Double-positive Research Articles

Epidemiology of Adult T-Cell Leukemia/Lymphoma in Martinique (1983-2013): Robustness of Classification of Lymphoma Study Group of Japa, Relevance of Cutaneous Lesions, Atypical Phenotype and Origin of the HTLV-1 Infected Cell

We describe the epidemiological, clinical and biological features of the patients with ATL (Acute T-cell Leukaemia), extracted from the Hematological Cancer Registry of Martinique, between January 1st, 1983 and March 31st, 2013 and confront our experience with the acquired data.One hundred and seventy-five new cases of ATL were listed. All the patients with ATL were of mixed African-Caucasian descent. There were 88 men and 87 women. The median age was 56 years (from 16 to 95). One hundred and forty six patients (83,4 %) were more than 40 years old. According to the classification of the Lymphoma Study Group of Japan ( LSG) (Shimoyama M and al, Br J Haematol on 1991), the distribution of the 3 clinical types, acute, lymphoma and chronic, was respectively 62.9 % (N=110), 29.1 % (N=51) and 8 % (N=14). None smoldering type was identified. Three cases presented exclusive skin lesions. The median survival time was 5,43 months for all the cases, 3.09 months for the acute type, 8.13 for the lymphoma type and 45.16 for the chronic type (fig 1, p value of log-rank test < 0.001). The survival was significantly higher for the lymphoma type with skin lesions (fig 2, median: 13.96 versus 6.06 months, p value of log-rank test < 0.035) and for the acute type without hypercalcemia (4 versus 2.4 months, p< 0.01). The symptoms associated with hypercalcaemia present in 82 patients (47 %) and skin lesions present in 74 patients(42 %) were the most successful clinical signs for the diagnosis of ATL. Forty two percent were infected by Strongyloïdes stercoralis (Ss). Over the studied period, 154 patients (104 acute, 41 lymphoma, 9 chronic) died with a median overall survival of 4.68 months, 6.22 for the lymphoma type and 2.86 months for the acute type. The hypercalcemia was the first cause of death. No strict lymphoma type (atypical lymphoïd cells < 1%) progressed to a leukaemia form. In most of the cases, the ATL cell presented the activated post-thymic T cell phenotype CD2, CD3, CD4, CD25. Weak expression of CD3 and TCR was observed in every case analyzed. The absence of CD7 membrane expression was observed in 75 among 96 cases. This typical phenotype applied to 79,6 % of the leukaemia patients. Twenty three patients had a different phenotype: double negative ( DN) CD4(-) CD8(-)( 14 cases), double positive ( DP) CD4(+)CD8(+) (4 cases) and negative CD3(-) (4 cases) and CD8(-) (1 case). Five DN cells expressed CD7. The patients with an atypical phenotype had a significantly lower survival (median: 2.0 versus 5.76 months, p=0.013). In our series, the epidemiological and clinical characteristics of patients with ATL were comparable with those of the series of Japan and Jamaica. Scarcity of evolution from a type to another testified to the robustness of the LSG and suggested that the natural history of the tumoral cells of the various types of ATL was different. Hypercalcaemia was a discrimination factor for severity, particularly in the acute type, and the major cause of death. Skin lesions conferring a better prognosis, in particular in the lymphoma type, required early detection of these lesions in indolent disease and in HTLV-1 healthy carriers, especially among relatives of patients with ATL and/or infected by Ss, and suggested that their treatment could limit transformation to the aggressive forms of ATL. The atypical phenotypes, in particular DN cases, CD7 (- ) or CD7 (+), DP cases and CD3(-) cases, could be interpreted as thymic progenitors. Also, weak expression of the complex CD3 / TCR observed on ATL cells suggested that the target cell had acquired an intermediate differentiation level between immature cortical thymocytes and mature thymocytes. The scarcity of ATL in adolescents, associated with the strong majority of the patients being more than 40 years old, suggested the hypothesis of the necessity of the "involution" of the thymus for an accomplished ATL cell. DisclosuresNo relevant conflicts of interest to declare.

