Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. To date, interactions between the immune system and the different stages of the virus life cycle remain poorly defined. We demonstrated for the first time that CHIKV Ags could be detected in vivo in the monocytes of acutely infected patients. Using in vitro experimental systems, whole blood and purified monocytes, we confirmed that monocytes could be infected and virus growth could be sustained. CHIKV interactions with monocytes, and with other blood leukocytes, induced a robust and rapid innate immune response with the production of specific chemokines and cytokines. In particular, high levels of IFN-α were produced rapidly after CHIKV incubation with monocytes. The identification of monocytes during the early phase of CHIKV infection in vivo is significant as infected monocyte/macrophage cells have been detected in the synovial tissues of chronically CHIKV-infected patients, and these cells may behave as the vehicles for virus dissemination. This may explain the persistence of joint symptoms despite the short duration of viremia. Our results provide a better understanding on the basic mechanisms of infection and early antiviral immune responses and will help in the development of future effective control strategies.

Human primary monocytes comprise a heterogeneous population that can be classified into three subsets based on CD14 and CD16 expression: classical (CD14high/CD16−), intermediate (CD14high/CD16+), and non-classical (CD14low/CD16+). The non-classical monocytes are the most pro-inflammatory in response to TLR stimulation in vitro, yet they express a remarkably high basal level of miR-146a, a microRNA known to negatively regulate the TLR pathway. This concurrence of a pro-inflammatory status and a high miR-146a level has been associated with cellular senescence in other cell types. Hence, we assessed the three monocyte subsets for evidence of senescence, including proliferative status, telomere length, cellular ROS levels, and mitochondrial membrane potential. Indeed, the non-classical subset exhibited the clearest hallmarks of senescence, followed by the intermediate and then the classical subset. In addition, the non-classical subset secreted pro-inflammatory cytokines basally in vitro. The highly pro-inflammatory nature of the non-classical monocytes could be a manifestation of the senescence-associated secretory phenotype (SASP), likely induced by a high basal NF-κB activity and IL-1α production. Finally, we observed an accumulation of the non-classical monocytes, in conjunction with higher levels of plasma TNF-α and IL-8, in the elderly. These factors may contribute to inflamm-aging and age-related inflammatory conditions, such as atherosclerosis and osteoarthritis. With our new understanding that the non-classical monocyte subset is a senescent population, we can now re-examine the role of this subset in disease conditions where this subset expands.

Flow cytometric analysis of lymphocyte subsets were evaluated in 391 healthy Asian subjects ranging in age from birth to 40 years. Lymphocyte subsets were analysed using specific monoclonal antibodies: CD20 (B cells), CD3 and CD2 (T cells), CD16 and CD56+ (NK cells), CD4/CD3+ (helper‐inducer T cells), CD8/CD3+ (suppressor/cytotoxic T cells), HLA‐DR expression on CD3 and CD25 (Tac) on CD3. The total white cell count, absolute lymphocyte counts, and B cell percentages peaked in infancy and declined steadily with age. Absolute counts of each subset, which were derived from absolute lymphocyte counts, also followed this trend. Increases with age were seen in the NK, T cell (CD2, CD3), and CD8 percentages. Males tended to have higher NK and CD8 percentages than females, and, conversely, females had higher CD3 and CD4 percentages than males. Comparison of our results with studies involving Caucasian subjects indicated higher NK percentages in our Asian population and lower CD4 absolute counts in the males of our population. These results indicate the presence of age, sex, and probable racial differences in lymphocyte subset expression. Our results may serve as reference standards for the Asian population. © 1996 Wiley‐Liss, Inc.

