Old questions, new tools: does next-generation sequencing hold the key to unraveling intestinal B-cell responses?

Analysis of the intestinal B-cell system and properties of immunoglobulin A, the main antibody isotype produced in the gut, has dominated the rise of mucosal immunology as a discipline. Seminal work established concepts describing the induction, transport, and function of mucosal antibodies. Still, open questions remain and we lack a comprehensive view of how the various sites and pathways of immunoglobulin A induction are integrated to respond to gut antigens. Next-generation sequencing (NGS) offers a novel approach to study B-cell responses, which might substantially enhance our tool box to answer key questions in the field and to take the next steps toward therapeutic exploitation of the mucosal B-cell system. In this review we discuss the potential, challenges, and emerging solutions for gut B-cell repertoire analysis by NGS.

Re-utilization of germinal centers in multiple Peyer's patches results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses.

Whereas gut IgA responses to the microbiota may be multi-centered and diverse, little is known about IgA responses to T-cell-dependent antigens following oral immunizations. Using a novel approach, gut IgA responses to oral hapten (4-hydroxy-3-nitrophenyl)acetyl-cholera toxin (NP-CT) conjugates were followed at the cellular and molecular level. Surprisingly, these responses were highly synchronized, strongly oligoclonal, and dominated by affinity matured cells. Extensive lineage trees revealed clonal relationships between NP-specific IgA cells in gut inductive and effector sites, suggesting expansion of the same B-cell clone in multiple Peyer's patches (PPs). Adoptive transfer experiments showed that this was achieved through re-utilization of already existing germinal centers (GCs) in multiple PPs by previously activated GC GL7(+) B cells, provided oral NP-CT was given before cell transfer. Taken together, these results explain why repeated oral immunizations are mandatory for an effective oral vaccine.

Inhibition of the proteasome reduces transfer-induced diabetes in nonobese diabetic mice.

Inhibition of the 26S proteasome reduces the severity of several immune-mediated diseases. Here, we report that the proteasome also regulates transfer-induced diabetes in nonobese mice. Treatment of recipient mice with the proteasome inhibitor N(alpha)-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (MG132) resulted in a 76% reduction in transfer-induced diabetes. The closely related inhibitor carbobenzoxy-l-leucyl-l-leucinal that inhibits calpains but not the proteasome had no protective effect, suggesting that MG132 acted via inhibition of the proteasome. MG132 decreased proliferation of transferred T cells in the pancreatic lymph nodes in vivo and prevented their expansion in a dose-dependent manner in vitro, consistent with a direct effect by MG132 on the T cells. MG132 did not prevent migration of transferred T cells into the islets but reduced the number of mice with severe infiltration. We suggest that MG132 prevents transfer-induced diabetes by directly targeting the autoreactive T cells and lowering their diabetogenic potential.

Effects of age on antibody affinity maturation.

The elderly are more susceptible to infectious diseases. Mortality and morbidity from infections increase sharply over the age of 65 years. At the same time, the efficacy of vaccinations in the elderly is decreased. The elderly also have an increased incidence of cancer and inflammatory diseases. All the above indicate an age-related dysregulation of the immune system. Evidence suggests that the change in the humoral immune response with age is a qualitative rather than a quantitative one, i.e. it is the affinity and specificity of the antibody that changes, rather than the quantity of antibody produced. There are a number of possible causes of this failure, one of which is a defect in the mechanism of hypermutation of immunoglobulin genes. We have studied individual clonal responses within germinal centres of spleen and Peyer's patches in young and old patient groups. Our results indicate that there is no difference in the actual mechanism of hypermutation with age. There are, however, differences that are due either to a change in selection processes or to a change in the founder cells available for activation.

Modeling the meta-dynamics of lymphocyte repertoires.

The complexity of biological systems, and the explosion of the quantity of biological information which is rapidly becoming available from experimental and clinical studies, necessitate the use of theoretical tools, namely, mathematical and computational modeling. The vertebrate adaptive immune system, with its learning and memory capabilities, is a particularly rich source of modeling challenges. Most difficult within this area is the study of lymphocyte repertoires--the generation of their diversity and the forces that shape the ever-changing dynamics of lymphocyte clones. I review several examples of problems in lymphocyte repertoire modeling, demonstrate the types of solutions employed, and highlight the contribution of these theoretical studies to immunological research.

CD1d endosomal trafficking is independently regulated by an intrinsic CD1d-encoded tyrosine motif and by the invariant chain.