Histone acetylation mediated by Brd1 is crucial for Cd8 gene activation during early thymocyte development.

During T-cell development, Cd8 expression is controlled via dynamic regulation of its cis-regulatory enhancer elements. Insufficiency of enhancer activity causes variegated Cd8 expression in CD4(+)CD8(+) double-positive (DP) thymocytes. Brd1 is a subunit of the Hbo1 histone acetyltransferase (HAT) complex responsible for acetylation of histone H3 at lysine 14 (H3K14). Here we show that deletion of Brd1 in haematopoietic progenitors causes variegated expression of Cd8, resulting in the appearance of CD4(+)CD8(-)TCRβ(-/low) thymocytes indistinguishable from DP thymocytes in their properties. Biochemical analysis confirms that Brd1 forms a HAT complex with Hbo1 in thymocytes. ChIP analysis demonstrates that Brd1 localizes at the known enhancers in the Cd8 genes and is responsible for acetylation at H3K14. These findings indicate that the Brd1-mediated HAT activity is crucial for efficient activation of Cd8 expression via acetylation at H3K14, which serves as an epigenetic mark that promotes the recruitment of transcription machinery to the Cd8 enhancers.

Developmental regulation of p53-dependent radiation-induced thymocyte apoptosis in mice

The production of T cell receptor αβ(+) (TCRαβ(+) ) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-β) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4(+) CD8(+) double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radio-sensitivity was already found to be high in immature CD4(-) CD8(-) (double-negative, DN) cKit(+) CD25(+) thymocytes, where TCR-β gene rearrangement is initiated. However, TCR-αβ(-) CD8(+) immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αβ(+) thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αβ(-) progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis.

Bcl11b prevents the intrathymic development of innate CD8 T cells in a cell intrinsic manner.

If Bcl11b activity is compromised, CD4(+)CD8(+) double-positive (DP) thymocytes produce a greatly increased fraction of innate CD8(+) single-positive (SP) cells highly producing IFN-γ, which are also increased in mice deficient of genes such as Itk, Id3 and NF-κB1 that affect TCR signaling. Of interest, the increase in the former two is due to the bystander effect of IL-4 that is secreted by promyelocytic leukemia zinc finger-expressing NKT and γδT cells whereas the increase in the latter is cell intrinsic. Bcl11b zinc-finger proteins play key roles in T cell development and T cell-mediated immune response likely through TCR signaling. We examined thymocytes at and after the DP stage in Bcl11b (F/S826G) CD4cre, Bcl11b (F/+) CD4cre and Bcl11b (+/S826G) mice, carrying the allele that substituted serine for glycine at the position of 826. Here we show that Bcl11b impairment leads to an increase in the population of TCRαβ(high)CD44(high)CD122(high) innate CD8SP thymocytes, together with two different developmental abnormalities: impaired positive and negative selection accompanying a reduction in the number of CD8SP cells, and developmental arrest of NKT cells at multiple steps. The innate CD8SP thymocytes express Eomes and secrete IFN-γ after stimulation with PMA and ionomycin, and in this case their increase is not due to a bystander effect of IL-4 but cell intrinsic. Those results indicate that Bcl11b regulates development of different thymocyte subsets at multiple stages and prevents an excess of innate CD8SP thymocytes.

