Self-tolerant Repertoire Research Articles

Evolution of thymopoietic microenvironments.

In vertebrates, the development of lymphocytes from undifferentiated haematopoietic precursors takes place in so-called primary lymphoid organs, such as the thymus. Therein, lymphocytes undergo a complex differentiation and selection process that culminates in the generation of a pool of mature T cells that collectively express a self-tolerant repertoire of somatically diversified antigen receptors. Throughout this entire process, the microenvironment of the thymus in large parts dictates the sequence and outcome of the lymphopoietic activity. In vertebrates, direct genetic evidence in some species and circumstantial evidence in others suggest that the formation of a functional thymic microenvironment is controlled by members of the Foxn1/4 family of transcription factors. In teleost fishes, both Foxn1 and Foxn4 contribute to thymopoietic activity, whereas Foxn1 is both necessary and sufficient in the mammalian thymus. The evolutionary history of Foxn1/4 genes suggests that an ancient Foxn4 gene lineage gave rise to the Foxn1 genes in early vertebrates, raising the question of the thymopoietic capacity of the ancestor common to all vertebrates. Recent attempts to reconstruct the early events in the evolution of thymopoietic tissues by replacement of the mouse Foxn1 gene by Foxn1-like genes isolated from various chordate species suggest a plausible scenario. It appears that the primordial thymus was a bi-potent lymphoid organ, supporting both B cell and T cell development; however, during the course of vertebrate, evolution B cell development was gradually diminished converting the thymus into a site specialized in T cell development.

Investigating T Cell Receptor Signals In Situ by Two-Photon Microscopy of Thymocytes Expressing Genetic Reporters in Low-Density Chimeras.

T cell development is a dynamic process accompanied by extensive thymocyte migration, cellular interactions, and T cell receptor (TCR) signaling. In particular, thymic selection processes that ensure a functional, self-tolerant repertoire require TCR interactions with self-peptide presented by major histocompatibility complex molecules expressed by specialized thymic antigen-presenting cells. The quantity and quality of these TCR signals influence T cell fate. Two-photon microscopy, which enables live imaging of cells in intact tissue, has emerged as a powerful tool to gain insights into thymocyte migration and TCR signaling during T cell development in situ. Here we describe the generation of non-irradiated, low-density chimeric mice by neonatal injection of adult bone marrow engineered to express fluorescent TCR signaling reporters for imaging by two-photon microscopy. We also describe how the thymic lobes from chimeric mice are prepared for live imaging of thymocyte behavior and TCR signaling events. While we focus on imaging TCR signals associated with T cell development in the thymus, these techniques can also be adapted to study TCR signaling in mature T cells in peripheral lymphoid organs.

Post-translational control of T cell development by the ESCRT protein CHMP5.

The acquisition of a protective vertebrate immune system hinges on the efficient generation of a diverse but self-tolerant repertoire of T cells by the thymus through mechanisms that remain incompletely resolved. Here we identified the endosomal-sorting-complex-required-for-transport (ESCRT) protein CHMP5, known to be required for the formation of multivesicular bodies, as a key sensor of thresholds for signaling via the T cell antigen receptor (TCR) that was essential for T cell development. CHMP5 enabled positive selection by promoting post-selection thymocyte survival in part through stabilization of the pro-survival protein Bcl-2. Accordingly, loss of CHMP5 in thymocyte precursor cells abolished T cell development, a phenotype that was 'rescued' by genetic deletion of the pro-apoptotic protein Bim or transgenic expression of Bcl-2. Mechanistically, positive selection resulted in the stabilization of CHMP5 by inducing its interaction with the deubiquitinase USP8. Our results thus identify CHMP5 as an essential component of the post-translational machinery required for T cell development.

