Optimal T-cell Receptor Research Articles

Antitumor progenitor exhausted CD8+ T cells are sustained by TCR engagement.

The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8+ T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure. Here we use a Lewis lung carcinoma model that elicits either optimal or attenuated TCR signaling in CD8+ T cells to show that formation of Tpex in tumor-draining lymph nodes and their intratumoral persistence is dependent on optimal TCR engagement. Notably, attenuated TCR stimulation accelerates the terminal differentiation of optimally primed Tpex. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cells and epigenetic imprinting involving increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, this study highlights the critical function of TCR engagement in sustaining Tpex during tumor progression.

Abstract 578: A functional approach to identifying and engineering TCRs results in highly-potent and specific TCRs for TCR-T cell therapy

Abstract Peptide-HLA (pHLA)- targeting therapeutics, such as T cell receptor-engineered T cells (TCR-T), have had clinical success in treating solid tumors. However, challenges related to safety exist: major concerns remain surrounding the cross-reactivity of T cell receptors (TCRs) as well as the ability of therapeutics to discriminate between on-target vs off-target pHLAs while maintaining high potency. Therefore, approaches that survey the diversity of the T cell repertoire to discover optimal TCRs, as well as platforms to comprehensively identify potential off-target liabilities, are critical to de-risking and accelerating the development of this promising class of pHLA-targeting therapeutics. We have developed a strategy that (1) queries the TCR repertoire to enrich and identify multiple active, sequence-distinct endogenous TCRs; (2) uses 3T-TRACE, a high-diversity pHLA library, to screen for cross-reactivity; and (3) exploits functional selections to simultaneously optimize for TCR potency and specificity. We applied this approach to identify TCRs of optimal specificity and potency targeting a peptide derived from the cancer-testis antigen NY-ESO-1 (SLLMWITQC) displayed by HLA-A2. We profiled 6 sequence-distinct TCRs using 3T-TRACE and validated potential endogenous off-target cross-reactivities. Many of the identified off-targets exhibited little to no sequence homology to the NY-ESO-1 epitope, highlighting the importance of diverse combinatorial libraries in identifying unexpected cross-reactivities. Leveraging the off-target liabilities identified by 3T-TRACE, we designed a functional library and selection scheme that enabled the identification of TCRs with increased potency and specificity. Optimized TCRs exhibited enhanced killing activity and improved safety against an NY-ESO-1-expressing melanoma cell line compared to benchmark TCRs, indicating that this approach has potential to improve clinical safety and efficacy. Using this multi-faceted and comprehensive approach we rapidly identified highly potent and specific TCRs against NY-ESO-1. Identifying cross-reactivities using 3T-TRACE proved to be critical in selecting TCRs suitable for engineering and functional selections. This approach can be extended to any pHLA target to create safe and effective TCR-Ts for clinical development. Citation Format: John D. Leonard, Alejandro Ramirez, Jason Romero, Jake Kleiner, Joanna Dreux, Bindu Hegde, Bryan Xie, Akshay Sharathchandra, Nathan Katz, Venita I. DeAlmeida, Hans-Peter Gerber, Marvin H. Gee, Leah V. Sibener. A functional approach to identifying and engineering TCRs results in highly-potent and specific TCRs for TCR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 578.

Identification of TCR repertoires in asymptomatic COVID-19 patients by single-cell T-cell receptor sequencing

The T cell-mediated immune responses associated with asymptomatic infection (AS) of SARS-CoV-2 remain largely unknown. The diversity of T-cell receptor (TCR) repertoire is essential for generating effective immunity against viral infections in T cell response. Here, we performed the single-cell TCR sequencing of the PBMC samples from five AS subjects, 33 symptomatic COVID-19 patients and eleven healthy controls to investigate the size and the diversity of TCR repertoire. We subsequently analyzed the TCR repertoire diversity, the V and J gene segment deference, and the dominant combination of αβ VJ gene pairing among these three study groups. Notably, we revealed significant TCR preference in the AS group, including the skewed usage of TRAV1-2-J33-TRBV6-4-J2-2 and TRAV1-2-J33-TRBV6-1-J2-3. Our findings may shed new light on understanding the immunopathogenesis of COVID-19 and help identify optimal TCRs for development of novel therapeutic strategies against SARS-CoV-2 infection.

T-cell receptors provide potential prognostic signatures for breast cancer.