Endosomal trafficking is an essential component of the CD1 pathway of lipid antigen presentation to T cells. We demonstrate that CD1d access to endosomal compartments is under dual regulation by an intrinsic tyrosine-based motif, which governs intense recycling between the plasma membrane and the endosome, and by the invariant chain, with which CD1d associates in the endoplasmic reticulum. Both pathways independently enhance antigen presentation to V(alpha)14(+) NKT cells, the main subset of CD1d-restricted T cells. These results reveal the complexity of CD1d trafficking and suggest that the invariant chain was a component of ancestral antigen presentation pathways prior to the evolution of MHC and CD1.

Deriving quantitative constraints on T cell selection from data on the mature T cell repertoire.

The T cell repertoire is shaped in the thymus through positive and negative selection. Thus, data about the mature repertoire may be used to infer information on how TCR generation and selection operate. Assuming that T cell selection is affinity driven, we derive the quantitative constraints that the parameters driving these processes must fulfill to account for the experimentally observed levels of alloreactivity, self MHC restriction and the frequency of cells recognizing a given foreign Ag. We find that affinity-driven selection is compatible with experimental estimates of these latter quantities only if 1) TCRs see more peptide residues than MHC polymorphic residues, 2) the majority of positively selected clones are deleted by negative selection, 3) between 1 and 3.6 clonal divisions occur on average in the thymus after completion of TCR rearrangement, and 4) selection is driven by 103-105 self peptides.

A quantitative theory of affinity-driven T cell repertoire selection.

Binding of the T cell antigen receptor (TCR) to peptides presented on molecules encoded by major histocompatibility complex (MHC) genes is the key event driving T cell development and activation. Selection of the T cell repertoire in the thymus involves two steps. First, positive selection promotes the survival of cells binding thymic self-MHC-peptide complexes with sufficient affinity. The resulting repertoire is self-MHC restricted: it recognizes foreign peptides presented on self, but not foreign MHC. Second, negative selection deletes cells which may be potentially harmful because their receptors interact with self-MHC-peptide complexes with too high an affinity. The mature repertoire is also highly alloreactive: a large fraction of T cells respond to tissues harboring foreign MHC. We derive mathematical expressions giving the frequency of alloreactivity, the level of self-MHC restriction, and the fraction of the repertoire activated by a foreign peptide, as a function of the parameters driving the generation and selection of the repertoire: self-MHC and self-peptide diversity, the stringencies of positive and negative selection, and the number of peptide and MHC polymorphic residues that contribute to T cell receptor binding. Although the model is based on a simplified digit string representation of receptors, all the parameters but one relate directly to experimentally determined quantities. The only parameter without a biological counterpart has no effect on the model's behavior besides a trivial and easily preventable discretization effect. We further analyse the role of the MHC and peptide contribution to TCR binding, and find that their relative, rather than absolute value, is important in shaping the mature repertoire. This result makes it possible to adopt different physical interpretations for the digit string formalism. We also find that the alloreactivity level can be inferred directly from data on the stringency of selection, and that, in agreement with recent experiments, it is not affected by thymic selection.

Models for antigen receptor gene rearrangement. I. Biased receptor editing in B cells: implications for allelic exclusion.

Recent evidence suggests that lymphocyte Ag receptor gene rearrangement does not always stop after the expression of the first productively rearranged receptor. Light chain gene rearrangement in B cells, and alpha-chain rearrangement in T cells can continue, which raises the question: how is allelic exclusion maintained, if at all, in the face of continued rearrangement? In this and the accompanying paper, we present comprehensive models of Ag receptor gene rearrangement and the interaction of this process with clonal selection. Our B cell model enables us to reconcile observations on the kappa:lambda ratio and on kappa allele usage, showing that B cell receptor gene rearrangement must be a highly ordered, rather than a random, process. We show that order is exhibited on three levels: a preference for rearranging kappa rather than lambda light chain genes; a preference to make secondary rearrangements on the allele that has already been rearranged, rather than choosing the location of the next rearrangement at random; and a sequentiality of J segment choice within each kappa allele. This order, combined with the stringency of negative selection, is shown to lead to effective allelic exclusion.

Models for antigen receptor gene rearrangement. II. Multiple rearrangement in the TCR: allelic exclusion or inclusion?

