CD19-targeted CAR regulatory T cells suppress B cell activities without GvHD

Regulatory T cells (Tregs) play essential roles in maintaining immunological self-tolerance and preventing autoimmunity. The adoptive transfer of antigen-specific Tregs has been expected to be a potent therapeutic method for autoimmune diseases, severe allergy, and rejection in organ transplantation. However, effective Treg therapy has not yet been established because of the difficulty in preparing a limited number of antigen-specific Tregs. Chimeric antigen receptor (CAR) T cells have been shown to be a powerful therapeutic method for treating B cell lymphomas, but application of CAR to Treg-mediated therapy has not yet been established. Here, we generated CD19-targeted CAR (CD19-CAR) Tregs from human peripheral blood mononuclear cells (hPBMCs) and optimized the fraction of the Treg source as CD4+CD25+CD127lowCD45RA+CD45RO–. CD19-CAR Tregs could be expanded in vitro while maintaining Treg properties, including a high expression of the latent form of TGF-β. CD19-CAR Tregs suppressed IgG antibody production from primary B cell differentiation in vitro via a TGF-β-dependent mechanism. Unlike conventional CD19-CAR CD8+ T cells, CD19-CAR Tregs suppressed antibody production in immunodeficient mice that were reconstituted with hPBMCs with reducing the risk of graft-versus-host disease. Therefore, the adoptive transfer of CD19-CAR Tregs may provide a novel therapeutic method for treating autoantibody-mediated autoimmune diseases. Research Therapeutics


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On the basis of these data, we conclude that CD45RA + Tregs are suitable for CD19-targeted CAR Treg generation.

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CD19-CAR Tregs also strongly suppressed IgG production from primary B cell 25 differentiation ( Figure 4B and Supplementary Figure 5B). TGF-β was reported to 26 induce IgA class-switching and reduce IgG production (35,36). Because CD19-CAR Tregs expressed high levels of TGF-β (see Figure 2D and Figure 3E), there is a 1 possibility that IgA class-switching is promoted by CD19-CAR Tregs. However, 2 CD19-CAR Tregs suppressed both IgG and IgA production ( Figure 4C). 3 It has been reported that CAR expression may produce tonic signals in the absence 4 of antigen binding (37). To examine whether CAR expression itself affects CAR Treg 5 functions, we generated HER2-targeted CAR Tregs (HER2-CAR Tregs) (38) and  11 Next, to investigate the mechanism of how CD19-CAR Tregs suppressed B cells, we 12 tested various antibodies against known effectors of Tregs including TGF-β, IL-10,

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Since some reports have shown that TGF-β suppresses IgG production in B cells in 25 vitro (39), these data strongly suggest that CD19-CAR-mediated TGF-β signals 26 suppress the proliferation and IgG production in B cells without promoting IgA class-switching.  (Supplementary Figure 7). These data indicate that CD19-CAR Tregs 20 specifically suppress Ig production from B cells in vivo.

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The transfer of human PBMCs resulted in severe GvHD, leading to weight loss hypotension, and respiratory insufficiency), which is associated with elevated serum 10 cytokines, including interleukin-6 (IL-6). Thus, we compared the effect of CD19-CAR 11 Tregs and CD19-CAR CD8 + T cells on GvHD effects in NSG mice reconstituted with 12 human PBMCs. CD19-CAR Tregs or CD19-CAR CD8 + T cells were transferred 7 days 13 after PBMC injection. Although CD19-CAR Tregs did not affect body weight by day 50 14 after transfer or reduce the GvHD score, CD19-CAR CD8 + T cells promoted weight loss 15 and did not reduce the GvHD score ( Figure 6E). Moreover, CD19-CAR CD8 + T cells, 16 but not CD19-CAR Tregs, increased mouse IL-6 levels and decreased human Tregs 6 17 days after CD19-CAR CD8 + T cells or CD19-CAR Tregs were simultaneously In this study, we optimized the conditions for generating stable CD19-targeted CAR 24 Tregs, which are suitable for the adoptive cell therapy of non-tumor, chronic 25 autoimmune diseases. We found that CD19-targeted CAR Tregs efficiently suppressed 26 primary human B cells compared to polyclonal Tregs both in vitro and in vivo. Unlike conventional CAR CD8 + T cells, CAR Tregs may be safer for chronic diseases because 1 CAR Tregs suppressed rather than resulted in GvHD.

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Our data is consistent with that of a previous report by Boroughs et al.(43). They 3 also used CD19-CAR for modifying co-stimulatory domains. They concluded that 4 CD19-CAR carrying CD28 signaling domains is superior to 4-1BB-based CD19-CAR, 5 which is consistent with our results. However, there is an important difference, namely, 6 they showed that CD19-targeted CAR Tregs have mild cytotoxicity, while our 7 CD19-CAR Tregs did not. This might be caused by the difference in Treg fraction 8 preparation strategy from PBMCs. They used the CD4 + CD25 hi CD127 low fraction as a 9 source of Tregs whereas we further purified CD45RA + cells from this fraction ( Figure   10 1A). The CD4 + CD25 hi CD127 low fraction contains both naïve/resting (CD45RA + ) Tregs 11 and CD45RO + effector/non Tregs (30,31). We showed that Foxp3 expression was 12 unstable in CD45RO + Tregs, and CD45RO + Tregs produce a greater amount of effector 13 cytokines and cytolytic mediators than CD45RA + Tregs (Figure 2). Although the 14 fraction of CD4 + CD25 hi CD127 low CD45RA + cells is small, we found that this fraction 15 can be expanded vigorously without losing Foxp3 expression and suppression functions, 16 and the retroviral gene transduction efficiency in this Treg fraction was higher than 17 Tconv cells, although the exact reason is not clear. Thus, we believe that the source of 18 Tregs is important in order to generate stable and non-cytolytic Tregs for Treg therapy.

