ABSTRACT
Tregs must be activated to suppress immune responses, but the transcriptional program controlling Treg activation remains incompletely understood. We previously found that Treg-specific deletion of the chromatin remodeling factor Brg1 impairs Treg activation and causes fatal autoimmunity in mice. Here, using a method that allows gene KO to be reversed in a Tamoxifen-dependent manner, we addressed whether reinstating Brg1 expression in the defective Tregs in the sick mice could restore Treg function, and if so, whether such Tregs could stop and resolve the fatal inflammation. We found that reexpressing Brg1 unexpectedly converted the defective Tregs into highly potent “SuperTregs”, which effectively rescued the dying mice. Remarkably, Brg1 reexpression in as little as 8% of the Tregs sufficed for the rescue in some cases. Brg1-deleted Tregs in the inflamed mice experienced excessive cytokine stimulation, became hyperactivated upon Brg1 reexpression and then deactivated as the inflammation subsided, suggesting that BRG1 acted in conjunction with inflammation to induce and maintain the SuperTreg phenotype. These data illustrate the power of reversible KO models in uncovering gene functions, and suggest a novel therapeutic strategy for IPEX(-related) disorders that exploits the defective Tregs and the inflammatory environment preexisting within the patients.
INTRODUCTION
Tregs are potent suppressors of immune responses1,2, and defects in Treg development and/or function can underlie devastating autoimmune disorders3–5. The majority of Tregs under physiological conditions are naïve, with little overt suppressor activity. Upon antigen and cytokine stimulation, naïve Tregs become activated and differentiated into effector cells expressing various cell surface and soluble molecules that mediate suppressor function1,6–9. It is therefore of great interest to characterize the mechanisms controlling Treg activation and effector function.
Brg1 is the catalytic subunit of the chromatin remodeling BAF (mSwi/snf) complex 10, which plays diverse roles in the immune system 11–16. We have identified Brg1 as a crucial regulator of Treg activation17. Specifically, Brg1 deletion in Tregs impairs Treg activation, concomitant with the onset of inflammation. Remarkably, as the inflammation progresses, Tregs become increasingly activated, but the activation levels are unable to catch up with the severity of inflammation, which fails to stop the progression of the disease, leading ultimately to the death of the KO mice. These data indicate that BRG1 acts to sensitize naïve Tregs to inflammatory cues, thus allowing them to promptly and effectively suppress autoimmunity17.
Our study described above is focused on the role of Brg1 in naïve Tregs in the healthy mice. To extend this line of investigation, we sought to determine whether in the Brg1 KO mice that have developed severe inflammation, reinstating Brg1 expression in the partially activated, Brg1-deleted Tregs could restore Treg function and even rescue the dying mice. There is no reason a priori to assume positive answers to these questions. To regulate target genes, Brg1 must act in conjunction with other transcription regulators including sequence-specific transcription activators and histone modifying enzymes. These other regulators provide the informational context for Brg1 function, which can dictate the outcome of Brg1 expression. As this context might differ in naïve vs. (partially) activated Tregs, it is difficult to infer, based on the role of Brg1 in naïve Tregs, the outcome of Brg1 reexpression in the partially activated, Brg1-deleted Tregs. Even if Brg1-reexpression can restore Treg function, it is unclear whether this is sufficient to resolve the severe inflammation and rescue the mice, given that the inflammation may have become overwhelming and/or tissue damages irreversible by the time of Brg1 reexpression. These considerations are not only important for understanding Brg1 function, but also have therapeutic implications for human autoimmune diseases resulting from Treg defects (see Discussion).
We have addressed these issues using LOFT, a reversible gene targeting strategy we previously developed18. The results reveal dramatic therapeutic effects of Brg reexpression on the sick mice, which is of both biological and clinical interest.
