https://orcid.org/0000-0002-1942-3674
Figures
Abstract
Leigh syndrome (LS) is the most common pediatric presentation of genetic mitochondrial disease and characterized by neurological and metabolic abnormalities. The hallmark of the disease is the presence of progressive, bilateral, symmetric neurodegenerative lesions in the brainstem and/or basal ganglia. Recent studies in the Ndufs4(-/-) mouse model of LS indicate that disease is causally driven by the immune system. Both microglia and peripherally originating macrophages are enriched in the lesions of Ndufs4(-/-) mice and pharmacologic elimination of these cell types prevents disease indicating a crucial role for innate immune cells. Here, we investigated the role of the adaptive immune system in Ndufs4(-/-) disease pathogenesis. We crossed Ndufs4(-/-) mice with mice expressing a null form of interleukin 2 receptor gamma (Il2rg) and monitored disease onset and progression. Il2rg knockout (KO) mice have dramatically depleted numbers of B-, T-, the adaptive immune system’s key cellular actors, and NK-cells. We observed no difference in neurological disease progression or overall survival between Ndufs4(-/-)/Il2rg(WT) and Ndufs4(-/-)/Il2rg(KO) mice, strongly suggesting that T cells, B cells, and NK cells do not play a significant role in CNS disease pathogenesis in Ndufs4(-/-) mice. Combined with previous studies indicating a causal role for macrophages, we conclude that LS CNS pathology is primarily driven by the monocyte/macrophage innate immune system.
Citation: Hanaford AR, Khanna A, Truong V, James K, Liao R, Chen Y, et al. (2025) Disruption of adaptive immunity does not attenuate disease in the Ndufs4(-/-) model of Leigh syndrome. PLoS One 20(6): e0324268. https://doi.org/10.1371/journal.pone.0324268
Editor: Metodi D. Metodiev, Imagine Institute, FRANCE
Received: November 26, 2024; Accepted: April 23, 2025; Published: June 10, 2025
Copyright: © 2025 Hanaford et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: US National Institutes of Health (NIH/GM R01 GM133865 to SCJ; NIH/GM R01 GM144368 to SCJ; NIH/NINDS R01 R01NS119426 to SCJ; and R35 GM139566 to PGM); Northwest Mitochondrial Research Guild (to SCJ, AH and BK); Northumbria University Internal Funding (SCJ); and an Academy of Medical Sciences (UK) Professorship (SCJ). Funding statement: we confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared no competing interests exist.
Introduction
Leigh syndrome (LS) is the most common clinical pediatric presentation of genetic mitochondrial disease (GMD). Also known as subacute necrotizing encephalomyopathy, the condition is characterized by symmetric, progressive, bilaterial necrotic lesions in the brainstem, basal ganglia, and occasionally cerebellum [1,2]. Brainstem lesions lead to respiratory failure — the most common cause of death for patients [3]. Symptoms typically begin in early childhood, often after a fever or viral infection [3–6]. Symptoms can include seizures, lactic acidosis, ataxia, and hypotonia.
LS is genetically diverse. Mutations in over 110 genes, in both the nuclear and mitochondrial genomes, have been causally linked to LS [7,8]. Mutations in NDUFS4, which encodes an electron transport chain complex I structural/assembly protein, are one cause of LS in humans [9]. Mice homozygous for Ndufs4 loss of function alleles develop a disease highly consistent with that observed in humans. These animals are born healthy but begin to show signs of progressive neurodegeneration early in life and have a significantly shortened life span (median survival is around 60 days) [10]. Ndufs4 null (termed knockout (KO), or (-/-)) mice develop bilateral, necrotic, inflammatory lesions in the brainstem, cerebellum and olfactory bulb, similar to those seen in humans [10].
Using the Ndufs4(-/-) mouse model, we have previously shown that these lesions are enriched with macrophages of peripheral origin in addition to microglia, brain resident macrophages [11]. Notably, while the exclusive genetic elimination of microglia only modestly improves the survival of Ndufs4(-/-) mice, combined pharmacologic depletion of both microglia and circulating macrophages with the Csf1r inhibitor pexidartinib fully suppresses neurodegenerative lesions, prevents neurodegenerative symptoms, and dramatically prolongs lifespan [11,12]. Pexidartinib treated animals do not present with LS-like symptoms before death, ultimately dying of apparent drug toxicity from chronic pexidartinib treatment [12]. Additionally, we recently showed that loss of interferon gamma (IFNγ) modestly improves survival and delays disease in Ndufs4(-/-) mice [13].
