Double-positive Subset Research Articles

Elevated checkpoint inhibitor expression and Treg cell number in autosomal dominant polycystic kidney disease and their correlation with disease parameters and hypertension.

Autosomal dominant polycystic kidney disease (ADPKD) has cancer-like pathophysiology. In this study, we aimed to investigate the phenotype of peripheral blood (PB) T cell subsets and immune checkpoint inhibitor expression of ADPKD patients across different chronic kidney disease (CKD) stages. Seventy-two patients with ADPKD and twenty-three healthy controls were included in the study. The patients were grouped into five different CKD stages, according to glomerular filtration rate (GFR). PB mononuclear cells were isolated and T cell subsets and cytokine production were examined by flow cytometry. CRP levels, height-adjusted total kidney volume (htTKV), rate of hypertension (HT) differed significantly across different GFR stages in ADPKD. T cell phenotyping revealed significantly elevated CD3+ T cells, CD4+, CD8+, double-negative, and double-positive subsets and significantly elevated IFN-γ and TNF-α producing subsets of CD4+, CD8+ cells. The expression of checkpoint inhibitors CTLA-4, PD-1, and TIGIT by T cell subsets was also increased to various extent. Additionally, Treg cell numbers and suppressive markers CTLA-4, PD-1, and TIGIT were significantly elevated in ADPKD patients' PB. Treg CTLA4 expression and CD4CD8DP T cell frequency in patients with HT were significantly higher. Lastly, HT and increased htTKV and higher frequency of PD1+ CD8SP were found to be risk factors for rapid disease progression. Our data provide the first detailed analyses of checkpoint inhibitor expression by PB T cell subsets during stages of ADPKD, and that a higher frequency of PD1+ CD8SP cells is associated with rapid disease progression.

Abstract 4476: PD-1 and TIGIT co-expression identifies a circulating CD8 T cell population predictive of response to anti-PD-1 therapy in melanoma and Merkel-cell carcinoma patients

Abstract Immunotherapies targeting the PD-1 pathway have profoundly transformed the clinical care of cancer patients for a growing variety of cancer types. However, most patients do not experience durable clinical benefit. The definition of robust and convenient biomarkers of PD-1 therapy efficacy to stratify patients beforehand or early after initiation of the therapy that could guide therapeutic management is still lacking while being a very active research field. Biomarkers described to date include tumor burden, neoantigen load, presence and number of PD-1+ CD8+ at the tumor margin, T-cell inflamed tumor microenvironment and PD-L1 expression by the tumor cells or other immune cells and composition of the gut microbiota. Most of these parameters are closely related/influenced by the presence, activation status and functional capacities of CD8+ T cells infiltrating the tumor site demonstrating their pivotal role for anti-PD-1 mediated anti-tumor efficacy. A population of PD-1high CD8 TILs was consequently described as predictive of PD-1 blockade in NSCLC. The exact contribution for clinical efficacy of TILs versus distinct CD8+ T cells from peripheral origins recirculating to the tumor site remains to be elucidated. Notably, immunological responses to PD-1 blockade at the periphery were described within the very first days following the first therapy dose. Therefore, describing circulating cellular population predictive of PD-1 inhibitor efficacy could represent a convenient, non-invasive and rapid method to assess anti-tumor benefits. Original findings reported in this study identified a circulating CD8 T cell population delineated by the co-expression of TIGIT and PD-1 inhibitory receptors as an early immune marker of anti-PD-1 efficacy in three independent cohorts of cancer patients (two melanoma patient's cohorts and one Merkel-cell carcinoma patient's cohort). The frequency of this double positive (DPOS) population even appeared predictive of PD-1 inhibitor therapy efficacy at baseline in the MCC cohort. Furthermore, to understand the mechanistical relevance of this subset for PD-1 blockade efficacy, we thoroughly described this DPOS T cell subset by flow cytometry, gene expression analysis, anti-tumor reactivity assay and TCR repertoire analysis, and compared it to its double negative (DNEG), PD-1 and TIGIT single positive counterparts. This DPOS subset was enriched in activated and proliferative T cells, retained expression of co-stimulatory molecules and was enriched for common features with Tfc. Moreover, this subpopulation exhibited a specific gene signature, strongly predictive of long-term survival in melanoma patients (TCGA analyses). We demonstrated that this subpopulation was enriched in tumor-specific T-cells (ELISPOT analysis against 11 antigen-derived peptides). Finally, clustering of TCR clonotypes revealed that the DPOS T cell population was significantly enriched in emerging clonotypes in responding patients, after 1 month of anti-PD-1 therapy echoing recent work from others. Our findings provide a compelling rationale to measure PD-1+TIGIT+ CD8 T-cell subset in the blood of cancer patients to monitor early anti-PD1 mediated clinical efficacy, and to use DPOS T cells as a window to study the dynamic changes that underly successful antitumor immunity. Citation Format: Sylvain Simon, Valentin Voillet, Virginie Vignard, Zhong Wu, Camille Dabrowski, Nicolas Jouand, Tiffany Beauvais, Amir Khammari, Cecile Braudeau, Regis Josien, Olivier Adotevi, Caroline Laheurte, Francçois Aubin, Charles Nardin, Samuel Rulli, Raphaël Gottardo, Nirasha Ramchurren, Cheever Martin, Steven P. Fling, Candice D. Church, Paul Nghiem, Brigitte Dreno, Stanley R. Riddell, Nathalie Labarriere. PD-1 and TIGIT co-expression identifies a circulating CD8 T cell population predictive of response to anti-PD-1 therapy in melanoma and Merkel-cell carcinoma patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4476.

