TCRα Chain Research Articles

Differential selection of Th1 effector and memory pools depends on the qualitative nature of TCR-driven CD4+ T cell activation (P1175)

Abstract Not all clones that participate in the effector Th1 response to intracellular viral or bacterial infection can give rise to memory T cells. In this study, we generated a mouse with a fixed TCRα chain of known specificity in order to characterize the evolution of effector and memory TCRβ repertoires following LCMV or Listeria infection by deep sequencing. The repertoire diversity of antigen-specific Th1 cells significantly decreased in the transition from effector to memory, and hierarchical cluster analysis revealed that the TCR repertoires of memory T cells, regardless of infectious model, were more similar to each other than to the effector Th1 populations from which they arose. We further generated a large panel of TCRs derived from our sequencing results that were cloned into retroviral expression vectors and used to generate TCR “retrogenic” bone marrow chimeras. Individual clonal T cell populations harvested from these mice were analyzed in adoptive transfer experiments to allow for their longitudinal analysis following infection. We found that the individual memory potential of each clone did not correspond to overall TCR avidity, as measured in tetramer equilibrium binding assays, but to the acquisition of high functional avidity and slow off-rates, as determined by tetramer decay. Our findings support a model in which differential selection of the Th1 effector and memory pools depends at least in part on the qualitative nature of TCR-driven CD4+ T cell activation.

Glycosylphosphatidylinositol-specific, CD1d-restricted T cells in paroxysmal nocturnal hemoglobinuria

AbstractThe mechanism of bone marrow failure (BMF) in paroxysmal nocturnal hemoglobinuria (PNH) is not yet known. Because in PNH the biosynthesis of the glycolipid molecule glycosylphosphatidylinositol (GPI) is disrupted in hematopoietic stem and progenitor cells by a somatic mutation in the PIG-A gene, BMF might result from an autoimmune attack, whereby T cells target GPI in normal cells, whereas PIG-A mutant GPI-negative cells are spared. In a deliberate test of this hypothesis, we have demonstrated in PNH patients the presence of CD8+ T cells reactive against antigen-presenting cells (APCs) loaded with GPI. These T cells were significantly more abundant in PNH patients than in healthy controls; their reactivity depended on CD1d expression and they increased upon coculture with CD1d-expressing, GPI-positive APCs. In GPI-specific T cells captured by CD1d dimer technology, we identified, through global T-cell receptor α (TCRα) analysis, an invariant TCRVα21 sequence, which was then found at frequencies higher than background in the TCR repertoire of 6 of 11 PNH patients. Thus, a novel, autoreactive, CD1d-restricted, GPI-specific T-cell population, enriched in an invariant TCRα chain, is expanded in PNH patients and may be responsible for BMF in PNH.

Ikaros to the rescue of TCR‐α chain gene rearrangement

Ikaros is a transcriptional regulator critical for B- and T-cell development. Recently, it has been shown to play a central role in facilitating rearrangement of antigen-receptor genes in B cells. Whether or not it had a similar function in this process in T cells, however, was a mystery. In this issue of the European Journal of Immunology, a role for Ikaros in T-cell receptor (TCR) rearrangement and expression of TCR-α chain genes is revealed in the study by Collins et al. [Eur. J. Immunol. 2013. 43: 521-532]. Ikaros functions in this capacity as an "accessibility factor," facilitating increased TCR-α chain gene transcription and accessibility of the locus to promote rearrangement. Interestingly, this study has also revealed differences in the mechanisms by which Ikaros promotes antigen-receptor rearrangement in B versus T cells, thereby suggesting that Ikaros may have lineage-specific functions in coordinating antigen-receptor rearrangement.

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.

