Expression of MHC II in DRG neurons attenuates paclitaxel-induced cold hypersensitivity in male and female mice

  1. Emily E. Whitaker
  2. Neal E. Mecum
  3. Riley C. Cott
  4. Diana J. Goode

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Abstract

Chemotherapy is often a life-saving treatment, but the development of intractable pain caused by chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting toxicity that restricts cancer survival rates. Recent reports demonstrate that paclitaxel (PTX) robustly increases anti-inflammatory CD4 + T cells in the dorsal root ganglion (DRG), and that T cells and anti-inflammatory cytokines are protective against CIPN. However, the mechanism by which CD4 + T cells are activated, and the extent cytokines released by CD4 + T cells target DRG neurons are unknown. Here, we are the first to detect major histocompatibility complex II (MHCII) protein in mouse DRG neurons and to find CD4 + T cells breaching the satellite glial cell barrier to be in close proximity to neurons, together suggesting CD4 + T cell activation and targeted cytokine release. MHCII protein is primarily expressed in small nociceptive neurons in male and female mouse DRG but increased after PTX in small nociceptive neurons in only female DRG. Reducing one copy of MHCII in small nociceptive neurons decreased anti-inflammatory IL-10 and IL-4 producing CD4 + T cells in naïve male DRG and increased their hypersensitivity to cold. Administration of PTX to male and female mice that lacked one copy of MHCII in nociceptive neurons decreased anti-inflammatory CD4 + T cells in the DRG and increased the severity of PTX-induced cold hypersensitivity. Collectively, our results demonstrate expression of MHCII protein in mouse DRG neurons, which modulates cytokine producing CD4 + T cells in the DRG and attenuates cold hypersensitivity during homeostasis and after PTX treatment.

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    Reply to the reviewers

    *We appreciate the valuable suggestions and the overall highly positive review of our manuscript. We have now included many suggestions provided by the reviewers, which have made our manuscript much stronger and more rigorous. One reviewer acknowledged, “This study uncovers sex-dependent mechanisms underlying cold sensitivity between male and female mice. The detailed IHC analysis of MHCII expression in DRG neurons is a clear strength of this study and supports flow cytometry results as well as existing literature. The specificity of MHCII expression on small diameter is well characterized and supported by conditional knockout mouse models of MHCII in TRVPV1-lineage neurons.” *

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Whitaker EE and co-authors implicate MHCII expression in DRG neurons in the resolution of pain following paclitaxel treatment. The authors demonstrate that CD4 T cells closely interact with DRG neurons, which also express MHCII proteins. They further characterize neuronal MHCII expression in naïve and paclitaxel treated mice in small diameter TRPV1+ neurons. Utilizing genetic animal models with MHCII knockout in TRPV1-lineage neurons, the authors highlight that loss of MHCII in TRPV1 neurons exaggerates cold sensitivity in naïve male mice, and in both sexes following paclitaxel treatment.

    Major concerns:

    The most pressing concern regarding this study is a lack of a vehicle control group. It is not appropriate to be comparing paclitaxel treated mice to naïve mice. Please include a vehicle treatment (cremophor:ethanol 1:1 diluted 1:3 in PBS) group for all experiments involving paclitaxel. This would also improve statistics as unpaired T tests comparing naïve vs paclitaxel is not convincing.

    Figure 1A only includes representative images of a small number of T cells in presumable contact with DRG neurons in female Day 14 paclitaxel mice, but does not include images from other groups. Similarly, B-D show a single CD4+ T cell in contact with DRG neurons in Day 14 paclitaxel and naïve female mice. Please include quantification of the frequency of CD4+ T cells interacting with DRG neurons in the different experimental groups utilized in this study.

    Please include entire blot for Figure 2A (or at least more of the blot). There is plenty of space in the figure and as it currently appears is not free from apparent manipulation.

    The authors conclude that MHCII helps to suppress chemotherapy-induced peripheral neuropathy, resolving cold allodynia following paclitaxel treatment. To support this conclusion, I think it is necessary to include a time-course experiment highlighting whether cKO of MHCII in TRPV1 neurons indeed increases the duration for cold hypersensitivity to resolve following paclitaxel treatment.

    Minor concerns:

    The graphical abstract is misleading. The authors suggest paclitaxel is acting exclusively via TLR4 and that signaling is resolved at Day 14 which their data does not support. Please adjust to reflect findings from the experiments included in this study.

    Figure 4 and 6 MHCII labelling is oversaturated in most of the images, creating a blurry hue in the representative images. This should be fixed

    The effects of the PTX cHET group are very mild in both the male and female cohorts, and specific to 1 trial. I believe these assessments were conducted at Day 6 post injection. Why was this timepoint chosen considering differences in MHCII expression in small neurons was only present at Day 14 relative to naïve? The statistical analysis should also have been a mixed-effects repeated measures between groups ANOVA.

