Eur. J. Immunol. 1990. 20: 2529-2.531
Correspondence Is the hypothesis alive that IgM anti-IgGl rheumatoid factor specificity is determined by framework regions ?
Sirs: To provide a structural explanation for the high frequency of IgM anti-IgG1 (rheumatoid factors, RF) among hybridomas from LPS-activated B cells [l] and hybrids derived from mice undergoing a secondary response to protein antigens [2],we determined the complete nucleic acid sequence of 24 monoclonal anti-IgGl RF (Of these 24 RF, 22 recognize only IgGl and 2 recognize IgGl and one other isotype [3,4]; D. A. Nemazee, personal communication). Their only common feature is V, usage: V, from 3 (out of the estimated 35) groups were significantly overrepresented. Otherwise V segment usage (VH, JH and DHand J,) ws highly diverse; no other bias than toV, groups 1 , s and 19 was detected [l, 21. Since the specificity for IgGl was indistinguishable regardless of V, group, we searched for sequence motifs common to all three. Not surprisingly, such motifs were not evident in the complementarity-determining regions (CDR), groups differ from each other extensively in CDR. Instead V,1, 8 and 19 showed homology in framework regions (FR) 2 and 3. For this reason we proposed that the IgGl specificity might be determined by these regions. In their recent article, Reininger et al. [5] challenge this idea (which they refer to as the FR hypothesis) arguing that their data favor “the involvement of CDR, but not light chain FR residues, in IgG binding”.We disagree with these authors for several reasons. (a) Most importantly, there is no relationship between their sample and ours. We focused on IgM RF that bind IgGI. This is the characteristic specificity of the LPS or secondary protein-derived RF. These RF are highly specific; only rarely do they bind to another isotype [3,4]. The RF described by Reininger et al. [S] are themselves IgG3 and appear to bind IgG1, IgGz,, IgG2b and IgG3. We see no reason why this sample should shed light on how the specificity of IgM anti-IgGl, RF is derived.
Correspondence
2529
They find that TNP competes with IgG binding, but IgG does not compete with TNP binding. Despite these conflicting results, they conclude: “the complete inhibition of the IgG-binding activity by the TNP hapten strongly suggests the existence of common or overlapping sites for anti-TNP and RF activities, rather than two distinct sites as proposed for the FR hypothesis”. On reading this paper we were tempted to choose the second result and to interpret the inability of IgG to compete with TNP binding as evidence for the FR hypothesis. Since this involved an arbitrary choice of the result that favored our model, we resisted the temptation. Furthermore, whether data derived from isotype-cross-reactive RF can be applied to isotype-specific (i.e.IgG1) RF awaits knowledge of how the specificity of such isotype-cross-reactive RF is determined. (c) Reininger et a!. argue that the FR sequences of the L chains in their survey provides further evidence against the FR hypothesis. The basis for this claim is that their antibodies do not bind IgG even though they have L chain FR sequences which are (in their words) “essentially identical” or “virtually identical” to the FR of the IgM anti-IgG1. Initially we stumbled over the expression, “essentially identical”, thinking that sequences are either identical or non-identical.We then realized that the authors actually mean homologous and indeed they are. CB-1 and CB-5, their non-RF, differ from RF in our report by only one residue in FR3 (part of the combining site in our model). We must note however that the difference is a substitution from alanine and proline hardly an innocent difference (see [6] as one example in the voluminous literature on the profound influence of proline residues on protein structure). It may be interesting (as the authors fail to point out) that the example, CB6, with an FR identical to the IgM anti-IgG1 RF is also an RF. Again, whether this is even relevant to the FR hypothesis must await knowing whehter CB6 and the IgM anti-IgM anti-IgGl RF bind the same antigenic site.