The Levels of a G-CSF-Inducible pSTAT3+pSTAT5+ Subpopulation of MDS Progenitors with Leukemic Stem Cell Phenotype Predict the Response to Azacytidine

AbstractAbstract 3795Both basal and cytokine-induced phosho-STAT3 and pSTAT5 levels are altered in leukemias and profiling the STAT signaling network at the single cell level provides prognostic information (Irish et al, 2004). As cell signaling interacts with the epigenome (Mohammad et al, 2010) and numerous genes involved in signaling are aberrantly methylated in MDS (Figueroa et al, 2010) we investigated the alterations of STAT3/5 signaling in MDS progenitors during azacitidine therapy.Bone marrow samples of 58 high-risk MDS and AML/MDS patients were obtained before (d0) and 15 days (d15) after azacytidine initiation and at the indicated time points. According to the IWG response criteria, patients were divided into 4 groups: response (CR and PR, n=11 and n=5, respectively), hematologic improvement (HI, n=8), stable disease (SD, n=11) and failure (F, n=23). Mononuclear cells were either left untreated or stimulated with G-CSF and GM-CSF for 15′ and then stained intracellularly with the indicated antibodies. The hematopoietic hierarchy and G-CSF receptor (CFS3R) transcripts by qPCR were assessed on immunomagnetically purified Lin-CD34+ and CD34+ cells, respectively. Statistical comparisons were done by ANOVA and unpaired or paired t-tests as appropriate. STAT signaling profiles (SPs) in CD34+ cells were grouped using complete linkage hierarchical clustering and correlated with treatment response, karyotype and transfusion rate by using χ2 or Fisher Exact tests.Unsupervised clustering of pretreatment SPs in CD34+ cells identified 2 clusters (Fig 1A) mainly differing in the degree of potentiated STAT3/5 phosphorylation. Patients in cluster I displayed weak potentiated expression of pSTAT3/5 and had marginally better response to azacytidine compared to the ones in cluster II (p=0.06), whereas there were no differences among the two groups regarding the MDS subtype (p=0.41), karyotype (p=0.45) and transfusion rate (p=0.33). We further identified a G-CSF-inducible pSTAT3/5 double positive (DP) subpopulation of MDS CD34+ cells, whose levels both at d0 and d15 of the 1st cycle were inversely associated with response (Fig 1B), whereas its kinetics were following the disease course and response to azacytidine (Fig 1C,D). The DP subset was enriched in GMPs compared to the G-CSF-unresponsive pSTAT3/5 double negative (DN) subpopulation, suggesting a higher leukemia initiating cell activity (Goardon et al, 2011), whereas the DN subset was marginally enriched in MEPs (Fig 2). Also, compared to the DN subset, the DP population exhibited decreased Ki67 expression and increased Bcl2 and p53 levels (Fig 3A), indicating quiescence and increased antiapoptotic and tumor suppressive properties, respectively. Of note, the levels of the above molecules in the two subsets remained unaltered both at d0 and d15, indicating that these subsets represent genuine cellular entities with solid properties. To determine if CSF3R expression and/or its modulation by azacitidine are responsible for the alterations of the DP population, we checked CSF3R levels in isolated CD34+ cells from patients with either absence or full expression of the DP subset because CSF3R is downregulated after G-CSF stimulation. The two groups showed identical protein and mRNA levels of CSF3R at both d0 and d15 (Fig 3B,C).In summary, we identified a G-CSF-inducible pSTAT3/5 DP subpopulation with leukemic stem cell properties, which is potentially involved in MDS biology and epigenetically modulated, as implied by its kinetics during hypomethylating therapy. More important, given the strong association of pretreatment levels of the DP subset with the response to azacitidine, the latter subset may serve both as a treatment target and response biomarker. [Display omitted] [Display omitted] [Display omitted] Disclosures:Kotsianidis:Genesis-Pharma: Honoraria, Research Funding.

