In a mouse model of experimental asthma, we found that CDR3 repertoires somatically diversified by N nucleotide addition are required for the development of a fully established allergic airway inflammation.
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 problem22. 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 identified in lambs, mice, and humans 23,24. This finding 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 defined, 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 benefits and detriments to lymphocyte development and immune function in TDT deficiency. The primary benefit to the absence of N nucleotides, and thus a focus on a germline-encoded TCR and Ig repertoire, is enhanced efficiency of positive selection and a more rapid population of lymphoid organs 31,32.
With two exceptions, the effect on antibody production, T cell function, specificity, 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 deficient 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 influenza 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 specific.
Here we demonstrate a role of N addition in the function of a TH2 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 inflammatory 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 specifically led to an altered TdT deficient TH2 type antibody response since total IgG1 levels were increased whereas total IgE levels were decreased when compared to wt mice.
Allergen-specific IgE levels proved similar in TdT−/− and wildtype mice indicating that the absence of N nucleotides did not entirely impair the absolute quantity of specific antibody production. This finding is in accordance with previous reports on other antigens 21,31,37−39. However, the TH2 cytokines IL-4 and IL-13 were reduced in the BAL fluids of TdT−/− mice, indicating that at least part of the problem reflected 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 influx 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 reflects an intrinsic difference in airway response between the two genotypes or experimental variance.
Increased total IgG1 levels and reduced total IgE levels indicate that in TdT−/− mice the cytokine milieu contains more TH1 components than in wt controls. The weakened TH2 cytokine bias might result from a disruption of the positive feedback loop that usually links the sensitization phase that yields specific IgE under the influence of TH2 cytokines and the effector phase that follows re-exposure to the allergen and TH2 cytokine release by mast cells and basophils 40–45, potentially due to changes in epitope recognition patterns.
The polyreactivity of the B-cell repertoire of TdT−/− mice is reduced compared to wt animals 38. Thus, it remains unclear if the allergen specific IgE antibodies expressed by TdT−/− mice have the same affinity as those of wildtype mice. Just like T cell patterns of epitope recognition, it can be speculated that specific IgE produced by TdT−/− mice might not recognize the complete range of epitopes of ovalbumin bound by wild type IgE, thereby narrowing its reactivity to the allergen 46. CDR-H3 length has significant influence on the tertiary structure of the antigen binding site 47 and the length of CDR-H3 regions correlates with the nature of recognized antigens 48. Thus TdT−/− mice might be able to produce a similar quantity of allergen specific IgE as wildtype mice, but a broader, wildtype spectrum of allergen specific 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-specific 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 fluids of TdT−/− mice. These observations support the hypothesis that the initial IgE-mediated inflammation was triggered without T-cell stimulation. Allergic asthma is not exclusively a TH2-dependent, IgE-mediated allergic inflammatory 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 49–52. Since the concentration of IL-5 in BAL fluids 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 influx of eosinophils into the BAL fluids 53,54. We favor the view that the reduced number of eosinophils in the BAL fluids can be attributed to the disturbed overall cytokine profile 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 deficient 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 reflect 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 findings 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 specific 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 inflammation in mice expressing “fetal like” Ig and TCR repertoires, as reflected by the absence of N nucleotides. We hypothesize that a type 1 allergy is not only mediated by the antibodies specifically reacting in the ELISA, but properties of the N deficient TCR repertoire may also be involved in establishing and maintaining a normal TH2 cytokine profile that leads to the influx of eosinophils and airway hyperreactivity.