ImmunologyToday, vol. 4, No. 3, 1983 restriction specificities (Table III) and capacity to respond correlates with the relatedness found earlier with respect to CTL allospecificity [C. Melief (Amsterdam), L. Sherman (La Jolla)]. This is compatible with the notion that the repertoires of ailospeeific and selfrestricted CTL are largely overlapping and detect similar sites on H-2 molecules. E. Rfisch and G. H/irnmerling (Heidelberg) have used the K b mutants to study the topographic arrangement of antigenic determinants on the K b molecule detected by monoclonal anti-H-2 antibodies [Proc. Natl Acad. Sci. USA (1982) 79, 4737]. At least two spatially distinct clusters of serologically detected K b
61 determinants were identified by antibody competition studies. The bm3 mutation (AA positions 77 and 89) affected determinants of one cluster, the b m l mutation (AA positions 152,155 and 156) those of the other cluster. CTL do not detect such distinct clusters on the K b molecule. For example, the diverse AA substitutions in b m l , bm5 and bm8 mutant K bmolecules resulted in their inability to serve as restriction elements for a single K brestricted influenza-specific CTL clone (Townsend eta/). Moreover, bin3 antiK b and bml anti-Kb C T L could not be selectively blocked at the target level by monoclonal antibodies to the two serologically detected K b clusters (Rfisch and
Monoclonal antibodies and human leucocyte antigens from Peter Beverley The conventional heteroantisera, ailoantisera and autoantisera which first defined human leucocyte differentiation antigens were often unsatisfactory and in the past few years the promise of monoclonal antibodies has been borne out in a flood of reports of new antibodies and the antigens they define. But each antibody usually acquires an arbitrary name from its originator and often a second name from a commercial company, leading to confusion with similar or identical antibodies appearing under different names and difficulties in deciding whether subsets of leucocytes defined by ostensibly similar antibodies are truly identical. Sixty research groups throughout the world have therefore collaborated in a workshop* involving exchanges of mouse monoclonal antisera, along histocompatibility workshop lines. O f the 300 or so antibodies submitted around 160 were divided grouped by specificity: T cell; B cell + common acute lymphocytic leukaemia antigen (CALLA); and monocyte/granulocyte. Participating groups received coded samples and in some cases frozen cells. For the first level of testing, participants assayed a small group of antibodies on a standard panel of targets and in the second level on a larger series of different target cells appropriate to the type of antibodies under test. The assay throughout was indirect immunofluorescenceby either microscopy or flow cytometry. The analyses and submission of data were done blind and all data were analysed in Paris. This included results on 1 367 separate target cells. According 'The firstinternationalworkshopon humanleucocyte differentiation antigens sponsored by INSERM, WHO and IUIS, was held in Paris on 7-11 November 1982. The proceedingswill be publishedby Springer-Verlag.
to my calculations, around 60 000 separate analyses were performed by the sixty participating groups. Since each series of antibodies contained duplicates it was possible to analyse the results for experimental variability of each group and some data were excluded on this basis. The remainder were examined by cluster analysis. For each determination the percentage of ceils stained was determined, a mean score for a given antibody on all targets was computed and antibodies were then assigned to clusters by examining differences between these scores (A. Bernard, L. Boumsell and C. Hill, Paris). Perhaps surprisingly the number of clusters defined in each workshop was small, 7 for T cells, 7 for B + CALLA, and 8 for monocytes and granulocytes but some antibodies could not readily be assigned to any cluster. What, however, is the significance of the clusters so far defined; do they represent groups of related antigens or do all the sera in a cluster see the same antigen if not the same epitope? At least for the T-cell
H/immerling), although this might be due to steric hindrance or the induction of conformational K b changes by the antibody. As succinctly summarized by R. Melvold at the meeting; the H-2 mutants have allowed: (1) assignment of traits/ functions to precise gene products; (2) insight in the correlation of structurefunction; (3) detailed analysis of T-cell fine specificityand T-cell repertoire; (4) a novel approach to the origin of H-2 polymorphism (gene conversion).
