: Studies in spontaneously non-obese diabetic mice. J Jap Diab Soc 21:295. 1978 71. Makino S, Kunimoto K, Muraoka T, et al: Breeding of a non-obese diabetic strain of mice. Exp Anita 29:1 - 13. ! 980 72. Festing MFW: Inbred strains of rats. Rat News Letter 10:19-26, 1982 73. Galfre G, liowc SC, Milstein C, e| ah Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266:550-552, 1977 74. Howard JC, Butcher GW, Galfre G, el ah Monoclonal antibodies as tools to anal)~e the serological and genetic complexities of major transplantation antigens, immunol Rev 47:139-174, 1979 75. ltoward JC, Butcher GW. Licence DR, et al: Isolation of six
JONATHAN HOWARD monoclonal alloantibodics against rat histocompatibility antigens: C]onal competition. Immunology 4hi 31-141, 1980 76. Gasser DL: Current ~tatus of rat immunogenetics. Adv lmmunol 25:95-140, 1977 77. Gill TJ IlL Gunther E, Stark O, et ah Alloantigenic systems in the rat. Second International Workshop, Frelburg, West Germany. May 14-16, 1979. Transplant Pro<: l 1:1549-1684, 1979 78. Gill TJ III, Kunz HW, Gasser DL: AIIoantigenic systems in the rat. Third International Workshop, Philadelphia (June 25-27, 1980). Transplant Proc 13:1307-1497, 1982 79. Gill TJ Ill, Kunz HW. [toward JC: Alloantigenic systems in the rat. Fourth International Workshop, Cambridge, U.K., August 18-20, 1982. Transplant Proc (in press) 80. Kohoutova M. Vojcik L, Gunther E, et ah Analysis of a new R T l t'¢combinant haplotyp¢. Transplant Pro¢ I 1:1588-1589. 1979 81. Butcher GW, iloward JC: r7 and rg: Two new recombinant R?'I haplotypcs. Rat News Letter 5:13-14. 1979 82. Natori T, Ohhashi T, lwabuchi K, et ah An intrn-RTl recombinant haplotyp¢. Rat News Letter 6:5-7. 1979 83. Monaco J J, McDevitt itO: Identification in a fourth class of proteins linked to the murine major histocompatihility complex. Proc Nail Acad Sci USA 79:3001-3005, 1982
A
SURVEY of immunogenetic studies among vertebrate species reveals a situation reminiscent of certain popular spectator sports, with the mouse and human in the role of the likely season finalists. But there is also a group of contenders in the semifinals, including rat, guinea pig, and chicken, and I believe that the rat is the top contender (though this could be interpreted as "rooting for the home team"). The Syrian hamster is the unchallenged occupant of the cellar position in this league, a position it occupies by virtue of displaying a major histocompatibility complex (MHC) so defective in conventional properties that it scarcely qualifies for the designation at all. It is widely believed; quite wrongly, that almost nothing is known about the rat MHC. What is true, however, is that until recently rat immunogeneticists had not combined their efforts into a common pool, and th:re was no common agreement on elementary nomenelatural problems. It is possible,for example, to find publications from distinguished scientists still using the obsolete Ag-B nomenclature for t h e rat MHC. We must hope that the founding of Rat News Letter by Michael Fasting in 1977J and the inauguration of the series of international workshops on rat histocompatibility systems by Tom Gill in the same year will finally provide the necessary solid center of agreement and up-to-date pooled information. From an immunogenetic point of view, the rat will never be the league champion, no matter how h a r d w e work, because the animals are so expensive to maintain. This is, obviously, quite apart from the clear preeminence that the mouse now enjoys from the extraordinary distinction Of its immunogenetic investigators over the past 60 years. But we certainly have only ourselves to blame if our colleagues remain ignorant of the: real state of affairs in rat immunogenetics. The BB rai provides an/inimal modelfor juvenile onset diabetes of quite exceptional interest, and f o r once there isno established rivalmodel in the mouse.lt will b e interesting t o : s e e Whether ~the ~utoimmune features Of the r a t disease andits association with the major ::hist~mpatibility::cgmp!ex ~ ! ! .bring: a new rgency [6 :rat: immunogenetiCS!:in!!gene~l. 'In a n y Met.sboF.c,m.Vet.32, N6.7.SuppL 1 (Jub/), 1983
event, the major concentrationof research interest that the BB rat is bound to generate must force rat immunogeneticists to put their house in good order. The major histocompatibility complex of the rat is now called R T I 3 (The italics conform with standard practice for genetic symbols in the mouse). This name replaces H-I ~ and Ag-B. 4 The chromosomal environment of R T I is not well known. Although the linkage group now contains the glyoxalase marker Glo-! s and otlter enzyme loci? a n apparent homology with the mouse 11-2 and human HLA linkage groups, the chromosome carrying R T I is not known. Fu;thermore, the position of the eentromere in relation to R T I is not known, so the orientation of the linkage group on the printed page is arbitrary. It is curious that the chromosome bearing R T I should still be unknown. The rat karyotype is well-differentiated, 7 unlike that of the mouse, and rat cells have be~n used extensively in somatic cell hybridization, particularly recently. THE FIRST RECOMBINANTS: A AND B REGIONS DEFINED
