Effects of Anti-CD19 CAR-T Cells in a Murine Model of IgG4- Related Disease

doi:10.21203/rs.3.rs-7951070/v1

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Effects of Anti-CD19 CAR-T Cells in a Murine Model of IgG4- Related Disease

https://doi.org/10.21203/rs.3.rs-7951070/v1

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Background

Chimeric antigen receptor T-cell (CAR-T) therapy, an emerging immunotherapy, has shown promising efficacy in several autoimmune diseases. In this study, we conducted a preclinical evaluation of the long-term therapeutic potential of CD19-specific CAR-T cell–mediated B-cell depletion in a mouse model that recapitulates key features of human IgG4-related disease (IgG4-RD).

Methods

B cell depletion strategies were evaluated in the Lat^Y136F mouse model, a spontaneous murine model of IgG4-RD. Anti-CD19 CAR-T cells or control cells were transferred into Lat^Y136F mice pretreated with total body irradiation. Lat^Y136F mice treated with anti-CD20 monoclonal antibodies (mAb) served as the positive control group.

Results

CD19-targeted CAR-T cell infusion resulted in a more profound depletion of B cells and plasmablasts compared to anti-CD20 mAb treatment. This depletion was observed in peripheral blood, spleen, lacrimal glands, lungs, and pancreas in Lat^Y136F mice. Moreover, CAR-T cell therapy significantly prolonged the survival of Lat^Y136F mice and improved clinical symptoms compared to anti-CD20 mAb treatment. However, while CAR-T cell therapy reduced inflammation and fibrosis in the lacrimal glands and pancreas, it did not improve these conditions in the lungs.

Conclusions

Our findings demonstrate that anti-CD19 CAR-T therapy effectively alleviates the progression of IgG4-RD, showing superior efficacy compared to anti-CD20 mAb in this preclinical model. These results support further investigation of CAR-T cells as a potential therapeutic option for IgG4-RD patients.

IgG4-RD

Autoimmune disease

CD19

CAR-T cells

IgG4-related disease (IgG4-RD) is a systemic immune-mediated condition characterized by a diverse range of clinical manifestations, including mass formation, organ dysfunction, and autoimmune inflammation^1,2. Patients often present with glandular enlargement, particularly affecting the pancreas, lacrimal glands, lung, salivary glands, and lymph nodes, accompanied by elevated IgG4 levels and lymphocytic infiltration^3,4. B cell plays a crucial role in the pathogenesis of IgG4-RD by producing IgG4 antibodies and secreting cytokines that promote pathological immune responses^5,6. Studies indicate that B cell activation is closely linked to inflammatory cell infiltration in affected tissues, making B cell-targeted therapeutic strategies a potential approach for treating IgG4-RD^2,7.

Currently, therapeutic strategies targeting B cells, such as anti-CD20 monoclonal antibodies, have shown promising efficacy in managing IgG4-RD^8–11. Clinical trials have revealed that anti-CD20 therapy effectively depletes B cells, alleviates clinical symptoms, and decreases pathological IgG4 levels^8–11. Notably, these therapies are particularly effective in glucocorticoid-resistant patients, offering a critical option for those who do not respond to conventional treatments^8–11. Nevertheless, some patients experience disease relapse shortly after treatment, likely due to incomplete B cell depletion^10,12,13. Approximately 45% of IgG4-RD patients fail to achieve complete clinical remission with current standard induction therapies, and 16.9% of those who achieve remission eventually experience disease relapses¹⁴. This underscores the urgent need for more potent and lasting therapeutic options to address this challenge comprehensively.

