Mature Αβ Research Articles

The Cyclin D3 Protein Enforces Monogenic TCRβ Expression by Mediating TCRβ Protein-Signaled Feedback Inhibition of Vβ Recombination.

In jawed vertebrates, adaptive immunity depends on the process of V(D)J recombination creating vast numbers of T and B lymphocytes that each expresses unique Ag receptors of uniform specificity. The asynchronous initiation of V-to-(D)J rearrangement between alleles and the resulting protein from one allele signaling feedback inhibition of V recombination on the other allele ensures homogeneous receptor specificity of individual cells. Upon productive Vβ-to-DβJβ rearrangements in noncycling double-negative thymocytes, TCRβ protein signals induction of the cyclin D3 protein to accelerate cell cycle entry, thereby driving proliferative expansion of developing αβ T cells. Through undetermined mechanisms, the inactivation of cyclin D3 in mice causes an increased frequency of αβ T cells that express TCRβ proteins from both alleles, producing lymphocytes of heterogeneous specificities. To determine how cyclin D3 enforces monogenic TCRβ expression, we used our mouse lines with enhanced rearrangement of specific Vβ segments due to replacement of their poor-quality recombination signal sequence (RSS) DNA elements with a better RSS. We show that cyclin D3 inactivation in these mice elevates the frequencies of αβ T cells that display proteins from RSS-augmented Vβ segments on both alleles. By assaying mature αβ T cells, we find that cyclin D3 deficiency increases the levels of Vβ rearrangements that occur within developing thymocytes. Our data demonstrate that a component of the cell cycle machinery mediates TCRβ protein-signaled feedback inhibition in thymocytes to achieve monogenic TCRβ expression and resulting uniform specificity of individual αβ T cells.

Early expression of mature αβ TCR in CD4−CD8− T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αβ T cell antigen receptor (TCR). We report that these early αβ TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αβ T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αβ TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors invivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Adapting T Cell Receptor Ligand Discrimination Capability via LAT.

Self- and non-self ligand discrimination is a core principle underlying T cell-mediated immunity. Mature αβ T cells can respond to a foreign peptide ligand presented by major histocompatibility complex molecules (pMHCs) on antigen presenting cells, on a background of continuously sensed self–pMHCs. How αβ T cells can properly balance high sensitivity and high specificity to foreign pMHCs, while surrounded by a sea of self-peptide ligands is not well understood. Such discrimination cannot be explained solely by the affinity parameters of T cell antigen receptor (TCR) and pMHC interaction. In this review, we will discuss how T cell ligand discrimination may be molecularly defined by events downstream of the TCR–pMHC interaction. We will discuss new evidence in support of the kinetic proofreading model of TCR ligand discrimination, and in particular how the kinetics of specific phosphorylation sites within the adaptor protein linker for activation of T cells (LAT) determine the outcome of TCR signaling. In addition, we will discuss emerging data regarding how some kinases, including ZAP-70 and LCK, may possess scaffolding functions to more efficiently direct their kinase activities.

Two Successive Inversional Vβ Rearrangements on a Single Tcrb Allele Can Contribute to the TCRβ Repertoire.

Mammalian TCRβ loci contain 30 Vβ gene segments upstream and in the same transcriptional orientation as two DJCβ clusters, and a downstream Vβ (TRBV31) in the opposite orientation. The textbook view is upstream Vβs rearrange only by deletion and TRBV31 rearranges only by inversion to create VβDJCβ genes. In this study, we show in mice that upstream Vβs recombine through inversion to the DJCβ2 cluster on alleles carrying a preassembled Trbv31-DJCβ1 gene. When this gene is in-frame, Trbv5 evades TCRβ-signaled feedback inhibition and recombines by inversion to the DJCβ2 cluster, creating αβ T cells that express assembled Trbv5-DJCβ2 genes. On alleles with an out-of-frame Trbv31-DJCβ1 gene, most upstream Vβs recombine at low levels and promote αβ T cell development, albeit with preferential expansion of Trbv1-DJβ2 rearrangements. Finally, we show wild-type Tcrb alleles produce mature αβ T cells that express upstream Vβ peptides in surface TCRs and carry Trbv31-DJβ2 rearrangements. Our study indicates two successive inversional Vβ-to-DJβ rearrangements on the same allele can contribute to the TCRβ repertoire.

