Cernunnos/Xlf Deficiency Results in Suboptimal V(D)J Recombination and Impaired Lymphoid Development in Mice.

Xlf/Cernunnos is unique among the core factors of the non-homologous end joining (NHEJ) DNA double strand breaks (DSBs) repair pathway, in the sense that it is not essential for V(D)J recombination in vivo and in vitro. Unlike other NHEJ deficient mice showing a SCID phenotype, Xlf-/- mice present a unique immune phenotype with a moderate B- and T-cell lymphopenia, a decreased cellularity in the thymus, and a characteristic TCRα repertoire bias associated with the P53-dependent apoptosis of CD4+CD8+ DP thymocytes. Here, we thoroughly analyzed Xlf-/- mice immune phenotype and showed that it is specifically related to the DP stage but independent of the MHC-driven antigen presentation and T-cell activation during positive selection. Instead, we show that V(D)J recombination is subefficient in Xlf-/- mice in vivo, exemplified by the presence of unrepaired DSBs in the thymus. This results in a moderate developmental delay of both B- and T-lymphocytes at key V(D)J recombination dependent stages. Furthermore, subefficient V(D)J recombination waves are accumulating during TCRα rearrangement, causing the typical TCRα repertoire bias with loss of distal Vα and Jα rearrangements.

Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire.

Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells.

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRß locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications.

High response rate after intratumoral treatment with interleukin-2: results from a phase 2 study in 51 patients with metastasized melanoma.

BACKGROUND: Systemic high-dose interleukin-2 (IL-2) achieved long-term survival in a subset of patients with advanced melanoma. The authors reported previously that intratumorally applied IL-2 induced complete local responses of all metastases in >60% of patients. The objectives of the current study were to confirm those results in a larger cohort and to identify patient or regimen characteristics associated with response. METHODS: Patients with melanoma who had a median of 12 injectable metastases received intratumoral IL-2 treatments 3 times weekly until they achieved clinical remission. The initial dose of 3 million international units was escalated, depending on the individual patient's tolerance. RESULTS: Forty-eight of 51 patients were evaluable. Only grade 1/2 toxicity was recorded. A complete response that lasted >/=6 months was documented in 70% of all injected metastases. A complete local response of all treated metastases was achieved in 33 patients (69%), including 11 patients who had between 20 and 100 metastases. Response rates were higher for patients who had stage III disease compared with patients who had stage IV disease. No objective responses of distant untreated metastases were observed. The 2-year survival rate was 77% for patients with stage IIIB/IIIC disease and 53% for patients with stage IV disease. Efficacy and survival did not differ between patients who had >/=20 lesions and patients who had <20 lesions. CONCLUSIONS: Intratumoral IL-2 treatment elicited complete local responses in a high percentage of patients. Further studies will be required to investigate the mode of action of this treatment and its impact on survival.

Age-dependent TCR revision mediated by interaction between alphabeta TCR and self-antigens.

Interactions between TCR and self-peptide/MHC complex play an important role in homeostasis and Ag reactivity of mature peripheral T cells. In this report, we demonstrate that the interactions between mature peripheral T cells and endogenous Ags have a negative impact on the maintenance of foreign Ag-specific T cells in an age-dependent manner. This is mediated by RAG-dependent secondary rearrangement of the TCR alpha-chain (receptor revision). The TCR revision in mature T cells is readily observed in mouse expressing transgenic TCR alpha-chain inserted into the physiological locus (knockin mouse) but not in conventional transgenic mouse with an identical TCR alpha-chain. Thus, our results suggest that under physiological conditions in which all TCR alpha-chains are susceptible to deletion by secondary rearrangement, TCR revision in mature peripheral T cells is an ongoing process in adult animals and contributes to age-dependent changes in T cell function and repertoire.

Multiple constraints at the level of TCRalpha rearrangement impact Valpha14i NKT cell development.

CD1d-restricted NKT cells that express an invariant Valpha14 TCR represent a subset of T cells implicated in the regulation of several immune responses, including autoimmunity, infectious disease, and cancer. Proper rearrangement of Valpha14 with the Jalpha18 gene segment in immature thymocytes is a prerequisite to the production of a TCR that can be subsequently positively selected by CD1d/self-ligand complexes in the thymus and gives rise to the NKT cell population. We show here that Valpha14 to Jalpha rearrangements are temporally regulated during ontogeny providing a molecular explanation to their late appearance in the thymus. Using mice deficient for the transcription factor RORgamma and the germline promoters T early-alpha and Jalpha49, we show that developmental constraints on both Valpha and Jalpha usage impact NKT cell development. Finally, we demonstrate that rearrangements using Valpha14 and Jalpha18 occur normally in the absence of FynT, arguing that the effect of FynT on NKT cell development occurs subsequent to alpha-chain rearrangement. Altogether, this study provides evidence that there is no directed rearrangement of Valpha14 to Jalpha18 segments and supports the instructive selection model for NKT cell selection.

Impact of cellular lifespan on the T cell receptor repertoire.