While these findings demonstrate that disease in the Ndufs4(-/-) mouse is directly mediated by immune cells, and most strongly implicate macrophages and monocytes (see Discussion), the role of other immune cells in disease pathogenesis remains unclear. In particular, the role, if any, of adaptive immune T-, B-, and NK cells has not yet been directly assessed. Abnormalities in humoral and T-cell responses have been identified in multiple patients with GMDs [4]. A case series of Leigh syndrome patients found that all had immunological deficiencies—including deficient response to vaccination, reduced class switched B-cells, and reduced memory T-cells [14].
IL2RG encodes the IL-2R gamma chain, a subunit common to multiple interleukin receptors (such as IL-2, IL-4, IL-7, IL-15, IL-9, IL-21). IL-2Rg is a critical component of receptors (such as IL-2, IL-15, IL-7) responsible for differentiation and survival of T-, B-, and NK- cells [15]. In humans, IL2RG defects can cause X-linked severe combined immunodeficiency [16]. Mice lacking functional Il2rg have significantly reduced levels of B, T, and NK-cells [17]. Il2rg loss of function can be combined with loss of function in other immune genes (such as Prkdc or Rag2) to generate highly immune deficient mouse strains for xenografting and generation of “humanized” mice [18]. Here, we generated Ndufs4(-/-)/Il2rg(KO) double knockout mice in order to directly assess whether NK cells and adaptive immune cells contribute to disease pathogenesis in LS.
Materials and methods
Animals
Il2rg knockout (KO) mice are from the Jackson Lab (strain 003174). Il2rg is on the X chromosome, so Il2rg(KO) indicates a homozygous mutant female (IL2rg(X+/X-)) or hemizygous mutant male (IL2rg(X-/Y)). Ndufs4(+/-) mice were originally obtained from the Palmiter laboratory at University of Washington, Seattle, Washington USA, and are available from the Jackson Laboratory (strain 027058). Strain details are described in Kruse et al [10]. Both lines are on the C57BL/6 background. Ndufs4(-/-) mice cannot be used for breeding due to their short lifespan and severe disease. Ndufs4(+/-) mice were bred with Il2rg(KO) mice to produce Ndufs4(+/-)/IL2rg(X+/X-) females and Ndufs4(+/-)/Il2rg(X-/Y) males which were then crossed to produce Ndufs4(-/-)/Il2rg(X-/X-) females, Ndufs4(-/-)/Il2rg(X+/X-) females, and Ndufs4(-/-)/Il2rg(X-/Y) males. Genotyping of the Ndufs4 and Il2rg alleles were performed according to the Jackson laboratory methods (strains 003174 and 027058). Only mice with PCR confirmed genotype were included in the study.
Mice were weaned at P20-22 days of age. Ndufs4(-/-) animals were housed with control littermates for warmth as Ndufs4(-/-) mice have low body temperature [10]. Mice were weighed and health assessed a minimum of 3 times a week. Following onset of Ndufs4(-/-) symptoms, wet food was provided in the bottom of the cage. Animals were euthanized if they lost 20% of maximum body weight for two consecutive days, were immobile, or were found moribund [12,19]. These endpoints have been developed to minimize suffering. Mice heterozygous for Ndufs4 loss of function have no reported phenotype, so controls consisted of both heterozygous and wild-type (WT) Ndufs4 animals. We refer to Ndfus4(Ctrl) mice here for clarity. The Ndufs4(Ctrl) and Ndufs4(-/-) mice wild type for Il2rg used in this study came from crosses of Ndufs4(+/-)/Il2rg(-/+) female mice with Ndufs4(+/-)/Il2rg(+/Y) male mice and our general Ndufs4 colony. Mice were fed PicoLab Diet 5058 and were on a 12-hour light-dark cycle. All animal experiments followed Seattle Children’s Research Institute (SCRI) guidelines and were approved by the SCRI IACUC. Euthanasia was performed by approved methods – cervical dislocation or CO2 asphyxiation.