Human blood monocytes can be broadly divided into two distinct subsets: CD14+CD16- and CD14+/lowCD16+ subsets. Perturbation in their proportions in the blood has been observed in several disease conditions. Although numerous phenotypic and functional differences between the two subsets have already been described, the roles contributed by each subset during homeostasis or disease conditions are still largely unclear. To uncover novel differences to aid in elucidating their functions, we perform a global analysis of the two subsets utilizing both proteomics and transcriptomics approaches. From the proteomics and transcriptomics data, the expression of 613 genes by the two subsets is detected at both the protein and mRNA levels. These 613 genes are assessed for up-regulation in each subset at the protein and mRNA levels using a cutoff fold change of > or =|1.5| between subsets. Proteins and mRNAs up-regulated in each subset are then mapped in silico into biological functions. This mapping reveals copious functional differences between the subsets, many of which are seen at both protein and mRNA levels. For instance, expression of genes involved in F(CY) receptor-mediated phagocytosis are up-regulated in the CD14+/lowCD16+ subset, while those involved in antimicrobial function are up-regulated in the CD14+CD16- subset. We uncover novel functional differences between the monocyte subsets from differences in gene expression at the protein and mRNA levels. These functional differences would provide new insights into the different roles of the two monocyte subsets in regulating innate and adaptive immune responses.

Regulation of adaptive immunity by innate immune cells is widely accepted. Conversely, adaptive immune cells can also regulate cells of the innate immune system. Here, we report for the first time the essential role of B cells in regulating macrophage (M/) phenotype. In vitro B cell/M/ co-culture experiments together with experiments in transgenic mice models for B-cell deficiency or overexpression showed B1 cells to polarize M/ to a distinct phenotype. This was characterized by downregulated TNF-a, IL-1b and CCL3, but upregulated IL-10 upon LPS stimulation; constitutive expression of M2 M/ markers (e.g. Ym1, Fizz1) and overexpression of TRIF-dependent cytokines (IFN-b, CCL5). Mechanistically, this phenotype was linked to a defective NF-jB activation, but a functional TRIF/STAT1 pathway. B1-cell-derived IL-10 was found to be instrumental in the polarization of these M/. Finally, in vivo relevance of B1-cell-induced M/ polarization was confirmed using the B16 melanoma tumor model where adoptive transfer of B1 cells induced an M2 polarization of tumor-associated M/. Collectively, our results define a new mechanism of M/ polarization wherein B1 cells play a key role in driving M/ to a unique, but M2-biased phenotype. Future studies along these lines may lead to targeting of B1 cells to regulate M/ response in inflammation and cancer. Supporting Information available online IntroductionMacrophages (Mf) are a heterogeneous cell population involved in diverse physiological processes including anti-microbial defence, wound resolution, inflammation, tissue remodeling, plaque formation in atherosclerosis and promotion of tumor growth [1][2][3][4][5][6][7]. Despite their heterogeneity, Mf can be broadly divided into M1 (classically activated) or M2 (alternatively activated) phenotypes [1,[8][9][10][11][12]. The M1 phenotype is induced in response to microbial products such as LPS or proinflammatory cytokines including IFN-g, IL-1b or TNF-a. M1 Mf produce further proinflammatory cytokines (TNF-a, IL-12 and CCL3) with reactive oxygen and nitrogen intermediates, which combine to give M1 Mf potent antimicrobial, tumoricidal and inflammatory properties [12]. In contrast, the M2 phenotype results from exposure to antiinflammatory molecules such as glucocorticoid hormones, IL-4, Ã These authors contributed equally to this work. 2296IL-13, IL-10 or immune complexes [10][11][12]. M2 Mf are antiinflammatory and immunosuppressive in nature. They are highly phagocytic, preferentially activate the arginase pathway, promote angiogenesis, tissue remodeling and have pro-tumoral activity [8,12]. However, in vivo, the division between M1 and M2 cells may be blurred, with the above phenotypes likely representing two extremes in a continuum of Mf functional states [9,[13][14][15].Transcriptome data demonstrate the existence of distinct polarization phenotypes for Mf associated with specific pathological conditions [4,7,[16][17][18]. Based on this, it is believed that the phenotype of an Mf is a reflection of its immediate microenvironment....

