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Differences with CARMA1-deficient mice.

Posted by benmed on 28 May 2009 at 12:27 GMT

To the editors:
Barnes et al. have provided interesting data on the role of CARMA1 in regulatory T cell (Treg) development utilizing a mouse with a mutant form of CARMA1 (so called king mutant) [1]. Their work confirms much of our findings in a traditional CARMA1 knockout mouse (CARMA1-/-) [2,3], and those of a different group [4]. One important difference in our report is that we did not find nearly as many peripheral Tregs as Barnes et al., nor did we see significant formation of adaptive Tregs after the induction of inflammation. One possible reason for this discrepancy is that the mutant form of CARMA1 in the king mouse used by Barnes et al. may retain some CARMA1 activity, allowing the mouse to have enough TCR induced signaling to develop some peripheral/adaptive Tregs. Although the authors did not report detectable CARMA1 protein in cell lysates from spleen and lymph node, this does not totally rule out low level protein expression. A second possibility is that the murine cytomegalovirus infection model used by Barnes et al. to induce adaptive Tregs provides a stronger stimulus to form Tregs than the allergic inflammation model we used in our studies. To better explore these possibilities we repeated the in vitro induction of Tregs performed by Barnes et al. (Figure 4 in their paper) using cells from our CARMA1-/- mice. We isolated CD4+ T cells from wild-type C57BL/6 and CARMA1-/- mice (in a C57BL/6 background), and then stimulated the T cells with anti-CD3/28 with IL-2 and TGFbeta. Although we were able to induce some Foxp3 expression in the CD4+ T cells isolated from the CARMA1-/- mice (see http://www.plosbiology.or...), the percentage of Foxp3+ cells induced was 2-fold lower than in CD4+ T cells from wild-type control mice. This differs from the results reported by Barnes et al. which reported induction of a similar percentage of Foxp3+ cells in cells taken from the king mutant mice when compared to wild-type cells. Similar to king mutant T cells., the CARMA1-/- T cells also underwent fewer rounds of proliferation following activation. These data suggest that although it is indeed possible to induce Foxp3+ expression in CARMA1-/- T cells with very strong stimuli, the defect in Treg formation may be more profound with complete absence of CARMA1 than seen in the king mutant mice. We propose that CARMA1 effectively serves to amplify TCR signaling and reduces the threshold for Treg development for both thymic and peripheral Tregs. The threshold may differ for these two Treg populations, but we do not believe that there is a true differential requirement for CARMA1 signaling for their formation as suggested in the manuscript and accompanying editorial [5]. Thus, it seems that the observed differences in Treg development seen in our paper when compared to Barnes et al. may be due to residual CARMA1 signaling in the king mutant mice.
Benjamin D. Medoff, MD1
Ramnik Xavier, MD2

1Center for Immunology and Inflammatory Diseases, Pulmonary and Critical Care Unit, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, USA

2Center for Computational and Integrative Biology, Gastrointestinal Unit, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA

1. Barnes MJ, Krebs P, Harris N, Eidenschenk C, Gonzalez-Quintial R, et al. (2009) Commitment to the regulatory T cell lineage requires CARMA1 in the thymus but not in the periphery. PLoS Biol 7: e51.
2. Medoff BD, Seed B, Jackobek R, Zora J, Yang Y, et al. (2006) CARMA1 is critical for the development of allergic airway inflammation in a murine model of asthma. J Immunol 176: 7272-7277.
3. Medoff BD, Sandall BP, Landry A, Nagahama K, Mizoguchi A, et al. (2009) Differential requirement for CARMA1 in agonist-selected T-cell development. Eur J Immunol 39: 78-84.
4. Molinero LL, Yang J, Gajewski T, Abraham C, Farrar MA, et al. (2009) CARMA1 Controls an Early Checkpoint in the Thymic Development of FoxP3+ Regulatory T Cells. J Immunol 182: 6736-6743.
5. Heller K (2009) Peripheral Regulatory T Cells Take the Road Less Traveled. PLoS Biology 7: e65.

No competing interests declared.

RE: Differences with CARMA1-deficient mice.

Hoebe replied to benmed on 02 Jun 2009 at 14:20 GMT

We thank the authors for their interest in our paper, but we respectfully disagree with their suggestion that our finding—the differential requirement for CARMA1 in thymic versus peripheral Treg formation [1]—simply reflects increased TCR signaling thresholds for Foxp3 induction in the absence of CARMA1 in both thymocytes and T cells. Based on the available data, we believe it is premature to conclude that CARMA1-deficient mice lack Foxp3+ thymocytes due to altered signaling thresholds, or lineage specification, or both. In either case, the phenotype of Carma1k/k and Carma1-/- mice clearly demonstrates a selective requirement for CARMA1-dependent signaling in the thymus.

