-->PPATHOGENS-D-26-00422

Central Nervous System T-cell immune architecture, and not HIV burden, tracks with cognition under long-term viral suppression

PLOS Pathogens

Dear Dr. Trunfio,

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Jason M. Brenchley

Academic Editor

PLOS Pathogens

Richard Koup

Section Editor

PLOS Pathogens-->--> -->-->Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497-->-->Michael Malim-->-->Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

The reviewers raised concerns that need to be addressed. If the authors can address the concerns, a revision could be considered.

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Mattia Trunfio, Gemma Caballero, Vanessa Gomez-Moreno, Simon A. Mallal, Celestine N. Wanjalla, Angela Jones, Karen Beeri, Alan Wells, Sarah LaMere, Ben Gouaux, Donald R. Franklin, Michael Corley, Ronald J. Ellis, David J. Moore, Scott L. Letendre, Davey Smith, Antoine Chaillon, and Sara Gianella. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plospathogens/s/authorship#loc-author-contributions

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Trunfio and colleagues examined how HIV-1 DNA and TCR repertoires across distinct CNS regions relate to cognitive impairment in post-mortem tissues from ART-suppressed PWH, with cognitive scores assessed within a year of death. Using multivariate regression, they found that TCR diversity was highest in blood and largely compartmentalized across CNS anatomical sites. Notably, greater TCR diversity in the hippocampus and thoracic spinal cord associated with worse verbal and motor function, and regional HIV-specific clonotype frequencies also correlated with cognitive outcomes. There were no differences in HIV-1 DNA between sites (excluding the blood) nor associations to cognitive scores. Strengths of the study include the unique and comprehensive sampling opportunity of the Last Gift Cohort. All subjects had sustained ART suppression, which is a strength relative to similar studies that have been limited by cohort heterogeneity in viremic status. The novelty in this study is that to my knowledge, TCR repertoires across distinct regions of the brain in PWH have not been characterized in PWH. The major driving weaknesses are that it is devoid of inflammatory measurements, either soluble in the plasma or CNS or by transcriptomics. This is important because most of these patients fit the definition of immune non-responders (CD4<350) which are well known to exhibit persistently high levels of inflammation. A second weakness is that the associations between TCRRs and specific cognitive scores are not necessarily found within the brain regions that govern those neurological functions, leaving the implications unclear. The authors do touch on this caveat in the discussion in sampling TCR-high parenchymal versus TCR-low tissue subregions, however the authors would need spatial profiling to confirm this.

Reviewer #2: Trunfio et al. compared metrics of HIV viral persistence and T cell receptor repertoire (TCRR) in multiple central nervous system (CNS) sites to cognitive measurements prior to death in 12 patients. While HIV metrics did not correlate with cognitive performance, some TCRR metrics correlated with some cognitive measures, with generally more richness/diversity tracking with worse cognitive scores.

Overall, the article is well-written and figures are clear.

Even under highly effective anti-retroviral therapy, cogitative pathology still occurs. This study seeks to determine if there are links between infection, T cell receptor repertoire, and cognitive performance.

A particular strength is the samples assessed as CNS tissue from people with HIV is relatively rare.

Reviewer #3: This is an exhaustive and comprehensive study exploring HAND pathogenesis in the context of ART using precious and very rare autopsy samples from the Last Gift Program. The authors have used state of the art methods and analyses. The weaknesses are detailed in the manuscript including the small number of brains but two exceptionally important issues are not discussed:

* the tissues that were analysed did not include deep white matter. The methods used bulk tissue with the authors explaining that there would be some adjacent tissue ie white matter but the precise neuropathology of HAND is not understood. Certainly in the past it was more of a deep white matter pathology involving especially the frontal white matter and part of the basal ganglia but not all of the basal ganglia. It would have been far better to have included both cortical tissue and deep white matter.

*the second issue relates to the cognitive impairment - it is not at all clear when this developed: we know when it was tested for but not its onset and progression. This is fundamental to understanding pathogenesis as at least some of the deficits likely reflected past damage with an appropriate current TCRR response now that has controlled the limited replication of whole virus or part thereof. This then changes the whole new suggested paradigm of pathogenesis and makes it only a possibility rather than a certainty.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: - The authors find associations between regional T cell receptor repertoires and worse outcomes in specific neurocognitive measures such as verbal and motor functions (Figure 4); however, the significance of these findings is unclear because the specific regions in which these associations are observed — the hippocampus and thoracic spinal cord — do not primarily govern these functions. The hippocampus is mostly involved with memory, and the thoracic spinal cord primarily controls trunk muscle movement, not verbal or motor neurocognitive performance.

- Similarly, the authors observed CMV-specific clonotypes in the TSC to correlate with worse learning scores, however the TSC does not control learning.

