Attenuated asthma phenotype in mice with a fetal-like antigen receptor repertoire

We hypothesized that the scarcity of N-nucleotides might contribute to the inability of the neonate to mount a robust allergic immune response. To test this, we used terminal deoxyribunucleotidyl Transferase deficient (TdT−/−) mice, which express “fetal-like” T cell receptor and immunoglobulin repertoires with largely germline-encoded CDR3 regions. Intraperitoneal sensitization was followed by aerosol provocation with either PBS or the allergen OVA in both TdT−/− mice and wild-type mice to develop allergic respiratory inflammation. The effects of this procedure were investigated by lung function test, immunological analysis of serum and brochoalveolar lavage. The local TH2 cytokine milieu was significantly attenuated in TdT−/− mice. Within this group, the induction of total IgE levels was also significantly reduced after sensitization. TdT−/− mice showed a tendency toward reduced eosinophilic inflow into the bronchial tubes, which was associated with the elimination of respiratory hyperreactivity. In conclusion, in a murine model of allergic airway inflammation, the expression of fetal-like antigen receptors was associated with potent indications of a reduced ability to mount an asthma phenotype. This underlines the importance of somatically-generated antigen-receptor repertoire diversity in type one allergic immune responses and suggests that the fetus may be protected from allergic responses, at least in part, by controlling N addition.


Introduction
Allergic asthma is a chronic in ammatory disease of the airways characterized by bronchial hyperreactivity and variable airway obstruction 1,2 .
Allergic sensitization is described as a misled classical a nity-driven immune response associated with an imbalance towards a T H 2 milieu 3,4 . T H 2-cell derived cytokines such as interleukin 4 (IL-4) promote B cell isotype switching to immunoglobulin E (IgE) which plays a key role in allergic asthma by acting as a link between an allergen and the mast cell to which the IgE is attached by its constant domain to membrane-bound Fcε receptors [5][6][7] .
We and others have previously shown that in type 1 allergic responses the third complementarity determining region of the H chain (CDR-H3), which encodes for the center of classic immunoglobulin antigen binding sites, often plays a key role in forming the interface between the allergen and IgE [8][9][10][11] . In contrast, in atopic dermatitis the circulating IgE repertoire re ects superantigen like activation 12 .
During fetal life, human and murine T cell receptor (TCR) and immunoglobulin (Ig) repertoires are restricted in diversity due to a reduction or absence of terminal deoxynucleotidyl transferase (TdT) activity in the fetal liver, fetal bone marrow, and fetal thymus; and thus a reduction or absence of N nucleotides within their CDR3 regions 13,14 . After birth, the diversity of both the TCR and Ig repertoires expands as a consequence of the contribution of N nucleotide addition to CDR3 [13][14][15][16][17][18][19] . The diversi cation of the adaptive antigen receptor repertoires in utero and after birth parallels the developing ability to respond to a broad range of antigens and to generate robust secondary immune responses, including allergic responses.
Although the classic immunoglobulin antigen binding site forms the Ig core of the allergen-antibody interaction, the importance of qualitative properties of the CDR-H3 region for the development of an allergic phenotype has not yet been conclusively clari ed. Previous studies show that gene targeted mouse lines with altered diversity gene loci region exhibit differing allergic phenotypes depending on the hydrophobicity of their CDR-H3 regions 8,20 . A mouse strain expressing predominantly neutral amino acids in CDR-H3 developed a strong allergic phenotype to ovalbumin 17 , whereas a repertoire enriched for positively charged CDR-H3 led to a weakened allergic phenotype. However, to the best of our knowledge the role of N regions on the development of allergic respiratory in ammation has not yet been characterized.
The aim of the present study was to investigate the in uence of CDR3 repertoires lacking N nucleotides on the development of the allergic phenotype. We found that in a murine model of allergic airway in ammation terminal deoxynucleotidyl transferase (TdT −/− ) de cient mice, which express "fetal-like" antigen receptor repertoires without N-nucleotides 21 , develop an impaired response.

Results
Induction of total IgE levels after sensitization with OVA is signi cantly reduced in TdT −/− mice. To assess allergic sensitization, serum concentrations of IgG 1 and IgE in the TdT −/− and wt mice were determined. Before sensitization, the serum levels of each of the corresponding antibodies were just above the limits of detection ( Supplementary Fig. S1). The same was shown for the levels of antibody classes of the animals exposed to PBS (control) nebulization (Fig. 2). In contrast, a signi cant increase was observed for both IgG 1 and IgE levels when sensitized with aerosolic OVA. This was true for both the overall levels and the OVA-speci c levels. When compared to controls, the TdT −/− mice demonstrated a signi cant increase of all analyzed antibody classes (p < 0.0001). The same tendency was observed for the increase in antibody concentrations due to sensitization to the wild type, (total IgG 1 p < 0.001, OVAspeci c IgG 1 , total IgE and OVA-speci c IgE p < 0.0001). However, in contrast to OVA-speci c antibody levels, total IgE levels in the TdT −/− mice were signi cantly lower than in wt (p < 0.0001).