Development of a Novel Method for in Vitro Analysis of CD8 Thymocyte Selection and Maturation,

Abstract 3235T cell development relies on the interaction between the T-cell receptor (TCR) on thymocytes and the self-major histocompatibility complex (MHC) expressed on thymic epithelial cells in the thymus. This process, called positive selection, rescues developing thymocytes from cell death while leading to their differentiation into mature T cells. Since it is believed that the proper development of CD8 T cells requires an intact thymus, several groups studied their development using fetal or reaggregation thymus organ cultures in vitro. Unfortunately, these models were shown to be cumbersome requiring a complicated set-up while generating limited cellular yield. Thus, we sought of developing a novel in vitro system using bone marrow-derived stromal cells to support CD8 T cell development and maturation in vitro. We selected the OTI system as a working model due to the availability of previously identified positively selecting peptides. Non-selected T-cell-committed double-positive (DP) OTI thymocytes (CD4+CD8+CD69−) were first fractionated based on the surface expression intensity of both TCR and CD5. These 3 subsets designated as TCRloCD5lo (DP1), TCRintCD5hi (DP2), and TCRhiCD5int (DP3) express different levels of ZAP70. Following fractionation, the DP subsets were co-cultured with bone marrow-derived stromal cells presenting OTI-selecting peptides. In the absence of cytokines, no CD8+ OTI cell development occurred in vitro. When repeated in the presence of γc-cytokines (IL2, IL4, IL7, IL9, IL15 and IL21) only rIL4 and rIL7 were able to induce CD8 T cell development. Supplementing the co-culture system with rIL4 led to the generation of 50–60% single-positive (SP) CD8 T cells only from the DP3 fraction whereas rIL7 induced the development of a minor fraction of CD8 T cells from DP2s (3–4%) and a major population from DP3 (50–76%). Furthermore, we found that rIL4 treatment triggers the development of 2 distinct populations of SP OTI cells (based on their CD8 expression intensity) which we termed CD8int and CD8hi. When analyzed by flow-cytometry, ex vivo generated CD8int, but not CD8hi, expressed high levels of CD69, PD-L1 and CD44. In contrast, SP CD8 T cells developed in the presence of rIL7 did not upregulate these markers. Since IL7 promotes survival and proliferation of TCR-triggered DPs while IL4 affects their differentiation, we admixed both cytokines during the co-culture and found a dominant rIL4 effect: the phenotype of SP CD8 T cells was similar to that induced by rIL4 alone. Taken together, our findings demonstrate that some DP thymocytes are efficiently selected in our system by OTI-specific positively selecting peptides. Notably, the addition of rIL7 leads to the development and maturation of classic CD8 T cells whereas rIL4 induces both classic and innate CD8 T cells.This work was supported by grant a from CIHR. Disclosures:No relevant conflicts of interest to declare.