RESULT
The LOFT strategy for Brg1 reversible KO (rKO)
Treg-specific Brg1 deletion followed by conditional restoration of Brg1 expression was achieved with the LOFT method 18 that requires a pair of alleles of the target gene (Brg1 in the current study): a floxed allele (Brg1F) and a reversibly trapped allele that is a null by default but can be conditionally converted to a wild-type (WT) allele. The latter allele is designated ΔR, where R denotes ‘reversible’ (Figure 1A, top left). The key component of the ΔR allele is a gene-trap cassette consisting of the neomycin phosphotransferase (Neo) and Ires-GFP. This cassette was inserted into intron #9 (Fig. 1B), thus capturing the upstream exon #8 (E8) to produce a fusion protein between the N-terminal 531 aa of BRG1 protein and the neomycin phosphotransferase, the former moiety being inactive, and the latter serving as the selection marker for successfully targeted embryonic stem (ES) cells. In addition, GFP was co-expressed with the fusion protein, which reported the status of ΔR allele. The gene-trap cassette was flanked by FLP recombination target (FRT) sites, allowing for conditional cassette excision in the presence of the FLP recombinase. The removal of the gene-trap cassette restores the expression of full-length BRG1, concomitant with the loss of GFP expression. Thus, in Brg1F/ΔR mice that also expressed Cre in Tregs (from the FoxP3YFP-Cre allele) and FlpoER (from the ubiquitous CAG promoter inserted into R26 locus), Brg1 expression is constitutively eliminated in Tregs but reinstated upon Tamoxifen (TAM) administration, the latter event reported by elimination of GFP fluorescence (Figure 1A, middle and bottom).
Characterization of the ΔR allele
We inserted the gene trap cassette into the ES cells using the traditional gene targeting method (Fig. 1B) to generate Brg1+/ΔR; R26CAG-FlpoER mice. PCR analysis confirmed that the mice carried ΔR (Fig. 1C). Following oral gavage of a full dose of TAM (500 ug/g, once daily for two consecutive days, termed the “full dose” regimen hereafter), GFP signal in the Tregs in the peripheral blood decayed gradually, disappearing almost completely on Day 7 after the gavage (Fig. 1F, left), the kinetics being comparable to that in the conventional CD4 cells (Fig. 1F, right). Finally, we bred the rKO mice by introducing Brg1Fand FoxP3YFP-Cre into the Brg1+/ΔR; R26CAG-FlpoER mice. As FoxP3YFP-Cre is located on the X chromosome randomly inactivated in females, the genotypes of rKO mice are gender-specific, being Brg1F/ΔR; FoxP3YFP-Cre; R26 CAG-FlpoER/CAG-FlpoER for males and Brg1F/ΔR; FoxP3YFP-Cre/YFP-Cre; R26 CAG-FlpoER/CAG-FlpoER for females. The rKO mice were fed with a low dose of TAM (12 ug/g, once only, termed the “low dose” regimen hereafter) to reverse Brg1 KO in a fraction of Tregs. The GFP+ and GFP- Treg subsets were then isolated by FACS. As expected, the gene trap cassette was lost in the GFP- subset (Fig. 1D) concomitant with the emergence of the functional Brg1 transcript (which contained E14-15; Fig. 1E). These data validated the functionality of the ΔR allele.
Dramatic effects of Brg1 reexpression on rKO mice
The severity of the inflammatory phenotypes was somewhat variable in different rKO mice, and tended to correlate with the frequencies of effector/memory-like (E/M) CD44hiCD62Llo CD4 cells in the peripheral blood. For convenience, we used the frequencies of the E/M CD4 cell at 3 weeks of age to divide the rKO mice into two groups: rKO1 (>65%) and rKO2 (< 65%), whose phenotypes are described in Fig. 2A-E and Fig. 2F-H, respectively. Of note, the majority (85%) of the rKO mice belonged to the rKO1 category.
We found that the rKO1 mice had developed severe inflammatory signs (including skin lesions, lymphoid organ enlargement and runting) by 3 weeks of age and died before Day 41, the median survival being 31 days (Fig. 2A, red line). To determine the consequences of Brg1 reexpression in rKO1 mice, mice were given TAM (full dose) around 3 weeks of age, namely ∼10 days before the predicted median death date. Remarkably, 55% of the mice (11/20) were rescued from death (Fig. 2A). Gross signs of inflammation disappeared within two months after TAM administration (Fig. 2B), and by 120 days, the runted mice had fully caught up in weight and size, revealing striking resilience of the mice (Fig. 2C). To directly examine the kinetics of inflammation resolution, we monitored the proportion of effector/memory-like (E/M, CD44hiCD62Llo) and naïve-like (Naïve, CD44loCD62Lhi) CD4 cells within the CD4 cell population in peripheral blood (Fig. 2D). In a 3-wks-old rKO1 mouse, the E/M and Naïve subset constituted 76% and 16% of total CD4 population, respectively (as opposed to 14% and 79% in the WT mice; Fig. 2D, top). TAM treatment (full dose) led to pronounced and progressive depletion of the E/M CD4 cells and simultaneous accumulation of the naïve CD4 cells, which became apparent within 2 weeks after the treatment (Fig. 2D-E). The reciprocal changes in the abundance of the E/M vs. naïve CD4 cells were not due to the conversion of the E/M to naïve CD4 cells (Fig. S1), and so might instead reflect the changes in their apoptosis/proliferation rates.