In the same vein, 9A6 is offered as evidence against the FR model.Their 9A6 and VS1 (from our survey) have identical FR sequences yet 9A6 does not have RF activity.To belabor the obvious: even though we demonstrated remarkable diversity of VH usage among RF, we did not provide an example associated with every VH.Therefore, as we noted (b) Reininger et al. make an arbitrary interpretation of their [2], we could not rule out the likely possibility that certain results to favor their model. Our model implies that the IgM VH might modify the postulated binding by FR2 and FR3 of anti-IgG1 should have at least two combining sites, one VL of 9A6, from the S107 family, could be such a case as we determined by V,FR2 and F R - another determined by VH in our survey did not find a member of this small family. and VLCDR. IN other words, if one knew the ligand bound by the CDR of such an RF, then this ligand should not The study by Reininger et al. raises interesting and compete with the RF activity and vice versa (barring any important questions. None of these, however, relates to the validity of the FR hypothesis as in our formulation.This is steric or allosteric effects). because, aside from the problems in data interpretation, the two sets of RF have different specificities. Ironically the Reininger et al. have found two antibodies which bind both authors believe our studies on yet another type of RF IgG (multiple isotypes) and the hapten TNP. They assume derived from MRLllpr mice [7] support their contention that TNP is bound in the CDR-encoded binding site. If so, that FWactivity is determined by CDR. However, MRLllpr they could test the model by performing competitive RF differ from IgM anti-IgG1 RF is multiple ways: they are inhibition studies. Nevertheless, they consider the possibil- isotype switched, somatically mutated, use different V, ity that the RF in their survey might also have an genes and, most importantly, are specific for the IgG2, FR-determined specificity. As these RF also bind TNP, the isotype only. Thus, we would not expect that the model prediction that RF and TNP activity is determined by based on IgM anti-IgGl should extend to the M R L l l p r RF different sites on the antibody can be neatly tested. and to make such a comparison would be meaningless.
2530
Correspondence
References 1 Shlomchik, M. J., Nemazee, D. A . , Sato,V. L.,Van Snick, J., Carson, D. A. and Weigert, M . G., J. Exp. Med. 1986. 164: 407. 2 Shlomchik, M. J, Nemazee, D. A.,Van Snick, J. and Weigert, M., J. Exp. Med. 1987. 165: 970. 3 Van Snick, J. and Coulie, l?, J. Exp. Med. 1982. 155: 219. 4 Van Snick, J. and Coulie, F’., Eur. J. Immunol. 1983. 13: 890. 5 Reininger, L., Spertini, F., Shibata.T., Jaton, J. C. and Izui, S., Eur. J. lmmunol. 1989. 19: 2123. 6 Wood, L. C., White, T. B., Ramdas, L. and Nall, B. T., Biochemistry 1988. 27: 8562. 7 Shlomchik, M. J., Marshak-Rothstein, A . , Wolfowicz, C. B.. Rothstein,T. L. and Weigert, M . G., Nature 1987. 328: 805. Mark Shlornchik Department of Pathology Hospital of the University of Pennsylvania Philadelphia, PA Martin Weigert Institute for Cancer Research Fox Chase Cancer Ccntcr Philadelphia, PA
Reply Sirs: On the basis of V, and VH sequence determination of 24 IgM anti-IgG1 mAb, Shlomchik et al. proposed a model according to which anti-IgG1 rheumatoid factor (RF) bind IgGl Fc via V, FR2 and FR3 regions, but not through the CDR binding site [ l , 21. This conclusion was based on the considerable homologies of FR2 and FR3, but not CDR of these L chains. However, they did compare these postulated RF-binding sequences with sequences of appropriate controls, i.e. non-RF mAb carrying the L chain encoded either by the V,l, V J o r VJ9gene family. This is exactly the point that we addressed in our recent study [3].
