Association of tuberculosis risk with genetic polymorphisms of the immune checkpoint genes PDCD1, CTLA-4, and TIM3

Chi-Wei Liu; Lawrence Shih-Hsin Wu; Chou-Jui Lin; Hsing-Chu Wu; Kuei-Chi Liu; Shih-Wei Lee

doi:10.1371/journal.pone.0303431

Peer Review History

Original SubmissionDecember 17, 2023
13 Mar 2024 Decision Letter - Afsheen Raza, Editor PONE-D-23-39192Association of tuberculosis risk with genetic polymorphisms of the immune checkpoint genes PD-1, CTLA-4, and TIM3PLOS ONE Dear Dr. Lee, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Apr 27 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This interesting study investigates the association between SNPs in the immunoregulatory genes with susceptibility to TB in a Taiwan population. The manuscript is well written. I have some minor suggestions Please remove the results from introduction section Please provide the chromosome locations of the studied genes in the introduction The authors have not mentioned about allele frequencies and its association with TB The authors have provided separate tables for frequency, and statistical results. It will be difficult for the readers to go back and forth In one table, they should provide frequencies for total TB and non-TB groups with statistical analysis results including different models. Likewise, a separate table for males and females can be provided The authors should correct the P values for multiple comparisons I suggest authors stratify the cases based on disease severity and perform association analysis with SNPs The age of controls is significantly higher than TB patients. How the authors justify this In addition to the models analyzed, I suggest analysis by overdominant model since heterozygotes show an association Reviewer #2: Summary: The authors tried to investigate the impact of polymorphism s in PDCD1, CTLA4, HAVCR2 on susceptibility for TB infection with total 555 cohort including 285 patients with TB infection. The authors genotyped 11 SNP sites and found that 11 SNPs did not differ significantly in frequency between the non-TB and TB groups. Then, the authors observed that rs2227982, rs13170556, rs231775, and rs231779 were sex-specifically associated with TB risk and concluded that the results may help explain the possibility of a difference in TB prevalence between non-TB and TB subjects in men and women in Taiwan cohort. Comment Both genetic susceptibility and impact of exhaustion related gene to TB infection is important topic as prevention medicine and interesting subject in immunology. However, this study has several issues for design, analyzing method and interpretation. Major 1, TB infection is a known as an opportunistic infection and well investigated the risk factor such as host status and environment for susceptibility. This study design does not care about non genetic factor at all. If authors try to achieve their purpose with current cohort, they should analyze with adjustment factors by generally acceptable cofounders or any other way to avoid the cofounder effects. Currently, the authors do not care a cofounder even the age factor for statistical analyses. The authors good to summarize the general information about risk factors of TB infection in introduction. 2. There are no description about how the SNPs affect to the susceptibility of TB infection. Some SNPs (or haplotype) must have been reported their functional effect on immune response or clinical outcome in previous reports. The author should try to describe the consistency or inconsistency with reasonable data or explanation. Why does some SNPs work as sex-specific manner? How can heterozygous genotype be impactful? Mechanism? Minor 1. I recommend using the proper symbol for each gene in scientific journal ; for example, PDCD1(italic) gene for PD-1 protein 2. The authors need to provide basic information about SNPs where do they located (intron, exon, or promoter region). The information helps to imagine the biological effect of SNPs. ****** 6. 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Revision 1
18 Apr 2024 Author Response Additional Editor Comments: The study is considered important in its field and can provide good background knowledge for scientists working in this field. However, the concerns raised by the reviewers is valid and needs to be addressed. Reply：Thank you for your kind comment. For your and reviewers’ comments, we modified the Introduction, Results, Discussion, Table 1-6, and S1-S6 Tables. We added two paragraphs in the next to the last paragraph of the Discussion (pages 36-39) in order to discuss the association between selected SNPs and clinical outcome of TB and the sex-dependent association of some selected SNPs with TB risk. In addition, we also modified our manuscript meets the style requirements of PLOS ONE. Responses to reviewer 1： Reviewer #1: This interesting study investigates the association between SNPs in the immunoregulatory genes with susceptibility to TB in a Taiwan population. The manuscript is well written. Reply：Thank you very much for your kind comment. We revised the text in the Introduction, Result, and Discussion and modified the Tables as you suggested. My response to your specific comment is as follows. In response to the minor comment： Comment 1: Please remove the results from introduction section. Reply: Thank you for your comment. As you suggested, we removed the results from introduction section. Comment 2: Please provide the chromosome locations of the studied genes in the introduction. Reply: Thank you for your comment. As you suggested, we added the statement about the chromosome locations of the PDCD1, CTLA4, and HAVCR2 genes in the introduction (page 3 and Lines 17-18; page 4 and Lines 10-11; page 5 and Lines 1-2). In addition, we added a new supplementary Table (S1 Table) for the characteristics of selected SNPs. Comment 3: The authors have not mentioned about allele frequencies and its association with