Retrogenic Mice Research Articles

Direct recognition of an intact foreign protein by an αβ T cell receptor

αβ T cell receptors (αβTCRs) co-recognise antigens when bound to Major Histocompatibility Complex (MHC) or MHC class I-like molecules. Additionally, some αβTCRs can bind non-MHC molecules, but how much intact antigen reactivities are achieved remains unknown. Here, we identify an αβ T cell clone that directly recognises the intact foreign protein, R-phycoerythrin (PE), a multimeric (αβ)6γ protein complex. This direct αβTCR–PE interaction occurs in an MHC-independent manner, yet triggers T cell activation and bound PE with an affinity comparable to αβTCR–peptide–MHC interactions. The crystal structure reveals how six αβTCR molecules simultaneously engage the PE hexamer, mediated by the complementarity-determining regions (CDRs) of the αβTCR. Here, the αβTCR mainly binds to two α-helices of the globin fold in the PE α-subunit, which is analogous to the antigen-binding platform of the MHC molecule. Using retrogenic mice expressing this TCR, we show that it supports intrathymic T cell development, maturation, and exit into the periphery as mature CD4/CD8 double negative (DN) T cells with TCR-mediated functional capacity. Accordingly, we show how an αβTCR can recognise an intact foreign protein in an antibody-like manner.

Machine learning analysis of the T cell receptor repertoire identifies sequence features of self-reactivity

The Tcell receptor (TCR) determines specificity and affinity for both foreign and self-peptides presented by the major histocompatibility complex (MHC). Although the strength of TCR interactions with self-pMHC impacts Tcell function, it has been challenging to identify TCR sequence features that predict Tcell fate. To discern patterns distinguishing TCRs from naive CD4+ Tcells with low versus high self-reactivity, we used data from 42 mice to train a machine learning (ML) algorithm that identifies population-level differences between TCRβ sequence sets. This approach revealed that weakly self-reactive Tcell populations were enriched for longer CDR3β regions and acidic amino acids. We tested our ML predictions of self-reactivity using retrogenic mice with fixed TCRβ sequences. Extrapolating our analyses to independent datasets, we predicted high self-reactivity for regulatory Tcells and slightly reduced self-reactivity for Tcells responding to chronic infections. Our analyses suggest a potential trade-off between TCR repertoire diversity and self-reactivity. A record of this paper's transparent peer review process is included in the supplemental information.

Homeostatic intestinal immunity is dominated by microbiota-independent cytotoxic Th1 cells

Abstract Barrier tissues such as the skin and gut form the first line of defense against pathogens and establish an immune dialogue with the resident microbiota. To that end, they harbor immune populations geared towards barrier function, tolerance, and repair. Of particular interest are the large numbers of T cells. Some are specific towards commensal microbes or recognize previously encountered pathogens. However, the origin, antigen specificity, and function of most barrier tissue-resident T cells remains elusive. We have identified a dominant CD4 T cell population in the small intestine lamina propria characterized by the expression of T-bet. Surprisingly, this population is present in both germ-free and conventionally reared (SPF) mice at homeostasis, and accounts for almost half of T cells at this site. Bulk and single-cell transcriptome analysis revealed that this population has cytotoxic potential. Furthermore, single-cell TCR sequencing showed parallel T cell repertoires in germ-free and SPF mice, suggesting shared antigen specificity. To explore the ontogeny of this population, we have identified clonally expanded TCRs and are generating low frequency retrogenic mice. Additionally, we have generated several T cell hybridomas from small intestine Th1 cells. Interestingly, preliminary results suggest that small intestinal Th1 TCRs exhibit some degree of self-reactivity, which may determine their development and differentiation in a foreign antigen-independent manner. Collectively, these studies will shed light on the function of a dominant intestinal T cell population and reveal novel insights into homeostatic tissue immune networks. This research was supported by the Intramural Research Program of NIAID, NIH.

Generation of Retrogenic Mice to Investigate T Cell Development.

T cells develop in the thymus from bone marrow precursors, and genetic manipulation is an indispensable tool to explore their development in vivo. Retroviral transduction of T cell precursors in the bone marrow can be used to specifically eliminate or enforce gene expression. Here, we describe a fast and efficient method to ectopically express a gene in T cell precursors through retroviral transduction and transplant into recipient mice, which will enable laboratories to evaluate gene function in T cell development in vivo.

The TCR Cα Domain Regulates Responses to Self-pMHC Class II.

