CD4+ T cells expressing CD40 (Th40 cells) constitute a pathogenic T-cell subset that is necessary and sufficient to transfer autoimmune disease. We have previously demonstrated that CD40 signals peripheral Th40 cells to induce RAG1 and RAG2 expression, proteins necessary for the expression of T-cell receptor (TCR), leading to TCR revision. The dependency of TCR expression in the thymus on RAG proteins has long been known. However, despite numerous publications, there is controversy as to whether TCR expression can be altered in the periphery, post-thymic selective pressures. Therefore, a better understanding of TCR expression in primary peripheral cells is needed. We now show that the CD40 protein itself interacts with RAG1 and RAG2 as well as with Ku70 and translocates to the nucleus in Th40 cells. This indicates that the CD40 molecule is closely involved in the mechanism of TCR expression in the periphery. In addition, Fas signals act as a silencing mechanism for CD40-induced RAGs and prevent CD40 translocation to the nucleus. It will be important to further understand the involvement of CD40 in peripheral TCR expression and how TCR revision impacts auto-antigen recognition in order to effectively target and tolerize autoaggressive T cells in autoimmune disease.
During a normal immune response, CD4+ T cells recognize foreign antigen presented in the context of MHC II utilizing antigen-specific T-cell receptors (TCRs). The generation of a functional TCR repertoire involves rearrangement of the TCR genes through activation of RAG1 and RAG21 early in thymic T-cell development. RAG1 and RAG2 translocate to the nucleus and induce rearrangements of the V, D and J regions of the TCR β genes then V and J of the TCR α genes.2 It has been demonstrated that the RAG proteins can be re-induced in more mature thymocytes to allow TCR editing which is thought to promote positive selection.3 For thymic cells, it was believed that RAGs are permanently inactivated once T cells exit the thymus; however, RAG expression has since been reported in both recent thymic emigrants and mature peripheral T cells.4,5,6,7,8,9,10,11 This process, taking place in the periphery, is known as TCR revision 4,5,12,13 which is suggested to play a role in the generation of TCRs necessary to recognize an antigen previously not encountered by the host.7,14 However, as TCR revision occurs in the periphery and as there are no known peripheral selective pressures, thymic selective pressures are not encountered and therefore the potential for the generation of an auto-aggressive TCR is higher.7,8,14 Despite numerous publications demonstrating TCR revision,4,5,6,7,8,9,10,11,12,13,15,16 the concept remains controversial. The mechanism of TCR expression was demonstrated in thymocytes as well as by using artificial substrates in vitro; however, the mechanism of TCR expression and revision in the periphery has not been studied in detail. Therefore, it is possible that different criteria are necessary for the mechanism of TCR expression and revision in the periphery.
CD40 signals are known to be critical in the establishment, fulmination and perpetuation of autoimmunity.7,17,18,19,20,21,22,23,24,25,26 We determined that CD40 signals directly to CD4+CD40+ T cells (Th40) induced RAG expression, altering the surface expression of TCR Vα protein molecules in these primary peripheral T cells.7,15 That highly auto-aggressive peripheral T-cell subset is necessary and sufficient to transfer type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model of that disease.15,24,27,28,29 Also, diabetogenic T-cell clones, such as the BDC-2.5 and BDC-6.3 clones, are CD40+, while non-diabetogenic ones are CD40−,27 demonstrating the importance of CD40 in establishing disease. Importantly, change in Vα surface expression was induced in the highly diabetogenic BDC2.5 T-cell clone7 and the 3A9 T-cell hybridoma,15 demonstrating that CD40 signals induced altered TCR surface expression and not clonal expansion of specific T cells. Peripherally expressed TCR Vβ is also known to undergo revision4,8,9,10,16,30 and it was demonstrated that some of the revising cells were CD40+.10 CD40, together with B-cell receptor, is known to signal B cells to activate RAG1 and RAG2 resulting in B-cell receptor revision.31 While it is known how to induce RAG expression, it is not known how to prevent it. Considering that CD40 plays such a prominent role in autoimmune disease and much study is devoted to prevention of CD40 signaling, it will be important to understand all aspects of CD40 signaling outcomes in order to properly target only those signals that are detrimental.
In this manuscript, we reveal new insight into the mechanism of TCR expression in peripheral T cells and demonstrate the involvement of CD40 in that mechanism. We also show that such CD40 involvement is evident in thymocytes, indicating that the whole picture has not yet been elucidated in regard to TCR expression.
MATERIALS AND METHODS
NOD mice from Jackson Laboratories and Taconic were housed at the University of Colorado Denver, AAALAC-approved facility. All experiments were carried out under an IACUC-approved protocol.