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

S114 Adaptive and Innate-Like T Cell Phenotypes in Asthma in Relationship to Compartment and Severity

Introduction and Objectives Asthma is a heterogeneous disease affecting 150–300 million people. Underlying mechanisms remain unclear. T H 17 cells expressing interleukin-17 are widely hypothesised to play a role, particularly in severe neutrophilic asthma. Mucosal associated invariant T (MAIT) cells are novel innate-like T-cells of unknown function which express CD161 and an invariant TCRα chain (Vα7.2-Jα33) and recognise the highly conserved restriction molecule MR1. We undertook to analyse IL-17 and key T-cell subsets, namely T H 17, T H 1, T H 2, T REG, and MAIT cells in relation to asthma severity and virus-induced exacerbations. Methods Cross sectional study: 76 subjects underwent detailed phenotyping, sputum induction, phlebotomy, and bronchoscopy. Samples were analysed by 9-colour flow-cytometry, RT-PCR, multiplex ELISA, microarray, and deep sequencing of the airway microbiome. Longitudinal study: 35 frequently exacerbating asthmatics: followed at 7 time-points during a naturally occurring cold. Results Contrary to initial hypotheses TH17 cell frequencies did not differ between health and any asthmatic phenotype, in any tissue compartment. TH2 cell frequencies were elevated in asthma in bronchoalveolar-lavage (BAL) (ANOVA p=0.041) and markedly in bronchial biopsies (p=0.048), as expected[1]. BAL TH1 cell frequencies were also increased in asthma (p=0.01) as described[2], whilst TREG frequencies were lower in severe asthma (p=0.019). T H 2 cytokines were increased in asthma in sputum (IL-5 p=0.005) and BAL (IL-5 p Longitudinal follow-up revealed no significant differences in T-cell frequencies during exacerbations, though sputum T H 17 cells tended to increase (NS). We observed that frequencies of Vα7.2+CD161+ (MAIT) cells in blood are lower in asthma than in health (p=0.013), and correlated with severity in blood (p for linear trend Conclusions A role for TH17 cells in asthma, particularly severe neutrophilic disease has been widely hypothesised, but is not supported by these data. High BAL IL-17 levels in older, steroid-naive, mild asthmatics may have a different cellular source. We describe a novel finding of deficient Vα7.2+CD161+ (MAIT) cells in severe asthma. References Robinson, DS, et al., Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med, 1992. 326(5): p. 298–304. Krug, N, et al., T-cell cytokine profile evaluated at the single cell level in BAL and blood in allergic asthma. Am J Respir Cell Mol Biol, 1996. 14(4): p. 319–26.

The Safety Evaluation of NKTT120, an Invariant NKT (iNKT) Cell Depleting Antibody in the Cynomolgus Monkey.

