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Cytological analysis of lymphocytes
Immunology Today, VoL 9, No. 1, 1988
!i!!iiiiii! ¸¸ Immunologists have been quick to exploit the opportunities offered by cytometric techniques. The 12th Annual Meeting of the Society for Analytical Cytology* heard, for instance, how the investigation of lymphocytes has benefited from the application of these techniques. Cytometric techniques, especially fluorescence-activated cell analysis and sorting, have contributed greatly to several areas of immunology, as was reflected in the diversity of immunological topics presented. This report concentrates on how analytical cytology has been applied to B and T lymphocytes and their products.
i::
:
Cytologicalanalysisof lymphocytes from H.SoMicklem Mature Ly-1+ B cells also carry Mac-l, the membrane expression of which is quantitatively related to that of IgM (Herzenberg).
B-ceUmemory and hypermutation Other interesting developments are taking place in the field of B-cell physiology, and promise to solve a number of old problems. Virgin B cells are grossly overproduced in the bone marrow of mice and rats, and under normal circumstances less than 10% survive for more than a Ly-1+ B lympho~tes R. Palacios (Basel) described the few hours after leaving the marrow. production and characteristics of Ly- However, in animals depleted of B 1+ 'pro-B' lymphocyte clones. These cells by anti-mu antibody, new B clones express the B lineage-specific cells can be incorporated in large antigens B-220 and Lyb-8, but lack numbers into the peripheral pool, T-cell and granulocyte lineage mar- where they enjoy an average lifespan kers; some also express Mac-1. They of 4-6 weeks (1. MacLennan, Birmlack surface or cytoplasmic Ig and ingham UK). This probably happens their heavy, K and X genes are in to a small extent normally and their germline configurations. Dif- appears to be independent of antiferentiation to surface Ig +, la + lym- gen; thus, presumably there must be phocytes could be induced in the some counting mechanism that regpresence of lipopolysaccharide, aza- ulates the rate of acceptance of new cytidine and accessory cells, and Ly- B cells into the pool. After leaving 1+ B-cell lines were established from the marrow, new B cells migrate as these differentiated cultures in the far as the T-cell area of the spleen, but very few normally proceed from presence of interleukins 4 and 5. Injection of pro-B clones into SCID there to the follicles and recirculate (severe combined immunodeficient) the others presumably dying in the mice also resulted in the production spleen, although there is little direct of donor-type Ly-I+B cells. These evidence for this. At least two pathdata support the view that the Ly-1 ÷ ways exist for the T-cell-dependent B-cell subset is separated from other activation of B cells. One of these B-cell subsets early in differentiation. involves rapid proliferation (a Ly-1 ÷ B cells are the predominant claimed cell cycle time of 4-5 hours) B-cell type in young mice, while in and hypermutation within the gerthe adult they have a =ories of in- minal centres of lymphoid follicles. teresting properties, including auto- As has long been known, many of antibody formation, primary resi- the lymphocytes produced in the dence in the peritoneal cavity and a follicle die there and are engulfed by tendency to produce chronic neo- macrophages. It is presumed that plasms similar to chronic lymphocytic there is strong selection for mutants leukaemia (CLL)in man (L. Herzen- with higher affinity for antigen, since berg, Stanford; J. Ansell, Edinburgh). the average antibody affinity inSuch neoplasms seem to be found creases during T-cell-dependent imparticularly frequently in autoim- mune responses. The recruitment of mune (NZB x NZW)F1 mice; this may additional high-affinity virgin B cells differ from humans, where chronic appears, in contrast, to be of negliautoimmune diseases such as gible importance for affinity maturrheumatoid arthritis do not seem to ation (MacLennan). Single point be strongly associated with CLL. mutations can change affinity and specificity dramatically, or, on the 'Held on 10-14 August 1987 in CambridgeUK," contrary, they may be without disabstracts have been published in the joumal cernible effect; this latter observation suggests that selection for Cytometry. (~ 1988, ElsevierPublications,Cambridge 0167 4919/88/$02 00
higher affinity is not necessarily stringent, despite the large-scale destruction of cells in germinal centres. The typical hypermutation rate of about one change in each thousand base pairs each generation is abolished after transfer of the cells to an irradiated recipient, again emphasizing the probable importance of microenvironment, presumably the germinal centre (K. Rajewsky, Cologne). A second T-cell-dependent activation pathway occurs outside the follicles and involves the interaction of B cells with interdigitating cells; it probably does not involve any hypermutation or affinity maturation (MacLennan). Hypermutation in anti-oxazolone responses produces point mutations in heavy and some K chain genes, while other K and • genes are unaffected. It is heavily dependent on the presence of L3T4+(CD4 +) T cells in the animal (C. Berek, Cambridge). There is some dispute whether hypermutation continues to occur with repeated immunizations (C. Milstein, Cambridge), or is a once-off preprogrammed step (Rajewsky). It is interesting that anti-digoxin antibodies produced by mutant hybridomas selected in a cell sorter show not only point mutations, but also blocks of changed heavy chain sequence suggestive of gene conversion (E. Haber, Boston).
