E protein binding at the Tcra enhancer promotes Tcra repertoire diversity

V(D)J recombination of antigen receptor loci is a highly developmentally regulated process. During T lymphocyte development, recombination of the Tcra gene occurs in CD4+CD8+ double positive (DP) thymocytes and requires the Tcra enhancer (Eα). E proteins are known regulators of DP thymocyte development and have three identified binding sites in Eα. To understand the contribution of E proteins to Eα function, mutants lacking one or two of the respective binding sites were generated. The double-binding site mutant displayed a partial block at the positive selection stage of αβ T cell development. Further investigation revealed loss of germline transcription within the Tcra locus at the Jα array, along with dysregulated primary and impaired secondary Vα-Jα rearrangement. Eα E protein binding increases Tcra locus accessibility and regulates TCRα recombination, thus directly promoting Tcra repertoire diversity.


Introduction
The adaptive immune system recognizes a wide variety of antigens by way of B cell receptors and T cell receptors (TCRs) on the surfaces of B and T cells, respectively. These highly diverse antigen receptors (AgRs) are generated by the process of V(D)J recombination during defined stages of B and T lymphocyte development (1). V(D)J recombination is catalyzed by the lymphoid-specific recombination activating gene (RAG) proteins, which act on AgR loci to create double-strand breaks between variable (V), diversity (D), and joining (J) gene segments and their respective recombination signal sequences. AgR diversity depends on combinatorial usage of V, D, and J segments, together with junctional heterogeneity introduced by non-homologous end joining DNA repair mechanisms (1,2). V(D)J recombination occurs in an ordered fashion during B and T cell development due to the activity of developmentally regulated enhancer and promoter elements (1). These elements drive germline transcription and chromatin accessibility at defined sites in AgR loci, which are then permissive for RAG binding. RAG proteins generally assemble on highly accessible D and J gene segment recombination signal sequences to form a chromatin structure referred to as the recombination center (RC). RC-bound RAG is then able to capture V gene segments to complete the recombination process (1,3).
Fetal liver or bone marrow origin precursors that migrate to the thymus can commit to the T cell fate and further differentiate into one of several mature T cell lineages. CD4 -CD8double negative (DN) thymocytes rearrange the Tcrg, Tcrd, and Tcrb genes, leading to TCRg/TCRd or TCRb/pre-Ta (pre-TCR) pairings. This allows adoption of the gd or ab T cell lineage fates, respectively (4). Having passed the b-selection checkpoint, developing ab T cells proceed to the CD4 + CD8 + double positive (DP) stage and Tcra rearrangement. Uniquely, Tcrd and Tcra gene segments are arranged in a single genetic locus. In the mouse, the Tcra-Tcrd locus is organized as an array of about 100 V gene segments, followed by Dd, Jd, and Cd gene segments, 60 Ja gene segments, and lastly Ca (5). This configuration leads to deletion of Tcrd upon Tcra rearrangement. Tcra rearrangement requires the Tcra gene enhancer (Ea), located directly downstream of Ca (6). Lymphoid specific transcription factors (TFs) bind adjacent to or within four defined proteinbinding elements (Ta1-Ta4) that make up Ea (7)(8)(9). These TFs include c-MYB, RUNX1, RUNX3, GATA3, ETS1/FLI1, SP1, CREB, LEF1/TCF1, CTCF, E2A/HEB, NFAT, AP1, and EGR1, with some having more than one identified binding site with the 275-bp element (10). TF occupancy and histone modification is observed as early as the DN stage, though Ea activation only occurs after pre-TCR signaling (11). Ea acts in cis to activate locus germline transcription, assemble an RC, and initiate Va-Ja rearrangement. Initially, Ea