Gasp,a Grb2-associating protein,is critical for positive selection of thymocytes

T cells develop in the thymus through positive and negative selection, which are responsible for shaping the T cell receptor (TCR) repertoire. To elucidate the molecular mechanisms involved in selection remains an area of intense interest. Here, we identified and characterized a gene product Gasp (Grb2-associating protein, also called Themis) that is critically required for positive selection. Gasp is a cytosolic protein with no known functional motifs that is expressed only in T cells, especially immature CD4/CD8 double positive (DP) thymocytes. In the absence of Gasp, differentiation of both CD4 and CD8 single positive cells in the thymus was severely inhibited, whereas all other TCR-induced events such as β-selection, negative selection, peripheral activation, and homeostatic proliferation were unaffected. We found that Gasp constitutively associates with Grb2 via its N-terminal Src homology 3 domain, suggesting that Gasp acts as a thymocyte-specific adaptor for Grb2 or regulates Ras signaling in DP thymocytes. Collectively, we have described a gene called Gasp that is critical for positive selection.     

MAP kinase phosphatase activity sets the threshold for thymocyte positive selection

Phosphorylation of MAP kinases is important for proper translation of T cell antigen receptor (TCR) signals into thymocyte cell fates, but the role of MAP kinase phosphatase (MKP) activity in thymocyte development has not been characterized. To explore the role of MKP in thymocytes, we constructed a double mutant MKP-3 (DM-MKP3) that acts as a dominant-negative inhibitor of ERK- and JNK-specific MKP. Thymocytes developing in the presence of DM-MKP3 have enhanced frequencies of both CD4 and CD8 mature, single-positive cells and no increase in apoptosis. Expression of DM-MKP3 also results in an increased proportion of thymocytes with high levels of both CD69 and TCRbeta, suggesting that the increased proportion of mature thymocytes is the result of an increased probability that CD4(+)CD8(+) cells will be positively selected. Thus, MKP activity controls thymocyte cell fate by regulating the threshold of TCR signaling that is able to induce positive selection.     

Trypanosoma brucei infection in nude mice: B lymphocyte function is suppressed in the absence of T lymphocytes

B lymphocyte function was assessed in outbred nude mice and nu/+controls infected with Trypanosoma brucei brucei. On day 10 of the infection in outbred nu/nu mice in which the initial wave of parasites was strongly controlled, B cell function was unaltered on enhanced compared with uninfected animals or infected nu/+. In other nu/nu mice unable to control the initial parasitaemia, thymidine incorporation and Ig secretion by spleen cells were increased on day 10 and their response to lipopolysaccharide in vitro negated. By day 15 however, even the spleen cells of infected nu/nu which controlled the initial wave of parasites were proliferating and secreting Ig on removal from the mice and they were unable to respond to LPS in vitro. These experiments confirm results of a previous study of B cell function in T cell-depleted mice (Askonas et al. 1979). T. b. brucei infection of mice causes both enhanced Ig production and suppression of the ability of B cells to respond to mitogen even in the absence of T cells, but the presence of T cells may accelerate the changes which occur in B lymphocytes following this infection.     

Alpha4beta1-integrin activation is necessary for high-efficiency T-cell subset interactions with VCAM-1 under flow