AbstractAbstract 2150Invariant Natural Killer T (iNKT) cells are a small subset of T lymphocytes (ranging from 0.01 – 0.1% of CD3+ T cells). iNKT cells recognize glycolipid antigens presented by the MHC class-I-like protein CD1d rather than peptide antigens. In contrast to most T cell subpopulations, which have diverse sequences for their T Cell Receptors (TCRs), iNKT cells express a uniquely rearranged, highly conserved, semi-invariant TCR-α chain (Vα24-Jα18 in humans), which preferentially pairs with specific TCR-β chains (Vβ11 in humans). iNKT cells are similar to innate cells in their rapid release of cytokines following iTCR antigen binding. They are also adaptive-like, with T cell properties including thymic positive selection and antigen recognition by CD1d presentation. iNKT cells are involved in mediating tissue injury and inflammation in multiple organ systems. Chronic inflammation is associated with the pathophysiology of Sickle Cell Disease (SCD) and our studies and those of others have found an increased ratio of activated iNKT cells in peripheral blood of patients with SCD. The role of iNKT cell activation in the pathology of SCD is supported by studies in a mouse model of SCD (Wallace et al. Blood 114:667, 2009). These data suggest that iNKT cell reduction and/or depletion would be effective in reducing the inflammatory state in SCD. To this end, we have developed a humanized monoclonal antibody (NKTT120) that exclusively binds to the CDR3 loop of the human and non-human primate (NHP) invariant T cell receptor and depletes iNKT cells. This antibody could provide an effective therapeutic intervention to modulate iNKT cell numbers, and thus their ability to mediate inflammation in SCD. The current study was designed to assess the overall safety of NKTT120 in the cynomolgus monkey (Macaca fascicularis). The cynomolgus monkey was selected for testing because NKTT120 is only active in human and old world NHP species. Thirty-two cynomolgus monkeys (3–5 kg) equally divided between males and females were studied. The groups consisted of a vehicle control and treatment groups that received 0.3 mg/kg, 3 mg/kg, and 10 mg/kg NKTT120 IV weekly for a total of 5 doses. Two animals per sex in the vehicle and the 10 mg/kg groups were recovered for an additional 2 months following the last dose. Animals were evaluated for food intake, body weight and general health. Standard hematology, coagulation testing and clinical chemistry testing were also performed. In addition, iNKT cell number and other lymphocytes were monitored during the study by FACS analysis. The repeat dose injections were well tolerated by all dose groups. No deaths or serious adverse events were reported during dosing or in the recovery period. Body weight, food intake and clinical evaluation, hematology, coagulation assays, and clinical chemistry were similar for vehicle control and all dosing groups. As expected, iNKT cells were depleted within 24 hours to below level of detection and remained depleted throughout the dosing period at all doses tested. The iNKT cell numbers of the 10 mg/kg recovery animals remained depleted throughout the 2 month recovery. However, there was no change in other cells of the lymphocytic series at any dose or at any time point evaluated. Overall our study showed that we can safely and specifically deplete iNKT cells in non-human primates following administration of NKTT120. These data support the use of NKTT120 as a therapeutic intervention in conditions such as sickle cell disease where iNKT modulation could be beneficial.Percent of Gated Cells (Mean ± SD) Estimated by FACS Analysis Before and Following NKTT120 IV Administration to Cynomolgus Macaques Shows Specific iNKT Cell Depletion at all Doses and Time pointsLymphocyte ProfilesPost Dose 1Post Dose 52 Months Post Dose 5Vehicle0.3 mg/kg3 mg/kg10 mg/kgVehicle0.3 mg/kg3 mg/kg10 mg/kgVehicle10 mg/kgiNKT Cells*0.05±.030.000.000.000.06±0.050.000.000.000.06±.010.00NK Cells**11±610±311±512±710±59±56±47±67±35±4Mature T cells**69±761±763±657±764±861±1059±1159±878±670±7CD8 T Cells**26±425±623±319±622±422±820±719±528±220±2CD4 T Cells**40±635±637±635±737±635±735±537±846±447±5B Cells**5±38±27±49±35±35±22±26±36±38±3*%CD3 Cells.**% Lymphocytes. Disclosures:Macdonald:NKT Therapeutics: Employment, Equity Ownership. Scheuplein:NKT Therapeutics: Employment, Equity Ownership. Thariath:NKT Therapeutics: Employment, Equity Ownership. LeBel:MPI Research: Employment; NKT Therapeutics: Research Funding. Zeigler:MPI Research: Employment; NKT Therapeutics: Research Funding. Truneh:NKT Therapeutics: Consultancy, Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. Schaub:NKT Therapeutics: Employment, Equity Ownership.

Preclinical Evaluation of Invariant Natural Killer T-Cells in the 5T33 Multiple Myeloma Model