Although T-cell receptors (TCRs) are related to the progression of breast cancer (BC), their prognostic values remain unclear. We downloaded the messenger RNA (mRNA) profiles and corresponding clinical information of 1413 BC patients from the Cancer Genome Atlas and Gene Expression Omnibus database, respectively. The different expression analysis of 104 TCRs in BC samples was performed, and the consensus clustering based on 104 TCRs was performed by using the K-mean method of R language. Univariate cox regression analysis was used to screen TCRs significantly associated with the prognosis of BC, and LASSO Cox analysis was applied to optimize key TCRs. The risk score was calculated using the prognostic model constructed based on six optimal TCRs, and multivariate Cox regression analysis was used to determine whether it was an independent prognostic signature. Finally, the nomogram was constructed to predict the overall survival of BC patients. Six optimal TCRs (ZAP70, GRAP2, NFKBIE, IFNG, NFKBIA, and PAK5), which were favorable for the prognosis of BC patients, were screened. Risk score could reliably predict the prognosis of BC patients as an independent prognostic signature. In addition, when bringing into two independent prognostic signatures, age and risk score, the nomogram model could better predict the overall survival of BC patients. Our results suggested that the poor prognosis of BC patients with high risk might be due to an immunosuppressive microenvironment. In summary, a prognostic risk model based on six TCRs was established and could efficiently predict the prognosis of BC patients.

Focused Regulatory T Cell Repertoires Are Indicative of Successful GvHD Control in Experimental and Clinical Stem Cell Transplantation

Acute Graft versus Host Disease (aGvHD) remains a major complication and leading cause of mortality after allogeneic stem cell or bone marrow transplantation (BMT). Current strategies for treatment are still based on unspecific immunosuppressive therapy. Over the last decade, there have been major advances in the field of adoptive immunotherapy using regulatory CD4+CD25+Foxp3+ T cells (Treg cells). Nonetheless, not much is known about the exact mechanisms of Treg-mediated suppression, and even less about the importance of T cell receptor (TCR) specificity and its diversity on the functionality of Tregs.We hypothesized that an optimal Treg TCR repertoire is necessary for successful prevention of aGvHD. To test this hypothesis, we sequenced the TCR repertoire of 8 patients who were diagnosed with aGvHD on day 30 post transplantation and compared it with the TCR repertoire of nine GvHD-free patients. Analysis of GvHD-free patients on day 30 (and 100 days-follow up) revealed a lower TCR diversity when compared to the patients suffering from GvHD. A more detailed analysis of the TCR repertoire showed that in patients without GvHD, fewer clonotypes were needed to comprise 50% of the whole repertoire as compared with samples from patients with GvHD (Figure 1A). Thus, expansion of protective clones indicates their potent immunosuppressive capabilities.Next, we employed a well-described murine model of allogeneic BMT (BL/6–>Balb/c) with co-injection of Tregs. Recipient Balb/c mice transplanted in this fashion were previously shown to be protected from aGvHD. However, the mechanisms involved in this Treg-mediated protection are not fully understood. Therefore, Tregs were FACS sorted from B6.Cg-Foxp3tm1Mal/J mice based on their Foxp3 expression. Recipient mice were transplanted with T-cell depleted bone marrow and a mixture of conventional T cells (Tconv) and Tregs in 1:1 ratio. Transferred Tregs were re-sorted on day 7 and day 14 from secondary lymphoid organs based on the congenic marker Thy 1.1 and Foxp3 expression. Using this model, we investigated the kinetics of the Treg TCR repertoire early after BMT in 5 independent experiments. We found a consistently similar narrowing of the repertoire and clonal expansion in mice protected from GvHD (Figure 1B). Diversity analysis using inverse Simpson Index also confirmed our findings. These data further support the notion that a clonal expansion of Tregs is necessary for an optimal immunosuppression of an allogeneic response, both in human and in mice. To test the functionality and phenotype of such expanded Tregs, they were re-sorted from BMT-recipient mice 14 days after transplantation. These Tregs were expanded using α-CD3 and α-CD28 antibodies and were functionality tested in an in vitro Treg suppression assay. Re-sorted Tregs after expansion showed expression of established Treg surface and intracellular markers such as Helios, CD25, GITR and CTLA-4. For the suppression assay, responder CD4 Tconv were stained with a proliferation tracking dye eFluor670 and stimulated in vitro with CD3 and CD28 beads in the presence of different ratios of re-sorted and expanded, or polyclonaly activated Tregs as the control. Allo-specific ex vivo Tregs exhibited a superior suppressive potential when compared with polyclonaly activated Tregs in vitro.Taken together, our current study highlights the importance of specific Treg driven allo-response in GvHD prevention. Further studies are needed, particularly in larger patient cohorts to confirm these findings. However, we propose that this approach might lead to identification and subsequent use of specific Treg clones with high immunosuppressive capacity for the prevention of aGvHD. [Display omitted] DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:abbvie: Consultancy; BMS: Consultancy; Roche: Consultancy; Amgen: Consultancy.