    Significance

    This study uncovers sex-dependent mechanisms underlying cold sensitivity between male and female mice. The detailed IHC analysis of MHCII expression in DRG neurons is a clear strength of this study, and supports flow cytometry results as well as existing literature. The specificity of MHCII expression on small diameter is well characterized and supported by conditional knockout mouse models of MHCII in TRVPV1-lineage neurons. The clear limitations of this study is the lack of a vehicle control group and limited behavioral analysis. They undermine the conclusions made by the author, and in extension, the significance of this study.

    This study adds to the understanding of neuro-immune signaling in peripheral neuropathic pain. As far as I am aware, this is the first study to investigate MHCII expression in DRGs in relation to development of chemotherapy-induced peripheral neuropathy. Thus this study provides an incremental advance in neuroimmune mechanisms contributing to the development of chemotherapy-induced peripheral neuropathy in mice.

    This study would be of interest to basic researchers interested in neuropathic pain, with particularly researchers with a focus on neuroimmunology and chemotherapy-induced peripheral neuropathy models. The sex differences observed in naïve mice would also be of interest to basic researchers within the wider pain field. Given the preliminary nature of the findings, I do not think this would be of interest to broader neuroimmunology or clinical audiences.

  5. Version published to 10.1371/journal.pone.0298396
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    Reply to the reviewers

    We appreciate the valuable suggestions and the overall highly positive review of our manuscript. We have now included many suggestions provided by the reviewers, which have made our manuscript much stronger and more rigorous. One reviewer acknowledged, “This study uncovers sex-dependent mechanisms underlying cold sensitivity between male and female mice. The detailed IHC analysis of MHCII expression in DRG neurons is a clear strength of this study and supports flow cytometry results as well as existing literature. The specificity of MHCII expression on small diameter is well characterized and supported by conditional knockout mouse models of MHCII in TRVPV1-lineage neurons.”

    R1: It is not, yet, possible to conclude that all experiments are adequately powered as N's for some studies are not provided.

    All experiments include N’s both in the text and in the figure legend.

    R1: It is unclear what is meant by "novel" expression, used throughout the manuscript.

    MHCII is traditionally thought to be constitutively expressed on antigen-presenting cells (APCs) and induced by inflammation on some non-APCs, including endothelial, epithelial, and glial cells (van Velzen et al., 2009). RNA seq data sets (Nguyen et al., 2021, Tavares- Ferreira et al., 2022, Usoskin et al., 2015, Lopes et al., 2017) demonstrate that mouse and human DRG neurons express transcripts for MHCII and MHCII-associated genes. However, there are no reports to date that demonstrate MHCII protein expression in terminally differentiated neurons. To the best of our knowledge, we are the first group to show that MHCII protein is expressed in DRG neurons.

    R1: The statement at the end of the abstract, "and that neuronal MHCII may also contribute to many other neurological disorders" seems premature, beyond the scope of the present study.

    We agree with the reviewer’s comment and have changed the sentence to the following: “Collectively, our results demonstrate expression of MHCII on DRG neurons and a functional role during homeostasis and inflammation” (pg. 1).

    R1: While cold allodynia (hypersensitivity) is a clinically important feature of CIPN, especially in CIPN associated with the platinum based chemotherapeutic agents, it is less so taxane CIPN. Do 60% of patients with PTX CIPN express cold allodynia or does that number refer to CIPN in general?

    This statistic is based on a study that conducted a meta-analysis of CIPN incidence and prevalence with paclitaxel, bortezomib, cisplatin, oxaliplatin, vincristine or thalidomide. However, we now include another reference (PMID: 15082135) that demonstrates patients receiving PTX experience cold hypersensitivity (pg.3).

    R1: Again, the future direction of expanding studies of the role of MHCII in other aspects of the CIPN phenotype might bear mention.

    We have included future directions regarding other aspects of CIPN phenotype in the discussion. We state, “Reducing the expression of MHCII in TRPV1-lineage neurons exacerbated PTXinduced cold hypersensitivity in both male and female mice. Future studies will evaluate the role of MHCII in PTX-induced mechanical hypersensitivity, another prominent feature of CIPN” (pg. 29).

    R1: Is there any evidence that IL-4 and/or IL-10 influence cold sensitivity?