AbstractAbstract 938Natural killer T (NKT) cells are T-lymphocytes that co-express conventional T-cell (CD3) and NK cell (NK1.1) surface receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain encoded by Vα14.Jα18 in mice and Vα24.Jα18 in human. These iNKTs can recognize glycolipid antigens such as α-Galactosylceramide (α-GalCer) presented by the class I-like major histocompatibility complex (MHC) molecule CD1d. Activation of iNKTs can lead to an anti-tumor Th1 (IFN-γ) response or an immunosuppressive Th2 (IL-4) response. Clinical studies in MM patients (1, 2) showed a low frequency and dysfunction of iNKTs which resulted in a low IFN-γ secretion. This defect could be overcome in vitro by stimulating the iNKTs with α-GalCer loaded DCs. Furthermore, when MM patients were injected with loaded DCs their iNKT pool expanded 100 fold. This make MM cells an interesting target for iNKT therapy. However, the data on NKT activity in MM patients is limited and the use of α-GalCer as a drug has not been preclinically evaluated yet. Therefore, in this study, we investigated the characteristics of iNKTs in the syngeneic 5T33MM murine model, which is an immunocompetent model of myeloma which mimics the human disease closely.We first investigated the frequency of iNKTs in the blood, BM, spleen and liver of both healthy and terminally diseased 5T33MM mice. The highest percentage of iNKTs was found in the liver (naive 7%) with a significant decrease in 5T33MM mice (2.6%). The percentage was also slightly decreased in spleen (from 1.5% in naive to 0.7% in 5T33MM mice) while no significant differences were observed in the other tissues. Next, we followed the frequency of iNKTs in the liver and spleen of MM mice during the development of the disease. We analyzed the number of iNKTs in the first, second, third and terminal week. We found that the percentage of iNKTs declined at the end stage of MM disease. To analyze the activity of iNKTs in vitro, liver iNKTs were cocultured with naive matured BM derived DCs in the presence or absence of 100 ng/ml α-GalCer. Naive iNKTs could secrete up to 2.3 ng/ml IFN-γ when stimulated with α-GalCer, and this level increased with the progression of MM to reach 3.3 ng/ml at week 2. However, the activity of the iNKTs dropped to undetectable levels upon further progression of the disease (week 4). In contrast, very slight IL-4 production was observed indicating that liver iNKTs are skewed to a Th1 profile and can therefore be used as an immunotherapeutic tool in MM. The activity of the NKTs was also followed in vivo. The serum level of IFN-γ peaked at 18h after α-GalCer injection in naive and non-terminal diseased mice and returned to baseline by 48h, however, the response of IFN-γ in diseased mice was twice (6 ng/ml) that measured in naive mice, confirming the possibility of inducing Th1 responses with α-GalCer in vivo in healthy and diseased mice. No response could be detected from terminally diseased mice. It has been described previously that CD1d is significantly downregulated in patients with advanced stages of MM (3). To investigate if this is similar in the 5T33MM model, we followed the expression of CD1d on spleen and BM cells during the course of the disease. Results showed a significant downregulation of CD1d expression on spleen cells from 93% CD1d (naive) to 68% at end stage. On BM cells, CD1d was less expressed compared to spleen cells, 52% in naive mice and expression declined significantly to 35%. CD1d expression on the MM cells themselves was high (79%) and did not alter during the course of the disease.We finally evaluated the effect of α-GalCer on the survival of MM mice. Survival was significantly increased when mice were injected with α-GalCer loaded DCs on the same day of 5T33MM inoculation (29 days survival) compared to mice injected with unloaded DCs (22 days survival).Taken together, our data demonstrate for the first time the possibility of using a murine model as a preclinical MM model to study the effects of α-GalCer on iNKTs and shows promising results of treating MM patients with a low tumorload. Disclosures:No relevant conflicts of interest to declare.

Cynomolgus Monkey Invariant NKT (iNKT) Cells Recover Following Depletion by Treatment with the Anti-Invariant T Cell Receptor Antibody NKTT120