Eosinophilic in ux into the airways is reduced in TdT −/− mice. Eosinophilic in ltration is associated with the development of local allergic airway in ammation. Thus, to assess the local in ammatory response in the airways, the in ux of eosinophilic granulocytes into the BAL uids was determined (Fig. 4). The number of eosinophils in BAL uids in non-sensitized mice of either genotype proved negligible. As expected, sensitization followed by aerosolic challenge with OVA resulted in a signi cant increase in the levels of eosinophils in the BAL uids of both wt (p < 0.05, 3.66 * 10 5 ± 0,37 *10 5 cells/mL; mean ± SEM) and TdT −/− mice (p < 0.0001, 2.04 * 10 5 ± 0,36 *10 5 cells/mL; mean ± SEM). However, when compared to wt, the eosinophilic in ux was markly reduced in the TdT −/− animals (p = 0.06).
Airway hyperresponsiveness is unset in TdT −/− mice. To assess airway hyperresponsiveness, the response to inhaled methacholine was investigated (

Discussion
In a mouse model of experimental asthma, we found that CDR3 repertoires somatically diversi ed by N nucleotide addition are required for the development of a fully established allergic airway in ammation.
During ontogeny, as new cell types appear and tissues and organs are created, the developing humoral immune system encounters a progressively increasingly complex array of self-antigens. Sequential exposure to these normal 'neo'antigens poses a risk for the development and survival of potentially pathogenic autoreactive immunoglobulins. The evolutionary adaptation in mammals that allows implantation of their embryos in the mother's womb creates an additional immunological problem 22 . Intimate contact with the mother's uterine tissue and leakage between the fetal and maternal circulations make maternal cells, tissues and organs yet another potential target for the developing immune system of the embryo and fetus. The infant inherits only half of maternal gene polymorphisms, thus the developing embryo and fetus can be considered a 'semi-allograft', with the potential for an attack on the mother by antibodies generated by her child.
Unsurprisingly, there is strong evidence that humoral immune responses in the mammalian embryo and fetus are selectively suppressed. This was made evident more than 40 years ago when the existence of a homologous, controlled, programmed hierarchy of antigen responsiveness was identi ed in lambs, mice, and humans 23,24 . This nding appeared paradoxical given that lymphocyte antigen receptor repertoires, immunoglobulin (Ig) 25 and T cell receptor (TCR) 26 , were presumed to be generated in a stochastic fashion through the process of random VDJ rearrangement and N addition. However, it was subsequently shown that both the process of VDJ rearrangement and the presence and extent of N addition are regulated during embryonic, fetal and neonatal development, restricting the diversity of both repertoires in the womb.
The focus of antigen receptor diversity is the third complementary determining region of the V domain (CDR3), which is somatically created by V(D)J rearrangement and N nucleotide addition. CDR3 regions are located at the center of the antigen binding site, as classically de ned, and thus typically play a commanding role in antigen recognition and binding 27 . Although restriction of antigen receptor CDR3 repertoires is common during mammalian ontogeny, the precise array and combination of mechanisms used vary by species. In mouse, the primary mechanism of repertoire control is the absence of terminal deoxynucleotidyl transferase (TdT) activity, the source of N nucleotide addition, until after birth, which restricts the diversity of both the Ig heavy (H) chain 28,29 and TCRβ 13 repertoires to germline content. Use of individual VH and, to a lesser extent, DH are also regulated 30 .
We and others have tested for bene ts and detriments to lymphocyte development and immune function in TDT de ciency. The primary bene t to the absence of N nucleotides, and thus a focus on a germlineencoded TCR and Ig repertoire, is enhanced e ciency of positive selection and a more rapid population of lymphoid organs 31,32 .
With two exceptions, the effect on antibody production, T cell function, speci city, and pathogen neutralization is either neutral (e.g. ovalbumin KLH) 33 or negative 34 . In T cells, the peripheral repertoire is more polyreactive and less peptide-oriented than is the N + repertoire 31 . Total antibody production to a large array of antigens is neutral or reduced 32 . N nucleotide de cient CD8 + memory T cells mediate poor recall responses compared to adults and are comprised of a repertoire of lower avidity 35,36 . Responses towards some epitopes are skewed 36 , and heterosubtypic immunity to in uenza virus is abrogated 34 . This manuscript was focused on testing one additional gap in our knowledge, i.e. the contribution of N nucleotide diversity to CD4 T cell mediated immune responses in general, and to allergens in speci c.
