- Email: [email protected]
Complexity of MHC class III genes and complement polymorphism
Immunology Today, Vol 10, No. 12, 1989
both high and low affinity IL-7 receptors. The Seattle group further reported the results of in-vivotrials using IL-7. Observed effects included an increase in the number of cells in the spleen and mesenteric lymph nodes and an increase in the number of marrow pre-B cells. It is unknown how many additional stromal cell-derived mediators that affect B-cell development remain to be identified. The details of the cellular interactions between stromal and B-lineage ceils are equally unknown.
Of interest in this regard was the description by P. Kincade (Oklahoma City) of a monoctonal antibody that inhibited B-cell production upon addition to long-term bone marrow cultures. It will also be important in the future to investigate the role of the extracellular matrix in B-cell development, although virtually nothing was reported on this topic.
that considerably more progress in the above areas will be reported at the time of the next congress in Budapest. It is expected that they will still be major topics on the agenda at that time. Progress in adapting the various systems which have been so successful with murine cells to human bone marrow will, it is hoped, also be reported.
Concludingremarks
K. Dorshkindis in the D/visionof Biomedical Science, University of Cafifomia, Riverside, CA 92521, USA.
In July, a comparatively small group of geneticists from 23 countries met, first in Mainz* and again in Berlin at the Immunology Congress, to have a closer look at the major histocompatibility complex (MHC) class III genes and other complement polvmorphisms. Class III genes indude not only complement but also other loci, like 21-hydroxylase (21-01-1)A and B, tumour necrosis factor (TNF) ~ and [3, and duplicated genes of heat shock protein 70 (HSP70), with no obvious functional relationship. On the other hand, complement loci are also found on chromosome 1 (H, C4BP, CR1, C8A and B), chromosome 9 (CSG: J. C~nd#t"7 Col,,rnhia), and ,-~,n,~,~,~,, l o
Complexityof MHCclassill genesaM---complementpolymorphism
It is appropriate to end this meeting summary on the optimistic note
i
!
iI i
from Christian Rittner and Peter M. Schneider gene products. A full account of these findings will be given at a later date (Complement Inflamm., in preparation). Suffice it to say that human C4 polymorphism has now reached a remarkable degree of resolution at the phenotypic as well as the molecular level. Th~ nn~ninn
¢,',¢¢inn r~f t h , , ~^,orlt-
shop on C4 polymorphism, spanned the evolutionary bow from mice to man, encompassing the chimpanzee. These three species possess duplicated C4 loci derived from recombinational events predating the separation of human and mouse ancestors. The two C4 loci of chimpanzees and humans share similarities in standard arrangement, with long (C4A)/(C4B) and short genes. To provide a solid basis for dis- Klein dates the duplication event at cussions, a round of reference ty- at least seven million years ago. Inpings on polymorphic complement terestingly, duplicated C4 genes in components preceded the confer- mice are flanked by retroviral inence. This is summarized in Table 1. sertiuns of unknown origin. The sigThe major emphasis was on human nificance of such duplication events, C4 polymorphisms, analysis of which named "converted evolution" by involved a large number of different Meo, is still a mystery. The search for 'new' class III genes, techniques, many reference laborato fill the 400 kb gap between TNF-e tores and the testing of a substantial and C2, by chromosome walking has number of samples. The aim was to give a better definition of variants, resulted in the isolation of Hpall tiny duplicated and converted genes and fragments (HTF) islands containing a high density of non-methylated CpG dinucleotides. When hybridizing * The Sixth ComplementGeneticsWorkshopwas genomic probes to northern blots of RNA, R.D. Campbell and colleagues held in Mainzon 27-29July1989.
(C3). Curiously enough, MHC-linked complement genes still attract most attention, probably because evolution, deficiency and disease seem to have a causal relationship. These observations have been the subject of speculations from both the neutralist 0. Klein, T~Jbingen) and selection,st (T. Meo, Paris)point of view.