(a) There is a lack of anti-lgGI RF activity in mb with the FR2 and FR3 V, sequences virtually identical to those claimed for the anti-IgGl binding site. Shlomchik et al. showed the considerable homologies in the FR2 and FR3 V, sequences of 18 of 24 IgM anti-IgG1 RF,which belong to theV,l,V,8 or V,19 groups [ l , 21. However,we believe that it is crucial to compare these sequences with those of non-RF mAb which utilize the above three V, groups. Although the number of mAb we studied was limited, three anti-TNP mAb (CB1, CB5 and 9A6) exhibited FR2 and FR3 V, sequences identical (9A6) or virtually identical, except for a single amino acid substitution in the FR3 (CB1 and CB5), to those described for IgM anti-IgG1 RF, and yet they have no RFactivities [ 3 ] .Drs. Shlomchik and Weigert argue that the VH of our 9A6 mAb derived from the S107 VH family, a family never encountered among their 24 anti-IgG1 R F mAb, might alter the IgG binding by the FR2/FR3 of VL. At present such an argument is difficult to prove or disprove. However, we would like to note two other examples which support our conclusion. First, Kofler et al. [4] have observed that an anti-DNA mAb devoid of RF activity (MRL-DNA10) exhibits FR2 and FR3 V, sequences virtually identical to an anti-IgG1 mAb (VSl), except for a single amino acid substitution (Phe instead of Tyr) at position 87 in FR3 (note that Phe is also observed at
Eur. J. Immunol. 1990. 20: 2529-2531
that position in several other anti-IgG1 mAb). Second, an IgG3 anti-DNA mAb (S7; [S]), which has FR2 and FR3 V, sequences identical to those of an anti-IgG1 mAb (A12; [2]), again lacks RF activity (Marshak-Rothstein, personal communication).What is important t o note is tht theVH of both MRL-DNA10 and S7 anti-DNA mAb are encoded by members of the 55.58 VH gene family, a family largely represented among anti-IgG1 mAb described by Shlomchik et al.
(b) Could a single amino acid substitution in the V , FR3 of CBI and CB5 m A b be responsible for the lack of RF activity ? Shlomchik and Weigert questioned that a single FR substitution at position 80 (Pro) in the non-RF/antiTNP CB1 and CB5 mAb, as compared with Ala in RF/anti-TNP CB6 mAb, might be responsible for the lack of RF activity in CB1 and CB5 mAb. Without access to known crystallographic structures of RF-IgG complexes, the contribution of the residue in position 80 cannot be unambiguously determined. However, it should be pointed out that a) there was no apparent effect on anti-IgG1 RF activity by another substitution at this position (Ser instead of Ala) among IgM anti-IgG1 mAb [ l , 21 and b) a single substitution at position 49 in FR2 (Pro/Ser) throughout V, FR2 and FR3 does not affect the RF activity of A12 and A17 anti-IgG1 mAb [2]. In addition, we would like to emphasize that the CB6 mAb exhibits one substitution in CDR1, four in CDR3, two in FR2 and three in FR3, when compared with the CB1 or CB5 VHregion [3]. In view of the largely documented contribution of CDR residues in the antibody binding site, we favored the involvement of CDR residues over V, FR2/FR3 residues in the RF activity exhibited by the CB6 mAb. (c) Do we make an arbitrary interpretation of our results ? The “FR hypothesis” predicts that anti-IgG1 RF should have two distinct combining sites. Therefore, the ligand for FR, i.e. IgGI, and the ligand for CDR should not compete with each other. We have shown that the hapten TNP competes with the IgG binding, but IgG does not compete with the TNP binding [3]. We interpreted the first part of these data as an indication for common or overlapping binding sites for anti-TNP and anti-IgG activities of our dual RF/anti-TNP mAb (CB6 and 4H10). The failure of reciprocal inhibition of the TNP binding by IgG is intriguing. As discussed in our paper, we think that the lack of reciprocal inhibition can be best explained by the possibility that the relative affinity of anti-TNP/RFmAb for the hapten TNP is higher than the relative affinity for IgG, which is in keeping