    IL-10 and IL-4 have been shown to suppress spontaneous activity from sensitized nociceptors (Krukowski et al., 2016; Laumet et al., 2020; Chen et al., 2020) and to reduce neuronal hyperexcitability (Li et al., 2018), respectively. In addition, IL-10 has been shown to reduce mechanical hypersensitivity (Krukowski et al., 2016); however, cold sensitivity has not been evaluated. IL-4 KO mice do not have an increase in tactile allodynia or cold sensitivity after CCI; however, there is an increase in anti-inflammatory cytokines, specifically IL-10, and opioid receptors, which may be a compensatory mechanism that protects against enhanced pain after injury (Nurcan Üçeyler et al. 2011).

    R1: Are these experiments run blinded?

    Yes, this is discussed in the materials and methods section (pg. 31).

    R1: The term "directly contacts" is unclear. No synaptic structure is identified. It might be more accurate to estimate the actual proximity between the two cells, especially as direct contact would not be necessary for the type of intercellular communication they are studying. This is not an EM study.

    We agree with the reviewer’s comment and have changed the wording to “in close proximity” (pgs. 1,5, 7, 27).

    R1: Two abbreviations are used for immunohistochemistry, ICC and IHC.

    IHC refers to immunohistochemistry, and ICC refers to immunocytochemistry. We accidently wrote ICC in the immunohistochemistry section in the materials and methods section. We have now corrected it to say IHC (pg. 32).

    R1: In some figure, group sizes are not indicated (e.g., Fig. 4D).

    All group sizes are indicated in the text and figure legends.

    R1: "small non-nociceptive neurons" - seems to refer to TRPV1+ neurons. There are, however, TRPV1-nociceptors. "Therefore, the majority of MHCII+ neurons in the DRG of naïve female mice were not TRPV1- lineage neurons but non-nociceptive C-LTMRs." Could use some clarification here. Are the authors suggesting that being TRPV1- defines a neuron a non-nociceptive?

    We never said small non-nociceptive neurons are TRPV1+ neurons. We crossed TRPV1 lineage mice with td-tomato to label TRPV1 lineage neurons, which include TRPV1 neurons, IB4, and a subset of Aẟ neurons. We found that TRPV1 lineage neurons comprise about 65% of small diameter neurons, so 35% of small diameter neurons are not TRPV1 lineage cells. These non- TRPV1 lineage small diameter neurons are non-nociceptive LTMRs, most likely TH and MrgB4 neurons.

    R2: The most pressing concern regarding this study is a lack of a vehicle control group. It is not appropriate to be comparing paclitaxel treated mice to naïve mice. Please include a vehicle treatment (cremophor:ethanol 1:1 diluted 1:3 in PBS) group for all experiments involving paclitaxel.

    We believe the most appropriate control to paclitaxel treatment is the naïve control because clinically, paclitaxel is always administered to the patient in a formulation of 50% Cremophor and 50% ethanol. In clinical studies, the controls are healthy no-pain individuals and patients receiving paclitaxel without pain. However, the percentage of patients receiving paclitaxel that do not develop CIPN is low, emphasizing the need for healthy individuals not taking paclitaxel.

    R2: Figure 1A only includes representative images of a small number of T cells in presumable contact with DRG neurons in female Day 14 paclitaxel mice but does not include images from other groups. Similarly, B-D show a single CD4+ T cell in contact with DRG neurons in Day 14 paclitaxel and naïve female mice. Please include quantification of the frequency of CD4+ T cells interacting with DRG neurons in the different experimental groups utilized in this study.

    We have now quantified the number of CD4+ T cells per mm2 of DRG tissue, which is found in the text (pg. 5) and figures (Fig. S1 and Fig. 1A). We plan to add the quantification of CD4+ T cells per mm2 of DRG tissue for naïve and day 14 PTX-treated male mice. This data will be included in the text (pg. 5) and in Fig. S1.

    R2: Please include entire blot for Figure 2A (or at least more of the blot). There is plenty of space in the figure and as it currently appears is not free from apparent manipulation.

    We included a larger area of the western blot in Fig 2A (pg. 9).

    R2: The authors conclude that MHCII helps to suppress chemotherapy-induced peripheral neuropathy, resolving cold allodynia following paclitaxel treatment. To support this conclusion, I think it is necessary to include a time-course experiment highlighting whether cKO of MHCII in TRPV1 neurons indeed increases the duration for cold hypersensitivity to resolve following paclitaxel treatment.