1989.ElsevierSciencePubhshersLid, UK.0167~1919/89/$02.00
(Oxford) identified 12 novel single copy genes. Localization of so many new genes within the MHC may nave a bearing on our understanding of the functions of the complex. Using a different approach but addressing the same question, Y. Morel (San Francisco) detected another a, ,nli,-~t~,4 ,~o,,., ,.,,'~'~,th~,~ DNA c,,=,,,~ opposite to that encoding the human E4 and 21-OH genes, again of unknown function. Obtnumbering all other issues, most abstracts submitted (published in Complement and Inflammation (1989) 6, 283-304) dealt with possible associations between complement polymorphgm~ a--l l u~ G" ~2&5~. Fifteen different posters described significant variations in the distribution of allele frequencies of MHClinked C2, BF, C4A and B genes in diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), chronic polyarthritis, multiple sclerosis, visceral leishmanias,s, chronic glumerulonephritis, leprosy, Brazilian paracoccidioidomycosis, common variable immunodeficiency, IgA deficiency, insulin-dependent diabetes mellitus (IDDM), Chagas disease and AIDS. The possibility that superhaplotypes (originally described by the late R. Ceppellini), rather than individual alleles, are associated with disease was also reported: the Caucasian
401
Immunology Today, VoL 10, No. 12, 1989
- erlin news
¢ie vs
Table 1. Typingof polymorphiccorrz',ementcomponents
Complement component
Technique a
C2
IEF/WB RFLP
FactorB
No. of participating laboratories
No. of submitted samples
Total no. of known alleles
5
30
< 10
HVAGE/IFX HVAGE/HOV IEF RFLP
11
100
> 20
C4
HVAGE/IFX HVAGE/HOV HVAGE/WB MAB SDS-PAGE(~/13chains) HAl (RodgersiChido) RFLP
14
140
> 40
C3
HVAGE/IFX
4
26
> 30
C6
IEF/WB
4
50
> 20
C7
IEF/WB IEF/HOV MAB
7
31
< 5
C8
IEF/HOV IEF/WB SDS-PAGE RFLP
3
20
< 10
Factor l
IEF
3
11
< 5
aAbbreviationsof typing techniques:HAl: haemagglutination-inhibitionwith humanalloantisera;HOV: complementcomponent-dependenthaemolyticoverlay;HVAGE:highvoltageagarosegel e!ectrophoresis; IEF:isoelectricfocusing;IFX:immunofixatio~,;MAB:monoclonalantibodies;RFLP:DNArestrictionfragment lengthpolymorphism;SDS-PAGE:SDS-polyacn/lamidegel electrophoresis;WB: westernblot.
402
superhaplotype B8, BFS, C4AQO, C4B1, DR3 is associated with diminished antibody response to the hepatitis B vaccine. However, the question that remained unaqswered was whether the complement genes themselves were responsible for the increased susceDtibi!ity to disease or whether the mapped genes on a limited number of ancestral hapiotypes (R.L. Dawkins, Perth) are only 'bystander' genes close to the unknown loci responsible for the diseases. Some of the formal presentations at both the Mainz Conference and the Berlin Congress provided new insights into the structure and function of C4A, C4B, FI and C8 polymorphisms. The structural basis for inherited deficiency of complement components was analysed at the gene and protein levels for factor I ~,,d the C8 ~ / chain. Using a
factor ! (FI) cDNA probe as well as synthetic oligonucleotides combined with conventional and large DNA fragment gel electrophoresis, it was possible to identify five characteristic haplotypes. This led to detection of the FI*QO atJeie in family studies of eight FI deficiency patients. Although no major structural aberrations of Lhe gene were found, no FI plasma levels were detectable. These results suggest a heterogeneity in the genetic basis of FI deficiency based possibly on independent mutational events (K. KOIble, Oxford). Similar evidence was presented for the deficiency of the C8 ~ / chain, although it was based on studies at the protein level. A monoclonai antibody and an affinity-purified rabbit antiserum directed against the subunit wer~ used to detect defective C8 ~ , protein in six patients carrying the deficiency, in these
