RESEARCH ARTICLE OPEN ACCESS Reduced Number of Thymoma CTLA4-Positive Cells Is Associated With a Higher Probability of Developing Myasthenia Gravis Rodrigo Álvarez-Velasco, MD, Oriol Dols-Icardo, PhD, Shaima El Bounasri, Laura López-Vilaró, Correspondence Dr. Gallardo Juan Carlos Trujillo, MD, David Reyes-Leiva, MD, Xavier Suárez-Calvet, PhD, Elena Cortés-Vicente, MD, PhD, egallardo@santpau.cat Isabel Illa, MD, PhD, and Eduard Gallardo, PhD Neurol Neuroimmunol Neuroinflamm 2023;10:e200085. doi:10.1212/NXI.0000000000200085 Abstract Background and Objectives Myasthenia gravis (MG) is an autoimmune disease associated with comorbid thymoma in 10%–15% of cases. Cytotoxic T lymphocyte–associated antigen 4 (CTLA4) expressed by T cells downregulates T-cell–mediated immune response. Polymorphisms in the CTLA4 gene have been associated with the development of MG. In this context, we aimed to determine whether CTLA4 expression in the thymoma differs between patients with and without MG and whether CTLA4 gene polymorphisms are associated with these differences. Methods This is a retrospective study of all patients, with and without MG, surgically treated at our institution for thymoma between January 2010 and December 2020. Ten samples were obtained from normal thymuses as controls. The number of CTLA4-positive cells in paraffin- embedded thymoma samples was determined by immunohistochemistry. The presence of follicular-center and regulatory T-cell lymphocytes was determined by immunohistochemistry (B-cell lymphoma [BCL]-6 expression) and double immunofluorescence–based staining of CD4-FOXP3, respectively. We evaluated the association between thymic expression of CTLA4 and the development of MG. We also determined the association between CTLA4 expression and various clinical and prognostic characteristics of MG. We sequenced the CTLA4 gene and evaluated possible associations between CTLA4 polymorphisms and thymic CTLA4 expres- sion. Finally, we assessed the potential association between these polymorphisms and the risk of MG. Results Forty-one patients with thymoma were included. Of them, 23 had comorbid MG (56.1%). On average, patients with MG had fewer CTLA4-positive cells in the thymoma than non-MG patients: 69.3 cells/mm2 (95% CIs: 39.6–99.1) vs 674.4 (276.0–1,024.0) cells/mm2; p = 0.001 and vs controls (200.74 [57.9–343.6] cells/mm2; p = 0.02). No between-group differences (MG vs non-MG) were observed in the number of cells positive for BCL6 or CD4-FOXP3. CTLA4 expression was not associated with differences in MG outcome or treatment re- fractoriness. Two polymorphisms were detected in the CTLA4 gene, rs231770 (n = 30 pa- tients) and rs231775 (n = 17). MG was present in a similar proportion of patients for all genotypes. However, a nonsignificant trend toward a lower CTLA4-positive cell count was observed among carriers of the rs231775 polymorphism vs noncarriers: 77.9 cells/mm2 (95% CI: −51.5 to 207.5) vs 343.3 cells/mm2 (95% CI: 126.2–560.4). From the Neuromuscular Diseases Unit (R.Á.-V., D.R.-L., E.C.-V., I.I.), Department of Neurology, Hospital de la Santa Creu i Sant Pau, Barcelona; Department of Medicine (R.Á.-V., D.R.-L.), Universitat Autónoma de Barcelona; Memory Unit (O.D.-I., S.E.B.), Neurology Department and Sant Pau Biomedical Research Institute, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona; Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED) (O.D.-I., S.E.B.), Madrid; Departments of Pathology (L.L.-V.) and Thoracic Surgery (J.C.T.), Hospital de la Santa Creu i Sant Pau, Barcelona; Neuromuscular Diseases Group (X.S.-C., E.G.), Sant Pau Biomedical Research Institute, Hospital de la Santa Creu i Sant Pau, Barcelona; and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) (E.G.), Instituto de Salud Carlos III, Madrid. Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2023 American Academy of Neurology 1 Glossary AChR = acetylcholine receptors; BCL = B-cell lymphoma; CTLA4 = cytotoxic T lymphocyte–associated antigen 4; EDTA = ethylene diamine tetraacetic acid; GWAS = genome-wide association study; IgG = immunoglobulin G; MG = myasthenia gravis; MGFA-PIS = MG Foundation of America postintervention status; PBS = phosphate buffer solution; Treg = T regulatory; WHO = World Health Organization. Discussion Reduced CTLA4 expression in thymoma may predispose to a higher risk of developing MG. Myasthenia gravis (MG) is an autoimmune disease charac- patients with thymoma to develop paraneoplastic MG.10 The terized by fatigability and weakness. In approximately 85% of findings of those studies suggest a potential association be- cases, the disorder is associated with autoantibodies that bind tween CTLA4 and the development of MG. Although this to acetylcholine receptors (AChRs) in the postsynaptic association seems to be especially relevant in patients with membrane of the neuromuscular junction.1 thymoma, the underlying mechanisms are still unclear.11 Thymomas are uncommon neoplasms derived from the epi- The major cell type expressing CTLA-4 are T regulatory thelial cells of the thymus. Comorbid thymoma and MG is (Treg) cells,12 which are CD4+ cells that specifically express common: up to 10%–15% of patients with MG have a thy- the FoxP3 transcription factor and play a key role in the moma, and 30%–45% of patients with thymoma develop MG. maintenance of immunologic self-tolerance.13 The functional The World Health Organization (WHO) classification sys- activity of Tregs seems to be inhibited in patients with MG, tem divides thymomas into 6 types according to epithelial cell although the underlying molecular basis remains unknown.14 morphology and the lymphocyte to epithelial cell ratio.2 The thymomas most frequently associated with MG are those with In this context, we postulate that differential expression of the highest lymphocytic component (types AB, B1, and B2).3 CTLA4 in the thymi of patients with MG would confer an Almost all patients with MG with thymoma have anti-AChR increased risk of MG. We conducted this study to analyze antibodies. Thymomas have also been associated with other differences in the number of CTLA4-positive thymic cells autoimmune diseases, although the relationship is not as between patients with and without thymoma-associated MG. strong as with MG.4 The pathophysiologic mechanisms by We then sought to determine whether CTLA4 polymor- which autoimmunity is triggered in patients with thymoma- phisms were responsible for the differential expression of associated MG are believed to differ from those of other MG CTLA4 in T lymphocytes inside the thymoma. subtypes; however, these mechanisms are not completely understood.5 At present, it is not clear why some patients with thymoma develop MG and others do not.6 Methods Patients Cytotoxic T lymphocyte–associated antigen 4 (CTLA4) is a The study population consisted of all consecutive patients CD28 antagonistic homolog receptor expressed by T cells. aged 18 years or older who underwent surgery for thymoma When CTLA4 interacts with CD80 or CD86 on antigen- between January 2010 and December 2020 at our center presenting cells, it exerts a downregulatory effect on T-cell– (Santa Creu i Sant Pau Hospital, Barcelona, Spain). We ex- mediated immune response, thus acting as an immune cluded patients who underwent preoperative chemotherapy checkpoint. CTLA4 is a key player in the control of self- or radiotherapy and those diagnosed with thymic carcinoma. reactive T-cell generation in the thymus. CTLA4-deficient For controls, 10 normal thymi were obtained from donors animals experience a lymphoproliferative disorder character- without autoimmune diseases (including MG) who had un- ized by polyclonal T-cell proliferation. Anti-CTLA4 anti- dergone cardiac or parathyroid surgery. bodies such as ipilimumab, which is standard of care in several cancer types, have been associated with the development of a Standard Protocol Approvals, Registrations, broad spectrum of autoimmune diseases, including MG.7 and Patient Consents All patients provided informed consent. The study was ap- CTLA4 