Response to Letter from Altman and Bécart

We are glad to have the opportunity to clarify the points that Drs. Altman and Becart have raised regarding our manuscript. Because Def6 is the official name of the gene, we will henceforth use this nomenclature rather than its alternate names, IBP and SLAT.Gene deletion that leads to autoimmunity such as lupus-like syndrome occur only in selected strains of mice because the development of autoimmunity usually requires interaction of the effects of the single gene deletion with those mediated by various strain-specific background loci (Jorgensen et al., 2004xJorgensen, T.N., Gubbels, M.R., and Kotzin, B.L. Curr. Opin. Immunol. 2004; 16: 787–793Crossref | PubMed | Scopus (45)See all ReferencesJorgensen et al., 2004). We were thus not surprised to observe, as Altman and Becart have, that Def6trap/trap mice on a C57BL/6 background do not develop a lupus-like syndrome. We have, however, observed that CD4+ T cells from Def6trap/trap mice backcrossed onto either a C57BL/6 or a BALB/c background (for > 10 generations) always exhibit deregulated expression of IL-17 and IL-21 under neutral conditions in vitro (although the absolute amounts can vary depending on the strain). Furthermore, deregulated expression of IL-17 and IL-21 can be observed in both transgenic (DO11.10) and nontransgenic Def6trap/trap T cells, indicating that this effect is not due to an artifact imposed by the DO11.10 transgene. For these reasons, we have felt comfortable using CD4+ T cells from Def6trap/trap mice on either a C57BL/6 or a BALB/c background in our manuscript. We believe that this is a strength of the system rather than a concern because the presence of similar abnormalities in CD4+ T cells from Def6trap/trap mice from multiple strains strongly supports the idea that this defect is truly due to the absence of Def6 and not to the presence of strain-specific modifiers (or to the presence of the DO11.10 transgene). Furthermore, our data on the role of Def6 on the production of IL-17 and IL-21 were backed by substantial mechanistic studies, which could not have been influenced by the strain background of our mice. In particular, these studies linked Def6 to the regulation of interferon-regulatory factor-4 (IRF-4), a transcription factor whose role in the regulation of IL-17 and IL-21 has been independently corroborated (Brustle et al., 2007xBrustle, A., Heink, S., Huber, M., Rosenplanter, C., Stadelmann, C., Yu, P., Arpaia, E., Mak, T.W., Kamradt, T., and Lohoff, M. Nat. Immunol. 2007; 8: 958–966Crossref | PubMed | Scopus (378)See all References, Huber et al., 2008xHuber, M., Brustle, A., Reinhard, K., Guralnik, A., Walter, G., Mahiny, A., von Low, E., and Lohoff, M. Proc. Natl. Acad. Sci. USA. 2008; 105: 20846–20851Crossref | PubMed | Scopus (147)See all References). We would not have been able to obtain all these mechanistic insights if we were solely dealing with epistatic interactions between the 129 and C57BL/6 strains.As discussed in our manuscript (Chen et al., 2008xChen, Q., Yang, W., Gupta, S., Biswas, P., Smith, P., Bhagat, G., and Pernis, A.B. Immunity. 2008; 29: 899–911Abstract | Full Text | Full Text PDF | PubMed | Scopus (101)See all ReferencesChen et al., 2008), although the absence of Def6 leads T cells to aberrantly produce IL-17 and IL-21 under neutral conditions in vitro, the ability of this abnormality to lead to pathophysiology in vivo will only be unmasked in the presence of additional interactions. This is where strain-specific modifiers (or the DO11.10 transgene as discussed) play a role. We believe that this situation, where multiple genes need to cooperate to unleash the pathogenic predisposition in vivo, is reflective of the pathophysiology seen in human autoimmune disorders. We would also like to stress that the development of lupus-like syndromes by many single-gene deficiencies (even on a mixed 129-BL6 strain) has provided important information regarding pathways, which are altered in patients with autoimmunity (Jorgensen et al., 2004xJorgensen, T.N., Gubbels, M.R., and Kotzin, B.L. Curr. Opin. Immunol. 2004; 16: 787–793Crossref | PubMed | Scopus (45)See all ReferencesJorgensen et al., 2004). The importance of our work thus goes beyond Def6 per se because it suggests that abnormalities in pathways regulating IRF-4 may play a role in the development of autoimmunity. Indeed, we have recently found that abnormalities in IRF-4 function can be detected in T cells from other spontaneous mouse models of autoimmunity such as MRL/lpr mice. Obviously, more studies will be needed to corroborate this notion but discounting the potential role of the Def6-IRF4 pathway in autoimmunity seems premature.Regarding the concern with DO11.10 transgene, we believe the disease results from the enhanced potency by which the DO11.10 T cell receptor (TCR), in the absence of Def6, recognizes self-peptides presented in the context of I-Ad. Thus, we are not surprised that Def6trap/trap OT-II mice do not develop a similar disease to Def6trap/trap DO11.10 mice because presentation of peptides to the OT-II TCR in these mice occurs in the context of I-Ab, which may not be able to present the same self-peptides and/or result in the same degree of heightened responses by the OT-II TCR compared to the DO11.10 TCR. Indeed, although the DO11.10 TCR and the OT-II TCR recognize the same core epitope of the OVA peptide, their ability to recognize secondary TCR contact residues and peptide-flanking residues differs (Robertson et al., 2000xRobertson, J.M., Jensen, J.E., and Evavold, B.D. J. Immunol. 