T-cell receptor affinity in thymic development

Understanding the thymic processes that support the generation of functionally competent and self-tolerant lymphocytes requires dissection of the T-cell receptor (TCR) response to ligands of different affinities. In spatially segregated regions of the thymus, with unique expression of proteases and cytokines, TCR affinity guides a number of cell fate decisions. Yet affinity alone does not explain the selection paradox. Increasing evidence suggests that the 'altered peptide' model of the 1980s together with the affinity model might best explain how the thymus supports conventional and regulatory T-cell development. Development of new tools to study the strength of TCR signals perceived by T cells, novel regulatory T-cell transgenic mice, and tetramer enrichment strategies have provided an insight into the nature of TCR signals perceived during thymocyte development. These topics are discussed and support for the prevailing hypotheses is presented.     

巨细胞病毒感染C57BL/6小鼠诱导NK细胞免疫应答的模型研究

目的:研究小鼠巨细胞病毒(murine cytomegalovirus,MCMV)感染C57BL/6小鼠诱导自然杀伤(natural killer,NK)细胞免疫应答的最佳剂量和最佳时间。方法:分别根据剂量和时间效应进行分组,剂量效应：无特定病原体( specific pathogen free,SPF)级8周龄C57...     

Contrasting phenotypes of liver-infiltrating leucocytes isolated from MCMV-infected BALB/c and C57 BL/6 mice

We characterized liver-infiltrating leucocytes (LIL) from BALB/c and C57BL/6 mice 0-56 days after murine cytomegalovirus (MCMV) infection. Inflammation clears from C57BL/6 mice 4-5 weeks post infection (pi), but persists for several months in BALB/c mice. The LIL obtained were 60-80% Thy 1.2+ by flow cytometry. The percentage of CD8+ cells rose sharply in all mice 7 days pi, with little decrease in BALB/c mice by day 56. CD4-CD8-Thy 1.2+/TCR alpha beta + cells were more prevalent in LIL than lymph node cells (LNC) irrespective of MCMV infection, whilst infection increased the proportion of CD8+ L-selectin- LIL (but not LNC). LIL from both mouse strains demonstrated cytotoxic activity against YAC-1 cells, but only LIL from BALB/c mice proliferated spontaneously ex vivo 21 days pi, as measured by tritiated thymidine incorporation. BALB/c LIL produced IFN gamma and IgG2a 7-21 days pi, whilst IL-10 secretion was similar in both strains. Thus, persistent hepatitis in BALB/c mice is associated with activation and proliferation of intrahepatic leucocytes with some bias towards a Th1 response.     

Ex‐vivo analysis of human Natural Killer T cells demonstrates heterogeneity between tissues and within established CD4+ and CD4− subsets

Our understanding of human type 1 natural killer T (NKT) cells has been heavily dependent on studies of cells from peripheral blood. These have identified two functionally distinct subsets defined by expression of CD4, although it is widely believed that this underestimates the true number of subsets. Two recent studies supporting this view have provided more detail about diversity of the human NKT cells, but relied on analysis of NKT cells from human blood that had been expanded in vitro prior to analysis. In this study we extend those findings by assessing the heterogeneity of CD4⁺ and CD4⁻ human NKT cell subsets from peripheral blood, cord blood, thymus and spleen without prior expansion ex vivo, and identifying for the first time cytokines expressed by human NKT cells from spleen and thymus. Our comparative analysis reveals highly heterogeneous expression of surface antigens by CD4⁺ and CD4⁻ NKT cell subsets and identifies several antigens whose differential expression correlates with the cytokine response. Collectively, our findings reveal that the common classification of NKT cells into CD4⁺ and CD4⁻ subsets fails to reflect the diversity of this lineage, and that more studies are needed to establish the functional significance of the antigen expression patterns and tissue residency of human NKT cells.     

Natural killer cell receptor expression reflects the role of human cytomegalovirus in the pathogenesis of a subset of CD4+ T-cell large granular lymphocytosis

A high frequency of CD4(+) T-cell large granular lymphocyte (T-LGL) lymphocytosis occurs in human leukocyte antigen (HLA) -DRB1*0701 individuals displaying monoclonal expansions of Vβ13.1+ CD4(+) T-cell clones, which specifically respond to human cytomegalovirus (HCMV) antigens. We previously reported the expression of natural killer (NK)-cell associated receptors (NKR) by HCMV-specific cytolytic CD4(+) T cells from healthy donors. In the present study a high expression of different NKR (i.e., NKG2D, killer Ig-like receptors (KIR), CD94, ILT2) was observed in CD4(+) T cells from both Vβ13.1- and Vβ13.1+ CD4(+) T-LGL cases. Remarkably, elevated numbers of CD94/NKG2C+ NK cells, previously shown to expand in association to HCMV infection, were preferentially found in Vβ13.1+ T-LGL, further supporting its role in the pathogenesis of a subset of CD4(+) T-LGL.     