CAR-T cell therapy, as an emerging immunotherapy, has demonstrated significant efficacy in various B cell-associated diseases, including diffuse large B cell lymphoma and acute lymphoblastic leukemia^15,16. By engineering T cells to specifically target and eliminate B cells, CAR-T therapy offers a high degree of specificity and sustained immune responses. In addition to its success in treating cancers, CAR-T therapy has also shown promising results in a range of autoimmune diseases, such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), ANCA-associated vasculitides (AAV), and systemic sclerosis (SSc)^17–24. Although preliminary studies have explored the efficacy of CAR-T therapy in murine models of IgG4-RD, these investigations were limited by relatively short observation periods²⁵. Given the chronic and relapsing nature of IgG4-RD, it is crucial to evaluate the long-term efficacy and durability of CAR-T–mediated B cell depletion. Such studies are essential to determine whether transient improvements translate into sustained clinical benefit and to provide insights into the long-term immunological consequences of CAR-T therapy in this setting.

In light of the promising potential of CAR-T therapy in clearing B cells and its success in treating other autoimmune diseases, we have initiated a study using the IgG4-RD mouse model, specifically the Lat^Y136F transgenic mice, to explore the effects of anti-CD-19 CAR-T cell therapy on IgG4-RD. This study aims to assess the therapeutic efficacy of CAR-T cells in targeting the underlying B cell-mediated mechanisms of IgG4-RD and explore their potential as a long-term treatment strategy for this refractory autoimmune condition.

Mice

Lat^Y136F mice were obtained from Shanghai Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China), while CD45.1 mice, which served as the donors, were kindly provided by Prof. Lai Guan Ng. All mice were bred under specific pathogen-free conditions. The animal procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.

Construction of CARs

MSGV1-1D3-28Z.1–3 mut plasmid was obtained from Addgene (plasmid #107227)²⁶. In our study, an enhanced green fluorescent protein (eGFP) sequence was inserted downstream of the CAR construct to serve as a marker for CAR expression.

Primary mouse CD8 CAR-T cell generation

293T cells were co-transfected with retroviral core vectors encoding the CAR and pCL-Eco using Lipo3000. After 48 to 72 hours, viral supernatants were collected and filtered through a 0.45 µm PES membrane before being used.

To generate primary CAR-T cells, CD8⁺ T cells were isolated from the spleen and lymph nodes of CD45.1 mice via negative selection and activated for 24 hours using plate-bound anti-CD3 and anti-CD28 antibodies in growth medium (RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, 2-mercaptoethanol, sodium pyruvate, HEPES and 50 U/mL IL-2). The cells were subsequently spinfected with viral supernatant containing polybrene (8 µg/mL) at 2000 rpm and 37°C for 2 hours. Control T cells underwent the same process using eGFP-transfected 293T cell culture medium. Twelve to sixteen hours post-infection, the supernatant was replaced with fresh growth medium. Two days later, CD8⁺ eGFP⁺ cells were sorted via FACS (BD Aria III).

Specific killing of CAR-T cells

After 4 days of culture, control T cells or CAR-T cells were cocultured with isolated CD19⁺ B cells to assess specific cytotoxicity of CAR-T cells. In each well, 5 × 10⁴ CD19⁺ B cells were used as target cells, and varying numbers of CAR-T cells or control T cells (1.25 × 10⁴, 2.5 × 10⁴, 5 × 10⁴, 1 × 10⁵, or 2.5 × 10⁵) were added, respectively. Following a 4-hour coculture period, cells were harvested, and CD19⁺ B cell lysis was analyzed by FACS.

Flow cytometry analysis

The surface staining was performed using the following monoclonal antibodies and reagents: Zombie Dye UV (BioLegend), BV650 anti-CD45 (BioLegend), BV421 anti-CD45.1 (Thermofish), FITC anti-CD19 (BioLegend), APC/Cyanine7 anti-CD138 (BioLegend), PE/Cyanine7 anti-B220 (BioLegend), APC anti-TCRβ (BioLegend), BV605 anti-CD8 (BioLegend), Alexa Fluor 700 anti-CD4 (BioLegend), BV421 anti-CD44 (BioLegend), BV786 anti-CD62L (Thermofish), BUV661 anti-CD69 (BD), PE anti-CXCR5 (BioLegend). Samples were analyzed using an LSR Fortessa (BD) or ZE5 (Bio-Rad) flow cytometer, and the data were processed with FlowJo v10.8.1 software.