OBJECTIVE: The purpose of this study was to examine the relationship between alpha4beta1-integrin state of activation on CD4+ T-cell subsets and their adhesive interaction to VCAM-1 under flow. METHODS: Human CD4+ memory and naive T-cells were freshly isolated and effector-helper T-cell subsets. Th1 and Th2 cells, were differentiated in vitro from CD4+ naive T-cells. The expression of activation/ligand induced epitopes on beta1-integrins of each T-cell subset was assessed using mAb HUTS21 and mAb 15/7. T-cell subsets attachment and rolling on VCAM-1 was determined under defined flow conditions and the rates of attachment (ka), accumulation, and instantaneous rolling velocities were correlated to their beta1-integrin activation epitope expression. RESULTS: A subset of memory T-cells constitutively express activation/ligand induced epitopes on beta1-integrins recognized by mAb HUTS21 and 15/7, whereas expression levels on naive T-cells is low or not detectable. Consistent with an activated phenotype, memory T-cells exhibit significantly higher rates of attachment and accumulation on VCAM-1 under flow as compared to naive T-cells. Interestingly, the expression of activation/ligand induced epitopes on beta1-integrins on Th2 cells and the ability of these cells to interact with VCAM-1 are comparable to memory T-cells. In contrast, Th1 cells did not interact as efficiently with VCAM-1, which correlated with lower expression of activation/ligand induced epitopes on these cells. VCAM-1 interactions are inhibited completely by pretreatment of the T-cells with blocking mAb to alpha4-integrins or beta1-integrins, indicating that alpha4beta1 is the predominant T-cell integrin involved. CONCLUSIONS: Memory T-cells express constitutively active alpha4beta1-integrins, as compared to naive T-cells, which mediate high rates of initial attachment and sustained high-affinity adhesive interactions with VCAM-1 under flow conditions in vitro. Similarly, in vitro differentiated Th2 cells but not Th1 cells, which also express elevated levels of activated alpha4beta1-integrins, are capable of sustaining high-affinity adhesive interactions with VCAM-1. The differences observed in beta1-integrin activation on T-cell subsets may underlie selective recruitment patterns of T-cell subsets in vivo.     

Many different Vβ CDR3s can reveal the inherent MHC reactivity of germline-encoded TCR V regions

We have hypothesized that in the prenegative selection TCR repertoire, many somatically generated complementary-determining region (CDR) 3 loops combine with evolutionarily selected germline Vα/Vβ CDR1/CDR2 loops to create highly MHC/peptide cross-reactive T cells that are subsequently deleted by negative selection. Here, we present a mutational analysis of the Vβ CDR3 of such a cross-reactive T-cell receptor (TCR), YAe62. Most YAe62 TCRs with the mutant CDR3s became less MHC promiscuous. However, others with CDR3s unrelated in sequence to the original recognized even more MHC alleles than the original TCR. Most importantly, this recognition was still dependent on the conserved CDR1/CDR2 residues. These results bolster the idea that germline TCR V elements are inherently reactive to MHC but that this reactivity is fine-tuned by the somatically generated CDR3 loops.     

Hard wiring of T cell receptor specificity for the major histocompatibility complex is underpinned by TCR adaptability

αβ T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR–pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR–pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR–pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.     

Human gammadelta T cells as mediators of chimaeric-receptor redirected anti-tumour immunity

Human peripheral blood gammadelta T cells (Vgamma9(+) Vdelta2(+)) can be selectively expanded in vivo by the systemic administration of aminobisphosphonates without prior antigen priming. To assess the potential of human gammadelta T cells to serve as effector cells of specific anti-tumour immunity, we expanded peripheral blood-derived gammadelta T cells and transduced them with recombinant retrovirus encoding G(D2)- or CD19-specific chimaeric receptors. Flow cytometric analysis of T cells from four individual donors cultured in the presence of zoledronate at day 14 of culture showed selective enrichment of the gammadelta T cell population (Vgamma9(+) Vdelta2(+) CD3(+) CD4(-) CD8(-)) to 73-96% of total CD3(+) T cells. Retroviral gene transfer resulted in chimaeric receptor surface expression in 73 +/- 12% of the population. Transduced gammadelta T cells efficiently recognized antigen-expressing tumour cell targets, as demonstrated by target-specific upregulation of CD69 and secretion of interferon-alpha. Moreover, transduced gammadelta T cells efficiently and specifically lysed the antigen-expressing tumour targets. They could be efficiently expanded in vitro and maintained in culture for prolonged periods. Zoledronate-activated human gammadelta T cells expressing chimaeric receptors may thus serve as potent and specific anti-tumour effector cells. Their responsiveness to stimulation with aminobisphosphonates may enable the selective re-expansion of adoptively transferred T cells in vivo, permitting long lasting anti-tumour immune control.     