Characterization of the recognition and functional heterogeneity exhibited by cytokine-induced killer cell subsets against acute myeloid leukaemia target cell

The polyclonal cytokine-induced killer (CIK) cells exhibit potent cytotoxicity against a variety of tumour cells including autologous and allogeneic acute myeloid leukaemic (AML) targets. At maturity, three lymphocyte subsets: CD3(-) CD56(+), CD3(+) CD56(-) and CD3(+) CD56(+), constitute the bulk of the CIK cell culture. The CD3(-) CD56(+) subset behaves like classical natural killer (NK) cells where cytotoxicity is potentiated by blocking the human leucocyte antigen Class I molecules in the AML targets. Both the CD3(+) CD56(+) and CD3(+) CD56(-) subsets, though known to kill autologous and allogeneic targets to a comparable degree and therefore non-major histocompatibility complex (MHC)-restricted, nevertheless require the presence of the MHC molecule on the target, which interacts with their CD3-T-cell receptor complex. Although CIK cells are often termed 'NK-like' T cells, we have demonstrated that the well-characterized NK receptors KIR, NKG2C/E, NKG2D and DNAM-1 are not involved in the process of AML recognition for the CD3(+) CD56(-) and CD3(+) CD56(+) subsets. The CD3(+) CD56(+) and CD3(+) CD56(-) subsets express a polyclonal and comparable TCRVbeta repertoire in a Gaussian distribution. The CD3(+) CD56(+) subset kills AML targets more efficiently than its CD3(+) CD56(-) counterpart because of the presence of a higher proportion of CD8(+) cells. The CD3(+) CD56(+) subset comprise more terminally differentiated late effector T cells that bear the CD27(+) CD28(-) or CD27(-) CD28(-) phenotype, with a higher granzyme A content. In comparison, the phenotype of the CD3(+) CD56(-) subset is consistent with early effector T cells that are CD27(+) CD28(+) and CD62L(+), known to be less cytotoxic but possess greater proliferative potential.     

Structure/function of human killer cell immunoglobulin-like receptors: lessons from polymorphisms, evolution, crystal structures and mutations

Stimulation or tolerance of natural killer (NK) cells is achieved through a cross-talk of signals derived from cell surface activating and inhibitory receptors. Killer cell immunoglobulin-like receptors (KIR) are a family of highly polymorphic activating and inhibitory receptors that serve as key regulators of human NK cell function. Distinct structural domains in different KIR family members determine function by providing docking sites for ligands or signalling proteins. Here, we review a growing body of literature that has identified important structural elements on KIR that contribute to function through studies of engineered mutants, natural polymorphic sequence variants, crystal structure data and the conservation of protein sequences throughout primate evolution. Extensive natural polymorphism is associated with both human KIR and their ligands, MHC class I (HLA-A, -B and -C) molecules, and numerous studies have demonstrated associations between inheritance of certain combinations of KIR and HLA genes and susceptibility to several diseases, including viral infections, autoimmune disorders and cancers. In addition, certain KIR/HLA combinations can influence pregnancy and the outcome of haematopoietic stem cell transplantation. In view of the significant regulatory influences of KIR on immune function and human health, it is essential to fully understand the impacts of these polymorphic sequence variations on ligand recognition, expression and function of the receptor.     

The natural killer T lymphocyte: a player in the complex regulation of autoimmune diabetes in non-obese diabetic mice

Manipulation of the immune response to specifically prevent autoaggression requires an understanding of the complex interactions that occur during the pathogenesis of autoimmunity. Much attention has been paid to conventional T lymphocytes recognizing peptide antigens presented by classical major histocompatibility complex (MHC) class I and II molecules, as key players in the destructive autoreactive process. A pivotal role for different types of regulatory T lymphocytes in modulating the development of disease is also well established. Lately, CD1d-restricted natural killer T (NKT) lymphocytes have been the subject of intense investigation because of their ability to regulate a diversity of immune responses. The non-classical antigen presenting molecule CD1d presents lipids and glycolipids to this highly specialized subset of T lymphocytes found in both humans and mice. From experimental models of autoimmunity, evidence is accumulating that NKT cells can protect from disease. One of the best studied is the murine type 1 diabetes model, the non-obese diabetic (NOD) mouse. While the NKT cell population was first recognized to be deficient in NOD mice, augmenting NKT cell activity has been shown to suppress the development of autoimmune disease in this strain. The mechanism by which CD1d-restricted T cells exert this function is still described incompletely, but investigations in NOD mice are starting to unravel specific effects of NKT cell regulation. This review focuses on the role of CD1d-restricted NKT cells in the control of autoimmune diabetes.     