γδ T cells shape memory-phenotype αβ T cell populations in non-immunized mice.

Size and composition of γδ T cell populations change dramatically with tissue location, during development, and in disease. Given the functional differentiation of γδ T cell subsets, such shifts might alter the impact of γδ T cells on the immune system. To test this concept, and to determine if γδ T cells can affect other immune cells prior to an immune response, we examined non-immunized mice derived from strains with different genetically induced deficiencies in γδ T cells, for secondary changes in their immune system. We previously saw extensive changes in pre-immune antibodies and B cell populations. Here, we report effects on αβ T cells. Similarly to the B cells, αβ T cells evidently experience the influence of γδ T cells at late stages of their pre-immune differentiation, as single-positive heat stable antigen-low thymocytes. Changes in these and in mature αβ T cells were most prominent with memory-phenotype cells, including both CD8+ and CD4+ populations. As previously observed with B cells, most of the effects on αβ T cells were dependent on IL-4. Unexpectedly, IL-4 seemed to be produced mainly by αβ T cells in the non-immunized mice, albeit strongly regulated by γδ T cells. Similarly to our findings with B cells, changes of αβ T cells were less pronounced in mice lacking all γδ T cells than in mice lacking only some, suggesting that the composition of the γδ T cell population determines the nature of the γδ-influence on the other pre-immune lymphocytes.

The UT family of MHC class I loci unique to non-eutherian mammals has limited polymorphism and tissue specific patterns of expression in the opossum.

BackgroundThe Major Histocompatibility Complex (MHC) class I family of genes encode for molecules that have well-conserved structures, but have evolved to perform diverse functions. The availability of the gray, short-tailed opossum, Monodelphis domestica whole genome sequence has allowed for analysis of MHC class I gene content in this marsupial. Utilization of a novel method to search for MHC related domain structures revealed a previously unknown family of MHC class I-related genes. These genes, named UT1-17, are clustered on chromosome 1 in the opossum, unlinked to the MHC region. UT genes are only found in marsupial and monotreme genomes, consistent with being ancient in mammals yet lost in eutherian mammals. This study investigates the expression and polymorphism of the UT loci in the opossum to gain insight into their possible function.ResultsOf the 17 opossum UT genes, most have restricted tissue transcription patterns, with the thymus and skin being the most common sites. Full-length structure of 11 UT transcripts revealed genes varying between five and eight exons, typical for class I family members. There were only two alternative splice variants found. The UT genes also have limited polymorphism and little evidence of positive selection. One locus, UT8, was chosen for further analysis due to its conservation amongst marsupials and generic characteristics. UT8 transcription is limited to developing αβ thymocytes, and is absent from mature αβ T cells in peripheral lymphoid tissues.ConclusionThe overall characteristics and features of UT genes including low polymorphism and restricted tissue expression make it likely that the molecules encoded by UT genes perform roles other than antigenic peptide presentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0181-9) contains supplementary material, which is available to authorized users.

Essential role for the histone acetyltransferase KAT7 in T cell development, fitness, and survival.

Histone acetylation has an important role in gene regulation, DNA replication, and repair. Because these processes are central to the development of the immune system, we investigated the role of a previously unstudied histone acetyltransferase named KAT7 (also known as Myst2 or HBO1) in the regulation of thymopoiesis and observed a critical role in the regulation of conventional and innate-like T cell development. We found that KAT7-deficient thymocytes displayed normal, positive selection and development into mature single-positive αβ thymocytes; however, we observed few peripheral CD4+ or CD8+ T cells. The observed effects did not appear to arise from alterations to DNA replication, the TCR repertoire, or a block in thymocyte maturation and, more likely, was linked to survival defects related to gene deregulation because KAT7 deficiency led to an almost complete and specific loss of global histone-H3 lysine 14 acetylation (H3K14ac). Overall, we demonstrated a nonredundant role for KAT7 in the maintenance of H3K14ac, which is intimately linked with the ability to develop a normal immune system.