    We conclude that neuronal MHCII suppresses cold hypersensitivity in naïve male mice and reduces the severity of PTX-induced cold hypersensitivity at the peak of the response (day 6) (pg. 1-2). In addition, knocking out one copy of MHCII in male TRPV1-lineage mice reduced total neuronal MHCII in naïve and PTX-treated mice (day 7 and 14) (pgs. 21-22; Fig.7). Moreover, knocking out one copy of MHCII in female TRPV1-lineage mice reduced surface- MHCII in female 7 days post-PTX (pgs. 19-20; Fig.6). Future studies will investigate the distinct roles of surface and intracellular neuronal MHCII and the contribution of MHCII to the resolution of CIPN.

    R2: The graphical abstract is misleading. The authors suggest paclitaxel is acting exclusively via TLR4 and that signaling is resolved at Day 14 which their data does not support. Please adjust to reflect findings from the experiments included in this study.

    We have removed TLR4 from our graphical abstract as we do not investigate the role of TLR4 in this manuscript. However, we do not suggest paclitaxel is acting exclusively through TLR4. We modified our wording to indicate both pro-inflammatory cytokines and PTX act on neurons to induce hyperexcitability and neurotoxicity: “Pro-inflammatory cytokines and PTX act on DRG neurons inducing hyperexcitability (Li et al., 2018, Boehmerle et al., 2006, Li et al., 2017) and neurotoxicity (Goshima et al., 2010, Flatters and Bennett, 2006), which manifests as pain, tingling, and numbness in a stocking and glove distribution (Rowinsky et al., 1993)” (pg. 9).

    R2: Figure 4 and 6 MHCII labelling is oversaturated in most of the images, creating a blurry hue in the representative images. This should be fixed.

    The signal intensity of immune cell MHCII is >5 times greater than neuronal MHCII; therefore, in order to visualize neuronal MHCII, the immune cell MHCII is oversaturated. We reference this in the discussion (pg. 26).

    R2: The effects of the PTX cHET group are very mild in both the male and female cohorts, and specific to 1 trial. R3: Furthermore, the behavioral effect is seemingly variable, with only one of the three trials being significantly different between groups. This variable response needs to be discussed further.

    This behavioral assay was developed by the UNE COBRE Behavior Core, under the guidance of Dr. Tamara King, who has extensive experience in using learning and memory measures to determine changes in pain such as development of thermal hypersensitivity (1-3, King et al, Nat Neuro 2009). Methodologically, the process is as follows: In the temperature placed preference assay, mice are placed on the reference plate (25 °C) to begin each 3-minute trial. For the habituation trial, both the test and reference plates are set to 25 °C, and the mice are allowed to explore for 3 minutes. The following 3 trials are the acquisition trials where the reference plate is set to 25 °C and the test plate to 20 °C. If the animals have cold hypersensitivity, modeling cold allodynia, then they will demonstrate faster acquisition of a learned avoidance response compared to the WT controls. For the results, we will clarify our findings, which are outlined below:

    1. We will change the axis labels to better distinguish BL/habituation trial from reference trials in the graphs.
    2. We will add graphs comparing naïve versus PTX for male and female WT mice.
    3. The changes in the graphs will now reflect 3 key findings: First, we note that PTX-treated mice learn to avoid the cold test plate faster than the naive controls in the WT mice reflecting PTX-induced cold hypersensitivity. Of interest, both males and females demonstrate learned avoidance by trial 2 and that the percent of time on the cold plate continued to decline only in the PTX-treated mice. We had not graphed this in the original figure and plan to add graphs for both male and female WT mice. These graphs are important to include as it validates that this TPP can capture the expected PTX-induced cold hypersensitivity in WT mice. Second, in terms of the naïve cHET mice, these data show that both female and male cHET mice demonstrate faster learning to avoid the cold (20 °C) plate compared to the WT mice (Fig. 8A, B. We note that the males demonstrate a more robust effect, (faster learned avoidance of the cold plate) with significant avoidance to the cold plate emerging in the cHET mice by trial 3 compared to trial 4 in the females (sig diff compared to BL trial). Third, we observed that cHET mice treated with PTX demonstrate even more accelerated learning to avoid the cold plate compared to WT mice treated with PTX. This observation suggests that PTX-treated cHET mice have heightened cold allodynia compared to the WT mice.

    R2: The statistical analysis (for the behavior) should also have been a mixed-effects repeated measures between groups ANOVA.

    We agree and re-analyzed our behavior data using repeated measures mixed-effects model (REML) with Dunnett’s multiple comparison test comparing trials 2-4 to trial 1 within same group, and Sidak’s multiple tests for significance between groups at the same trial (pgs. 23-25; Fig. 8)

    R3: Presented in Figure 3, the authors present data to show surface expression of MHCII, along with the ability of MHCII to present OVA peptide, on naïve and PTX-treated DRG neurons. These data are probably the most relevant in terms of expression as they look at the surface expression of MHCII along with the potential of MHCII to function; therefore, it is unclear why the authors only conducted this analysis on female neurons, and not both male and female neurons. Given the claims of the paper in terms of sex differences for MHC expression, I strongly suggest this is done in order to put the other observations into context.

