Combination therapy strategies for improving PD-1 blockade efficacy: a new era in cancer immunotherapy.

Programmed death 1 (PD-1) is an immune checkpoint molecule that negatively regulates T-cell immune function through the interaction with its ligand PD-L1. Blockage of this interaction unleashes the immune system to fight cancer. Immunotherapy using PD-1 blockade has led to a paradigm shift in the field of cancer drug discovery, owing to its durable effect against a wide variety of cancers with limited adverse effects. A brief history and development of PD-1 blockade, from the initial discovery of PD-1 to the recent clinical output of this therapy, have been summarized here. Despite its tremendous clinical success rate over other cancer treatments, PD-1 blockade has its own pitfall; a significant fraction of patients remains unresponsive to this therapy. The key to improve the PD-1 blockade therapy is the development of combination therapies. As this approach has garnered worldwide interest, here, we have summarized the recent trends in the development of PD-1 blockade-based combination therapies and the ongoing clinical trials. These include combinations with checkpoint inhibitors, radiation therapy, chemotherapy and several other existing cancer treatments. Importantly, FDA has approved PD-1 blockade agent to be used in combination with either CTLA-4 blockade or chemotherapy. Responsiveness to the PD-1 blockade therapy is affected by tumour and immune system-related factors. The role of the immune system, especially T cells, in determining the responsiveness has been poorly studied compared with those factors related to the tumour side. Energy metabolism has emerged as one of the important regulatory mechanisms for the function and differentiation of T cells. We have documented here the recent results regarding the augmentation of PD-1 blockade efficacy by augmenting mitochondrial energy metabolism of T cell.

Ischemic Preconditioning Enhances Muscle Endurance during Sustained Isometric Exercise.

Ischemic preconditioning (IPC) enhances whole-body exercise endurance. However, it is poorly understood whether the beneficial effects originate from systemic (e. g., cardiovascular system) or peripheral (e. g., skeletal muscle) adaptations. The present study examined the effects of IPC on local muscle endurance during fatiguing isometric exercise. 12 male subjects performed sustained isometric unilateral knee-extension exercise at 20% of maximal voluntary contraction until failure. Prior to the exercise, subjects completed IPC or control (CON) treatments. During exercise trial, electromyography activity and near-infrared spectroscopy-derived deoxygenation in skeletal muscle were continuously recorded. Endurance time to task failure was significantly longer in IPC than in CON (mean±SE; 233±9 vs. 198±9 s, P<0.001). Quadriceps electromyography activity was not significantly different between IPC and CON. In contrast, deoxygenation dynamics in the quadriceps vastus lateralis muscle was significantly faster in IPC than in CON (27.1±3.4 vs. 35.0±3.6 s, P<0.01). The present study found that IPC can enhance muscular endurance during fatiguing isometric exercise. Moreover, IPC accelerated muscle deoxygenation dynamics during the exercise. Therefore, we suggest that the origin of beneficial effects of IPC on exercise performance may be the enhanced mitochondrial metabolism in skeletal muscle.

Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-kappa b-inducing kinase.

The alymphoplasia (aly) mutation of mouse is autosomal recessive and characterized by the systemic absence of lymph nodes (LN) and Peyer's patches (PP) and disorganized splenic and thymic structures with immunodeficiency. Although recent reports have shown that the interaction between lymphotoxin (LT) and the LT beta-receptor (Ltbeta r, encoded by Ltbr) provides a critical signal for LN genesis in mice, the aly locus on chromosome 11 is distinct from those for LT and its receptor. We found that the aly allele carries a point mutation causing an amino acid substitution in the carboxy-terminal interaction domain of Nf-kappa b-inducing kinase (Nik, encoded by the gene Nik). Transgenic complementation with wild-type Nik restored the normal structures of LN, PP, spleen and thymus, and the normal immune response in aly/aly mice. In addition, the aly mutation in a kinase domain-truncated Nik abolished its dominant-negative effect on Nf-kappa b activation induced by an excess of Ltbeta r. Our observations agree with previous reports that Ltbeta r-deficient mice showed defects in LN genesis and that Nik is a common mediator of Nf-kappa b activation by the tumour necrosis factor (TNF) receptor family. Nik is able to interact with members of the TRAF family (Traf1, 2, 3, 5 and 6), suggesting it acts downstream of TRAF-associating receptor signalling pathways, including Tnfr, Cd40, Cd30 and Ltbeta r. The phenotypes of aly/aly mice are more severe than those of Ltbr-/- mice, however, indicating involvement of Nik in signal transduction mediated by other receptors.

