- Email: [email protected]
Final round comments on the issue of haplotype exclusion
seminars in IMMUNOLOGY, Vol. 14, 2002: pp. 243–244 doi:10.1016/S1044–5323(02)00053-2, available online at http://www.idealibrary.com on
Final round comments on the issue of haplotype exclusion Michael J. H. Ratcliffe ∗ and Kelly A. Pike
predictions based on an equal ability of sIg+ and Tµ+ cells to support bursal follicle formation. Proof that the expression of Tµ is sufficient to support follicle formation clearly does not require that “all H and L loci are uniquely in the unrearranged configuration in the Tµ only follicles,” simply that these cells do not express endogenous sIg. Indeed we have shown that Tµ+ cells can contain VDJH and/or VJL rearrangements, at greatly reduced frequencies, but that the majority of these rearrangements are non-productive. Langman and Cohn again raise the issue of the role of D regions in RF2. They propose that VDJ with the D in RF1 are selectively eliminated, whereas those VDJ with D in FR2 are selected, expanded and their D regions converted to RF1 by gene conversion. This simply does not fit the available sequence data, where selection for RF1 occurs prior to the onset of gene conversion. We agree that if two and only two rearrangements were permitted, subsequent to DJH rearrangement, then selection could account for a H+/0 /L+/0 phenotype. However, that also does not fit the available data. There are examples from day 13 embryo bursa of DDJH , DDDJH , VDDJH and even VDDDJH sequences. Thus, at a given point in time in the bursa, the number of post-DJH rearrangements is heterogeneous, with three or four post-DJH rearrangements occurring among some B cell precursors. Under these circumstances, it is difficult to envisage why would one not observe H+/− and/or L+/− B cells being selected in bursal follicles, given that the colonization of the approximately 10,000 bursal follicles takes place over a period of several days. Monoallelic locus accessibility would account for these configurations since the rate-limiting step would be the induction of locus availability for rearrangement rather than the rearrangement event itself. We agree that the analysis of B cells from embryo bursectomized chicks would be informative. However, at this point too little is known about how these cells develop in an extra-bursal environment for them to be fitted unequivocally into one or other model.
Langman and Cohn raise a number of issues that should be addressed here. In essence, they argue that in the chicken system, the data can be accounted for by proposing, in accordance with the views of Weill and Reynaud, that rearrangement is sufficiently slow that among those cells that could colonize bursal follicles each cell could only have undergone two rearrangements. Among these, only those cells that have undergone one productive VJL and one productive VDJH would be selected in bursal follicles. We will return to this issue, but Langman and Cohn also raise a number of points that need to be addressed first. The ability of the Tµ construct to support B cell development in cells lacking endogenous VDJH and/or VJL rearrangements proves that there is no obligate requirement for recognition of the prediversified sIg receptor on that B cell by a bursal ligand. We agree that these experiments do not rule out the possibility that there is a ligand for the Cµ domain(s), or the extracellular domains of the Igα/β chains of the sIg receptor complex. However, recent experiments in which bursal colonization is supported by expression of a chimeric receptor in which the extracellular and transmembrane of mouse CD8α and CD8β are fused to the cytoplasmic domains of chicken Igα and Igβ, respectively, do indeed rule out obligate recognition of any extracellular domain of the sIg receptor complex (Pike et al., manuscript in preparation). It remains possible that the formation of a bursal follicle might require recognition of a bursal ligand by the prediversified receptor specificity. Should this be the case, however, the presence of follicles that do not contain cells expressing sIg+ cells needs be explained. While the frequency of bursal follicles containing exclusively Tµ+ cells in chicks expressing Tµ is typically low, the frequency is exactly in line with From the Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, Ont., Canada M5S 1A8. * Corresponding author. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1044–5323 / 02 / $– see front matter
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M. J. H. Ratcliffe and K. A. Pike
We do not agree with the tenet that monoallelic accessibility is equivalent to the ABORT function in HAPEX. Nonetheless we agree that the origin of the asymmetry is an important issue and recent evidence that the Ig loci physically change location within the nucleus may provide insights. Monoallelic accessibility to rearrangement may not be equivalent to monoallelic accessibility to transcription in a cell with two rearranged VDJH or VJL . Under these circumstances, rearrangement would be required to maintain haplotype exclusion. Conversely, monoallelic accessibility may be equivalent to monoallelic expression of IL-4. In this situation, monoallelic IL-4 expression appears to be reset stochastically after each cell division. Monoallelic rearrangement would then be required to obviate the possibility that the daughter cells of a B
cell undergoing clonal expansion would reset monoallelic expression to the other allele. The issue of why the fusion efficiency is ∼0.3 as opposed to 1 may simply reflect the mechanism of rearrangement, as may the issue of D reading frame usage. The use of VDH , as opposed to VH donor segments might be expected to confer extended CDR3 diversity in a situation where the chicken DH sequences have rather limited intrinsic diversity. Lastly, we have argued that the selective pressure maintaining the acceptor VH and VL sequences is simply the requirement for maintaining a gene conversion substrate. Thus, we have demonstrated significant levels of acceptor VH and VL polymorphism, most of which results in amino acid replacement in the CDRs. Indeed the level of polymorphism in the acceptor VL gene is higher than that seen in the donor VL genes.
