The possibility that idiotype/anti-idiotype interactions play a significant role in physiological immunoregulation has been examined in many previous studies. While it is well established that purposeful immunization with purified monospecific idiotype-bearing antibodies may reproducibly lead to anti-idiotype antibody production, and that this phenomenon can be experimentally extended to the second and even third generations of antibodies, it has been difficult to explain why a potentially unlimited sequence of idiotype/anti-idiotype inductions is not a commonplace finding in the setting of natural immunity. Previous reviewers of this manuscript pointed out to very significant features of the idiotype/anti-idiotype interaction modes and of Complement activation that must be addressed in detail before the hypothesis is taken at its face value. Not being in the Complement or Idiotype fields, which are admittedly very complex, I will refrain from raising these types of objections. Instead, having a bias toward simple approaches in a more physiopathological setting, I would rather propose that the hypothesis can be boiled down to the idea that in vivo interactions between idiotypes and anti-idiotypes are self-limited because immune complex formation will lead to demise and clearance of the B cells displaying the corresponding epitopes. While two different effector mechanisms (Complement and cells capable of ADCC) are advanced, I would favor Complement as the variable that requires immediate experimental testing, because it is ubiquitous in the circulation, and because its depletion can be conveniently achieved by Cobra Venom Factor administration. If the authors can demonstrate that expansion of the idiotype network beyond its original limits occurs when Complement is no longer available, then a significant finding may be at hand. This is, of course, not identical with the demonstration of a novel pathway for complement activation, since there is no evidence to support the idea that the classical complement activation pathway is unable to carry out this function.

I work in experimental models in immunopathology.

Idiotype (Id) – anti-Id network theory is based on the fact that an Id is not a self determinant, as it is generated only following entry of an extraneous antigen into a host- body on induced antibody against the antigen (1st antibody). The paratopes of complementarity determining regions (CDRs) of first antibody (anti- antigen antibody) act as Id, which, in turn, induces 2nd antibody (anti-Id antibody) or 3rd antibody (anti- anti- Id antibody) etc., all of which have the potential to act as extraneous antigens. On binding of an epitope with its specific paratope, the CH2 domain of IgG and CH3 domain of IgM are exposed, enabling  binding of 2 or more globular heads of C1q to allow assembly of activated C1qr2s2 that activates classical C3 convertase (C4b2b), and classical C5 convertase (C4b2b3b). If an extraneous antigen on occupying the CDRs of an antibody can activate classical complement pathway, an idiotope on occupying the CDRs of anti-Id antibody (2nd antibody), can as well stimulate a classical complement pathway, instead of proposal of a Neo-classical complement pathway, unless the authors have enough proof to present a new C3 or C5 convertase, other than the classical C3 and C5 convertase, the C4b2b and C4b2b3b, respectively.  Similar situations will arise when the epitopes of 1st antibody (Id) will bind with the paratopes of CDRs of 2nd antibody (anti-Id antibody) or the epitopes of 2nd antibody (anti-Id antibody) with the paratopes of CDRs of 3rd antibody (anti- anti-Id antibody) and so on. The most important step is occupation of paratopes of CDRs of an antibody (Id) with its specific epitopes (idiotopes), so that the CH2 domain of IgG and CH3 domain of IgM antibodies are exposed for subsequent activation of classical C3 convertase.                                                                             If at all an abrogation of immune response is visualized, then even 1st antibody (eliciting idiotype or Id) onwards, the immune response should be terminated. Because, 1st antibody (Id) will provide the unique and specific CDRs on which not only an epitope binds, but also new antigenic determinants (idiotopes) will induce generation of 2nd antibody (anti-Id antibody), and 2nd antibody (anti-Id antibody) will induce 3rd antibody (anti-anti-Id antibody) and so on. Further, on binding of second antibody with the first antibody bearing B cells, the ADCC may also get activated against the first antibody bearing B lymphocytes, hence even the first antibody bearing B cell may be destroyed, thus, abrogating the immune response even against the original antigen.  Thus, in principle, no immune response against a foreign antigen will be elicited. However, this does not happen in routine procedures.
To me frankly, anti-anti-idiotypic antibodies appear just as a conceptualized phenomenon, which may not be occurring at all in the host, as under :
1. If at all, anti-anti-idiotypic or even anti-idiotypic antibody network exists, then how unique specificity of B lymphocytes are maintained? So, do all B lymphocytes have some kind of more than 1 specificity?
2. If at all, anti-anti-idiotypic antibody network exists, then complement / ADCC activation will kill even first antibody bearing lymphocytes creating tremendous ‘holes’ in B cell repertoire of an individual,  but this is not a usual case of complement activation.
3. If at all membrane attack complex (MAC) kills anti-anti-idiotypic antibodies (third antibody onwards) bearing B cells, what stops it not to kill a 2nd antibody and 1st antibody bearing B cells?
Hence, it may be concluded that the proposed theory of Neo-classical pathway of complement activation appears similar just as the classical pathway of complement activation, and may not be acceptable as of now, until and unless enough proof is provided for Neo- C3 or C5 convertases for establishing the theory.

The manuscript provides a novel perspective on in vivo complement activation as a result of id-anti-id interactions with profound implications on immunoregulation. While the thesis is plausible, there remain some important unanswered questions as recent studies on idiotypy have been few, given the complexity of idiotype-mediated immunoregulation. For example, dynamics of required hinge flexibility of B-cell surface immunoglobulin for complement fixation dependent upon C1q binding-site exposure is not yet defined; an outcome of cell signalling subsequent to 'id-anti-id interaction' remains an enigma as threshold  and the resulting outcome is yet to be understood. In fact, id-anti-id interaction may not be 'endless' but may have a gradual ripple effect ensuring immunoregulation based homeostasis. it should be noted that for classical pathaway to be activated, at least 1000 IgM or IgG monomers need to be present in close proximity, during capping, to permit firm C1q binding to initiate the classical complement cascade. This likely would dtermine the required threshold for complement activation. The configuration of monomer IgM and IgG expressed on B cell surface needs to be determined if it is planar (C1q -binding site not exposed) or not (C1q -binding site exposed). Further, id-anti-id complexes are normally present in blood circulation and these should normally permit c1q biding resulting in complement cascade which must be tightly regulated given pathological consequences. Most of these areas are yet to be fully elucidated by experimentation. Nevertheless, the outlined thesis is much relevant to disease state, e.g., systemic autoimmune disease, where these idiotypic- anti-idiotypic poulations are likely to be dysregulated. hence, the proposed hypothesis needs to be tested experimentally in health and disease state. The hypothesis provides a logical and rational perspective that must be tested experimentally before any firm conclusions could be made.

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