T cells play a central role in adaptive immunity by recognizing peptide Ags presented by MHC molecules (pMHC) via their clonotypic TCRs. αβTCRs are heterodimers, consisting of TCRα and TCRβ subunits that are composed of variable (Vα, Vβ) and constant (Cα, Cβ) domains. Whereas the Vα, Vβ, and Cβ domains adopt typical Ig folds in the extracellular space, the Cα domain lacks a top β sheet and instead has two loosely associated top strands (C- and F-strands) on its surface. Previous results suggest that this unique Ig-like fold mediates homotypic TCR interactions and influences signaling in vitro. To better understand why evolution has selected this unique structure, we asked, what is the fitness cost for development and function of mouse CD4+ T cells bearing a mutation in the Cα C-strand? In both TCR retrogenic and transgenic mice we observed increased single-positive thymocytes bearing mutant TCRs compared with those expressing wild-type TCRs. Furthermore, our analysis of mutant TCR transgenic mice revealed an increase in naive CD4+ T cells experiencing strong tonic TCR signals, increased homeostatic survival, and increased recruitment of responders to cognate pMHC class II upon immunization compared with the wild-type. The mutation did not, however, overtly impact CD4+ T cell proliferation or differentiation after immunization. We interpret these data as evidence that the unique Cα domain has evolved to fine-tune TCR signaling, particularly in response to weak interactions with self-pMHC class II.

Signatures of recent activation identify a circulating T cell compartment containing tumor-specific antigen receptors with high avidity.

T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen-specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma-combined with transgenic reexpression of identified TCRs by CRISPR-Cas9-mediated orthotopic TCR replacement-revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.

065 Longitudinal analysis of T cell dynamics in alopecia areata at single-cell resolution

Alopecia areata (AA) is defined by autoreactive T cells that attack hair follicles and lead to non-scarring hair loss. We previously demonstrated that AA is driven by cytotoxic CD8+ T cells that undergo clonal expansion. However, the relationship between CD8+ T cell pathogenicity and clonality in AA, as well as their temporal changes throughout disease course, remain poorly understood. Here, we conducted a time-course study in which we performed concomitant single-cell RNA and T cell receptor (TCR) sequencing analysis of T cells in both the skin and skin-draining lymph nodes (SDLNs) prior to, during, and after AA onset in the graft-induced C3H/HeJ mouse model. We observed a striking increase in CD8+ T cell clonality in both the skin and SDLNs during AA onset, with little to no clonal expansion in other T cell subsets. Clonality increased throughout AA progression, but later decreased in chronic AA, when the mice exhibited total body hair loss. Although we identified shared TCR sequences between AA skin and SDLNs, the degree of T cell clonality was increased in the skin, suggesting a two-step mechanism in which activated CD8+ T cells undergo an initial round of expansion in the SDLNs, followed by subsequent round(s) upon antigen exposure in the skin. We functionally investigated the causal role of specific TCR αβ pairs by generating retrogenic TCR mice expressing an expanded TCR sequence identified in our single-cell studies. Mice expressing expanded TCRs developed AA-like hair loss, whereas control mice expressing unexpanded TCR sequences did not. Our results demonstrate that individual expanded CD8+ T cell clones are sufficient to cause disease induction. These findings have broad implications for the development of novel therapeutics in AA aimed at the depletion of expanded pathogenic T cells, as well as clinical immunomonitoring in AA patients by following the dynamics of T cell clonality.

Thymic Interferon Impacts T cell Selection

Abstract Autoimmune diseases are triggered by the loss of immunological tolerance to self-antigens. Although previous work has characterized mechanisms driving immune tolerance to tissue restricted self-antigens, less is understood about how tolerance is established for inflammation-associated self-antigens that are expressed transiently during acute infection. This is particularly important with the discovery that autoantibodies to type I interferons (IFN) are commonly found in elderly individuals, and predispose to severe COVID19. Recent work from several groups has found that Type I and Type III IFN are expressed in the thymus at steady state (in the absence of infection) and drive expression of interferon stimulated genes in antigen presenting cells and developing thymocytes. We hypothesize that IFN is present in the thymus in order to tolerize developing T cells to self-antigens presented during acute infection (such as those derived from interferon and interferon stimulated genes.) In line with this, T cell receptor (TCR) repertoire analysis showed that mice lacking the type I IFN receptor (Ifnar KO) have distinct T conventional (Tconv) and T regulatory (Treg) TCR repertoires from wild type (WT) mice. We have identified two groups of interesting receptors from this TCR repertoire analysis; 1) IFN-deleted receptors(~50 TCRs) that are present in the Ifnar KO Tconv repertoire and absent from the WT repertoire, and 2) IFN-dependent Treg receptors(~750 TCRs) that are present in the WT Treg repertoire but lost from the Ifnar KO Treg repertoire. We have expressed TCRs from each group in hybridomas and retrogenic mice in order to determine the specificity of these TCRs and characterize the development of thymocytes expressing them. Supported by grants from NIH (T32 AI007313)