For a long time, breeding experiments calculated to reveal genetic fine structure in the rat M H C produced no recombinantsbetween recognized MHC functions. In I977 the first two recombinants within the rat M H C were described from two different laboratories.S~9.In each case a "conventional '~ serologic marker was recombined from markers determining other typical MHC-specified functions, such as mixed lymphocyte response (MLR). ~(The term Conventional in this context always refers to antigens of the class ltype, which are carriedon all lymphooytes and probably on all tissue cells. In mice and rats they are expressed on From the Agrtcuhural Research-Council :lr~tttute of Animal Physiology. Babraham.':Cambridge..UK. Supported in part~by:)VIH gront~Al 13162 and by MRC grants 978/120~C and 979/4.~6/C. Reprint ~,eque~lxthouldbe'addresxed to Dr J,C: Howard. A.R.C
znsmute of Ailimai e~yaOto~i:'~iibmham.:!Ca~b~dge;CRZ ~a Z UK © 1983 by Grune K Straiten. Inc. 0026:.0495/320;92000951.00/0 41
42
JONATHAN HOWARD
erythrocytes, and this provides a quick and convenient means of typing for those antigens). The MLRassociated region was also shown to specify histocompatibility antigens causing skin graft rejection, R immune response genes, ~°and, more usefully, serologic naarkers carried on a lymphocyte subpopulation and B cells,s and absent from T cells, platelets, and erythrocytes." in other words, single recombination events were apparently separating all the conventional serologic alloantigen-specifying regions from regions specifying in-like antigens. It was obvious that this genetic structure was unlike that of the mouse (Fig 1). Because mouse la antigens are specified by genetic regions lying between regions coding for the conventional K and D ailoantigens, no single recombination event can separate all la antigens from all conventional ailoantigens. The rat MHC, on the other hand, looked in 1978 much more like the HLA region of humans, where H LA-DR representing In-like antigens, clearly lies to one side of the region specifying conventional HLA-A,B,C antigens and can be separated from it by single recombination events (Fig 1).
RT1 RAT
A
¢I~ ,=
The discovery of intra-MHC recombinants led to a new seriousness in trying to deal with nomenclatura| problems. In particular, the two regions defined by recombination were named A (conventional, class l, K,D-like, HLA-A,B,C-Iike) and B (B lymphocytes, class II, MLC-stimulating, la-likc, HLA-DR-Iike), 1° and a numerical series of defined laboratory recombinants was inaugurated, to which all rat immunogeneticists now subscribe. THE SECOND PHASE: t-I-6 AND THE C REGION
To the right of the B region there exists another histocompatibility locus. Originally named 1t-6 by Stark and colleagues, t2 this locus was noted to determine relatively slow first-set skin graft rejection and to be closely linked to the conventionally defined MHC, but to be recombinable from it. Two apparently reciprocal recombinants, r3 and r4, between the a and u haplotypes, were recovered from 67 F2 progeny. The curious genetic region defined by r3 and r4 has been named C, and has been further characterized by recombinants r5, r14 and r15. '3a' Immunization
'0
a
E
¢
glo grc
J H-2
HLA
MAN
K1 K2
A
~
S
B .......
J m
.....
O
C
A
mi
am
,
U
Os2Qa3
11. Q a l
Fig. 1, A scflemati~ comparison of the MHCs of rat, mouse, and man. The centromare is drawn to the left for mouse and man. The rat MHC is aligned relative to the glyoxaLsse-1 locus glo-l, Regions specifying class I polypeptides are indicated by squares, and class II polypept|des by circles. T h e Imnail circles in 14-2 called LIMP represent the low molecular weight pclypeptldoa recently described,s~Shaded areas show the presumed location o f class ll| products, complement components C4. C2, Factor B in mouse and man. Polymerphlc complement componer~ts |ir~ked to MHC have not been described In the rat. References to features of the rat MHC can bs found in the text. Squares denoting class I-determinlng loci are represented in three ways. Serial squares indicate the presence of a locus specifying -; known major hlst0competibilRy antigen capable of inducl,~g • plimary allogenelc cytotoxic T cell response in vitro and capable o f forming restricting assoc~Jtor~ with minor histocompat|bi|tW antigens or virus antigens. Stipphxd squares Indicate the presence of probable presence of other ¢~Jalm! I o ~ known am'y from sero~gt¢ or structural properties. Open squares :,ndlcate t h e location o f know n "'medial'" histo¢ompstibittty afltigeaS,tO capsble of evoking secondary unrestricted 8|logene|c cytntox|c T cellS,but Probably incapable of f ~ l r d n g restricted associations with minor histocompatibllity antigens or viruses.