410 HER2 as a relevant molecule in tumour initiating cells

Background. Anterior mediastinal masses derive from a variety of diseases. Thymomas have been shown to commonly hold CD4+CD8+ double-positive (DP) lymphocytes, and identification of this subset by two-color flow cytometric study was suggested to help diagnosis of thymoma. Several other thymic diseases, however, possibly hold CD4+CD8+ DP lymphocytes. In this study, we utilized the three-color flow cytometric method for further examination of the phenotypes of lymphocytes in the thymic diseases.Materials and methods. One hundred eight specimens (77 primary and 10 metastatic thymomas, 10 thymic carcinomas, 2 thymic carcinoids, 4 malignant lymphomas, 2 seminomas, an inflammatory pseudotumor, and 2 nonneoplastic thymic hyperplasias) were subjected to the study. The expressions of CD3, CD4, and CD8 on tumor-associated lymphocytes were evaluated by three-color flow cytometric study.Results. The proportion of the CD4+CD8+ DP subset was more than 30% in all 78 lymphocyte-rich thymomas, in 2 malignant lymphomas, and in both thymic hyperplasias. CD3 expression of the CD4+CD8+ DP subset ranged from a negative to a high level in thymomas and thymic hyperplasias, while it was restricted to a particular level in CD4+CD8+ DP-type malignant lymphomas. The proportion of CD3+ cells in the CD4+CD8− single-positive subset was consistently less than 90% in the lymphocyte-rich thymomas, while it was more than 90% in the thymic hyperplasias.Conclusion. Although identification of the CD4+CD8+ DP subset in the tumor-associated lymphocytes does not necessarily indicate thymoma, a further characterization of thymic neoplasms possessing the CD4+CD8+ DP subset was enabled by three-color flow cytometric study, suggesting the utility of this method as an ancillary tool for differential diagnosis of these diseases.

ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

The first definitive cell fate decision in development occurs at the blastocyst stage with establishment of the trophoblast and embryonic cell lineages. In the mouse, lineage commitment is achieved by epigenetic regulation of a critical gatekeeper gene, the transcription factor Elf5, that reinforces placental cell fate and is necessary for trophoblast stem (TS) cell self-renewal. In humans, however, the epigenetic lineage boundary seems to be less stringent since human embryonic stem (ES) cells, unlike their murine counterparts, harbour some potential to differentiate into trophoblast derivatives. Here, we show that ELF5 is expressed in the human placenta in villous cytotrophoblast cells but not in post-mitotic syncytiotrophoblast and invasive extravillous cytotrophoblast cells. ELF5 establishes a circuit of mutually interacting transcription factors with CDX2 and EOMES, and the highly proliferative ELF5(+)/CDX2(+) double-positive subset of cytotrophoblast cells demarcates a putative TS cell compartment in the early human placenta. In contrast to placental trophoblast, however, ELF5 is hypermethylated and largely repressed in human ES cells and derived trophoblast cell lines, as well as in induced pluripotent stem cells and murine epiblast stem cells. Thus, these cells exhibit an embryonic lineage-specific epigenetic signature and do not undergo an epigenetic reprogramming to reflect the trophoblast lineage at key loci such as ELF5. Our identification of the trophoblast-specific transcriptional circuit established by ELF5 will be instrumental to derive human TS cell lines that truly reflect early placental trophoblast and that will be most beneficial to gain insights into the aetiology of common pregnancy complications, including intra-uterine growth restriction and pre-eclampsia.

Regulation of Zap70 Expression During Thymocyte Development Enables Temporal Separation of CD4 and CD8 Repertoire Selection at Different Signaling Thresholds

To investigate the temporal regulation of the commitment of immature thymocytes to either the CD4(+) or the CD8(+) lineage in the thymus, we developed a transgenic mouse that expressed a tetracycline-inducible gene encoding the tyrosine kinase zeta chain-associated protein kinase of 70 kD (Zap70), which restored development in Zap70(-/-) thymocytes arrested at the preselection, CD4(+)CD8(+) double-positive (DP) stage. After induction of the expression of Zap70 and the production of Zap70 protein, CD4(+) single-positive (SP) cells that expressed Zbtb7b (which encodes the CD4(+) T cell-associated transcription factor ThPOK) became abundant within 30 hours, whereas CD8(+) SP cells were not detectable until day 4. We found that mature CD4(+) and CD8(+) cells arose from phenotypically distinct subsets of DP thymocytes that developed with different kinetics and contrasting sensitivities to stimulation of the T cell antigen receptor (TCR). In wild-type mice, expression of endogenous Zap70 progressively increased during maturation of the DP subsets, and the abundance of Zap70 protein determined the sensitivity of the cells to stimulation of the TCR. This temporal gradient in the amount of Zap70 protein enabled the selection of CD4(+) and CD8(+) repertoires in separate temporal windows and at different TCR signaling thresholds, thereby facilitating discrimination of distinct positive selection signals in these lineages.