Adoptive transfer of CAR-T cells

For the indicated groups, total body irradiation (TBI) at 1.5 Gy was administered on day 0. On day 1, the mice were injected via the tail vein with 1 × 10⁶ CAR-T cells, control cells, or 200 µg of anti-CD20 antibody (BioLegend) in 100 µL PBS. For the antibody group, injections were repeated weekly for two additional doses.

Enzyme linked immunosorbent assay (ELISA)

Serum IgG1 levels were quantified using a commercial ELISA kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Serum concentrations of IL-6, IFN-γ, and TNF-α were measured using DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA).

Histology

The lungs, lacrimal glands, and pancreases from the mice were collected, fixed in 4% PFA, and paraffin-embedded. Five-micrometer sections were then stained using H&E and Masson's trichrome. The inflammation was scored on a scale from 0 to 4 (0 = no changes; 1 = mild inflammation with single-cell infiltration; 2 = moderate inflammation; 3 = more severe inflammation; 4 = severe inflammation with atrophy) as described previously^27,28. Fibrosis was evaluated on a scale of 0–3 (0 = no changes; 1 = mild fibrosis; 2 = more severe fibrosis; 3 = presence of burned-out lesions accompanied by parenchymal atrophy) as described previously^27,28.

Immunohistochemistry

Tissue sections embedded in paraffin were first rehydrated and permeabilized. After performing antigen retrieval and blocking, the sections were incubated overnight at 4°C with primary antibodies targeting CD19 (Abcam). To visualize bound antibodies, a horseradish peroxidase-conjugated secondary antibody was applied for one hour at room temperature, followed by incubation with diaminobenzidine solution to develop the immunoreactivity.

Statistical analysis

The data are presented as the mean ± standard deviation (SD). Multiple group comparisons were performed using one-way analysis of variance (ANOVA), followed by Dunn’s correction. Comparisons between two groups were made using an unpaired Student’s t-test. All P values are two-tailed, with values less than 0.05 considered statistically significant. Statistical analyses were conducted using GraphPad Prism 9.

Generation of Mouse Anti-CD19 CAR-T Cells

We designed a retroviral expression vector for murine anti-mouse CD19 CAR-eGFP, based on the 1D3 anti-mouse CD19 CAR. This construct includes the extracellular scFv domain of 1D3, linked to the transmembrane and cytoplasmic signaling domains of CD28, along with a variant of the CD3ζ C terminus (Supplementary Fig. 1A). The CD3ζ variant carries alanine substitutions for the conserved tyrosine residues in two of the three cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs)²⁶. These mutations reduce cellular activation and prevent the exhaustion of CAR-T cells, promoting their prolonged survival in vivo²⁶. Following retroviral transduction, CAR expression was confirmed through eGFP expression in primary CD8 + T cells from CD45.1 mice (Supplementary Fig. 1B). Live eGFP⁺ T cells were sorted by FACS with approximately 90% purity (Supplementary Fig. 1C). After 3–5 days of culture, the cytotoxicity of these sorted cells was assessed. The CAR-T cells exhibited highly specific cytotoxic activity, achieving nearly 100% target cell killing at an E:T ratio of 1:1 (Supplementary Fig. 1D).

Efficient and Sustained B-Cell Depletion In Vivo by Mouse Anti-CD19 CAR-T Cells

We first confirmed that B cells were overexpressed in the lacrimal gland, lung and pancreas of Lat^Y136F mice, a spontaneous murine model of IgG4-RD, compared to wild-type mice (Supplementary Fig. 2A-2C). To test CD19-targeted CAR T cells in this model, we employed a protocol similar to standard CAR-T approaches, with the notable adaptation of using purified CD8 + T cells to mitigate the potential disease-enhancing effects of autoreactive CD4 + T cells. The therapeutic interventions were applied to 6-week-old Lat^Y136F mice, which were randomly assigned to four experimental groups (Fig. 1A). For CAR-T infusion preconditioning, low-dose total body irradiation (TBI) was used, as previous studies have indicated its necessity in mouse models of leukemia. Anti-CD20 antibody treatment, administered weekly for three weeks, served as a positive control, while CAR-T groups received a single transfer (Fig. 1A).