patients, the subunit is functionally inactive as shown by hemolytic overlay. In addition, C8 ~/appears to be polymorphic, indicating a heterogeneous basis for the deficiency. Evidence was also obtained for decreased serum levels of the 13subunit in patients with ~/deficiency, poiniing to a possible major role for the ~ / subunit in the secretion of the complete C8 molecule (L. Roncelli, F. Tedesco, Trieste). For the C8 13 gene, the first two DNA restriction fragment length polymorphisms (RFLPs) were reported. They are strongly associated with each other, but do not correlate with the protein variants defined by isoelectric focusing. These RFLPs may prove useful in the analysis of the genetic basis of C8 13 deficiency as well as in genetic linkage studies (D. Herrmann, Mainz; S. Rogde, Oslo). The two isotypes of the fourth component, C4A and C4B, exhibit striking functional and biochemical differences in spite of 99% identity. Using site-dire~ed mutagenesis and expression of a variety of mutated C4 gene constructs in fibroblasts, the molecular basis for these differences was localized to a sequence of four amino acids between residues 1101 and 1106 in the C4d region. This sequence exclusively comprises the serological determinant Chido 4 on the C4B molecule. It was possible to show that the substitution of a single amino acid at position 1106 (Asp to His) changed the hemolytic activity from C4B to C4A. C4B usually has a three- to four-fold higher hemolytic activity than C4A. In the same way, the mobility of the C4 ~ chains in SDS-PAGE could be changed from C4B to C4A~ an apparent molecular weight change of 2 kDa, by exchanging L for P at position 110!. ,,,is did not affect the hemolytic activity of the protein. The fum~ional differences seem to be singularly affected by the presence of His or Asp side chains in conformational position to the internal thiolester bond of C4 (M. Carroll, Boston; D. Isenman, Toronto). C4A and C4B deficiency is caused in about 50% of all affected haplotypes by gene deletions encompassing a single C4 and an adjacent 21-0H gene. On the other C4 null haplotypes, it was suggested that the respective genes were either not expressed, or that these genes express a homoduplicated product
Immunology Today, Vol. 10, No. 12, 1989 i
~ 'r
.r
identical to the C4 isotype already present. In a study of 26 individuals with homozygous C4A or C4B deficiency, it was possible to show that all C4A 'null' genes carried all sequences typical for a C4A gene, although no gene product was detectable. In contrast, all C4B 'null' genes had only sequences characteristic for a C4A gene. Therefore, these genes may express the C4A isotype. The molecular mechanism leading to this altered sequence could be either gene conversion or multiple recombination. The results were obtained by analysing the isotype-speciflc C4d region with oligonucleotides, restric-
tion enzymes and genomic sequencIn summary, complement genetics ing after DNA amplification using has evolved to a considerable degree the polymerase chain reaction (L. and has left its egg shells behind. Braun, P.M. Schneider, C. Rittner, Major contributions to clarify the Mainz). molecular basis of genetic variations Evidence for separate control of have been made. Even some sites C4A and C4B gene expression has resDorsible for functional diversity been obtained by studying synowal have been elucidated. The most immacrophages of RA and osteo- portant question: why a given variarthritis patients. Using northorn blot ation, for example deficiency, may or analysis and C4 isotype-specific may not lead to disease still remains o!igonucleotides directed against the to be answered. sequence coding for the Chido 4 determinant, C4B was found to be C Rittner and P.M. Schneider are at predominantly expressed only in the Institut for Rechtsmedizin, Johannes macrophage~ of RA patients (A. Gutenberg-Universit~t Mainz, 6500 Mainz, Falus, Budapest). FRG.
Two of the highlights of the T-cellrelated sessions in Berlin were, not surprisingly, the thymic education of ~ T-cell receptor-positive (TCR)T lymphocytes and the role(s) of TCR ~18 /ymphocytes. Judging from the tremendous progress, it seems unbelievable that the first genes for these receptors were cloned barely six years ago2,3 and that it was only during the 1986 congress that M. Brenner (Harvard Univ.) announced that, after all, the T-cell ~1 chain4 does associate with
Thymicon|ogenyand seledion of and T cells
~nofh~r rhMn ~, to ~r~rnn a rlictinr'f r,,~.,
LI
I~
*
~l
11.4:e
eI
, I.#1
I I I
lt.,illJl,'I
11,. t
from DavidA. Ferridl, Pamela S. Ohaslli,Valerie Wallace, Marco Sdlilhm anli Tall W. tlall "We dance round in a ring and suppose but the secret sits in the middle and knows" 1
I I,..