with the elegant study on dual anti-DNA/antiarsonate mAb [6]. Along the same line, we have recently observed that one of the IgM anti-IgG1 R F mAb,VSl, studied by Shlomchik et al., has anti-DNP activity. Crossinhibition studies have shown that unlike our anti-TNP/RF mAb, DNP is not able to compete withn RF IgG binding (RF activity determined by ELISA: in the absence of DNP, = 1.404; in the presence of DNP, A405 = 1.393), while IgGl competes with DNP binding (anti-DNP activity: in the absence of non-anti-DNPaggregated IgG1, Am5 = 1.487; in the presence of non-anti-DNP aggregated IgGl, A J ~= S 0.020). We believe that this is consistent with the idea that the affinity of VS1 mAb for IgGl may be far higher than that for DNP hapten, since this mAb, generated during the secondary immune responses against KLH, is likely to be the result of specific B cell stimulation with
Eur. J. Imrnunol. 1990. 20: 2529-2531
autoantigen (probably immune complexes; [7]).Therefore, as we discussed in our paper in conjunction with the work of Naparstek et al. [6], antibodies t o both self and non-self epitopes may be derived from the same germ-line Ig gene segments, and as a result of the maturation of antibodies following immunization with self antigens (as for VS1 or non-self antigens (as for CB6 and 4H10),they acquire a higher affinity for self epitopes (as for VS1) or non-self antigens (as for CB6 and 4H10). We agree that MRLllpr IgGzaRFdiffer from IgM anti-IgG1 RF in multiple ways. However, the important point is that as Shlomchick et al. demonstrated [8], MRLllpr anti-IgG2, exhibited somatic mutations in the CDR at a rate higher than in the FR. Does it mean that MRLllpr anti-IgG2, RF utilize the CDR for IgG2, binding site,while IgM anti-IgG1 RF use the FR for their RF activity ? This would imply that depending on the specificity of RF, RF can bind to Fcy either through FR or CDR. Shlomchick et al. do not exclude the possible usage of CDR for some anti-IgG1 RF, because 6 of 24 IgM anti-IgG1 RF [ l , 21 do not belong to the VJ, 8 and 19 groups. In this context, we would like to note that Shlomchick et al. discussed that the VH might shift specificity of RF from anti-IgG1 to anti-IgGl,, because IgM anti-IgG2, RF (JV12) and IgM anti-IgG1 RF (JV4) carry identical L chain sequences, but different members of the 5558 VH family and CDR3 of different length [2]. All these arguments appear difficult to follow. Clearly, the interpretation of their data and ours is a matter of speculation. We, however, believe that the inspection of the crystallographic models of RF-IgGI and RF-IgG2, complexes should definitely answer this question, if this is practically feasible.
Correspondence
2531
References 1 Shlomchik, M. J., Nemazee, D. A.,Van Snick, J.. Carson, D. A. and Weigert, M. G., J. Exp. Med. 1986. 164: 407. 2 Shlornchik, M. J., Nemazee, D. A.,Van Snick, J. and Wcigert. M., J. Exp. Med. 1987. 165: 970. 3 Reininger, L., Spertini, F., Shibata,T., Jaton, J. C. and Izui, S.. Eur. J. lrnrnunol. 1989. 19: 2123. 4 Kofler, R.. Noonan, D. J.. Strohal, R., Balderas, R. S., Moller, N. P. H., Dixon, F. J. and Theofilopoulos, A. N., Eur. J. Irnrnunol. 1987. 17: 91. 5 Shlomchik, M., Mascelli, M., Shan, H., Radic, M. Z., Pisetzsky, D., Marshak-Rothstein, A. and Weigert, M. G., J. Exp. Med. 1990. 171: 265. 6 Naparstek,Y., Andrk-Schwartz, J., Manser, T., Wysocki. L. J.. Breitman, L., Stollar, B. D., Gefter, M. and Schwartz, R. S., J. Exp. Med. 1986. 164: 614. 7 Nemazee, D., J. Exp. Med. 1985. 161: 242. 8 Shlomchik, M. J., Marshak-Rothstein, A., Wolfowicz, C. B., Rothstein,T. L. and Weigert, M. G., Nature 1987. 328: 805.
Luc Reininger Basel Institute for Immunology Basel Jean-Claude Jaton Department of Medical Biochemistry Centre MCdical Universitaire University of Geneva Geneva
Shozo Izui Department of pathology Centre MCdical Universitaire University of Geneva Geneva
Correspondence Is the hypothesis alive that IgM anti-IgGl rheumatoid factor specificity is determined by framework regions ?