C. ,f M. Melief is in the Department of Tumor Immunology, CentralLaboratoryof the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands. workshop the answers are fairly clear because in addition to the serological studies required by the workshop several groups had performed a variety of other assays of selected antibodies. T-cell antigens Each cluster appears to identify either families of closely related molecules (probably the case for the corticothymic antigens; R. W. Knowles et al., New York) or more commonly the same molecule or group of associated polypeptide chains (probably true for most other T-cell antigens so far defined). The evidence for these statements derives principally from immunoprecipitation and SDS polyacrylamide gel analysis showing similar molecular weight antigens brought down by antibodies of a cluster (Table I) (R. W. Knowles et al., New York;J, Ledbetteretal., Seattle). In most cases more definitive evidence of the identity of the molecules defined by different antibodies, for example by sequential immunoprecipitation, has not been obtained. Within each cluster there is considerable heterogeneity when the antibodies are examined by other techniques; for example, not all antibodies within a cluster block the binding of all other
TABLE I. T-cell dusters Cluster
Number of antibodies in cluster
Corticothymic PanT + BCLL Pan T, E receptor Pan T, mitogenic Pan T + T precursors Helper inducer Suppressor cytotoxic No clustering
3 18 3 4 4 3 14 11 antigens present on T SD on normal cells
Approximate molecular weight of antigen 42 000 + 12 000 67 000 50 000 19 000-29 000 41 000 55 000 32 000, 47 000 blasts or malignancies only or with high
CLL: chronic lymphocyte leukaemia; E: sheep erythrocyte; SD: standard deviation. The biochemistry of several of these antigens is still the subject of much dispute. In many cases other bands are seen on gels and molecular weights in non-reduced gels imply the existence of polymers.
62 members of that cluster. Similarly when antibodies within a cluster are examined for their reactivity with cells of a series of non-human primates, very few show identical patterns of binding (P. J. Martin et al., Seattle). In addition functional tests, for example the ability to block T-cell cytotoxicity, also demonstrate considerable heterogeneity within clusters (A. McMichael et aL, Oxford). Thus the T-cell clusters probably represent families of antibodies which most often identify different epitopes on the same molecule (or complex of polypeptide chains in the case of T3 and T8). In the case ofT cells it is possible both to define stages of differentiation within the T-cell lineage (for example, cortical thymocytes or peripheral T cells), and to separate functionally distinct subsets of peripheral lymphocytes (T4 and T8) as well as to begin to define molecules which clearly have functionally significant roles (T3, T4, T8). While many anti-B and monocyte/granulocyte sera are now available, much less is known of the differentiation of cells of these lineages. It is still unclear, for example, whether there are different B-cell subsets rather than stages of differentiation and very little is known with regard to the function of any B or myeloid antigens other than surface Ig and HI_,A-DR. The availability of antibodies which clearly subdivide these types of cells, however, is likely to promote a search for better functional assays for the subsets which can be isolated using monoclonal antibodies. The workshop also illustrated the varied uses of monoclonal antibodies in everything from studies of the molecules of cell surfaces to treatment of patients. For me at least, a highlight was the unveil-
Immunology Today, vol. 4, No. 3, 1983 ing of a new model for the elusive T-cell receptor (E. Reinhertz etal., Boston) (see Immunol. Today(1983)4, 5-8) whichlinks a newly defined clonally expressed molecule with T-cell surface antigens (T3, 4 and 8) that have been shown by many groups to be important in T-cell function. While objections to the model can be raised the studies elegantly illustrated the power of monoclonal antibodies combined with monoclonal IL2-dependent T cells in the analysis of lymphocyte function.
B-cell ~antigens Several studies in the B-cell workshop illustrated another point of more immediate practical importance, namely that many antigens which are restricted in distribution within the haemopoietic system may be widely distributed elsewhere (G. R. Pilkington et al., Melbourne. P. M. Lansdorp et al., Amsterdam; J. Brochier TABLE II. Terminology for humanleucocyte antigens
]7,X~AI3IlDlc CD # (T gp 19-29) uCHT1
Interpretation CD
#
T gP 19-29 UCHT1
Cluster, differentiation Workshop-assigned number for the cluster* Cell type on which antigen defined Nature of antigen, protein, glycoprotein, etc. Molecular weight in kilodaltons Laboratory name for a particular antiserum (optional)
* Workshop CD numbers will be assigned by a committee designated at the workshop.
et al., Lyon). Curiously many anti B + C A L L A antibodies stain frozen sections of kidney, different antibodies each giving different and characteristic patterns of tubular and glomerular staining. Whether these antibodies identify the same molecules in the kidney and haemopoietic tissues is not known but these additional reactivities should give pause to enthusiastic therapists with antibody in vivo and complicate the use of such antibodies for immunohistological studies. The wide range of topics covered in the free communication sessions suggested that indirect immunofluorescence even on a large panel of target cells is not an adequate method for classifying differentiation antigens. So what did the first workshop achieve? To reduce the chaos of the literature to 22 clusters of antibodies is a considerable achievement even though an over-simplification since several antibodies did not cluster. Furthermore, a terminology was proposed and agreed without dissent, though the assignment of CD numbers has yet to be made (Table II). The first workshop provided a framework for future studies and a much clearer picture of what is known and not known about h u m a n leucocyte differentiation. This is perhaps much more than those of us who ploughed through the drudgery of the serology expected. For this successful outcome much of the credit must go to the workshop organizers and particularly the tireless secretaries, Alain Bernard and Laurence Boumsell. Peter Beverl(y is in the ICRF Human Tumour Immunology Group, Foz'ulty of Clinical Sciences, School of Medicine, University College of London, London WC1E 6JJ, UK.