MHC OF THE RAT
between C region-incompatible recombinants generates a potent cytotoxie T lymphocyte (CTL) response specific for C-region productsJ r~5 The properties of this response are most unusual and need emphasis. A typical MHC incompatibility will evoke a primary CTL response in vitro. C-region incompatibility induces no in vitro primary response: It is, in this sense, a weak incompatibility. The typical C-region CTL response is seen only after in vivo printing and in vitro boosting with C-incompatible cells. Unlike a response against a typical minor histocompatibility antigen, however, CTL against C-region products are not restricted in their specificity to the products o f any other MHC region. The quality of C-region antigens is therefore equivocal: They are weak in the sense of requiring prior immunization to evoke an in vitro CTL response, while the response has the typical specificity of major alloantigens in not being MHC-restricted. Ailoantigens determined by the C-region are probably responsible at least in part for the remarkable data of Marshak et al, who found a weak but complex alloantigenic system closely linked to the MHC, requiring priming and boosting to evoke an unrestricted CTL r e s p o n s e . 16.t7
Serologic investigations of C-region products have recently demonstrated lymphocyte alloantigens apparently structurally related to those specified by the A region. =~ The constellation of properties displayed by the C region are so strongly reminiscent of those of the Q a / T l a region of the mouse tt-2 complex ~9''° that the possibility of homology cannot be excluded. There is no known comparable MHC region in HLA. HETEROGENEITY OF la ANTIGENS: DISTINCTION BETV'¢EEN B AND D REGIONS BY RECOMBINATION
One ~.'xpects an MHC to specify more than one la glycoprotein. In the mouse the l-Aand I-E molecules are now well-defined, with four loci specifying their constituent a and B chains. 2~The original definition of the rat t~ region was identical to that of the whole mouse ! region in the sense that it included lr-gene function, strong MLC-stimulating activity, and serologic antigens expressed predominantly by B lymphocytes.9"2ZIt was therefore reasonable that, as defined, the B region might prove complex, and specify more than one+Ia-like glycoprotein. :This has provedto be the case. Evidence that the rat M H C as a whole specified giycoproteins corresponding to I-A and I-E came from immunoprecipitation studies using mouse alloantisera specific for mouse I-A or I-E products. These reagents cross-reacted extensively onrat cells(without marked polymorphicspecificity) and wereshown+t0 precipitate two independent la-like glycoproteins discriminableby two-dimensional p01yaerylamide gel eleetroph0resis. 2'
43
Partial N-terminal sequences of a and/~ polypeptides precipitated by rat alloantisera indicated that both I-A and I-E homologues were coded in R T I . 24Rat alloantisera also discriminated two molecules using sequential precipitation in appropriate cross-reactive strain combinationsY Finally, mouse monoclonal antibodies have been raised+ against rat niembrane glycoproteins and shown clearly to be specific for two independent la-like molecules. :+,27 The loci specifying these two serologieally defined la-likemolecules have now been separated by the r12 recombination. 2s The two la loci defined by the r12 recombinant haplotype have been named R T I B and R T I D . Combined serologic and immunochemicai studies have shown that the molecule apparently homologous with mouse I-A is specified by R T I B , and that homologous with mouse I-E by R T I D . 29The order of loci is R T I A - B - D . It should be noted that the present use of R T I B refers only to the locus specifying the I-A homologue, while the former use of the same term referred to the homologue of the whole mouse /-region. It would seem reasonable to introduce the term "/-region" into the rat MHC to cover both RTI B- and RTI D-specifying loci. In this case the loci can be referred to as I-B (I-A homologue) and I-D (I-E homologue), and their products similarly. The only known recombinant within the rat/-region is rl 2, and it separates the ot and/~ chain loci of I-B and the ~ and B chain loci of 1-D. in the mouse nearly all intra-l-region recombinations fall between I-E/3 and I-E c~. One might hazard to guess that the highfrequency recombination site recently postulated within 1-E in the mouse3° may not be present in the rat MHC. The arrangement of the la loci in the rat appears therefore to be more symmeti'ical than those of the mouse, where three of the four la polypeptides are specified in the region conventionally termed 1-,4, while only the c~ chain of the I-E product is actually coded in 1-E. In the mouse a number of defects in the transcription and expression of the I-E molecule have been recorded. As a result, many 1-1-2 haplotypes have no detectable I-E expression. There is no evidence for this anomaly in the rat. Every haplotype tested with the /-D-specific xenogeneic monoelonal antibody MRC OX 17 has proved positive.27'3~ Formal evidence that/-B and I-D both lie between R T I , 4 and R T I C i s not available at the moment. From the indirect evidence available, however, there seems tobe little doubt that this isthe ease. R T I E A N D THE:GROVVTH AND REPRODUCTION COMPLEX
The growth and repr0duction ~ m p l e x ( G R C ) was named t0 describe a mutantphenotype-irlvolving low
44
JONATHAN HOWARD