Mutated and hybrid Ig molecules How interactions between different parts of immunoglobulin molecules distant in terms of primary structure affect the conformation and specificity of the molecule were also explored (Haber; V. Oi, Mountain View). Haber exploited the variants of murine anti-digoxin hybridomas mentioned above to assess the relationship between antibody specificity, primary structure and three-dimensional structure. Digoxin is a valuable model antigen since it evokes antibodies of high affinity, fills the combining site of the antibody and possesses many structurally defined congeners. Spontaneous mutants were isolated on the basis of inhibition of antigen binding by digitoxin. The data revealed precise interaction between
ImmunologyToday,Vol. 9, No. ................
:
positions 10 2, 94 and 101 of the heavy chain in determining the fine specificity of the antibody. Oi used a series of engineered hybrid IgGl/IgG2a anti-dansyl antibodies to investigate the structural determinants of Fab segmental flexibility. Although in general such flexibility is correlated with the length of the upper hinge region, this study showed that the structure of part of the CH1 region has an important influence. The belief that complement fixation depends on segmental flexibility is contradicted by the observation of three rigid hybrids that show good complement fixation. The structure of CH3 is, however, capable of influencing complement fixation, presumably through a conformational change in the molecule affecting the Cl-binding site. Such findings are not only of intrinsic interest. They also form an important background for attempts to 'humanize' mouse hybridoma antibodies, for example by inserting mouse complementarity-determining regions (CDRs) into a human VH framework, in order to render useful therapeutic antibodies non-immunogenic in man. This can be done without necessarily damaging antibody function, as exemplified by the behaviour of a hybrid molecule consisting of the murine K and heavychain CDRs of the therapeutic monoclonal antibody Campath-1 on a human IgG1 background. This molecule binds antigen, mediates antibody-dependent cell-mediated cytotoxicity and fixes complement (M. Verhoeyen, Cambridge). It remains to be seen whether such hybrid antibodies will be significantly less immunogenic in humans than the original mouse antibody.
T-cell derived cytokines and B cells Much recent work has centred on IL-4 (alias B-cell stimulatory factor 1 (BSF-1) etc.) and IL-5, now that recombinant sources of these factobs have become available (E. Severinson, Stockholm; A. Radbruch, Cologne; J. Aubry, Dardilly; E. Vitetta, New York). IL-4 was repeatedly found to promote Ig class switching to G1 at the expense of G3 and G2 in mice. When IL-4 was added to cultures grown in the presence of bacterial lipopolysaccharide a substantial number of IgE-producing cells emerged (Severinson). IL-4 also induced the expression of CD23 antigen (a receptor for e chain Fc) on
....
I, 1988
.........................................................................................................................................................................................
human B cells and enhanced the expression of HLA-DR. The relationship between CD23 and IL-4 may be important, since CD23- cells showed no proliferative response to IL-4 in combination with anti-lgM antibody (Aubry). How IL-4 and other exogenous agents influence class switching is still unknown, but the cytokines seem to 'instruct' cells to switcl, to a particular class, rather than select cells precommitted to a given switch. Specific recombinases appear not to be involved. More probably, particular genes may be rendered available for recombination, and changes in their patterns of DNA methylation may be important (Radbruch). T-cell-derived cytokines alone cannot induce proliferation and differentiation in antigen (TNP)specific B cells: a preliminary period of contact with helper T cells is also required. Two helper T-cell clones specific for keyhole limpet haemocyanin showed very different characteristics. One made IL-2, IL-3 and gamma interferon, was unresponsive to IL-1, promoted delayed-type hypersensitivity (DTH) and provided relatively little help to B cells; addition of these cells to antigen-specific B cells induced the production of IgG3 and IgG2a antibodies. The other helper clone made IL-3, IL-4 and IL-5, responded to IL-1, helped B cells efficiently and switched them to IgG1 production, but produced no DTH (Vitetta).