We conclude that reversing Brg1 KO in all of the Brg1-deficient Tregs as late as 10 days before the predicted median death date rescued 55% of the dying mice. However, in clinical settings, it is unfeasible to repair genetic defects in all of the target cells. Therefore, we repeated with the rescue experiment using the low-dose TAM regimen, which resulted in Brg1-reexpression in variable fractions (10%-50%) of Tregs among different individuals (not shown). Under this condition, 18% (3/17) of the dying rKO1 mice were rescued (Fig. 2A, low dose), with their inflammation resolved and body weight (largely) recovered (Fig. S2A).
Brg1-reexpression proved more effective in rescuing the rKO2 mice, where inflammation was somewhat less devastating. In the absence of TAM, all but one (11/12) rKO2 mice died before Day 42 and the remaining mouse died on Day 67, with the median survival being 38 days, which was only mildly longer than that rKO1 mice (Fig. 2F). Furthermore, the rKO2 mice were nearly as runted as rKO1 (Fig. 2G). Thus, rKO2 mice were also very sick. Nevertheless, following the low-dose TAM treatment in 3-wks-old mice, which restored Brg1 expression in 8% to 68% Tregs (measured on Day 14 after the treatment; Fig. 4A, right plot), 100% (5/5) of the rKO2 mice survived (Fig. 2F, blue line), with their body weights catching up and inflammation subsiding over time (Fig. 2H). Remarkably, these changes were observed even in the mouse where Brg1 expression was restored in only 8% of the Tregs, despite quite severe inflammation before TAM treatment (Fig. 2H, thick blue line). Note that his body weight might never fully catch up, remaining slightly lower than an age- and sex-matched littermate control even on Day 251 after TAM (25.2 vs. 28.8g). Nevertheless, by Day 251, this mouse seemed to have become otherwise perfectly healthy, devoid of any overt sign of illness such as skin lesions and lethargy (not shown).
Collectively, these data reveal powerful effects of Brg1 reexpression on the sick mice, with as little as 8% of Brg1-reexpressed Tregs sufficient for the rescue in some cases.
Brg1 reexpression, presumably in conjunction with excessive cytokine stimulation, produced hyperactivated, highly suppressive “SuperTregs”
To characterize Brg1-reexpressed Tregs, we treated 3-wks-old rKO1 mice with the low dose of TAM and compared gene expression patterns in Brg1-deleted (GFP+) vs. Brg1-reexpressed (GFP-) Treg subsets isolated 7 days after TAM, when the two populations were cleanly distinguishable (Fig. 1F). This analysis would reveal the role of Brg1 in partially activated Tregs exposed to inflammation. As a control, we addressed the role of Brg1 in Tregs under the physiological condition. To this end, we compared Brg1-deleted (YFP+) and Brg1-sufficient (YFP-) Tregs from the healthy, mosaic females (Brg1F/ΔR; FoxP3YFP- Cre/+; R26 CAG-FlpoER/CAG-FlpoER, where YFP-Cre was expressed in only half of the Tregs due to random X-inactivation; these mice also carried R26 CAG-FlpoER/CAG-FlpoER just as the rKO1 mice in order to control for any potential nonspecific confounding effects of FlpoER expression when comparing differentially expressed genes between the two strains). As additional controls, we used Tregs isolated from WT mice and from rKO1 mice not treated with TAM, the former being Brg1-sufficient while the latter Brg1-deficient, therefore comparable to Brg1-sufficient Tregs from the mosaic females and the Brg1-deficient Tregs from TAM-treated rKO1 mice, respectively. All the mice were 3-4 weeks old when sacrificed.