Pro-survival members of the Bcl-2 family are potent inhibitors of cell death and determine the lifespan of immature thymocytes by counteracting the intrinsically active apoptotic program in these cells. BH3-only proteins are potent antagonists of Bcl-2-like molecules and regulate death and survival of lymphocytes during their development and homeostasis. The intrinsic lifespan of CD4(+)8(+) double-positive thymocytes was reported to actively shape the diversity of the immune repertoire, since mice overexpressing Bcl-x(L) were reported to show a bias towards the usage of distal 3' Jalpha elements 1. To gain support for this concept, we analyzed TCRalpha rearrangements in T lymphocytes that show an extended lifespan due to either loss of the BH3-only protein Bim or overexpression of Bcl-2. A minor but reproducible skewing towards the usage of the more distal 3' Jalpha elements was observed in developing thymocytes and mature T cells from bim(-/-) and vav-bcl-2 transgenic mice, indicating that prolonged survival of double-positive thymocytes does have a significant impact on the selected TCRalpha repertoire. However, the changes that we observed were less pronounced than those found in lck-bcl-x(L) transgenic mice, pointing towards qualitative differences between Bcl-2- and Bcl-x(L)-mediated cell death inhibition during T cell development.

Analysis of TCRalphabeta combinations used by simian immunodeficiency virus-specific CD8+ T cells in rhesus monkeys: implications for CTL immunodominance.

Immunodominance is a common feature of Ag-specific CTL responses to infection or vaccines. Understanding the basis of immunodominance is crucial to understanding cellular immunity and viral evasion mechanisms and will provide a rational approach for improving HIV vaccine design. This study was performed comparing CTLs specific for the SIV Gag p11C (dominant) and SIV Pol p68A (subdominant) epitopes that are consistently generated in Mamu-A*01(+) rhesus monkeys exposed to SIV proteins. Additionally, vaccinated monkeys were used to prevent any issues of antigenic variation or dynamic changes in CTL responses by continuous Ag exposure. Analysis of the TCR repertoire revealed the usage of higher numbers of TCR clones by the dominant p11C-specific CTL population. Preferential usage of specific TCRs and the in vitro functional TCR-alpha- and -beta-chain-pairing assay suggests that every peptide/MHC complex may only be recognized by a limited number of unique combinations of alpha- and beta-chain pairs. The wider array of TCR clones used by the dominant p11C-specific CTL population might be explained by the higher probability of generating those specific TCR chain pairs. Our data suggest that Ag-specific naive T cell precursor frequency may be predetermined and that this process dictates immunodominance of SIV-specific CD8(+) T cell responses. These findings will aid in understanding immunodominance and designing new approaches to modulate CTL responses.

Genetic evidence supporting selection of the Valpha14i NKT cell lineage from double-positive thymocyte precursors.

Invariant Valpha14i NKT (iNKT) cells are a specialized subset of T lymphocytes with regulatory functions. They coexpress TCRalphabeta and natural killer cell markers. They differentiate through interaction of their Valpha14-Jalpha18 invariant TCRalpha chains with CD1d expressed on double-positive (DP) thymocytes. Although their development has been shown to be thymus dependent, their developmental pathway has not been definitively established. By using genetic analyses, we show here that all iNKT cells are selected from a pool of DP thymocytes. Their development is absolutely dependent on Runx1 and ROR(gamma)t, transcription factors that influence, but are not required for, development of conventional T cells. Our results indicate that even though CD1d binding DP thymocytes have yet to be observed, Valpha14-Jalpha18 rearrangement in these cells is required for development of iNKT cells.

Multiple T-cell clones specific for the same foreign pMHC ligand can be generated from a single, ancestral TCR-VDJbeta precursor.

Owing to ordered, stage-specific T-cell receptor (TCR) gene rearrangements and cell division during T-cell development, small cohorts of "half-sibling" T cells sharing an ancestral TCR VDJbeta rearrangement but expressing different TCR alpha-locus rearrangements may be selected into the mature T-cell repertoire. We wondered whether different alphabetaTCRs expressed by T cells from the same ancestral VDJbeta cohort might be capable of recognizing the same foreign peptide-major histocompatibility complex complex (pMHC). By a combined flow cytometric and single-cell polymerase chain reaction (PCR) approach to analyze TCRs selected by the previously defined foreign antigen, pCW3170-179/H-2Kd, we were able to identify cohorts of half-sibling antigen-specific CD8 T cells after their expansion in immunized mice. We amplified residual DJbeta rearrangements as clonal markers to confirm that the shared VDJbeta sequences represent ancestral rearrangements rather than identical but independent ones. An intriguing explanation of our findings would be that only a very limited repertoire of TCR alpha-chains is selected to pair with a given TCR beta-chain during T-cell development.

Diverse TCRs recognize murine CD1.