activates the T early alpha (TEA) and Traj49 promoters associated with the most Va-proximal Ja gene segments (12)(13)(14). The assembled RC then directs an initial round of Va-Ja rearrangement, referred to as a primary rearrangement, to nearby Ja segments. However, owing to the lack of D segments, Tcra is capable of undergoing multiple rounds of Va-Ja rearrangement, and these secondary rearrangements will continue until a TCR is created that can mediate positive selection. Secondary rearrangements are thought to depend on RCs created by Ea and the promoter of the rearranged V gene segment (15). Following positive selection, Ea activity is downregulated in single-positive thymocytes and mature ab T lymphocytes (16). E proteins are a class of basic helix-loop-helix (bHLH) proteins that recognize a canonical CANNTG DNA sequence, also referred to as an E-box (17). In developing T cells, the E2A (Tcf3) and HEB (Tcf12) E proteins have wide-ranging targets including genes regulating cell survival and cell-cycle progression, control of developmental checkpoints, and stage-specific chemokine expression (17). E proteins are important regulators of V(D)J recombination. In both developing B and T cells, E proteins regulate the stage-specific expression of the RAG genes (18,19). E proteins also regulate germline transcription at the Igh, Igk, Igl, Tcrg, Tcrd, and Tcrb genes leading to recombination permissive chromatin environments (17,(20)(21)(22). Furthermore, E proteins are essential regulators of T cell development checkpoints, and have been previously shown to control DP cell development and transition to the SP stage (23)(24)(25)(26). Of the multiple TFs binding at Ea, E proteins are notable in that they occupy three identified binding sites during thymocyte development and show diminished binding in mature ab T lymphocytes (11, 16,27,28). However, it is not known whether E proteins have direct effects on Ea activity and Tcra recombination, in part due to the broad impacts, noted above, on the DP thymocyte population in mouse E protein knockout models.
To assess the role of E proteins in Tcra rearrangement, E-boxes at Ea were deleted. Loss of the 5′ E-box (E1) had no discernable effect on thymocyte development. However, the additional deletion of the 3′ E-box (E3) impaired positive selection of DP thymocytes, with a concordant loss of CD4 + and CD8 + single positive (SP) thymocytes. The double E-box deletion reduced germline transcription across Ja segments, which is expected to result in reduced accessibility for Va-Ja recombination. Consistent with this, alterations in Ja segment usage and invariant natural killer T (iNKT) cell development were detected, suggestive of dysregulated primary and impaired secondary Va-Ja rearrangement. Therefore, E protein binding to Ea increases Tcra locus germline transcription, regulates Ja segment recombination, and promotes Tcra repertoire diversity.
Single-guide (Supplementary Table 1) CRISPR/Cas9 electroporation of Ea DE1/DE1 was used to generate alleles with two mutated Ea E-boxes. Founders were screen by Sanger sequencing. The DE1DE3 and DE1DE3(1) alleles were detected in different founders and maintained separately by crossing to lab-maintained C57BL/6. The mutation was introduced onto a RAG-deficient background by crossing DE1DE3 to Rag1 tm1Mom /J (RRID : IMSR_JAX:002216).
Except where noted, all analyses were performed on mice 3-4 weeks of age obtained from heterozygous by heterozygous Ea allele breeding. All mice were of mixed C57BL/6 and SJL/J strain background.
The Duke University Cancer Institute Transgenic and Knockout Mouse Shared Resource carried out the above CRISPR/ Cas9-mediated mutagenesis. All mice were bred in a Duke University Division of Laboratory Animal Resources specific pathogen-free facility and handled in accordance with protocols approved by the Duke University Institutional Animal Care and Use Committee.