gene polymorphisms have been shown to predispose proved by the Institutional Ethics Committees at the Hospital to several autoimmune diseases.8 A genome-wide association de la Santa Creu i Sant Pau. study (GWAS) conducted in a North American population with AChR antibody–positive MG found that MG was asso- Clinical Variables ciated with the rs231770 variant of the CTLA4 gene.9 Another Patients with thymoma were classified into 2 subgroups study found that the CTLA4 49A/A genotype predisposed according to the presence or not of comorbid MG. MG was 2 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 2 | March 2023 Neurology.org/NN Table 1 Clinical Characteristics of Patients With Thymoma With and Without Associated Myasthenia Gravis No MG (n = 18), n (%)a MG (n = 23), n (%)a p Value Sex, male 12 (66.7%) 10 (43.5%) 0.14 Mean age (SD) at thymectomy, y 66.9 (11.4) 53.4 (2.9) 0.002 Immunosuppressant treatment before thymectomy 2 (11.1%) 16 (70.0%) <0.001 Thymoma histologic subtype (WHO) 0.003 A/AB 14 (77.8%) 7 (30.4%) B1/B2/B3 4 (22.2%) 16 (69.6%) Thymoma stage (Masaoka) 0.14 Localized (I & II) 17 (94.4%) 18 (78.3%) Invasive (III & IV) 1 (5.56%) 5 (21.7%) Other autoimmune diseases 6 (33.3%) 5 (21.7%) 0.41 Abbreviations: MG = myasthenia gravis. a All data are given as n (%) unless otherwise indicated. defined as the presence of the usual symptoms of MG and a ethylene diamine tetraacetic acid (EDTA). Goat nonimmune positive test for anti-AChR antibodies (>0.4 nmol/L by ra- serum was used to block the tissue for 1 hour. The sections dioimmunoassay). All patients with thymoma were followed were incubated with the following monoclonal antibodies: up preoperatively and postoperatively for a minimum period mouse antihuman CTLA-4 (clone BNI3, isotype immuno- of a year by a neurologist specialized in MG. Anti-AChR globulin G [IgG]2a, 1:100; Novus Biologicals, Minneapolis, antibodies and neurologic examination was performed in all MN), mouse antihuman B-cell lymphoma (BCL)-6 (clone patients to ensure they did not develop MG. GI191E, provided by Dr. Roncador CNIO, isotype IgG1, 1: 350), mouse antihuman CD20 (clone L26, isotype IgG2a, The following variables were recorded and assessed: de- nondiluted; Dako, Glostrup, Denmark), and mouse antihu- mographic characteristics (sex, age at onset, and date of di- man CD8 (clone C8/144 B, isotype IgG1, non-diluted; agnosis); follow-up time; anti-AChR titer at thymectomy (by Dako). After 3 washes with phosphate buffer solution radioimmunoassay); distribution of muscle weakness (ocular, (PBS), a peroxidase-labeled goat antimouse secondary bulbar, or limb predominance); thymoma histology (WHO antibody was applied (1:50; Jackson Immunoresearch classification)2; thymoma stage (Masaoka staging system)15; Laboratories, West Grove, PA). Sections were developed frequency of myasthenic crisis; mortality and cause of death; with DAB (3,39-Diaminobenzidine) staining (Vector Lab- treatment required; clinical outcome at last visit according to oratories, Burlingame, CA). To label the Treg cells, sam- MG Foundation of America postintervention status (MGFA- ples were permeabilized with 0.5% Tween 20 (Sigma PIS)16; and treatment refractoriness, which was defined as Aldrich, Saint Louis, MO), and then, double immunoflu- either unchanged or worse MGFA-PIS after treatment with orescence was performed with rabbit antihuman CD4 corticosteroids and ≥2 other immunosuppressive agents at (clone SP35, isotype IgG, 1:50; Abcam, Cambridge, UK) adequate dose levels for a sufficient duration.17 and mouse antihuman FOXP3 (clone 236A, isotype IgG1, 1:10; Abcam). After 3 washes with PBS, secondary anti- Treatment outcomes were classified as good or poor. A good bodies were added: goat antirabbit IgG-AlexaFluor 594 outcome was defined as an MGFA-PIS score of minimal manifestations or better, which indicates an asymptomatic Table 2 Autoimmune Diseases Present in Patients patient with a normal or only minimally altered physical ex- Diagnosed With Thymoma With and Without amination. Poor