
==== Front
J Clin Immunol
J Clin Immunol
Journal of Clinical Immunology
0271-9142
1573-2592
Springer US New York

38578340
1690
10.1007/s10875-024-01690-0
Letter to Editor
Monozygotic Twins with MAGT1 Deficiency and Epstein–Barr virus-positive Classic Hodgkin Lymphoma Receiving anti-CD30 CAR T-cell Immunotherapy: A case Report
Wang Jiachen 12
Zhou Mi 12
Zhou Jianfeng 12
http://orcid.org/0000-0001-9421-6344
Xiao Min xiaomin@tjh.tjmu.edu.cn

12
Huang Liang lhuang@tjh.tjmu.edu.cn

12
1 grid.33199.31 0000 0004 0368 7223 Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
2 Immunotherapy Research Center for Hematologic Diseases of Hubei Province, Wuhan, China
5 4 2024
5 4 2024
2024
44 4 9122 10 2023
13 3 2024
© The Author(s) 2024, corrected publication 2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
http://dx.doi.org/10.13039/100014718 Innovative Research Group Project of the National Natural Science Foundation of China No. 82270203 and No. 81770211 No. 81670152 81800162 Zhou Mi Xiao Min Huang Liang issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2024
==== Body
pmcTo the Editor,

Epstein-Barr virus (EBV)-positive classical Hodgkin lymphoma (cHL) is the most common malignancy in patients with “X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia” (XMEN). Aberrant expression of MAGT1 leads to XMEN, a congenital disorder of combined primary immunodeficiency (PID) characterized by increased susceptibility to chronic EBV infection and EBV-associated lymphoproliferation [1]. Patients with variants in MAGT1 suffer from an N-linked glycosylation defect, resulting in low CD4+ cell counts with an inverted CD4:CD8 ratio, reduced expression of NKG2D (a natural killer (NK)-cell activating receptor) on NK cells and cytotoxic T lymphocytes (CTLs), and impaired T-cell activation through NKG2D [2].

A variety of approaches have been used to control this disease. However, the therapeutic effects are limited [3]. A new approach involving chimeric antigen receptor (CAR) T cells specific for CD30 can be used to treat relapsed or refractory (r/r) HL. Previously, we reported the safety, efficacy and robust long-term performance of CD30 CAR T-cell immunotherapy in our center [4]. However, there have been no reports on CAR T-cell therapy for lymphoma in XMEN thus far. Here, we present the first case of an identical twin with MAGT1-deficient cHL receiving murine CD30 CAR T-cell therapy and hope to provide insight into a therapeutic strategy for such patients.

Case Presentation

The monozygotic twins were diagnosed with the cHL (mixed cellularity) at three and nine years of age. Twin 1 and twin 2 developed progressive disease (PD) after receiving chemotherapy, autologous HSCT, anti-CD30 antibodies, and anti-PD-1 monoclonal antibodies. Supplemental Table 1 shows the therapeutic clinical therapy and disease state timeline of this case.

The twins were referred to our hospital to receive murine anti-CD30 CAR T cell therapy. Before treatment, CD30 target antigen expression was confirmed by immunohistochemical staining of the initially diagnosed lymph nodes (Supplemental Fig. 1). The structure (Fig. 1A) and manufacturing of CAR T cells are described in the Supplemental Methods as previously described [4]. The twins were given a standard dose of the FC regimen on days − 5 to -3 as lymphodepletion. Anti-CD30 CAR T cells (4 × 106 kg/day) were infused on days 0–3 and days 0–5 for twins 1 and 2, respectively (Fig. 1B). T cells with the anti-CD30 CAR transgene expanded and persisted well in the twins compared with the previously reported average level in our center [4]. The key factors related to the therapeutic effect of CAR-T cells are presented in Supplemental Table 2. Compared to those of twin 2, twin 1 had a higher level of CAR T-cell expansion and longer persistence (Fig. 1C and D, Supplemental Table 3), and the level of interleukin-6 (IL-6) was greater in twin 1 (Fig. 1E). Grade 1 and grade 0 cytokine release syndrome (CRS) were observed in twin 1 and twin 2, respectively (Fig. 1F). Neither of the twins experienced any significant infections or neurological symptoms before or after CAR-T cell therapy. The twins achieved complete remission (CR) at + 3 months after CAR T-cell infusion. Twenty-three months later, twin 1 developed PD, but twin 2 remained in CR (Fig. 1G). Then, twins 1 and 2 received 200 mg of anti-PD-1 antibody every 1–2 months and every 2 months, respectively. As of December 2023, Twin-1 was maintained in stable disease (SD), and Twin-2 remained in CR.