plex formed by the noncovalent association of the o~13 heterodimer with five other polypeptides) was discussed by R. Klausner (Bethesda). Although various partial complexes can be found intracellularly, only the complete complex is efficiently transported to the cell surface. The rate of production of the ~ (16kDa) chain is probably limiting, other components generally being overproduced and degraded. The ontogeny of T-cell receptor expression is still far from clear, but minor populations of cells with different receptors are found in both mouse (R.Macdonald, Epalinges; J. Owen, Birmingham UK) and chicken (C. Chen, Birmingham USA). For example, expression of the Vl38 gene family is twice as frequent in a small population of murine cortical thymocytes as in cortisone-resistant thymocytes and T cells, suggesting possible developmental regulation of gene expression. Rearrangement of both "y and ~ chain genes occurs early in thymic ontogony and is suggested to be a relatively primitive recognition mechanism. Possibly, productive rearrangements of ~/ and/or a preclude subsequent rearrangement of (~ and 13genes (Macdonald; Owen). The thymus is still seen as a site for the development of tolerance by clonal deletion. Elimination of I-E-reactive thymocytes depended on bone marrow-derived cells, not thymic epithelial cells (M. Blackman, Denver).
T.cell antigen receptors The biosynthesis and expression of the T-cell antigen receptor (the corn-
Spedding Micklem is in the Department of Zoology, Universityof Edinburgh, Edinburgh EH93JT, UK.
Reprints We can supply reprints of any article appearing in ImmunologyTodayat very reasonable rates. Pleaseapply for detailsto: ProductionManager, ElsevierPublicationsCambridge, 68 HillsRoad, Cambridge CB2 lEA, UK. (Minimum order 100)
!i!!iiiiii! ¸¸ Immunologists have been quick to exploit the opportunities offered by cytometric techniques. The 12th Annual Meeting of the Society for Analytical Cytology* heard, for instance, how the investigation of lymphocytes has benefited from the application of these techniques. Cytometric techniques, especially fluorescence-activated cell analysis and sorting, have contributed greatly to several areas of immunology, as was reflected in the diversity of immunological topics presented. This report concentrates on how analytical cytology has been applied to B and T lymphocytes and their products.
i::
:
Cytologicalanalysisof lymphocytes from H.SoMicklem Mature Ly-1+ B cells also carry Mac-l, the membrane expression of which is quantitatively related to that of IgM (Herzenberg).
B-ceUmemory and hypermutation Other interesting developments are taking place in the field of B-cell physiology, and promise to solve a number of old problems. Virgin B cells are grossly overproduced in the bone marrow of mice and rats, and under normal circumstances less than 10% survive for more than a Ly-1+ B lympho~tes R. Palacios (Basel) described the few hours after leaving the marrow. production and characteristics of Ly- However, in animals depleted of B 1+ 'pro-B' lymphocyte clones. These cells by anti-mu antibody, new B clones express the B lineage-specific cells can be incorporated in large antigens B-220 and Lyb-8, but lack numbers into the peripheral pool, T-cell and granulocyte lineage mar- where they enjoy an average lifespan kers; some also express Mac-1. They of 4-6 weeks (1. MacLennan, Birmlack surface or cytoplasmic Ig and ingham UK). This probably happens their heavy, K and X genes are in to a small extent normally and their germline configurations. Dif- appears to be independent of antiferentiation to surface Ig +, la + lym- gen; thus, presumably there must be phocytes could be induced in the some counting mechanism that regpresence of lipopolysaccharide, aza- ulates the rate of acceptance of new cytidine and accessory cells, and Ly- B cells into the pool. After leaving 1+ B-cell lines were established from the marrow, new B cells migrate as these differentiated cultures in the far as the T-cell area of the spleen, but very few normally proceed from