TB. Reply: Thank you for your comment. As you suggested, we respectively used χ2 test and odds ratio analysis to evaluate the difference in allele frequencies between TB and non-TB groups and the association between allele of SNPs and TB risk. In addition, we added the statement about these results to the text in the Results, Tables 2-4, and S4-S5 Tables. Comment 4: The authors have provided separate tables for frequency, and statistical results. It will be difficult for the readers to go back and forth. Reply: Thank you for your comment. As you suggested, to reduce reading difficulties of the readers, we modified Tables 2-5 and S4-S5 Tables and provided genotype frequencies for total TB and non-TB groups with statistical analysis results including different models in one table. Comment 5: In one table, they should provide frequencies for total TB and non-TB groups with statistical analysis results including different models. Likewise, a separate table for males and females can be provided. Reply: Thank you for your comment. As you suggested, we added the genotype frequencies for total TB and non-TB groups with statistical analysis results including different models in one table. In addition, we provided separate tables for the statistical analysis results in the overall, male, and female participants. Comment 6: The authors should correct the P values for multiple comparisons. Reply: Thank you for your comment. As you suggested, we added the Bonferroni correction of P values in Tables 2-6 and S4-S6 Tables. Comment 7: I suggest authors stratify the cases based on disease severity and perform association analysis with SNPs. Reply: Thank you for your valuable comments. As you suggested, we evaluated the percentage of TB severity (cavitation and pleural effusion) in patient with TB. However, the number of subjects with different TB severity (cavity and pleural effusion) is not sufficient to evaluate the association of selected SNPs with TB severity. We added the percentage of TB severity (cavitation and pleural effusion) in Table 1 and the statement about the limitations in our study to the Discussion (page 37). Comment 8: The age of controls is significantly higher than TB patients. How the authors justify this? Reply: Thank you for your question. In our study, to avoid the interference of some antiviral and anti-cancer drugs, patients with cancer or other immune-related diseases and viral infections (e.g., hepatitis B, hepatitis C, HIV) were excluded. Because young participants rarely have clinical evidence for the exclusion criteria, it resulted in the age of subjects enrolled in control group was older than TB group. In order to reduce the influence of age of subjects on association of target SNPs with TB susceptibility, we used the logistic regression to simultaneously adjust for age of subjects. In addition, the older people without a history of TB disease seem to be more likely to represent as a control group than young people because they still did not have TB infection. We added the statement about the limitations in our study to the Discussion (page 39) as your suggestion. Comment 9: In addition to the models analyzed, I suggest analysis by overdominant model since heterozygotes show an association. Reply: Thank you for your suggestion. According to the definition of various genetic models, the additive model in our study represents overdominant model (wild type homozygous + minor allele homozygous versus heterozygous). As you suggested, we modified the model name in our manuscript. Responses to reviewer 2： Reviewer #2: Both genetic susceptibility and impact of exhaustion related gene to TB infection is important topic as prevention medicine and interesting subject in immunology. However, this study has several issues for design, analyzing method and interpretation. Reply: Thank you very much for your kind comment. We appreciate your providing us the second opportunity to revise the manuscript. We added one new supplementary Table (S1 Table) and two paragraphs in the next to the last paragraph of the Discussion (pages 36-39) as you suggested. My response to your specific comment is as follows. In response to the major comment： Comment 1: TB infection is a known as an opportunistic infection and well investigated the risk factor such as host status and environment for susceptibility. This study design does not care about non genetic factor at all. If authors try to achieve their purpose with current cohort, they should analyze with adjustment factors by generally acceptable cofounders or any other way to avoid the cofounder effects. Currently, the authors do not care a cofounder even the age factor for statistical analyses. The authors good to summarize the general information about risk factors of TB infection in introduction. Reply: Thank you for your comment. In our study, we majorly investigated the association of host genetic polymorphisms with susceptibility to TB. To avoid the interference of non-genetic factors for TB susceptibility, subjects with cancer or other immune-related diseases and viral infections (e.g., hepatitis B, hepatitis C, HIV) were excluded. In addition, the selected participants were majorly enrolled in northern Taiwan, which means our study also excluded the effects of some environmental conditions on susceptibility to TB. In this study, we did not evaluate the association of household contacts of TB patients with TB risk. Some TB subjects were household contacts of TB patients. However, it is difficult to clarify whether participants are non-household contacts of TB patients because of consideration of patient confidentiality restraints. In addition, significant differences in age between non-TB and TB groups were found. Because young participants rarely have clinical evidence for the exclusion criteria, it resulted in the age of subjects enrolled in control group was older than TB group. In order to reduce the influence of age