Circulating CD19 + B-cell depletion was assessed at two time points post-treatment. Two weeks after CAR-T cell transfer with TBI preconditioning, circulating CD19 + B cells were nearly eradicated, with depletion levels comparable to those achieved by anti-CD20 antibody treatment (Fig. 1B). Notably, CAR-T therapy demonstrated superior durability, maintaining low B-cell levels for up to 10 weeks post-transfer, whereas B-cell counts rebounded significantly in the antibody-treated group (Fig. 1C). CAR-T treatment also resulted in a modest decrease in CD138 + plasma cells after two weeks, with a more pronounced reduction observed at 10 weeks (Fig. 1D). In contrast, anti-CD20 antibody treatment caused a transient decline in CD138 + cells (Fig. 1E).

After 10 weeks, tissue-specific analysis revealed that CD19 + B-cells were reduced in all examined tissues (spleen, lung, pancreas and lacrimal gland) following both CAR-T cell therapy and anti-CD20 mAb treatment, with more extensive depletion in the CAR-T group (Fig. 2A). Only CAR-T therapy effectively reduced CD138 + plasma cell frequencies across various tissues, while anti-CD20 treatment achieved a reduction solely in the pancreas (Fig. 2B).

Therapeutic effects of anti-CD19 CAR-T cells in Lat^Y136F mice

The mice treated with CAR-T therapy showed no significant differences in body weight changes compared to those treated with Control-T or Anti-CD20 (Fig. 3A). However, 80% of mice treated with Control-T cells developed skin lesions within three months post-treatment, while only 25% in the CAR-T group and 40% in the anti-CD20 group experienced such issues (Fig. 3B).

Generally, female Lat^Y136F mice in the control group succumbed at an average age of 16 weeks, which was consistent with our observations. However, CAR-T therapy significantly improved mouse survival rates compared to the control group, survived the 24th week (Fig. 3C). Mice treated with the antibody exhibited a survival rate comparable to that of the CAR-T group. Additionally, at 16 weeks of age, mice in the CAR-T group showed a slight reduction in spleen weight (Fig. 3D). Histologically, CAR-T therapy significantly reduced immune cells infiltration and fibrosis in lacrimal gland and pancreas, but not in the lung of Lat^Y136F mice (Fig. 3E, 3F and Fig. 4). Furthermore, CAR-T therapy significantly lowered serum IgG1 levels in Lat^Y136F mice (Fig. 3G).

Anti-CD19 CAR-T Cells Do Not Have an Effect on Endogenous T Cells

To assess the effects of CD19-targeted CAR-T cells on T cell responses, flow cytometry analysis was conducted by gating on CD4 + and CD8 + cell populations from different tissues of Lat^Y136F mice. The majority of infiltrating T cells were CD44 + CD62L−, indicative of an activated phenotype (data not shown). CAR-T cell injection increased the proportion of CD4 + T cells while slightly reducing the percentage of CD4 + Cxcr5 + cells in peripheral blood (Supplementary Fig. 3A). However, there were no significant differences in the proportions of CD4 + T cells, CD8 + T cells, Treg cells, and Tfh cells in the spleen, lungs, lacrimal glands, or pancreas between the CAR-T cell and control T cell treatment groups (Supplementary Fig. 3B–3E). We further assessed the serum cytokine profile related to CD19-targeted CAR-T cell activation. Notably, a substantial elevation of the pro-inflammatory cytokine IL-6 was observed in the CAR-T–treated group (Fig. 5A–C).