Combination Therapy of Allogeneic HSCT and In Vivo Activated iNKT Cells Prevents Acute Gvhd While Augmenting GVL Effect

Adult acute lymphoblastic leukemia (ALL) is a highly aggressive cancer with poor clinical prognosis and high relapse rate. Whereas allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for highly differentiated hematologic malignancies, allo-HSCT provides only limited benefits for treating ALL with relapses frequent. Additionally, the development of graft-versus-host-disease (GVHD) remains a major concern for allo-HSCT recipients. Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that express both a semi-invariant T cell receptor and natural killer cell markers. They recognize lipid antigens presented by non-polymorphic CD1d and have been reported to directly target CD1d+ solid tumors. Many B-ALL, including the P210 B-ALL cell line, are also CD1d+. Clinically, increased iNKT cells have simultaneously been correlated with reduced risk of developing acute GVHD and lower rates of malignant relapse. Because iNKT cells comprise only a tiny fraction of total T cells, we established a protocol to insert an iNKT TCR into murine HSC, thereby generating retrogenic mice with 5-10x expansion of iNKT cells in spleen and liver. Although engineered T cell therapy, including CD19-CART therapy, has proven to be effective in inducing remission in many B-ALL patients, when used as a monotherapy, relapse remains a major obstacle. We therefore tested whether a combination of allo-HSCT and iNKT cell therapy could robustly prevent GVHD while preserving graft-versus-leukemia effects (GVL). To test whether the addition of iNKT cells into allo-graft was capable of preventing acute GVHD (aGVHD), lethally irradiated BALB/C were given B6 bone marrow (2.5 x 106) and whole splenocytes (2.5 x 106, 5 x 106, or 10 x 106) from either B6 or retrogenic B6-iNKT mice. Mice that received 5 x 106 B6 splenocytes developed lethal aGVHD and became moribund by Day 10. Half of the mice that received 2.5 x 106 B6 splenocytes develop lethal aGVHD and died by Day 20. In contrast, only half of the mice that received 5 x 106 or 10 x 106 B6-iNKT splenocytes developed lethal aGVHD by Day 20, and all recipients of 2.5 x 106 B6 splenocytes remained alive by Day 20, suggesting the addition of iNKT cells into allograft may reduce the severity of aGVHD. To test whether adding iNKT cells to allograft could enhance GVL without causing GVHD, BALB/C mice with pre-established P210 B-ALL leukemia were given allo-HSCT with or without iNKT cell addition. Initially, our data showed freshly isolated iNKT cells were rapidly cleared following transplantation. We therefore included lethally irradiated P210 loaded with α-Galactosylceramide (α-Galcer) as adjuvant, and were able to see robust expansion of iNKT cells in vitro and in vivo (>20% vs <1% in WT in BM and Liver). Additionally, we found that HSCT recipients that also received iNKT cells were able to fully clear bone marrow residing B-ALL cells by Day8, compared to less than half in control, suggesting an important role of iNKT cells for targeting bone marrow residing B-ALL cells to prevent relapse. Taken together, combination therapy of allogeneic HSCT with infusion of donor-type iNKT cells activated via irradiated α-Galcer-loaded host-type ALL represent a novel approach for preventing GVHD and augmenting GVL effect against ALL. Disclosures No relevant conflicts of interest to declare.

Generation of T Cell Receptor Retrogenic Mice.

The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus-mediated stem cell-based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor. After successful transduction, bone marrow is injected into recipient mice, in which T cell development is driven by expression of the vector-encoded TCR. This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followed in most laboratories with standard biomedical equipment, and is supported by a troubleshooting guide that covers potential pitfalls and unexpected results. © 2019 by John Wiley & Sons, Inc.