outcome was defined as MGFA-PIS worse Myasthenia Gravis than minimal manifestations status. No comorbid MG (6/18) Comorbid MG (5/23) Immunohistochemistry CIDP (n = 2) Multiple sclerosis (n = 1) Formaldehyde-fixed paraffin-embedded tissues were rou- Erythroblastopenia (n = 1) Bone marrow aplasia (n = 1) Goods syndrome (n = 1) Sjögren disease (n = 1) tinely obtained from all surgically removed thymomas. Sec- Aplastic anemia (n = 1) Graves’ disease (n = 1) tions measuring 2–4 μm were collected from the thymoma Bullous pemphigoid(n = 1) Pernicious anemia (n = 1) tissues and processed. Samples were dewaxed, hydrated Abbreviations: CIDP = chronic idiopathic demyelinating polyneuropathy; through a graded ethanol series into water, and washed in MG = myasthenia gravis. double-distilled water. Antigen retrieval was performed with Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 2 | March 2023 3 Table 3 CTLA4-Positive Cell Count in Thymomas of Patients With and Without Myasthenia Gravis, Stratified by Sex, Histologic Subtype, and Previous Immunosuppressive Treatment No MG, cells/mm2 MG, cells/mm2 p Value Sex Female 888.9 83.2 0.009 Male 557.4 51.3 0.02 Thymoma histologic subtype (WHO) A/AB 824.9 62.5 0.03 B1/B2/B3 185.5 72.3 0.03 Patients without immunosuppressive treatment before thymectomy 649.33 48.1 0.04 Abbreviations: MG = myasthenia gravis; WHO = World Health Organization. (Invitrogen, Waltham, MA) and goat antimouse IgG-Alexa Results Fluor 488 (Invitrogen). Forty-one patients with thymoma met the inclusion criteria All samples were evaluated by a pathologist specialized in chest and agreed to participate in the study; of them, 23 (56.1%) pathology. Five random fields of each tissue specimen were had MG. Of the 41 patients, 22 (53.7%) were female. The analyzed under ×10 magnification using the Microscope Zeiss mean (SD) age at thymectomy was 59.3 (14.3) years. In Axioskop 2 plus. Quantitative analysis of immunohistochemical general, patients with MG were younger, and a higher pro- staining was conducted using the image J imaging analysis portion was receiving immunosuppressants, had B-type thy- software. A customized algorithm for each marker was used to momas (WHO classification), and more advanced thymoma determine the number of positive cells per field. The average (Masaoka staging system). Table 1 summarizes the clinical value of the 5 fields was taken. Pathologist classifying thymo- characteristics of patients with and without thymoma. Eleven mas and investigators performing immunohistologic analysis patients (27%) had another autoimmune disease, 6 in the were blinded to whether the patient had MG or not. non-MG group and 5 in the MG group (Table 2). No dif- ferences in the prevalence of other autoimmune diseases were DNA Extraction and Genotyping observed in patients with and without MG. Genomic DNA was extracted from EDTA-anticoagulated peripheral blood. All coding exons and rs231770 were ana- CTLA4-Positive Cell Count lyzed by Sanger sequencing using the Big-Dye Terminator The number of thymic cells expressing CTLA4 was significantly sequencing kit (v. 3.1, Applied Biosystems), run on an ABI lower in patients with MG vs those without MG, with a mean 3730xl DNA analyzer, and analyzed with Sequencer software CTLA4-positive cell count of 69.3 cells/mm2 (95% CI: (v. 4.2; Gene Codes). Primers are listed in eTable 1, links.lww. 39.6–99.1) vs 674.4 cells/mm2 (95% CI: 276.0–1,024.0), re- com/NXI/A795, in supplementary material. spectively (p = 0.001). Patients with thymoma also had fewer thymic CTLA4-positive cells than controls without thymoma Statistical Analysis (mean 1,702.7 cells/mm2 [95% CI: 1,062.6–2,340.7]; p = 0.01). Data are summarized as percentages for categorical variables and CTLA4 was more frequently expressed by medullar than mean values with SD for continuous variables. Differences be- cortical lymphocytes of control thymuses (3,330.0 cells/mm2 tween groups were assessed with the χ 2 test for categorical var- iables and the 2-sided t test for continuous