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Final round comments on the issue of haplotype exclusion Michael J. H. Ratcliffe ∗ and Kelly A. Pike
predictions based on an equal ability of sIg+ and Tµ+ cells to support bursal follicle formation. Proof that the expression of Tµ is sufficient to support follicle formation clearly does not require that “all H and L loci are uniquely in the unrearranged configuration in the Tµ only follicles,” simply that these cells do not express endogenous sIg. Indeed we have shown that Tµ+ cells can contain VDJH and/or VJL rearrangements, at greatly reduced frequencies, but that the majority of these rearrangements are non-productive. Langman and Cohn again raise the issue of the role of D regions in RF2. They propose that VDJ with the D in RF1 are selectively eliminated, whereas those VDJ with D in FR2 are selected, expanded and their D regions converted to RF1 by gene conversion. This simply does not fit the available sequence data, where selection for RF1 occurs prior to the onset of gene conversion. We agree that if two and only two rearrangements were permitted, subsequent to DJH rearrangement, then selection could account for a H+/0 /L+/0 phenotype. However, that also does not fit the available data. There are examples from day 13 embryo bursa of DDJH , DDDJH , VDDJH and even VDDDJH sequences. Thus, at a given point in time in the bursa, the number of post-DJH rearrangements is heterogeneous, with three or four post-DJH rearrangements occurring among some B cell precursors. Under these circumstances, it is difficult to envisage why would one not observe H+/− and/or L+/− B cells being selected in bursal follicles, given that the colonization of the approximately 10,000 bursal follicles takes place over a period of several days. Monoallelic locus accessibility would account for these configurations since the rate-limiting step would be the induction of locus availability for rearrangement rather than the rearrangement event itself. We agree that the analysis of B cells from embryo bursectomized chicks would be informative. However, at this point too little is known about how these cells develop in an extra-bursal environment for them to be fitted unequivocally into one or other model.
Langman and Cohn raise a number of issues that should be addressed here. In essence, they argue that in the chicken system, the data can be accounted for by proposing, in accordance with the views of Weill and Reynaud, that rearrangement is sufficiently slow that among those cells that could colonize bursal follicles each cell could only have undergone two rearrangements. Among these, only those cells that have undergone one productive VJL and one productive VDJH would be selected in bursal follicles. We will return to this issue, but Langman and Cohn also raise a number of points that need to be addressed first. The ability of the Tµ construct to support B cell development in cells lacking endogenous VDJH and/or VJL rearrangements proves that there is no obligate requirement for recognition of the prediversified sIg receptor on that B cell by a bursal ligand. We agree that these experiments do not rule out the possibility that there is a ligand for the Cµ domain(s), or the extracellular domains of the Igα/β chains of the sIg receptor complex. However, recent experiments in which bursal colonization is supported by expression of a chimeric receptor in which the extracellular and transmembrane of mouse CD8α and CD8β are fused to the cytoplasmic domains of chicken Igα and Igβ, respectively, do indeed rule out obligate recognition of any extracellular domain of the sIg receptor complex (Pike et al., manuscript in preparation). It remains possible that the formation of a bursal follicle might require recognition of a bursal ligand by the prediversified receptor specificity. Should this be the case, however, the presence of follicles that do not contain cells expressing sIg+ cells needs be explained. While the frequency of bursal follicles containing exclusively Tµ+ cells in chicks expressing Tµ is typically low, the frequency is exactly in line with From the Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, Ont., Canada M5S 1A8. * Corresponding author. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1044–5323 / 02 / $– see front matter
243
M. J. H. Ratcliffe and K. A. Pike
We do not agree with the tenet that monoallelic accessibility is equivalent to the ABORT function in HAPEX. Nonetheless we agree that the origin of the asymmetry is an important issue and recent evidence that the Ig loci physically change location within the nucleus may provide insights. Monoallelic accessibility to rearrangement may not be equivalent to monoallelic accessibility to transcription in a cell with two rearranged VDJH or VJL . Under these circumstances, rearrangement would be required to maintain haplotype exclusion. Conversely, monoallelic accessibility may be equivalent to monoallelic expression of IL-4. In this situation, monoallelic IL-4 expression appears to be reset stochastically after each cell division. Monoallelic rearrangement would then be required to obviate the possibility that the daughter cells of a B
cell undergoing clonal expansion would reset monoallelic expression to the other allele. The issue of why the fusion efficiency is ∼0.3 as opposed to 1 may simply reflect the mechanism of rearrangement, as may the issue of D reading frame usage. The use of VDH , as opposed to VH donor segments might be expected to confer extended CDR3 diversity in a situation where the chicken DH sequences have rather limited intrinsic diversity. Lastly, we have argued that the selective pressure maintaining the acceptor VH and VL sequences is simply the requirement for maintaining a gene conversion substrate. Thus, we have demonstrated significant levels of acceptor VH and VL polymorphism, most of which results in amino acid replacement in the CDRs. Indeed the level of polymorphism in the acceptor VL gene is higher than that seen in the donor VL genes.
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