High-rate quantum key distribution over 100 km using ultra-low-noise, 2-GHz sinusoidally gated InGaAs/InP avalanche photodiodes.

We have demonstrated quantum key distribution (QKD) over 100 km using single-photon detectors based on InGaAs/InP avalanche photodiodes (APDs). We implemented the differential phase shift QKD (DPS-QKD) protocol with electrically cooled and 2-GHz sinusoidally gated APDs. The single-photon detector has a dark count probability of 2.8 × 10(-8) (55 counts per second) with a detection efficiency of 6 %, which enabled us to achieve 24 kbit/s secure key rate over 100 km of optical fiber. The DPS-QKD system offers better performances in a practical way than those achieved using superconducting single-photon detectors. Moreover, the distance that secure keys against the general individual attacks can be distributed has been extended to 160 km.

Field test of quantum key distribution in the Tokyo QKD Network.

A secure communication network with quantum key distribution in a metropolitan area is reported. Six different QKD systems are integrated into a mesh-type network. GHz-clocked QKD links enable us to demonstrate the world-first secure TV conferencing over a distance of 45km. The network includes a commercial QKD product for long-term stable operation, and application interface to secure mobile phones. Detection of an eavesdropper, rerouting into a secure path, and key relay via trusted nodes are demonstrated in this network.

Facilitation of beta selection and modification of positive selection in the thymus of PD-1-deficient mice.

PD-1 is an immunoglobulin superfamily member bearing an immunoreceptor tyrosine-based inhibitory motif, and disruption of the PD-1 gene results in the development of lupus-like autoimmune diseases. In this study, we examined effects of the PD-1 deficiency on the thymocyte differentiation at the clonal level using T cell receptor (TCR)-beta (Vbeta8) and TCR-alpha/beta (H-Y and 2C) transgenic mice. In these TCR transgenic lines, PD-1 expression in the thymus was variably augmented, but as in the normal mice, confined largely to the CD4(-)CD8(-) thymocytes. The transgenic mice crossed with PD-1(-/)- mice in the neutral genetic backgrounds exhibited selective increase in the CD4(+)CD8(+) (DP) population with little effect on other thymocytes subsets. Similarly, the absence of PD-1 facilitated expansion of DP thymocytes in recombination activating gene (RAG)-2(-/)- mice by anti-CD3epsilon antibody injection. On the other hand, H-Y or 2C transgenic PD-1(-/)- mice with the positively selecting background showed significantly reduced efficiency for the generation of CD8(+) single positive cells bearing the transgenic TCR-alpha/beta in spite of the increased DP population. These results collectively indicate that PD-1 negatively regulates the beta selection and modulates the positive selection, and suggest that PD-1 deficiency may lead to the significant alteration of mature T cell repertoire.

The bcl-2 gene product inhibits clonal deletion of self-reactive B lymphocytes in the periphery but not in the bone marrow.

To test whether the product of the bcl-2 proto-oncogene blocks clonal deletion of self-reactive B cells, we have generated transgenic mice carrying the bcl-2 gene and the immunoglobulin genes for the anti-erythrocyte 4C8 antibody. In these transgenic mice, clonal deletion of self-reactive immature B cells in the bone marrow was not inhibited in spite of expression of the bcl-2 gene. In contrast, self-antigen-induced clonal deletion of mature self-reactive Ly-1 B (B1) cells in the peritoneal cavity was inhibited in the transgenic mice. These results indicate that the mechanism for clonal deletion of immature self-reactive B cells in the bone marrow differs from that of mature self-reactive B cells in the periphery.