Sirs: To provide a structural explanation for the high frequency of IgM anti-IgG1 (rheumatoid factors, RF) among hybridomas from LPS-activated B cells [l] and hybrids derived from mice undergoing a secondary response to protein antigens [2],we determined the complete nucleic acid sequence of 24 monoclonal anti-IgGl RF (Of these 24 RF, 22 recognize only IgGl and 2 recognize IgGl and one other isotype [3,4]; D. A. Nemazee, personal communication). Their only common feature is V, usage: V, from 3 (out of the estimated 35) groups were significantly overrepresented. Otherwise V segment usage (VH, JH and DHand J,) ws highly diverse; no other bias than toV, groups 1 , s and 19 was detected [l, 21. Since the specificity for IgGl was indistinguishable regardless of V, group, we searched for sequence motifs common to all three. Not surprisingly, such motifs were not evident in the complementarity-determining regions (CDR), groups differ from each other extensively in CDR. Instead V,1, 8 and 19 showed homology in framework regions (FR) 2 and 3. For this reason we proposed that the IgGl specificity might be determined by these regions. In their recent article, Reininger et al. [5] challenge this idea (which they refer to as the FR hypothesis) arguing that their data favor “the involvement of CDR, but not light chain FR residues, in IgG binding”.We disagree with these authors for several reasons. (a) Most importantly, there is no relationship between their sample and ours. We focused on IgM RF that bind IgGI. This is the characteristic specificity of the LPS or secondary protein-derived RF. These RF are highly specific; only rarely do they bind to another isotype [3,4]. The RF described by Reininger et al. [S] are themselves IgG3 and appear to bind IgG1, IgGz,, IgG2b and IgG3. We see no reason why this sample should shed light on how the specificity of IgM anti-IgGl, RF is derived.
Correspondence
2529
They find that TNP competes with IgG binding, but IgG does not compete with TNP binding. Despite these conflicting results, they conclude: “the complete inhibition of the IgG-binding activity by the TNP hapten strongly suggests the existence of common or overlapping sites for anti-TNP and RF activities, rather than two distinct sites as proposed for the FR hypothesis”. On reading this paper we were tempted to choose the second result and to interpret the inability of IgG to compete with TNP binding as evidence for the FR hypothesis. Since this involved an arbitrary choice of the result that favored our model, we resisted the temptation. Furthermore, whether data derived from isotype-cross-reactive RF can be applied to isotype-specific (i.e.IgG1) RF awaits knowledge of how the specificity of such isotype-cross-reactive RF is determined. (c) Reininger et a!. argue that the FR sequences of the L chains in their survey provides further evidence against the FR hypothesis. The basis for this claim is that their antibodies do not bind IgG even though they have L chain FR sequences which are (in their words) “essentially identical” or “virtually identical” to the FR of the IgM anti-IgG1. Initially we stumbled over the expression, “essentially identical”, thinking that sequences are either identical or non-identical.We then realized that the authors actually mean homologous and indeed they are. CB-1 and CB-5, their non-RF, differ from RF in our report by only one residue in FR3 (part of the combining site in our model). We must note however that the difference is a substitution from alanine and proline hardly an innocent difference (see [6] as one example in the voluminous literature on the profound influence of proline residues on protein structure). It may be interesting (as the authors fail to point out) that the example, CB6, with an FR identical to the IgM anti-IgG1 RF is also an RF. Again, whether this is even relevant to the FR hypothesis must await knowing whehter CB6 and the IgM anti-IgM anti-IgGl RF bind the same antigenic site.