Clasping and ataxia were assessed by visual scoring and analyzed as previously described [19]. During disease progression, Ndufs4(-/-) animals can display intermittent/transient improvement of symptoms, so here we report whether the animal ever displayed the symptoms for two or more consecutive days. Onset of weight loss is reported as the age of maximum body weight.
A Med Associates ENV-571M single-lane rotarod was used for the rotarod performance test. A mouse was placed on the rod already rotating at 6 rpm and latency to fall was timed for a maximum of 600 seconds while rotation remained constant. For each mouse, three trials were performed with a minimum of 5 minutes between each trial. The best of three trials was reported.
Lymphocyte analysis
Blood from adult Ndufs4(+/+) mice between ages P39 and P83 was collected by terminal cardiac puncture and placed in 0.4% sodium citrate to prevent coagulation. Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium from Corning. Isolated PBMCs were stained for surface antigens (CD45, NK1.1, B220, and CD3) using fluorescent antibodies and dead cells were stained with Zombie Live/Dead dye from BioLegend (cat. #423101, used at a 1:200 dilution). Antibody clones, fluorochromes, and dilutions for staining are provided in Table 1. Staining was performed on ice for 45 minutes in the dark. Stained cells were acquired using a LSRII Fortessa Cell Analyzer (BD Biosciences). Data was analyzed using FlowJo V9.0 software.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 10.0.0 with statistical test results and replicate numbers detailed in figure legends. Error bars represent the standard error of the mean (SEM). Analyses included pairwise unpaired parametric t-test (Figs 1A–1C and S1B–S1D); two-way ANOVA followed by all possible pairwise t-tests with Tukey’s multiple testing correction (Fig 2F); and pairwise log-rank tests (Figs 2B–2D, 3A and 3B).
Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of adult mice of the indicated genotype, stained with fluorescent antibodies, and analyzed by flow cytometry. Within the live cell population, leukocytes were identified by positive staining for CD45, the common leukocyte antigen. Among CD45 positive cells, T-, B-, and NK-cells were identified by staining for CD3, B220 or NK1.1, respectively. B- (A), T- (B), and NK- cells (C) were significantly depleted in ll2rg(KO) mice compared to Il2rg(WT) mice (data shown are cells per 1mL of blood; for gating and normalization to CD45 positive cell counts see S1 Fig). (A-C) *p < 0.05 by unpaired parametric t-test (see Methods). n = 3 animals per genotype, each datapoint represents an individual mouse. Bars show mean and error bars standard error of the mean (SEM). See S1–S3 Files for raw data.
(A) Weight of Ndufs4(-/-)/Il2rg(WT), Ndufs4(-/-)/Il2rg(+/-), and Ndufs4(-/-)/Il2rg(KO) animals. Weight from our complete Ndufs4(-/-) colony—Ndufs4(-/-)/Il2rg(WT) mice from our Ndufs4(-/-)/Il2rg(+/-) colony and those never crossed with Il2rg(KO)--provided for reference. Data shown Locally Weighted Scatterplot Smoothing (LOWESS) curves with all datapoints (descriptive only, not statistical comparisons). (B) Age of onset of weight loss (see Methods). Pairwise log-rank comparisons = no significant findings between any pairwise comparison. (C) Onset of ataxia onset. Ndufs4(-/-)/Il2rg(+/-) vs Ndufs4(-/-)/Il2rg(KO) *p < 0.05 by Log-rank test, other comparisons not significant. (D) Onset of forelimb clasping. Pairwise log-rank comparisons = no significant findings between any pairwise comparison. (E) Summary of median age of symptom onset. (F) Rotarod performance as assessed by latency to fall (see Methods). Comparisons were made by two-way ANOVA followed by all possible pairwise t-tests with Tukey’s multiple testing correction. No significant differences between genotypes at any age. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. Note that comparisons have been made to the larger Ndufs4(-/-) cohort which includes Ndufs4(-/-)/Il2rg(WT) mice and those never crossed with Il2rg(KO). (A-E) No significant differences were observed between Ndufs4(-/-)/Il2rg(WT) (mice birthed from Ndufs4(-/-)/Il2rg(+/-) colony)) and our complete Ndufs4(-/-) colony--Ndufs4(-/-)/Il2rg(WT) mice and those never crossed with Il2rg(KO).