AbstractAbstract 4836Invariant Natural Killer T (iNKT) cells are a small subset of T lymphocytes (ranging from 0.01 – 0.1% of CD3+ T cells) that share surface markers and functional characteristics with T cells and natural killer (NK) cells. Unlike other T cells, they recognize glycolipid antigens presented by the MHC class-I-like protein CD1d rather than peptide antigens. In contrast to most T cells which express diverse T cell receptor (TCR) sequences, iNKT cells express a unique, highly conserved, semi-invariant TCR-α chain (Vα24-Jα18 in humans), which preferentially pairs with specific TCR-β chains (Vβ11 in humans). Like cells of the innate immune system, iNKT cells are rapid-onset cells with a universal receptor. They also share properties of T cells like requiring thymic positive selection and recognition of antigen presented on the MHC-I like molecule CD1d. As such, they serve as a bridge between the innate and adaptive immune systems. They can play either a pro-inflammatory role to enhance or an immuno-regulatory role to attenuate a developing immune response. iNKT cells have been shown to be involved in mediating tissue injury and inflammation in multiple organ systems. There is a growing recognition that chronic inflammation is associated with the pathophysiology of Sickle Cell Disease (SCD). Wallace et al. (Blood 114:667, 2009) found an increased ratio of activated iNKT cells in peripheral blood of patients with SCD compared to normal volunteers. Our study of peripheral blood iNKT cells from 5 SCD patients and 10 unaffected African American volunteers found that an average of 50.5% of the iNKT cells of the SCD patients were activated as indicated by up regulation of the lymphocyte activation marker CD69. In the healthy controls an average of only 8.9% of iNKT cells expressed CD69. A key role of iNKT cell activation in the pathology of SCD is supported by studies in a mouse model of SCD (Blood 114:667, 2009) that suggest iNKT cell depletion could reduce inflammation in the SCD patient. We have developed a humanized monoclonal antibody (NKTT120) that binds the CDR3 loop of the human and the old-world non-human primate invariant TCR with high specificity. NKTT120 depletes iNKT cells in human iTCR transgenic mice and cynomolgus monkeys (Macaca fascicularis). In the transgenic mice, iNKT cells reappear in the peripheral circulation within weeks after complete depletion. We conducted a study in cynomolgus monkeys to explore the relationship between dose and duration of iNKT cell depletion. Each group of animals (n=3) received a single dose of NKTT120. The dosing groups were 10, 30, 100 and 300 ug/kg, respectively. iNKT cells and other lymphocytes were monitored 24, 48, 72, 96 and 168 hours following dosing and weekly thereafter using flow cytometry. In all dose groups, iNKT cells were depleted within 24 hours with no significant changes in the other cells of the lymphocytic series. No adverse events have been noted to date. iNKT cells in the 10 ug/kg and the 30 ug/kg dose group were recovered by week 5 and week 7 respectively. As of week 12 post-dosing, iNKT cells in the 100 and the 300 ug/kg dose groups have not recovered. The kinetics of recovery in these higher dose animals will shed light on the impact of what is expected to be full tissue depletion on iNKT cell recovery. Overall, our study has shown that we can safely deplete iNKT cells in non-human primates and that iNKT cells can recover after depletion.Percent of Cells (Mean±SD) Estimated by FACS Analysis Before and Following NKTT120 IV Administration Shows Recovery Following DepletionLymphocyte ProfilesPre SampleDay 1 Post DoseWeek 5 Post DoseWeek 7 Post Dose10 ug/kg30 ug/kg100 ug/kg300 ug/kg10 ug/kg30 ug/kg100 ug/kg300 ug/kg30 ug/kg100 ug/kg300 ug/kgiNKT Cells*0.29±0.170.02±0.030000.14±0.120.01±0.005000.05±0.0100.01±0.01NK Cells**11.6±3.98.1±1.88.5±2.17.8±411.1±2.511.4±2.616.2±4.38.9±3.114.3±4.313.2±3.19.2±4.113.2±2.5Mature T cells**73.1±5.569.6±470±7.575.9±4.365.1±3.670.6±4.567.4±5.178.2±5.766.5±4.472.3±4.980.8±6.369.3±3.2CD8 T Cells**26.0±4.722.1±4.220.7±2.827.8±3.522.4±4.126.2±5.524.1±6.331.8±3.726.5±6.125.4±5.832.7±5.127.2±6.9CD4 T Cells**42.0±444.3±7.945.2±5.243.2±1.538.3±4.141.1±6.939.5±1.740.9±2.635.4±3.343.2±7.742.2±0.737.4±5.4B Cells**3.4±2.18.9±2.46.8±1.65.1±18.5±5.17±1.17.4±2.94.3±0.67.8±4.95.5±1.52.4±0.26.1±3.5*% of CD3**% of Lymphocytes Disclosures:Scheuplein:NKT Therapeutics: Employment, Equity Ownership. Macdonald:NKT Therapeutics: Employment, Equity Ownership. Zeigler:MPI Research: Employment; NKT Therapeutics: Research Funding. LeBel:MPI Research: Employment; NKT Therapeutics: Research Funding. Thariath:NKT Therapeutics: Employment, Equity Ownership. Truneh:NKT Therapeutics: Consultancy, Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. Nathan:NKT Therapeutics: Consultancy. Schaub:NKT Therapeutics: Employment, Equity Ownership.