    We completely agree and have added male mice data in Figs. 2 and 3. By western blot, we show that PTX increased the amount of MHCII protein 14 days post-PTX in DRG neurons from female mice, but there’s no change in MHCII protein after PTX in male mice (Fig. 2). In agreement with the western blot, surface-MHCII determined by flow cytometry did not increase after PTX on DRG neurons from male mice (Fig. 3B). Moreover, the frequency of DRG neurons from male mice with surface-MHCII (determined by ICC) and OVA peptide did not change after PTX treatment (Fig. 3D, E). However, the percent area with polarized MHCII on DRG neurons from male mice increased 14 days post-PTX, indicating a modest PTX-induced response in males (Fig. 3F). We have now included the frequency of surface-MHCII on DRG neurons from male and female mice after PTX treatment, and again there was no change in surface-MHCII in male mice (Fig. 6). Collectively, our data demonstrates that neuronal MHCII in male mice is not strongly regulated by PTX treatment.

    R3: Given the data presented in Figure 3, it is not clear what the relevance of investigating the subcellular puncta expression of MHCII neurons is, particularly when considering the sex differences observed, and how this was not been performed for surface expression.

    We now include surface and total MHCII quantification for male and female WT and cHET mice (Figs. 6,7). In the text, we describe the significance of surface versus endosomal MHCII. “While endosomal MHCII can promote TLR signaling events(Liu et al., 2011), expression of MHCII on the cell surface is required to activate CD4+ T cells.” (pg. 10). “Although the major role for surface MHCII is to activate CD4+ T cells, cAMP/PKC signaling occurs in the MHCII-expressing cell(Harton, 2019). In addition, it has recently been shown that endosomal MHCII plays an important role in promoting TLR responses(Liu et al., 2011), and since DRG neurons are known to express TLRs (Lopes et al., 2017, Wang et al., 2020, Cameron et al., 2007, Barajon et al., 2009, Xu et al., 2015, Zhang et al., 2018), this suggests the potential for T-independent responses in MHCII+ neurons. Knocking out one copy of MHCII in TRPV1- lineage neurons (cHET) from female mice did not change total MHCII 7 days post-PTX but reduced surface-MHCII. Accordingly, PTX-treated cHET female mice were more hypersensitive to cold than PTX-treated WT female mice, suggesting a role for neuronal MHCII in CD4+ T cell activation and/or neuronal cAMP/PKC signaling. In contrast, knocking out one copy of MHCII in TRPV1-lineage neurons (cHET) from male mice did not change surface-MHCII in naïve or PTX-treated mice but reduced total MHCII, indicating endosomal MHCII and potentially a role in TLR signaling. Future studies are required to delineate MHCII surface and endosomal signaling mechanisms in naïve and PTX-treated female and male mice.” (pg. 28).

    R3: Furthermore, the authors should provide details of what the abundant non-neuronal structures are within the DRG images that appear positive for MHCII staining.

    We now include an image of the high MHCII+ cells in mouse DRG co-stained with macrophage and dendritic cell markers (CD11b/c), indicating the presence of immune cells (Fig. S6).

    R3: The behavioral data presented in Figure 7 is somewhat confusing. Can the authors confirm how many alleles of MHCII were knocked out from the Trpv1-lineage neurons for these experiments? In Figure 7, it states cKO Het, which suggests that only one allele was deleted within the Trpv1 population. If this is the case, this needs to be clearly outlined within the results section and not simply referred to as "knocking out MHCII in Trpv1-lineage neurons". In addition, an explanation as to why heterozygous cKO were used rather than homozygous cKO needs to be provided. This is particularly relevant when discussing potential sex differences.

    The mouse behavior is performed in wild type and TRPV1lin MHCII+/- heterozygote mice (Fig 8). Instead of saying we knocked out MHCII, we changed the text to “knocking out one copy of MHCII in TRPV1-lineage neurons” (pgs. 23, 29). In the methods section, we state that “cHET×MHCIIfl/fl crosses only yielded 8% cKO mice (4% per sex) instead of the predicted 25% (12.5% per sex) based on normal Mendelian genetics. Thus, cKO mice were only used to validate MHCII protein in small nociceptive neurons” (pg. 30) (Fig 7).

    R3: A significant gap in the current manuscript is the functional assessment of MHCII protein expressed on DRG neurons in terms of T cell activity. I would suggest the authors consider performing a co-culture DRG-T cell (i.e. Treg) assay where anti-inflammatory cytokine release can be measured in the presence and absence of MHCII on DRG neurons.