Antibodies
Fluorescently conjugated antibodies used in flow cytometry and cell sorting are commercially available and have been previously validated (Supplementary Table 2). CD1d tetramer was obtained from the National Institutes of Health Tetramer Core Facility.

Flow cytometry and cell sorting
Thymus was harvested and dissociated to single-cell suspension in FACS buffer (2.5% FBS and 2 mM EDTA supplemented PBS) and filtered using 70 nm nylon mesh. For analysis by flow cytometry, 3 x 10 6 cells were stained with fluorescently labeled antibodies and loaded CD1d-tetramer for 30 minutes at 4°C and then washed with excess FACS buffer. Samples were re-suspended in FACS buffer containing 7-Aminoactinomycin D (7-AAD) (ThermoFisher Scientific, Cat. A1310) or DAPI (Sigma-Aldrich, Cat. D9542) and analyzed on FACSCantoII or Fortessa X20 (BD Biosciences) cytometers available via the Duke University Cancer Institute Flow Cytometry Shared Resource. Analysis was performed using FlowJo (version 10.8.1) software. Gating scheme is shown in Supplementary Figure 1.

Tcra repertoire library preparation
Three to five million pre-selection DP thymocytes (CD4 + CD8 + CD3 lo ) were sorted from Ea +/+ and Ea DE1DE3/DE1DE3 thymuses. Sorted cells were re-suspended in TRIzol and stored at -80°C for later RNA extraction. RNA was purified using Direct-Zol RNA Microprep (Zymo Research) kit with on-column DNase digestion. Sequencing libraries were prepared with modification of previously published methods (29, 30). Briefly, 5 ng of total RNA was reverse transcribed using SmartScribe Reverse Transcriptase (Takara Bio, Cat. 639538) with Trac-RT and SMARTnnnA template switch oligo (Supplementary Table 1). Samples were then treated for 40 min at 37°C with 5 units uracil DNA glycosylase (NEB). cDNA purification was carried out using Ser-Mag Carboxylate-Modified Magnetic SpeedBeads (GE Healthcare Life Sciences). Q5 high-fidelity polymerase (NEB) was used to amplify cDNA (18 cycles), which was then purified using Ser-Mag Carboxylate-Modified Magnetic SpeedBeads. Q5 high-fidelity polymerase was used to perform dual-indexing of amplified cDNA, followed again by Ser-Mag Carboxylate-Modified Magnetic SpeedBeads purification. Sample quality control was performed by agarose gel electrophoresis. Replicate Ea +/+ and Ea DE1DE3/ DE1DE3 indexed samples were pooled at equal concentration and final library preparation carried out using NEBNext Ultra II DNA library preparation kit (NEB). Library was gel extracted using Zymoclean Gel DNA recovery kit (Zymo Research). Sequencing was performed on a MiSeq sequencer (Illumina) (300 x 300 bp) by the Duke University Cancer Institute Sequencing and Genomic Technologies Shared Resource.

Generation of RAG-deficient DP thymocytes
Experimental mice were injected intraperitoneally with 150 µg anti-CD3ϵ (BioLegend, Cat. 145-2C11) at 3 weeks of age. At 10 days post-injection, thymus was harvested and dissociated to single-cell suspension in FACS buffer. Whole thymocytes were re-suspended in TRIzol and stored at -80°C for later RNA extraction.

Reverse transcription
RNA was purified using Direct-Zol RNA Microprep (Zymo Research) kit with on-column DNase digestion. cDNA synthesis was performed using SuperScript III Reverse Transcriptase (Invitrogen, Cat. 108080093) with random hexamers as per manufacturer instructions.

RT-qPCR
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed with PowerTrack SYBR Green Master Mix (ThermoFisher Scientific, Cat. A46109) as per manufacturer instructions, using 5 ng of cDNA per 20

Statistical analysis
All reported data are from individual mice, with no repeated measurements from the same sample. Sample size was not predetermined by statistical methods. GraphPad Prism (version 9.5.0) software was used for all statistical analyses and generation of graphs.

Normal ab T cell development upon deletion of a single Ea E-box
To assess the impact of E protein binding at Ea, the E-box upstream of Ta1 (E1) was targeted by CRISPR/Cas9. The resulting 13 base pair (bp) deletion (DE1) eliminates the 6 bp E-box; although the upstream 7 bp were also removed and base pair changes occur at the gRNA recognition sequence ( Figure 1A, Supplementary Figure 3A), no transcription factor binding was detected at these sites in previous dimethylsulfate genomic footprinting experiments (27). The generated allele is hereafter denoted as Ea DE1 .
Ea deletion blocks thymocyte development at the DP stage, albeit with normal thymus cellularity (6). Using flow cytometry, no significant difference was detected in the numbers and proportions of total, DP, and SP thymocytes in the Ea DE1/DE1 mutants when compared to Ea +/+ littermates ( Figures 1B-E, Supplementary  Figure 3B). Further analysis of CD3 and CD69 expression did not show a significant difference in DP cells that are positively selected in Ea DE1/DE1 mice ( Figures 1F, G).