In summary, the combination of high densities of intra-tumoural Tregs, CD8 T cells and CD20 B cells represents a favourable prognostic panel in TNBCs. These data also indicate new avenues for further investigation on the interaction between immune cell types within the tumour microenvironment and highlight the potential of Treg density and localisation within tumours to affect clinical outcome.

High macrophage infiltration into tumours often correlates with poor prognoses; in colorectal, stomach and skin cancers, however, the opposite is observed but the mechanisms behind this phenomenon remain unclear. Here, we sought to understand how tumour-associated macrophages (TAMs) in colorectal cancer execute tumoursuppressive roles. We found that TAMs in a colorectal cancer model were pro-inflammatory and inhibited the proliferation of tumour cells. TAMs also produced chemokines that attract T cells, stimulated proliferation of allogeneic T cells and activated type-1 T cells associated with anti-tumour immune responses. Using colorectal tumour tissues, we verified that TAMs in vivo were indeed pro-inflammatory. Furthermore, the number of tumour-infiltrating T cells correlated with the number of TAMs, suggesting that TAMs could attract T cells; and indeed, type-1 T cells were present in the tumour tissues. Patient clinical data suggested that TAMs exerted tumour-suppressive effects with the help of T cells. Hence, the tumour-suppressive mechanisms of TAMs in colorectal cancer involve the inhibition of tumour cell proliferation alongside the production of pro-inflammatory cytokines, chemokines and promoting type-1 T-cell responses. These new findings would contribute to the development of future cancer immunotherapies based on enhancing the tumour-suppressive properties of TAMs to boost anti-tumour immune responses. To elucidate the roles of TAMs, we first used an in vitro model known as the multi-cellular tumour spheroid (MCTS) model. This model has been proven to exhibit micro-environmental heterogeneity comparable to that of tumours in vivo, in terms of oxygen, nutrient, catabolite and metabolite gradients, resulting in sub-populations of proliferative and necrotic tumour cells typical of non-vascular tumour micro-regions [9,10]. Compared with using animal models, this MCTS model offers the advantages of studying the interactions between tumour cells and TAMs without confounding factors from other cell types, and in a 'human' microenvironment. In this study, we used colorectal cancer as a model to study the mechanisms underlying the tumour-suppressive effects of TAMs. We co-cultured primary human monocytes with human colorectal tumour cells for 8 days as MCTSs, during which time the monocytes would differentiate into TAMs. We performed global gene expression profiling to obtain an overview of the biological functions of TAMs, followed by validation with functional assays. Subsequently, we verified the in vitro findings with tumour tissues from colorectal cancer patients.The TAMs in the colorectal cancer model were pro-inflammatory and inhibited the proliferation of tumour cells. The TAMs also secreted chemokines that attract T cells and expressed surface molecules for antigen presentation and T-cell co-stimulation. In a mixed lymphocyte reaction (MLR) assay, the TAMs stimulated proliferation of allogeneic T cells and activated type-1 T cells, which are associated with anti-tumour immune responses [11]. To co...

The analysis of neoantigen-specific CD8+ T cells in tumour-bearing individuals is challenging due to the small pool of tumour antigen-specific T cells. Here we show that mass cytometry with multiplex combinatorial tetramer staining can identify and characterize neoantigen-specific CD8+ T cells in mice bearing T3 methylcholanthrene-induced sarcomas that are susceptible to checkpoint blockade immunotherapy. Among 81 candidate antigens tested, we identify T cells restricted to two known neoantigens simultaneously in tumours, spleens and lymph nodes in tumour-bearing mice. High-dimensional phenotypic profiling reveals that antigen-specific, tumour-infiltrating T cells are highly heterogeneous. We further show that neoantigen-specific T cells display a different phenotypic profile in mice treated with anti-CTLA-4 or anti-PD-1 immunotherapy, whereas their peripheral counterparts are not affected by the treatments. Our results provide insights into the nature of neoantigen-specific T cells and the effects of checkpoint blockade immunotherapy.