Genetic Ablation of Cereblon (CRBN) Increases Long-Lived Memory T Cells

Background: Immunomodulatory drugs (IMiDs) consist of thalidomide and derivatives, pomalidomide and lenalidomide. IMiDs target the E3 ubiquitin ligase substrate receptor, cereblon (CRBN), which forms a complex with DDB1-Cul4A, to induce anti-proliferative effects in tumor cells and increase the activation of T cells. Using crbn deficient T-cells, we have shown that CRBN is a negative regulator of T cell activation in mice. Dynamic changes in the T-cell repertoire occur after thymic involution. With the reduction of thymic output with age, there is a loss of naïve T-cells in the peripheral blood, and the accumulation of a memory-like T-cell population that arises through homeostatic proliferation. In this study, we demonstrate the role of CRBN in peripheral homeostatic regulation during aging. Methods: In this study, C57BL6 crbn -/- mice were housed in pathogen-free conditions to limit foreign antigen exposure. Splenocytes were stained with T cell markers to define various memory populations from 3, 8, 10 and 13 month old crbn -/- and age-matched wild-type mice. Expression of CD8, CD44, CD127 (IL-7 receptor) and KLRG1 were determined by flow cytometry. CD44-CD127+ KLRG1- CD8+ T cells were considered naive in this analysis. Consistent with previous reports, CD44+ CD127+ KLRG1- T cells represent long-term memory T-cells while CD44+CD127-KLRG1+ T cells are effector memory cells. To better understand age-related changes in the thymus, the thymus was dissected and the absolute number of thymocytes was determined by trypan blue staining. Thymic subpopulations were defined using CD4 and CD8 as single positive, double positive, and double negative cells in 13 month old crbn -/- and WT mice. We further characterized double negative (DN) populations using CD44 and CD25 to define DN1, DN2, DN3, and DN4. Results: Splenocytes in 3 and 8 month old crbn -/- and WT mice have no significant differences in CD44+ memory cells, naïve cells (p=0.7850 and p=0.5061) or recently activated cells [CD69+ or CD25+]. Changes related to thymic involution in WT mice become evident at 10 months of age. Crbn -/- mice exhibit a similar percentage and absolute number of total memory cells (p=0.8194) at 10 months of age, as defined by CD44 expression. Evaluation of long-lived versus short-lived sub-populations of memory cells in these older mice showed that crbn deficiency is associated with significantly more CD44+ CD127+ KLRG1- CD8+ [long-lived] memory T cells compared to WT mice (p<0.001). Evaluation of the thymus revealed no difference in absolute numbers of thymocytes in younger mice. Moreover, crbn deficient mice have normal distribution of SP, DP, and DN populations, with only slight changes in DN1, DN2, DN3, and DN4 subpopulations. Absolute numbers of thymocytes, however, were significantly higher in the crbn -/- mice at 10 months of age compared to age-matched controls (p=0.0182). Conclusions: The fate of developing T cells is regulated by positive and negative selection in the thymus where the coordination and selection of self-tolerant repertoire is maintained. Age-related changes caused by thymic involution impacts the protective responses of these cells against foreign pathogens and tumor cells. Our data show that the genetic germline depletion of crbn leads to an increase in long-lived memory T cells in naturally aged animals. Universally in cancer patients, and especially in MDS, the lymphocyte compartment is characterized by premature age-related changes due possibly to reduction in lymphopoiesis at the stem cell level or due to chronic antigen stimulation. Given that the CD44+ CD127+ KLRG1- population is significantly higher in aged crbn -/- mice, these mice may be more resistant to viral infection and or development of malignancy. The mechanism responsible for this phenotypic difference could be due to cell intrinsic signaling or metabolic properties. However, a role for CRBN in thymic epithelial cells must also be considered given the use of germline deficient animals in this study. Taken together, we have demonstrated that ablation of crbn results in an increase in long-lived CD8+ T cells during aging, which suggests that targeting CRBN and/or treating with lenalidomide may functionally improve memory T cell capacity in the elderly. Disclosures No relevant conflicts of interest to declare.

CCRL1 marks heterogeneity in cortical and medullary thymic epithelial cells.

Cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs), which play essential roles in the establishment of a functionally competent and self-tolerant repertoire of T cells, are derived from common thymic epithelial progenitor cells (pTECs). Recent findings indicate that mTECs are derived from cells that express molecules that are abundant in cTECs rather than mTECs, and provide fresh insight into the characteristics of pTECs and their diversification pathways into TEC subpopulations. In this issue of the European Journal of Immunology, Ribeiro et al. [Eur. J. Immunol. 2014. 44: 2918-2924] focus on CCRL1, an atypical chemokine receptor that is highly expressed by cTECs rather than mTECs, and show that CCRL1-expressing embryonic TECs can give rise to mTECs. Interestingly, Ribeiro et al. further report that a fraction of postnatal mTECs express CCRL1 at a low level, suggesting novel complexity in mTECs.

