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Disruption of the CDCD40 ligand system prevents an oxygen-induced respiratory distress syndrome. Am J Pathol , ; — CD40 plays a crucial role in lipopolysaccharide-induced acute lung injury. Up-regulation of surfactant protein production in a mouse model of secondary pulmonary alveolar proteinosis. Support Center Support Center. External link. Please review our privacy policy. B cells are involved in the production of proteins called antibodies or immunoglobulins that help protect the body against infection.
There are several classes of antibodies, and each one has a different function in the immune system. B cells are able to mature into the cells that produce immunoglobulin M IgM without any signals from other cells. In order for B cells to mature into the cells that produce antibodies of a different class, the CD40 receptor must interact with CD40 ligand. When these two proteins are connected, they trigger a series of chemical signals that instruct the B cell to start making immunoglobulin G IgG , immunoglobulin A IgA , and immunoglobulin E IgE.
CD40 ligand is also necessary for T cells to interact with other cells of the immune system, and it plays a key role in T cell differentiation the process by which cells mature to carry out specific functions. These mutations lead to the production of an abnormal CD40 ligand or prevent production of this protein. Mutations in the CD40LG gene also impair the T cell's ability to differentiate and interact with immune system cells. People with X-linked hyper IgM syndrome are more susceptible to infections because they do not have a properly functioning immune system.
To address this issue, iMB cells were generated by transferring iGB-lo and iGB-hi cells on day 2 of culturing, when these cells had expanded to similar levels. These data indicate that the bifurcated B mem cell fate was not determined by different levels of cell proliferation. However, they also demonstrated that the frequency of the CD80 hi population in iMB-hi cells increased as the culture period of iGB-hi cells became longer i. Regardless of their BCR isotype, iMB-lo cells preferentially differentiated toward GC B cells, as clearly seen on day 2, whereas iMB-hi cells preferentially differentiated into plasmablasts or PCs, becoming evident on day 3.
The behavior of the iMB-mid cells was intermediate Figure 4a,b and Figure 4—figure supplement 1. Next, we investigated the in vivo fate of these iMB cells in response to immunization with a cognate antigen. Four days after immunization, most donor-derived, NP-binding plasmablasts were derived from iMB-hi cells.
Taken together, these data indicate that the quantity of CD40 signaling in B cells determines their differentiation fate toward phenotypically and functionally distinct B mem cell subsets. The recipient mice were then immunized with NP-CGG in alum and analyzed 4 or 10 days after immunization. The mean of the values in each group is indicated by a horizontal bar f. All data are representative of two independent experiments.
On the basis of the data described above, it is likely that the CD40 signaling quantity is primarily determined by the expression level of CD40L on cognate T cells. As CD40L on T cells was shown to be induced in an antigen-dose-dependent manner Jaiswal and Croft, , it seemed plausible that the quantity of antigen presented on B cells would determine the expression level of CD40L on cognate T cells, which in turn determines the differentiation fate toward each B mem subset.
CD40L expression on the T cells was rapidly induced and its levels positively correlated with antigen dose Figure 5b , as was also the case for CD80 expression on B cells on day 2 Figure 5c.
Data are shown as in c. The mean of the values in each group is indicated by a horizontal bar f, h, j. All data are representative of two independent experiments except f, h, j , where data from two independent experiments are combined. During an immune response, B cells expressing a high-affinity BCR would take up more of the cognate antigen, and thus would present a larger amount of antigenic peptide—MHC complex to cognate T cells Schwickert et al.
B cells from B hi ki and B ki mice, expressing discriminating allotypic markers, were co-transferred into B6 mice, and immunized with NP-CGG in alum. These data indicated that B cells with higher antigen affinity preferentially differentiate into CD80 hi B mem cells rather than CD80 lo B mem cells, probably through more extensive antigen presentation to cognate T cells, which results in greater induction of CD40L.
In any case, BCR affinity appears to be a primary determinant for the differential B mem subset development that is dependent on CD40 signaling quantity. We next investigated the CD40 signaling mechanisms that are responsible for the development of the CD80 hi B mem cell subset.
Furthermore, knockdown of Rel and Rela gene expression in iGB-hi cells resulted in downregulation of surface CD80 expression Figure 6c,d and Figure 6—figure supplement 1a. We next examined whether a similar signaling pathway is involved in the generation of B mem cells.
