Cell Repertoire In Mice Research Articles

Antigen-presenting autoreactive B cells activate regulatory T cells and suppress autoimmune arthritis in mice.

B cells undergo several rounds of selection to eliminate potentially pathogenic autoreactive clones, but in contrast to T cells, evidence of positive selection of autoreactive B cells remains moot. Using unique tetramers, we traced natural autoreactive B cells (C1-B) specific for a defined triple-helical epitope on collagen type-II (COL2), constituting a sizeable fraction of the physiological B cell repertoire in mice, rats, and humans. Adoptive transfer of C1-B suppressed arthritis independently of IL10, separating them from IL10-secreting regulatory B cells. Single-cell sequencing revealed an antigen processing and presentation signature, including induced expression of CD72 and CCR7 as surface markers. C1-B presented COL2 to T cells and induced the expansion of regulatory T cells in a contact-dependent manner. CD72 blockade impeded this effect suggesting a new downstream suppressor mechanism that regulates antigen-specific T cell tolerization. Thus, our results indicate that autoreactive antigen-specific naïve B cells tolerize infiltrating T cells against self-antigens to impede the development of tissue-specific autoimmune inflammation.

Pre-existing T Cell Memory to Novel Pathogens.

Immunological experiences lead to the development of specific T and B cell memory, which readies the host for a later pathogen rechallenge. Currently, immunological memory is best understood as a linear process whereby memory responses are generated by and directed against the same pathogen. However, numerous studies have identified memory cells that target pathogens in unexposed individuals. How "pre-existing memory" forms and impacts the outcome of infection remains unclear. In this review, we discuss differences in the composition of baseline T cell repertoire in mice and humans, factors that influence pre-existing immune states, and recent literature on their functional significance. We summarize current knowledge on the roles of pre-existing T cells in homeostasis and perturbation and their impacts on health and disease.

Abstract 2282: Immune modulation of the T cell repertoire in mice following immunotherapy and chemotherapy combination treatments

Abstract Background: Immune checkpoint blockade therapeutics have revolutionized cancer therapy. However, most patients are either unresponsive or develop acquired resistance. Understanding patient-specific temporal T cell repertoires in the context of immune interventions will contribute to a better understanding of potential improvement strategies for better T cell anti-tumor immunity. Current methods to understand the T cell repertoire changes over time are limited due to the limited amount of material and prohibitive cost. In this study, we designed and performed longitudinal TCR sequencing in mice treated with multiple therapies. Methods: C57BL/6 mice were used in each treatment group, with blood drawn overtime before, during and after treatment. Mice were treated with a combination of immunotherapeutic agents and OVA peptide immunization, 100µl blood was drawn at multiple pre-, on- and post-treatment time points followed subsequently by TCR sequencing. Cyclophosphamide (CTX) is known to cause lymphodepletion followed by homeostatic proliferation. Thus, we also treated B16-F10 tumor bearing mice with CTX, and performed TCR sequencing on blood draws pre-, on-, and post-treatment to track TCR clone diversity and clonality over time. Results: We find that our method is sensitive enough to detect effects of immunotherapies on TCR diversity in small sample quantities. Using our method, we discovered that certain immunotherapeutic agents, most evidently CD40, have a significant but transient effect on the diversity of the T cell repertoire. We observed an initial increase in repertoire clonality, seven days post CD40 treatment followed by contraction. We show how we can identify antigen specific T cells and follow reacting T cell clones. In addition, we find that CTX treatment is associated with increased clonality in tumor bearing mice particularly in the week following treatment reflecting changes in the TCR repertoire. Conclusion: We have developed a method by which we can follow the TCR repertoire in mice treated with immunotherapy or chemotherapy. This enabled us to isolate and study the effects of different immune modulatory drugs of the endogenous T cell repertoire overtime. Citation Format: Yuval Elhanati, Mariam M. George, Hyejin Choi, Rachana Maniyar, Linda Hamadane, Neeman Mohibullah, Benjamin D. Greenbaum, Taha Merghoub. Immune modulation of the T cell repertoire in mice following immunotherapy and chemotherapy combination treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2282.