Biased distribution of recombination sites within S regions upon immunoglobulin class switch recombination induced by transforming growth factor beta and lipopolysaccharide.

We have characterized extrachromosomal circular DNAs from adult mouse spleen cells that were induced to switch to immunoglobulin A (IgA) with bacterial lipopolysaccharide (LPS) and transforming growth factor beta (TGF-beta), and identified breakpoints of S mu/S gamma 3, S mu/S gamma 2, S mu/S alpha, S gamma 3/S alpha, and S gamma 2/S alpha recombinants. The S mu recombination donor sites clustered in the 3' half of the S mu region, while the S alpha recombination acceptor sites clustered in the 5' half of the S alpha region. In addition, donor and acceptor sites of S gamma regions also clustered in the 3' and 5' parts, respectively. These site preferences are in sharp contrast to the dispersed distribution of S mu/S gamma 1 breakpoints within both S mu and S gamma 1 regions upon IgG1 switch induced by LPS and interleukin 4. Our results support the hypotheses that TGF-beta increases the frequency of switch recombination events to IgA and that the switch recombination to IgA often proceeds by successive recombination of S mu/S gamma and S gamma/S alpha.

Trans-splicing as a possible molecular mechanism for the multiple isotype expression of the immunoglobulin gene.

We analyzed the molecular mechanism for the immunoglobulin (Ig) multiple isotype expression using a transgenic mouse (TG.SA) model system. Though most of the endogenous mu chain expression was excluded by the expression of the human rearranged mu transgene in the TG.SA mouse, a significant portion of splenic B lymphocytes could express the transgenic human IgM and endogenous mouse IgG simultaneously after stimulation with lipopolysaccharide and interleukin 4. The fluorescence-activated cell sorter-purified population of the human IgM+/mouse IgG+ cells expressed mRNA that consisted of properly spliced sequences of the transgenic VHDJH and the endogenous mouse C gamma genes (trans-mRNA), together with the transgenic human mu mRNA and germline transcripts of the mouse C gamma gene, without apparent rearrangement of the transgene. We also found that a lymphoma tumor, derived from the cross between the TG.SA mouse and another transgenic mouse carrying Ig H chain enhancer-driven c-myc oncogene, expressed about equal levels of the trans-mRNA and the transgenic mu mRNA without DNA rearrangement in either the transgene or the endogenous mouse switch region. These findings strongly support our previous proposal that the trans-splicing can account for the multiple isotype expression in this transgenic model and also suggest that novel molecular mechanism(s) might be involved in this reaction.

Different stromal cell lines support lineage-selective differentiation of the multipotential bone marrow stem cell clone LyD9.

An interleukin 3-dependent multipotential stem cell clone, LyD9, has been shown to generate mature B lymphocytes, macrophages, and neutrophils by coculture with primary bone marrow stromal cells. We report here that coculture with the cloned stromal cell lines PA6 and ST2 can support differentiation of LyD9 cells predominantly into granulocyte/macrophage colony-stimulating factor (GM-CSF)- and granulocyte (G)-CSF-responsive cells, respectively. However, these stromal cell lines were unable to support lymphopoiesis of LyD9 cells. The GM-CSF-dependent line, L-GM, which was derived from LyD9 cells cocultured with PA6 stromal cells, could differentiate into macrophages and granulocytes in the presence of GM-CSF. The L-GM line can further differentiate predominantly into neutrophils by coculture with ST2 stromal cells. The G-CSF-dependent line, L-G, which was derived from LyD9 cells cocultured with ST2 stromal cells, differentiated into neutrophils in response to G-CSF. Although the stromal cell-supported differentiation of LyD9 cells required the direct contact between LyD9 and stromal cells, a small fraction of LyD9 cells that were pretreated with 5-azacytidine could differentiate into neutrophils and macrophages without direct contact with stromal cells. These results indicate that different stromal cell lines support lineage-selective differentiation of the LyD9 stem cell and that 5-azacytidine treatment can bypass the requirement of direct contact with stromal cells, albeit with a lower frequency.