Expression of T-bet, Eomesodermin and GATA-3 in porcine αβ T cells

The transcription factors GATA-3, T-bet and Eomesodermin play important roles in T-cell development, differentiation and memory formation. However, their expression has not been studied in great detail in porcine T cells. We report on protein expression at the single cell-level of these transcription factors in thymocytes and mature αβ T cells. GATA-3 expression was found in γδ− thymocytes, with decreasing expression from the CD4−CD8α− stage towards single-positive stages. Extra-thymic CD4+ T cells but not CD8β+ T cells expressed low levels of GATA-3, which decreased with age. CD4+ and CD8β+ T-bet+ cells mainly displayed a CD8α+CD27− and perforin+CD27dim/− phenotype, respectively and had the capacity for IFN-γ production; indicative of an effector/effector memory phenotype. Eomesodermin+ αβ T cells had mixed phenotypes in regard to CD8α, CD27 and perforin expression. In conclusion, our data so far support the hitherto reported roles for GATA-3 in T-cell development and T-bet for Th1 effector-differentiation, but question the role of Eomesodermin for memory formation of porcine T-cells.

Insights into the Transcriptional Regulation of the Unrearranged and Rearranged Tcra and Tcrd Genes

The combined T-cell receptor α and δ locus, Tcra/Tcrd, encodes the TCRα and TCRδ chains of the αβ or γδ T-cell receptors (TCRαβ and TCRγδ), respectively, which define the two distinct T-cell lineages, αβ and γδ T lymphocytes. Like other antigen receptor loci, this locus must recombine its variable (V), diversity (D), and joining (J) gene segments to generate a diverse range of TCR that allow vertebrates to respond to an unlimited number of antigens. The Tcra/Tcrd germline transcription and subsequent V(D)J gene segment rearrangements are strictly regulated by two distant transcriptional enhancers, Eα and Eδ, respectively, during thymocyte development. Once the Tcra locus is productively rearranged, it is assumed Eα remains active for the transcription of the rearranged locus and the expression of the functional TCRα chain in αβ T lymphocytes. However, our recent experiments have shown Eα is significantly inhibited during the final stage of thymocyte development, concomitantly with the expression of the rearranged Tcra locus, and remains inhibited in αβ T lymphocytes. These results imply the existence of an Eα-independent mechanism to activate transcription of the rearranged Tcra locus in αβ T lymphocytes. Interestingly, Eα is essential for the normal expression of the rearranged Tcrd locus in γδ T lymphocytes. In this review, the current knowledge about the regulation of Tcra/Tcrd germline transcription and gene segment rearrangement during thymocyte development and the possible mechanisms for transcription of the rearranged Tcra locus in mature αβ T lymphocytes are discussed. The knowledge of the detailed mechanisms involved in the regulation of transcription at the Tcra/Tcrd locus by distant enhancers is important to understand the cases in which deregulation this process resulta in disease.

Human regulatory T-cell development is dictated by Interleukin-2 and -15 expressed in a non-overlapping pattern in the thymus

Thymus-derived FOXP3-expressing regulatory T-cells (tTregs) are master orchestrators of physiological and pathological immune responses, thus constituting ideal targets for the treatment of autoimmunity. Despite their clinical importance, the developmental program governing their differentiation in the human thymus remains poorly understood.Here, we investigated the role of common gamma-chain cytokines in human tTreg differentiation, by performing gain- and loss-of-function experiments in 3D and 2D postnatal thymic cultures. We identified IL-2 and IL-15 as key molecular determinants in this process and excluded a major function for IL-4, IL-7 and IL-21. Mechanistically, IL-2 and IL-15 were equally able to drive tTreg precursor differentiation into FOXP3+ cells, and promote tTreg proliferation and survival. Both cytokines also increased the expression levels of molecules associated with effector function within FOXP3+ subsets, supporting their involvement in tTreg functional maturation. Furthermore, we revealed that IL-2 and IL-15 are expressed in a non-overlapping pattern in the human thymus, with the former produced mainly by mature αβ and γδ thymocytes and the latter by monocyte/macrophages and B lymphocytes.Our results identify core mechanisms dictating human tTreg development, with IL-2 and IL-15 defining specific niches required for tTreg lineage stabilization and differentiation, with implications for their therapeutic targeting in autoimmune conditions.