variables (or Wilcoxon test in cases of non-normal distribution). Allele and genotype Table 4 Differences in the Number of CD4+, CD4-FOXP3+ frequencies were compared between groups using the Fisher and BCL6+ Cells in the Thymomas of Patients exact test. We performed an receiver operating characteristic With and Without Associated Myasthenia Gravis analysis to determine the best CTLA4+ cell count cutoff that No MG allows to predict MG occurrence with optimal sensitivity and Cells/mm2 (95% CI) MG p Value specificity. CIs of odd ratios (ORs) were calculated using the Woolf method. Pairwise deletion was used for treating missing Total CD4+ cells 245.1 (221.1–269.1) 252.7 (213.0–292.5) 0.75 values. All statistical analyses were performed using STATA 15. CD4-FOXP3+ cells 556.3 (185.1–927.6) 556.3 (350.2–762.5) 1 BCL6+ cells 107.82 (6.80–208.8) 498.94 (0–1,328.6) 0.38 Data Availability The anonymized data that support the findings of this study Abbreviations: MG = myasthenia gravis. are available on request from the corresponding author. 4 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 2 | March 2023 Neurology.org/NN Figure 1 Thymoma Sections of A, AB, and B2 Thymomas of 3 Patients With Myasthenia Gravis (MG), 3 Patients Without MG, and 1 Normal Thymus Sections were processed for CTLA4 and BCL6 by immunohistochemistry and FOXP3 (green)-CD4 (red) using double immunofluorescence (×10 magnification). CTLA4 = Cytotoxic T lymphocyte–associated antigen 4. [95% CI: 2,434.7–4,224.3] vs 761.6 [95% CI: 418.5–1,104.8]; the corresponding values were 69.2 (95% CI: 24.40–114) vs p = 0.02). No differences in CTLA4-positive cell counts were 69.5 (95% CI: 22.81–116.22), p = 0.99. No association was observed between patients with thymoma who presented with detected between CTLA4-positive cell count and treatment comorbid autoimmune disease (other than MG) and patients refractoriness in patients with MG, which are as follows: no without any comorbid autoimmune disease, which are as treatment vs treatment refractoriness: 62.54 cells/mm2 (95% follows: 660.1 cells/mm2 (95% CI: 182.1–1,138.0) vs 708.9 CI: 29.13–95.96) vs 101.61 (95% CI: 0–205.39), p = 0.31. cells/mm2 (95% CI: 90.8–1,279.0) (p = 0.9). CTLA4 Gene Sequencing Stratified by sex, thymoma histologic subtype, and previous We genotyped all coding exons and the single nucleotide immunosuppressive treatment, the differences in the number polymorphism rs231770 in a noncoding region in patients of cells expressing CTLA4 between patients with and without with thymoma with (n = 19) and without (n = 16) associ- MG remained significant (Table 3). ated MG. We performed a receiver operating characteristic analysis to The rs231770 polymorphism was present in 30 of these 35 assess the validity of the CTLA4-positive cell count to predict patients (85.7%), and a second polymorphism, rs231775, was MG occurrence in patients with thymoma. We obtained an present in 17 of these patients (48.6%). The genotype distri- area under the curve of 0.85 (95% CI: 0.71–0.94) and a value bution of CTLA4 polymorphisms in patients with thymoma of 170 cells/mm2 below which MG occurs with Se of 87% and with and without MG was in accordance with the Hardy- Sp of 78%. Weinberg equilibrium (p > 0.05). In patients with thymoma, the prevalence of paraneoplastic MG did not differ significantly No association was observed between the number of CTLA4- positive cells and the number of either CD4-FOXP3–positive Figure 2 Prevalence of Paraneoplastic Myasthenia Gravis or BCL6-positive cells (r2 = 0.011 and r2 = 0.0015, re- (MG) in Patients With Thymoma With Different spectively). Similarly, no differences in the number of positive CTLA4 rs231775 Genotypes CD4, CD4-FOXP3, or BCL6 cells were observed between patients with and without MG (Table 4). Figure 1 shows examples of CTLA4, BCL6, and CD4-FOXP3 expression in the thymomas of patients with and without MG. No association was found between CTLA4-positive cell count and anti-AChR antibody titers in patients with MG during thymectomy (r2 = 0.042). No association was found between CTLA4-positive cell count and MG outcomes (MGFA-PIS) at 1 year post-thymectomy or at the final follow-up. At 1 year, patients with a good outcome had a mean of 52.8 cells/mm2 (95% CI: 12–93.52) vs 78.4 (95% CI: 28.83–127.9) in pa- CTLA4 = Cytotoxic T lymphocyte–associated antigen 4. tients with a poor outcome (p = 0.38). At the final follow-up, Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 2 | March 2023 5 Table 5 Differences in the Prevalence of Myasthenia Gravis Between Patients With Thymoma According to CTLA4 rs231770 Genotypes and Alleles rs231770 MG (+) thymoma (n = 19) MG (2) thymoma (n = 16) OR (95% CI) p Value Genotype TT 3 (60%) 2 (40%) 1.31 (0.13–17.7) 0.78 TC 6 (43%) 8 (57%) 0.46 (0.09–2.23) 0.27 CC 10 (63%) 6 (37%) 1.85 (0.40–8.93) 0.37 Allele T 12 (50%) 12 (50%) 0.77 (0.29–2.07) 0.60 C 26 (57%) 20 (43%) 1.3 (0.48–3.50) Abbreviations: MG = myasthenia gravis; OR = odds ratio. among the 3 rs231770 genotypes. In addition, no differences in of the CTLA4 gene polymorphisms were associated with a the prevalence of MG were found among patients with the 3 higher probability of developing MG nor with a difference in rs231775 genotypes. However, a trend toward a higher prev- the number of CTLA4-positive cells in the thymoma. How- alence of MG was found in patients with the G/G genotype ever, carriers of the rs231775 variant showed a trend toward compared with those with the A/A or A/G phenotype (OR: 4; lower CTLA4 expression. CTLA4 is constitutively expressed 95% CI: 0.4–40.1). See Figure 2 and Tables 5 and 6. by Treg cells and after T-cell receptor–mediated activation in a subset of CD4+ helper T cells. However, in contrast to None of the polymorphisms were associated with differences previous reports,18 the number of Treg cells did not differ in the number of CTLA4-positive cells within the thymoma, between patients with and without MG, suggesting that the although a trend was observed in patients carrying the lower number of CTLA4-positive cells in patients with MG is rs231775 variant toward a lower cell count compared with due to the reduced expression of the protein and not to a noncarriers: 77.9 cells/mm2 (95% CI: −51.5 to 207.5) vs decrease in the number of Treg cells. We hypothesize that a 343.3 cells/mm2 (95% CI: 126.2–560.4). dysfunction of Treg lymphocytes that would lead to a lower expression of CTLA4 in some patients with thymoma would confer an increased risk of developing MG. Indeed, an in- Discussion ducible deletion of CTLA-4 on Tfr cells results in decreased suppressive function in vitro and in vivo.19 In this study, the number of thymoma cells expressing CTLA4 were markedly reduced in patients with MG compared with We found a higher expression of CTLA4 in the medulla of those without MG (69.3 vs 674.4 cells/mm2, p = 0.001), and control thymuses compared with that in the cortex. Likewise, these differences were independent of sex, age, histologic we found a higher expression of CTLA4 in type A/AB thy- subtype, and previous immunosuppressive treatment. None momas (medullary or mixed component) compared with that Table 6 Differences in the Prevalence of Myasthenia Gravis Between Patients With Thymoma With Different CTLA4 rs231775 Genotypes and Alleles rs231775 MG (+) thymoma (n = 19) MG (2) thymoma (n = 16) OR (95% CI) p Value Genotype AA 10 (56%) 8 (44%) 1.1 (0.29–4.20) 1 AG 5 (42%) 7 (58%) 0.46 (0.11–1.9) 0.23 GG 4 (80%) 1 (20%) 4 (0.4–40.1) 0.23 Allele A 25 (52%) 23 (48%) 0.75 (0.27–2.1) 0.58 G 13 (59%) 9 (41%) 1.33 (0.48–3.69) Abbreviations: GG = guanine guanine; MG = myasthenia gravis; OR = odds ratio. 