male fertility and reduced body size) "~'3~The trait was shown to map dose to the MHC, and the recombinant r l l showed the gene order to be A-(B-D).GRC) 4 From the low recombination frequency between RTIA and GRC ~2it is tempting to locate GRC to the:left of RTIC. Recently, the rl I recombinant has been used to demonstrate the close linkage of a new cell-surface ~dloantigen, RTI E, with the GRC. ~ Overall, the properties of the R T I E product resenable those of a class I molecule. Evidence has been presented for the expression of R T I E on erythrocyte membranes, and immunoprecipitation studies have shown heavy and light polypeptides corresponding tolerably well in apparent molecular weight to those of conventional class ! glycoproteins) ~ R T I E products seem also to excite cellular immunity, as judged by the development of cytotoxic efl'eetor cells:'6 RT1C, RTIE AND THE MOUSE PARADIGM
On the strength of all the evidence presently available it is reasonable to describe the order of loci in RT! as A-B-D-E(GRC)-C (Fig 1). Because R T I E has class I-like properties and lies to the opposite side of the typical /-region from RTIA, Gill and his colleagues ~s'-~7have been tempted to draw a formal analogy between the K-I-Dstructure of 1t-2 and the A-I-E structure of RTI. in both cases, loci specifying class 11 molecules are flanked by loci specifying class ! molecules. The main objection to this simplifying proposition is that there is no evidence that R T I E specifies a major transplantation antigen rather than another antigen of the R T I C type. it may be significant that experiments demonstrating the generation of cytotoxic cells specific for R T I E products were conducted using the protocol required for R T I C antigens, namely in vivo priming and in vitro boost. Furthermore, exten~sive experiments in the author'slaboratory have failed to provide any unequivocal evidence for either primary CTL or MHC restriction of secondary CTL induced by the products of any locus other than R T I A (A.M. Livingstone, unpublished results). Similar results are reported from Gunther and colleagues) s For the moment it is rather important that R T I E and R T I C be compared formally. Although the recombination frequencies separating these two loci from R T I A seem fairly different at present (about 3% for R T I A - R T I C "compared with about 0.5% for R T I A - R T I E ) , similar variations in recombination frequency have been reported between homol0gous pairs of loci in !t-2)~ The expression of RTI E products on erythrocytes~ is a further difference from the reported properties of R T I C products. Curiously, however, R T I E products are not detectable on erythrocytes of certain haplotypes (although present in an
crythrocyte lysate)) ~ It remains possible, therefore, that R T I C products are also present but have not yet been detected. My own strong preference is to consider R F I E to be more closely related to R T I C than to RTIA. If the homology between R T I C and the Q a / T l a region of mouse !1-2 is correct, then we should expect to find several linked loci with similar properties. It should be emphasized, though, that until laboratories carrying the informative R T I E and R T I C recombinants compare their results formally, it remains perfectly possible that R T I E and R T I C are literally one and the same thing. It is natural to attempt a formal comparison between RT1 and H-2. The main source of omfusion lies in the lack of a well-defined sec,~nd locus determining strong class I products in the rat. Allth~ classic class i alloantigenie activity.is concentrated in the products of the A region, while R T I E and R T I C determine weak and equivocal antigens of uncertain status. There is some evidence for more than one distinct antigenic:product from RTIA. Microsequeneing of the 45Kpolypeptides precipitated by alloantisera from normal iymphocytes suggested a limited heterogeneity: ° The data were compatible with two polypeptides present in equivalent amounts and presenting strikingly similar but not identical N-terminal sequences within each haplotype. Between haplotypes the sequences were more distinct. Such limited sequence heterogeneity between products of distinct genes on.a single chromosome is typical of very closely linked members of a multigene family. It is more reminiscent, for example, of the relationship within the tt-2D family, ~* than between K and D:" A similar conclusion has been reached recently on serologic grounds using a panel of monoclonal alloantibodies. The results suggest the expression by RTI of a series of at least three very similar molecules, all of which share one common specificity, two of which share a second, and one of which carries a unique specificity: 3 Again, this situation is reminiscent of the H-2K or H-2D families, where members of each closely linked series share most but not all the specificities characteristic of the particular haplotype: ~ Finally, two conventional immunopreeipitation studies have demonstrated structural heterogeneity among class I alloantigens in the rat. zS"4s'~ Unfortunately, none of these studes has combined serologic, immunochemical, and immunogenetic techniques to demonstrate formally what the genetic basis may be for the observed heterogeaeity or which structures correspond to the class 1 major histocompatibility antigens defined a s such on the basts of T cell-mediated responses. Until these comparisons a r e made the organization of the rat M H C and its formal relationship with tl-2 remains sub judice.