HIV感染者/AIDS患者外周血T细胞抗原受体γδT细胞的检测及其临床意义

目的　探讨外周血T细胞抗原受体 (TCR)γδT细胞在艾滋病病毒 (HIV)感染者 /艾滋病 (AIDS)患者疾病进程中的作用。方法　应用流式细胞仪采用三色荧光抗体染色技术分别检测 18例HIV感染者、2 9例AIDS患者、2 5例伴有机会性感染的AIDS患者、2 0例高效抗逆转录病毒联合疗法 (HAART)治疗后的AIDS患者及 2 0例正常对照者外周血的TCRγδT细胞与TCRαβT细胞。 结果　HIV感染者组和AIDS患者组的外周血TCRγδT细胞的百分率比正常对照组明显地增高 (P <0 0 1) ,而TCRαβT细胞的百分率明显地低于正常对照组 (P <0 0 1) ,AIDS患者组的CD+ 3 细胞的百分率明显地低于HIV感染者组和正常对照组 (P <0 0 1)。AIDS患者伴机会性感染组的外周血TCRγδT细胞的百分率明显地高于HAART治疗后的AIDS患者组 (P <0 0 1) ,该组的TCRαβT细胞的百分率及CD+ 3 T细胞的百分率则明显地低于经HAART治疗后的AIDS患者组 (P <0 0 1)。结论　TCRγδT细胞数量增多是机体受到病毒感染时的一种早期非特异性免疫反应 ,在AIDS的疾病进程中及机会性感染时起一定的免疫防御作用。     

IL-15 as a mediator of CD4+ help for CD8+ T cell longevity and avoidance of TRAIL-mediated apoptosis

CD4+ helper T cells contribute to the induction and maintenance of antigen-specific CD8+ T cells. Their absence results in short-lived antigen-specific CD8+ T cells and defective secondary CD8+ T cell responses because of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Here, we show that IL-15 codelivered with vaccines can overcome CD4+ T cell deficiency for promoting longevity of antigen-specific CD8+ T cells and avoidance of TRAIL-mediated apoptosis. In both priming and secondary responses, IL-15 down-regulates proapoptotic Bax, an intermediate in TRAIL-mediated apoptosis, and increases anti-apoptotic Bcl-X(L) in CD8+ T cells. Thus, IL-15 is sufficient to mimic CD4+ T cell help. Antigen-specific CD4+ T cells induce dendritic cells (DCs) to produce IL-15. IL-15 is also necessary for optimal help, because helper cells do not deliver effective help through IL-15-/- DCs. Therefore, IL-15 codelivered with vaccines can overcome CD4+ helper T cell deficiency for induction of functionally efficient CD8+ T cells and maintenance of CD8+ cytotoxic T lymphocytes (CTLs), and IL-15 is probably one of the natural mediators of help. These findings suggest new vaccine strategies against infections and cancers, especially in individuals with CD4-deficiency.     

Development of a diverse human T-cell repertoire despite stringent restriction of hematopoietic clonality in the thymus