Here we demonstrate a role of N addition in the function of a T H 2 CD4 T cell-dependent IgE immune response to an allergen. We observe that the absence of N nucleotides leads to a complex disturbance of an allergen-induced in ammatory network, indicating a differentially altered sensitization phase and effector phase of this CD4 T cell dependent allergic response. The initial sensitization with ovalbumin using Alum as an adjuvant speci cally led to an altered TdT de cient T H 2 type antibody response since total IgG 1 levels were increased whereas total IgE levels were decreased when compared to wt mice.
Allergen-speci c IgE levels proved similar in TdT −/− and wildtype mice indicating that the absence of N nucleotides did not entirely impair the absolute quantity of speci c antibody production. This nding is in accordance with previous reports on other antigens 21,31,37−39 . However, the T H 2 cytokines IL-4 and IL-13 were reduced in the BAL uids of TdT −/− mice, indicating that at least part of the problem re ected an effect of the altered repertoire on the intrinsic function of CD4 T cells, themselves, potentially due to the skewing of epitope recognition that occurs in the absence of N addition 36 . Unexpectedly, in comparison to wt, in the TdT −/− mice the in ux of eosinophils was weaker and the bronchial hyperresponsiveness to methacholine was attenuated. However head-out body plethysmography must be interpreted carefully and it remains unclear if the apparent difference between the wt and TdT −/− control groups re ects an intrinsic difference in airway response between the two genotypes or experimental variance.  48 . Thus TdT −/− mice might be able to produce a similar quantity of allergen speci c IgE as wildtype mice, but a broader, wildtype spectrum of allergen speci c antibodies might be required to yield a clinically relevant allergic airway response. This would support the hypothesis that allergies may represent a misled oligoclonal allergen-speci c immune response.
The most obvious difference between the phenotypes of TdT −/− mice and wildtype mice was the reduction of OVA-induced airway hyperresponsiveness to methacholine. The expression of IL-5 is normally triggered by elevated levels of IL-4, IL-13 and IgE. Intriguingly, the concentration of IL-5 was not reduced in BAL uids of TdT −/− mice. These observations support the hypothesis that the initial IgEmediated in ammation was triggered without T-cell stimulation. Allergic asthma is not exclusively a T H 2dependent, IgE-mediated allergic in ammatory disease. Other factors, such as innate lymphoid cells (ILC2s), can also contribute by secreting IL-5 and IL-13, which in turn contribute to the adaptive type 2 immune response [49][50][51][52] . Since the concentration of IL-5 in BAL uids in TdT-/-mice was similar to wildtype, but the number of eosinophils was reduced, mediators other than IL-5 must be involved in regulating the in ux of eosinophils into the BAL uids 53,54 . We favor the view that the reduced number of eosinophils in the BAL uids can be attributed to the disturbed overall cytokine pro le in TdT −/− mice.
Although the diversity of both the Ig and TCR repertoire is restricted during ontogeny in both human and mouse, the precise timing and details differ 55 . The range of CDR-H3 lengths in humans is much larger compared to mice, although the murine CDR-H3 repertoire does not represent a subset of that of humans 56 . Moreover, in mouse the Ig and TCR repertoires remain de cient in N addition until days after birth; whereas in human N addition is limited in the embryo, begins to increase in the fetus in the second trimester of pregnancy, and achieves an adult phenotype by six months after birth 57 . Thus, it is unclear if these results can be transferred to human on a one-for-one basis. It also needs to be taken into account that these results might not necessarily re ect all type one allergies other than OVA. However, recent studies have shown that protection against allergy may begin in the womb, with maternal IgG being transferred to the embryo from the second trimester on, offering protection against allergen sensitization 58 . Thus, it is possible that exposure to allergens in the human womb in mothers who lack protective IgG may promote expression of a 'locked-in' allergen-sensitive Ig or TCR repertoire that will result in an increased risk of allergy after birth. Consequently, the differences in repertoire pre and post birth could lead to markedly different outcomes.
Studies of T cell function in the absence of TdT have either been general (e.g. 33 , or focused on CD8 T cell function 35,36,59,60 . Our ndings suggest that equal attention should be placed on the study of the effects of the role of N addition in CD4 T cell epitope recognition and function 61 .