MHC OF THE RAT
IMMUNE RESPONSE GENES IN THE RAT
Some of the earliest studie~ on Ir gene control were performed using synthetic polypeptides in congenic rat strains..High and low antibody responsiveness to HGAL were shown to be linked to the MHC, 47 and immunization of recombinant animals showed that differential responsiveness mapped to the I (BID) region, t° Both I-B and I-D subregions probably control differential responsiveness. The rl 2 (BID) recombination was, in fact, discovered by a discordance between RTIA and immune responsiveness to GLT. :a In this case differential responsiveness mapped to I-D. More recently the in vitro proliferative responses to bovine and pork insulins have been shown to be under MHClinked genetic control, 4"'49 and differential responsiveness apparently maps to I-B (D.V. Cramer, personal communication). It was, of course, to be expected by analogy with mouse data that both the la molecules of the rat should be involved in antigen presentation. COMPLEMENTATION IN Ir GENE SYSTEMS
There is no molecular or genetic evidence yet that I-B or I-D region products display novel specificities in FI hybrid cells because of formation of hybrid a/3 dimers. It would, however, be extraordinary if this effect did not occur, as it is well known in the mouse.-'° Furthermore, Gunther and colleagues s~ observed anomalous high responsiveness to TGAL in Ft hybrids between R T ! congenic rats carrying low and intermediate response alleles. Although the molecular basis for this effect was not demonstrated, it seems likely from mouse studies that hybrid c~/~/-region dimers, causing a novel specificity of antigen presentation, were responsible. A start has been made in defining the association between la polypeptide structure and immune responsiveness in the rat. Massey and Wilson 5' have shown that in vitro proliferative responses to GT are under R T l - t i n k e d control. Unexpectedly, the two highresponder haplotypes, R T I = and R T I ¢, appear to be equivalent in the sense that cells bearing either of these haplotypes are able to present GT indiscriminately to each other. The implication that these two haplotypes might share identical class 11 polypeptides has apparently been confirmed by studies on the structures recognized by an RT! De-specific monoclonal antibody.at This antibody precipitated RTI D molecules carrying an apparently common B chain from the a. c, o, and f haplotypes. Subsequent work has shown that R T I f also belongs to the G T reciprocal presentation group (G. Massey,' persOnal communication); The RTI D ~8 chain involved in this cross, presentation group is also assc,e!ated with the/r,gene:colttrolled
45
immune response against the class l M H C molecule. RTIA=. s3.s4 Inthis case, however, the association is with low responsiveness. Positive correlation between an la polypeptide and unresponsiveness is suggestive that the immune response in question may be regulated in part by suppression. It is tempting to relate these findings in the rat to the 1-E-suppressed immune response to LDH isozymes and other antigens recently illuminated by Baxevanis et al. ~5 EXPERIMENTAL AUTOIMMUNE DISEASE
The classic experimental investigation of autoimmune disease in rats is the experimental allergic encephalomyelitis (EAE) induced in LEW (formerly known as Lewis) rats by footpad injection of guinea pig spinal cord or\myelin basic protein in Freund's complete adjuvant. -~ As with most autoimmune diseases, EAE has resisted a simple immunogenetic analysis, while still presenting tantalizing evidence that genetic factors in general, and the MHC i'n particular, play an important predeterminative role. There is no doubt that the LEW rat is pecu|iarly~susceptible to the induction of EAE, but it has been difficult to apportion responsibility betweea IVlHC and background genes. 57-5q The study of EAE in rats has been particularly effective: although a similar condition can be induced in mice, no mouse strain is as susceptible to the severe disease as the LEW rat. Nevertheless, some ~mmunogenetic studies have confirmed an association between H-2 and EAE, ~° and, most imp¢,rtantly, an indication that the disease process entails l~ecognition of antigen in association with I-A products has been obtained by McDevitt and colleagues.6~Theit" investigation showed that the induction of EAE could'be prevented by passive transfer of high-titred anti-l-A monoclonal antibodies into mice at risk. This important and rational protocol seems likely to be of considerable value in the analysis of experimenial fiutoimmune disease, and may even contain the germ Of a valid clinical procedure. Perhaps the most important theoretical implication to develop from these:studies on EAE in mice and rats is that the disease state contains a t least two components at the immunologic level: First, from the evidence for a positive association between particular M H C alleles and disease, even v,;~,en the al/ele is in the heterozygous condition, sTJs from the'direct evidence for MHC-restrieted presentation of the sensitizing antigen u and from the protection experiments using in vivo administration of anti-l-A antibodies, 6t it is clear that t h e development of t h e disease S t a t e involves a conventional form of MHC-restrieted T c e l l induction on antigen-presenting cells.It is likely that this process
46
JONATHAN HOWARD
is subject t o / r gone control such that presentation of the encephalitogenic peptides in ass~)ciat~on with some class !1 antigens will not lead to an immune response. Susceptible animals are therefore high responders, and the healthy or resistant animals are the low responders. Arguably, the resistant animals are those that have what is sometimes called an "It-gone defect." (When the problems of autoimmunity are put into this context, one can readily see the merit in an explanation for lr genes in terms of cross-reactive self-tolerance). 6~'~ Secondly, there is convincing evidence that under certain conditions a state of active immunosuppression can be established in susceptible animals? ~ In general this state follows immunization with nonencephalitogenie forms of the antigen, and can be ab~gated by treatment with cyclophosphamide.~ The immunogenetics of illduction of suppression are not known but are an obvious avenue of investigation, it would be of great interest to know whether a "resistant" genotype may not only represent one in which the MHC (or other factors) does not predispose to induction of a pathogenic response, but also one that predisposes to the induction of suppression. IMMUNOGENETICS OF BB RATS