This series of papers addresses the effects of continuous Ag receptor gene rearrangement in lymphocytes on allelic exclusion. The previous paper discussed light chain gene rearrangement and receptor editing in B cells, and showed that these processes are ordered on three different levels. This order, combined with the constraints imposed by a strong negative selection, was shown to lead to effective allelic exclusion. In the present paper, we discuss rearrangement of TCR genes. In the TCR alpha-chain, allelic inclusion may be the rule rather than the exception. Several previous models, which attempted to explain experimental observations, such as the fractions of cells containing two productive TCRalpha rearrangements, did not sufficiently account for TCR gene organization, which limits secondary rearrangement, and for the effects of subsequent thymic selection. We present here a detailed, comprehensive computer simulation of TCR gene rearrangement, incorporating the interaction of this process with other aspects of lymphocyte development, including cell division, selection, cell death, and maturation. Our model shows how the observed fraction of T cells containing productive TCRalpha rearrangements on both alleles can be explained by the parameters of thymic selection imposed over a random rearrangement process.

Reconciling repertoire shift with affinity maturation: the role of deleterious mutations.

The shift in Ab repertoire, from Abs dominating certain primary B cell responses to genetically unrelated Abs dominating subsequent "memory" responses, challenges the accepted paradigm of affinity maturation. We used mathematical modeling and computer simulations of the dynamics of B cell responses, hypermutation, selection, and memory cell formation to test hypotheses attempting to explain repertoire shift. We show that repertoire shift can be explained within the framework of the affinity maturation paradigm, only when we recognize the destructive nature of hypermutation: B cells with a high initial affinity for the Ag are less likely to improve through random mutations.

MHC-linked syngeneic developmental preference in thymic lobes colonized with bone marrow cells: a mathematical model.

Reconstitution of the T-cell compartment after bone marrow transplantation depends on successful colonization of the thymus by bone-marrow-derived progenitor cells. Recent studies compared the development of syngeneic and allogeneic bone-marrow-derived cells in co-cultures with lymphoid-depleted fetal thymus explants, leading to the discovery of MHC-linked syngeneic developmental preference (SDP) in the thymus. To determine the nature of cell interactions among the bone marrow and thymic elements that might underlie SDP, we analyzed this phenomenon by mathematical modeling. The results indicate that syngeneic mature T cells, responsible for inducing this preference, probably interfere both with the seeding of allogeneic bone-marrow-derived thymocyte progenitors in the thymic stroma and with their subsequent proliferation. In addition, the possibility of augmented death among the developing allogeneic thymocytes cannot be ruled out.

Role of the thymus in pediatric HIV-1 infection.

Several lines of evidence suggest that HIV-1 is present in the thymus during HIV-1 infection. Precursors to mature CD4+ T lymphocytes develop in the thymus, which suggests that thymic infection may play a role in the CD4+ T-cell decline observed during the course of pediatric HIV-1 infection. We illustrate, through mathematical modeling, the potential effects of thymic infection on the course of pediatric AIDS disease progression. We find that infection in the thymus not only can supplement peripheral infection but can help explain the faster progression in pediatric cases, as well as the early and high viral burden.

Blind T-cell homeostasis and the CD4/CD8 ratio in the thymus and peripheral blood.

We present a model of the dynamics of CD4 and CD8 T-cell subsets in the thymus and peripheral blood and use it to study the blind homeostasis hypothesis, which states that the total T-cell population in the periphery is subject to regulation rather than regulation of the CD4 or CD8 subsets individually. Our model reconstructs experimental observations by Adleman and Wofsy on the effects of CD4+ T-cell depletion in mice. Our results point to the importance of the thymus in recovery from CD4+ T-cell depletion and particularly to the need to hypothesize an intrathymic feedback regulation of T-cell production exerted by CD4+ T cells. Our results support the blind homeostasis hypothesis for regulation of the peripheral blood levels of CD4+ and CD8- T cells.

Modelling Trypanosoma congolense parasitaemia patterns during the chronic phase of infection in N'Dama cattle.