High-Throughput T Cell Receptor (TR) Repertoire Analysis of Virus-Specific T Cells: Implications for T Cell Immunotherapy and Viral Infection Risk Stratification

▪Viral infections, mainly by cytomegalovirus (CMV), Epstein Barr virus (EBV) and polyomavirus type I (BKV), are major causes of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). As effective immune responses against human viruses rely on an armamentarium of T-cell receptor (TR) repertoire capable of recognizing a broad range of antigenic peptides of those pathogens, reconstitution of antiviral immunity, either by spontaneous generation of endogenous virus-specific T cells (VSTs) or by adoptive immunotherapy with VSTs, plays a critical role to fight infections.We here evaluated the diversity and clonality of TR repertoire of functional tri-virus-specific T cell products generated from immunocompetent donors (n=10) and compared their TR gene repertoire to that of peripheral blood mononuclear cells (PBMCs) from patients who had undergone allo-HSCT (n=5). To generate tri-VSTs, PBMCs derived from 15-20ml of peripheral blood of normal donors, were exposed to EBV, CMV and BKV overlapping peptides and cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors. Specificity of donor-derived VSTs and patient-derived PBMCs was measured by IFN-γElispot. TR diversity was investigated by next-generation sequencing on a MiSeq Sequencer, after amplification of TR beta chain gene rearrangements by RT-PCR with the BIOMED-2 protocol. Raw NGS reads were filtered based on their length and quality and the filtered-in sequences were submitted to IMGT/HighVQUEST. Metadata analysis and clonotype computation were performed using a validated in-house bioinformatics platform. As clonotype we defined sequences carrying the same TRBV gene and identical CDR3 amino acid sequence.Tri-VSTs provided 947,298 productive TRBV-TRBD-TRBJ rearrangements and a polyclonal and highly diverse TR gene repertoire, consisting of a total of 169,502 unique clonotypes (average: 16,950/sample, range 4,057-45,602), 64,971 (38.3%) of which were expanded (corresponding to more than one sequence). In terms of clonality, the mean relative frequency of the major clonotype in all tri-VSTs was 12.6% (range 3.3-29.2%). Interestingly, among tri-VST cell lines, 637 clonotypes were shared (present in >2/10 samples), 80 were highly shared (present in >3/10 samples) while 7 were present in 6-8 different VST lines and largely expanded, accounting for up to 29.2% of all sequences.Importantly, there were 65 of 96 major VST clonotypes shared, thus suggesting that they were potentially associated with recognition of the targeted viruses. Given that 4/10 VSTs cell lines were not specific for CMV, while being EBV-and BKV-specific, dominant TRs in those 4 cell lines can potentially be associated with EBV- or BKV-activity. By searching a public database of TR clonotypes with known reactivity against EBV and/or CMV (ShugayM, Nucleic Acids Research, 2018), we found 8 shared EBV-specific and 4 shared CMV-specific clonotypes among our VSTs and the 499 public clonotypes. When we compared the produced VSTs with PBMCs from 3 allo-grafted patients with circulating CMV-, BKV- and EBV-specific T cells and previous viral reactivation, we detected 163 shared clonotypes. Likewise, we observed 21 and 23 shared clonotypes in similar frequencies, between VSTs and PBMCs from 2 patients with CMV- or BKV-specific T cell immunity. These data identify clones that potentially expand in vivo and protect patients from viral infections.Overall, our findings reveal high levels of TR clonality in cell lines enriched for T cells reactive against EBV and/or CMV and/or BKV and provide insights into the TR repertoire of ex vivo- or endogenously-generated VSTs. Our approach may help to identify optimal TRs for immunotherapy as well as TRs which can be used as a tool for risk stratification of viral infections. DisclosuresAgathangelidis:Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Abbvie: Research Funding; Janssen: Honoraria, Research Funding.