Deletion of two Ea E-boxes impairs positive selection
To further impact E protein binding at Ea, the E3 E-box (located in Ta4) was targeted for deletion in the Ea DE1 allele. The generated Ea DE1DE3 allele precisely eliminates the 6 bp E3 E-box (Figure 2A). A second allele, Ea DE1DE3(1) , disrupts E3 by a 1 bp deletion (Supplementary Figure 4A). As in Ea DE1/DE1 , thymic cellularity in Ea DE1DE3/DE1DE3 double mutants did not differ from that of wild-type littermates (Supplementary Figure 4B). However, analysis by flow cytometry revealed clear reductions of CD4 + and CD8 + thymocytes in the double mutants ( Figures 2B-E). This suggests reduced positive selection, a supposition confirmed by the substantial reductions in both the frequencies and numbers of CD3 + CD69 + DP thymocytes (Figures 2F, G). The developmental block was similarly observed in Ea DE1DE3(1)/DE1DE3(1) double mutants (Supplementary Figures 4C-I).
To understand the molecular basis for this block, TF binding at Ea was assessed by ChIP-qPCR. As per expectations, E2A and HEB binding was significantly reduced (Figures 3A-C). That reductions were only partial and were detected at all three E protein binding sites likely reflects residual E protein binding at the intact E2 site (in Ta3), coupled with the inability of ChIP to resolve binding signals at E protein binding sites separate by no more than 150 bp. Interestingly, ablation of E-boxes in Ta1 and Ta4 provoked mildly reduced binding of GATA3 (whose defined binding site is in Ta3), but not RUNX1 (whose defined binding sites are in Ta2) ( Figures 3A, D, E) (27). In contrast, no changes in transcription factor binding were detected at the RAG anti-silencer element (ASE), which contains binding sites for all of these factors. These results highlight that loss of E protein binding may mediate effects in part by destabilizing the binding of other components of the Ea enhanceosome, with some transcription factors having greater dependence on E-protein binding than others. Figures 5A-C).

Ea E-box deletion reduces Tcra-Tcrd germline transcription
Ea activity was evaluated by using RT-qPCR to assess transcription of known Ea targets in DP thymocytes from Rag1deficient Ea DE1DE3/DE1DE3 and Ea +/+ littermates ( Figure 4A). Ea acts in cis to activate the T early-a (TEA) promoter immediately 5′ of the Ja array, and the Ja49 promoter 15 kb downstream of TEA. These promoters target primary Va-Ja rearrangements to the most proximal Ja segments, after which they are excised (12, 13,35,36). Ja accessibility in secondary rearrangements is driven by promoters of rearranged Va segments (15). The double E-box mutant showed  Loss of E1 does not impair T cell development. (A) Diagram of relative positions of Ta1-Ta4 protein binding regions and E1-E3 E protein binding sites within Ea, with sequences of wild-type (+) and DE1 mutation indicated below. E protein binding motifs are highlighted in red. (B) Representative flow cytometry plots, displayed as CD4 versus CD8, of live thymocytes from Ea +/+ , Ea +/DE1 , and Ea DE1/DE1 mice. Frequencies of gated populations are shown. (C-E) Frequencies and numbers of (C) DP thymocytes, (D) CD4 + SP thymocytes, and (E) CD8 + SP thymocytes, in Ea +/+ , Ea +/DE1 , and Ea DE1/DE1 mice, with gating as shown in Supplementary Figure 1. Note that CD8 + SP are gated as CD8 + TCRb + CD24to exclude CD8 immature single positives. (F) Representative flow cytometry plots, displayed as CD69 versus CD3, of DP thymocytes (gated as shown in (B)) from Ea +/+ , Ea +/DE1 , and Ea DE1/DE1 mice. Frequencies of gated populations are shown. (G) Frequency and number of CD3 + CD69 + DP cells in Ea +/+ , Ea +/DE1 , and Ea DE1/DE1 , following gating as shown in (F). Data are pooled from 3 independent experiments and are plotted as mean ± SD. Ea +/+ (n = 4), Ea +/DE1 (n = 5), and Ea DE1/DE1 (n = 5). Statistical analysis: one-way ANOVA with correction for multiple comparison using Tukey's post hoc testing. Significant differences were not detected. a significant loss of germline transcription for the region spanning TEA to Ca (Trac) ( Figure 4B). Upstream of TEA, the Jd segments showed loss of transcription while proximal Va segments had no significant difference in expression (Figures 4C, D). Expression of genes downstream of Tcra-Tcrd likewise remained largely unperturbed ( Figure 4E). Thus, in agreement with the partial development block, DE1DE3 causes reduced transcription of a subset of Ea targets.