Brg1-deletion in the mosaic females and Brg1-reexpression in rKO1 mice on Day 7 after TAM treatment affected 618 and 1352 genes, respectively, with only 241 genes shared, suggesting divergent roles of Brg1 under the physiological vs. inflammatory conditions (Fig. 3A; see Supplemental Data for complete list of these genes; raw data already deposited). Brg1-target genes are of diverse functions, a conspicuous group being related to Treg function (Fig. 3B). These genes can be divided into two categories: the “naïve genes” that are predominantly expressed in naïve Tregs (Bach2 and Ccr7) 8,19, and “activation/effector function genes” preferentially expressed in activated/effector Tregs, including Icos 8, Tigit 20,Cxcr321, Klrg122, Prdm123 and Gzmb24. In the Brg1 KO Tregs within the mosaic females, the “naïve genes” were upregulated, while most of the “activation/effector function genes” repressed, relative to the Brg1-sufficient Tregs in both the mosaic females and the WT mice (lane 3 vs. 1-2), confirming that the direct effect of Brg1 deletion was to inhibit Treg activation 17. Interestingly, in the rKO1 mice with severe inflammation, the Brg1 KO Tregs were partially/weakly activated, with the “naïve genes” repressed and some of the “activation/effector function genes” (i.e.,Cxcr3, Gzma, Gzmb,Gzmf) upregulated relative to the Brg1-sufficient controls (lane 4 vs. 1-2). These data reinforce the notion that Brg1 KO impairs Treg activation, which triggers inflammation, leading to a secondary partial/weak Treg activation 17. As expected, in the rKO1 mice, following the low-dose TAM treatment which restored Brg1 expression in a subset of Tregs, the Brg1-deficient subset remained mostly unaffected, with the expression pattern comparable to that in the rKO1 mice without TAM treatment (lane 5 vs. 4). In sharp contrast, the Brg1-reexpressed Treg subset in these mice became dramatically activated, as revealed by 5-10x repression of naïve genes and 2-14x upregulation of all the activation/effector function genes relative to the partially activated, Brg1-deleted subset (lane 6 vs. 5). Thus, Brg1 reexpression in the rKO1 mice led to Treg super-activation, the resultant super-activated Tregs (“SuperTregs”) presumably highly suppressive. The data also demonstrate that although the Brg1 target genes were in general highly divergent in the mosaic (healthy) vs. rKO1(inflamed) mice (Fig. 3A), the Brg1-controlled transcription program underlying Treg activation was conserved between the two distinct conditions, but with a twist: in the rKO1 mice, BRG1 was able to upregulate the activation markers to much higher levels than in mosaic mice (lane 6 vs. 2), which seemed to reflect (in part) a synthetic effect of cytokine stimulation in the rKO1 mice (see further).
Activated Tregs are more apoptotic and proliferative than naïve Tregs 8. Indeed, in SuperTregs, the prosurvival gene Bcl2 was repressed whereas the pro-apoptosis gene Casp3 and many cell cycle promoting genes upregulated when compared with all other Treg types examined (Fig. 3C, lane 6 vs 1-5). Consistent with the increased proliferation, Brg1-reexpressed Tregs became somewhat more abundant after TAM treatment (not shown). Curiously, the pro-survival Birc5 was also upregulated in SuperTregs, perhaps reflecting a negative feedback effect (Fig. 3C).
We next used FACS to validate the RNA-seq results. SuperTregs were indeed more proliferative and more apoptotic (Fig. 3D). To assay their suppressive function, dye-labeled conventional CD4 cells (Tconv) were stimulated with antigen presentation cells and anti-CD3 for 5 days in the presence or absence of Tregs before the FACS analysis. In the absence of Tregs, 72% of the Tconv survived and had undergone multiple rounds of division as revealed by progressive dye dilution (Fig. 3E, left FACS plot). Tregs from WT mice, Brg1- deleted Tregs from rKO1 mice and SuperTregs all inhibited proliferation and promoted apoptosis in a dose-dependent manner, but importantly, SuperTregs was the most effective (Fig. 3E-F; see Fig. S3 for a replica experiment).