(A) Survival curves for Ndufs4(-/-)/Il2rg(WT), Ndufs4(-/-)/Il2rg(+/-), and Ndufs4(-/-)/Il24g(KO) animals, with our larger Ndufs4(-/-) cohort - Ndufs4(-/-)/Il2rg(WT) mice and those never crossed with Il2rg(KO) – provided for reference. See (D) for statistical information (p-values by pairwise log-rank comparisons). (B) Data from (A), plotting Ndufs4(-/-)/Il24g(+/-) (all female as Il2rg is X-linked, see description of genotype in Methods) plotted against sex-split curves for Ndufs4(-/-) mice. Statistical comparison between female groups provided in (D), no significant differences observed. (C) The primary cause of death in Ndufs4(-/-)/Il2rg(WT), Ndufs4(-/-)/Il2rg(+/-), Ndufs4(-/-)/Il2rg(KO), and the larger Ndufs4(-/-) cohort was euthanasia due to weight loss, with similar patterns observed in all groups (n’s in (D)). FDIC – found dead in cage (cause of death not known). (D) Summary of median survival, number of animals per group, and statistical comparisons by Log-rank test. Only Ndufs4(-/-)/Il24g(KO) vs Ndufs4(-/-)/Il2rg(+/-) reached statistical significance by Log-rank test, *p = 0.034. Ndufs4(-/-)/Il2rg(WT) and Ndufs4(-/-) survival were not significantly different.
Scientific rigor
Sex-Both male and female animals were used in these experiments. No significant sex differences have been reported in Ndufs4(KO)s or Il2rg(KO)s and none were observed.
Exclusion criteria
Only animals with a PCR confirmed genotype were included in the study. Animals euthanized prior to the age of disease onset in the Ndufs4(-/-) were excluded from study. Our criteria for early life euthanasia and study exclusion include severe weaning stress (significant weight loss or death prior to P30), runts (defined as ≤5 g body weight at weaning age), or health issues unrelated to the Ndufs4(-/-) phenotype (such as hydrocephalus). These criteria are applied to all genotypes as part of our standard animal care. Exclusion due to these criteria occurs in up to ~10% of Ndufs4(-/-) animals in our prior studies. From Ndufs4(+/-)/Il2rg(+/-) x Ndufs4(+/-)/Il2rg(-/Y) and Ndufs4(+/-)/Il2rg(+/-) x Ndufs4(+/-)/Il2rg(+/Y) matings, 1 of 10 Ndufs4(-/-)/Il2rg(KO) and 1 of 8 Ndufs4(-/-)/Il2rg(WT) were excluded, similar to our overall exclusion rate for Ndufs4(-/-) mice.
Results
Il2rg knockout mice have reduced lymphocyte numbers
We first quantified B-, T-, and NK cells in the blood of adult Il2rg(KO) and Il2rg(WT) mice by flow cytometry in order to validate the previously reported impact of Il2rg depletion. Il2rg is encoded on the X chromosome, so Il2rg(KO) indicates homozygous knockout female (IL2rg(X+/X-)) or hemizygous knockout male (IL2rg(X-/Y)). Consistent with prior reports, Il2rg(KO) mice had a dramatic reduction in B-, T-, and NK-cells as indicated by a reduction in the absolute number of B220 positive, CD3 positive, and NK1.1 positive cells, respectively (Fig 1A–1C). Flow cytometry gating and cell frequency within the CD45 positive leukocyte population is shown in S1 Fig (see S1–S3 Files for raw cytometry data).
Reduction of lymphocytes does not impact neurological phenotype in Ndufs4(-/-) mice
To investigate the contribution of lymphocytes to Ndufs4(-/-) pathology, we crossed Ndufs4(+/-) mice with Il2rg(KO) mice to generate an Ndufs4(+/-)/Il2rg(+/-) line for production of double knockouts. As in humans, mouse Il2rg is an X-linked gene; here, we use Il2rg(KO) to indicate both female homozygous knockouts and hemizygous Il2rg knockout Il2rg males, and Il2rg(WT) for homozygous wildtype females and hemizygous wildtype males. All Il2rg(+/-) heterozygotes are female.