6 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 2 | March 2023 Neurology.org/NN in type B thymomas (cortical component) of patients without primary biliary cirrhosis,24 type 1 diabetes,25 and other autoim- MG. However, this finding is not replicated in patients MG in mune diseases. A previous study found that the GG genotype was whom the expression of CTLA4-positive cells is equally low in unexpectedly protective against paraneoplastic MG in patients both histologic subtypes. In the stratified study, the differences with thymoma, in contrast to the significant association between between CTLA4 cells between patients with and without MG this genotype and all other non-MG autoimmune diseases.10 We remain significant regardless of the histologic subtype. also sequenced the rs231770 polymorphism, which was associ- ated with MG in a GWAS involving 1,032 patients with MG in CTLA4 knock out results in increased T helper differentia- North America.9 Another study found that this polymorphism tion, increased germinal center B cells, and higher serum was associated with a lower expression of CTLA4.26 immunoglobulin levels in mice.19 We did not find any dif- ferences in the number of follicular center cells (BCL6- Although we found no increased risk of developing MG in positive cells) in the thymomas of patients with and without patients carrying the rs231770 or rs231775 polymorphisms, MG. Similarly, we found no association between the number we did observe a trend toward a higher risk in patients with of CTLA4-positive cells and the number of follicular cells. the +49 GG genotype. However, this genotype was not as- Although the thymus is considered the site of induction for sociated with the number of CTLA+ cells, although the lack loss of self-tolerance in patients with MG, altered peripheral of statistical association could be due to an insufficient immunoregulatory mechanisms such as defective immuno- sample size. In addition, other variants (apart from those suppressive function of Tregs may help to maintain the evaluated in this study) in the noncoding region of the AChR-specific autoimmune response. In this regard, it would CTLA4 gene could also be implicated in the lower number be valuable to conduct a study to evaluate CTLA4 and BCL6 of CTLA4+ cells in the thymomas of patients with MG. expression at the periphery to better understand the role of Finally, nongenetic factors could also explain these differ- CTLA4 in the pathogenesis of thymoma-associated MG. ences, with a lower CTLA4 expression in patients with MG being an epiphenomenon and not the primary cause of the A previous study shows an increase in germinal center number loss of immune self-tolerance. in adjacent thymic tissue but not in the thymomas of patients with thymoma-associated MG compared with nonmyasthenic A better understanding of the mechanisms by which auto- thymoma patients.20 In addition, recently, Song et al.21 dem- immunity is triggered in patients with MG is essential to onstrated that thymoma-associated patients with MG have a develop specific therapies designed to minimize or even higher percentage of BCL6-positive cells in adjacent thymic prevent the frequent side effects of current immunosuppres- tissue around thymoma than non-MG thymoma patients or sants. Confirmation of CTLA4 involvement in MG would controls. Because we analyzed exclusively thymoma tissue and represent a potential new therapeutic target. not adjacent tissues, this could explain the lack of differences in BCL6 expression between patients with and without MG. In conclusion, in this study, the thymomas of patients with MG had fewer CTLA4-positive cells than those without Our data show that the number of thymic CTLA4-positive MG. However, no clear association was found between cells does not seem to influence MG prognosis. However, CTLA4 gene polymorphisms and the number of positive cells given the small sample size of our study, we cannot rule out or the development of MG in patients with thymoma. Larger this potential association. In addition, several different studies are warranted to clarify this potential association. factors—such as age, immunosuppressive treatment, and/or histologic subtype—can act as confounders between the Acknowledgment number of CTLA4-positive cells and prognosis. D. Reyes-Leiva, X. Suárez-Calvet, E. Cortés-Vicente and Eduard Gallardo are members of the European Reference Patients with loss-of-function