TCR repertoire evolution during maintenance of CMV-specific T-cell populations.

During infections and cancer, the composition of the T-cell receptor (TCR) repertoire of antigen-specific CD8+ T cells changes over time. TCR avidity is thought to be a major driver of this process, thereby interacting with several additional regulators of T-cell responses to form a composite immune response architecture. Infections with latent viruses, such as cytomegalovirus (CMV), can lead to large T-cell responses characterized by an oligoclonal TCR repertoire. Here, we review the current status of experimental studies and theoretical models of TCR repertoire evolution during CMV infection. We will particularly discuss the degree to which this process may be determined through structural TCR avidity. As engineered TCR-redirected T cells have moved into the spotlight for providing more effective immunotherapies, it is essential to understand how the key features of a given TCR influence T-cell expansion and maintenance in settings of infection or malignancy. Deeper insights into these mechanisms will improve our basic understanding of T-cell immunology and help to identify optimal TCRs for immunotherapy.

Fine-Tuning of Optimal TCR Signaling in Tumor-Redirected CD8 T Cells by Distinct TCR Affinity-Mediated Mechanisms.

Redirecting CD8 T cell immunity with self/tumor-specific affinity-matured T cell receptors (TCRs) is a promising approach for clinical adoptive T cell therapy, with the aim to improve treatment efficacy. Despite numerous functional-based studies, little is known about the characteristics of TCR signaling (i.e., intensity, duration, and amplification) and the regulatory mechanisms underlying optimal therapeutic T cell responses. Using a panel of human SUP-T1 and primary CD8 T cells engineered with incremental affinity TCRs against the cancer-testis antigen NY-ESO-1, we found that upon activation, T cells with optimal-affinity TCRs generated intense and sustained proximal (CD3ζ, LCK) signals associated with distal (ERK1/2) amplification-gain and increased function. In contrast, in T cells with very high affinity TCRs, signal initiation was rapid and strong yet only transient, resulting in poor MAPK activation and low proliferation potential even at high antigen stimulation dose. Under resting conditions, the levels of surface TCR/CD3ε, CD8β, and CD28 expression and of CD3ζ phosphorylation were significantly reduced in those hyporesponsive cells, suggesting the presence of TCR affinity-related activation thresholds. We also show that SHP phosphatases were involved along the TCR affinity gradient, but displayed spatially distinct regulatory roles. While PTPN6/SHP-1 phosphatase activity controlled TCR signaling initiation and subsequent amplification by counteracting CD3ζ and ERK1/2 phosphorylation, PTPN11/SHP-2 augmented MAPK activation without affecting proximal TCR signaling. Together, our findings indicate that optimal TCR signaling can be finely tuned by TCR affinity-dependent SHP-1 and SHP-2 activity, and this may readily be determined at the TCR/CD3 complex level. We propose that these TCR affinity-associated regulations represent potential protective mechanisms preventing high affinity TCR-mediated autoimmune diseases.

NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease

AbstractTransfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and β genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR “single editing” (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165–specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.

Optimal T-cell receptor affinity for inducing autoimmunity

T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity.

Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections

Restoration of antigen-specific T cell immunity has the potential to clear persistent viral infection. T cell receptor (TCR) gene therapy can reconstitute CD8 T cell immunity in chronic patients. We cloned 10 virus-specific TCRs targeting 5 different viruses, causing chronic and acute infection. All 10 TCR genetic constructs were optimized for expression using a P2A sequence, codon optimization and the addition of a non-native disulfide bond. However, maximum TCR expression was only achieved after establishing the optimal orientation of the alpha and beta chains in the expression cassette; 9/10 TCRs favored the beta-P2A-alpha orientation over alpha-P2A-beta. Optimal TCR expression was associated with a significant increase in the frequency of IFN-gamma+ T cells. In addition, activating cells for transduction in the presence of Toll-like receptor ligands further enhanced IFN-gamma production. Thus, we have built a virus-specific TCR library that has potential for therapeutic intervention in chronic viral infection or virus-related cancers.

NY‐ESO‐1 antigen‐reactive T cell receptors exhibit diverse therapeutic capability

The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy.