Antibodies and reagents
Microbeads for cell-sorting were from Miltenyi Biotec (Auburn, CA, USA). CD40 antibodies 1C1032 and FGK4533 were produced in house. CD40 (sc-975 and sc-977), RAG1 (sc-363), RAG2 (sc-5600), Ku70 (sc-9033) and Ku86 (sc-9034) antibodies were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) Fas antibody (554256; clone Jo2) was from BD Biosciences (San Jose, CA, USA). Dulbecco's modified Eagle's medium was from HyClone (Waltham, MA, USA) and fetal calf serum from Gemini Bio-Products (West Sacramento, CA, USA). All other reagents were from Sigma-Aldrich (St. Louis, MO, USA).
T-cell purification and cell culture
Splenic Th40 cells and thymocytes from 10- to 16-week-old, female NOD mice (sorted magnetically as described23) and BDC2.5 and BDC 6.3 T-cell clones were cultured as described34 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 5 µM β-mercaptoethanol. Cells were either isotype treated or CD40 crosslinked using 10 µg/ml of biotinylated anti-CD40, 1C10, followed by 1 µg/ml of streptavidin. In cases where Fas was crosslinked, it was done either alone or simultaneously with CD40 using 10 µg/ml of biotin-anti-Fas antibody followed by 1 µg/ml of streptavidin.
Protein preparations and immunoprecipitations
Whole-cell, cytoplasmic and nuclear extracts were prepared as described.23 Immunoprecipitation with CD40 antibody FGK45 was done as described35 using 1.5×107 cells per sample and assaying one-third of the resulting eluates in western blots. Great care was taken to start with equal numbers of cells and to treat all samples exactly the same as it is not possible to assay an internal standard in immunoprecipitations.
Western blots were performed as described35,36 with antibodies diluted at 1∶500 in 6% non-fat dry milk (Carnation, Nestle USA Inc., Solon, OH, USA) in TBST except in the case of western blots for Ku proteins where 9% non-fat dry milk was used.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
CD40 interacts with both Ku70 and RAG1/RAG2.
Ku protein is involved in the processing and joining of DNA ends in the V(D)J-recombination process38 and CD40 is known to interact with Ku protein in B cells.39 Therefore, we determined whether CD40 could interact with Ku protein in T cells as well. Ku70, but not Ku86, was found to associate with CD40 in the unique Th40 effector cell population and, in contrast to what was reported in B cells,39 it was associated in a CD40 stimulation-dependent manner (Figure 1a). Since CD40 interacted with Ku70, which is important in RAG-mediated DNA rearrangement,38 we speculated that CD40 may associate with RAG1 and RAG2 as well. In western blots following CD40 immunoprecipitation, CD40 was found to associate with both RAG1 and RAG2 (Figure 1a). To confirm the CD40–RAG1/RAG2 co-immunoprecipitation, we immunoprecipitated RAG1 and performed western blots for CD40, RAG2 and Ku70. RAG1 co-immunoprecipitated an approximately 55-kD CD40 band (Figure 1b). The interaction between CD40 and RAG1 was stronger when performed this way rather than co-immunoprecipitating using an antibody to CD40. We have demonstrated that CD40 in actuality consists of several constellations composed of differentially glycosylated CD40 molecules.35 Therefore, several CD40 constellations35 may interact with RAG1, but the one immunoprecipitated by the CD40 antibody used here, dissociates from RAG1 during CD40 stimulation. CD40 constellations that stay associated with RAG1 throughout CD40 stimulation would be detected after RAG1 immunoprecipitation followed by western blot using an antibody that recognizes denatured CD40 protein. In addition, RAG1 co-immunoprecipitated RAG2 and Ku70 (Figure 1b).
RAG proteins are synthesized in the cytoplasm and are then translocated to the nucleus to perform their function. We further tested whether the CD40 interaction with RAG1 and RAG2 as well as Ku70 took place in the cytoplasm or in the nucleus. When CD40 was immunoprecipitated from cytoplasmic and nuclear extracts of treated NOD Th40 cells, it was apparent that CD40 interacted with RAG1 in the nucleus and that the interaction diminished after overnight CD40 engagement (Figure 1c). RAG2 interaction took place only in the cytoplasm.
CD40 stimulation alters both TCR Vα and Vβ mRNA expression.