- Do the authors have CD8 T cell counts as well? Many of these subjects likely have inverted CD4/CD8 ratios, and it would be helpful to know whether TCRs are more reflective of CD4 versus CD8 repertoires.

- The association between higher TCR diversity and worse neurocognitive outcomes is somewhat contrary to the broader literature. Oligoclonality or skewed repertoire diversity — rather than expanded diversity — has been associated with neurocognitive impairment. The significance of this finding and the potential mechanism underlying it is somewhat uncertain.

- The positive association between HIV-specific T cell clonotype frequency and better GDS and processing speed scores is intriguing but difficult to interpret. One possibility is that a higher proportion of HIV-specific clonotypes reflects more active immune surveillance of the tissue. However, neither the HIV-specific clonotype frequency nor the neurocognitive outcomes correlated with HIV DNA levels in these regions, leaving the biological significance of this association uncertain.

- Lines 395-397 regarding the underlying nature of the high TCR richness. I do agree that leukocytosis in the CNS could plausibly explain the findings. It is unfortunate that the authors do not have the cellular or transcriptomic data to test this hypothesis.

Reviewer #2: 1: A key aspect of the study is correlating measures of viral persistence and T cell receptor repertoire with cognitive performance scores. There will necessarily be a gap of time between biological measurements performed after the patient’s death and cognitive assessment prior to death. How stable were the cognitive metrics over time in this cohort? Alternatively, is there data on expected progression in a similar cohort? Was the length of time between biological measurements and cognitive assessment considered in any of the modeling?

2: Similarly, by necessity sampling of many CNS sites cannot be performed longitudinally and must be a snapshot of a particular time. How much variation in T cell receptor repertoire metrics is seen in sites that can be sampled over time (eg. PBMCs, cerebrospinal fluid) either in this cohort or in other published work, even from animal models if necessary?

In both cases, any additional data or references could strengthen the paper.

Reviewer #3: Please see above - white matter needs to be obtained and more precise details for the cognitive impairment

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: - Minor: it would improve readability if the acronyms were also spelled out in the text, when they are first referred to.

- Minor: lines 207-212, the authors should refer to the specific figure legend in the text.

Reviewer #2: 1: Though this is an acknowledged limitation of the study, a biological mechanism for the observed correlations is lacking. Some method of assessing inflammation could significantly improve the link.

2: Do the multiple measures of the HIV reservoir correlate with each other in each tissue? For example, is more HIV DNA associated with more 2LTR, usGag, or msTat/Rev? If not, (for example a lot of DNA but not ms transcripts) does that suggest something about the amount of active viral replication in the particular tissue?

3: Line 121: “emerged as an important contributor…” How accurate is the term contributor vs. correlate. Is there direct evidence of contribution in all cited cases?

4: Line 140 cites perturbed and abnormal TCRR in PWH in peripheral blood. Is that the case in this study if the peripheral blood TCRR were compared to uninfected controls?

5: Line 176. First use of abbreviations in the results eg. FMC, HPC, usGAG, etc. should be defined. Figure legends do a good job of this, though.

6: Figure 5 radial diagrams. The % labels are hard to read, could use larger and/or darker font.

7: Line 504-505 Sentence beginning “Although substantial inter-host variability,” is lacking a word. Maybe, “Although there was substantial…”

8: Methods 3.4 for the TCR sequencing could use more detail on the IMMUNOVerse platform.

Reviewer #3: Because of the issues discussed above the figure showing the sites of tissue that were analysed is inaccurate.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Submitted filename: PPATHOGENS-D-26-00422_reviewer comments.docx

Dear Dr. Trunfio,

We are pleased to inform you that your manuscript 'Central Nervous System T-cell immune architecture, and not HIV burden, tracks with cognition under long-term viral suppression' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Jason M. Brenchley

Academic Editor

PLOS Pathogens

Richard Koup

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

The reviewers believe their concerns have been addressed.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #2: Trunfio et al. compared metrics of HIV viral persistence and T cell receptor repertoire (TCRR) in multiple central nervous system (CNS) sites to cognitive measurements prior to death in 12 patients. While HIV metrics did not correlate with cognitive performance, some TCRR metrics correlated with some cognitive measures, with generally more richness/diversity tracking with worse cognitive scores.

Overall, the article is well-written and figures are clear.

Even under highly effective anti-retroviral therapy, cogitative pathology still occurs. This study seeks to determine if there are links between infection, T cell receptor repertoire, and cognitive performance.

Reviewer #3: The authors ahve adequately addressed the issues raised

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #2: The authors have sufficiently addressed the potential major issues by adding to the text.

Reviewer #3: Not applicable

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #2: The authors have sufficiently addressed the minor issues.

Reviewer #3: Not applicable

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No