of subjects on association of target SNPs with TB susceptibility, we used the logistic regression to simultaneously adjust for age of subjects. We added the statement about the limitations in our study to the Discussion (page 39). Comment 2: There are no description about how the SNPs affect to the susceptibility of TB infection. Some SNPs (or haplotype) must have been reported their functional effect on immune response or clinical outcome in previous reports. The author should try to describe the consistency or inconsistency with reasonable data or explanation. Why does some SNPs work as sex-specific manner? How can heterozygous genotype be impactful? Mechanism? Reply: The important effects of several host genetic factors on tuberculosis infection have been reported [Reference 41]. PD-1, CTLA4, and TIM3 were associated with host immune response against Mtb infection [Reference 6]. In this study, we indicated the associations of PDCD1 rs2227982, HAVCR2 rs13170556, CTLA4 rs231775, and CTLA4 rs231779 with TB risk. Though the number of subjects with different TB severity (cavitation and pleural effusion) is not sufficient to evaluate the association of selected SNPs with TB severity in our study, some previous studies have reported the association of these SNPs with clinical outcome of diseases, such as TB, primary biliary cirrhosis, and Hepatitis C [References 26, 42-44]. In PDCD1 rs2227982, TB patients with TC or CC genotype have higher rate of tuberculous cavity than those with TT genotype [Reference 42]. In previous study of African Population, the CTLA4 haplotype (rs11571315-rs733618-rs4553808-rs231774-rs231775-rs231777-rs3087243; G-A-A-A-G-C-A) of was associated with cavities in patients with TB [Reference 26]. The CTLA4 haplotype (rs231775-rs231777-rs3087243-rs231725; G-C-G-A) was a protective factor for progression of primary biliary cirrhosis in Japanese patients [Reference 43]. Though the association between HAVCR2 rs13170556 with clinical outcomes of TB is unknown, the protective effects of rs13170556 TC/CC genotypes with the F protein on the outcomes of HCV infection was reported [Reference 44]. In addition, the GTExPortal database indicates an association of PDCD1 rs2227982, HAVCR2 rs13170556, CTLA4 rs231775, and CTLA4 rs231779 with their gene expression in different tissues. Taken together, these finding may suggest that different genotypes in these SNPs may affect the host immune response against Mtb infection and clinical outcome of TB by regulating their gene expression. The sex-dependent association of PDCD1, CTLA4, and HAVCR2 polymorphisms with TB susceptibility were found in our study. Some previous studies also indicated that PDCD1, CTLA4, and HAVCR2 polymorphisms were sex-dependently associated with MAC-LD risk, the resolution of hepatitis C virus infection, and the outcomes of HCV infection, respectively [References 32, 44, 45]. In addition, previous study indicated that the effects of some sex-specific factors on the immune response and women have a higher prevalence of autoimmune diseases compared with men [Reference 46]. The difference in function of CD4+CD25+ regulatory T-cells was observed in male and female mice [Reference 47]. Taken together, these observations may suggest that the difference in sex-dependent effect of PDCD1, CTLA4, and HAVCR2 polymorphisms on immune response against Mtb infection. In our study, odds ratio analysis showed the significant associations of heterozygous genotype in PDCD1 rs2227982, HAVCR2 rs13170556, CTLA4 rs231775, and CTLA4 rs231779 with TB risk. In previous studies, the significant associations of heterozygous genotypes of PDCD1 rs2227982 and HAVCR2 rs13170556 with the risk for breast cancer and the outcomes of HCV infection were reported, respectively [References 44, 48]. Though the transcriptional regulatory mechanism of different genotypes in these SNPs did not evaluate in our study, the differences in gene expression between heterozygous and homozygous genotypes of these SNPs in different tissues were found in the GTExPortal database [Reference 30]. Based on these findings, we hypothesize that the difference in gene expression between heterozygous and homozygous genotype of these SNPs may influence immune response against Mtb infection. However, our findings still need more researches with large sample sizes to validate. In future, the transcriptional regulatory mechanism of these SNPs and serum levels of soluble PD-1, CTLA4, and TIM3 should be investigated. We added the statement in the Discussion (page 36-39). In response to the Minor comment： Comment 1: I recommend using the proper symbol for each gene in scientific journal; for example, PDCD1(italic) gene for PD-1 protein. Reply: Thank you for your comment. As you suggested, we used the proper symbol for each gene in the text of our manuscript, Figure legend, and Tables. Comment 2: The authors need to provide basic information about SNPs where do they located (intron, exon, or promoter region). The information helps to imagine the biological effect of SNPs. Reply: Thank you for your comment. As you suggested, we added a new supplementary Table (S1 Table) for the characteristics of selected SNPs. Attachments Attachment Submitted filename: Responses to reviewers 20240403.docx https://doi.org/10.1371/journal.pone.0303431.r002
25 Apr 2024 Decision Letter - Afsheen Raza, Editor Association of tuberculosis risk with genetic polymorphisms of the immune checkpoint genes PDCD1, CTLA-4, and TIM3 PONE-D-23-39192R1 Dear Dr. Shih-Wei Lee We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Afsheen Raza, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0303431.r003
Formally Accepted
29 Apr 2024 Acceptance Letter - Afsheen Raza, Editor PONE-D-23-39192R1 PLOS ONE Dear Dr. Lee, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Afsheen Raza Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0303431.r004

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