Zfat‐Deficient CD4+CD8+ Double‐Positive Thymocytes Are Susceptible to Apoptosis With Deregulated Activation of p38 and JNK

Zfat, which is a nuclear protein harboring an AT-hook domain and 18-repeats of C2H2 zinc-finger motif, is highly expressed in immune-related tissues, including the thymus and spleen. T cell specific deletion of the Zfat gene by crossing Zfat(f/f) mice with LckCre mice yields a significant reduction in the number of CD4(+) CD8(+) double-positive (DP) thymocytes. However, physiological role for Zfat in T cell development in the thymus remains unknown. Here, we found that Zfat-deficient DP thymocytes in Zfat(f/f)-LckCre mice were susceptible to apoptosis both at an unstimulated state and in response to T cell receptor (TCR)-stimulation. The phosphorylation levels of p38 and JNK were elevated in Zfat-deficient thymocytes at an unstimulated state with an enhanced phosphorylation of ATF2 and with an over-expression of Gadd45α⋅ On the other hand, the activation of JNK in the Zfat-deficient thymocytes, but not p38, was strengthened and prolonged in response to TCR-stimulation. All these results demonstrate that Zfat critically participates in the development of DP thymocytes through regulating the activities of p38 and JNK.

BI-31 * ANALYSIS AND QUANTIFICATION OF MULTIPLE ANTIGEN EXPRESSION IN GLIOBLASTOMA

Glioblastoma (GBM), one of the most common and fatal types of brain tumor, is marked by significant antigenic heterogeneity. Identification and quantification of tumor related antigens in the context of GBM tissue is an essential step towards developing antigen- targeted therapies. Immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) clinical specimens is a valuable technique for evaluating antigen expression in large study cohorts. To overcome the limitations of manual semi-quantitative scoring and subjectivity in the evaluation of IHC staining, we analyzed and quantified multiple antigens across entire tumor sections using Image Pro Premier v9.1 (DAB plug-in). For each slide, dense tumor regions (DTRs, tumor cells >60%), tumor infiltration regions (TIRs, tumor cells <50%) and pseudopalisading necrosis regions (PPNs) were defined from HE section by a neuropathologist. We quantified the expression of tumor-associated antigens IL13Rα2, HER2, EGFR and the proliferation marker Ki67 within these defined regions for 44 brain tumor samples (35 stage IV and 9 stage III). Our results demonstrate the heterogeneous expression patterns of IL13Rα2, HER2 and EGFR in GBMs. For example, in dense tumor regions 52%, 61% and 77% of samples showed IL13Rα2, HER2 or EGFR positivity, respectively. In correlation studies, 25% of samples were triple positive, 11% of samples showed double positivity for IL13Rα2 and HER2 or IL13Rα2 and EGFR, and 25% of samples were double positive of EGFR and HER2. Less than 7% of samples were negative for all three antigens. Interestingly, a higher percentage of samples showed triple positive expression in PPN regions (43%) versus the DTR (25%) and TIR (25%) regions. Also, Ki67 positivity was higher in PPN and DTR regions. In this study we developed methods for combining pathological annotations with DAB-capturing software, which allowed us to quantify protein expression in a more precise, consistent and efficient manner.

Prognostic significance of neural stem cell markers, Nestin and Musashi-1, in oral squamous cell carcinoma: expression pattern of Nestin in the precancerous stages of oral squamous epithelium.

Besides the tissue-specific stem cell markers, neural and hematopoietic stem cell markers were found to play an important role in carcinogenesis. Based on this background, we have investigated the expression pattern and prognostic significance of neural stem cell markers, Nestin and Musashi-1, in oral cancer. We used immunohistochemistry and immunofluorescence analyses to study the expression pattern and correlation between Nestin and Musashi-1 in oral squamous cell carcinoma. The Kaplan-Meier method was used to construct overall and disease-free survival curves, and the differences were calculated using log-rank test. Nestin expression was gradually increased in the transformation stages of oral cancer. Both Nestin and Musashi-1 expressions were associated with higher stage and poorly differentiated status of oral carcinoma. Interestingly, Nestin and Musashi-1 double positive cases showed statistically highly significant correlation with poorer survival of oral carcinoma patients. Expression of Nestin in the preneoplastic lesions indicates its role in the transformation of oral squamous epithelium. Clinicopathological and survival analyses suggest that Nestin and Musashi-1 might be associated with invasion, differentiation and poorer survival in oral squamous cell carcinoma. In addition to their role as independent prognostic indicators, Nestin and Musashi-1 double positivity can be used to select high-risk cases for effective therapy and this is the novel finding of this study. Nestin and Musashi-1 are found to be independent prognostic markers of oral cancer, and they might be used as molecular targets for effective therapy.