Distinct functional programs in fetal T and myeloid lineages.

Pregnancy poses a challenge to normal mechanisms of immune recognition and rejection: both the mother and her fetus are exposed to allogeneic cells from one to the other. In the case of the mother, these cells are fetal cells carrying paternal antigens; in the case of the fetus, they are maternal cells expressing non-inherited maternal alloantigens (1, 2). Since adaptive immune recognition of these alloantigens could result in mutual rejection and an end to the pregnancy, there are extensive mechanisms in place to inhibit such responses, including poor antigen presentation (3), non-canonical MHC expression, and unique placental and decidual immunomodulatory cell populations (4). The reader is referred to several excellent reviews on this subject (4–7). Given the inherent difficulties attending experiments in humans, studies of the fetal–maternal interface have focused primarily on inbred strains of laboratory mice. There are, however, major differences between the biology of immune system development of such mice and that found in humans, making it challenging to relate findings in one species to the other. In mice, by example, mature αβ T cells colonize peripheral lymphoid organs during very late gestation and do not fully populate the periphery until after birth (8). By contrast, mature αβ T cells can be found in the periphery of the human fetus as early as 10–12 gestational weeks (5, 9). Thus, early hypotheses posited that in utero tolerance was maintained by a passive or inert fetal immune system (similar to that found in the mouse) (Figure ​(Figure1A).1A). However, current research suggests that there exist distinct fetal programs both in the T and myeloid compartments that contribute to the unique environment in utero, both in mice and in humans (Figures ​(Figures11B,C). Figure 1 Models of human immune development. Throughout different stages of development, fetal T and myeloid cells, as compared to their adult counterparts, specifically populate a subset of tissues including the epithelium (DETC, non-DETC cells, and Langerhans ... Fetal T Cell Development and Function Early work in quail chick embryos demonstrated that thymic T cell development occurs in sequential waves, each of which can be identified by differential stem cell colonization of thymic tissue and by unique TCRs (10). These waves appear to be developmentally regulated as they wax and wane according to embryonic gestational age (10), and further work in mice has identified discrete TCR (γδ) utilization during fetal and neonatal development as compared to the adult TCR (αβ) (11–15). Fetal-derived γδ T cells have limited TCR diversity, suggesting a distinct and limited antigen recognition repertoire (12). Furthermore, these cells appear to localize to specific tissues, including the epithelium (16) and the intestine (17). This localization and restricted TCR repertoire suggest that these fetal-derived cells may play a unique role in barrier sites and, as they are developmentally restricted, may be important for promoting tolerance to skin and gut microbiota in early life. Because of their distinct TCR repertoire and anatomical location, multiple fetal-derived functional populations have been characterized in mice, including dendritic epidermal T cells (DETCs) and non-DETC γδ T cell populations found in the dermis (18, 19). DETCs are the first T cells and seed the epidermis early in development (20). These cells have been implicated in the inhibition of inflammatory skin conditions (21), protection against cutaneous malignancies (22, 23), and wound repair (24, 25). Non-DETC γδ T cell populations have been shown to be the primary producers of IL-17 (18, 19) in the skin and may play a role in response to infection. These functions may be indicative of a fetal-specific program, ontologically geared toward appropriate development and maintenance of in utero tolerance. Work in humans has demonstrated that while fetal T cells are capable of recognizing and responding to alloantigen in utero (1), these cells preferentially differentiate into T regulatory (Treg) cells, capable of suppressing immune responses (1, 26). Furthermore, these studies show that the fetal T cells are derived from a fetal hematopoietic stem/progenitor cell (HSPC) in the fetal liver and fetal bone marrow, which gives rise to downstream progeny that are distinct from those generated by adult bone marrow-resident HSPC. Taken together, these data suggest that there are developmentally restricted windows of T cell development in which fetal T cells, functionally distinct from their adult counterparts, arise from discrete HSPC, and seed specific anatomical locations (Figure ​(Figure11B,C).