CCR6 is expressed on an IL-10–producing, autoreactive memory T cell population with context-dependent regulatory function

Interleukin (IL)-10 produced by regulatory T cell subsets is important for the prevention of autoimmunity and immunopathology, but little is known about the phenotype and function of IL-10–producing memory T cells. Human CD4+CCR6+ memory T cells contained comparable numbers of IL-17– and IL-10–producing cells, and CCR6 was induced under both Th17-promoting conditions and upon tolerogenic T cell priming with transforming growth factor (TGF)–β. In normal human spleens, the majority of CCR6+ memory T cells were in the close vicinity of CCR6+ myeloid dendritic cells (mDCs), and strikingly, some of them were secreting IL-10 in situ. Furthermore, CCR6+ memory T cells produced suppressive IL-10 but not IL-2 upon stimulation with autologous immature mDCs ex vivo, and secreted IL-10 efficiently in response to suboptimal T cell receptor (TCR) stimulation with anti-CD3 antibodies. However, optimal TCR stimulation of CCR6+ T cells induced expression of IL-2, interferon-γ, CCL20, and CD40L, and autoreactive CCR6+ T cell lines responded to various recall antigens. Notably, we isolated autoreactive CCR6+ T cell clones with context-dependent behavior that produced IL-10 with autologous mDCs alone, but that secreted IL-2 and proliferated upon stimulation with tetanus toxoid. We propose the novel concept that a population of memory T cells, which is fully equipped to participate in secondary immune responses upon recognition of a relevant recall antigen, contributes to the maintenance of tolerance under steady-state conditions.

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

In human gene therapy applications, lentiviral vectors may have advantages over gamma-retroviral vectors in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and a potentially safer integration site profile. However, unlike gamma-retroviral vectors it has been problematic to drive the expression of multiple genes efficiently and coordinately with approaches such as internal ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1 were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG) before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor gene expression. Furthermore, we determined that the furin cleavage site was recognized in lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with an efficiency of 20-30%, which could not be increased by addition of multiple furin cleavage sites. The novel bicistronic lentiviral vector developed herein afforded robust anti-melanoma activities to engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic activity. Such optimal vectors may have immediate applications in cancer gene therapy.

Requirements for Retroviral Targeting of a Suicide Gene to Alloreactive Memory Stem T Cells for Adoptive Immunotherapy of Leukemia.

In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA–CD62L−, CD28±CD27+, IL-2±IFN–γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA–CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generates central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHag-specific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect.

Rip2 Participates in Bcl10 Signaling and T-cell Receptor-mediated NF-κB Activation

Engagement of the T-cell receptor (TCR) initiates a signaling cascade that ultimately results in activation of the transcription factor NF-kappaB, which regulates many T-cell functions including proliferation, differentiation and cytokine production. Herein we demonstrate that Rip2, a caspase recruitment domain (CARD)-containing serine/threonine kinase, plays an important role in this cascade and is required for optimal TCR signaling and NF-kappaB activation. Following TCR engagement, Rip2 associated with Bcl10, a CARD-containing signaling component of the TCR-induced NF-kappaB pathway, and induced its phosphorylation. Rip2-deficient mice were defective in TCR-induced NF-kappaB activation, interleukin-2 production, and proliferation in vitro and exhibited defective T-cell-dependent responses in vivo. The defect in Rip2-/- T-cells correlated with a lack of TCR-induced Bcl10 phosphorylation. Furthermore, deficiency in Bcl10-dependent NF-kappaB activation could be rescued in Rip2-/- embryonic fibroblasts by exogenous wild-type Rip2 but not a kinase-dead mutant. Together these data define an important role for Rip2 in TCR-induced NF-kappaB activation and T-cell function and highlight the significance of post-translational modification of Bcl10 by Rip2 in T-cell signaling.

Molecular and functional bases of self-antigen recognition in long-term persistent melanocyte-specific CD8+ T cells in one vitiligo patient.