61 determinants were identified by antibody competition studies. The bm3 mutation (AA positions 77 and 89) affected determinants of one cluster, the b m l mutation (AA positions 152,155 and 156) those of the other cluster. CTL do not detect such distinct clusters on the K b molecule. For example, the diverse AA substitutions in b m l , bm5 and bm8 mutant K bmolecules resulted in their inability to serve as restriction elements for a single K brestricted influenza-specific CTL clone (Townsend eta/). Moreover, bin3 antiK b and bml anti-Kb C T L could not be selectively blocked at the target level by monoclonal antibodies to the two serologically detected K b clusters (Rfisch and
Monoclonal antibodies and human leucocyte antigens from Peter Beverley The conventional heteroantisera, ailoantisera and autoantisera which first defined human leucocyte differentiation antigens were often unsatisfactory and in the past few years the promise of monoclonal antibodies has been borne out in a flood of reports of new antibodies and the antigens they define. But each antibody usually acquires an arbitrary name from its originator and often a second name from a commercial company, leading to confusion with similar or identical antibodies appearing under different names and difficulties in deciding whether subsets of leucocytes defined by ostensibly similar antibodies are truly identical. Sixty research groups throughout the world have therefore collaborated in a workshop* involving exchanges of mouse monoclonal antisera, along histocompatibility workshop lines. O f the 300 or so antibodies submitted around 160 were divided grouped by specificity: T cell; B cell + common acute lymphocytic leukaemia antigen (CALLA); and monocyte/granulocyte. Participating groups received coded samples and in some cases frozen cells. For the first level of testing, participants assayed a small group of antibodies on a standard panel of targets and in the second level on a larger series of different target cells appropriate to the type of antibodies under test. The assay throughout was indirect immunofluorescenceby either microscopy or flow cytometry. The analyses and submission of data were done blind and all data were analysed in Paris. This included results on 1 367 separate target cells. According 'The firstinternationalworkshopon humanleucocyte differentiation antigens sponsored by INSERM, WHO and IUIS, was held in Paris on 7-11 November 1982. The proceedingswill be publishedby Springer-Verlag.
to my calculations, around 60 000 separate analyses were performed by the sixty participating groups. Since each series of antibodies contained duplicates it was possible to analyse the results for experimental variability of each group and some data were excluded on this basis. The remainder were examined by cluster analysis. For each determination the percentage of ceils stained was determined, a mean score for a given antibody on all targets was computed and antibodies were then assigned to clusters by examining differences between these scores (A. Bernard, L. Boumsell and C. Hill, Paris). Perhaps surprisingly the number of clusters defined in each workshop was small, 7 for T cells, 7 for B + CALLA, and 8 for monocytes and granulocytes but some antibodies could not readily be assigned to any cluster. What, however, is the significance of the clusters so far defined; do they represent groups of related antigens or do all the sera in a cluster see the same antigen if not the same epitope? At least for the T-cell
H/immerling), although this might be due to steric hindrance or the induction of conformational K b changes by the antibody. As succinctly summarized by R. Melvold at the meeting; the H-2 mutants have allowed: (1) assignment of traits/ functions to precise gene products; (2) insight in the correlation of structurefunction; (3) detailed analysis of T-cell fine specificityand T-cell repertoire; (4) a novel approach to the origin of H-2 polymorphism (gene conversion).
C. ,f M. Melief is in the Department of Tumor Immunology, CentralLaboratoryof the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands. workshop the answers are fairly clear because in addition to the serological studies required by the workshop several groups had performed a variety of other assays of selected antibodies. T-cell antigens Each cluster appears to identify either families of closely related molecules (probably the case for the corticothymic antigens; R. W. Knowles et al., New York) or more commonly the same molecule or group of associated polypeptide chains (probably true for most other T-cell antigens so far defined). The evidence for these statements derives principally from immunoprecipitation and SDS polyacrylamide gel analysis showing similar molecular weight antigens brought down by antibodies of a cluster (Table I) (R. W. Knowles et al., New York;J, Ledbetteretal., Seattle). In most cases more definitive evidence of the identity of the molecules defined by different antibodies, for example by sequential immunoprecipitation, has not been obtained. Within each cluster there is considerable heterogeneity when the antibodies are examined by other techniques; for example, not all antibodies within a cluster block the binding of all other
TABLE I. T-cell dusters Cluster
Number of antibodies in cluster
Corticothymic PanT + BCLL Pan T, E receptor Pan T, mitogenic Pan T + T precursors Helper inducer Suppressor cytotoxic No clustering
3 18 3 4 4 3 14 11 antigens present on T SD on normal cells
Approximate molecular weight of antigen 42 000 + 12 000 67 000 50 000 19 000-29 000 41 000 55 000 32 000, 47 000 blasts or malignancies only or with high
CLL: chronic lymphocyte leukaemia; E: sheep erythrocyte; SD: standard deviation. The biochemistry of several of these antigens is still the subject of much dispute. In many cases other bands are seen on gels and molecular weights in non-reduced gels imply the existence of polymers.