The Fate of Low Affinity Tumor-Specific CD8+ T Cells in Tumor-Bearing Mice

A major challenge in tumor immunology is how best to activate the relatively low avidity self-specific and tumor-specific T cells that are available in the self-tolerant repertoire. To address this issue, we produced a TCR transgenic mouse expressing a class I-restricted hemagglutinin (HA)-specific TCR (clone 1 TCR) derived from a mouse that expressed HA as a self-Ag in the insulin-producing beta cells of the pancreatic islets (InsHA) mice. Upon transfer of clone 1 TCR CD8(+) T cells into InsHA mice, very few cells were activated by cross-presented HA, indicating that the cells were retained in InsHA mice because they ignored the presence of Ag, and not because they were functionally inactivated by anergy or tuning. Upon transfer into recipient mice in which HA is expressed at high concentrations as a tumor-associated Ag in spontaneously arising insulinomas (RIP-Tag2-HA mice), a high proportion of clone 1 cells were activated when they encountered cross-presented tumor Ag in the pancreatic lymph nodes. However, the activated cells exhibited very weak effector function and were soon tolerized. The few activated cells that did migrate to the tumor were unable to delay tumor progression. However, when HA-specific CD4 helper cells were cotransferred with clone 1 cells into RIP-Tag2-HA recipients and the mice were vaccinated with influenza, clone 1 cells were found to exert a significant level of effector function and could delay tumor growth. This tumor model should prove of great value in identifying protocols that can optimize the function of low avidity tumor-specific T cells.

Genetic dissection of thymus development in mouse and zebrafish.

Lymphoid organs represent a specialized microenvironment for interaction of stromal and lymphoid cells. In primary lymphoid organs, these interactions are required to establish a self-tolerant repertoire of lymphocytes. While detailed information is available about the genes that control lymphocyte differentiation, little is known about the genes that direct the establishment and differentiation of principal components of such microenvironments. Here, we discuss genetic studies addressing the role of thymic epithelial cells (TECs) during thymopoiesis. We have identified an evolutionarily conserved key regulator of TEC differentiation, Foxn1, that is required for the immigration of prothymocytes into the thymic primordium. Because Foxn1 specifies the prospective endodermal domain that gives rise to thymic epithelial cells, it can be used to identify the evolutionary origins of this specialized cell type. In the course of these studies, we have found that early steps of thymus development in zebrafish are very similar to those in mice. Subsequently, we have used chemical mutagenesis to derive zebrafish lines with aberrant thymus development. Strengths and weaknesses of mouse and zebrafish models are largely complementary such that genetic analysis of mouse and zebrafish mutants may lead to a better understanding of thymus development.

Apoptosis in the development of the immune system: growth factors, clonal selection and bcl-2.

The mammalian immune system is essential for surviving challenge infections with a great range of potential pathogens. The protective effect produced is dependent on many different types of cells which require flexible and independent production and regulation. In particular, many important responses are carried out by lymphocytes, which recognise foreign antigen through exquisitely specific receptors: i.e. surface immunoglobulin (sIg) on B lymphocytes and the T cell receptor (TCR) on T lymphocytes. Each lymphocyte displays receptors with a single specificity, allowing cells with particular specificities to be regulated independently. Since millions of different Igs and TCRs are expressed, the precise selection and regulation of each T and B cell population to produce a useful self-tolerant repertoire is a very complex process. Control of cell populations can, in theory, be exercised at a number of levels, including modulation of active cell death by apoptosis. Recent research has demonstrated that regulation of apoptosis is indeed a crucial element in the control of the immune system in general, and in the development of the TCR and Ig repertoires in particular. The molecular analysis of apoptosis now takes a high priority and the proto-oncogene bcl-2 appears to be responsible for specific suppression of apoptosis in several important situations. It is also clear that malfunctions affecting apoptosis, and in particular bcl-2, can result in significant progression towards malignancy.