Highly diverse TCRα chain repertoire of pre-immune CD8+T cells reveals new insights in gene recombination

Highly diverse TCRα chain repertoire of pre-immune CD8⁺T cells reveals new insights in gene recombination

The Molecular Basis for Recognition of CD1d/α-Galactosylceramide by a Human Non-Vα24 T Cell Receptor

Author SummaryCertain lineages of T cells can recognize lipids as stimulatory antigens when presented in the context of CD1 molecules. We know how most Natural Killer T (NKT) cells react with this unusual ligand because they use a single invariant T cell receptor (TCR) alpha chain to do the job. NKT cells place particular emphasis on their CDR3α and CDR2β loops in recognition of antigen—these complementarity determining regions (CDRs) are the hypervariable parts of the TCR that “complement” an antigen's shape. How do these other T cells recognize closely related yet distinct lipid antigens? Here we show that human CD1d-restricted T cells, typically called Vα24− T cells due to their use of diverse Vα domains in their TCRs, use similar molecular strategies to respond to lipid antigens presented by CD1d. To this end we present a 2.5 Å complex structure of a Vα24− TCR complexed with CD1d presenting the protypical lipid, α-galactosylceramide (αGalCer). The TCR examined in this study notably shifts its binding slightly, placing more emphasis on the interaction with the CDR1α loop as revealed through alanine scanning mutagenesis. This shift explains the inability of these T cells to respond to lipids that vary at this site of contact (the 4'OH), like the related α-linked glucosylceramide. These results provide a molecular basis for the fine-specificity of different CD1d-restricted T cell lineages.

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) α and β loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders(1-4). T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the αβTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αβTCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC(5). Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events(6). For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes(7-9). Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HY(cd4) model, in which the transgenic HY TCRα is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice(10). Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HY(cd4) mouse model. While negative selection in HY(cd4) mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

A new method for quantitative analysis of the T cell receptor V region repertoires in healthy common marmosets by microplate hybridization assay

The common marmoset, Callithrix jacchus, is one of the smallest primates and is increasingly used for an experimental nonhuman primate model in many research fields. Analysis of T cell receptor (TCR) repertoires is a powerful tool to investigate T cell immunity in terms of antigen specificity and variability of TCR expression. However, monoclonal antibodies specific for many TCR Vα or Vβ chains have not been created. We have recently identified a large number of TCRα chain variable (TRAV) and TCRβ chain variable (TRBV) sequences from a cDNA library of common marmosets. The purpose of this study is to develop a new method for analysis of TCR repertoires in the common marmoset using this sequence information. This method is based on a microplate hybridization technique using 32 TRAV-specific and 32 TRBV-specific oligoprobes following an adaptor-ligation PCR. This enables the easy quantitation of the respective TRAV and TRBV expression levels. No cross-hybridization among specific-oligoprobes and very low variances in repeated measures of the same samples was found, demonstrating high specificity and reproducibility. Furthermore, this method was validated by an antihuman Vβ23 antibody which specifically bound to marmoset Vβ23. Using this method, we analyzed TCR repertoires from various tissue samples (PBMCs, spleen, lymph node and thymus) and isolated T cell subpopulations (CD4+CD8+, CD4+CD8− and CD4−CD8+) from the thymus of 10 common marmosets. Neither tissue-specific nor T cell subpopulation-specific differences was found in TRAV and TRBV repertoires. These results suggest that, unlike mice, TCR repertoires in the common marmoset are not affected by endogenous superantigens and are conserved among individuals, among tissues, and among T cell subpopulations. Thus, TCR repertoire analysis with high specificity and reproducibility is a very useful technique, with the potential to replace flow cytometric analysis using a panel of TRV-specific antibodies, many of which remain unavailable.