Onset of weight loss is one of the first signs of disease observable in the Ndufs4(-/-) mouse model. Il2rg disruption was associated with a modest reduction in weight, in both Ndufs4(-/-) (Fig 2A). Ndufs4(Ctrl)/Il2rg(KO) mice are also smaller in size than Ndufs4(Ctrl)/Il2rg(WT) mice (S2 Fig). There was no difference in age of weight loss onset between the three genotypes, Ndufs4(-/-)/Il2rg(KO), Ndufs4(-/-)/Il2rg(+/-), and Ndufs4(-/-)/Il2rg(WT), and each displayed similar weight gain/loss patterns (Fig 2A and 2B). Weight from our Ndufs4(-/-) colony--Ndufs4(-/-)/Il2rg(WT) mice and those Ndufs4(-/-) mice never crossed with Il2rg(KO))-- is provided for reference
Ataxia and limb clasping are signs of neurological dysfunction assessed by visual scoring of symptoms in the Ndufs4(-/-) line. Il2rg deficiency had no impact on the age of onset of these symptoms (Fig 2C and 2D). The difference between age of onset of ataxia in Ndufs4(-/-)/Il2rg(KO) mice and Ndufs4(-/-)/l2rg(WT) mice was statistically significant, but so small it is unlikely to be biologically meaningful. The median age of onset of weight loss, ataxia, and clasping for each genotype is summarized in Fig 2E including the sample size (n). Data from our Ndufs4(-/-) colony--Ndufs4(-/-)/Il2rg(WT) mice and those Ndufs4(-/-) mice never crossed with Il2rg(KO))-- is provided for reference.
Finally, Ndufs4(-/-) mice experience a progressive decline in rotarod performance, a behavioral test measuring overall coordination and muscle strength, as disease progresses. Decline in rotarod performance was not attenuated by Il2rg loss (Fig 2F).
Reduction of lymphocytes very modestly impacts survival in Ndufs4(-/-) mice
Complete loss of Il2rg in Ndufs4(KO) mice did not impact overall survival (Fig 3A and 3B). Ndufs4(-/-)/Il2rg(+/-) animals had modestly, but statistically significant, increased survival compared to Ndufs4(-/-)/Il2rg(KO) mice. There is no statistically significant difference between Ndufs4(-/-)/Il2rg(+/-) mice compared with Ndufs4(-/-)/Il2rg(WT) mice or our larger Ndufs4(-/-) colony. Il2rg(+/-) mice are not known to phenotypically differ from Il2rg(WT) mice (17). Thus, it is unlikely that Il2rg heterozygosity provides a survival benefit.
Median survival, sample size, and statistical comparisons are shown in Fig 3D. Data from our Ndufs4(-/-) colony - Ndufs4(-/-)/Il2rg(WT) mice and those Ndufs4(-/-) mice never crossed with Il2rg(KO)) – are provided for reference.
The overall cause of death distribution, which can reveal subtle changes in disease progression, was similar among all groups (Fig 3C). The primary cause of death in Ndufs4(-/-) mice was euthanasia due to weight loss, regardless of Il2rg status. Survival data and statistics are summarized in Fig 3D.
Discussion
Here, we used a genetic model to assess the role of lymphocytes in the onset and progression of CNS disease in the Ndufs4(-/-) model of LS. We found that severe depletion of NK cells, and B-, T-, representing the primary cellular actors of the adaptive immune system, did not impact overall disease onset or progression in the Ndufs4(-/-) mouse model of LS.
Our previous studies have demonstrated a causal role for immune cells in the pathogenesis of LS, with observations to date most strongly implicating circulating and tissue resident macrophages [11–13]. It has long been recognized that microglia (the brain resident macrophage) are highly abundant in LS CNS lesions; we have recently demonstrated that macrophages of peripheral origin are also highly enriched in the lesions observed in diseased Ndufs4(-/-) mice [11]. In addition, we have found that depletion of microglia alone only modestly impacts disease course in the Ndufs4(-/-) model [11], whereas pan-macrophage ablation via high dose pexidartinib prevents disease onset and dramatically extends survival [12]. Together with our findings here, these data strongly suggest that adaptive immune cells are not a significant component of CNS disease pathogenesis in Ndufs4(-/-) mice. This is consistent with the model that CNS pathology in LS is driven by actions of the macrophage/monocyte innate immune system.