Role of type I interferon receptor signaling on NK cell development and functions.

Type I interferons (IFN) are unique cytokines transcribed from intronless genes. They have been extensively studied because of their anti-viral functions. The anti-viral effects of type I IFN are mediated in part by natural killer (NK) cells. However, the exact contribution of type I IFN on NK cell development, maturation and activation has been somewhat difficult to assess. In this study, we used a variety of approaches to define the consequences of the lack of type I interferon receptor (IFNAR) signaling on NK cells. Using IFNAR deficient mice, we found that type I IFN affect NK cell development at the pre-pro NK stage. We also found that systemic absence of IFNAR signaling impacts NK cell maturation with a significant increase in the CD27+CD11b+ double positive (DP) compartment in all organs. However, there is tissue specificity, and only in liver and bone marrow is the maturation defect strictly dependent on cell intrinsic IFNAR signaling. Finally, using adoptive transfer and mixed bone marrow approaches, we also show that cell intrinsic IFNAR signaling is not required for NK cell IFN-γ production in the context of MCMV infection. Taken together, our studies provide novel insights on how type I IFN receptor signaling regulates NK cell development and functions.

Exacerbation of autoimmune neuro-inflammation in mice cured from blood-stage Plasmodium berghei infection.

The thymus plays an important role shaping the T cell repertoire in the periphery, partly, through the elimination of inflammatory auto-reactive cells. It has been shown that, during Plasmodium berghei infection, the thymus is rendered atrophic by the premature egress of CD4+CD8+ double-positive (DP) T cells to the periphery. To investigate whether autoimmune diseases are affected after Plasmodium berghei NK65 infection, we immunized C57BL/6 mice, which was previously infected with P.berghei NK65 and treated with chloroquine (CQ), with MOG35–55 peptide and the clinical course of Experimental Autoimmune Encephalomyelitis (EAE) was evaluated. Our results showed that NK65+CQ+EAE mice developed a more severe disease than control EAE mice. The same pattern of disease severity was observed in MOG35–55-immunized mice after adoptive transfer of P.berghei-elicited splenic DP-T cells. The higher frequency of IL-17+- and IFN-γ+-producing DP lymphocytes in the Central Nervous System of these mice suggests that immature lymphocytes contribute to disease worsening. To our knowledge, this is the first study to integrate the possible relationship between malaria and multiple sclerosis through the contribution of the thymus. Notwithstanding, further studies must be conducted to assert the relevance of malaria-induced thymic atrophy in the susceptibility and clinical course of other inflammatory autoimmune diseases.

The effects of variations in genomic modules on breast cancer phenotype

The primary aim of this study is to present how expression heterogeneity is reflected in biological processes. We hypothesize that loss of normal regulatory mechanism is related with changed characteristics of a genomic module, which emerges its own phenotypes as a whole. In breast cancer, receptor status is related to degree of cancer variations. In this regard, comparing expression data according to receptor status is a valuable process to prove the hypothesis. For this reason, we investigated differences of genomic modules according to the receptor status. As a result, 4 and 3 genomic modules, which represent distinct biological processes, were obtained from estrogen receptor (ER)-/ progesterone receptor (PR)-positive double positive (DP) and ER-/PR-negative double negative (DN) breast cancer groups, respectively. Comparing the genomic modules between the groups, we found that there were differences of functional roles, which were mainly caused by DP-exclusive genes. The DP-exclusive genes showed variable expressions in the DN group but not in the DP group, and these expression heterogeneities in the DN group reflected transition of the genomic modules to have more malignant properties than in the DP group. In addition, the DP-exclusive genes themselves have a higher possibility to be involved in oncogenic processes in the DN group. In conclusion, this study suggests that expression heterogeneity of the DP-exclusive genes is not a result of deregulation, but a process that drives emergence of novel traits.