Vitiligo patients possess high frequencies of circulating CD8+ T lymphocytes specific for the melanocyte differentiation antigen Melan-A/MART-1. These self-specific T cells exhibit intact functional properties and their T cell receptors are selected for a narrow range of high affinities of antigen recognition, suggesting their important role in the pathogenesis of vitiligo. In order to understand the molecular base for this unexpected, optimal T cell receptor recognition of a self-antigen, a tetramer-guided ex vivo analysis of the T cell receptor repertoire specific for the Melan-A antigen in a patient affected by vitiligo is reported. All T cell receptors sequenced corresponded to different clonotypes, excluding extensive clonal expansions and revealing a large repertoire of circulating Melan-A-specific T lymphocytes. A certain degree of T cell receptor structural conservation was noticed, however, as a single AV segment contributed to the alpha chain rearrangement in 100% of clones and a conserved amino acid sequence was found in the beta chain complementarity determining region 3 of various high affinity cells. We suggest that the conserved alpha chain confers self-antigen recognition, necessary for intrathymic selection and peripheral homeostasis, to many synonymous T cell receptors, whereas the beta chain fine tunes the T cell receptor affinity of the specific cells. In addition, we demonstrate that many high avidity T cell clones from this patient were capable of specifically lysing normal, HLA-matched melanocytes. These autoreactive clones persisted for more than 3 y in the patient's peripheral blood. These data, together with the skin-homing potential of the clones, directly point to the in vivo pathogenic role of melanocyte-specific cytotoxic T lymphocytes in vitiligo.

CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling.

The transmembrane phosphatase CD45 regulates both Lck activity and T cell receptor (TCR) signaling. Here we have tested whether the large ectodomain of CD45 has a role in this regulation. A CD45 chimera containing the large ectodomain of CD43 efficiently rescues TCR signaling in CD45-null T cells, whereas CD45 chimeras containing small ectodomains from other phosphatases do not. Both basal Lck activity in unstimulated cells and the TCR-induced increase in tyrosine phosphorylation of the TCR zeta-chain and in Lck activity depend on the expression of CD45 with a large ectodomain. Unlike CD45 chimeras containing small ectodomains, both the CD45 chimera with a large ectodomain and wild-type CD45 itself are partially localized to glycosphingolipid-enriched membranes (GEMs). Taken together, these data show that the large CD45 ectodomain is required for optimal TCR signaling.

RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems.

The immune system consists of two evolutionarily different but closely related responses, innate immunity and adaptive immunity. Each of these responses has characteristic receptors-Toll-like receptors (TLRs) for innate immunity and antigen-specific receptors for adaptive immunity. Here we show that the caspase recruitment domain (CARD)-containing serine/threonine kinase Rip2 (also known as RICK, CARDIAK, CCK and Ripk2) transduces signals from receptors of both immune responses. Rip2 was recruited to TLR2 signalling complexes after ligand stimulation. Moreover, cytokine production in Rip2-deficient cells was reduced on stimulation of TLRs with lipopolysaccharide, peptidoglycan and double-stranded RNA, but not with bacterial DNA, indicating that Rip2 is downstream of TLR2/3/4 but not TLR9. Rip2-deficient cells were also hyporesponsive to signalling through interleukin (IL)-1 and IL-18 receptors, and deficient for signalling through Nod proteins-molecules also implicated in the innate immune response. Furthermore, Rip2-deficient T cells showed severely reduced NF-kappaB activation, IL-2 production and proliferation on T-cell-receptor (TCR) engagement, and impaired differentiation to T-helper subtype 1 (TH1) cells, indicating that Rip2 is required for optimal TCR signalling and T-cell differentiation. Rip2 is therefore a signal transducer and integrator of signals for both the innate and adaptive immune systems.

Zooming in on the hydrophobic ridge of H-2Db: implications for the conformational variability of bound peptides

Class I major histocompatibility complex (MHC) molecules, which display intracellularly processed peptides on the cell surface for scanning by T-cell receptors (TCRs), are extraordinarily polymorphic. MHC polymorphism is believed to result from natural selection, since individuals heterozygous at the corresponding loci can cope with a larger number of pathogens. Here, we present the crystal structures of the murine MHC molecule H-2Db in complex with the peptides gp276 and np396 from the lymphocytic choriomeningitis virus (LCMV), solved at 2.18 Å and 2.20 Å resolution, respectively. The most prominent feature of H-2Db is a hydrophobic ridge that cuts across its antigen-binding site, which is conserved in the Ld-like family of class I MHC molecules. The comparison with previously solved crystal structures of peptide/H-2Db complexes shows that the hydrophobic ridge focuses the conformational variability of the bound peptides in a “hot-spot”, which could allow optimal TCR interaction and discrimination. This finding suggests a functional reason for the conservation of this structural element.