presence of interleukins 4 and 5. Injection of pro-B clones into SCID there to the follicles and recirculate (severe combined immunodeficient) the others presumably dying in the mice also resulted in the production spleen, although there is little direct of donor-type Ly-I+B cells. These evidence for this. At least two pathdata support the view that the Ly-1 ÷ ways exist for the T-cell-dependent B-cell subset is separated from other activation of B cells. One of these B-cell subsets early in differentiation. involves rapid proliferation (a Ly-1 ÷ B cells are the predominant claimed cell cycle time of 4-5 hours) B-cell type in young mice, while in and hypermutation within the gerthe adult they have a =ories of in- minal centres of lymphoid follicles. teresting properties, including auto- As has long been known, many of antibody formation, primary resi- the lymphocytes produced in the dence in the peritoneal cavity and a follicle die there and are engulfed by tendency to produce chronic neo- macrophages. It is presumed that plasms similar to chronic lymphocytic there is strong selection for mutants leukaemia (CLL)in man (L. Herzen- with higher affinity for antigen, since berg, Stanford; J. Ansell, Edinburgh). the average antibody affinity inSuch neoplasms seem to be found creases during T-cell-dependent imparticularly frequently in autoim- mune responses. The recruitment of mune (NZB x NZW)F1 mice; this may additional high-affinity virgin B cells differ from humans, where chronic appears, in contrast, to be of negliautoimmune diseases such as gible importance for affinity maturrheumatoid arthritis do not seem to ation (MacLennan). Single point be strongly associated with CLL. mutations can change affinity and specificity dramatically, or, on the 'Held on 10-14 August 1987 in CambridgeUK," contrary, they may be without disabstracts have been published in the joumal cernible effect; this latter observation suggests that selection for Cytometry. (~ 1988, ElsevierPublications,Cambridge 0167 4919/88/$02 00
higher affinity is not necessarily stringent, despite the large-scale destruction of cells in germinal centres. The typical hypermutation rate of about one change in each thousand base pairs each generation is abolished after transfer of the cells to an irradiated recipient, again emphasizing the probable importance of microenvironment, presumably the germinal centre (K. Rajewsky, Cologne). A second T-cell-dependent activation pathway occurs outside the follicles and involves the interaction of B cells with interdigitating cells; it probably does not involve any hypermutation or affinity maturation (MacLennan). Hypermutation in anti-oxazolone responses produces point mutations in heavy and some K chain genes, while other K and • genes are unaffected. It is heavily dependent on the presence of L3T4+(CD4 +) T cells in the animal (C. Berek, Cambridge). There is some dispute whether hypermutation continues to occur with repeated immunizations (C. Milstein, Cambridge), or is a once-off preprogrammed step (Rajewsky). It is interesting that anti-digoxin antibodies produced by mutant hybridomas selected in a cell sorter show not only point mutations, but also blocks of changed heavy chain sequence suggestive of gene conversion (E. Haber, Boston).
Mutated and hybrid Ig molecules How interactions between different parts of immunoglobulin molecules distant in terms of primary structure affect the conformation and specificity of the molecule were also explored (Haber; V. Oi, Mountain View). Haber exploited the variants of murine anti-digoxin hybridomas mentioned above to assess the relationship between antibody specificity, primary structure and three-dimensional structure. Digoxin is a valuable model antigen since it evokes antibodies of high affinity, fills the combining site of the antibody and possesses many structurally defined congeners. Spontaneous mutants were isolated on the basis of inhibition of antigen binding by digitoxin. The data revealed precise interaction between
ImmunologyToday,Vol. 9, No. ................