In their online commentary, Medoff and Xavier present data that confirms our finding that Foxp3 can be induced in peripheral CARMA1-deficient CD4+ T cells, but with less efficiency than in wild-type cells. Indeed, in Figure 4B we demonstrate that the TCR signaling threshold for in vitro Foxp3 induction is increased in Carma1k/k CD4+ T cells [1]. In vivo, it is known that certain components of the gut flora can promote peripheral Treg accumulation [2]. Despite the increased TCR signaling threshold, a substantial Treg cell population exists in the gut of healthy Carma1k/k mice (see Table 1) [1]. This finding demonstrates that Carma1k/k Treg cells can accumulate in the gut under steady-state conditions, not only after the strong systemic stimulus of MCMV infection. Additionally, we note that Treg expansion in Carma1k/k splenocytes did not occur at 7 or 14 days after LPS injection or Listeria monocytogenes infection, and seems not to be simply induced by any inflammatory stimulus (unpublished data). Developing thymocytes with self-reactive TCRs might need to activate CARMA1 in order to avoid deletion, and we note this possibility in the discussion (although positive and negative selection of conventional thymocytes occurs normally without CARMA1-dependent signaling [3,4]). Alternatively, Treg precursors may require CARMA1-dependent activation of NF-B to develop. We found that CD4 SP CD25+ thymocytes (a population that contains Treg precursors) are absent in the Carma1k/k thymus (see Figure 3A) [1], and elegant studies by Molinero and colleagues further support this possibility [5]. Notably, they find that RIP-OVA mice reconstituted with OT-II; Carma1-/- bone marrow cells, in which developing thymocytes encounter cognate antigen expressed by medullary thymic epithelial cells, also lack thymic Treg cells [5]. One might expect at least a partial rescue of Foxp3 induction in these mice if reduced TCR signal strength were solely responsible for the thymic Treg deficiency. In sum, the available data fully support our conclusion that CARMA1 is essential for thymic Treg development, but not peripheral Foxp3 induction.

Medoff and Xavier also speculate that Carma1k/k mice retain residual CARMA1 activity based on somewhat more reduced peripheral Treg cell numbers in their Carma1-/- mice [6] and in their commentary they show a figure with slightly less efficient in vitro Foxp3 induction in Carma1-/- CD4+ T cells. Again, we respectfully disagree with this suggestion. First of all, Carma1k/k mice have identical numbers of Foxp3 expressing CD4+ splenocytes as reported for the Carma1-knockout mice from Molinero and colleagues [5]. Secondly, subtle changes in serum or cell culture media conditions can greatly affect in vitro Foxp3 induction [7,8], as does the presence of antigen experienced CD4+ T cells (which secrete IFN and other polarizing cytokines upon activation with CD3/28) [9]. To draw conclusions with regard to Foxp3 induction in Carma1k/k versus Carma1-/- CD4+ T cells, Medoff and Xavier would need to compare naive CD4+ T cells from these mouse strains in the same assay (our mice are available through MMRRC). Extensive analysis of Carma1k/k mice (see Figures S2 and S3) [1] showed that they phenocopy Carma1-/- mice [3], and differed from mice homozygous for the hypomorphic unmodulated mutation [10] (e.g. compare the basal serum Ig concentrations). Therefore, there is no evidence to contradict the conclusion that Carma1k/k T cells completely lack CARMA1 activity.

Although Medoff and Xavier conclude in their original paper [6] that CARMA1 has an essential role in the development of both adaptive and natural Treg cells, their enclosed figure confirms our data and conclusions that conversion of naive CD4+ T cells into Foxp3+ Treg cells can still occur (albeit with less efficiency) in Carma1-deficient mice.

Michael Barnes and Kasper Hoebe

1. Barnes MJ, Krebs P, Harris N, Eidenschenk C, Gonzalez-Quintial R, et al. (2009) Commitment to the regulatory T cell lineage requires CARMA1 in the thymus but not in the periphery. PLoS Biol 7: e51.
2. Mazmanian SK, Round JL, Kasper DL (2008) A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453: 620-625.
3. Hara H, Wada T, Bakal C, Kozieradzki I, Suzuki S, et al. (2003) The MAGUK family protein CARD11 is essential for lymphocyte activation. Immunity 18: 763-775.
4. Jost PJ, Weiss S, Ferch U, Gross O, Mak TW, et al. (2007) Bcl10/Malt1 signaling is essential for TCR-induced NF-kappaB activation in thymocytes but dispensable for positive or negative selection. J Immunol 178: 953-960.
5. Molinero LL, Yang J, Gajewski T, Abraham C, Farrar MA, et al. (2009) CARMA1 controls an early checkpoint in the thymic development of FoxP3+ regulatory T cells. J Immunol 182: 6736-6743.
6. Medoff BD, Sandall BP, Landry A, Nagahama K, Mizoguchi A, et al. (2009) Differential requirement for CARMA1 in agonist-selected T-cell development. Eur J Immunol 39: 78-84.
7. Maynard CL, Hatton RD, Helms WS, Oliver JR, Stephensen CB, et al. (2009) Contrasting roles for all-trans retinoic acid in TGF-beta-mediated induction of Foxp3 and Il10 genes in developing regulatory T cells. J Exp Med 206: 343-357.
8. Veldhoen M, Hirota K, Christensen J, O'Garra A, Stockinger B (2009) Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells. J Exp Med 206: 43-49.
9. Hill JA, Hall JA, Sun CM, Cai Q, Ghyselinck N, et al. (2008) Retinoic acid enhances Foxp3 induction indirectly by relieving inhibition from CD4+CD44hi Cells. Immunity 29: 758-770.
10. Jun JE, Wilson LE, Vinuesa CG, Lesage S, Blery M, et al. (2003) Identifying the MAGUK protein Carma-1 as a central regulator of humoral immune responses and atopy by genome-wide mouse mutagenesis. Immunity 18: 751-762.

No competing interests declared.