mutations in the CTLA4 gene Network for Neuromuscular Diseases (EURO-NMD), in- develop inflammatory lesions containing activated T cells and cluding CSUR (a national reference center for rare neuromus- macrophages affecting multiple organs. Polymorphisms in the cular diseases), and XUECs (Networks of Specialized Centers CTLA4 gene are also associated with several autoimmune dis- of Excellence in Minority Health). Coauthor Professor Isabel eases in humans.22 After a complete sequencing of all CTLA4 Illa, MD, PhD, passed away on March 20, 2022. coding regions, we found the presence of a previously described polymorphism (rs231775) in exon 1 of the CTLA4 gene in a Study Funding significant proportion of the patients. This A/G polymorphism This work was funded by the “Instituto de Salud Carlos III” at position +49 results in an amino acid change (p.Thr17Ala) in through project PI19/01,774 (cofunded by the European the leader sequence. The rs231775 polymorphism leads to in- Union ERDF), PI I. Illa, and E. Gallardo. E. Cortés-Vicente efficient processing in the endoplasmic reticulum, a differential was supported by a “Juan Rodés” grant (JR19/00,037) from glycosylation pattern, and decreased trafficking to the cell surface the “Fondo de Investigación en Salud, Instituto de Salud of CTLA4.23 The guanine guanine (GG) genotype in the Carlos III” and cofunded by the European Union (ERDF/ CTLA4 rs231775 polymorphism has been associated with an ESF, “A way to make Europe”/“Investing in your future”), increased risk of several conditions, including Graves’ disease, Ministry of Health (Spain). Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 2 | March 2023 7 Disclosure The authors report no disclosures relevant to the manuscript. Appendix (continued) Go to Neurology.org/NN for full disclosure. Name Location Contribution Elena Neuromuscular Diseases Drafting/revision of the Publication History Cortés- Unit, Department of article for content, including Vicente, Neurology, Hospital de la medical writing for content; Received by Neurology: Neuroimmunology & Neuroinflammation MD, PhD Santa Creu i Sant Pau, major role in the acquisition September 7, 2022. Accepted in final form November 23, 2022. Barcelona, Spain of data; study concept or Submitted and externally peer reviewed. The handling editor was design; and analysis or Associate Editor Marinos C. Dalakas, MD, FAAN. interpretation of data Isabel Illa, Neuromuscular Diseases Drafting/revision of the MD, PhD Unit, Department of article for content, including Neurology, Hospital de la medical writing for content; Appendix Authors Santa Creu i Sant Pau, major role in the acquisition Barcelona, Spain of data; study concept or Name Location Contribution design; and analysis or interpretation of data Rodrigo Neuromuscular Diseases Drafting/revision of the Álvarez- Unit, Department of article for content, including Eduard Neuromuscular Diseases Drafting/revision of the Velasco, Neurology, Hospital de la medical writing for content; Gallardo, Group, Sant Pau Biomedical article for content, including MD Santa Creu i Sant Pau, major role in the acquisition PhD Research Institute, Hospital medical writing for content; Barcelona; Department of of data; study concept or de la Santa Creu i Sant Pau, major role in the acquisition Medicine, Universitat design; and analysis or Barcelona; Centro de of data; study concept or Autónoma de Barcelona, interpretation of data Investigación Biomédica en design; and analysis or Barcelona, Spain Red de Enfermedades Raras interpretation of data (CIBERER), Instituto de Salud Oriol Dols- Memory Unit, Neurology Drafting/revision of the Carlos III, Madrid, Spain Icardo, PhD Department and Sant Pau article for content, including Biomedical Research medical writing for content; Institute, Hospital de la Santa major role in the acquisition Creu i Sant Pau, Universitat of data; and analysis or Autònoma de Barcelona; interpretation of data References Network Center for 1. 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