Fig. 1 Clinical examination of the response to the infusion of murine anti-30 CAR T cell therapy. (A) Schematic diagram of the murine CAR30 vectors. The third-generation CARs were composed of a single chain variable fragment (scFv), two costimulatory domains from CD28 and 4-1BB, and CD3ζ chain as the activation domain. The scFv was derived from a murine monoclonal antibody against human CD30. Abbreviations: SP, signal peptide; VL, variable L chain; L, linker; VH, variable H chain. (B) Lymphodepleting chemotherapy regimen and CAR T-cell infusion dose used in this case. Twin 1 and Twin 2 are represented in red and blue, respectively. (C) CAR30 transgene copy numbers detected by droplet digital polymerase chain reaction (ddPCR). (D) CAR30+ T-cell percentages among CD3+T cells detected by flow cytometry. (E) Levels of IL-6 after CAR T-cell therapy. (F) Levels of ferritin after CAR T-cell therapy. (G) FDG-PET/CT images obtained at 0 (left), + 3 months (middle), and + 20 months (right) after murine anti-CD30 CAR T-cell immunotherapy. Twin 1 and Twin 2 are represented in red and blue, respectively. (H) Pedigrees showing the families of the affected individuals (twins) harboring the MAGT1 alteration. Solid symbols indicate affected persons who were hemizygous for the mutant allele; solid center symbols indicate unaffected persons hemizygous for the mutant allele; circles indicate female family members; squares indicate male family members. (I) Sanger sequencing of the region surrounding the missense MAGT1 mutation (c.131_134delinsGTGGTGGTTTTGGTGTGT, p.Val44Glyfs*38) in a reference control subject and the patients. (J) Location of MAGT1 variants in previously reported cases (represented in black) and in our patients (represented in red). (K) NKG2D protein expression in activated CD8+ T cells and activated CD56+ NK cells in twin-1, twin-2, mother and HDs, as measured by flow cytometry. (L) Perforin release in activated CD8+ T cells, and activated CD56+ NK cells in the twin-1, twin-2, mother and HDs, demonstrating markedly reduced expression in the twins’ cells, as measured by flow cytometry, was markedly reduced. (M) NK-cell cytotoxicity was measured by an enhanced green fluorescent protein (EGFP)-K562 flow cytometric method, and T-cell cytotoxicity was measured by CD8+ T cells against the Nalm6-luciferase cell line, which demonstrated markedly reduced NK-cell and T-cell cytotoxicity in the twins. (N) Degranulation was measured by flow cytometry in CD8+ T cells stimulated with CD3/CD28 Dynabeads and NK cells stimulated with the K562 cell line through CD107a expression, demonstrating defect in the degranulation of CD8+ T cells and NK cells in the twins. The Supplemental Materials and Methods section contains the contain details of the methodology. Due to the limited number of primary specimens, experiments in K.L.M.N. was performed only once

In contrast, the twins’ half-brother, who shares the same mother and is 10 years older, currently remain healthy. The patients did not have a family history of cancer. To explore potential inborn errors of immunity (IEIs) underlying the persistent refractoriness of these young twins, whole-genome sequencing (WGS) was retrospectively performed on the peripheral blood mononuclear cells (PBMCs) of the twins and their older half-brother. WGS data have been uploaded to the National Center for Biotechnology Information (NCBI): PRJNA809536. The 2022 Immunological Societies Expert Committee (IUIS) updated gene list was included in the analysis. The filtered nonsynonymous variants are showed in Supplemental Table 4. Alterations that the twins shared but the half-brother lacked were selected. Among these, a complex hemizygous frameshift variant in MAGT1 (c.131_134delinsGTGGTGGTTTTGGTGTGT, p.Val44Glyfs*38, NM_032121.5), which has never been reported in public databases (1000G, ExAC, and GenomAD), was identified (Fig. 1J). Sanger sequencing confirmed that the twins were hemizygous for the MAGT1 frameshift variant, while their half-brother and mother carried the wild-type gene (Fig. 1H, I). In addition, XMEN-related clinical manifestations and laboratory findings of the twins were identified [1] and shown in Supplemental Fig. 2A-3 H.

Furthermore, several functional experiments in vitro were performed to explore the characteristics of XMEN. As the CAR30 transgene was detected at zero copy number by droplet digital polymerase chain reaction (ddPCR) in both twins, there was no impact on the activity of CAR T-cells in vitro. First, NKG2D, the best biomarker of XMEN disease, was assessed, and it was found to be decreased in both CD8+ T cells and NK cells from twins compared those from mothers and two healthy donors (Fig. 1K, Supplemental Fig. 3). Second, the twins’ CD8+ T cells and NK cells had impaired expression of perforin (Fig. 1L, Supplemental Fig. 4) and diminished cytotoxicity when stimulated (Fig. 1M). Third, degranulation assays indicated that activated CTLs and NK cells were normal, while resting CTLs and NK cells were deficient (Fig. 1N, Supplemental Fig. 5).