A larger number of L chains (Tac) enhance the association rate of interleukin 2 to the high affinity site of the interleukin 2 receptor.

The IL-2-R is composed of at least two proteins, that is, a 55-kD protein (p55, the L chain, or Tac) and a 75-kD protein (p75, the H chain, or converter). The high affinity binding of IL-2 results in the formation of the ternary complex consisting of IL-2, and the L and H chains. To distinguish the affinity conversion model and the binary complex model we have carried out kinetic studies on the IL-2 binding to the high affinity IL-2-R on T lymphocytes expressing various numbers of L chains and a relatively constant number of H chains. We found that expression of a larger number of L chains accelerated the association of IL-2 to the high affinity receptor. The results are not compatible with the binary complex model that assumes a fixed number of high affinity sites determined by the numbers of a limiting chain. Instead, the results are consistent with the prediction of the affinity conversion model that assumes association of IL-2 to the L chain as the first step of the ternary complex formation and they indicate that the possible role of excess L chains is to accelerate the formation of the ternary complex. The reaction rate constants calculated from the affinity conversion model were reasonably constant.

Expression and rearrangement of the alpha, beta, and gamma chain genes of the T cell receptor in cloned murine large granular lymphocyte lines. No correlation with the cytotoxic spectrum.

Using cloned murine large granular lymphocyte (LGL) lines, the expression and the rearrangement of the alpha, beta, and gamma chain genes of the T cell receptor (TCR) were analyzed. Morphological, phenotypical, as well as functional studies indicated that the LGL lines were identical to normal, endogenous NK cells. Northern blot hybridization analysis indicated that the full-length transcripts of all the alpha, beta, and gamma chain genes were expressed in most of the LGL lines, including two lines derived from athymic nude mice. In one line, SPB, however, no transcript of the gamma chain gene was detected, whereas the alpha and beta chain genes were clearly expressed. In every LGL line studied, all of the alpha, beta, and gamma chain genes were rearranged. Conforming to the results of Northern blot hybridization study, the gamma chain gene of the SPB line was aberrantly rearranged, whereas those of all the other lines were productively rearranged. The results clearly revealed that NK cells represented a population of lymphocytes genetically committed to the T cell lineage. It was also suggested that the expression and rearrangement of the TCR genes of NK cells might occur in a thymus-independent fashion. An SPB line without expression of the gamma chain gene could exhibit NK activity indistinguishable from other NK lines. Furthermore, the rearrangement patterns of the beta chain gene did not correlate with the specificity of the cytotoxic activity. These results strongly suggested that the cytotoxic activity in NK cells was not directly mediated by TCR on them. We particularly noted that the beta chain gene of most independently established LGL lines showed identical patterns of rearrangement, indicating that they used the same V beta and J beta gene segments. The significance of the restricted pattern of rearrangement of the beta chain gene in LGL lines, as well as the possible functional roles of TCR on NK cells, was discussed.

A transgenic model of autoimmune hemolytic anemia.

We made double transgenic mice bearing immunoglobulin heavy and light chain genes encoding an autoantibody against the mouse erythrocyte by the cross of C57BL/6 mice carrying the transgene for each chain of the immunoglobulin. Although no obvious disorders were found in the single-chain transgenic mice, severely anemic symptoms were found in some of the double transgenic mice, in which most B cells express, at least on their surface, the autoantibody reactive to self-antigens on the erythrocyte. Individual double-transgenic mice showed a wide variation of phenotypes between severe anemia and no symptoms. Both deletion and anergy of autoreactive B cells were seen in each individual mouse, but their relative contribution to self-tolerance was variable and not directly related to the severity of anemia or the amount of the autoantibody produced. This transgenic system provides a good autoimmune disease model for exploring its onset mechanism, and means of its treatment and prevention.