ceptor expressed on a subset of r cellss. The new data presented at the congress (four symposia, 19 workshops and hundreds of posters), together with the •recently published work, allow the synthesis of a clearer picture of the ontogeny and selection of T cells (see
shrouded manner by which the thy- fic for a male-specific antigen, H-Y mus selects T cells for both tolerance plus H-2D b (Ref. 7), an allo-antigen, to self antigens (negative selection) Ld (Ref. 8), and lymphocytic cilerio~ ~ and acquisition of restriction to self meningitis virus (LCMV) plus ~" i ~~'~ major histocompatibility complex (R. Zinkernagel, Univ. Hosp., Zurich); (MHC) molecules (positive selection), and t'~¢o express (x~ TCRs from CD4 + Fig. 1). has recently yielded significantly to clones specific for cytochrome c and the efforts of both molecular and restricted by I-Ek (M. Davis, Stanford Thymic selectior,of T cells Univ.; S. Hedrick, Univ. of CaliforJ. Owen (Birmingham Univ., UK) cellular immunologists. nia). In the appropriate MHC ensummarized a scheme for T-cell ~,i o.qment these transgenic mice dedevelopment in the thymus. The Positive selection veloped a mature T-cell repertoire The roots of analysis of positive CDa-CDS- double-negative (DN), mainly composed of transgenic reTCR- T-cell precursors develop selection (which skews the T-cell repceptors and greatly skewed towards through a complicated series of in- ertoire to recognize foreign antigen termediates, including a transient in the context of 'self' MHC mol- the expression of CD8 or CD4 (dedouble-positive (DP) CD4+CD8 + ecules) can be traced to studies pending on whether the transgene stage, to emerge as m~ture of ra,diation-induced bone marrow receptor was MHC class-I or class-II single-positive (SP), CD4+CD8 - or chimeras and in-vivo antibody block- restricted, respectively). To test for positive selection in a CD4-CD8 + T-cell subsets that pos- ing experiments (see Ref. 6 for rehighly controlled manner, the H-Y sess .~ selected TCR repertoire. The view). Recently T-cell receptor transgenic plus H-2D b specific transgene recepcloning and sequencing of TCR genes has allowed the application of mice have been used to investigate tor (H. von Boehmer, Basel Inst.) was highly defined research tools (most positive selection of the T-cell reper- bred into the TCR-deficient severe notably transgenic mice and mono- toire. Five transgenic models have combined immunodeficienoj (SOD) clonal antibodies) to T-cel! bio!ooy. been g~,lerated: three express eLI3 mouse. In a non-relevant MHC enOwing to t;,ese recer,t advances, the ICRs trom CD8 + T-cell clones speci- vironment, thymocytes expressing © 1989,Elsevier Science PublishersLtd, UK. 0167-4919/89/502.00
403
both high and low affinity IL-7 receptors. The Seattle group further reported the results of in-vivotrials using IL-7. Observed effects included an increase in the number of cells in the spleen and mesenteric lymph nodes and an increase in the number of marrow pre-B cells. It is unknown how many additional stromal cell-derived mediators that affect B-cell development remain to be identified. The details of the cellular interactions between stromal and B-lineage ceils are equally unknown.
Of interest in this regard was the description by P. Kincade (Oklahoma City) of a monoctonal antibody that inhibited B-cell production upon addition to long-term bone marrow cultures. It will also be important in the future to investigate the role of the extracellular matrix in B-cell development, although virtually nothing was reported on this topic.
that considerably more progress in the above areas will be reported at the time of the next congress in Budapest. It is expected that they will still be major topics on the agenda at that time. Progress in adapting the various systems which have been so successful with murine cells to human bone marrow will, it is hoped, also be reported.
Concludingremarks
K. Dorshkindis in the D/visionof Biomedical Science, University of Cafifomia, Riverside, CA 92521, USA.
In July, a comparatively small group of geneticists from 23 countries met, first in Mainz* and again in Berlin at the Immunology Congress, to have a closer look at the major histocompatibility complex (MHC) class III genes and other complement polvmorphisms. Class III genes indude not only complement but also other loci, like 21-hydroxylase (21-01-1)A and B, tumour necrosis factor (TNF) ~ and [3, and duplicated genes of heat shock protein 70 (HSP70), with no obvious functional relationship. On the other hand, complement loci are also found on chromosome 1 (H, C4BP, CR1, C8A and B), chromosome 9 (CSG: J. C~nd#t"7 Col,,rnhia), and ,-~,n,~,~,~,, l o
Complexityof MHCclassill genesaM---complementpolymorphism
It is appropriate to end this meeting summary on the optimistic note
i
!