Ribosomal protein L22 (Rpl22) controls the development of αβ lineage T cells by regulating ER stress pathways (HEM4P.239)

Abstract While mature αβ and γδ T cells arise from a common CD4-CD8- thymocyte progenitor, the pathways controlling separation of these lineages remain poorly understood. We have identified the ribosomal protein Rpl22, a novel effector that plays a crucial role in αβ T cell development, but is dispensable for γδ T cell development. Indeed, Rpl22-deficiency selectively blocks the development of αβ T cells at the β-selection checkpoint in a p53-dependent manner. Here, we investigate the basis for p53 induction caused by Rpl22-deficiency and its restriction to αβ T cell progenitors. We found that Rpl22 can directly bind and suppress the translation of p53 mRNA, and that the absence of Rpl22 induces p53 through translational de-repression. Rpl22 appears to specifically regulate p53 in αβ T cell progenitors by controlling the activity of signaling processes that induce p53, such as endoplasmic reticulum (ER) stress. αβ T cell development is dependent on the activation of ER stress responses, which are known to induce p53. Pharmacologic exacerbation of ER stress blocks development of αβ, but not γδ T cells. Moreover, Rpl22-inactivation exacerbates signals from all three known ER stress sensors (PERK, Ire1α, and ATF6). Finally, inhibiting the ER stress response mediated by PERK partially restores development of Rpl22-deficient αβ progenitors. Together, our data suggest that Rpl22 regulates αβ T cell development by controlling the physiologic ER stress upon which their development depends.

Significant involvement of nuclear factor‐κB‐inducing kinase in proper differentiation of αβ and γδ T cells

Nuclear factor-κB-inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune-compromised and autoimmune-prone. To investigate the role of NIK in αβ T-cell repertoire formation, we analysed T-cell development in aly/aly mice bearing a transgenic T-cell receptor (TCR). Although there were no apparent abnormalities in the mature αβ T cells of non-transgenic aly/aly mice, the maturation efficiency of idiotype(high+) T cells in the TCR-transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αβ T-cell repertoire could be altered by the absence of functional NIK. In one strain of TCR-transgenic aly/aly mice with a negatively selecting H-2 background, the proportion of CD8(low+) idiotype(high+) cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non-transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one-half to one-fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αβ T cells and that NIK in non-haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.

From pre-DP, post-DP, SP4, and SP8 Thymocyte Cell Counts to a Dynamical Model of Cortical and Medullary Selection

Cells of the mature αβ T cell repertoire arise from the development in the thymus of bone marrow precursors (thymocytes). αβ T cell maturation is characterized by the expression of thousands of copies of identical αβ T cell receptors and the CD4 and/or CD8 co-receptors on the surface of thymocytes. The maturation stages of a thymocyte are: (1) double negative (DN) (TCR−, CD4− and CD8−), (2) double positive (DP) (TCR+, CD4+ and CD8+), and (3) single positive (SP) (TCR+, CD4+ or CD8+). Thymic antigen presenting cells provide the appropriate micro-architecture for the maturation of thymocytes, which “sense” the signaling environment via their randomly generated TCRs. Thymic development is characterized by (i) an extremely low success rate, and (ii) the selection of a functional and self-tolerant T cell repertoire. In this paper, we combine recent experimental data and mathematical modeling to study the selection events that take place in the thymus after the DN stage. The stable steady state of the model for the pre-DP, post-DP, and SP populations is identified with the experimentally measured cell counts from 5.5- to 17-week-old mice. We make use of residence times in the cortex and the medulla for the different populations, as well as recently reported asymmetric death rates for CD4 and CD8 SP thymocytes. We estimate that 65.8% of pre-DP thymocytes undergo death by neglect. In the post-DP compartment, 91.7% undergo death by negative selection, 4.7% become CD4 SP, and 3.6% become CD8 SP. Death by negative selection in the medulla removes 8.6% of CD4 SP and 32.1% of CD8 SP thymocytes. Approximately 46.3% of CD4 SP and 27% of CD8 SP thymocytes divide before dying or exiting the thymus.