The knockdown of Rela selectively suppressed the development of CD80 hi B mem cells among the B cells that had responded to an immunized antigen Figure 6g,h and Figure 6—figure supplement 1c. The number on each histogram indicates gMFI. Tubulin and Lamin B were used as loading controls for cytoplasmic or nuclear proteins, respectively. Splenocytes from these mice were analyzed by FCM at 10 days after immunization. The mean of the values in each group is indicated by a horizontal bar f, h.
The expression of CD80 on these cells was analyzed on day 5, and shown as in a. The CD80 expression on these cells is shown as in c. Expression of the indicated proteins in these cells was analyzed by FCM. The shadowed histograms represent the cells cultured with medium alone. At 10 days after immunization, splenocytes from the recipients were analyzed by FCM. Two weeks after the transfer, spleen cells of the recipient mice were analyzed by FCM.
The mean of the values in each group is indicated by a horizontal bar g, j. All data are representative of two independent experiments except j , in which data from two independent experiments are combined. The regulation of the bidirectional response of B mem cells, either to PCs or to GC B cells upon secondary antigen challenge, has recently been explained by defining the functionally different B mem cell subsets.
However, our in vitro culture system without specific antigens clearly showed that the distinct fates of these B mem subsets upon re-stimulation are not determined by the BCR affinity or isotype, although they might be affected by slightly different levels of CD40 expression on CD80 hi and CD80 lo B mem cells Figure 2—figure supplement 1j. Thus, cell status represented by, for example, transcriptomic or epigenetic profiles may largely define the function of each B mem subset He et al.
As CD40L in the recipient mice was essential for the generation of both types of B mem cells from transferred B cells, we hypothesized that the generation of either CD80 hi or CD80 lo B mem cells is determined by a difference in the quantity of CD40 signaling.
This hypothesis was strongly supported by our simplified experimental system, which enables in vivo generation of memory-like B iMB cells without immunization from naive B cells cultured on feeder cells 40LB and transferred into mice. The commitment to differentiate into either CD80 hi or CD80 lo iMB cells, as determined by the different quantities of CD40 signaling in B cells, was made within two days of culture when the proliferation did not differ among the conditions, and partially made in just one day when CD80 expression was hardly detectable on B cells.
Therefore, it seems that the quantity of CD40 signaling in B cells directs cellular programming, which determines the differentiation into distinct B mem cell subsets. Therefore, our data describing the in vivo differentiation of iGB cells into B mem -like iMB cells probably explain the mechanism for the induction of bidirectional B mem cell differentiation upon T-B interactions, during both the early phase and the GC phase of the primary immune response.
Our additional data suggest that, in the early phase, B cells expressing BCR with higher affinity to antigen present more antigenic peptide to cognate T cells, thus inducing CD40L on these T cells more strongly so that they acquire stronger CD40 signaling than lower affinity B cells. Top In the pre-GC phase of the primary response, the BCR affinity to antigen or the amount of available antigen determine the quantity of antigen presentation to T cells, and the extent of the induction of CD40L on T cells.
Thus, the strength of CD40 signaling in B cells is determined by the interacting T cells, which then directs the differentiation fate to distinct B mem subsets: relatively stronger CD40 signal commits B cells towards CD80 hi B mem cells, whereas weaker CD40 signal commits B cells towards CD80 lo B mem cells. Supposing that T FH cells are heterogeneous in terms of cytokine production Weinstein et al.
Thus, a combination and integration of signaling pathways, one from the strong CD40 stimulation, the other from a particular cytokine, and maybe more, may ultimately determine the fate of GC B cells. In addition, we previously reported that iGB cells that were secondarily cultured with IL preferentially develop into bone marrow PCs but not B mem cells in vivo after adoptive transfer Nojima et al.
Given that low-affinity B cells do enter into the GC reaction and could maintain their low affinity even after mutation, why are only few CD80 lo B mem cells generated during the GC phase? It has been shown that half of GC B cells undergo apoptosis every 6 hr Mayer et al. These data suggest that weaker CD40 signaling in the GC phase may not be enough to prevent the apoptosis of B cells and therefore could fail to induce CD80 lo B mem cell development. Alternatively, lower-affinity B cells may be excluded from the GC in a competitive situation Schwickert et al.