1108 Preliminary evidences for recruitment of innate responses to rectal cancer cell death elicited by neo-adjuvant radio-chemotherapy

Introduction: Rectal cancer patients with a cT3N+M0 tumor stage responds to the neo-adjuvant therapy, which causes necrosis and inflammation in situ. We cannot predict which patients will response. Methods: We collected blood and tissue samples at three time points: at diagnosis, at the end of the first CT cycle and at 8 week after the end of the therapy (coinciding with the surgical resection time of the tumour). At each time point we characterized circulating monocytes by flow cytometry, infiltrating macrophages by immunohistochemistry and selected inflammatory molecules in serum and plasma. Results: We recruited 28 pts, with No substantial changes were detectable in the number of circulating monocytes. In contrast we observed a clear expansion of CD14/CD86 and CD14/CD163 double positive subsets. In fact we observed that in the responder patients the expansion of the CD14/86 subset was clear in the first weeks of treatment and decreased there after. The immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative M2 polarization. Conclusion: These data suggest that neoadjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors.

Characterization of innate responses elicited by neoadjuvant radio-chemotherapy for rectal cancer

e15044 Background: The neoadjuvant chemo-radiotherapy (CT-RT) has improved the treatment of locally advanced rectal cancer reducing the local recurrence. However a survival benefit has not been reached yet. In order to increase the rate of pathological complete remissions in our Institution we intensified both the CT schedule adding oxaliplatin to 5-FU and the RT program with tomotherapy. The aim of this study was to verify: whether the pattern of innate response elicited by the neoadjuvant CT-RT is heterogeneous among pts and whether this information can be used to identify which pts will benefit from the treatment. Methods: We collected samples of T3N+M0 rectal cancer pts before, during and after neoadjuvant CT-RT (3 cycles of oxaliplatin + 5-FU; 45 Gy). At each time point we characterized circulating monocytes by flow cytometry, infiltrating macrophages by immunoistochemistry (IHC) and selected inflammatatory molecules by ELISA.Results: We recruited so far 25 pts, of whom 10 have reached the surgery with three pathological complete remission and four down staging. No substantial changes were detectable in the number of circulating monocytes. In contrast we observed a clear expansion of CD14/CD86 and CD14/CD163 double positive subsets. This event was transient and apparently causally related to the treatment since it abated at the later time point. Moreover, it correlated with sensitivity to the treatment: 5/7 pts who underwent disease regression had an early and transitory increase of the number of CD14/CD86 and CD14/CD163 positive cells, which was absent or negligible in non responder pts. The IHC study revealed a massive tumoral infiltration by macrophages which displayed clear features of alternative M2 polarization as assessed by expression of the CD163 and 206 scavenger receptors. A subset of pts had elevated PTX3 and low CRP concentration at the onset of treatment. PTX3 concentration abated after the first CT cycle. Conclusions: These data suggest that neoadjuvant CT-RT modulates the cellular components of innate immune responses, that could represent valuable predictive factors. No significant financial relationships to disclose.

Human thymic epithelial cell (TEC) culture system for promoting T lineage commitment and development of T cells from cord blood and bone marrow stem cells (86.6)

Abstract The discovery of Delta-like-1 (Dl1)/Notch signaling as the earliest requirement for T lineage commitment and development makes it possible to generate T cells from hematopoietic stem cells in vitro. The mouse bone marrow stroma OP9 cells that overexpress Dl1is the first stroma cell line that is capable of promoting the development of both mouse and human T cells; however, a similar approach with human stroma cells has not been established. To develop a similar culture system to closely emulate human T cell development, we have generated a human TEC line from a pediatric thymus and expressed the mouse Dl1 in this line (TEC84-Dl1). The TEC84-Dl1 expresses CD104, CD29, CD49f, CD54 (ICAM-1), and CD106 (VCAM-1); these cells also express HLA-ABC and HLA-DR. When TEC84-Dl1 were co-cultured with cord blood CD34posCD45RAposCD38neg cells, we were able to generate preT/NK (CD34posCD7posCD1aneg), preT (CD34posCD7posCD1apos), immature single positive and double positive subsets; the co-culture system also generated a CD3pos subset. Similar results were also obtained with CD34pos cells isolated from adult bone marrow. Thus, our co-culture system with the human TEC provides a complete human system for replicating thymopoiesis in vitro and offers a novel tool to optimize the development of T lineage cells that would benefit cord blood and bone marrow transplant recipients (Supports: AG023809 to PL, IDPH to PS, AI068390 to KK).

MicroRNA-Mediated Gene Regulation Effectively Restricts In Vivo Transgene Expression in Hematopoietic Stem Cell Progeny.