Subcutaneous panniculitis‐like T‐cell lymphoma in the pediatric age group: A lymphoma of low malignant potential

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare neoplasm of mature αβ cytotoxic T-cells. Most commonly occurring in young adults, few reports are described in children. A separate analysis of a significant cohort of pediatric patients has not previously been performed. We analyzed the pathology including molecular results as well as available clinical data from 16 pediatric patients (age 5 months to 21 years) who had a total of 19 biopsies submitted to the National Cancer Institute from 1999 to 2011. This included 6 males and 10 females. Most patients (10/16, 62.5%) had multiple skin lesions at the time of biopsy. Histologic features included rimming of adipocytes by atypical lymphocytes, fat necrosis, and karyorrhectic debris. Four biopsies showed only partial involvement by lymphoma; and plasma cells were identified in 14/19 (74%) cases, including three in which they were focally prominent. The neoplastic cells in general were positive for CD3, CD8, TIA-1, and βF1 and were negative for CD4 and CD56. CD5 expression was weak to negative in 5/8 cases (63%). A clonal T-cell receptor gene rearrangement was demonstrated in 11/17 (65%). Patients were treated with a variety of agents. While 5/9 (56%) patients had evidence of recurrent skin lesions, no deaths were attributed to disease for the seven patients with follow-up information. Pediatric SPTCL shares many clinical and pathologic features with adult SPTCL. The presence of partial involvement or admixed plasma cells makes the differential diagnosis with reactive conditions challenging in some cases.

Abstract PR12: Early TCRα expression during T cell development for the generation of enhanced-affinity TCRs for TCR gene therapy.

Abstract Thymic selection eliminates T cells expressing TCRs with a high affinity for self-antigen. However, many of the most promising tumor antigens for T cell-based cancer immunotherapies are unmodified self-antigens. Therefore, tumor-specific T cells often have a low avidity for tumor antigens, thus limiting the efficacy of this approach. TCR gene therapy using TCR genes that have been modified for increased affinity can overcome this limitation. The generation of enhanced affinity TCRs generally involves saturation mutagenesis targeting the complementarity determining regions of the TCR genes to be modified. However, this approach requires considerable trial and error, and is still not always successful at generating a higher affinity TCR without promiscuous off-target activity. We have developed a system for selecting high affinity tumor antigen-specific TCRs from a population of T cells selected by agonist signaling during T cell development in vitro. The αβ/γδ lineage choice is in part dictated by the strength of TCR/pre-TCR signaling during early T cell development, such that the pre-TCR provides a weak signal that drives αβ T cell development while the mature γδ TCR provides a stronger signal that drives γδ T cell development. In TCR transgenic mice, early expression of the transgenic TCRα chain prior to β-selection can also provide a stronger γδ-like signal through the mature αβ TCR, leading to the development of lineage redirected TCRαβ+ CD4/CD8 double negative (DN) γδ wanna-be” cells. The development of these DN TCRαβ+ cells is enhanced in the presence of cognate antigen or TCR crosslinking; suggesting that early expression of an antigen-specific TCRα chain before β-selection could promote the generation of DN TCRαβ+ cells in the presence of antigen, through a process in which the introduced TCRα chain pairs with an endogenous TCRβ chain to produce an αβ TCR with high affinity for the target antigen. To test this hypothesis, the TCRα chain from a tumor antigen-specific TCR was transduced into hematopoietic progenitor cells and a library of TCRβ chains was isolated from the lineage redirected DN TCRαβ+ T cells that developed. Several TCRβchains of varying CDR3 lengths were isolated that confer enhanced affinity for the target antigen when paired with the introduced TCRα chain. This abstract is also presented as Poster A13. Citation Format: Thomas M. Schmitt, Philip D. Greenberg, David H. Aggen, David Kranz, Sebastian Ochsenreither. Early TCRα expression during T cell development for the generation of enhanced-affinity TCRs for TCR gene therapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR12.