Direct detection of FoxP3 expression in thymic double-negative CD4-CD8- cells by flow cytometry.

Foxp3 expression is a marker of regulatory T cells (Treg), but how early it is expressed in the thymus is still not fully defined. In this study, we examined Foxp3 expression in double-negative (DN) CD4−CD8− T cell precursors in the thymus by flow cytometry. By increasing the number of collected cells from the conventional 104 cells up to more than 106 cells during flow cytometry, we found that DN cells exhibited higher Foxp3 expression than double-positive (DP) CD4+CD8+ and single-positive (SP) CD4+ or CD8+ (SP) T cells. CD44+ expression positively correlated with Foxp3 in thymic DN cells. Furthermore, TCR-β−CD25+ DN cells exhibited the highest frequency of Foxp3-expressing cells. Almost all Foxp3+ cells expressed CD25in DN cells. These results suggest that Foxp3 expression in DN cells can directly be detected by flow cytometry and it was positively corelated with CD25 and CD44 in DN cells.

Fli-1 regulates the DN2 to DN3 thymocyte transition and promotes γδ T-cell commitment by enhancing TCR signal strength.

Friend leukemia integration 1 (Fli-1) is a member of the Ets transcription factor family and is expressed during T-cell development; however, the role Fli-1 plays in early T-cell differentiation has not been elucidated. In this report, we demonstrate that in mouse, Fli-1 overexpression retards the CD4(-) CD8(-) double-negative (DN) to CD4(+) CD8(+) double-positive (DP) transition by deregulating normal DN thymocyte development. Specifically, Fli-1 expression moderates the DN2 and DN3 developmental transitions. We further show that Fli-1 overexpression partially mimics strong TCR signals in developing DN thymocytes and thereby enhances γδ T-cell development. Conversely, Fli-1 knockdown by small hairpin RNA reverses the lineage bias from γδ T cells and directs DN cells to the αβ lineage by attenuating TCR signaling. Therefore, Fli-1 plays a critical role in both the DN2 to DN3 transition and αβ/γδ lineage commitment.

Expression pattern of Notch intracellular domain (NICD) and Hes-1 in preneoplastic and neoplastic human oral squamous epithelium: their correlation with c-Myc, clinicopathological factors and prognosis in Oral cancer.

Notch pathway molecules crosstalk with Wnt/β-catenin signaling cascade in stem cells and tumors. However, the correlation between the expression pattern of Notch intracellular domain NICD, Hes-1 and c-Myc has not been studied in oral squamous cell carcinoma. The aim of this study is to investigate the correlation and prognostic significance of NICD, Hes-1 and c-Myc in oral cancer. Immunohistochemistry was used to study the expression pattern of NICD, Hes-1 and c-Myc in oral preneoplastic and neoplastic tissues. In addition, double immunofluorescence was used to examine the co-localization of NICD, Hes-1 and c-Myc in H314 cells. The expression pattern of NICD and Hes-1 was gradually increased from normal to dysplasia to carcinoma. Interestingly, statistically significant correlation was not observed between NICD, Hes-1 and c-Myc in oral squamous cell carcinoma. Furthermore, NICD+/c-Myc+ and Hes-1+/c-Myc+ double positive cases showed worst survival when compared with other cases in oral cancer. Notch signaling molecules, NICD and Hes-1, are found to be involved in the progression of oral squamous cell carcinoma. Interestingly, NICD, Hes-1 and c-Myc may have independent roles in oral cancer. On the other hand, we have demonstrated that NICD+/c-Myc+ and Hes-1+/c-Myc+ double positivity might be used as independent prognostic indicator of oral carcinoma.

Analysis of CD4+ CD8+ double-positive T cells in blood, cerebrospinal fluid and multiple sclerosis lesions.