Previously we demonstrated CD40-induced alterations in the TCR protein expressed on the surface of Th40 cells.7,15 However, altered surface expression of TCR in response to CD40 stimulation could result from already produced intracellular TCR protein being routed to the surface of the cell and therefore, any alteration seen on the surface is not necessarily due to actual alterations in TCR mRNA expression. Also, only four TCR Vα antibodies are available for flow cytometric staining, limiting the scope of detection. To address this we performed RT-PCR to achieve a more comprehensive picture of the TCR Vα species represented as well as to assess whether the actual TCR Vα mRNA species are altered by CD40 engagement. We began with the well characterized diabetogenic T-cell clone BDC2.5 and found that while other Vα species were present, the most prominent, canonical TCR Vα27 mRNA in isotype-treated BDC2.5 T cells was Vα1 (Figure 2a). When CD40 was stimulated overnight, the Vα mRNA expression pattern changed with several original mRNAs no longer detectable. Importantly, a new mRNA species, Vα3, appeared. In all, 10 different Vα species were represented in the BDC2.5 T-cell clone. To verify the results, we examined TCR expression in another diabetogenic T cell clone, BDC6.3 that also expresses CD40.27 Several Vα mRNAs were detectable in isotype treated cells (Figure 2b) and when CD40 was stimulated two new Vαs, Vα6 and Vα19, appeared. Vβ expression has been extensively studied3,4,5,8,9,10,12,13 and it was shown that a population of TCR Vβ revising T cells in germinal centers are CD40+.10 When TCR Vβ was assayed, it was evident that CD40 engagement led to alterations also in the TCR Vβ mRNA expression (Figure 2a and b).
Further, we determined whether primary, peripheral T cells would alter TCR mRNA expression in a similar manner. Like the T-cell clones, Vβ4+-sorted NOD Th40 cells demonstrated expression of several Vα mRNAs with several mRNA species disappearing, while several new ones appeared when CD40 was stimulated (Figure 2c). The time frame (18 h) in these experiments was such that cell proliferation would not account for the changes detected.23
CD40-induced increase in RAG1 and RAG2 expression is attenuated by Fas in autoimmune derived Th40 cells
We have previously shown the induction of RAG1 and RAG2 protein expression by CD40 stimulation of NOD splenic, total T cells as well as of diabetogenic T-cell clones.7 We also demonstrated that CD40 stimulation rescues Th40 cells from Fas-induced death.23,28 However, when Fas was engaged in addition to CD40, the cells did not proliferate as did cells stimulated by CD40 alone.23 Therefore, we confirmed RAG1 and RAG2 protein expression in the CD40-stimulated sorted Th40 cells and determined whether Fas could affect CD40-induced RAG expression. As RAG proteins exert their function in the nucleus, we determined the levels in nuclear extracts from treated cells. CD40 engagement induced a high level of RAG1 protein in the nucleus, while Fas engagement overnight had no effect (Figure 3a). Surprisingly, if Fas was engaged simultaneously with CD40, the CD40-induced RAG1 expression was prevented (Figure 3a). Low levels of RAG2 were induced when CD40 was engaged overnight (Figure 3a) and again, as for RAG1, Fas engagement simultaneously with CD40 prevented the RAG2 induction (Figure 3a).
CD40 localizes to the nucleus in Th40 cells
CD40 has been shown to localize to the nucleus of B cells where it binds to and stimulates the BLyS/BAFF promoter.40 Therefore, we determined whether CD40 localizes to the nucleus in Th40 cells as well, as would be indicated by the results in Figure 1c. Clearly, CD40 was present in nuclear extracts from NOD Th40 cells and increased in response to CD40 engagement (Figure 3b). While CD40 was present in the nucleus of all samples, CD40 stimulation itself significantly upregulated the level (Figure 3b). Several bands were present when detecting CD40 with a C-terminally reactive anti-CD40 antibody. Interestingly, when an N-terminally reactive anti-CD40 antibody was utilized, a larger band was better detected at approximately 55 kD (Figure 3b). We determined whether Fas engagement could impact nuclear levels of CD40. Fas engagement prevented the CD40-induced upregulation of CD40 in the nucleus (Figure 3b). CD40 is a highly abundant protein in Th40 cells from autoimmune NOD background.23 Therefore, we determined whether the presence of CD40 in the nucleus could be due to contamination from the cytoplasmic fraction. Another highly abundant protein, Hsp70, is ubiquitously expressed and localizes to the cytoplasm. When Hsp70 was analyzed in the cytoplasmic and nuclear extracts, it was localized exclusively to the cytoplasm, demonstrating the purity of the nuclear fraction (Figure 3b).