Recognition of CD1d-sulfatide mediated by a type II natural killer T cell antigen receptor

Natural killer T cells (NKT cells) are divided into type I and type II subsets on the basis of differences in their T cell antigen receptor (TCR) repertoire and CD1d-antigen specificity. Although the mode by which type I NKT cell TCRs recognize CD1d-antigen has been established, how type II NKT cell TCRs engage CD1d-antigen is unknown. Here we provide a basis for how a type II NKT cell TCR, XV19, recognized CD1d-sulfatide. The XV19 TCR bound orthogonally above the A' pocket of CD1d, in contrast to the parallel docking of type I NKT cell TCRs over the F' pocket of CD1d. At the XV19 TCR-CD1d-sulfatide interface, the TCRα and TCRβ chains sat centrally on CD1d, where the malleable CDR3 loops dominated interactions with CD1d-sulfatide. Accordingly, we highlight the diverse mechanisms by which NKT cell TCRs can bind CD1d and account for the distinct antigen specificity of type II NKT cells.

Oligodendrocytes Enforce Immune Tolerance of the Uninfected Brain by Purging the Peripheral Repertoire of Autoreactive CD8+ T Cells

Myelin-specific CD8(+) Tcells are thought to contribute to the pathogenesis of multiple sclerosis. Here we modeled this contribution in mice with CD8(+) Tcells recognizing ovalbumin (OVA) expressed in oligodendrocytes (ODCs). Surprisingly, even very high numbers of activated OVA-reactive CD8(+) Tcells failed to induce disease and were cleared from the immune system after antigen encounter inthe centralnervous system (CNS). Peripheral infection with OVA-expressing Listeria (Lm-OVA) enabled CD8(+) Tcells to enter the CNS, leading to the deletion of OVA-specific clones after OVA recognition on ODCs. In contrast, intracerebral infection allowed OVA-reactive CD8(+) Tcells to cause demyelinating disease. Thus, in response to infection, CD8(+) Tcells also patrol the CNS. If the CNS itself is infected, they destroy ODCs upon cognate antigen recognition in pursuit of pathogen eradication. In the sterile brain, however, antigen recognition on ODCs results in their deletion, thereby maintaining tolerance.

The role of ID3 in iNKT cell development (115.12)

Abstract Invariant NKT cells comprise an unconventional T cell population with innate characteristics. Like the conventional αβ T cell lineage, they develop in the thymus from DP precursors after receiving TCR-dependent positive selection signals, although utilizing different pathways. iNKT cells further mature through a series of differentiation stages and acquire their effector properties intrathymically. The ID3 protein is required to reduce E protein activity for proper selection of DP progenitors towards conventional thymocyte lineages. Here, we investigate the requirement for ID3 in the thymic development of iNKT lineage, in vivo. We show that the frequency of iNKT cells in the Id3-/- thymus is higher than in WT littermates, in a cell-intrinsic manner. Id3-deficient post-selected DP cells express higher levels of the canonical Vα14-Jα18 TCRα chain transcripts. Additionally, the transcription factor PLZF, a molecular determinant of the NKT lineage, is expressed in a subset of post-selected DP cells in the Id3-/- mice. However, further thymic iNKT differentiation is blocked at an immature stage. Taken together these results suggest that ID3 restricts the innate T cell potential of DP precursors thus contributing to the lineage fate decisions at the DP stage. However, ID3 is necessary for the completion of the innate T cell program during iNKT differentiation. Ongoing gene expression analysis will identify potent E protein target genes essential for NKT development