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It has been demonstrated that "exTregs" can be pathogenic and that the stability of 20 Tregs is a bottleneck for Treg cell therapy (15). In this study, we showed that 21 CD19-CAR CD45RA + Tregs did not lose Foxp3 expression after long-term expansion in 22 vitro ( Figure 2B), retained Foxp3 expression and persisted for 1 month in NSG mice, 23 and did not exhibit any harmful GvHD effects (Figure 6 and Supplementary Figure 7). 24 Since CD19-CAR CD8 + T cells persist in the human body in CAR-T therapy, we expect 25 that CD19-CAR Tregs also persist for a long time and maintain Treg functions. conventional CD19-CAR CD8 + T cells that kill B cells (28). Apparently, CD19-CAR 1 CD8 + T cells have the potential to induce GvHD or cytokine release syndrome. In 2 contrast, our CD19-CAR CD45RA + Tregs suppress B cell proliferation and IgG 3 production but do not kill B cells and have little risk of harmful effects. Anti-CD20 4 antibody therapy is another way to control B cells in autoimmune diseases (27).

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Anti-CD20 antibodies also deplete B cells and cause hypogammaglobulinemia, thereby 6 increasing the risk of infection. CD19-CAR Tregs may also have similar adverse 7 effects; however, we expect that the recovery of immunoglobulin after CD19-CAR Treg 8 therapy is faster than anti-CD20 depleting antibodies, because CD19-CAR Tregs might 9 just suppress B cells rather than deplete them. To improve the safety of CAR Treg 10 therapy, engineered CARs that are equipped with a suicide gene or work only in the 11 presence of drugs may be made available (18).

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In this study, we propose that TGF-β is a major mechanism for B cell suppression by 13 CD19-CAR Tregs. Although TGF-β is a secreted cytokine, we showed that Treg-B cell  Although our data strongly support that TGF-β from CAR Tregs plays a major role in 24 the suppression of B cells, it has not yet been clarified how TGF-β signals suppress the 25 proliferation and IgG production of B cells. Although it is well known that TGF-β 26 signals induce IgA class-switching (35, 36), we have not observed IgA induction by co-culture with CD19-CAR Tregs. Exogenous TGF-β has been shown to suppress IgG1 1 or IgG2a production from murine B cells induced by IL-4 and IFN-γ, respectively (44).

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Exogenous TGF-β has been shown to decrease B lymphocyte Ig secretion by inhibiting 3 the synthesis of Ig mRNA and inhibiting the switch from the membrane form to the 4 secreted forms of Ig mRNAs (45). Further study is necessary to clarify the precise 5 effects of CD19-CAR Tregs on B cells.

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CAR-T cells could be therapeutic not only for cancer but also for various diseases in 7 which pathogenic cells are involved. A recent paper showed that the adoptive transfer of 8 cardiac fibroblast-specific CD8 + T cells results in a significant reduction in cardiac 9 fibrosis and restoration of function after injury in mice (46). CAR Tregs could be an 10 alternative way to suppress, but not eliminate, pathogenic cells without harmful side 11 effects. The usefulness of CAR-Tregs should be confirmed by using immunocompetent 12 murine models of autoimmunity. However, it is always important to use human T cells 13 to confirm the therapeutic value of CAR Tregs because methods for isolation and 14 expansion of pure human Tregs have not been completely established (14).    Supplementary Table. 25 The virus was prepared as previously described (47). Briefly, the cDNA expression 26 vector was transduced into HEK293T cells along with the VSV-G expression vector and packaging vector. Eighteen hours after transduction, the vector-containing culture 1 medium was changed into fresh culture medium; 48 h later, the virus-containing 2 medium was collected, passed through a 0.45 μm filter, and concentrated using 3 centrifugation (8,000 g at 4°C for 16 h). The virus pellets were resuspended in culture 4 medium. K562 cells were transduced to express human CD19 (hCD19-K562) or human 5 HER2 (HER2-K562) with lentivirus. antigen-expressing K562 cells were added 3 or 4 days after the first co-culture.

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Polyclonal Tregs that were transduced with an empty gene or not transduced were 21 cultured with anti-CD3/CD28 dynabeads in the presence of 100 ng/mL human IL-2.

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Tconvs and CD8 T cells were cultured in the presence of 10 and 30 ng/mL human IL-2, 23 respectively, and gene transduction was carried out on day 1. All T cells were cultured at

Xenograft mouse model for antibody production in vivo 21
The 8-to 11-week-old male and female NSG mice were intravenously injected with 2 × 22 10 6 CD19-targeted CAR Tregs or empty Tregs 4 to 6 h or 7 days after intravenous 23 injection of 5 × 10 6 hPBMCs. Saline-injected mice served as controls. On days 14, 21, 24 and 28, peripheral blood and spleens were collected, and then erythrocytes were lysed.

Conflict of interest statement 22
The authors have declared that no conflicts of interest exist.  Student's t-test or (E) a one-way analysis of variance (ANOVA) (*p < 0.05, **p < 0.01).