Identification of specific T lymphocytes against to an IFNg inducible lysosomal thiol reductase (GILT)-dependent, class II MHC-associated epitope in GILT−/− mice

Abstract IFNγ inducible lysosomal thiol reductase (GILT) is the only thiol reductase localized in lysosomes and late endosomes, with its optimal activity at acidic pH. GILT is constitutively expressed in professional APC, further upregulated by IFNγ, and participates in Ag processing by catalyzing disulfide (S-S) bond reduction in proteins thus facilitating unfolding of native S-S bond containing Ag for further processing. It was reported that GILT−/− mice mounted significantly reduced T cell proliferative responses to S-S bond-containing protein Ags in vitro, but responded normally to a non-S-S bond containing protein. In addition, GILT−/− APC showed a selective deficiency in presenting some cysteine-containing epitopes within a model protein Ag, hen egg white lysozyme. Using HPLC-MS/MS, we first identified one group of unique GILT-dependent, class II MHC-associated peptides from B6 mice, but not from GILT−/− mice. Binding of these GILT dependent, class II MHC-associated peptides to I-Ab proteins was confirmed in vitro. Furthermore, T cells specific to one of the unique GILT-dependent peptides, BP7, was detected in GILT−/− mice, but not found in B6 mice. BP7 peptide specific T cell hybridoma clones were established as well. Taken together, our results indicate that GILT−/− mice have a deficiency in generation of certain T cell epitopes due to loss of GILT activity. Missing of GILT dependent epitopes might lead to survival of the T cells specific to these GILT-dependent epitopes (escaping from the negative selection) in thymus of GILT−/− mice. We are in progress of cloning the BP7 specific TCRs from T cell hybridoma clones. The potential role of the BP7 specific T lymphocytes in autoimmunity will be examined in BP7 specific T cell retrogenic mice.

TCR Retrogenic Mice as a Model To Map Self-Tolerance Mechanisms to the Cancer Mucosa Antigen GUCY2C.

Characterizing self-tolerance mechanisms and their failure is critical to understand immune homeostasis, cancer immunity, and autoimmunity. However, examination of self-tolerance mechanisms has relied primarily on transgenic mice expressing TCRs targeting well-characterized, but nonphysiologic, model Ags, such as OVA and hemagglutinin. Identifying TCRs directed against bona fide self-antigens is made difficult by the extraordinary diversity of TCRs and the low prevalence of Ag-specific clones (<10-100 naive cells per organism), limiting dissection of tolerance mechanisms restricting immunity to self-proteins. In this study, we isolated and characterized TCRs recognizing the intestinal epithelial cell receptor and colorectal cancer Ag GUCY2C to establish a model to study self-antigen-specific tolerance mechanisms. GUCY2C-specific CD4+ effector T cells were isolated from immunized, nontolerant Gucy2c -/- mice. Next-generation sequencing identified GUCY2C-specific TCRs, which were engineered into CD4+ T cells in vitro to confirm TCR recognition of GUCY2C. Further, the generation of "retrogenic" mice by reconstitution with TCR-transduced hematopoietic stem cells resulted in normal CD4+ T cell development, responsiveness to immunization, and GUCY2C-induced tolerance in recipient mice, recapitulating observations in conventional models. This retrogenic model can be employed to define self-tolerance mechanisms restricting T and B cell responses to GUCY2C to optimize colorectal cancer immunotherapy without autoimmunity.