In the same vein, 9A6 is offered as evidence against the FR model.Their 9A6 and VS1 (from our survey) have identical FR sequences yet 9A6 does not have RF activity.To belabor the obvious: even though we demonstrated remarkable diversity of VH usage among RF, we did not provide an example associated with every VH.Therefore, as we noted (b) Reininger et al. make an arbitrary interpretation of their [2], we could not rule out the likely possibility that certain results to favor their model. Our model implies that the IgM VH might modify the postulated binding by FR2 and FR3 of anti-IgG1 should have at least two combining sites, one VL of 9A6, from the S107 family, could be such a case as we determined by V,FR2 and F R - another determined by VH in our survey did not find a member of this small family. and VLCDR. IN other words, if one knew the ligand bound by the CDR of such an RF, then this ligand should not The study by Reininger et al. raises interesting and compete with the RF activity and vice versa (barring any important questions. None of these, however, relates to the validity of the FR hypothesis as in our formulation.This is steric or allosteric effects). because, aside from the problems in data interpretation, the two sets of RF have different specificities. Ironically the Reininger et al. have found two antibodies which bind both authors believe our studies on yet another type of RF IgG (multiple isotypes) and the hapten TNP. They assume derived from MRLllpr mice [7] support their contention that TNP is bound in the CDR-encoded binding site. If so, that FWactivity is determined by CDR. However, MRLllpr they could test the model by performing competitive RF differ from IgM anti-IgG1 RF is multiple ways: they are inhibition studies. Nevertheless, they consider the possibil- isotype switched, somatically mutated, use different V, ity that the RF in their survey might also have an genes and, most importantly, are specific for the IgG2, FR-determined specificity. As these RF also bind TNP, the isotype only. Thus, we would not expect that the model prediction that RF and TNP activity is determined by based on IgM anti-IgGl should extend to the M R L l l p r RF different sites on the antibody can be neatly tested. and to make such a comparison would be meaningless.
2530
Correspondence
References 1 Shlomchik, M. J., Nemazee, D. A . , Sato,V. L.,Van Snick, J., Carson, D. A. and Weigert, M . G., J. Exp. Med. 1986. 164: 407. 2 Shlomchik, M. J, Nemazee, D. A.,Van Snick, J. and Weigert, M., J. Exp. Med. 1987. 165: 970. 3 Van Snick, J. and Coulie, l?, J. Exp. Med. 1982. 155: 219. 4 Van Snick, J. and Coulie, F’., Eur. J. Immunol. 1983. 13: 890. 5 Reininger, L., Spertini, F., Shibata.T., Jaton, J. C. and Izui, S., Eur. J. lmmunol. 1989. 19: 2123. 6 Wood, L. C., White, T. B., Ramdas, L. and Nall, B. T., Biochemistry 1988. 27: 8562. 7 Shlomchik, M. J., Marshak-Rothstein, A . , Wolfowicz, C. B.. Rothstein,T. L. and Weigert, M . G., Nature 1987. 328: 805. Mark Shlornchik Department of Pathology Hospital of the University of Pennsylvania Philadelphia, PA Martin Weigert Institute for Cancer Research Fox Chase Cancer Ccntcr Philadelphia, PA
Reply Sirs: On the basis of V, and VH sequence determination of 24 IgM anti-IgG1 mAb, Shlomchik et al. proposed a model according to which anti-IgG1 rheumatoid factor (RF) bind IgGl Fc via V, FR2 and FR3 regions, but not through the CDR binding site [ l , 21. This conclusion was based on the considerable homologies of FR2 and FR3, but not CDR of these L chains. However, they did compare these postulated RF-binding sequences with sequences of appropriate controls, i.e. non-RF mAb carrying the L chain encoded either by the V,l, V J o r VJ9gene family. This is exactly the point that we addressed in our recent study [3].