This study was able to shed light on just the section of the complex immune network that depends on the activity of TdT. The results that we obtained indicate that the absence of TdT leads to an alteration in the pattern of cytokine production after allergic sensitization and challenge; a feature that had not been described previously. Further studies are therefore necessary in order to present a detailed mechanism in this context. To identify further TdT-affected key components of the immune system, -omics experiments should be considered. In addition, it is necessary to investigate which speci c genetic changes concerning the TCR might lead to a rescue of the TdT phenotype.
In conclusion, we found that the allergic phenotype was abolished in a murine model of allergic airway in ammation in mice expressing "fetal like" Ig and TCR repertoires, as re ected by the absence of N nucleotides. We hypothesize that a type 1 allergy is not only mediated by the antibodies speci cally reacting in the ELISA, but properties of the N de cient TCR repertoire may also be involved in establishing and maintaining a normal T H 2 cytokine pro le that leads to the in ux of eosinophils and airway hyperreactivity.
Mice. We have used a previously described TdT −/− mouse strain on a BALB/c background 21,31 using wildtype mice as controls (Harlan Winkelmann (Borchen, Germany)). All animals were kept under pathogen-free conditions in single ventilated cage systems. At a constant temperature of 20 °C, the mice were exposed to an arti cial light-dark rhythm of 12/12 hours. The animals were offered an ovalbuminfree diet and water ad libitum for food and drinking water intake. The study was carried out in compliance with ARRIVE guidelines.
Test Protocol. The chronological sequence of operations and analyses was designed as follows (Fig. 1). Before starting of the test series, the concentration of individual antibody classes was measured in serum by ELISA. On days 1, 14, and 21, the mice were intraperitoneally sensitized with either the allergen ovalbumin (OVA) or with PBS as a control. On days 26, 27, and 28, the aerosol provocation was again performed with either OVA or PBS. The resulting effects on serum antibody concentration were subsequently determined by ELISA. In addition, lung function was examined. Bronchoalveolar lavage (BAL) was performed to assess both the number of eosinophils and the amount of characteristic cytokines in the BAL uids. Mice were sensitized to ovalbumin (OVA) as previously described 8,20 . 10 µg of OVA grade IV (Sigma, Germany) were adsorbed to 1.5 mg Al(OH) 3 (Table 1). Determination of Antibody Titers. To quantify the immunoglobulin levels in serum, blood samples were taken on the day before the rst sensitization and on day 30. Serum concentrations of total and allergenspeci c IgE and IgG 1 were measured by ELISA as previously described 20 . Antibodies and standards were purchased from BD (Heidelberg, Germany). Serum samples were diluted 1:500 (IgG 1 ) and 1:100,000 (IgE) for OVA sensitized mice. For non-sensitized controls, samples were diluted 1:10 (IgG 1 ) and 1:2 (IgE), respectively. Determination of OVA-speci c IgE was performed as described in 8 .
Assessment of Lung Function. As previously described, head-out body plethysmography was used to assess lung function on day 29 40 . Methacholine provocation was performed in the headout body plethysmograph to evaluate airway responsiveness (AR). After a period of acclimatization and recording of the baseline, the mice inhaled aerosol PBS. Methacholine was then applied in ascending concentration (6.25 mg/mL to 100 mg/mL). The mean expiratory ow at which 50 % of the tidal volume in ml/s has been exhaled (MEF 50 ) was used to assess air ow limitation. The PC 50  Serum immunoglobulin levels In serum of wt and TdT-/-mice, both sensitized and non-sensitized, immunoglobulin levels of (a) total IgG1, (b) total IgE, (c) OVA-speci c IgG1 and (d) OVA-speci c IgE were measured using ELISA. Sensitization induced a signi cant rise in OVA-speci c IgG1 and IgE levels. No signi cant differences between wt and TdT-/-mice were observed in OVA-speci c IgE levels. Total IgE and total IgG1 levels were signi cantly increased after sensitization for both strains. The increase in IgG1 levels was signi cantly higher and the increase in IgE levels was signi cantly lower in TdT-/-mice when compared to wt (mean shown as blue lines, SEM shown as black bars). TdT-/-(p = 0.06) compared to non-sensitized mice. A signi cant rise was observed in IL-4 levels in wt (p < 0.001), in IL-13 (Wt p < 0.0001; TdT-/-p < 0.01) and IL-5 (Wt p < 0.0001; TdT-/-p < 0.001) compared to non-sensitized mice. However, this increase was attenuated in sensitized TdT-/-mice compared to wt mice (p < 0.05 (IL13 and IL-4)).
(d-f) The levels of the cytokines IL-6, IL-10 and INFy showed no differences between the two genotypes. All levels were normalized to wt control (mean shown as blue lines, SEM shown as black bars).