Abstract A039: FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1

Abstract The outcome of cancer patients has recently been improved by utilizing novel immunotherapies that inhibit the regulatory immune system. Regulatory T-cells (Treg) contribute not only to suppression of excessive immune responses in the steady state but also to reduction of antitumor immune responses in cancer patients. We have shown that down-regulation of the Helios transcription factor undermines CD4 Treg stability inducing these cells to convert into effector cells that enhance the antitumor immune response. Helios is also the canonical transcription factor of CD8 Treg and essential for CD8 Treg function. CD8 Treg are Qa-1 (HLA-E in human)–restricted CD8 T-cells that mainly regulate Qa-1+ immune cells. The peptide FAM49B(p190-198) is a Qa-1 binding peptide derived from ubiquitously expressed FAM49B. Although FAM49B(p190-198) is regularly degenerated in the endoplasmic reticulum normal cells, we found that FAM49B(p190-198) is stably presented in some tumor cells. Here, we investigate the potential contribution of FAM49B(p190-198)/Qa-1 specific CD8 Treg to cancer.FAM49B(p190-198)/Qa-1 specific CD8 Treg were detected by FAM49B(p190-198)/Qa-1 tetramer after immunization with FAM49B(p190-198) peptide–loaded dendritic cells in CD8 Treg (i.e., CD44+CD122+Ly49+ CD8 T-cells). T-cell receptor cDNAs were amplified from the sorted single cells and inserted into retrovirus expression vectors for transduction in 58α–β– hybridoma cells and bone marrow cells. EL4-Qa-1 KO cells and EL4-FAM49B KO cells were generated using CRISPR/Cas9 technology. These tools allowed validation of the specificity of the TCRs and their Qa-1–restriction. For in vivo studies, TCR retrogenic mice were developed by transferring TCR-transduced Rag2–/– bone marrow hematopoietic stem cells into lethally irradiated CD45.1 mice. Retrogenic T-cells were prepared by CD8 enrichment of retrogenic mouse splenocytes and in vitro culture for 3 days in the presence of IL15c. For in vivo studies, we use an adoptive transfer model of retrogenic T-cells for EL4 tumor bearing mice. Rag2–/–γC–/– mice were inoculated with 0.5 x 10^6 EL4 WT-cells and 0.5 x 10^6 EL4 Qa-1 KO cells on each lateral flank, then intravenously injected with retrogenic T-cells (on day 13 and 17). FAM49B(p190-198)/Qa-1 specific CD8 T-cells preferentially use TCR Vα3.2 (TRAV9N-3) and Vβ5.1/5.2 (TRBV12-1/12-2) chains independent of their expression of Ly49+. We tested 17 pairs obtained from Ly49+ cells using the in vitro system and found that 11 pairs bound strongly to Fam49b(p190-198)/Qa-1 tetramer while 8 out of the 11 pairs exhibited antigen-specific activation. Interestingly, one TCR pair (No.8) responded to WT EL4 cells without antigen peptides, which was abolished in coculture wells with Qa-1 KO EL4 cells and FAM49B KO EL4 cells. This TCR hybridoma was also activated by stimulated WT B6 splenocytes but not by Qa-1 KO splenocytes suggesting its regulatory potential. Adoptive transfer of TCR No.8-expressing retrogenic T-cells delayed EL4 WT tumor growth but not EL4 Qa-1 KO tumor growth when inoculated in the same hosts. Tumor-infiltrating CD8 T-cells displayed a more activated phenotype in WT tumors compared to KO tumors, indicating that retrogenic T-cells recognize and target EL4 cells in a Qa-1-dependent manner. Citation Format: Hidetoshi Nakagawa, Hye-Jung Kim, Harvey Cantor. FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A039.

Genetically modified hematopoietic stem/progenitor cells that produce IL-10–secreting regulatory T cells

Random amino acid copolymers used in the treatment of multiple sclerosis in man or experimental autoimmune encephalomyelitis (EAE) in mice [poly(Y,E,A,K)n, known as Copaxone, and poly(Y,F,A,K)n] function at least in part by generation of IL-10-secreting regulatory T cells that mediate bystander immunosuppression. The mechanism through which these copolymers induce Tregs is unknown. To investigate this question, four previously described Vα3.2 Vβ14 T cell receptor (TCR) cDNAs, the dominant clonotype generated in splenocytes after immunization of SJL mice, that differed only in their CDR3 sequences were utilized to generate retrogenic mice. The high-level production of IL-10 as well as IL-5 and small amounts of the related cytokines IL-4 and IL-13 by CD4+ T cells isolated from the splenocytes of these mice strongly suggests that the TCR itself encodes information for specific cytokine secretion. The proliferation and production of IL-10 by these Tregs was costimulated by activation of glucocorticoid-induced TNF receptor (GITR) (expressed at high levels by these cells) through its ligand GITRL. A mechanism for generation of cells with this specificity is proposed. Moreover, retrogenic mice expressing these Tregs were protected from induction of EAE by the appropriate autoantigen.

A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45

The Src Family kinase Lck sets a critical threshold for Tcell activation because it phosphorylates the TCR complex and the Zap70 kinase. How a Tcell controls the abundance of active Lck molecules remains poorly understood. We have identified an unappreciated role for a phosphosite, Y192, within the Lck SH2 domain that profoundly affects the amount of active Lck in cells. Notably, mutation of Y192 blocks critical TCR-proximal signaling events and impairs thymocyte development in retrogenic mice. We determined that these defects are caused by hyperphosphorylation of the inhibitory C-terminal tail of Lck. Our findings reveal that modification of Y192 inhibits the ability of CD45 to associate with Lck in cells and dephosphorylate the C-terminal tail of Lck, which prevents its adoption of an active open conformation. These results suggest a negative feedback loop that responds to signaling events that tune active Lck amounts and TCR sensitivity.