(a) There is a lack of anti-lgGI RF activity in mb with the FR2 and FR3 V, sequences virtually identical to those claimed for the anti-IgGl binding site. Shlomchik et al. showed the considerable homologies in the FR2 and FR3 V, sequences of 18 of 24 IgM anti-IgG1 RF,which belong to theV,l,V,8 or V,19 groups [ l , 21. However,we believe that it is crucial to compare these sequences with those of non-RF mAb which utilize the above three V, groups. Although the number of mAb we studied was limited, three anti-TNP mAb (CB1, CB5 and 9A6) exhibited FR2 and FR3 V, sequences identical (9A6) or virtually identical, except for a single amino acid substitution in the FR3 (CB1 and CB5), to those described for IgM anti-IgG1 RF, and yet they have no RFactivities [ 3 ] .Drs. Shlomchik and Weigert argue that the VH of our 9A6 mAb derived from the S107 VH family, a family never encountered among their 24 anti-IgG1 R F mAb, might alter the IgG binding by the FR2/FR3 of VL. At present such an argument is difficult to prove or disprove. However, we would like to note two other examples which support our conclusion. First, Kofler et al. [4] have observed that an anti-DNA mAb devoid of RF activity (MRL-DNA10) exhibits FR2 and FR3 V, sequences virtually identical to an anti-IgG1 mAb (VSl), except for a single amino acid substitution (Phe instead of Tyr) at position 87 in FR3 (note that Phe is also observed at
Eur. J. Immunol. 1990. 20: 2529-2531
that position in several other anti-IgG1 mAb). Second, an IgG3 anti-DNA mAb (S7; [S]), which has FR2 and FR3 V, sequences identical to those of an anti-IgG1 mAb (A12; [2]), again lacks RF activity (Marshak-Rothstein, personal communication).What is important t o note is tht theVH of both MRL-DNA10 and S7 anti-DNA mAb are encoded by members of the 55.58 VH gene family, a family largely represented among anti-IgG1 mAb described by Shlomchik et al.
(b) Could a single amino acid substitution in the V , FR3 of CBI and CB5 m A b be responsible for the lack of RF activity ? Shlomchik and Weigert questioned that a single FR substitution at position 80 (Pro) in the non-RF/antiTNP CB1 and CB5 mAb, as compared with Ala in RF/anti-TNP CB6 mAb, might be responsible for the lack of RF activity in CB1 and CB5 mAb. Without access to known crystallographic structures of RF-IgG complexes, the contribution of the residue in position 80 cannot be unambiguously determined. However, it should be pointed out that a) there was no apparent effect on anti-IgG1 RF activity by another substitution at this position (Ser instead of Ala) among IgM anti-IgG1 mAb [ l , 21 and b) a single substitution at position 49 in FR2 (Pro/Ser) throughout V, FR2 and FR3 does not affect the RF activity of A12 and A17 anti-IgG1 mAb [2]. In addition, we would like to emphasize that the CB6 mAb exhibits one substitution in CDR1, four in CDR3, two in FR2 and three in FR3, when compared with the CB1 or CB5 VHregion [3]. In view of the largely documented contribution of CDR residues in the antibody binding site, we favored the involvement of CDR residues over V, FR2/FR3 residues in the RF activity exhibited by the CB6 mAb. (c) Do we make an arbitrary interpretation of our results ? The “FR hypothesis” predicts that anti-IgG1 RF should have two distinct combining sites. Therefore, the ligand for FR, i.e. IgGI, and the ligand for CDR should not compete with each other. We have shown that the hapten TNP competes with the IgG binding, but IgG does not compete with the TNP binding [3]. We interpreted the first part of these data as an indication for common or overlapping binding sites for anti-TNP and anti-IgG activities of our dual RF/anti-TNP mAb (CB6 and 4H10). The failure of reciprocal inhibition of the TNP binding by IgG is intriguing. As discussed in our paper, we think that the lack of reciprocal inhibition can be best explained by the possibility that the relative affinity of anti-TNP/RFmAb for the hapten TNP is higher than the