Discussion and Conclusions

XMEN is caused by loss-of-function variants in MAGT1. In the present case, identical twins suffered from the same type of disease due to the same MAGT1 hemizygous deletion. In addition, next-generation sequencing (NGS) revealed typical genetic aberrations (Supplemental Table 4) in cHL according to the initial diagnosis via formalin-fixed, paraffin-embedded (FFPE) sequencing, indicating that germline MAGT1 alteration was the pathogenic driving factor [5].

In the present patients, MAGT1 germline mutation screening was not performed during diagnosis or at the beginning of CAR T-cell therapy. Our case underscores the importance of identifying MAGT1 deficiency in young patients with EBV-positive lymphoproliferative disease through high-throughput sequencing.

There is no international consensus on the treatment of XMEN. Anti-CD20 therapy with rituximab for EBV control has not been recommended because of its inconsistent efficacy and lack of effect on chronic EBV infection [1]. Magnesium supplementation therapy has been proven ineffective in a clinical trial (US National Institutes of Health ClinicalTrials.gov#NCT02496676). HSCT has also been attempted in some patients, but posttransplant mortality remains high [3]. Recently, Brault et al. presented data on a novel gene-editing approach that utilizes CRISPR-Cas9 to compensate for the deletion of the MAGT1 gene [6]. Despite this exciting progress, there is still a long way to go for clinical applications of this new technique.

This case provides evidence for the use of anti-CD30 CAR T-cell therapy in hematologic malignancy patients with germline MAGT1 variants. Although XMEN patients had significant CTL dysfunction, both twins achieved CR after CAR T-cell immunotherapy (twin 1 developed PD twenty-three months later). We suspect that the CAR T-cell component might compensate for the T-cell defects, and that other cytotoxic mechanisms of CAR-T cell, such as cytokine-mediated killing (e.g., killing via IFNγ), may compensate for the perforin-deficient effects on cytotoxicity. Since this was a retrospective study, CAR T-cell functional experiments were not performed in vitro. It took twenty-three months for twin 1 to relapse, while twin 2 remained in CR. Compared to twin 2, twin 1 had a greater disease load, lower infusion dose of CAR T cells, stronger CAR transgene amplification, and greater CRS. This result suggested that a low tumor load and an adequate infusion dose of CAR T cells are necessary for prolonged CR.

Although allo-HSCT can be curative for immunodeficient patients, most XMEN patients die from transplant-related complications [3]. The decision for allo-HSCT in patients with XMEN, should be balanced against the risks and the availability of a suitable donor. In this case, we recommended that Twin-1 received allo-HSCT from an unrelated healthy donor as a salvage approach when the disease progressed after CAR T-cell therapy. However, his parents repeatedly declined HSCT and requested that he take anti-PD-1 antibodies for maintenance.

In conclusion, if lymphoma is diagnosed at a young age or has a poor therapeutic outcome, it should be suspected to be a possible IEI. A novel inherited germline alteration in MAGT1 was identified, and this case is the first time CAR T-cell immunotherapy has been used in XMEN. CD30 CAR T-cell therapy may be a viable option for XMEN patients with r/r HL. More prospective experimental data are needed to explore the potential of bridging HSCT with CAR T-cell therapy.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Abbreviations