Ea E-box deletion impairs TCR expression in thymic DP cells
Flow cytometry was also used to analyze surface TCR expression in recombinase-sufficient thymocytes. Notably, DP thymocytes of DE1DE3 mice displayed a substantial reduction in TCRb surface expression ( Figure 5A), with a much smaller reduction apparent on CD3 + CD69 + DP thymocytes ( Figure 5B). There was no difference in TCRb surface expression on CD4 + and CD8 + SP thymocytes (Figures 5C, D). Because TCR expression is upregulated during positive selection, the reduced frequency of CD3 + CD69 + DP thymocytes (Figures 2F, G) could account in part for the overall reduction in TCRb expression in DP thymocytes. Alternatively, or in addition, the reduction in TCRb surface expression could reflect an increase of pre-TCR expression in D E1D E3 DP thymocytes due to impaired Tcra gene rearrangement and TCRab surface assembly, or diminished TCRab expression due to reduced transcription of rearranged Tcra genes. That there is no change in TCRb surface expression in more mature thymocyte populations may reflect selection for higher TCR expression during positive selection, or diminished effects of E proteins on Ea in mature cells (16,37).

Ea E-box deletion impairs TCRa rearrangement
As noted previously, TEA and Ja49 promoter-driven transcription normally target primary rearrangements to Ja segments proximal to these promoters. Once these promoters are deleted by primary rearrangement, secondary rearrangement is thought to be directed by the promoter of the rearranged Va gene segment. However, in mice with genetic deletion of the TEA (or TEA and Ja49) promoter(s), the activation of cryptic downstream promoters causes dysregulated primary rearrangement directed more broadly across the central and distal Ja segments. As such, changes in Ja transcriptional activity are reflected by changes to the TCRa repertoire (12, 13,15,35,36).
To assess how E protein binding at Ea impacts TCRa rearrangement, Ja segment usage was evaluated by performing 5′ rapid amplification of cDNA ends (5′RACE) on preselection DP thymocytes (CD4 + CD8 + CD3 lo ) sorted from Ea DE1DE3/DE1DE3 and Ea +/+ littermates. The double E-box mutants showed a significantly altered Ja repertoire. While proximal and distal Ja segments were underrepresented, most segments from Ja49 to Ja31 were significantly overrepresented ( Figure 6, Supplementary Figure 6). This suggests a modest defect in primary rearrangements that preferentially affects TEA-dependent Ja segments, coupled with a substantial defect in secondary rearrangements required for usage of distal Ja segments.

iNKT cells are reduced in double Ea E-box mutant
DP thymocytes of Rorc -/mice are short-lived and the TCRa repertoire is consequently limited to primary Va-Ja rearrangements (38). Invariant natural killer T cells (iNKT) are absent in Rorc -/mice and thus their characteristic Va14-Ja18 TCRa chain is considered to be the product of secondary TCRa recombination (39,40). iNKT cellularity and development was therefore assessed to provide additional evidence that Ea E-box mutations impact secondary Va-Ja rearrangement.
While there was no observed change in Ea D E 1 / D E 1 (Supplementary Figures 7A, B), flow cytometry analysis determined iNKTs to be significantly reduced in both frequency and number in double E-box mutants (both Ea DE1DE3/DE1DE3 and Ea DE1DE3(1)/DE1DE3(1) ) ( Figures 7A, B, Supplementary Figure 7C). Because all iNKT developmental stages were reduced numerically and proportionally ( Figure 7C, Supplementary Figure 7D), iNKT development is obstructed prior to TCR surface expression and subsequent lineage commitment. This suggests diminished rearrangement of the Va14-Ja18 invariant Tcra chain in double E-box mutants, and is in accord with reduced usage of Ja18 observed in repertoire analysis. In conjunction with the broadly reduced use of distal Ja segments, this result indicates an impairment of secondary TCRa recombination upon Ea Ebox deletion. Prior work demonstrated a deficiency in iNKT cells in mice lacking E protein HEB (26). Moreover, mice deficient in E protein inhibitors (Id2-Id3 double deficiency) were shown to have an increase in iNKT cells as well as elevated Va14-Ja18 rearrangement in preselection DP thymocytes (41). However, in neither case was a mechanism clearly established. The present work makes clear that one mechanism by which E proteins control iNKT cell development is by direct effects on the Tcra locus.