Human and murine T cells that specifically recognize CD1d and produce IL-4 and IFN-gamma play a role in immunoregulation and tumor rejection. In the mouse, most CD1d1-reactive T cells described express an invariant Valpha14-Jalpha281 TCR associated with TCR beta-chains of limited diversity. Similarly, human CD1d-reactive T cells express a highly restricted TCR repertoire. Here we report the unexpected result that in mice immunized with CD1d1-bearing transfectant cells, a diverse repertoire of TCRs was expressed by CD1d1-reactive T cell clones isolated by limiting dilution without preselection for NK1 expression. Only 3 of 10 CD1d1-reactive T cell clones expressed the invariant Valpha14-Jalpha281 TCRalpha rearrangement. T cells expressing Valpha10, -11, -15, and -17, and having non-germline-encoded nucleotides resulting in diverse V-J junctions were identified. Like CD1d1-reactive T cells expressing the invariant Valpha14-Jalpha281 TCR alpha-chain, CD1d1-reactive clones with diverse TCRs produced both Type 1 (IFN-y) and Type 2 (IL-4, IL-10) cytokines. This establishes the existence of significant diversity in the TCRs directly reactive to the CD1d1 protein. Our findings reveal that CD1d interacts with a broad array of TCRs, suggesting substantial redundancy and flexibility of the immune system in providing T cells serving the role(s) mediated by CD1d reactivity.

Central T cell tolerance in lupus-prone mice: influence of autoimmune background and the lpr mutation.

Lupus-prone mice develop a systemic autoimmune disease that is dependent upon the B cell help provided by autoreactive alphabeta CD4+ T cells. Since autoreactive T cells with high affinity for self peptides are normally deleted in the thymus, their presence in these mice suggests the possibility that intrathymic negative selection may be defective. Here, we directly compared central T cell tolerance in response to a conventional peptide Ag in lupus-prone MRL/MpJ mice with a nonautoimmune strain using an MHC class II-restricted TCR transgene. Our results did not demonstrate any defects after Ag exposure in the induction of intrathymic deletion of immature CD4+ CD8+ thymocytes, in TCR down-regulation, or in the number of apoptotic thymocytes in MRL/MpJ compared with nonautoimmune mice. Furthermore, we found that the lpr mutation had no influence upon the Ag-induced thymic deletion of immature thymocytes. These data support the notion that T cell autoreactivity in MRL/MpJ mice is caused by defects in peripheral control mechanisms.

TCR transgenic mice in which usage of transgenic alpha- and beta-chains is highly dependent on the level of selecting ligand.

We have produced a TCR transgenic mouse that uses a TCR derived from a Th1 clone that is specific for residues 64 to 76 of the d allele of murine hemoglobin presented by I-Ek. Examination of these TCR transgenic mice on an H-2(k/k) background that expressed the nonstimulatory s allele of murine hemoglobin revealed that these mice express many endogenous TCR chains from both alpha and beta loci. We found that this transgenic TCR is also very inefficient at mediating beta selection, thereby showing a direct linkage between beta selection and allelic exclusion of TCR beta. We have also examined these mice on MHC backgrounds that have reduced levels of I-Ek and found that positive selection of cells with high levels of the transgenic TCR depends greatly on the ligand density. Decreasing the selecting ligand density is a means of reducing the number of available selecting niches, and the data reveal that the 3.L2 TCR is used sparingly for positive selection under conditions where the number of niches becomes limiting. The results, therefore, show a way that T cells may get to the periphery with two self-restricted TCRs: one that efficiently mediates positive selection, and another that is inefficient at positive selection with the available niches.

Flow-microfluorometric monitoring of oligoclonal CD8+ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo.

The TCR repertoire of CD8+ T cells specific for Moloney murine leukemia virus (M-MuLV)-associated Ags has been investigated in vitro and in vivo. Analysis of a large panel of established CD8+ CTL clones specific for M-MuLV indicated an overwhelming bias for V beta4 in BALB/c mice and for V beta5.2 in C57BL/6 mice. These V beta biases were already detectable in mixed lymphocyte:tumor cell cultures established from virus-immune spleen cells. Furthermore, direct ex vivo analysis of PBL from BALB/c or C57BL/6 mice immunized with syngeneic M-MuLV-infected tumor cells revealed a dramatic increase in CD8+ cells expressing V beta4 or V beta5.2, respectively. M-MuLV-specific CD8+ cells with an activated (CD62L-) phenotype persisted in blood of immunized mice for at least 2 mo, and exhibited decreased TCR and CD8 levels compared with their naive counterparts. In C57BL/6 mice, most M-MuLV-specific CD8+ CTL clones and immune PBL coexpressed V alpha3.2 in association with V beta5.2. Moreover, these V beta5.2+ V alpha3.2+ cells were shown to recognize the recently described H-2Db-restricted epitope (CCLCLTVFL) encoded in the leader sequence of the M-MuLV gag polyprotein. Collectively, our data demonstrate a highly restricted TCR repertoire in the CD8+ T cell response to M-MuLV-associated Ags in vivo, and suggest the potential utility of flow-microfluorometric analysis of V beta and V alpha expression in the diagnosis and monitoring of viral infections.