iI i
from Christian Rittner and Peter M. Schneider gene products. A full account of these findings will be given at a later date (Complement Inflamm., in preparation). Suffice it to say that human C4 polymorphism has now reached a remarkable degree of resolution at the phenotypic as well as the molecular level. Th~ nn~ninn
¢,',¢¢inn r~f t h , , ~^,orlt-
shop on C4 polymorphism, spanned the evolutionary bow from mice to man, encompassing the chimpanzee. These three species possess duplicated C4 loci derived from recombinational events predating the separation of human and mouse ancestors. The two C4 loci of chimpanzees and humans share similarities in standard arrangement, with long (C4A)/(C4B) and short genes. To provide a solid basis for dis- Klein dates the duplication event at cussions, a round of reference ty- at least seven million years ago. Inpings on polymorphic complement terestingly, duplicated C4 genes in components preceded the confer- mice are flanked by retroviral inence. This is summarized in Table 1. sertiuns of unknown origin. The sigThe major emphasis was on human nificance of such duplication events, C4 polymorphisms, analysis of which named "converted evolution" by involved a large number of different Meo, is still a mystery. The search for 'new' class III genes, techniques, many reference laborato fill the 400 kb gap between TNF-e tores and the testing of a substantial and C2, by chromosome walking has number of samples. The aim was to give a better definition of variants, resulted in the isolation of Hpall tiny duplicated and converted genes and fragments (HTF) islands containing a high density of non-methylated CpG dinucleotides. When hybridizing * The Sixth ComplementGeneticsWorkshopwas genomic probes to northern blots of RNA, R.D. Campbell and colleagues held in Mainzon 27-29July1989.
(C3). Curiously enough, MHC-linked complement genes still attract most attention, probably because evolution, deficiency and disease seem to have a causal relationship. These observations have been the subject of speculations from both the neutralist 0. Klein, T~Jbingen) and selection,st (T. Meo, Paris)point of view.
1989.ElsevierSciencePubhshersLid, UK.0167~1919/89/$02.00
(Oxford) identified 12 novel single copy genes. Localization of so many new genes within the MHC may nave a bearing on our understanding of the functions of the complex. Using a different approach but addressing the same question, Y. Morel (San Francisco) detected another a, ,nli,-~t~,4 ,~o,,., ,.,,'~'~,th~,~ DNA c,,=,,,~ opposite to that encoding the human E4 and 21-OH genes, again of unknown function. Obtnumbering all other issues, most abstracts submitted (published in Complement and Inflammation (1989) 6, 283-304) dealt with possible associations between complement polymorphgm~ a--l l u~ G" ~2&5~. Fifteen different posters described significant variations in the distribution of allele frequencies of MHClinked C2, BF, C4A and B genes in diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), chronic polyarthritis, multiple sclerosis, visceral leishmanias,s, chronic glumerulonephritis, leprosy, Brazilian paracoccidioidomycosis, common variable immunodeficiency, IgA deficiency, insulin-dependent diabetes mellitus (IDDM), Chagas disease and AIDS. The possibility that superhaplotypes (originally described by the late R. Ceppellini), rather than individual alleles, are associated with disease was also reported: the Caucasian
401
Immunology Today, VoL 10, No. 12, 1989
- erlin news
¢ie vs
Table 1. Typingof polymorphiccorrz',ementcomponents
Complement component
Technique a
C2
IEF/WB RFLP
FactorB
No. of participating laboratories
No. of submitted samples
Total no. of known alleles
5
30
< 10
HVAGE/IFX HVAGE/HOV IEF RFLP
11
100
> 20
C4
HVAGE/IFX HVAGE/HOV HVAGE/WB MAB SDS-PAGE(~/13chains) HAl (RodgersiChido) RFLP
14
140
> 40
C3
HVAGE/IFX
4
26
> 30
C6
IEF/WB
4
50
> 20
C7
IEF/WB IEF/HOV MAB
7
31
< 5
C8
IEF/HOV IEF/WB SDS-PAGE RFLP
3
20
< 10
Factor l
IEF
3
11
< 5
aAbbreviationsof typing techniques:HAl: haemagglutination-inhibitionwith humanalloantisera;HOV: complementcomponent-dependenthaemolyticoverlay;HVAGE:highvoltageagarosegel e!ectrophoresis; IEF:isoelectricfocusing;IFX:immunofixatio~,;MAB:monoclonalantibodies;RFLP:DNArestrictionfragment lengthpolymorphism;SDS-PAGE:SDS-polyacn/lamidegel electrophoresis;WB: westernblot.