The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ^−/− xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.Hematopoietic stem cell transplantation (HSCT) has become common clinical practice in the treatment of leukemia, lymphoma, and certain inherited immune and metabolic disorders. After transplantation there is an immediate need for de novo production of granulocytes, erythrocytes, and platelets, referred to as “hematopoietic reconstitution,” later followed by recovery of lymphocyte numbers, termed “immune reconstitution.” Although often successful, HSCT is associated with a number of complications arising from poor immune reconstitution, which presents one of the most important causes of morbidity after HSCT (1, 2). Poor myeloid reconstitution is directly linked to low numbers of HSCs in the transplant, but the reasons for poor immune and, in particular, T-lymphocyte reconstitution are much less clear.A study on the application of antithymocyte globulin in cord blood transplantation showed that antithymocyte globulin administration in pretransplantation conditioning results in decreased T-cell reconstitution and survival (3), indicating the need for a better understanding of de novo T-cell development after HSCT. As T cells develop in the thymus, in contrast to all other blood lineages that develop in the bone marrow (BM), HSC-derived progenitors need to seed the thymus. Earlier work in mice has indicated that relatively few progenitors seed the thymus (4, 5), yet their numbers and the subsequent fate of the progeny derived from an HSC clone has remained elusive. The nature of the thymus-seeding cell has been subject of much debate, certainly in humans. CD34⁺CD38⁻CD7⁺ cells (6, 7), CD34⁺CD38⁻CD10⁻CD62L⁺ cells (8), and others have been proposed as thymus-seeding cells in the human situation. In contrast to mice, the earliest human thymocytes show a much broader lineage developmental potential, including low levels of erythroid and megakaryocytic potential (9), and may therefore be much closer related to HSCs or the proposed myeloid lymphoid precursors.In this study, we aimed to address the functional relationship between HSCs and developing T cells by using DNA barcoding. This method allows marking of each stem cell and its progeny by a unique, neutral DNA sequence that can be retrieved by next-generation sequencing. Here we used replication-deficient lentiviruses to transfer a DNA barcode and the GFP into phenotypically defined human HSCs. These marked HSCs were transplanted, using an optimized protocol (10), into NOD/SCID/Il-2Rγ^−/− (NSG) mice (11), which allowed us to determine the dynamics of clonal reconstitution in the thymus in great detail. We used umbilical cord blood (UCB) as source of human stem cells, because UCB is frequently used as a readily available HSC source with less stringent HLA (human leukocyte antigen)-matching requirements (12). However, T-cell immune reconstitution, a general problem in allogeneic HSCT, is more problematic in UCB. Therefore, UCB provides an appropriate HSC source to address questions of lineage tracing and clonality in the blood and immune system. As we demonstrate here, only a fraction of HSC clones in UCB contributes to the T-cell pool, yet a fully diverse T-cell receptor (TCR) repertoire can be generated in the thymus.     

HIV-1 Virological Synapse is not Simply a Copycat of the Immunological Synapse

The virological synapse (VS) is a tight adhesive junction between an HIV-infected cell and an uninfected target cell, across which virus can be efficiently transferred from cell to cell in the absence of cell-cell fusion. The VS has been postulated to resemble, in its morphology, the well-studied immunological synapse (IS). This review article discusses the structural similarities between IS and VS and the shared T cell receptor (TCR) signaling components that are found in the VS. However, the IS and the VS display distinct kinetics in disassembly and intracellular signaling events, possibly leading to different biological outcomes. Hence, HIV-1 exploits molecular components of IS and TCR signaling machinery to trigger unique changes in cellular morphology, migration, and activation that facilitate its transmission and cell-to-cell spread.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y₁ receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y₁ receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y₁ antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 μM ) totally inhibited the [Ca²⁺]_i rise induced by ADP (0.1 μM ) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM ) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM ). A3P5P (1 m M ) reduced the number of [³³P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [³³P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M ). In summary, these results demonstrate that the platelet P2Y₁ receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Dendritic cell-based tumor vaccine for cervical cancer I: in vitro stimulation with recombinant protein-pulsed dendritic cells induces specific T cells to HPV16 E7 or HPV18 E7