    The functional implication of MHCII protein on DRG neurons in terms of T cell activity is out of the scope of this manuscript. We currently have another manuscript in progress investigating CD4+ T cell signaling and cytokine production when co-cultured with DRG neurons. R3: Within the first paragraph of the results section, the authors reference Goode et al, 2022, stating that they have previously shown that CD4+ T cells in the DRG secrete anti-inflammatory cytokines. I have read this paper and could not find any data that showed increased secretion of cytokines, only that there is an increase in T-cell populations that contain anti-inflammatory markers. Please consider rewording to reflect the observations made in the original paper. We have changed “secrete” to “produce” (pg. 5) because we detected anti-inflammatory cytokines (IL-10 and IL-4) within CD4+ T cells using intracellular staining and multi-color flow cytometry.

    R3: Figure 1A states that it is "day 14 PTX", however, there is no reference to this in the corresponding text - please state what Figure 1A is showing in the main text and legend regarding PTX treatment.

    We have now included text and Fig. 1. legend that states that the images in Fig1A are of DRG tissue collected from female mice 14 days after PTX treatment (pg. 5).

    R3: Throughout the results section (Figure 3-Figure 6), the authors provide percentage changes in observed difference in expression, however, in addition to this, it would be valuable to have the actual number of neurons analysed for each group and sex.

    We now report in the materials and methods section the number of neurons that were analyzed (pg. 33).

    R3: For Figure 5, can the authors confirm whether this was performed on tissue sections or dissociated cell culture?

    This analysis was performed in DRG tissue sections. The legend now states, “Gaussian distribution of the diameter of MHCII+ DRG neurons in DRG tissue from naïve (pink), day 7 (orange) and day 14 PTX-treated (blue) (A) female and (E) male mice (n=8/sex, pooled neurons).”

    R3: Can the authors comment on why surface expression for MHCII was not performed on the these reporter neurons?

    In the future, we plan to delineate which subsets of neurons express MHCII by co-staining for MHCII and specific neuronal markers. However, these studies are beyond the scope of the current manuscript.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript sets out to investigate the mechanism by which the previously reported infiltration CD4+ T cells into the DRG parenchyma can mediate analgesia in the paclitaxel (PTX) model of chemotherapy-induced neuropathic pain (CIPN). The authors provide good rationale for the purpose of the study and make a number of interesting observations, including the expression of MHCII on DRG neurons, the effect of PTX on MHCII expression on DRG neurons, and the effect of targeted deletion of MHCII on Trpv1-expressing putative nociceptive neurons in exacerbating the effect of PTX-induced cold hypersensitivity. These data culminate to a hypothesis that MHCII expression on DRG neurons may drive T-cell mediated anti-inflammatory effects (and analgesia) in models where their recruitment is notable. Overall I enjoyed reading the manuscript, however, I believe there are a number of points that need to be considered further.

    Major comments.

    • Presented in Figure 3, the authors present data to show surface expression of MHCII, along with the ability of MHCII to present OVA peptide, on naïve and PTX-treated DRG neurons. These data are probably the most relevant in terms of expression as they look at the surface expression of MHCII along with the potential of MHCII to function; therefore, it is unclear why the authors only conducted this analysis on female neurons, and not both male and female neurons. Given the claims of the paper in terms of sex differences for MHC expression, I strongly suggest this is done in order to put the other observations into context.
    • Given the data presented in Figure 3, it is not clear what the relevance of investigating the subcellular puncta expression of MHCII neurons is, particularly when considering the sex differences observed, and how this was not been performed for surface expression. Furthermore, the authors should provide details of what the abundant non-neuronal structures are within the DRG images that appear positive for MHCII staining.
    • The behavioural data presented in Figure 7 is somewhat confusing. Can the authors confirm how many alleles of MHCII were knocked out from the Trpv1-lineage neurons for these experiments? In Figure 7, it states cKO Het, which suggests that only one allele was deleted within the Trpv1 population. If this is the case, this needs to be clearly outlined within the results section and not simply referred to as "knocking out MHCII in Trpv1-lineage neurons". In addition, an explanation as to why heterozygous cKO were used rather than homozygous cKO needs to be provided. This is particularly relevant when discussing potential sex differences. Furthermore, the behavioural effect is seemingly variable, with only one of the three trials being significantly different between groups. This variable response needs to be discussed further.
    • A significant gap in the current manuscript is the functional assessment of MHCII protein expressed on DRG neurons in terms of T cell activity. I would suggest the authors consider performing a co-culture DRG-T cell (i.e. Treg) assay where anti-inflammatory cytokine release can be measured in the presence and absence of MHCII on DRG neurons.