Stem cell engineering and targeted in vivo gene delivery increasingly require tight control of transgene expression. Lineage- and differentiation stage-specific gene regulation is classically afforded by pol-II-dependent transcript regulation. A super-imposed layer of post-transcriptional control would be valuable to correct undesirable expression patterns or fine-tune developmentally regulated or inducible gene expression. microRNAs (miRNAs) have recently emerged as potent repressors of gene expression at the post-transcriptional level. In this study, we investigate the potential of using miRNA regulation to provide lineage-restricted expression, exploiting miRNAs with distinctive expression patterns in hematopoietic tissue. miR-223 is preferentially expressed in granulocytes and monocytes (80-and 110-fold, respectively). miR-181a is highly expressed in B-lymphocytes and, particularly, in thymocytes, but down-regulated (∼1000-fold) in post-thymic T cells. We, therefore, constructed lentiviral vectors encoding either green fluorescent protein (GFP) or an antigen-specific receptor, placed under the transcriptional control of the ubiquitous EF1a promoter and tagged with miRNA-recognition elements (MREs) complementary to the mature miR-223 or miR-181a. In a panel of murine and human cell lines expressing varying levels of the two miRNAs we find that GFP knockdown is dependent on the presence of the miRNA and directly proportional to the number of MRE repeats. Four copies of the repeat permit better down-regulation than 2 copies (albeit depending on the level of endogenous miRNA). Two different MREs can be combined in tandem, resulting in additive down-regulation. In vivo, in mouse bone marrow chimeras harboring the miR-223-responsive vector, GFP expression is specifically repressed in myeloid cells (>85% compared to the control vector lacking miRNA target sequences). Reciprocally, chimeras harboring the miR-181a-regulated vector express GFP in myeloid and erythroid lineages, but transgene expression is profoundly repressed in thymocytes and B-cell progenitors. A vector harboring a composite MRE confers GFP expression almost exclusively confined to the erythroid lineage. These results demonstrate for the first time that transgene expression can be selectively regulated at the post-transcriptional level within the hematopoietic tissue. In mouse chimeras expressing a CD19-specific antigen receptor (which we and others are currently using in clinical trials in B cell malignancies), we analyze receptor expression in the 4 double negative subsets (DN1, DN2, DN3 and DN4), the double-positive subset (DP), and the CD4+ and CD8+ single positive (SP) subsets. We detail and quantify receptor knock-down at each developmental stage, and show that miR-181a-mediated regulation prevents receptor expression at critical stages of positive and negative thymic selection. Antigen receptor expression is dramatically repressed in DN and DP cells, while rising in CD4+ and CD8+ SP thymocytes. Importantly, expression is fully restored in post-thymic T cells, and maintained in activated T cells. miRNA-mediated post-transcriptional regulation is thus proving to be a powerful means to direct lineage- and differentiation stage-specific transgene expression in genetically modified stem cells.

Antagonistic Interactions between Ikaros and the Chromatin Remodeler Mi-2β Determine Silencer Activity and Cd4 Gene Expression

Lineage commitment is induced by changes in gene expression dictated by the intimate interaction between transcription factors and chromatin regulators. Here, we revealed the antagonistic interplay between Ikaros and its associate the chromatin remodeler Mi-2beta during T cell development, as exemplified by the regulation of Cd4 expression. Loss of Ikaros or Mi-2beta led to activation or repression, respectively, of the Cd4 locus at inappropriate stages of development. Their combined mutation reverted to normal CD4 expression. In double-negative thymocytes, Ikaros binding to the Cd4 silencer contributed to its repressive activity. In double-positive thymocytes, concomitant binding of Mi-2beta with Ikaros to the Cd4 silencer caused silencer inactivation, thereby allowing for CD4 expression. Mi-2beta facilitated recruitment of histone acetyl transferases to the silencer. This recruitment possibly antagonized Ikaros and associated repressive activities. Thus, concomitant interactions between functionally opposing chromatin-regulating machineries are an important mode of gene regulation during lineage determination.

Notch-induced T cell development requires phosphoinositide-dependent kinase 1

Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.

Three-color flow cytometric study on lymphocytes derived from thymic diseases.