Purpose Human papillomavirus (HPV) type 16 and 18 are the most prevalent genotypes in cervical cancers. The viral oncoproteins E6 and E7 are considered to be tumor-specific targets for immunotherapy. HPV E7 antigen-loaded dendritic cells (DC) were evaluated as cellular tumor vaccine.Methods Autologous monocyte-derived DCs loaded with recombinant HPV16 or HPV18 E7 oncoprotein were used to induce in vitro a specific T cell response. Specificities of activated T cells were determined.Results E7-specific T cells could be identified in 18/20 T cell lines from healthy blood donors. CD4⁺ T cell responses (13/16) were found by proliferation assay. CD8⁺ CTLs (12/18) were detectable by interferon-gamma (IFN-) ELISpot analysis. Seven donors reacted in both assays and only 2/20 T cell lines did not react in any assay. Thus, specific T cells could be activated in >80% of healthy individuals. T cell lines from suitable donors were specific for HLA-A*0201-restricted epitopes. Furthermore, HPV E7 antigen-loaded DC stimulated specific responses in freshly isolated tumor infiltrating lymphocyte (TIL) populations of cervical cancer patients.Conclusion Autologous dendritic cells loaded with HPV E7 protein can induce T cell responses in healthy individuals by in vitro stimulation and evoke responses in TIL from cervical cancer biopsies. Since there are no limitations with respect to specific HLA-haplotypes, these findings may be a basis for the development of a therapeutic protein-based DC tumor vaccine against cervical cancer for HPV16- and HPV18-positive patients.Abbreviations B-LCL B lymphoblastoid cell line - CTL Cytotoxic T lymphocyte - DC Dendritic cell - IFN- Interferon-gamma - IL Interleukin - GM-CSF Granulocyte/macrophage-colony stimulating factor - HPV Human Papillomavirus - HSA human serum albumin - PBL Peripheral blood lymphocytes - PBMC Peripheral blood mononuclear cells - SD standart deviation - TIL Tumor infiltrating lymphocytes - TNF- Tumor necrosis factor-alpha     

Crystal structure of a human CD3-epsilon/delta dimer in complex with a UCHT1 single-chain antibody fragment

The alpha/beta T cell receptor complex transmits signals from MHC/peptide antigens through a set of constitutively associated signaling molecules, including CD3-epsilon/gamma and CD3-epsilon/delta. We report the crystal structure at 1.9-A resolution of a complex between a human CD3-epsilon/delta ectodomain heterodimer and a single-chain fragment of the UCHT1 antibody. CD3-epsilon/delta and CD3-epsilon/gamma share a conserved interface between the Ig-fold ectodomains, with parallel packing of the two G strands. CD3-delta has a more electronegative surface and a more compact Ig fold than CD3-gamma; thus, the two CD3 heterodimers have distinctly different molecular surfaces. The UCHT1 antibody binds near an acidic region of CD3-epsilon opposite the dimer interface, occluding this region from direct interaction with the TCR. This immunodominant epitope may be a uniquely accessible surface in the TCR/CD3 complex, because there is overlap between the binding site of the UCHT1 and OKT3 antibodies. Determination of the CD3-epsilon/delta structure completes the set of TCR/CD3 globular ectodomains and contributes information about exposed CD3 surfaces.     

Tie2cre-induced inactivation of the miRNA-processing enzyme Dicer disrupts invariant NKT cell development

MicroRNAs (miRNAs) are a class of evolutionarily conserved small noncoding RNAs that are increasingly being recognized as important regulators of gene expression. The ribonuclease III enzyme Dicer is essential for the processing of miRNAs. CD1d-restricted invariant natural killer T (iNKT) cells are potent regulators of diverse immune responses. The role of Dicer-generated miRNAs in the development and function of immune regulatory iNKT cells is unknown. Here, we generated a mouse strain with a tissue-specific disruption of Dicer, and showed that lack of miRNAs after the deletion of Dicer by Tie2-Cre (expressed in hematopoietic cells and endothelial cells) interrupted the development and maturation of iNKT cells in the thymus and significantly decreased the number of iNKT cells in different immune organs. Thymic and peripheral iNKT cell compartments were changed in miRNA-deficient mice, with a significantly increased frequency of CD4⁺CD8⁺ iNKT cells in the thymus and a significantly decreased frequency of CD4⁺ iNKT cells in the spleen. MiRNA-deficient iNKT cells display profound defects in α-GalCer-induced activation and cytokine production. Bone marrow (BM) from miRNA-deficient mice poorly reconstituted iNKT cells compared to BM from WT mice. Also, using a thymic iNKT cell transfer model, we found that iNKT cell homeostasis was impaired in miRNA-deficient recipient mice. Our data indicate that miRNAs expressed in hematopoietic cells and endothelial cells are potent regulators of iNKT cell development, function, and homeostasis.     