relative affinity for IgG, which is in keeping with the elegant study on dual anti-DNA/antiarsonate mAb [6]. Along the same line, we have recently observed that one of the IgM anti-IgG1 R F mAb,VSl, studied by Shlomchik et al., has anti-DNP activity. Crossinhibition studies have shown that unlike our anti-TNP/RF mAb, DNP is not able to compete withn RF IgG binding (RF activity determined by ELISA: in the absence of DNP, = 1.404; in the presence of DNP, A405 = 1.393), while IgGl competes with DNP binding (anti-DNP activity: in the absence of non-anti-DNPaggregated IgG1, Am5 = 1.487; in the presence of non-anti-DNP aggregated IgGl, A J ~= S 0.020). We believe that this is consistent with the idea that the affinity of VS1 mAb for IgGl may be far higher than that for DNP hapten, since this mAb, generated during the secondary immune responses against KLH, is likely to be the result of specific B cell stimulation with
Eur. J. Imrnunol. 1990. 20: 2529-2531
autoantigen (probably immune complexes; [7]).Therefore, as we discussed in our paper in conjunction with the work of Naparstek et al. [6], antibodies t o both self and non-self epitopes may be derived from the same germ-line Ig gene segments, and as a result of the maturation of antibodies following immunization with self antigens (as for VS1 or non-self antigens (as for CB6 and 4H10),they acquire a higher affinity for self epitopes (as for VS1) or non-self antigens (as for CB6 and 4H10). We agree that MRLllpr IgGzaRFdiffer from IgM anti-IgG1 RF in multiple ways. However, the important point is that as Shlomchick et al. demonstrated [8], MRLllpr anti-IgG2, exhibited somatic mutations in the CDR at a rate higher than in the FR. Does it mean that MRLllpr anti-IgG2, RF utilize the CDR for IgG2, binding site,while IgM anti-IgG1 RF use the FR for their RF activity ? This would imply that depending on the specificity of RF, RF can bind to Fcy either through FR or CDR. Shlomchick et al. do not exclude the possible usage of CDR for some anti-IgG1 RF, because 6 of 24 IgM anti-IgG1 RF [ l , 21 do not belong to the VJ, 8 and 19 groups. In this context, we would like to note that Shlomchick et al. discussed that the VH might shift specificity of RF from anti-IgG1 to anti-IgGl,, because IgM anti-IgG2, RF (JV12) and IgM anti-IgG1 RF (JV4) carry identical L chain sequences, but different members of the 5558 VH family and CDR3 of different length [2]. All these arguments appear difficult to follow. Clearly, the interpretation of their data and ours is a matter of speculation. We, however, believe that the inspection of the crystallographic models of RF-IgGI and RF-IgG2, complexes should definitely answer this question, if this is practically feasible.
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References 1 Shlomchik, M. J., Nemazee, D. A.,Van Snick, J.. Carson, D. A. and Weigert, M. G., J. Exp. Med. 1986. 164: 407. 2 Shlornchik, M. J., Nemazee, D. A.,Van Snick, J. and Wcigert. M., J. Exp. Med. 1987. 165: 970. 3 Reininger, L., Spertini, F., Shibata,T., Jaton, J. C. and Izui, S.. Eur. J. lrnrnunol. 1989. 19: 2123. 4 Kofler, R.. Noonan, D. J.. Strohal, R., Balderas, R. S., Moller, N. P. H., Dixon, F. J. and Theofilopoulos, A. N., Eur. J. Irnrnunol. 1987. 17: 91. 5 Shlomchik, M., Mascelli, M., Shan, H., Radic, M. Z., Pisetzsky, D., Marshak-Rothstein, A. and Weigert, M. G., J. Exp. Med. 1990. 171: 265. 6 Naparstek,Y., Andrk-Schwartz, J., Manser, T., Wysocki. L. J.. Breitman, L., Stollar, B. D., Gefter, M. and Schwartz, R. S., J. Exp. Med. 1986. 164: 614. 7 Nemazee, D., J. Exp. Med. 1985. 161: 242. 8 Shlomchik, M. J., Marshak-Rothstein, A., Wolfowicz, C. B., Rothstein,T. L. and Weigert, M. G., Nature 1987. 328: 805.
Luc Reininger Basel Institute for Immunology Basel Jean-Claude Jaton Department of Medical Biochemistry Centre MCdical Universitaire University of Geneva Geneva
Shozo Izui Department of pathology Centre MCdical Universitaire University of Geneva Geneva