We reanalyzed parasitaemia profiles of the trypanotolerant N'Dama cattle (Bos taurus), consecutively infected with the same four clones of Trypanosoma congolense. Our analysis shows that each individual parasitaemia is characterized by progressively longer intervals between parasites waves. This pattern is most visible during the chronic phase of infection. In addition, the last of the four infections had a significantly larger overall duration of inter-wave intervals. We retrieved these patterns by numerical simulations of a mathematical model, which incorporates assumptions about the molecular basis of antigenic variation and about the anti-parasitic major immune processes. Six potential factors that may determine parasitaemia pattern were studied: carrying capacity of the host environment, intrinsic growth rate of the parasite, affinity maturation of the immune response, immune cell birth and death rate, levels of antibodies to variant surface glycoprotein and levels of antibodies to invariant antigens. Our simulations suggest that the first five factors are not likely to determine the chronic phase parasitaemia pattern whereas the sixth one, namely, antibody response to invariant antigens, yielded profiles consistent with the experimental data. Being cumulative, the immune response to anti-invariant antigens may be increasingly effective as infection proceeds and in successive infections. Comparisons between N'Dama and Zebu and between chronic and acute phases will be needed to make a statement on the role of this phenomenon in trypanotolerance.

Regulatory feedback pathways in the thymus.

The idea that thymocytopoiesis may be subject to feedback regulation by mature lymphocytes is proposed on the basis of recent data from in vitro experimental models. Analysis of the data using mathematical models, presented here by Ramit Mehr and colleagues, suggests possible feedback control mechanisms in T-cell development.

Feedback regulation of T cell development: manifestations in aging.

Recent findings have indicated that mature T cells may regulate thymocytopoiesis in an age-related differential manner. The studies were based on T lymphocyte development in mouse fetal thymus stroma colonized with immature thymocytes and CD4+ T cells from young or old donors. In the present study, we used mathematical modeling and computer simulations in order to identify the thymocyte subsets that are targets for this type of regulation, and the processes affected by it. Our results suggest that thymocyte development is subject to regulation through 2 feedback loops: mature CD4+ cells exert a negative feedback on the double-negative to double-positive transition and on double-positive subset growth, and a positive feedback on the double-positive to CD4 single-positive transition. These effects may operate, in young mice, through a reduction in the rate of death of CD4+8- thymocytes, and a faster maturation of double-positive cells. In old mice, our simulations suggest that there may additionally be a reduction in double-positive proliferation rate. In some, but not all, of the simulations of old donor- derived thymocytes, we also had to assume a reduction in double-negative to double-positive differentiation, an increase in double-positive death rates, an increase of CD4+8- cell division rate, and a decrease of differentiation to the CD8 lineage.

Feedback regulation of T cell development in the thymus.

Recent findings suggest that mature T cells in the thymus may regulate the growth and differentiation of immature thymocytes. Here we use mathematical modeling and computer simulations to identify the thymocyte subsets that might serve as targets for regulation, and the processes that might be affected by regulation. Our results suggest that thymocyte development is subject to regulation through two feedback loops: mature CD4+ T cells exert a positive feedback on the single positive CD4+8- thymocyte compartment, by reducing CD4+8- cell death and possibly accelerating the differentiation of CD4+8+ thymocytes into CD4+8- thymocytes; they may also exert a negative feedback on the double-positive CD4+8+ thymocyte compartment, by reducing the proliferation or accelerating the maturation of these cells.

Lymphocyte development in irradiated thymuses: dynamics of colonization by progenitor cells and regeneration of resident cells.

Lymphocyte development in irradiated thymuses was analyzed using two complementary strategies: an in vitro experimental model and computer simulations. In the in vitro model, fetal thymus lobes were irradiated and the regeneration of cells that survived irradiation were examined, with the results compared to those of reconstitution of the thymus by donor bone marrow cells and their competition with the thymic resident cells. In vitro measurements of resident cell kinetics showed that cell proliferation is slowed down significantly after a relatively low (10 Gy) irradiation dose. Although the number of thymocytes that survived irradiation remained low for several days post-irradiation, further colonization by donor cells was not possible, unless performed within 6 h after irradiation. These experimental results, coupled with the analysis by computer simulations, suggest that bone marrow cell engraftment in the irradiated thymus may be limited by the presence of radiation-surviving thymic resident cells and the reduced availability of seeding niches.

Modeling positive and negative selection and differentiation processes in the thymus.

T cells begin their development as precursor cells in the bone marrow. These cells migrate to the thymus, where they further divide, differentiate, and mature into functional T cells. Most thymocytes (95-99%) die in the course of this process, and only relatively few exit the thymus as mature cells. Here we develop a differential equation model of cell proliferation, differentiation and death in the thymus that can account for both the total number of thymus cells and the fractions of various types of immature and mature thymocytes. Our model suggests that positive and negative selection may have more complex effects than simply deleting some cells and allowing others to survive.