Fas engagement prevents CD40 from translocating to the nucleus
We determined whether Fas engagement had an impact on CD40 levels in the cytoplasm as well. CD40 was immunoprecipitated from cytoplasmic and nuclear extracts. Interestingly, Fas had no impact on the cytoplasmic levels of CD40 (Figure 3c). However, Fas co-engagement with CD40 almost completely prevented the translocation of CD40 itself into the nucleus (Figure 3c). The major band detected was a band at approximately 55 kD, as demonstrated by the N-terminally reactive antibody in Figure 3b. It is interesting that Fas prevented much of the CD40-induced nuclear expression of RAG1 and RAG2 as well as nuclear translocation of CD40 itself.
We have determined that Th40 cells occur at greatly expanded numbers in T1D15,24,27,28,29 and that Fas can be induced on Th40 cells.28 CD154 also is hyperexpressed in autoimmunity.41 Because FasL is activation-induced, it necessarily is hyperexpressed during autoimmunity. Thus, since Fas prevents CD40-induced RAG expression, when Th40 cells encounter antigen in the milieu of increased CD154 and FasL, the ability to alter TCR may be hampered. During foreign antigen exposure, this action would preserve the antigen-specific response. As antigen is cleared, FasL levels decrease allowing potential TCR revision if CD154 levels remain high. This would serve to expand the T-cell repertoire. As CD154 levels decrease, no further TCR alteration would occur. The problem within the autoimmune milieu is that self-antigen preserves CD154 and FasL expression. As we show here, Fas and CD40 co-engagement prevents RAG nuclear translocation thus preserving clonality.
CD40 associates with RAG1 and Ku80 in thymocytes
Considering that CD40 associates with RAG1 and RAG2 in peripheral Th40 cells, we determined whether a similar association could be found in thymocytes. Indeed, when CD40 was immunoprecipitated from NOD thymocytes, a clear association with RAG1 was found (Figure 4). Interestingly, the association was biphasic in that immediately ex vivo, there was a strong association but after 3 h of CD40 stimulation, that association was not evident. However, after overnight CD40 stimulation, there was again a strong association (Figure 4). When RAG2 was analyzed, there was no association of this molecule with CD40 (data not shown). We then determined whether there was an interaction between CD40 and Ku70/Ku80 in thymocytes. Interestingly, the major and sustained interaction was occurring with Ku80 (Figure 4). A low interaction with Ku70 was apparent in the unstimulated sample which disappeared when CD40 was stimulated (Figure 4).
Autoimmune disease ensues when self-tolerance by the immune system is breached. The etiology of that breach is not clearly understood, but numerous studies have identified the CD40–CD154 dyad as critical in the establishment and perpetuation of autoimmune disease.17,20,22,23,24,25,27 It was previously thought that once a T cell exits the thymus, the TCR expressed by that T cell was permanent. It was subsequently demonstrated that TCR revision occurs in peripheral T cells;4,7,9,10,15,16 however, despite numerous publications, there is still controversy about the veracity of this finding.
Many auto-antigens have been identified in T1D such as chromogranin A,42 the long sought after BDC2.5 auto-antigen, insulin B9-23,43,44,45 zinc transporter 8,46 IA-247 and Hsp60.48 While it is not known whether all these antigens drive disease concurrently, it is plausible that one antigen initiates breach of tolerance with subsequent antigens taking over the role of driver antigens once TCR revision and recognition of additional antigens occurs. Presumably, to breach self-tolerance, a T cell must acquire a self-reactive TCR. Thymic selective pressures ensure that T cells carry TCRs that are appropriate, but once a T cell reaches the periphery, there are no known selective pressures that guide TCR revision.
The dependency of TCR expression on RAGs has long been known; however, the experiments establishing this were performed on thymocytes and by using artificial substrates in vitro. Therefore, a better understanding of TCR expression mechanisms in primary peripheral cells is needed. In this manuscript, we begin to characterize the direct involvement of CD40 in TCR expression in peripheral auto-aggressive Th40 cells as well as a mechanism to prevent the induction of RAGs.
Clearly, CD40 interacts with both RAG1 and RAG2 in peripheral Th40 cells, but it is interesting that it interacts with RAG1 in the nucleus while interacting with RAG2 only in the cytoplasm. It is possible that RAG2 functions in the nucleus without the need for interaction with CD40 and that CD40 perhaps acts as a chaperone for RAG2 in the cytoplasm, releasing it once the proteins translocate to the nucleus. It should be noted that the CD40–RAG2 interaction was not as strong when assayed in the cytoplasmic fraction as seen in whole-cell extract. It is conceivable that this is due to separation of the fractions somehow disrupting the compartment in which most of the interaction takes place, at the interface of cytoplasm and nucleus. CD40 interaction with Ku70 took place mainly in the nucleus with an increase in the level of interaction as CD40 surface receptor was engaged. When thymocytes were examined, CD40 interacted strongly with RAG1 but not with RAG2. Also, conversely to the interaction observed in peripheral Th40 cells, CD40 mainly interacted with Ku80 in thymocytes instead of Ku70. This suggests that there are differences in the mechanisms of TCR expression comparing central thymic events and peripheral T-cell events. The data also suggest that CD40 may be closely involved in the actual processes executed by RAG1 and RAG2 leading to altered TCR expression, assigning yet another role to this versatile molecule. As it was shown previously in B cells that CD40 binds the BLyS/BAFF promoter,40 it will be important to determine whether CD40 binds specific DNA sequences in Th40 cells and, if so, what consequences such binding may have on transcriptional events.