Diverse clinical outcomes of eosinophilic patients with T-cell receptor gene rearrangements: the emerging diagnostic importance of molecular genetics testing

Abnormal clones of clusters of differentiation (CD)3(-)CD4(+) or CD3(+)CD4(-)CD8(-) phenotypically abnormal lymphocytes have been identified in some patients who have the idiopathic hypereosinophilic syndrome. This report illustrates the disparate clinical courses of six eosinophilic patients with evidence of abnormal T-cell clones based on the finding of a T-cell receptor gene rearrangement. The data suggest that molecular genetics testing for T-cell receptor gene rearrangements should be included in the routine work-up of patients with idiopathic eosinophilia.     

An anergic immune signature in the tumor microenvironment of classical Hodgkin lymphoma is associated with inferior outcome

Objective

The classical Hodgkin lymphoma (cHL) tumor microenvironment shows an ongoing inflammatory response consisting of varying degrees of infiltrating eosinophils, mast cells, macrophages, regulatory T lymphocytes (Tregs), and activated lymphocytes surrounding the malignant cells. Herein, different immune signatures are characterized and correlated with treatment outcome.

Methods

Tumor‐infiltrating leukocytes were phenotyped in biopsies from 459 patients with cHL. Time to progression (TTP) (primary progression, relapse, or death from cHL) and overall survival were analyzed using Cox proportional hazards regression.

Results

The leukocyte infiltration in the microenvironment was highly diverse between patients and was categorized in 4 immune signatures (active, anergic, innate, or mixed). A high proportion of Tregs (anergic) resulted in shorter TTP (median 12.9‐year follow‐up) in age‐adjusted analyses (hazard ratio = 1.82; 95% confidence interval 1.05‐3‐15). Epstein‐Barr virus (EBV)‐positive cases had higher proportions of macrophages and activated lymphocytes than EBV negative, but neither of those leukocytes predicted prognosis.

Conclusions

Abundant Tregs (anergic signature) indicate a shorter TTP, particularly in younger patients. This is probably due to a reduced ability of the immune system to attack the tumor cells. Our data warrant further investigation if these suggested immune signatures could predict outcome of immunotherapy such as immune checkpoint inhibitors.     

Cyclin-dependent kinase 1 (Cdk1) is essential for cell division and suppression of DNA re-replication but not for liver regeneration

Cyclin-dependent kinase 1 (Cdk1) is an archetypical kinase and a central regulator that drives cells through G2 phase and mitosis. Knockouts of Cdk2, Cdk3, Cdk4, or Cdk6 have resulted in viable mice, but the in vivo functions of Cdk1 have not been fully explored in mammals. Here we have generated a conditional-knockout mouse model to study the functions of Cdk1 in vivo. Ablation of Cdk1 leads to arrest of embryonic development around the blastocyst stage. Interestingly, liver-specific deletion of Cdk1 is well tolerated, and liver regeneration after partial hepatectomy is not impaired, indicating that regeneration can be driven by cell growth without cell division. The loss of Cdk1 does not affect S phase progression but results in DNA re-replication because of an increase in Cdk2/cyclin A2 activity. Unlike other Cdks, loss of Cdk1 in the liver confers complete resistance against tumorigenesis induced by activated Ras and silencing of p53.     

Crystal structure of the complex between programmed death-1 (PD-1) and its ligand PD-L2

Programmed death-1 (PD-1) is a member of the CD28/B7 superfamily that delivers negative signals upon interaction with its two ligands, PD-L1 or PD-L2. The high-resolution crystal structure of the complex formed by the complete ectodomains of murine PD-1 and PD-L2 revealed a 1:1 receptor:ligand stoichiometry and displayed a binding interface and overall molecular organization distinct from that observed in the CTLA-4/B7 inhibitory complexes. Furthermore, our structure also provides insights into the association between PD-1 and PD-L1 and highlights differences in the interfaces formed by the two PD-1 ligands (PD-Ls) Mutagenesis studies confirmed the details of the proposed PD-1/PD-L binding interfaces and allowed for the design of a mutant PD-1 receptor with enhanced affinity. These studies define spatial and organizational constraints that control the localization and signaling of PD-1/PD-L complexes within the immunological synapse and provide a basis for manipulating the PD-1 pathways for immunotherapy.