Stress-induced facilitation of host response to bacterial challenge in F344 rats is dependent on extracellular heat shock protein 72 and independent of alpha beta T cells

Activation of the in vivo stress response can facilitate antibacterial host defenses. One possible mechanism for this effect is stress-induced release of heat shock protein 72 (Hsp72) into the extracellular environment. Hsp72 is a ubiquitous cellular protein that is up-regulated in response to cellular stress, and modulates various aspects of immune function including macrophage inflammatory/bactericidal responses and T-cell function when found in the extracellular environment. The current study tested the hypothesis that in vivo extracellular Hsp72 (eHsp72) at the site of inflammation contributes to stress-induced restricted development of bacteria, and facilitated recovery from bacteria-induced inflammation, and that this effect is independent of alpha beta (αβ) T cells. Male F344 rats were exposed to either inescapable electrical tail-shocks or no stress, and subcutaneously injected with Escherichia coli (ATCC 15746). The role of eHsp72 was investigated by Hsp72-immunoneutralization at the inflammatory site. The potential contribution of T cells was examined by testing male athymic (rnu/rnu) nude rats lacking mature αβ T cells and heterozygous thymic intact control (rnu/+) rats. The results were that stressor exposure increased plasma concentrations of eHsp72 and facilitated recovery from bacterial inflammation. Immunoneutralization of eHsp72 at the inflammatory site attenuated this effect. Stressor exposure impacted bacterial inflammation and eHsp72 equally in both athymic and intact control rats. These results support the hypothesis that eHsp72 at the site of inflammation, and not αβ T cells, contributes to the effect of stressor exposure on subcutaneous bacterial inflammation.

The Transcription Factor PU.1 Regulates γδ T Cell Homeostasis

BackgroundT cell development results in the generation of both mature αβ and γδ T cells. While αβ T cells predominate in secondary lymphoid organs, γδ T cells are more abundant in mucosal tissues. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi1) is essential for early stages of T cell development, but is down regulated during the DN T-cell stage.Methodology/Principal FindingsIn this study, we show that in mice specifically lacking PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1 lck−/−) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild-type mice. The increase in γδ T cell numbers in PU.1-deficient mice is consistent in γδ T cell subsets identified by TCR variable regions. PU.1-deficient γδ T cells demonstrate greater proliferation in vivo and in vitro.Conclusions/SignificanceThe increase of γδ T cell numbers in Lck-Cre deleter strains, where deletion occurs after PU.1 expression is diminished, as well as the observation that PU.1-deficient γδ T cells have greater proliferative responses than wild type cells, suggests that PU.1 effects are not developmental but rather at the level of homeostasis. Thus, our data shows that PU.1 has a negative influence on γδ T cell expansion.

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature αβ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu(159) and Gly(160), and detected the corresponding 10-aa spacer (160)GDPPDLADQG(169) of CA mutants. The site of the second cleavage depended on Glu(159): moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a first-order, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser(170) of the β-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intra-molecular process performed by an N-terminal nucleophile, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between Glu(159) and Ser(170).

Cytokines and culture medium have a major impact on human in vitro T-cell differentiation

An important proof of principle has been achieved with the development of an in vitro T-cell differentiation assay based on the coculture of hematopoietic progenitors with the OP9-Delta1 stromal cell line. The original murine T cell differentiation assay has since been adapted for human T-cell differentiation, however with lower efficiency. The choice of both medium and cytokines is crucial in this assay, therefore our work has been focused on these two factors. The use of freshly reconstituted medium, the optimization of interleukine-7 (IL-7) concentration, and the addition of stem cell factor (SCF) have allowed to improve the proliferation of progenitors and T-cell precursors as well as the yield of double positive CD4 + CD8+ T cells, and mature γδ and αβ T cells. These optimizations make the OP9-Delta1 system sensitive enough to perform both quantitative and qualitative assays with various type of progenitors, including those transduced by a retroviral vector. The improved OP9-Delta1 assay therefore constitutes an extremely useful test for basic research purposes and for translational medicine.