In recent work, we found that loss of IFNγ provided a modest but statistically significant and gene dose-dependent improvement in survival and delay in symptom onset in Ndufs4(-/-) mice [13]. Notably, cytotoxic lymphocytes, including T- and NK- cells, and cytotoxic macrophages are the major producers of IFNγ [20,21]. Given that IFNγ depletion attenuates disease progression, but B-, T-, and NK- cell depletion fails to alter disease course, it seems likely that disease-promoting IFNγ is macrophage-derived, representing positive feedback.
In the cohorts tested here, we observed a modest but statistically significant reduction in Ndufs4(-/-)/Il2rg(KO) survival compared to Ndufs4(-/-)/Il2rg(+/-) animals. This might indicate that the Ndufs4(-/-)/Il2rg(KO) mice are more frail than Ndufs4(-/-)/Il2rg(+/-) animals, which would be consistent with the possibility that adaptive immune depletion not only fails to prevent disease but is actively harmful to the Leigh syndrome model. It is also possible that loss of Il2rg impacts the phenotype of macrophages, possibly slightly accelerating disease through increased activation. This may be worth further study.
Recent evidence supports the notion that activation of the innate immune system may occur in multiple forms of mitochondrial disease [4,22,23]. Various contributing pathways have been implicated, such as type I interferon signaling, but no clear single immune-activating mediator has yet been identified. Interestingly, while CNS lesions in LS appear to be an innate immune phenomenon, clinical immunodeficiencies appear to be common among GMD patients, including those with LS [24]. In one cohort of GMD patients, almost half were diagnosed with an immune deficiency (25). Evidence indicates that GMD patients have an impaired humoral response to infection, and failure to develop and/or maintain antibodies titers in response to vaccination is common in this patient population [25,26]. T-cell mediated immune alterations have also been reported in GMD patients, such a reduction in T-cell repertoire in MELAS patients [27,28]. A patient with GMD caused by mtDNA deletion had reduced T, B, and NK cell levels and no evidence of multimorbidity with other immune deficiency syndromes [29]. These defects in adaptive immunity might be related to dysregulation of the monocyte/macrophage population. Recurrent infections are common in GMD patients, particularly in the respiratory tract [24]. Infection has been associated with episodes of underlying disease progression and can be life threatening—sepsis and pneumonia are the two of the most common causes of death in pediatric GMD patients [3].
Integrating findings in human patients with our current and previous findings in the Ndufs4(-/-) mouse suggests that innate and adaptive immune actors are both differentially impacted in GMD, and play distinct roles in disease onset and progression. Our results support efforts to identify targeted therapies aimed at suppressing CNS disease driving macrophages/monocytes, while suggesting that patient benefits may depend on the ability to limit off-target suppression of already disrupted adaptive immune functions.
Supporting information
S1 Fig. Supplemental flow cytometry data.
(A) Gating used to identify leukocyte population. Leukocytes are identified by CD45 expression. B-cells are characterized by high B220 expression and negative CD3 expression. T-cells are characterized by high CD3 expression and negative B220 expression. NK cells are identified by high NK1.1 expression and negative CD3 expression. (B) Frequency of B220 positive cells among the CD45 cell population (B-cells). (C) CD45/CD3 positive cells (T-cells) and D) CD45/NK1.1 positive cells (NK cells). While raw NK cell counts are significantly reduced in Il2rg(KO) mice (Fig 1C) their frequency within the overall leukocyte population is increased.
https://doi.org/10.1371/journal.pone.0324268.s001
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S2 Fig. Ndufs4(Ctrl) mouse weights.
Weight of male Ndufs4 control mice knockout or WT for Il2rg (all from the Ndufs4(+/-)/Il2rg colony). Ndufs4 controls are genotype Ndufs4(+/+) or Ndufs4(+/-) as previous work has shown no differences between the two (see Methods). Il2rg is an X-linked gene, so male heterozygotes do not exist. Data shown are average with SEM and Locally Weighted Scatterplot Smoothing (LOWESS) curves to show overall trends.
https://doi.org/10.1371/journal.pone.0324268.s002
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S1 File. FCS files - first batch containing source data for flow cytometry.
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S2 File. FCS files - second batch containing source data for flow cytometry.
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S3 File. FCS files - third batch containing source data for flow cytometry.
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S1 Data. This file contains all raw data used for generation of the figures appearing in the manuscript.
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