Mice With Chronic Kidney Disease Display Premature Immune Senescence.

Background: Accumulation of effector memory T cells and T cell senescence have been described in elderly humans and animals. These changes have been implicated in many age-associated disease processes. Our group recently reported that children with chronic kidney disease (CKD) also show signs of premature immune senescence. Mice are typically studied without long-standing CKD. We aimed to determine whether CKD alters the T cell repertoire in mice, and thus could be a variable worthy of consideration in murine models of transplant biology. Methods: CKD was induced in 129X1/SvJ mice through five-sixths nephrectomy (5/6Nx) in a two-stage surgery. Age-matched mice that underwent unilateral partial nephrectomy resulting in overall two-sixths renal mass reduction (2/6Nx) or no surgeries (NS) served as controls. Renal function was assessed by measuring blood urea nitrogen (BUN), plasma cystatin c (CysC), and urinary albumin to creatinine ratio (ACR). T-cell memory subtypes were assessed via multicolor flow cytometry of blood and spleen 8 weeks following CKD induction. Activation markers CD25, CD69, and CD27 and senescence markers PD1 and KLRG1 were assessed. T cell memory subsets were determined by expression of CD44, CD62L, and CCR7. Results: Mice undergoing 5/6Nx had significantly higher BUN, CysC and urinary ACR than the 2/6Nx and NS age-matched controls. Total peripheral white blood cell count was not significantly different among the groups, but the 5/6Nx mice had significantly lower percentage of lymphocytes and absolute number of CD3+ T cells compared to both control groups. The frequency of peripheral blood CD4+ and CD8+ T cells as well as the CD4+:CD8+ ratio did not vary significantly among the groups. Mice with CKD had significantly higher frequency of both CD4+ and CD8+ splenic T cells expressing PD1 and KLRG1. The PD1+ and KLRG1+ cells were primarily CD62L-CCR7-CD44high effector memory T cells. Conclusions: Mice with CKD display signs of premature immune senescence with lymphopenia, reduced number of peripheral T cells, and increased expression of the senescence-associated markers PD-1 and KLRG1 on splenic T cells. These data suggest that CKD can promote significant alterations in the immune repertoire independent of age or environmental antigen exposure. The impact of these alterations on alloimmunity is under investigation.

Multiple Cytokines Regulate the NK Gene Complex-Encoded Receptor Repertoire of Mature NK Cells and T Cells

Mature NK cells comprise a highly diverse population of lymphocytes that express different permutations of receptors to facilitate recognition of diseased cells and perhaps pathogens themselves. Many of these receptors, such as those belonging to the NKRP1, NKG2, and Ly49 families are encoded in the NK gene complex (NKC). It is generally thought that these NKC-encoded receptors are acquired by a poorly understood stochastic mechanism, which operates exclusively during NK cell development, and that following maturation the repertoire is fixed. However, we report a series of observations that demonstrates that the mature NK cell repertoire in mice can in fact be radically remodeled by multiple cytokines. Thus, both IL-2 and IL-15 selectively induce the de novo expression of Ly49E on the majority of mature NK cells. By contrast, IL-4 not only blocks this IL-2-induced acquisition of Ly49E, but reduces the proportion of mature NK cells that expresses pre-existing Ly49 receptors and abrogates the expression of NKG2 receptors while leaving the expression of several NKRP1 receptors unaltered. IL-21 also abrogates NKG2 expression on mature NK cells and selectively down-regulates Ly49F. IL-4 and IL-21 additionally cause dramatic and selective alterations in the NKC-encoded receptor repertoire of IL-2-activated T cells but these are quite different to the changes induced on NK cells. Collectively these findings reveal an unexpected aspect of NKC receptor expression that has important implications for our understanding of the function of these receptors and of the genetic mechanisms that control their expression.

Differences in the avidity of TCR interactions with a superantigenic ligand affect negative selection but do not allow positive selection.