62 members of that cluster. Similarly when antibodies within a cluster are examined for their reactivity with cells of a series of non-human primates, very few show identical patterns of binding (P. J. Martin et al., Seattle). In addition functional tests, for example the ability to block T-cell cytotoxicity, also demonstrate considerable heterogeneity within clusters (A. McMichael et aL, Oxford). Thus the T-cell clusters probably represent families of antibodies which most often identify different epitopes on the same molecule (or complex of polypeptide chains in the case of T3 and T8). In the case ofT cells it is possible both to define stages of differentiation within the T-cell lineage (for example, cortical thymocytes or peripheral T cells), and to separate functionally distinct subsets of peripheral lymphocytes (T4 and T8) as well as to begin to define molecules which clearly have functionally significant roles (T3, T4, T8). While many anti-B and monocyte/granulocyte sera are now available, much less is known of the differentiation of cells of these lineages. It is still unclear, for example, whether there are different B-cell subsets rather than stages of differentiation and very little is known with regard to the function of any B or myeloid antigens other than surface Ig and HI_,A-DR. The availability of antibodies which clearly subdivide these types of cells, however, is likely to promote a search for better functional assays for the subsets which can be isolated using monoclonal antibodies. The workshop also illustrated the varied uses of monoclonal antibodies in everything from studies of the molecules of cell surfaces to treatment of patients. For me at least, a highlight was the unveil-
Immunology Today, vol. 4, No. 3, 1983 ing of a new model for the elusive T-cell receptor (E. Reinhertz etal., Boston) (see Immunol. Today(1983)4, 5-8) whichlinks a newly defined clonally expressed molecule with T-cell surface antigens (T3, 4 and 8) that have been shown by many groups to be important in T-cell function. While objections to the model can be raised the studies elegantly illustrated the power of monoclonal antibodies combined with monoclonal IL2-dependent T cells in the analysis of lymphocyte function.
B-cell ~antigens Several studies in the B-cell workshop illustrated another point of more immediate practical importance, namely that many antigens which are restricted in distribution within the haemopoietic system may be widely distributed elsewhere (G. R. Pilkington et al., Melbourne. P. M. Lansdorp et al., Amsterdam; J. Brochier TABLE II. Terminology for humanleucocyte antigens
]7,X~AI3IlDlc CD # (T gp 19-29) uCHT1
Interpretation CD
#
T gP 19-29 UCHT1
Cluster, differentiation Workshop-assigned number for the cluster* Cell type on which antigen defined Nature of antigen, protein, glycoprotein, etc. Molecular weight in kilodaltons Laboratory name for a particular antiserum (optional)
* Workshop CD numbers will be assigned by a committee designated at the workshop.
et al., Lyon). Curiously many anti B + C A L L A antibodies stain frozen sections of kidney, different antibodies each giving different and characteristic patterns of tubular and glomerular staining. Whether these antibodies identify the same molecules in the kidney and haemopoietic tissues is not known but these additional reactivities should give pause to enthusiastic therapists with antibody in vivo and complicate the use of such antibodies for immunohistological studies. The wide range of topics covered in the free communication sessions suggested that indirect immunofluorescence even on a large panel of target cells is not an adequate method for classifying differentiation antigens. So what did the first workshop achieve? To reduce the chaos of the literature to 22 clusters of antibodies is a considerable achievement even though an over-simplification since several antibodies did not cluster. Furthermore, a terminology was proposed and agreed without dissent, though the assignment of CD numbers has yet to be made (Table II). The first workshop provided a framework for future studies and a much clearer picture of what is known and not known about h u m a n leucocyte differentiation. This is perhaps much more than those of us who ploughed through the drudgery of the serology expected. For this successful outcome much of the credit must go to the workshop organizers and particularly the tireless secretaries, Alain Bernard and Laurence Boumsell. Peter Beverl(y is in the ICRF Human Tumour Immunology Group, Foz'ulty of Clinical Sciences, School of Medicine, University College of London, London WC1E 6JJ, UK.