Abstract 4406: Clinical efficacious combined chemo/immunotherapy differently activates AKT pathway and functionality of gp100 and Melan-A specific T cell clones

Abstract A phase II randomized clinical study is ongoing in our Institution to prove the clinical efficacy of dacarbazine (DTIC) one day before peptide-vaccination (Melan-A and NY-ESO-1) in the prevention of melanoma relapse in clinically disease-free HLA-A2 patients. In a previous pilot study, this combination therapy (using Melan-A and gp100 peptides) increased the number of tumor-reactive long-lasting effector-memory CD8+ lymphocytes. To identify the mechanisms enhancing the immune response, induced by DTIC combined with peptide-vaccination, we analyzed the endogenous and treatment-induced antigen specific CD8 T-cell response at the clonal level. We analyzed the sequence of the TCR α-chain of these clones and the molecular results were correlated with the expression of CD27/CD28 co-stimulatory molecule, AKT activation and anti-tumor lytic activity. The combination of chemo/immunotherapy elicited in Melan-A-specific, but not in gp100 clones, a renewal of high-avidity/tumor-reactive T-cell clones, with a broadening TCR diversity in long-surviving patients, suggesting that the selection of immune-resistant tumor variants may be circumvented by this combination, and thus prevent tumor recurrence in melanoma. In gp100 clones, AKT activation (pSer473-AKT) canonically correlates with CD28 and/or CD27 expression, independent of the treatment while in Melan-A clones, lacking CD27 and CD28 expression, non-canonical AKT activation was only observed after the combination therapy. The identification of the extracellular stimuli and signaling pathway responsible for DTIC-mediated activation of the AKT signaling are currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4406. doi:1538-7445.AM2012-4406

Highly diverse TCRα chain repertoire of pre-immune CD8+T cells reveals new insights in gene recombination

Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8(+) T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity.

504 Participation of Invariant NKT Cells (Vα24Jα18) during Asthma Exacerbation in Children

BackgroundInvariant NKT cells (or type 1 NKT cells) co-express CD3 marker and NK receptors (CD56, CD161) and use a single type of TCRα chain (Vα24Jα18 for humans), comprising CD4-CD8-, CD4+ and CD8+ subsets. Participation of these cells and their cytokines in asthmatic children, in stable conditions and under exacerbation, was studied.MethodsThree groups on children (6–12 years old) were selected: 1) asthmatics under exacerbation attack (AE) within the first 24 hours after the attack and before starting any treatment; 2) asthmatics with stable asthma (SA), without symptoms for at least a month before bleeding; and 3) healthy controls (HC) without history of asthma, atopy and with normal lung function were selected in the Allergy and Clinical Immunology Service, Hospital Infantil de Mexico. Invariant NKT cells and subset levels as well as intracellular cytokines were evaluated in whole blood by 4-color flow cytometry (antibodies against CD3, CD4, CD8, CD161, Va24, IL-4 and IFN-g).ResultsProportion of iNKT cells among total CD3+ cells in HC group was 0.9%, while in SA patients they were increased up to 2.6%; interestingly, during exacerbation such cells were dimished (1.8%). Concerning iNKT CD4+ cells were 0.6% in HC, 1.8% in SA, and 0.7% in AE, while iNKT CD8+ cells were 0.1% in HC, 0.7% in SA, and 0.4% in AE. Both iNKT cell subsets expressed intracellular IFN-g and IL-4 cytokines in AE, SA and HC but predominantly IFN-g in iNKT CD8+ cells from AE patients.ConclusionsiNKT cells participation in asthma pathogenesis was confirmed. Increase of IFN-g production in patients with exacerbations, may provide a regulatory environment to stabilize the condition.