BB rats are Wistar-derived albino rats carrying the R T ! ~ allele typical of this provenance. There is no
reported evidence that the line is heterogeneous at the MHC. When spontaneous diabetes mellitus (IDDM) was first observed in the colony maintained at BioBreeding Laboratories of Canada Ltd., theL:olony had been closed to new stock for 6 years (Chappell, personal communication). In view of the high standard of maintenance and observation at BioBreeding Laboratories, it seems unlikely that the diabetic condition was in fact present for 6 years before it was noticed. Fur=hermore, IDD M has not been reported from the many other laboratories holding Wistar-derived albino colonies. The single reasonable explanation for the sudden onset of IDDM in t h e BB colony is that a mutation occurred that, in association with the BB genetic background, predisposed strongly to the disease. That some single event occurred that spread through only a part of the colony is suggested by the fact that lines of rats from the 'BB colony with no IDDM are widely used as forms of control:for the typical diabetic BB rat. Such are, for example the W and V lines segregated from the colony by Butler at the University'of Massachusetts (L. Butler, personaLc~mmunication). A similar nondiabetie line of BB rat is maintained by Thibert in Ottawa. Both susceptible and control lines of BB rat are apparently homozygous for the R T ! = haplotypc. One would, therefore, be strongly inclined to belicve that the mutation that predisposes
to IDDM occurred at another locus. Before assuming this, however, one should remember that the H , 2 K b mutation H-2 t"'~ predisposes the mouse carrying it to the induction of autoimmune thyroiditis, 67 a susceptibility characteristic of the 11-2 k and 11-2 d haplotypes a:~d not of the H-2 b. o n ~ with the use of a panel of monoclonal antibodies hiis-it been possible to show convincingly that the H - 2 K pr~uct of H-2 b"l is serologically distinct from that of tl-2"b. ~ One wonders, therefore, whether a mutation at R T I " would have been noticed yet. An argument against the relevant mutation of the BB rat having been at the MHC concerns the susceptibility of segregat;ng populations to IDDM. In the studies of Colic and her colleagues69 it is clear that IDDM is associated with R T I " , whether it is in the homozygous or heterozygous condition. It is also the case, however, that F~ hybrids between IDDM-suseeptible and nonsusceptible control BB rats are invariably nondiabetic, arguing strongly that the relevant differonce between these two rat lines is not at the MHC, which would, of course, be heterozygous for the putative dominant R T l - l i n k e d susceptibility marker in the Ft hybrids. It seems likely that the principal differences between diabetic land nondiabetic BB rats lies at a single locus unrelated to the MHC. The outstanding characteristic of the normal allele at this locus seems to be that it protects ~,the BB rat from a susceptibility to IDDM that it possesses by virtue of other genetic characteristics. Of these characteristics only one is seriously recognized, namely, the dominant susceptibility associated with R T I ~ . This susceptibility is only apparent if the rat is also homozygous for the mutant allele at the "protective" locus. Striking evidence that the difference between a vulnerable and invulnerable subline of BB ratsmay be due to the activity of ff single locus emerges from the genetic studies of Butler "at the University of. Massachusetts (personal communication). This investig~/t0r has developed inbred lines of IDDl~l-susceptible arid control BB rats. The most interesting case is that of the W control line, which was extracted from the fifth generation of brother x sister mating of the susceptible BA line. Presumably, at the 5th B x S generation the BA line was still segregating for the "protective" allele. However, at the 12th B x S generation the susceptible allele had evidently been fixed in the BA line, and the protective allele fixed in the W line. At this time the incidence'of IDDM in the BA line was about 6.0%, a figure typical of all BB lines from which segregating genetic factors have been eliminated b y inbreeding:The incidence of IDDM in the W line was zero. All F~ progeny in the BA x W cross were normal,
MHC OF THE RAT
47 Table 1, R T f Haplotypos and RT1 Congentc Strains
Typical I.trsins
RT1 a b c d e f g h
ACI, DA, AVN BUF, A L B AUG, PVG BD'V BD"V AS2 KGH HW
i
BI
k
SHR, WKA LEW
I
m n
o p t
u x y z
MNR BN MR RP TO WF, AO nes~ved for wilds Reserved for unknowns Reserved for unknowns
LEW congdsnic LEW.1A(AVN) LEW. 1a LEW. 1C (AUG) LEW. 1D(BDEt LEW.1E(BDVII) LEW. 1F(AS2)
PVG ¢ongenic
PVG-RTf"(DA) PVG
BN congenic
Othol's
BN.B4(DA) BN. 1B(BUR BN. I C(AUG|
BN, 1G(KGH) LEW. 1H(HW) DA. 1l(Bi) PVG. 1K(VVKA) PVG-RTI~{AGUS) PVG-RT;*(F344) PVG~RTtt(LEW)
BN. IK(WKA) BN. 1L(LEW)
LEW.1N(BN)
PVG-RTf"(BN) PVG-RT I*(IC1)
BN
LEW. 1U(WP)
PVG.RTt"(AO)
BN. 1U(WF)
LEW
AUG.1N(BN)
Inforrne, tion concerning the origins and availability of the rats listed on this table can be obtained as follows. LEW strains: Dr O. Stark, Department of Biology. Facult! of General Medicine, Albertov 4, Prague 2. Czechoslovakia; and Dr E. GiJnther0 Mex-Planckqnstitut f~r Immunbiologie, 78 Freiburg-Zahringen, St~beweg 51, W. Ge~'many. PVG strains: Dr G.W. Butcher, Department of Immunology, Agricultural Research Council, Institute of AnimrJl Physio~'3y, Bsbrahem, Cambridge CB2 4AT, UK; BN stratus: Dr T.J. Gill IlL Department'of Pathology, Unwersity of Pittsburgh School of Me.d~cine. Pittsburgh, PA 15261. Dr Gill also maintains the largest collection of inbred rat strains in the world. His colony provides an invaluable and uni~m resource.