CAR-T therapies have demonstrated significant therapeutic potential in treating various autoimmune diseases, including SLE and other autoimmune diseases²⁹. These treatments work by reprogramming T cells to target specific antigens on B cells, which are pivotal in the pathogenesis of autoimmune conditions. In this study, we present the preclinical evaluation focusing on the long-term efficacy of anti-CD19 CAR-T therapy in IgG4-RD, an immune-mediated fibro-inflammatory disorder characterized by tissue infiltration by IgG4-positive plasma cells and fibrosis³⁰. We utilized the Lat^Y136F mice, which closely mimics the human manifestation of IgG4-RD, including multi-organ involvement, B-cell infiltration, fibrosis and elevated IgG1 levels, which are analogous to human IgG4^27,28,31. Importantly, the therapeutic effects of both anti-CD19 CAR-T cells and anti-CD20 antibodies were evaluated not only to assess their capacity for B-cell depletion but also to determine whether CAR-T therapy could provide more durable and sustained benefits compared to conventional B-cell–targeted approaches.

Anti-CD20 treatment has already demonstrated efficacy in clinical studies, showing a beneficial effect in IgG4-RD patients through B-cell depletion^8–11. Our results were consistent with these findings in Lat^Y136F mice, where anti-CD20 antibodies extended survival and slowed fibrosis progression. However, the anti-CD19 CAR-T treatment showed superior and more durable effects. While anti-CD20 treatment led to a rebound in peripheral B cells ten weeks post-treatment, anti-CD19 CAR-T cells maintained long-lasting B-cell suppression. Importantly, CAR-T therapy also resulted in a significant reduction in B-cell infiltration in key organs, such as the lungs, pancreas, and lacrimal glands. These effects were not observed in the anti-CD20-treated group, highlighting the enhanced tissue penetration and retention of CAR-T cells compared to antibody-based therapies. This advantage has been previously observed in studies of SLE, where CAR-T therapy demonstrated prolonged B-cell depletion and improved disease outcomes²².

IgG4-RD is associated with the accumulation of plasma cells, which produce the pathogenic autoantibody mouse IgG1 or human IgG4^27,28. In our study, we also observed that CAR-T therapy led to a reduction in mouse IgG1 levels and significant alleviation of disease symptoms. However, the effect on CD138⁺ plasma cells was modest. This result may be attributed to the indirect mechanism of action of anti-CD19 CAR-T cells, which primarily target B cells but not plasma cells directly^32,33. It is also possible that the short lifespan of the Lat^Y136F mice limited our ability to fully assess the long-term impact of CAR-T therapy on plasma cell populations. Nevertheless, our findings suggest that inhibiting plasma cell accumulation, even if not through direct depletion, could be sufficient to reduce disease symptoms and halt disease progression. Interestingly, previous studies have demonstrated that engineered T cells co-expressing anti-CD19 CAR and anti-BCMA CAR structures can effectively suppress B-cell lymphoma progression by targeting both B cells and plasma cells, compared to single-targeted CAR-T cells³⁴. This insight raises the possibility that combining B-cell depletion with strategies targeting plasma cells might further enhance the therapeutic efficacy of CAR-T therapy in autoimmune diseases.

Sun et al. further demonstrated that CAR-T therapy markedly reduced plasma cell frequencies and alleviated pulmonary inflammation and fibrosis in Lat^Y136F mice within three weeks of treatment²⁵. However, from a long-term perspective, anti-CD19 CAR-T treatment did not improve lung inflammation or reduce the severity of fibrosis. Previous studies have shown that CAR-T accumulation in tissues, particularly the lungs, can exacerbate pulmonary fibrosis and hypertension in models of systemic sclerosis³⁵. However, in our study, Lat^Y136F mice showed increased CD4 + T cells post-treatment, which may hinder CAR-T expansion and infiltration. The rapid disease progression in Lat^Y136F mice, characterized by severe multi-organ fibrosis that peaks by eight weeks^27,28, may also limit the window for effective therapeutic intervention. A disease model with slower progression, more closely mirroring the clinical course of human IgG4-RD, would offer a more suitable platform for evaluating the long-term efficacy of CAR-T therapy.