Finally, we have begun to define the mechanism underlying gene hyperactivation in SuperTregs, namely, how BRG1 in the inflamed rKO1 mice could upregulate the activation markers to much higher levels than BRG1 in the healthy mosaic or WT mice (lane 6 vs. 1-2). To address this issue, we used Cxcr3 as a model. Cxcr3 marks the Treg subset specialized in suppressing the Th1 response21. It is a direct target of BRG1 17 and hyperactivated in SuperTregs (Fig. 3B). Importantly, Cxcr3 is induced in response to IFNγ stimulation, perhaps in conjunction with TCR signaling21. Given that Cxcr3 is subject to joint regulation by BRG1 and IFNγ /TCR, our hypothesis is that in rKO1 mice with severe inflammation, Tregs experience enhanced IFNγ /TCR stimulation, which can conceivably complement BRG1 to induce strong Cxcr3 expression. Indeed, in ∼3 weeks-old rKO1 mice with severe inflammation, STAT1 phosphorylation in Tregs was markedly elevated, indicating excessive IFNγ signaling (Fig. 3G, left and middle). Interestingly, TCR signaling seemed unaltered in these Tregs (Fig. 3G, right).
Our data collectively suggest that Brg1 reexpression acted (partly) in conjunction with inflammatory cytokines to convert Brg1-deleted Tregs into hyperactivated Tregs endowed with potent suppressive activity.
The fate of SuperTreg in vivo
We have followed SuperTregs in the five TAM-treated rKO2 mice (Fig. 2H); these mice, treated with the low-dose TAM regimen, harbored both GFP- and GFP+ Treg subsets, the former being SuperTregs while the latter serving as an internal control for FACS analysis. Peripheral blood was drawn and crucial Treg markers (KLRG1, ICOS, TIGIT, CXCR3, all induced on SuperTregs; Fig. 3B) monitored over time.
In the five mice, SuperTregs comprised 8% to 68% of total Tregs in the blood on Day 14 after TAM (Fig. 2H). We were especially intrigued in the mouse harboring the least (8%) amount of SuperTregs (thick blue line, Fig. 2H). In this particular mouse, the KLRG1+ Treg subset, barely detectable within Brg1-deleted (GFP+) Treg subset, accounted for as much as 21% of the SuperTreg population on Day 14 after TAM, which remained elevated thereafter, presumably reflecting the persistence of certain degree of inflammation (Fig. 4A, row 2-4; Fig. 4B, top left, pink line). By Day 14 after TAM, ICOS had been dramatically induced in SuperTregs, being expressed on (almost) all KLRG1+and KLRG1- subsets (as opposed to 32% in Brg1-deleted Tregs; Fig. 4A, row2, column 4). Of note, in the ICOS+ subset of SuperTregs, the level of ICOS expression was also elevated relative to ICOS+ Treg subset in the WT mice, suggesting that SuperTregs were more active than the activated Treg subset in WT mice on the single cell basis (Fig. 4A, row 2, column 3 vs. 1). Interestingly, in contrast to KLRG1, ICOS expression in SuperTregs (and in Brg1-deleted Tregs) declined over time to the baseline by Day 251 after TAM, occurring faster in the KLRG1- subset (Fig. 4A, row 2, column 3-8; Fig. 4B, bottom left), suggesting (partial) resolution of inflammation. Indeed, TIGIT and CXCR3, also induced on SuperTregs (albeit to less extents than ICOS), had similarly declined to (near) basal levels by Day 251 (Fig. 4A, row 3-4; Fig. 4B, bottom), as were the abundance of the E/M CD4 cells in the peripheral blood (Fig. 2H, right). Of note, on Day 151 after TAM, the frequency of GFP- Treg subset within the Treg population was markedly increased (to 20.6% from 9.5% on Day 56, Fig. 4A, row 1). To determine whether this increase was due to the accumulation of the GFP- Treg subset and/or depletion of the GFP+ Treg subset, we examined the abundance of the two Treg subsets relative to that of conventional CD4 cells, finding that the increased frequency of the GFP- subset was due to its accumulation, as the abundance of the GFP+ subset remained constant as compare with Day 56 (Fig. 4B, right, heavy lines). Interestingly, by Day 251, GFP+ subset had become partially depleted while the GFP+ subset further accumulated (Fig. 4B, right, heavy lines, last time point). The mechanisms underlying the reciprocal changes in the two Treg subsets are unclear, but might involve competition between the Brg1-sufficient and Brg1-deficient Tregs.