as were all backeross progeny in the direction (BA x W) × W. in the backeross (BA x W) x BA, however, the incidence of IDDM was about 30%, compatible with a single segregating recessive factor in th~ BA:line with a penetrance of about 60%. If the BB rat is to develop its full potential as a model for ht man juvenile onset diabetes 'mellitus, it is essential that inbred lines, preferably congenie for relevant genetic markers, be used and become widely available. The situation at present is unsatisfactory, with no general agreement to use only manifestly inbred lines and congenicr,: The situation is; of course, complicated by the lx~r health of IDDM rats; particularly in conjunction with the IDDM-associated immunodefieiency, and inbreeding depression is a major obstacle. Nevertheless, if genetically well-defined animals are not made available quickly, the emphasis of research interest is likely to move rapidly and irretrievably over to the N O D mouse2°'~z and pressure will grow to make this interesting but little known model for IDDM generally available. APPENDIX 1::INBRED STRAINS OF RATS Contrary to popular belief, there arc large numbers
of inbred rat strains, :Indeed, a *:list containing more
than ! 50 strains has recently been compiled and published by Festing72in Rat News Letter 10.* APPENDIX 2: R T I CONGENIC SERIES There are three major congenic series of rats available at pr,;sent, based on the LEW, PVG, and BN
backgrounds (Table 1). The LEW series was established by Stark and his colleagues in Prague and is also maintained by Gunther at Freiburg. The PVG series was established by Butcher and Howard in Cambridge, England. The BN series was begun by Joy Palm in Philadelphia and has been maintained and extended by Gill and his colleagues in Pittsburgh. This last laboratory has an extensive collection of other congenics. APPENDIX 3 : R T 1 RECOMBINANTS
T~bl¢ 2 ~ s all the laboratory recombinant haplof type, o:..-~TI presently recorded. Thts hst is constantly ~:,lg extended.
*(Ra'~:NewsLetter isavailable from Dr. M,F.W~ Fcsting, Medical Research CoUnCil Laboraiory Animals Centre,. Woodmanstcrne Road, C.arshalton, Srai-rcy~SM5 4EF, UK)
48
JONATHAN HOWARD
Table2. Recombinant RT1Hlplotypes
rI
r2 r3 r4 t5 r6
rT" rB r9 t rlO
Suam
A
B
FVG.R 1 LEW.1AR1 LEW.1WR ! LEW. 1AR2
a n u a n u a a
c u
a c u
n
I
LEW, 1WR2 PVG.R7 PVG.R8
r11
--
I
r ~2(p3)
V~'RC
n u I
r14 r15
D
E
C
a u I
n u I
Refe+enCe 8 9 11 t1 13 80 81 81 82 32
i
34
!
28 14 14
a u
• The PVG,R7 line is about to become extinct. Liver DNA and RT f I--cont~tining ted! hybrids h~tve been p~esetved. tAn r9 recombinant line was never established and the recombinant chrt~--.,c~ne is extinct (T. Nero
APPENDIX
4:
MONOCLONAL
ANTIBODIES
AGAINST
RT ! PRODUCTS
The first use of antibody-secreting plasma cell hybrids to prepare monoclonal antibodies against
MHC alloantigens in any species, was in the rat. ~3 Since then, it has become apparent that ailoimmtmizations in the rat provide a particularly favorable system for generating anti-MHC monoclonals in large num~ r s : *s5 Xenoimntunization of mice with rat cells or cell products has also been a useful method of generating monocional antibodies specific for RTI products, usually but not always without ailelie specificity. Xenoantibodies against class ! ~7and class 1126'27products have been described. A compilation of monoclonal allo- and xenoantibodies specific for RTI products is presently in preparation by Butcher and Howard for publication in lmmut~ogenetics. ACKNOWLEDGMENT
i should like to record a particular debt of gratitude to Dr. Errol Marliss and to the Juvenile Diabetes Foundation for the invitation to attend the workshop on the BB rat, and for the intensiveintroduction to the pressing problems of juvenile onset diabetes. I am also grateful to Dr. G.W. Butcher for helpful conversations and for reading the manuscript, and to Jenny Richards for typing the manuscript.