T cells with a CD4(+) CD8(+) double-positive (DP) phenotype are present in small numbers in the peripheral blood of healthy humans and may have anti-viral capacities. Here we investigate numbers and function of DP T cells in patients with relapsing-remitting multiple sclerosis (MS), either treatment-naive or under therapy with natalizumab. Flow cytometry analysis revealed that frequencies of circulating DP T cells in treatment-naive and natalizumab-treated MS patients are comparable to healthy controls. These cells have a memory phenotype with cytotoxic potential, express high levels of CD49d and are similarly functional in treatment-naive as well as natalizumab-treated MS patients. DP T cells were enriched in the cerebrospinal fluid, but do not invade acutely inflamed MS lesions. In conclusion, DP T cells are functional in MS and may play a role in the immune surveillance of the central nervous system, but do not display functional impairment under natalizumab therapy.

IL-23 promotes TCR-mediated negative selection of thymocytes through the upregulation of IL-23 receptor and RORγt.

SummaryTransient thymic involution is frequently found during inflammation, yet the mode of action of inflammatory cytokines is not well defined. Here we report that interleukin-23 (IL-23) production by the thymic dendritic cells (DCs) promotes apoptosis of the CD4hiCD8hi double positive (DP) thymocytes. A deficiency in IL-23 signaling interferes with negative selection in the male Db/H-Y T-cell receptor (TCR) transgenic mice. IL-23 plus TCR signaling results in significant up-regulation of IL-23 receptor (IL-23R) expressed predominantly on CD4hiCD8hiCD3+αβTCR+ DP thymocytes, and leads to RORγt dependent apoptosis. These results extend the action of IL-23 beyond its peripheral effects to a unique role in TCR mediated negative selection including elimination of natural T regulatory cells in the thymus.

Electromagnetic TE scattering by a conducting cylinder coated with an inhomogeneous dielectric/magnetic material

The problem of electromagnetic TE scattering by an infinitely long conducting cylinder coated with an inhomogeneous dielectric/magnetic material is analyzed. A volume-surface integral equation (VSIE) approach is utilized to model the problem. By imposing the boundary conditions on the conducting surface, a surface magnetic field integral equation (MFIE) is developed. Inside the volume of the coating region, volume MFIEs are applied. The resultant integral equations are solved using the moment method. Numerical results for the bistatic radar cross section for different structures are presented. The results are validated using the exact series solution for a conducting circular cylinder coated with multilayers of homogeneous materials. Two types of coating materials are studied: the conventional or double positive (DPS) materials and the double negative (DNG) materials.

TCF-1 and LEF-1 act upstream of Th-POK to promote the CD4(+) T cell fate and interact with Runx3 to silence Cd4 in CD8(+) T cells.

TCF-1 and LEF-1 are essential for early T cell development, but their roles beyond the CD4+CD8+ double positive (DP) stage are unknown. By specific ablation in DP thymocytes, we demonstrated that deficiency in TCF-1 and LEF-1 diminished CD4+ T cell output and redirected CD4+ T cells to a CD8+ T cell fate. The role of TCF-1 and LEF-1 in CD4-CD8 lineage choice was partly mediated by direct positive regulation of Th-POK. Furthermore, loss of TCF-1 and LEF-1 unexpectedly caused CD4 derepression in CD8+ lineage-committed T cells without affecting the expression of Runx factors. Instead, TCF-1 physically interacted with Runx3 to cooperatively silence the Cd4 gene. Thus, TCF-1 and LEF-1 adopt distinct genetic wiring to program CD4+ fate decision and establish CD8+ T cell identity.