Discussion
TCRa rearrangement is a highly regulated and ordered process that takes place during the DP stage of thymocyte development (5). Ea is a required cis-acting element which plays crucial roles in promoting locus accessibility and establishing the RC (14,42). Among the TFs binding at Ea, E proteins have three identified binding sites and show recruitment as early as the DN stage (11, 16,27). E2A and HEB carry out a myriad of roles crucial to lymphocyte development, including mediating germline transcription and recombination accessibility at other antigen receptor loci (17,22,43). The findings shown here indicate that Ea E protein binding contributes to the regulation of TCRa recombination and repertoire diversity.
Although there was no effect of single E-box deletion (DE1), double E-box disruption [DE1DE3 and DE1DE3(1)] produced a partial block in ab T cell development at the DP stage, and thus impaired generation of SP thymocytes. The present data cannot distinguish whether the effects of E protein binding to E1 and E3 are distinct or redundant. Generation of DE3 and DE2 single mutants, as well as combinatorial E-box deletions, would be needed to more fully understand the contributions of the three E protein binding sites. We did not disrupt E2 in the present study because mutation of two E-protein binding sites proved sufficient to test the central hypothesis of the study, namely that E protein binding to Ea is important for Ea activity and Tcra recombination. The finding that Ea GATA3 binding is at least partially dependent on E protein binding suggests that one function of E proteins is to stabilize the binding of other transcription factors on Ea, and that reduced binding of other transcription factors may contribute to impaired Ea activity when E protein binding is prevented.
Intriguingly, germline transcription in DE1DE3 was primarily reduced across the Ja array, even though Ea is known to regulate chromatin structure and transcription over hundreds of kb upstream and downstream. Interaction with and accessibility at these other regions may be regulated or compensated by other Ea binding TFs. Furthermore, while transcript abundance is reduced at both TEA and Ja49, the overrepresentation of central Ja segments in the TCRa repertoire indicates that recombination is being preferentially directed by the latter promoter (12). Consistent with this, Ja49 is the most proximal Ja segment to be present at a higher proportion in the repertoire. Collectively, these data suggest that E proteins binding at Ea has a prominent role in regulating TEA functionality and thus the accessibility of TEA-dependent Ja segments.
The TEA and Ja49 promoters are crucial to locus accessibility and directing primary recombination to proximal Ja elements. In the , Ea +/DE1DE3 (n = 8), and Ea DE1DE3/DE1DE3 (n = 9). In all cases, summary graph data for all genotypes are presented with normalization to the average value for Ea +/ + (set to 1) within individual experiments. Data are plotted as mean ± SD. Statistical analysis: one-way ANOVA with correction for multiple comparison using Tukey's post hoc testing. *p < 0.05, **p < 0.01, ****p < 0.0001.
absence of the TEA promoter, or the TEA and Ja49 promoters, primary recombination is dysregulated and there is elevated usage of Ja gene segments across the central and distal portions of the Ja array (12, 15). However, relative usage of Ja segments distal to Ja31 was reduced in DE1DE3 mice. This, together with the reduction in iNKT cells, indicates a substantial impairment of secondary Va-Ja recombination in DE1DE3 mice. This suggests that E protein binding at Ea may additionally regulate the promoters of rearranged Va gene segments, even though there is no effect on those promoters when they are more distant in the unrearranged locus. The reported findings show that E protein binding at Ea is important to but not necessary for ab T cell development. Ablation of all Ea E-boxes may produce a more prominent developmental block at DP, but this may not be equivalent to DEa given that other TFs may retain binding ability and thus maintain sufficient Ea function to permit TCRa chain recombination, albeit at reduced levels. The importance of E protein binding is further emphasized by the broad conservation of E-boxes at Ea in mammals (Supplementary Figure 8). The present results add to the literature on E protein control of AgR gene recombination, with E proteins now shown to regulate all AgR loci. In contributing to locus accessibility and regulation of Va-Ja rearrangement, Eabound E proteins increase TCRa repertoire diversity and the potential for antigen recognition by ab T cells.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/geo/, GSE227164.

Ethics statement
The animal study was reviewed and approved by Duke University Institutional Animal Care and Use Committee. presented as mean ± SD. Statistical analysis: one-way ANOVA with correction for multiple comparison using Tukey's post hoc testing. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Author contributions
AM, SR, MK, and YZ contributed to conception and design of the study. Investigation and analysis was carried out by AM. AM and MK contributed to writing of manuscript original draft. AM, SR, MK, and YZ contributed to review, editing, and approval of submitted manuscript.

Funding
This study was funded by the NIH (R01-GM059638 and P01-AI102853 to YZ, and R35 GM136284 to MK).