REFERENCES
I. Festing MFW: Rat News Letter. Medical Research Council Labora!ory Anima|s ('entre. ISSN 0309-1848 2. Gill T J. et al: Report of the First International Workshop on Alloantigcnic Systems ;~ the Rat. Pittsburgh. June 6-8, 1977. Transpla+,t Proc i0:271-285, 1978 3, Stark O, Kre.n V: Erythrocytc and transplantation antigens in inbred.strains of raL~. VI. The histocompatibility-! system and its alleles. Folia Biol (Praha) ! 3:356--360. 196"1 4. Elkins WL, Palm J: Identification of a single strong histocompatibility locus in the rat by normal spleen-cell transfer. Ann NY Acad Sci :2~:~,73--580, 1966 5. Stoic V+ Kunz HW, Gill TJ 11|: The linkage ofglyoxalase-I to the major histocompatibility complex in the rat. J Immunol 125:! 167-1 ! 70, 1980 6. VandeBerg JL, Bittner GN, Meyer GS, et ah Linkage of neuraminida~- and a-mnnnosidase to the major histocompatibility complex in the rat. J lmmur=ogenct 8:239-242, 198 ! 7. tlungcrford DA, Nowel! PC: J Morphol ! 13:275, 1963 8. Butcher GW, Howard JC: A recombinant in the major histocompatibility complex of the rat. Nature 226;362-364, 1977 9. Stark O, Gtlnther E, Kohoutova M, ct ah Genetic recombination in the major histocompatibility complex. (H-I, An-B) of the rat. immunogenetics 5:1993-1997, 1977 !0. GIlnthcr E, Stark O, Koch C: Genetic definition of I regiondetermined antigens of the rat major histocompatibility complex. Eur J lmmunot 8:206-2|2, 1978 t I. Davies liftS, Butcher GW: Kidney alloantigens determined by two regions Of the rat major histocompatibility complex, lmmunogenetics 6:!71-18! 1":)78 12. Kohoutova ',~ ";unther E, Stark O, Vojcik L; Further two rccombinants at t : -ajor histocompabitility complex of tbo rat. Folia Biol (Praha) ,",+. ~ - 4 0 7 , 1978 13. Lynch DH, IP,.~ ,. CW:Genetie identificztlon of a locus
linked to the rat M H C that codes for a membrane antigen detectable
with cytotoxic lymphocytcs. J Immunol 121:2367-2375, 197g 14. Wonigcit K, Hanisch L, Steiniger B. et ah Analysis of the two new R T I C region recombinants" r14 and r15 and a LEW subline lacking expression of C-region antigens. (unpublished o~¢rvations) t5. Gilnther E, Wurst W. Stock W: Specificity of anti-RTlC alloantisera and cytotoxic T lymphocytes. (unpublished observations) 16. Marshak A, Doherty PC, Wilson DB: The control of spocificity of cytotoxic T !ymphocytes by the major histocompatibility complex (An-B) in rats and identification of a new alloantigen ~ystem showing no Ag-B restriction. J Exp Med 146:1773-1790, 1977 17. Sieck T, Wilson DB: Studies of the MHC-linked "CT" alloantigenicsystem in rats. II. Evidence for more than one locus and several different determinants. Immunogen©tics 14:293-302, 198 i I 8. Haustein D, Stock W, Gflnther E: Rat major histocompatibility RTI.C antigens of restricted tissue distributionconsist of two polypcptide chains with molecular weights of about 42000 and 12500. Immunogenet[cs 15:27l=278, 1982 19. Fischer Lindahl X, Langhornc J: Medial histocompatibility antigens, Scand J immunol 14:643--654, 1981 20. Flaherty L: The " T t a , region antigens, in Doff M (¢d): The Role of the Major Histocompatibility Complex in Immunobiology. New" York, Garland Prccs, 1981, pp 33-57 21. Klein J, Juretic A, Baxcvanis CN, et ah The tradhional and a new version of the mouse 11-2 complex. Nature 291:455-460, 198 ! 22. Howard JC: Recombinants tn the major histocompatibility comp|¢X. Rat News Letter 1:8-9,1977 23: Blankenhorn EPo Cecka .!M, Fre|inger J, et ah Structure of la antigens from the rat, Mouse :alloantiscra dcmonstrat© at least two
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MHC OF TttE RAT
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