The transcriptional repressor NKAP is required for iNKT cell development and maturation (HEM3P.274)

Abstract Invariant Natural Killer T (iNKT) cells are an innate lineage of lymphocytes that can produce copious amount of cytokines within hours of stimulation. In the thymus, positive selection into the iNKT cell lineage occurs at the double positive (DP) stage, before maturation through various stages (stage 0-3) to complete development. NKAP, a transcriptional repressor, is required for conventional T lymphocyte development and maturation. NKAP interacts with Hdac3 for its repressor function. Previously, using CD4-cre NKAP cKO mice, we have shown that NKAP is required for positive selection into the iNKT cell lineage. To study the role of NKAP in later developmental stages, we generated PLZF-cre NKAP cKO mice with NKAP deletion occurring after entry into the iNKT lineage at stage 0. In the PLZF-cre NKAP cKO mice we observed a significant decrease in the absolute number of iNKTs with a 20-fold reduction at stage 2 and 200-fold reduction at stage 3. This defect is cell intrinsic and cannot be rescued by over-expression of Bcl-XL. Our preliminary results indicate that there may be a block in iNKT cell maturation, as expression of transcription factors Tbet, Gata3, and ROR-γt that define NKT1, NKT2 and NKT17 lineages respectively, are decreased in PLZF-cre NKAP cKO. PLZF-cre Hdac3 cKO mice have a similar defect in iNKT development. Thus, NKAP together with Hdac3 may regulate iNKT cell development and maturation.

Hybrid Vγ-Cβ TCR chains support T cell development and an immunocompetent T cell repertoire (LYM7P.713)

Abstract Developing T cells undergo VDJ recombination at four T cell receptor (TCR) loci (γ, δ, β and α) to generate a highly diverse γδ or αβ TCR. During the CD4-CD8- (DN) stage of development, simultaneous recombination at the TCRγ, TCRδ and TCRβ loci can result in unusual interchromosomal trans rearrangements between these loci, particularly between TCRβ and TCRγ, creating hybrid TCR genes encoded by two different chromosomes. We have found that in-frame trans rearrangements between the Vγ2 gene and a variety of Dβ and Jβ genes are transcribed, spliced and expressed on the surface of a small population of peripheral T cells. Vγ2-Cβ hybrid TCR chains are capable of mediating β-selection and require the expression of TCRα for subsequent developmental transitions. However, the relatively low frequency of these cells has made analysis of their function difficult. Here we report that transgenic expression of a Vγ2-Jβ2.4 trans rearrangement is capable of driving T cell development in the thymus through the CD4/CD8 double positive (DP) and single positive (SP) stages as well as maintaining a robust population of CD4+ and CD8+ T cells in the periphery. These mice are also capable of mounting a T-dependent IgG1 response to peptide immunization. These data suggest that non-conventional Vγ-Cβ TCR chains are sufficient to support the development of an immunocompetent T cell repertoire.

Gene targeting of RhoA reveals its essential role in thymocyte development and metabolism (LYM7P.718)

Abstract RhoA GTPase is an intracellular signal transducer capable of regulating a wide range of cell functions including cytoskeleton dynamics, proliferation, and survival. In T lymphocytes, studies by using dominant negative mutant or C3 transferase expressing transgenic mice suggest that RhoA is involved in TCR signaling and related T cell functions such as polarization, migration, survival, and proliferation. To date, the physiological role of RhoA in T cell development remains unclear. In this study, we have achieved T cell-specific deletion of RhoA and found that RhoA gene disruption caused (1) a drastic decrease in the numbers of all thymocyte subsets including CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), CD4+CD8- single positive (SP), and CD4-CD8+ SP T cells, (2) an impaired β-selection at DN stage and positive selection at DP stage associated with altered pre-TCR and TCR signaling transduction, respectively, (3) a diminished thymocyte proliferation which could be due to impaired cytokinesis, (4) an increased apoptosis, and (5) an enhanced mitochondrial function as manifested by increased oxidative phosphorylation, glycolysis, mitochondrial membrane potential, mitochondrial numbers, mitochondrial DNA and ROS production. Thus, RhoA critically regulates T cell development by impacting on β-selection, positive selection, cell proliferation and survival through mediating pre-TCR signaling, TCR signaling, cytokinesis, and metabolism.