Previous studies and our observations imply transcriptomic predisposition of CD80 hi and CD80 lo B mem cells that may account for their preferential differentiation upon re-stimulation, and suggest that the transcriptomic statuses may be established during the primary response as proposed for the T cell memory. What would be the survival advantage for individuals of generating bifurcated B mem cell subsets upon infection?
On the other hand, CD80 lo B mem cells mainly express low-affinity BCRs, but have a greater proliferative potential and preferentially generate secondary GC upon reencounter with antigen, when they can further diversify their BCR repertoire. Therefore, CD80 lo B mem cells, unlike high-affinity CD80 hi B mem cells, can cope with a broader array of epitopes that may be generated by pathogens through mutations. Taken together, robust and rapid high-affinity Ab production by CD80 hi B mem cells eliminates the majority of reinfecting pathogens, whereas pathogens that escape destruction because of epitope changes resulting from mutations are eliminated by Abs derived from CD80 lo B mem cells that evolved in GCs.
Thus, if we could dissect the CD40 signaling pathways that direct differentiation into CD80 hi B mem cells and those promote cell proliferation or survival, selective suppression of the former pathways during vaccination might convert the generation of CD80 hi B mem cells into CD80 lo B mem cells that would eventually produce broadly reactive Abs. Alternatively, additive stimulation of CD40 with proper timing would facilitate the generation of CD80 hi B mem cells that rapidly produce highly specific Abs upon actual infection.
All of the following mice were backcrossed to B6 or congenic B6 CD Mice were immunized i. Sex-matched, 7-week-old or older mice were used for all experiments. All mice were bred and maintained under specific pathogen-free conditions, and all animal experiments were performed under protocols approved by the Animal Care and Use Committee of the Tokyo University of Science Approval No. The data were analyzed using Flowjo Tree Star. For intracellular staining, cells were fixed and permeabilized using a Foxp3 staining kit eBioscience before staining.
Sorted T cell subsets 2. Four weeks after the immunization, B mem cells were purified from pooled spleens through two-step negative sorting and final positive sorting: cells stained with biotinylated antibodies against CD4, CD8a, CD11b, CD43, CD To inhibit CD40 signaling, mice were injected s. To activate CD40 signaling, mice were injected i. Production of cell lines, iGB cell culture and iMB cell generation were performed as previously described Nojima et al.
Splenocytes from mice that had been transfected with iGB cells 2 week earlier were analyzed by FCM to estimate the numbers of donor-derived CD Retroviral transduction of iGB cells was performed as previously described Haniuda et al. Retroviral transduction of in-vivo-activated primary B cells and their transfer into mice were performed as previously described Inoue et al. In brief, B hi ki mice were injected i. Cultured B cells were spin-infected with retroviral vectors and further cultured for 3 hr.
The cell lysates were centrifuged and supernatants were collected as the cytosolic fraction. The lysates were centrifuged and the supernatants were used as the nuclear fractions. Gene expression levels were determined by the relative standard curve method and normalized to that of Gapdh. All data generated or analysed during this study are included in the manuscript and supporting files.
Source data files have been provided for Figures In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "The quantity of CD40 signaling determines the differentiation of B cells into functionally distinct memory cell subsets" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Tadatsugu Taniguchi as the Senior Editor. The reviewers have opted to remain anonymous. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
As you might see from their reports, all three reviewers have found the manuscript interesting and worthy of inviting for a re-submission. Moreover, they have raised several points that if addressed will certainly increase the general impact. The consensus was that there needs to be an effort to better characterize this population not only in terms of markers such as surface markers or Bcl6 and Blimp but also in terms of their time of generation in relation B cell division or after transfer.
It will also be beneficial, if within the reach of the authors, to validate their observations in an infectious model and not only with model antigens. In parallel, the text also needs to be softened in several parts. In line with this, the manuscript will also benefit from a robust discussion on how the in vitro system does not completely recapitulate the results observed in vivo. This has to be addressed and discussed extensively. As the manuscript stands, the authors views on this point and the role of CD40 in B cell differentiation appears to contrast with some of the current literature, and this needs to be discussed thoroughly.
Please find below the full reviewers' comments so that you get an idea of their different views as well as experimental work that you might carry out to add to accommodate their concerns.
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