The products of the sag genes of the exogenous mouse mammary tumor virus (MMTV) genome and of endogenous Mtv integrants have been demonstrated to affect the T cell repertoire in mice by causing the deletion of T cells expressing receptors encoded by particular V beta gene segments. Since these deletions affect large populations of T cells with receptors of heterogeneous specificity, they serve as an important model for the study of T cell development in normal mice. Using several C3H/HeN-based strains that express different MMTV(C3H) transgenes, we demonstrate here that the stage of development at which T cell deletion occurs is determined by the level of ligand expression. Although at low levels of ligand expression in the thymus some signs of activation were observed in immature thymocytes, we were unable to detect a level of Sag expression that led to net positive selection. Moreover, we detected a level of Sag-transgene expression that did not cause negative selection in the thymus; no signs of positive selection were observed either. Inclusion of the env gene in the construct, earlier shown to markedly potentiate stimulation by Sag in mixed lymphocyte reactions, also markedly increased the ability of Sag to drive negative selection. These data are interpreted as showing that marked quantitative differences in expression of superantigens does not reveal a level at which only positive selection occurs. This, in turn, suggests that positive selection will occur on ligands distinct from those that drive clonal deletion.

Compartmentalization of lambda subtype expression in the B cell repertoire of mice with a disrupted or normal C kappa gene segment.

The establishment of the B cell repertoire depends on two major parameters. The first is determined by mechanistic processes that give rise to a great diversity of B cell receptors from a combination of multiple gene segments. The second is dominated by selective processes that recruit B cell clones via their immunoglobulin receptors. To assess the impact of these parameters on the composition of B cell repertoire, we constructed a mouse model displaying a B cell repertoire limited in its diversity. To this end, we disrupted the C kappa segment by gene targeting. B cells from such mutant mice do not express the kappa light chain. Their light chain repertoire is therefore limited by the expression of only four main lambda light chains: lambda 1, lambda 2(V2), lambda 2(Vx) and lambda 3. In this study we described the proportions of each lambda subtype in various lymphoid compartments. Our results show that the lambda 1 subtype is dominant in the spleen and the bone marrow. Moreover, lambda 1 prevalence is independent of the wild or mutant C kappa genotype. These results suggest that the mechanistic processes are mainly responsible for the bias in lambda subtype expression. On the other hand, the lambda 2(V2) and/or lambda 3 subtypes are expressed at higher levels in the peritoneal cavity. Their prevalence is again observed regardless of the C kappa genotype and seems to be due to B1 cells. These results suggest that different mechanistic processes could control lambda subtype expression in B1 and B2 cell lineages.

Dominant determinants in hen eggwhite lysozyme correspond to the cryptic determinants within its self-homologue, mouse lysozyme: implications in shaping of the T cell repertoire and autoimmunity.

We have studied the mouse lysozyme (ML) peptide-specific T cell repertoire in mice of five different major histocompatibility complex (MHC) class II haplotypes. 14 ML peptides were tested in a lymph node T cell proliferation assay. Upon immunization of diverse mouse strains with native ML, there was no response to any of the ML peptides tested. However, nine peptides were immunogenic, although there was no consistent pattern of reactivity toward any peptide among these strains. Thus, an autoreactive T cell repertoire directed against cryptic self(ML)-determinants exists, and it is different in mice of different MHC haplotypes. Moreover, our results demonstrate that crypticity is MHC associated and not merely a structural attribute of the determinant. On comparison of the pattern of response of various peptides of ML and that of its foreign homologue, hen eggwhite lysozyme (HEL) in H-2k, H-2b, and H-2d strains of mice, a striking correlation was evident. The stretches of amino acid sequences of determinants within HEL that were dominant in each of these three strains, almost exactly overlapped in position with those of the cryptic ML determinants against which self-reactivity was demonstrated in the same strain. These results demonstrate that the dominance-crypticity relationship between HEL and ML resulting from differential processing of these two proteins is critical in determining the response to HEL rather than the degree of sequence difference between them. These observations have important implications in the shaping of the T cell repertoire for foreign proteins and in the pathogenesis of autoimmunity.