In the remaining four rKO2 mice (#2-4), where Brg1 was reexpressed in more (25% to 68%) Tregs (Fig. 2H), the E/M CD4 cells were depleted far more rapidly (Fig. 2H, bottom), and all the activation markers (including KLRG1) decayed over time (Fig. 4B), consistent with more effective resolution of inflammation. The reciprocal changes in the abundance of the GFP- vs GFP+ Treg subsets were also observed (Fig. 4B, top right, thin lines). Finally, we also followed the fate of the 3 rKO1 mice treated with the low-dose TAM regimen, with similar findings (Fig. S2B).
We conclude that SuperTregs tended to lose the hyper-activated phenotype as the inflammation subsided, suggesting that the inflammatory environment was essential for maintaining Treg hyperactivation. Our data also support the notion that the enhancement of Treg suppressive function in response to inflammation is “memory-less”, a feature important for avoiding generalized immunosuppression that could otherwise result from repeated activation9.
DISCUSSION
Using conventional gene KO technologies, many genes have been identified that affect Treg function and immune tolerance. The current work is the first to address the effects of reversing the KO, which provides insights hard to obtain using conventional KO models, as discussed below.
Consequences of Brg1 KO and reexpression in Tregs
These are summarized in Fig. 5, which is based on the current and the previous work17. Specifically, in WT mice, when antigens activate conventional T cells, Tregs also get activated to restrict the immune response. In rKO mice, Brg1 KO impairs Treg activation, leading to the onset of inflammation. As the inflammation intensifies, Tregs get partially activated (partly) by inflammatory stimuli such as IFNγ, but this is insufficient to stop the ongoing inflammation (dotted line). Importantly, at this point, when Brg1 is re-expressed upon TAM administration, it acts in conjunction with the inflammatory stimuli to convert the functionally compromised Tregs into hyperactivated “SuperTregs”, which overwhelm the inflammation. As the inflammation is resolved, SuperTregs reverse activation-induced changes (not depicted).
A few issues are noteworthy regarding this model.
First, we wish to reiterate that compared with the activated Tregs in the WT mice, the activated Tregs in the SuperTreg population were not only more abundant, but also expressed higher levels of some activation markers (like ICOS). Thus, SuperTregs showed both quantitative and qualitative differences from the activated Tregs in the WT mice.
Second, TAM treatment should also lead to Brg1 reexpression in the Treg precursors in the thymus and bone marrow, and the nascent, Brg1-sufficient Tregs might also contribute to the resolution of inflammation. However, this contribution might be minimal, given the low rate of T cell production in adult mice, especially in the sick mice where the thymi were profoundly atrophic as a result of inflammatory stress (not shown).
Third, Brg1-reexpressed Tregs had markedly accumulated by 5 months after the low-dose TAM treatment, which should ensure permanent benefit of the treatment.
Finally, excessive STAT1 signaling (caused e.g. by Treg-specific SOCS1 ablation) is known to cause CXCR3 overexpression, which paradoxically impairs the ability of Tregs to control Th1 response, in apparent conflict with our observation25. We note that the CXCR3 is much more overexpressed in the SOCS1-deleted Tregs than in our SuperTregs (Fig. 6H in reference vs. Fig. 4 in this study). Perhaps STAT1 signaling can produce opposite effects when elevated to different levels.
Value of reversible KO mice
In contrast to our previous study which used conventional gene KO model to address the role of Brg1 in naïve Tregs in normal mice, the current study used a reversible KO method to explore the consequence of Brg1 reexpression in the sick mice. Obviously, the effect of Brg1 reexpression on the partially activated Tregs was hard to predict from the known roles of Brg1 in naïve Tregs, partly because of the change in the information context of Brg1 action under the two distinct conditions (see Introduction); indeed, the majority of Brg1-affected genes in SuperTregs were different from that affected by Brg1-deletion in naïve Tregs (Fig. 3A). It is even harder to predict that Brg1-reexpression in as little as 9% of defective Tregs would suffice to resolve inflammation in the sick mice; indeed, to our knowledge, the efficacy of Tregs to stop severe ongoing systemic inflammation in adult/adolescent mice remains largely unexplored, although it is well known that adoptive transfer of Tregs into neonatal Scurfy mice (which lack Tregs) can prevent the onset of lethal autoimmunity (see, e.g, 21. Our study thus illustrates the value of reversible KO methods in uncovering gene functions. Unfortunately, such methods remain way underutilized, despite brilliant successes in a few isolated cases published in high profile journals26–28.