Quantitative regulation of class switch recombination by switch region transcription.

The isotype specificity of immunoglobulin (Ig) class switching is regulated by a cytokine which induces transcription of a specific switch (S) region, giving rise to so-called germline transcripts. Although previous studies have demonstrated that germline transcription of an S region is required for class switch recombination (CSR) of that particular S region, it has not been shown whether the level of S region transcription affects the efficiency of CSR. We addressed this question by using an artificial DNA construct containing a constitutively transcribed mu switch (Smu) region and an alpha switch (Salpha) region driven by a tetracycline-responsive promoter. The construct was introduced into a switch-inducible B lymphoma line and the quantitative correlation between Salpha region transcription and class switching efficiency was evaluated. The level of Salpha transcription was linearly correlated with CSR efficiency, reaching a plateau at saturation. On the other hand, we failed to obtain the evidence to support involvement of either RNA-DNA heteroduplex or trans germline transcripts in CSR. Taken together, it is likely that S region transcription and/or transcript processing in situ may be required for CSR. We propose that because of the unusual properties of S region DNA, transcription induces the DNA to transiently be single stranded, permitting secondary structure(s) to form. Such structures may be recognition targets of a putative class switch recombinase.

Expression of different combinations of interleukins by human T cell leukemic cell lines that are clonally related.

We have analyzed expression patterns of 7 lymphokine mRNAs by Northern blot analyses in 19 different human T cell clones derived from patients with adult T cell leukemia. However, we were not able to reveal particular combinations of lymphokine production that allowed classification of human T cells. Especially, four clonally related leukemic lines that were established independently from the same patient with adult T cell leukemia expressed different combinations of lymphokine mRNAs, indicating that the expression of various lymphokines is not fixed but rather variable even among progenies of a single T cell clone.

Migration and differentiation of autoreactive B-1 cells induced by activated gamma/delta T cells in antierythrocyte immunoglobulin transgenic mice.

Using normal and transgenic (Tg) mice, we have shown that peritoneal B-1 cells are activated by administration of cytokines or lipopolysaccharide and migrate to other lymphoid organs where they differentiate into antibody-secreting cells. However, little is known about the process of B-1 cell migration and differentiation in vivo. We developed a mouse line by crossing the antierythrocyte antibody Tg mice (HL mice) with TCR-gamma/delta Tg mice specific for a self-thymus leukemia (TL) antigen in the recombination activating gene (RAG)2(-/-) background. In the presence of the self-antigen, Tg gamma/delta T cells increased in number and manifested activated phenotypes. Peritoneal B-1 cells in these mice migrated into mesenteric lymph nodes and differentiated into autoantibody-secreting cells, resulting in strong autoimmune hemolytic anemia. Furthermore, transfer of RAG2(-/-) x HL bone marrow or peritoneal cells into the peritoneal cavity of RAG2(-/-) x TCR-gamma/delta Tg mice gave rise to donor-derived B-1 cells in mesenteric lymph nodes, and these cells produced the autoantibody. Thus, this study demonstrates that the migration of B-1 cells and differentiation into the antibody-secreting cells can be induced by noncognate T cell help and implies the possibility that gamma/delta T cells may induce B-1 cell differentiation in vivo.

Mapping of the nu gene using congenic nude strains and in situ hybridization.