:
positions 10 2, 94 and 101 of the heavy chain in determining the fine specificity of the antibody. Oi used a series of engineered hybrid IgGl/IgG2a anti-dansyl antibodies to investigate the structural determinants of Fab segmental flexibility. Although in general such flexibility is correlated with the length of the upper hinge region, this study showed that the structure of part of the CH1 region has an important influence. The belief that complement fixation depends on segmental flexibility is contradicted by the observation of three rigid hybrids that show good complement fixation. The structure of CH3 is, however, capable of influencing complement fixation, presumably through a conformational change in the molecule affecting the Cl-binding site. Such findings are not only of intrinsic interest. They also form an important background for attempts to 'humanize' mouse hybridoma antibodies, for example by inserting mouse complementarity-determining regions (CDRs) into a human VH framework, in order to render useful therapeutic antibodies non-immunogenic in man. This can be done without necessarily damaging antibody function, as exemplified by the behaviour of a hybrid molecule consisting of the murine K and heavychain CDRs of the therapeutic monoclonal antibody Campath-1 on a human IgG1 background. This molecule binds antigen, mediates antibody-dependent cell-mediated cytotoxicity and fixes complement (M. Verhoeyen, Cambridge). It remains to be seen whether such hybrid antibodies will be significantly less immunogenic in humans than the original mouse antibody.
T-cell derived cytokines and B cells Much recent work has centred on IL-4 (alias B-cell stimulatory factor 1 (BSF-1) etc.) and IL-5, now that recombinant sources of these factobs have become available (E. Severinson, Stockholm; A. Radbruch, Cologne; J. Aubry, Dardilly; E. Vitetta, New York). IL-4 was repeatedly found to promote Ig class switching to G1 at the expense of G3 and G2 in mice. When IL-4 was added to cultures grown in the presence of bacterial lipopolysaccharide a substantial number of IgE-producing cells emerged (Severinson). IL-4 also induced the expression of CD23 antigen (a receptor for e chain Fc) on
....
I, 1988
.........................................................................................................................................................................................
human B cells and enhanced the expression of HLA-DR. The relationship between CD23 and IL-4 may be important, since CD23- cells showed no proliferative response to IL-4 in combination with anti-lgM antibody (Aubry). How IL-4 and other exogenous agents influence class switching is still unknown, but the cytokines seem to 'instruct' cells to switcl, to a particular class, rather than select cells precommitted to a given switch. Specific recombinases appear not to be involved. More probably, particular genes may be rendered available for recombination, and changes in their patterns of DNA methylation may be important (Radbruch). T-cell-derived cytokines alone cannot induce proliferation and differentiation in antigen (TNP)specific B cells: a preliminary period of contact with helper T cells is also required. Two helper T-cell clones specific for keyhole limpet haemocyanin showed very different characteristics. One made IL-2, IL-3 and gamma interferon, was unresponsive to IL-1, promoted delayed-type hypersensitivity (DTH) and provided relatively little help to B cells; addition of these cells to antigen-specific B cells induced the production of IgG3 and IgG2a antibodies. The other helper clone made IL-3, IL-4 and IL-5, responded to IL-1, helped B cells efficiently and switched them to IgG1 production, but produced no DTH (Vitetta).
plex formed by the noncovalent association of the o~13 heterodimer with five other polypeptides) was discussed by R. Klausner (Bethesda). Although various partial complexes can be found intracellularly, only the complete complex is efficiently transported to the cell surface. The rate of production of the ~ (16kDa) chain is probably limiting, other components generally being overproduced and degraded. The ontogeny of T-cell receptor expression is still far from clear, but minor populations of cells with different receptors are found in both mouse (R.Macdonald, Epalinges; J. Owen, Birmingham UK) and chicken (C. Chen, Birmingham USA). For example, expression of the Vl38 gene family is twice as frequent in a small population of murine cortical thymocytes as in cortisone-resistant thymocytes and T cells, suggesting possible developmental regulation of gene expression. Rearrangement of both "y and ~ chain genes occurs early in thymic ontogony and is suggested to be a relatively primitive recognition mechanism. Possibly, productive rearrangements of ~/ and/or a preclude subsequent rearrangement of (~ and 13genes (Macdonald; Owen). The thymus is still seen as a site for the development of tolerance by clonal deletion. Elimination of I-E-reactive thymocytes depended on bone marrow-derived cells, not thymic epithelial cells (M. Blackman, Denver).
T.cell antigen receptors The biosynthesis and expression of the T-cell antigen receptor (the corn-
Spedding Micklem is in the Department of Zoology, Universityof Edinburgh, Edinburgh EH93JT, UK.
Reprints We can supply reprints of any article appearing in ImmunologyTodayat very reasonable rates. Pleaseapply for detailsto: ProductionManager, ElsevierPublicationsCambridge, 68 HillsRoad, Cambridge CB2 lEA, UK. (Minimum order 100)