2000; 164: 4706–4712PubMedSee all ReferencesRobertson et al., 2000). We thus believe that not all TCRs can lead to pathophysiology in the absence of Def6 and that this effect may be restricted to TCRs in which the lack of Def6 “tips” the balance toward T cell activation. Testing this possibility will require crossing the Def6trap/trap mice to a large number of TCR-transgenic mice in different genetic backgrounds. In support of our idea that the pathophysiology observed in Def6trap/trap DO11.10 mice is due to a dual effect of alterations in TCR responsiveness and effector function, we have found that the deregulated expression of IL-17 and IL-21 by nontransgenic Def6trap/trap T cells is augmented by repetitive stimulation. Understanding the relationship between the antigen specificity of the T cells and the putative self-antigen(s) that elicits the autoimmune-like inflammatory response will require the identification of the self-antigen(s).Similar to Altman and Becart, we have also found that the lack of Def6 renders mice less susceptible to experimental autoimmune encephalomyelitis (EAE). However, we do not believe that such a result invalidates the findings in Def6trap/trap DO11.10 mice. Although both the pathophysiology observed in Def6trap/trap DO11.10 and that observed in EAE involve IL-17-producing T cells, these two models are drastically different. Indeed, although the arthritis in Def6trap/trap DO11.10 mice develops spontaneously in the absence of strong inflammatory stimuli, EAE is an induced model of autoimmunity, which requires immunization with complete Freund's adjuvant, i.e., a strong inflammatory milieu. As we have stated in our manuscript, the aberrant IL-17 and IL-21 production by Def6trap/trap T cells occurs under neutral conditions, i.e., in the absence of an inflammatory milieu. We favor the possibility that these are T helper 0 (Th0) cells that have only acquired a partial Th17 cell program and that this may actually further contribute to the pathophysiology because the regulatory loops that may be in place in “fully differentiated” Th17 cells to control their function may not be operative in these “partially differentiated Th17-like” cells. In support of this idea, in vitro Th17 cell differentiation occurs rather normally in the absence of Def6. We thus suspect that although Def6 controls IRF-4 function under Th0 cell conditions, its ability to inhibit IRF-4 will be blocked upon exposure to a Th17 cell-inducing inflammatory milieu. Thus, the effects of Def6 deficiency on IL-17-mediated autoimmune responses will vary depending on the precise pathway that has gone awry.Regarding the production of IL-17 in Figure 7A versus that in Figure 4A (Chen et al., 2008xChen, Q., Yang, W., Gupta, S., Biswas, P., Smith, P., Bhagat, G., and Pernis, A.B. Immunity. 2008; 29: 899–911Abstract | Full Text | Full Text PDF | PubMed | Scopus (101)See all ReferencesChen et al., 2008), the scales of the two figures are different because of the presence of Th17 cell-skewing conditions in Figure 4A. This may lead to over-interpretation of the magnitude of discrepancy between the two figures. We do not believe there is a significant difference between the two figures, and any differences in baseline IL-17 production may be due to the different purification methods. Nevertheless, it does not invalidate the key point of Figure 7A, which is to demonstrate that concomitant lack of IRF-4 can abolish any aberrant production of IL-17 and IL-21 detected in the absence of Def6.In summary, we believe that our study provides new insights into the pathophysiology of autoimmune disorders. As for many other molecules, much more work will be required to fully understand the role of Def6 in autoimmunity. We became involved in characterizing Def6-dependent pathways because of our interest in IRF-4, which has complex regulatory functions. We thus expect the biology of Def6 to exhibit an even more complex role in T cell physiology and pathophysiology given its additional effects on cytoskeletal reorganization, NFAT (Becart et al., 2008xBecart, S., Balancio, A.J., Charvet, C., Feau, S., Sedwick, C.E., and Altman, A. Immunity. 2008; 29: 704–719Abstract | Full Text | Full Text PDF | PubMed | Scopus (25)See all ReferencesBecart et al., 2008), and other targets.