    Minor comments.

    • Within the first paragraph of the results section, the authors reference Goode et al, 2022, stating that they have previously shown that CD4+ T cells in the DRG secrete anti-inflammatory cytokines. I have read this paper and could not find any data that showed increased secretion of cytokines, only that there is an increase in T-cell populations that contain anti-inflammatory markers. Please consider rewording to reflect the observations made in the original paper.
    • Figure 1A states that it is "day 14 PTX", however, there is no reference to this in the corresponding text - please state what Figure 1A is showing in the main text and legend regarding PTX treatment.
    • Throughout the results section (Figure 3-Figure 6), the authors provide percentage changes in observed difference in expression, however, in addition to this, it would be valuable to have the actual number of neurons analysed for each group and sex.
    • For Figure 5, can the authors confirm whether this was performed on tissue sections or dissociated cell culture? In addition, can the authors comment on whey surface expression for MHCII was not performed on the these reporter neurons?

    Significance

    This paper presents interesting data on the expression of MHCII on DRG neurons, which corroborates existing and published RNA expression data from the literature. In addition, this paper builds on our current understanding of how T-cells may be able to interact with DRG neurons in order to modulate their responses in instances of nerve injury. However, there are significant gaps in the data presented which prevent a more informative conclusion being drawn regarding the role of MHCII in modulating neuronal responses following PTX-induced CIPN.

    Audience: I would suggest basic scientists working within the field of pain and neuroimmunology would be interested in this work.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Whitaker EE and co-authors implicate MHCII expression in DRG neurons in the resolution of pain following paclitaxel treatment. The authors demonstrate that CD4 T cells closely interact with DRG neurons, which also express MHCII proteins. They further characterize neuronal MHCII expression in naïve and paclitaxel treated mice in small diameter TRPV1+ neurons. Utilizing genetic animal models with MHCII knockout in TRPV1-lineage neurons, the authors highlight that loss of MHCII in TRPV1 neurons exaggerates cold sensitivity in naïve male mice, and in both sexes following paclitaxel treatment.

    Major concerns:

    The most pressing concern regarding this study is a lack of a vehicle control group. It is not appropriate to be comparing paclitaxel treated mice to naïve mice. Please include a vehicle treatment (cremophor:ethanol 1:1 diluted 1:3 in PBS) group for all experiments involving paclitaxel. This would also improve statistics as unpaired T tests comparing naïve vs paclitaxel is not convincing.

    Figure 1A only includes representative images of a small number of T cells in presumable contact with DRG neurons in female Day 14 paclitaxel mice, but does not include images from other groups. Similarly, B-D show a single CD4+ T cell in contact with DRG neurons in Day 14 paclitaxel and naïve female mice. Please include quantification of the frequency of CD4+ T cells interacting with DRG neurons in the different experimental groups utilized in this study.

    Please include entire blot for Figure 2A (or at least more of the blot). There is plenty of space in the figure and as it currently appears is not free from apparent manipulation.

    The authors conclude that MHCII helps to suppress chemotherapy-induced peripheral neuropathy, resolving cold allodynia following paclitaxel treatment. To support this conclusion, I think it is necessary to include a time-course experiment highlighting whether cKO of MHCII in TRPV1 neurons indeed increases the duration for cold hypersensitivity to resolve following paclitaxel treatment.

    Minor concerns:

    The graphical abstract is misleading. The authors suggest paclitaxel is acting exclusively via TLR4 and that signaling is resolved at Day 14 which their data does not support. Please adjust to reflect findings from the experiments included in this study.

    Figure 4 and 6 MHCII labelling is oversaturated in most of the images, creating a blurry hue in the representative images. This should be fixed

    The effects of the PTX cHET group are very mild in both the male and female cohorts, and specific to 1 trial. I believe these assessments were conducted at Day 6 post injection. Why was this timepoint chosen considering differences in MHCII expression in small neurons was only present at Day 14 relative to naïve? The statistical analysis should also have been a mixed-effects repeated measures between groups ANOVA.

    Significance

    This study uncovers sex-dependent mechanisms underlying cold sensitivity between male and female mice. The detailed IHC analysis of MHCII expression in DRG neurons is a clear strength of this study, and supports flow cytometry results as well as existing literature. The specificity of MHCII expression on small diameter is well characterized and supported by conditional knockout mouse models of MHCII in TRVPV1-lineage neurons. The clear limitations of this study is the lack of a vehicle control group and limited behavioral analysis. They undermine the conclusions made by the author, and in extension, the significance of this study.