402
superhaplotype B8, BFS, C4AQO, C4B1, DR3 is associated with diminished antibody response to the hepatitis B vaccine. However, the question that remained unaqswered was whether the complement genes themselves were responsible for the increased susceDtibi!ity to disease or whether the mapped genes on a limited number of ancestral hapiotypes (R.L. Dawkins, Perth) are only 'bystander' genes close to the unknown loci responsible for the diseases. Some of the formal presentations at both the Mainz Conference and the Berlin Congress provided new insights into the structure and function of C4A, C4B, FI and C8 polymorphisms. The structural basis for inherited deficiency of complement components was analysed at the gene and protein levels for factor I ~,,d the C8 ~ / chain. Using a
factor ! (FI) cDNA probe as well as synthetic oligonucleotides combined with conventional and large DNA fragment gel electrophoresis, it was possible to identify five characteristic haplotypes. This led to detection of the FI*QO atJeie in family studies of eight FI deficiency patients. Although no major structural aberrations of Lhe gene were found, no FI plasma levels were detectable. These results suggest a heterogeneity in the genetic basis of FI deficiency based possibly on independent mutational events (K. KOIble, Oxford). Similar evidence was presented for the deficiency of the C8 ~ / chain, although it was based on studies at the protein level. A monoclonai antibody and an affinity-purified rabbit antiserum directed against the subunit wer~ used to detect defective C8 ~ , protein in six patients carrying the deficiency, in these
patients, the subunit is functionally inactive as shown by hemolytic overlay. In addition, C8 ~/appears to be polymorphic, indicating a heterogeneous basis for the deficiency. Evidence was also obtained for decreased serum levels of the 13subunit in patients with ~/deficiency, poiniing to a possible major role for the ~ / subunit in the secretion of the complete C8 molecule (L. Roncelli, F. Tedesco, Trieste). For the C8 13 gene, the first two DNA restriction fragment length polymorphisms (RFLPs) were reported. They are strongly associated with each other, but do not correlate with the protein variants defined by isoelectric focusing. These RFLPs may prove useful in the analysis of the genetic basis of C8 13 deficiency as well as in genetic linkage studies (D. Herrmann, Mainz; S. Rogde, Oslo). The two isotypes of the fourth component, C4A and C4B, exhibit striking functional and biochemical differences in spite of 99% identity. Using site-dire~ed mutagenesis and expression of a variety of mutated C4 gene constructs in fibroblasts, the molecular basis for these differences was localized to a sequence of four amino acids between residues 1101 and 1106 in the C4d region. This sequence exclusively comprises the serological determinant Chido 4 on the C4B molecule. It was possible to show that the substitution of a single amino acid at position 1106 (Asp to His) changed the hemolytic activity from C4B to C4A. C4B usually has a three- to four-fold higher hemolytic activity than C4A. In the same way, the mobility of the C4 ~ chains in SDS-PAGE could be changed from C4B to C4A~ an apparent molecular weight change of 2 kDa, by exchanging L for P at position 110!. ,,,is did not affect the hemolytic activity of the protein. The fum~ional differences seem to be singularly affected by the presence of His or Asp side chains in conformational position to the internal thiolester bond of C4 (M. Carroll, Boston; D. Isenman, Toronto). C4A and C4B deficiency is caused in about 50% of all affected haplotypes by gene deletions encompassing a single C4 and an adjacent 21-0H gene. On the other C4 null haplotypes, it was suggested that the respective genes were either not expressed, or that these genes express a homoduplicated product
Immunology Today, Vol. 10, No. 12, 1989 i
~ 'r
.r
identical to the C4 isotype already present. In a study of 26 individuals with homozygous C4A or C4B deficiency, it was possible to show that all C4A 'null' genes carried all sequences typical for a C4A gene, although no gene product was detectable. In contrast, all C4B 'null' genes had only sequences characteristic for a C4A gene. Therefore, these genes may express the C4A isotype. The molecular mechanism leading to this altered sequence could be either gene conversion or multiple recombination. The results were obtained by analysing the isotype-speciflc C4d region with oligonucleotides, restric-
tion enzymes and genomic sequencIn summary, complement genetics ing after DNA amplification using has evolved to a considerable degree the polymerase chain reaction (L. and has left its egg shells behind. Braun, P.M. Schneider, C. Rittner, Major contributions to clarify the Mainz). molecular basis of genetic variations Evidence for separate control of have been made. Even some sites C4A and C4B gene expression has resDorsible for functional diversity been obtained by studying synowal have been elucidated. The most immacrophages of RA and osteo- portant question: why a given variarthritis patients. Using northorn blot ation, for example deficiency, may or analysis and C4 isotype-specific may not lead to disease still remains o!igonucleotides directed against the to be answered. sequence coding for the Chido 4 determinant, C4B was found to be C Rittner and P.M. Schneider are at predominantly expressed only in the Institut for Rechtsmedizin, Johannes macrophage~ of RA patients (A. Gutenberg-Universit~t Mainz, 6500 Mainz, Falus, Budapest). FRG.