Anterior mediastinal masses derive from a variety of diseases. Thymomas have been shown to commonly hold CD4(+)CD8(+) double-positive (DP) lymphocytes, and identification of this subset by two-color flow cytometric study was suggested to help diagnosis of thymoma. Several other thymic diseases, however, possibly hold CD4(+)CD8(+) DP lymphocytes. In this study, we utilized the three-color flow cytometric method for further examination of the phenotypes of lymphocytes in the thymic diseases. One hundred eight specimens (77 primary and 10 metastatic thymomas, 10 thymic carcinomas, 2 thymic carcinoids, 4 malignant lymphomas, 2 seminomas, an inflammatory pseudotumor, and 2 nonneoplastic thymic hyperplasias) were subjected to the study. The expressions of CD3, CD4, and CD8 on tumor-associated lymphocytes were evaluated by three-color flow cytometric study. The proportion of the CD4(+)CD8(+) DP subset was more than 30% in all 78 lymphocyte-rich thymomas, in 2 malignant lymphomas, and in both thymic hyperplasias. CD3 expression of the CD4(+)CD8(+) DP subset ranged from a negative to a high level in thymomas and thymic hyperplasias, while it was restricted to a particular level in CD4(+)CD8(+) DP-type malignant lymphomas. The proportion of CD3(+) cells in the CD4(+)CD8(-) single-positive subset was consistently less than 90% in the lymphocyte-rich thymomas, while it was more than 90% in the thymic hyperplasias. Although identification of the CD4(+)CD8(+) DP subset in the tumor-associated lymphocytes does not necessarily indicate thymoma, a further characterization of thymic neoplasms possessing the CD4(+)CD8(+) DP subset was enabled by three-color flow cytometric study, suggesting the utility of this method as an ancillary tool for differential diagnosis of these diseases.

Alterations during Positive Selection in the Thymus of nackt CD4‐Deficient Mice

The T‐cell repertoire is shaped by the positive and negative selection of immature CD4+ CD8+ double positive (DP) thymocytes. Positive selection of DP T cells to the CD4+ CD8− and CD4− CD8+ simple positive (SP) lineages is a multistep process which involves cellular interactions between thymocytes and stromal cells. Mutant nackt (nkt/nkt) mice have been shown to have a deficiency in the CD4+ CD8− T‐cell subset both in the thymus and in the periphery. The present report suggests that nkt/nkt mice present alterations in early steps of positive selection because they show decreases in the percentages of CD69+ and CD5+ cells within the DP subset. Experiments involving bone marrow transfer and thymic chimeras demonstrate that the thymic epithelium of nkt/nkt mice is involved in the alterations registered during positive selection and dictates the ultimate fate of CD4+ SP cells.

Alterations during positive selection in the thymus of nackt CD4-deficient mice.

The T-cell repertoire is shaped by the positive and negative selection of immature CD4(+) CD8(+) double positive (DP) thymocytes. Positive selection of DP T cells to the CD4(+) CD8(-) and CD4(-) CD8(+) simple positive (SP) lineages is a multistep process which involves cellular interactions between thymocytes and stromal cells. Mutant nackt (nkt/nkt) mice have been shown to have a deficiency in the CD4(+) CD8(-) T-cell subset both in the thymus and in the periphery. The present report suggests that nkt/nkt mice present alterations in early steps of positive selection because they show decreases in the percentages of CD69(+) and CD5(+) cells within the DP subset. Experiments involving bone marrow transfer and thymic chimeras demonstrate that the thymic epithelium of nkt/nkt mice is involved in the alterations registered during positive selection and dictates the ultimate fate of CD4(+) SP cells.

Response Differences between Human CD4+ and CD8+ T-Cells during CD28 Costimulation: Implications for Immune Cell-Based Therapies and Studies Related to the Expansion of Double-Positive T-Cells during Aging

Since CD28 costimulation is critical for T-cell activation, there is great interest in CD28 as a target for immuntherapeutic approaches. We show that stimulation of human CD4(+) and CD8(+) T-cells differs in their responsiveness to stimulation with anti-CD3/CD28-coated beads, as surrogate antigen-presenting cells. While the CD4(+) subset responded with sustained proliferation, CD8(+) T-cells grew for a limited period only and failed to produce IL-2 beyond the first few days in culture. This decrease is accompanied with an increased rate of apoptosis in CD8(+) T-cells despite Bcl-x(L) expression. The CD8(+) but not the CD4(+) subset developed a reversible double-positive phenotype during CD28 costimulation. This finding may have some bearing on the appearance of double-positive T-cells in human peripheral blood. This double-positive subset was shown to undergo a statistically significantly increase during aging in humans. Taken together, the above data have important implications for immunotherapy and immune senescence.