    This study adds to the understanding of neuro-immune signaling in peripheral neuropathic pain. As far as I am aware, this is the first study to investigate MHCII expression in DRGs in relation to development of chemotherapy-induced peripheral neuropathy. Thus this study provides an incremental advance in neuroimmune mechanisms contributing to the development of chemotherapy-induced peripheral neuropathy in mice.

    This study would be of interest to basic researchers interested in neuropathic pain, with particularly researchers with a focus on neuroimmunology and chemotherapy-induced peripheral neuropathy models. The sex differences observed in naïve mice would also be of interest to basic researchers within the wider pain field. Given the preliminary nature of the findings, I do not think this would be of interest to broader neuroimmunology or clinical audiences.

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    Referee #1

    Evidence, reproducibility and clarity

    Section A:

    The key conclusions of this study are quite robust and compelling.

    While no claims need qualification clarification of some conclusions could improve the impact of this study.

    Additional experiments are not essential to support the claims of this study.

    Sufficient details are provided to allow reproduction of the key findings of this study.

    It is not, yet, possible to conclude that all experiments are adequately powered as N's for some studies are not provided.

    Significance

    Section B:

    • State what audience might be interested in and influenced by the reported findings.

    This study should be of broad interest not only in the field of the neurobiology of pain but in broader issues related to neuroimmunology.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    I am a clinician-scientist with clinical responsibilities in immunologic disorders and a basic scientist with expertise in the area of pain, including chemotherapy-induced painful peripheral neuropathies and neuroimmune mechanisms.

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
    • Place the work in the context of the existing literature (provide references, where appropriate).

    The authors provide compelling evidence that MHCII expression of MHCII in primary sensory neurons, is regulated in painful chemotherapy-induced peripheral neuropathy (CIPN) induced by the commonly used taxane class of chemotherapy drugs, paclitaxel (PTX).

    The present studies build on recent literature demonstrating that PTX CD4+ T cells in DRG. This is key to their hypothesis as T cells and anti-inflammatory cytokines protect against CIPN. In the present study these investigators studied how CD4+ T cells are activated role of cytokines released from these cells on CIPN. To key findings of the present study: the expression of functional MHCII protein in DRG neurons and the proximity of the DRG neurons and CD4+ T cells. While the MHCII protein was expressed in small-diameter, nociceptive, DRG neurons, in male mice, in females it was induced by PTX. Compatible with the hypothesis that the anti-inflammatory CD4+ T cells attenuate CIPN. Finally, in support of the contribution of this mechanism to CIPN pain, they demonstrated that attenuation of MHCII protein from nociceptors produced the predicted increase in cold hypersensitivity. Taken together their findings support suppression of CIPN by MHCII

    While the experiments are well designed and executed and the results clearly presented, I have some relatively minor concerns that, if addressed, might improve the ability of a general scientific audience to appreciate the impact of the findings presented (possibly a penultimate paragraph covering caveats and limitations of the present study).

    It is unclear what is meant by "novel" expression, used throughout the manuscript.

    The statement at the end of the abstract, "and that neuronal MHCII may also contribute to many other neurological disorders" seems premature, beyond the scope of the present study.

    While cold allodynia (hypersensitivity) is a clinically important feature of CIPN, especially in CIPN associated with the platinum based chemotherapeutic agents, it is less so taxane CIPN. Do 60% of patients with PTX CIPN express cold allodynia or does that number refer to CIPN in general? Again, the future direction of expanding studies of the role of MHCII in other aspects of the CIPN phenotype might bear mention. Is there any evidence that IL-4 and/or IL-10 influence cold sensitivity? Are these experiments run blinded?

    The term "directly contacts" is unclear. No synaptic structure is identified. It might be more accurate to estimate the actual proximity between the two cells, especially as direct contact would not be necessary for the type of intercellular communication they are studying. This is not an EM study.

    Two abbreviations are used for immunohistochemistry, ICC and IHC.

    In some figure, group sizes are not indicated (e.g., Fig. 4D).

    "small non-nociceptive neurons" - seems to refer to TRPV1+ neurons. There are, however, TRPV1-nociceptors.

    "Therefore, the majority of MHCII+ neurons in the DRG of naïve female mice were not TRPV1-lineage neurons but non-nociceptive C-LTMRs." Could use some clarification here. Are the authors suggesting that being TRPV1- defines a neuron a non-nociceptive?

  10. Version published to 10.1101/2023.03.31.535136v3 on bioRxiv
  11. Version published to 10.1101/2023.03.31.535136v2 on bioRxiv
  12. Version published to 10.1101/2023.03.31.535136v1 on bioRxiv