Medical relevance of the current study
Our study has therapeutic implications for heritable autoimmune disorders resulting from Treg defects, the best defined being the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX)resulting from FOXP3 mutations3–5. The IPEX phenotypes tend to vary with the nature of the mutations. For example, missense mutations and promoter mutations can be associated with normal Treg numbers (but compromised Treg suppressive function) and a milder phenotype. In addition to FoxP3, mutations at a number of other genes important for Treg function (including CD25, STAT5b, ITCH and STAT1) are known to cause IPEX-like disorders 5. Treatment options for the IPEX disorder are limited mainly to immunosuppressive drugs and allogeneic hematopoietic stem cell transplantation (HSCT). Immunosuppressive therapy is beneficial only temporarily, as it fails to prevent disease progression in most patients, with the overall survival rate being only 65% at 24 years of age4. HSCT does not improve the survival rate, and furthermore, some patients cannot undergo HSCT due to limited donor availability or because their clinical manifestations are not severe enough to justify HSCT 4,29. Effective therapies for IPEX-like disorders similarly remain elusive.
We envision an alternative strategy for treating IPEX(-related) disorders. In contrast to HSCT, our strategy exploits preexisting defective Tregs. Specifically, we propose to correct the genetic defects in the Tregs in vivo, thus restoring their function and even converting them into SuperTregs. This conversion is plausible if the mutations compromise Treg activation in a reversible manner as in the case of Brg1 KO. Alternatively, the mutations might not affect Treg activation but block some other aspects of Treg function. In this case, the defective Tregs should already be activated in the inflammatory environment prior to gene therapy, and if the particular Treg defects are (partially) reversible, then repairing the mutations might suffice to convert the Tregs into SuperTregs, which seems feasible at least for the IPEX patients with normal numbers of Tregs mentioned above.
The efficiency of gene-editing determines the therapeutic efficacy. Gene editing tools vary in efficiency. Fortunately, the highly effective “base editors” that can change A>G or C>T have been developed 30,31, which is applicable to, for example, the many IPEX patients carrying a single G>A substitution at FoxP34. The base editor together with relevant gRNA expression cassette might be delivered systemically into such patients using a lentiviral vector, such as the CD4-targeted lentiviral vector that transduces up to 7% of human CD4 cells in mice following a single i.p injection 32,33. This strategy may particularly benefit patients with the Tregs mildly compromised in function but normal in numbers, where correction of the mutations in a small fraction of these Tregs might suffice to effect a cure. This gene-editing based strategy may not be far-fetched. Indeed, In animal models, gene editing has shown great promises for treating monogenic diseases (via simple injection of gene editing components) such as Duchenne Muscular Dystrophy and Leber Congenital Amaurosis 10, the latter already approved for Phase ½ trials34,35. IPEX(-related) disorders represent valid candidates for gene editing-based therapies, as previously proposed 3.
MATERIALS AND METHODS
Mice
Brg1ΔR allele was generated using traditional gene targeting strategy as described for the Baf57ΔR allele18, except that the homology arms in the Baf57ΔR targeting construct were replaced with the sequences from the Brg1 locus (Fig. 1A). The rKO mice were then created by introducing BrgF36, R26CAG-FlpoER37 and FoxP3YFP-Cre38 into the Brg1ΔR/+ mice. Of note, this breeding scheme also generated conventional, irreversible KO littermates, whose genotypes were identical to rKO except that both alleles of Brg1 were floxed. Interestingly, the phenotype of these littermates were generally weaker than rKO mice (but similar to the conventional Brg1 KO mice previously described17, presumably because the conventional KO mice carried two copies of BrgF, both of which must be deleted to eliminate BRG1, whereas in rKO mice, Cre only needed to delete a single BrgF. The mice were maintained on C57/B6 background. All the experiments were approved by the animal ethical committees at ShanghaiTech and Yale University, and were performed in accordance with institutional guidelines.