Abstract A074: A Zap70-dependent negative feedback loop governs Lck activation by CD45

Abstract The kinase Lck phosphorylates the T cell antigen receptor (TCR) complex when antigen is encountered and therefore its activity sets a critical threshold for T cell activation. Yet how Lck activity is regulated in resting or stimulated cells remains poorly understood. We have identified that a cryptic phosphosite within Lck, Y192, profoundly affects Lck activity when mutated. Notably, mutation of Y192 blocks critical TCR proximal signaling events including phosphorylation of the effector kinase Zap70 and subsequent calcium response. Accordingly, thymocyte development in retrogenic mice harboring a Y192 mutation is severely impaired. These mice are unable to produce mature T cells and accumulate double negative and double positive (CD4/CD8) thymocytes. We determined that these defects are caused by hyperphosphorylation of the canonical inhibitory phosphorylation site (Y505) within Lck's C-terminus. Lck's C-terminus is phosphorylated by the inhibitory kinase Csk and we have employed a regulatable reconstituted system to acutely inhibit Csk activity. Upon Csk inhibition, the phosphatase CD45 dephosphorylates Lck's C-terminus (Y505). Interestingly, mutation of Y192 disrupts CD45's ability to dephosphorylate Y505 and activate Lck. Because Y192 phosphorylation decreases in response to small molecule inhibition of Zap70, an Lck substrate, our findings support a mechanism of negative feedback. We predict that tonic or basal signaling events that occur in the absence of TCR ligand use negative feedback, including Y192 phosphorylation, to tune Lck activity. Because modulation of Lck activity impacts TCR sensitivity, feedback mechanisms are likely critical to establishing a threshold for T cell activation. Citation Format: Adam H. Courtney, Theresa A. Kadlecek, Byron Au-Yeung, Arthur Weiss. A Zap70-dependent negative feedback loop governs Lck activation by CD45 [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A074.

Rapid identification and expression of human TCRs in retrogenic mice

Single-cell paired TCR identification is a powerful tool, but has been limited in its previous incompatibility with further functional analysis. The current protocol describes a method to clone and functionally evaluate in vivo TCRs derived from single antigen-responsive human T cells and monoclonal T cell lines. We have improved upon current PCR-based TCR sequencing protocols by developing primers that allow amplification of human TCRα and TCRβ variable regions, while incorporating specific restriction cut sites for direct subcloning into the template retroviral vector. This streamlined approach for generating human:mouse chimeric TCR vectors allows for rapid TCR expression in humanized-retrogenic (hu-Rg) mice through retroviral mediated stem cell gene transfer. Using widely available techniques and equipment, this method is easily adaptable by most laboratories. This is the first TCR identification protocol that is efficiently combined with subsequent in vivo TCR expression.

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCRα and TCRβ chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Autoimmune T cells binds to self antigen weakly in the thymus but greatly upregulate the binding strength in the periphery

Abstract How autoimmune TCRs progress through thymocyte selection to egress in the periphery and induce disease is unclear. Autoimmune TCRs are believed to be of low affinity for self antigens due to the process of thymocyte negative selection. Using an experimental autoimmune encephalomyelitis (EAE) mouse model, we have cloned two tetramer positive myelin oligodendrocyte glycoprotein (MOG)35-55-specific TCRs (named B8 and 1C9) from polyclonal CD4+ T cells; both TCRs have a high two dimensional (2D) affinity for MOG (similar to a pathogen-specific TCR SMARTA for an LCMV epitope gp66-77) by the micropipette adhesion frequency assay. A second critical feature of antigen recognition is the bond lifetime between the TCR and pMHC that occurs under applied force. To this end, we have produced retrogenic mice for the two TCRs and used their thymocytes on biomembrane force probe (BFP) to measure single TCR-pMHC bond lifetimes. Despite their high affinity, both TCRs had short bond lifetimes with the MOG antigen displaying a slip bond where bond lifetimes decrease with increasing force. In contrast, the same TCRs on naïve T cells in the periphery displayed much longer bond lifetimes (up to 100 fold) and strong catch bonds (a counterintuitive type of molecular bond that strengthens under force), suggesting physiological regulation of TCR kinetics within single T cell clones. In comparison, the SMARTA TCR formed indistinguishable catch bonds with gp66-77 on thymocytes and naïve T cells. Therefore, these data identify bond lifetime under force as a unique kinetic property of autoimmune TCRs where weak interactions in the thymus potentially avoid negative selection yet in the periphery the TCRs bind with sufficiently long lifetime to promote autoimmunity.