The chromosomal location of the nu gene, which is responsible for hairlessness and athymus, was determined using six DNA markers (interleukin 3 [Il-3], Myhs, Acrb, Evi-2, Mpo, and Hox-2) on mouse chromosome 11. We constructed the high-resolution physical mapping of the six DNA markers on chromosome 11 by in situ hybridization using fluorescence-labeled cosmid probes. The results indicate the order of centromere-(41cM)-Il-3-(3cM)-Myhs- (4cM)-Acrb-(6cM)-Evi-2-(3cM)-Mpo-(5cM)- Hox-2. We have used congenic nude strains and examined which of the six DNA markers were derived from the original nude mouse. We found the Evi-2 locus is linked to the nu gene in all the informative, independent congenic nude strains. From these data, we could estimate the location of the nu gene, not only genetically but also physically within a region that spans approximately 17 megabases (9 cM) between the Acrb and Mpo genes.

Effects of breeding environments on generation and activation of autoreactive B-1 cells in anti-red blood cell autoantibody transgenic mice.

In anti-red blood cell autoantibody transgenic (autoAb Tg) mice almost all B cells are deleted except for B-1 cells in the peritoneal cavity and the gut. About one-half of the auto Ab Tg mice suffer from autoimmune hemolytic anemia (AIHA) in the conventional condition. Oral administration of lipopolysaccharides activates B-1 cells and induces autoimmune symptoms in the Tg mice, suggesting that the autoimmune disease in anti-RBC autoAb Tg mice is triggered by infections. To examine the association of bacterial infections with the generation of B-1 cells and the occurrence of the autoimmune disease, we analyzed anti-RBC autoAb Tg mice bred in germ-free and specific pathogen-free conditions. In germ-free conditions, few peritoneal B-1 cells were detected, while a significant number of peritoneal B-1 cells existed in specific pathogen-free conditions. In both conditions, no mice suffered from AIHA. However, when these Tg mice were transferred to the conventional condition or injected with lipopolysaccharide, peritoneal B-1 cells expanded and some of these mice suffered from AIHA. These results clearly showed that bacterial infections are responsible for both the expansion of B-1 cells and the onset of the autoimmune disease in these Tg mice.

Alymphoplasia (aly)-type nuclear factor kappaB-inducing kinase (NIK) causes defects in secondary lymphoid tissue chemokine receptor signaling and homing of peritoneal cells to the gut-associated lymphatic tissue system.

Alymphoplasia (aly) mice, which carry a point mutation in the nuclear factor kappaB-inducing kinase (NIK) gene, are characterized by the systemic absence of lymph nodes and Peyer's patches, disorganized splenic and thymic architectures, and immunodeficiency. Another unique feature of aly/aly mice is that their peritoneal cavity contains more B1 cells than normal and aly/+ mice. Transfer experiments of peritoneal lymphocytes from aly/aly mice into recombination activating gene (RAG)-2(-/-) mice revealed that B and T cells fail to migrate to other lymphoid tissues, particularly to the gut-associated lymphatic tissue system. In vivo homing defects of aly/aly peritoneal cells correlated with reduction of their in vitro chemotactic responses to secondary lymphoid tissue chemokine (SLC) and B lymphocyte chemoattractant (BLC). The migration defect of aly/aly lymphocytes was not due to a lack of expression of chemokines and their receptors, but rather to impaired signal transduction downstream of the receptors for SLC, indicating that NIK is involved in the chemokine signaling pathway known to couple only with G proteins. The results showed that the reduced serum levels of immunoglobulins (Igs) and the absence of class switch to IgA in aly/aly mice are due, at least in part, to a migration defect of lymphocytes to the proper microenvironment where B cells proliferate and differentiate into Ig-producing cells.

Lineage-restricted function of nuclear factor kappaB-inducing kinase (NIK) in transducing signals via CD40.

CD40 signaling in B cells and dendritic cells (DCs) is critical for the development of humoral and cell-mediated immunity, respectively. Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK) has been implicated as a central transducing kinase in CD40-dependent activation. Here, we show that although NIK is essential for B cell activation, it is dispensable for activation of DCs. Such data provide compelling evidence that different intermediary kinases are used by different cellular lineages to trigger NF-kappaB activation via CD40.