Tamoxifen (TAM) treatment
For full dose regimen, 50 mg TAM (Sigma Aldrich) was added to 900ul corn oil plus 100ul 100% ethanol (50 mg/ml final concentration), and dissolved by incubation at 55°C for 30 min. The solution can be stored at −20°C. The drug was delivered (typically into 3-wks-old mice) via oral gavage at 10 ul/g body weight, once a day for two consecutive days. Low-dose regimen was identical except that the drug was at lower concentration (1.25 mg/ml) and delivered by a single gavage, translating to 40x less TAM as compared with the full dose regimen. While the full dose regimen invariably caused complete deletion of the gene-trap cassette, the low-dose regimen produced variable, highly unpredictable deletion, with the efficiencies ranging from 7% to 70% in difference individuals.
Flow cytometry
Lymphocytes were stained with antibodies and analyzed using FACS fortessa (BD Biosciences). Phospho-STAT1 and phospho-AKT in splenic Tregs were detected using the following cocktail and the Transcription Factor Buffer Set (BD Pharmingen, 562574):CD4-BV650 or CD4-APC (Biolegend), FoxP3-Percp5.5(BD), Stat1 (pY701)-PE-Texas Red (BD) and AKT (pS473)-BV421 (BD). To minimize sample-to-sample variation of Phospho-STAT1 and phospho-AKT signals, WT and rKO1 splenocytes were stained with CD4-BV650 and CD4-APC respectively before the cells were pooled and stained with the remaining antibodies. The cells in Fig. 4 were analyzed with the following cocktail: CD4-BV650 (Biolegend), CD25-BV605(Biolegend), CD278-PE(Biolegend), CXCR3-APC (eBiosciences), KLRG1-BV421 (BD), TIGIT-APC-R700 (BD).
Gene expression profiling by RNA-seq
Lymphocytes from lymph nodes and spleens from 3-4-wks-old mice were first magnetically depleted of non-CD4 cells before electronic sorting of Tregs (CD4+CD25+YFP+). Total RNA was isolated from 0.1 million Tregs using RNAprep Pure Micro Kit (TIANGEN), and cDNA synthesized from mRNA using SMART-Seq® v4 Ultra™ Low Input RNA Kit (Clontech). Library was then constructed and sequenced on Illumina HiSeq platform with the PE150 strategy, which yielded 25∼ 60 million reads per sample. To identify differentially expressed (DE) genes between the Brg1+ and Brg1- Treg subsets in mosaic mice and TAM-treated rKO1 mice, the count data were TMM normalized, the genes < 5 cpm for both subsets filtered out, and the p values adjusted by the Benjamini and Hochbergare method. DE genes are defined as those with absolute fold-changes ≥2 and padj<0.05. The data have been deposited (BioProject ID PRJNA547476):https://dataview.ncbi.nlm.nih.gov/object/PRJNA547476?reviewer=r0talgg7e1c3r0nmh68gm2r8bj
In vitro suppression assay
Conventional CD4 cells and Tregs were isolated from PLN and spleens from 3 to 4-wks-old mice. CD4+ cells were first enriched using Mouse CD4 T Cell Isolation Kit (Biolegend) before electronic sorting. Brg1 KO Tregs and SuperTregs were isolated from rKO1 mice before and 7 days after TAM, respectively, while Brg1-sufficient littermates (Brg1F/+; FoxP3YFP-Cre(/YFP-Cre); R26CAG-FlpoER/CAG-FlpoER) used as the source of conventional CD4 cells (CD4+CD25- YFP-) and WT Tregs (CD4+CD25+YFP+). The purity of conventional CD4 and Tregs exceeded 95% and 90%, respectively. To assess Treg function, conventional CD4 cells (5×104) were labeled with CellTrace Violet (GIBCO) and stimulated with Rag1-/- splenocytes(5×104) plus 1ug/ml anti-CD3e in the presence of indicated numbers of Tregs. Five days later, the cells were stained with 7-AAD and anti-CD4 APC before flow cytometrical analysis of proliferation and survival of the conventional CD4 cells.
STUDY APPROVAL
All mouse studies were approved by the IACUC at the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and conducted in an AAALAC-accredited facility in compliance with the relevant regulations.
CONFLICTS OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENT
We thank T. Chatila, L. Lu, D. Rudra and Y. Wan for advice.