Two of the highlights of the T-cellrelated sessions in Berlin were, not surprisingly, the thymic education of ~ T-cell receptor-positive (TCR)T lymphocytes and the role(s) of TCR ~18 /ymphocytes. Judging from the tremendous progress, it seems unbelievable that the first genes for these receptors were cloned barely six years ago2,3 and that it was only during the 1986 congress that M. Brenner (Harvard Univ.) announced that, after all, the T-cell ~1 chain4 does associate with
Thymicon|ogenyand seledion of and T cells
~nofh~r rhMn ~, to ~r~rnn a rlictinr'f r,,~.,
LI
I~
*
~l
11.4:e
eI
, I.#1
I I I
lt.,illJl,'I
11,. t
from DavidA. Ferridl, Pamela S. Ohaslli,Valerie Wallace, Marco Sdlilhm anli Tall W. tlall "We dance round in a ring and suppose but the secret sits in the middle and knows" 1
I I,..
ceptor expressed on a subset of r cellss. The new data presented at the congress (four symposia, 19 workshops and hundreds of posters), together with the •recently published work, allow the synthesis of a clearer picture of the ontogeny and selection of T cells (see
shrouded manner by which the thy- fic for a male-specific antigen, H-Y mus selects T cells for both tolerance plus H-2D b (Ref. 7), an allo-antigen, to self antigens (negative selection) Ld (Ref. 8), and lymphocytic cilerio~ ~ and acquisition of restriction to self meningitis virus (LCMV) plus ~" i ~~'~ major histocompatibility complex (R. Zinkernagel, Univ. Hosp., Zurich); (MHC) molecules (positive selection), and t'~¢o express (x~ TCRs from CD4 + Fig. 1). has recently yielded significantly to clones specific for cytochrome c and the efforts of both molecular and restricted by I-Ek (M. Davis, Stanford Thymic selectior,of T cells Univ.; S. Hedrick, Univ. of CaliforJ. Owen (Birmingham Univ., UK) cellular immunologists. nia). In the appropriate MHC ensummarized a scheme for T-cell ~,i o.qment these transgenic mice dedevelopment in the thymus. The Positive selection veloped a mature T-cell repertoire The roots of analysis of positive CDa-CDS- double-negative (DN), mainly composed of transgenic reTCR- T-cell precursors develop selection (which skews the T-cell repceptors and greatly skewed towards through a complicated series of in- ertoire to recognize foreign antigen termediates, including a transient in the context of 'self' MHC mol- the expression of CD8 or CD4 (dedouble-positive (DP) CD4+CD8 + ecules) can be traced to studies pending on whether the transgene stage, to emerge as m~ture of ra,diation-induced bone marrow receptor was MHC class-I or class-II single-positive (SP), CD4+CD8 - or chimeras and in-vivo antibody block- restricted, respectively). To test for positive selection in a CD4-CD8 + T-cell subsets that pos- ing experiments (see Ref. 6 for rehighly controlled manner, the H-Y sess .~ selected TCR repertoire. The view). Recently T-cell receptor transgenic plus H-2D b specific transgene recepcloning and sequencing of TCR genes has allowed the application of mice have been used to investigate tor (H. von Boehmer, Basel Inst.) was highly defined research tools (most positive selection of the T-cell reper- bred into the TCR-deficient severe notably transgenic mice and mono- toire. Five transgenic models have combined immunodeficienoj (SOD) clonal antibodies) to T-cel! bio!ooy. been g~,lerated: three express eLI3 mouse. In a non-relevant MHC enOwing to t;,ese recer,t advances, the ICRs trom CD8 + T-cell clones speci- vironment, thymocytes expressing © 1989,Elsevier Science PublishersLtd, UK. 0167-4919/89/502.00
403