CD40-induced alteration in TCR expression was evident in established T-cell clones and interestingly, within those clones, several other Vα mRNAs were present, suggesting that TCR revisions have occurred over time. In the BDC2.5 T-cell clone, the Vα1 mRNA expression was drastically decreased in response to CD40 stimulation, indicating that this TCR is less available to recognize antigen (Figure 2a). In fact, CD40-stimulated BDC2.5 T cells are significantly less diabetogenic.49 As TCR Vβ expression was also altered in response to CD40 signals, the data support a role for CD40 in the expression of both TCR Vα and Vβ; however, it is not clear whether actual recombination of the TCR loci occurred as we were not able to purify any excision circles (data not shown). This then suggests that more than one mechanism (i.e., actual rearrangement of the TCR DNA loci) exist, and that perhaps additional mechanisms preserve the remaining TCR gene segments such that a T cell is capable of utilizing several different TCRs as the situation may dictate. Regardless, actual immediate alteration of TCR expression was brought about by CD40. Additionally, the TCR revision in the T-cell clones was consistent at 24 h when resting clones were CD40-stimulated, but it would be interesting to elucidate whether different activation states of the cells as well as longer CD40 stimulation or culture would further alter TCR expression.
Another interesting aspect is that CD40-induced RAG expression was prevented by co-engagement of Fas. To our knowledge, this is the first demonstration of how to prevent RAG induction which could have important implications for maintaining certain TCR specificities in a situation requiring the complete elimination of an antigen. We have determined that Th40 cells occur at greatly expanded numbers in T1D15,24,27,28 and that Fas can be induced on Th40 cells.24 CD154 also is hyperexpressed in T1D.41 Because FasL is activation-induced, it necessarily is hyperexpressed during autoimmunity. Thus, since Fas prevents CD40-induced RAG expression, when Th40 cells encounter antigen in the milieu of increased CD154 and FasL, the ability to alter TCR may be hampered. During foreign antigen exposure, this action would preserve the antigen-specific response. As antigen is cleared, FasL levels decrease allowing potential TCR revision if CD154 levels remain high. This would serve to expand the T-cell repertoire. As CD154 levels decrease, no further TCR alteration would occur. The problem within the autoimmune milieu is that self-antigen preserves CD154 and FasL expression. As we show here, Fas and CD40 co-engagement prevents RAG nuclear translocation thus preserving clonality.
Several CD40 bands were present in the nucleus of the Th40 cells. While it remains to be determined whether the bands in the nuclear extracts represent different glycoforms of CD40, we have demonstrated the presence of such bands in whole-cell extracts and raft fractions previously.35
The fact that thymocytes demonstrated CD40 association with RAG1 but not RAG2 and stronger association with Ku80 than with Ku70 is intriguing. It indicates that although the mechanism of TCR expression has been well studied in thymocytes, there may be additional components of that mechanism that are unknown or perhaps there is a secondary mechanism that can be employed for TCR expression.
Our present data indicate a need for a better understanding of the processes that lead to TCR revision in the periphery. Are those processes necessarily the same as those in the thymus? Are there selective pressures in the periphery? Answers to these questions may explain the etiology of the breach of tolerance in autoimmunity.
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This work was supported by grants from the American Diabetes Association, the Juvenile Diabetes Research Foundation, the Kleberg Foundation and an R01 from NIDDK awarded to DHW. We thank Dr Kathryn Haskins and Dr Rocky Baker, University of Colorado at Denver and National Jewish Health, for generously supplying BDC T-cell clones.
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Journal of Neuroimmunology (2019)
The Journal of Clinical Endocrinology & Metabolism (2019)
CD40-targeted peptide proposed for type 1 diabetes therapy lacks relevant binding affinity to its cognate receptor. Reply to Pagni PP, Wolf A, Lo Conte M et al [letter]
Clinical Immunology (2018)
The Journal of Clinical Endocrinology & Metabolism (2018)