In addition to B-cell involvement, CD4 + T cells play a central role in driving inflammation and fibrosis in IgG4-RD^36–38. Th1 cells are elevated, producing IFN-γ and TGF-β, while Th2 cells dominate affected tissues, releasing IL-4, IL-5, and IL-13^39–42. Circulating CD4 + cytotoxic T lymphocytes (CTLs) contribute to fibrosis through granzyme, TGF-β, and IL-1β production^38,43,44. Follicular helper T cells (Tfh), including Tfh1 and Tfh2 subsets, are increased, with enhanced expression of CXCL13, BCL6, IL-21, and CD40L, driving IgG production and exacerbating disease activity^45–48. Lat^Y136F mice demonstrate a predominance of CD4 + T cells in affected tissues, such as the lungs and pancreas, emphasizing their significant role in disease pathogenesis. Interestingly, Lat^Y136F mice exhibit a lack of regulatory T cells (Tregs), which are known to suppress CD4 + T-cell proliferation and mitigate inflammation in autoimmune models. Based on these insights, targeting CD19 with CAR-engineered Tregs (CAR-Tregs) may offer a more effective therapeutic approach for IgG4-RD and other autoimmune diseases, given their dual ability to deplete pathogenic cells and modulate the immune response⁴⁹.

Despite the challenges observed in this study, the findings provide valuable insights into the potential of CAR-T therapy for IgG4-RD. The success of anti-CD19 CAR-T cells in depleting B cells and reducing disease symptoms in Lat^Y136F mice lays the groundwork for further investigation of CAR-T as a treatment option for autoimmune diseases. However, translating these preclinical results to human patients will require careful consideration of several factors, including the potential for T-cell-mediated toxicity and the long-term effects of CAR-T therapy on plasma cell populations. Future studies should focus on optimizing CAR-T cell designs, possibly incorporating strategies to target both B cells and plasma cells more effectively. Additionally, clinical trials should aim to assess the safety and efficacy of CAR-T therapy in patients with refractory IgG4-RD, particularly those who have failed conventional therapies.

In conclusion, CAR-T cell therapy holds significant promise for the treatment of autoimmune diseases, including IgG4-RD. The preclinical results presented in this study suggest that anti-CD19 CAR-T cells could offer a new avenue for treating refractory autoimmune conditions by effectively depleting B cells and mitigating disease symptoms. While further research is needed to refine CAR-T therapy for this purpose, these findings highlight the potential of CAR-T cells as a transformative treatment for autoimmune diseases.

Ethics approval and consent to participate

The animal procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.

Consent for publication

Not applicable.

Funding

This work was supported by the Innovative Research Team of High-level Local Universities in Shanghai (SHSMUZDCX20210902); the National Ten Thousand Talent Programme (SQ2022RA2C000263).

Contribution

J.H., A.Z., X.S. and R.J. conceived this study and designed the experiments. J.H., Y.Y, Y.Y. and Y.F. performed the experiments, analyzed the data. J.H., A.Z., X.S. and R.J. wrote the paper. All authors critically reviewed the manuscript for important intellectual content.

Competing interests

The authors declare no competing interests.

Data availability

Data will be made available on request.

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Effects of Anti-CD19 CAR-T Cells in a Murine Model of IgG4- Related Disease

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Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Mice

Construction of CARs

Primary mouse CD8 CAR-T cell generation

Specific killing of CAR-T cells

Flow cytometry analysis

Adoptive transfer of CAR-T cells

Enzyme linked immunosorbent assay (ELISA)

Histology

Immunohistochemistry

Statistical analysis

Results

Generation of Mouse Anti-CD19 CAR-T Cells

Efficient and Sustained B-Cell Depletion In Vivo by Mouse Anti-CD19 CAR-T Cells

Anti-CD19 CAR-T Cells Do Not Have an Effect on Endogenous T Cells

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1