1000G 1000 Genomes Project

allo-HSCT allogeneic hematopoietic stem cell transplantation

anti-PD-1 anti-programmed cell death protein 1 antibody

autoHSCT autologous hematopoietic stem cell transplantation

Bio-RAID Biology Research and Development,chimeric antigen receptor

CD107a cluster of differentiation 107a

CD3ζ CD3-zeta chain

ChiCTR Chinese clinical trial registry

cHL classic Hodgkin lymphoma

CR complete remission

CRS cytokine release syndrome

CTL cytotoxic T lymphocyte

delins deletion/insertion

ddPCR droplet digital polymerase chain reaction

DNA deoxyribonucleic acid

DNM de novo mutation

Dr. doctor

EBV Epstein-Barr virus

EGFP enhanced green fluorescent protein

FDG-PET/CT fluorodeoxyglucose positron emission tomography/computed tomography

ExAC Exome Aggregation Consortium

Prof professor

fs frame shift

FFPE formalin-fixed,paraffin-embedded

GenomAD Genome Aggregation Database

Gly glycine

HD healthy donor,IEI,Inborn Errors of Immunity

IFNγ Interferon-gamma

IL-6 interleukin-6

IUIS Union of Immunological Societies Expert Committee

MAF minor allele frequency

MAGT1 magnesium transporter 1

NCBI National Center for Biotechnology Information

NGS next-generation sequencing

No number

NK natural killer

NKG2D natural killer group 2,member D

PET/CT positron emission tomography/computed tomography

PBMC peripheral blood mononuclear cell

PD progressive disease

PID primary immunodeficiency

r/r relapsed/refractory

SD stable disease

scFv single chain variable fragment

SP signal peptide

TCR T-cell receptor

VL variable L chain

Val Valine

VH variable H chain

WGS whole-genome sequencing

XMEN X-linked immunodeficiency with magnesium defect,EBV infection,and neoplasia

Acknowledgements

Firstly, we thank the patients and their family for their participation in this study and all the faculty and staff at our Clinical and Laboratory Unit for clinical and technical support. Secondly, the authors wish to acknowledge Dr. Huimin Zhou and Dr. Sijuan Zou for their contribution of the PET/CT images, Prof. Qilin Ao for conducting the pathology experiments, Dr. Kefeng Shen, Dr. Zhang Meilan, Dr. Zhang Wei and Dr. Zhu Zhoujie for their guidance in the WGS data analysis, Dr. Xia Mao for conducting CAR-T cell flow cytometry experiments, Dr. Liting Chen and Dr. Jia Gu for performing the droplet digital PCR experiment, Dr. Jie Xiong, Dr. Li Zhe, Dr. Ge Tong, and Dr. Deng Xinyue for their assistance in specimen collection. Thirdly, we would also like to thank the Bio-RAID Company for their support in preparing CAR T cells, PerfectMedical Diagnostics (Ezhou, Hubei Province, China), and Novogene Company for providing the DNA sequencing service.

Author Contributions

Jiachen Wang conceptualized and designed the study, conducted the experiments, analyzed the data, drafted the manuscript, and revised it. Mi Zhou provided patient care and contributed to manuscript revisions. Jianfeng Zhou and Liang Huang supervised the research and patient care. Min Xiao led the research activities and obtained the funding for the project leading to this publication. All authors have reviewed and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82270203 and No. 81770211 granted to Min Xiao), the National Natural Science Foundation of China (No. 81670152 granted to Liang Huang), and the National Natural Science Foundation of China (No. 81800162 granted to Mi Zhou). The authors would like to thank all the study team members, the patient, and his family.

Data Availability

The WGS datasets presented in this study have been uploaded to the NCBI under the accession number PRJNA809536 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA809536). The cytologic data of the patients can be available by contacting the corresponding author upon reasonable request.

Declarations

Ethics Approval and Consent Statement

This study received approval from the institutional review board of Tongji Hospital, Tongji Medical College, and Huazhong University of Science and Technology. It was registered with the Chinese Clinical Trial Registry (ChiCTR) under the registration number ChiCTR-OPN16009069.

Patient Consent

Two patients were children younger than 16, the patient consent and permission for publication were obtained from the children’s mother.

Conflict of interest

The authors have no competing interests to declare.

Trial Registration

This study received approval from the institutional review board of Tongji Hospital, Tongji Medical College, and Huazhong University of Science and Technology. It was registered with the Chinese Clinical Trial Registry (ChiCTR) under the registration number ChiCTR-OPN16009069.

The original online version of this article was revised: There was an overlap in the original Figure 1.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jiachen Wang and Mi Zhou contributed equally as first authors to this work. Min Xiao and Liang Huang contributed equally as corresponding authors.

Change history

5/25/2024

A Correction to this paper has been published: 10.1007/s10875-024-01723-8
==== Refs
References

1. Ravell JC Chauvin SD He T Lenardo M An update on XMEN Disease J Clin Immunol 2020 40 5 671 81 10.1007/s10875-020-00790-x 32451662
2. Chaigne-Delalande B Li FY O’Connor GM Lukacs MJ Jiang P Zheng L Mg2 + regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D Science 2013 341 6142 186 91 10.1126/science.1240094 23846901
3. Klinken EM Gray PE Pillay B Worley L Edwards ESJ Payne K Diversity of XMEN Disease: description of 2 novel variants and analysis of the lymphocyte phenotype J Clin Immunol 2020 40 2 299 309 10.1007/s10875-019-00732-2 31865525
4. Wang D Zeng C Xu B Xu JH Wang J Jiang LJ Anti-CD30 chimeric antigen receptor T cell therapy for relapsed/refractory CD30(+) lymphoma patients Blood Cancer J 2020 10 1 8 10.1038/s41408-020-0274-9 31974371
5. Brune MM, Juskevicius D, Haslbauer J, Dirnhofer S, Tzankov A. Genomic Landscape of Hodgkin Lymphoma. Cancers (Basel). 2021;13(4).
6. Torgerson TR Is this a cure for XMEN? Blood 2021 138 26 2743 4 10.1182/blood.2021012755 34967867