In vitro differentiation of a CD4/CD8 double-positive equivalent thymocyte subset in adult Xenopus.

The thymus is the major site of selection and differentiation of T cells in mammals and birds. To begin to study the evolution of thymocyte differentiation, we have developed, in the frog Xenopus, an in vitro system that takes advantage of cortical thymocyte antigen (CTX), a recently discovered T cell antigen whose expression is restricted to Xenopus cortical thymocytes. Upon transient stimulation with suboptimal mitogenic concentrations of the phorbol ester phorbol myristate acetate (PMA) plus ionomycin, Xenopus thymocytes are induced to differentiate into cycling T lymphoblasts that actively synthesize and express high levels of surface MHC class I and class II molecules. This appearance of T lymphoblasts correlates with a rapid down-regulation of both surface CTX protein and CTX mRNA. A thymocyte subset with an immature phenotype (CTX+, CD8+, class II- or class II low and class I-) was characterized by depleting class II+ cells or by panning with anti-CTX mAb. This immature CTX+ thymocyte subset displays a limited proliferative capacity compared to total, class II+ or to CTX- thymocytes, and can be induced, by PMA/ionomycin, to differentiate into more mature T lymphoblasts expressing surface class II and class I molecules. These results provide the first in vitro evidence in an ectothermic vertebrate of a conserved intrathymic pathway of thymocyte differentiation. In addition, our data reveal that CTX can serve as a differentiation surface marker of a population of immature thymocytes that appears to be the equivalent of the mammalian CD4/CD8 double-positive subset.

Oligoclonality of Rat Intestinal Intraepithelial T Lymphocytes: Overlapping TCR β-Chain Repertoires in the CD4 Single-Positive and CD4/CD8 Double-Positive Subsets

AbstractPrevious studies in humans and mice have shown that gut intraepithelial lymphocytes (IELs) express oligoclonal TCR β-chain repertoires. These studies have either employed unseparated IEL preparations or focused on the CD8+ subsets. Here, we have analyzed the TCR β-chain repertoire of small intestinal IELs in PVG rats, in sorted CD4+ as well as CD8+ subpopulations, and important differences were noted. CD8αα and CD8αβ single-positive (SP) IELs used most Vβ genes, but relative Vβ usage as determined by quantitative PCR analysis differed markedly between the two subsets and among individual rats. By contrast, CD4+ IELs showed consistent skewing toward Vβ17 and Vβ19; these two genes accounted collectively for more than half the Vβ repertoire in the CD4/CD8 double-positive (DP) subset and were likewise predominant in CD4 SP IELs. Complementarity-determining region 3 length displays and TCR sequencing demonstrated oligoclonal expansions in both the CD4+ and CD8+ IEL subpopulations. These studies also revealed that the CD4 SP and CD4/CD8 DP IEL subsets expressed overlapping β-chain repertoires. In conclusion, our results show that rat TCR-αβ+ IELs of both the CD8+ and CD4+ subpopulations are oligoclonal. The limited Vβ usage and overlapping TCR repertoire expressed by CD4 SP and CD4/CD8 DP cells suggest that these two IEL populations recognize restricted intestinal ligands and are developmentally and functionally related.

Double-positive thymocytes resistant to antigen-MHC-induced negative selection lack active caspase.

Thymocytes bearing autoreactive TCR are eliminated from the organism by a process termed negative selection. The molecular basis of this deletion has been recently shown to be a consequence of TCR-triggered activation of a caspase by certain peptide-MHC ligands in the immature CD4+CD8+ double-positive (DP) thymocyte subpopulation. Of note, the numerically minor TCRhigh DP thymocyte subpopulation, unlike the major TCRlow DP subset, is resistant to negative selection. Despite exposure to cognate peptide, TCRhigh DP thymocytes mature into single-positive thymocytes and are exported into the periphery. Here we investigated the mechanism by which these thymocytes escape negative selection. Using a cytochemical assay in conjunction with a caspase-specific affinity ligand, we demonstrate that the resistance of the TCRhigh DP thymocytes to negative selection correlates with the disappearance of TCR-triggered caspase activity in these cells. Thus thymocytes which have presumably begun the positive selection process inactivate the thymic caspase pathway and are no longer susceptible to negative selection.