Strategy of lipid recognition by invariant natural killer T cells: 'one for all and all for one'

Invariant natural killer T (iNKT) cells are evolutionarily conserved lipid-reactive T cells that bridge innate and adaptive immune responses. Despite a relatively restricted T-cell receptor (TCR) diversity, these cells respond to a variety of structurally distinct foreign (i.e. microbial or synthetic) as well as host-derived (self-) lipid antigens presented by the CD1d molecule. These multi-tasking lymphocytes are among the first responders in immunity, and produce an impressive array of cytokines and chemokines that can tailor the ensuing immune response. Accordingly, iNKT cells play important functions in autoimmune diseases, cancer, infection and inflammation. These properties make iNKT cells appealing targets in immune-based therapies. Yet, much has to be learned on the mechanisms that allow iNKT cells to produce polarized responses. Responses of iNKT cells are influenced by the direct signals perceived by the cells through their TCRs, as well as by indirect co-stimulatory (and potentially co-inhibitory) cues that they receive from antigen-presenting cells or the local milieu. A decade ago, biochemists and immunologists have started to describe synthetic lipid agonists with cytokine skewing potential, paving a new research avenue in the iNKT cell field. Yet how iNKT cells translate various antigenic signals into distinct functional responses has remained obscure. Recent findings have revealed a unique and innate mode of lipid recognition by iNKT cells, and suggest that both the lipid antigen presented and the diversity of the TCR modulate the strength of CD1d-iNKT TCR interactions. In this review, we focus on novel discoveries on lipid recognition by iNKT cells, and how these findings may help us to design effective strategies to steer iNKT cell responses for immune intervention.     

Distinct granuloma responses in C57BL/6J and BALB/cByJ mice in response to pristane

Granuloma formation is an inflammatory response of the host against invading pathogens or indigestible substances. We generated mesenteric oil granulomas by injecting pristane into the peritoneal cavity (PC) of mice, and compared oil granuloma formation in the C57BL/6J and BALB/cByJ strains of mice. The formation and kinetics of oil granulomas were distinct between the two strains. In C57BL/6J mice, injected pristane induced oil granuloma formation at both the mesenteric centers (MG) and margins (SG). MG was resolving by 11 weeks, and SG persisted. In BALB/cByJ mice, MG developed slower but persisted longer than in C57BL/6J mice, and SG resolved sooner than in C57BL/6J mice. Injection of India ink revealed that phagocytes were localised mainly to the SG in C57BL/6J mice, but were located diffusely in both MG and SG of BALB/cByJ mice. SG cells expressed more monocyte chemotactic protein‐1 (MCP‐1) mRNA than MG cells in C57BL/6J mice, but there was no difference in MCP‐1 expression between the MG and SG in BALB/cByJ mice. These observations suggest that the recruitment of inflammatory leucocytes under the direction of chemokines differentiates the patterns of granuloma responses to pristane in C57BL/6J and BALB/cByJ mice.     

Redundancy in the immune system restricts the spread of HSV-1 in the central nervous system (CNS) of C57BL/6 mice

Resistance to lethal encephalitis in mice infected with HSV-1 via the oral mucosa is mouse strain dependent. In susceptible BALB/c, HSV-1 spreads throughout the CNS but in resistant BL/6 mice, virus is restricted to the brainstem. To examine the contribution of cellular immunity in restricting viral spread, we used a combination of antibody depleted and KO mice. Individually, NK/NKT, iNKT, CD4⁺, CD8⁺, and γδ T-cells do not restrict HSV-1 spread. In contrast, virus spreads throughout the CNS of BL/6 CL I KO mice and BL/6 mice treated with either anti-asialoGM1 Ab or both anti-CD8 and anti-NK1.1 mAbs. The results highlight the importance of redundancy in the immune system in restricting viral spread in the CNS, argue for a role of NK/NKT and CD8⁺ T-cells in mediating the restriction, and provide a hierarchical order of the individual elements in controlling virus in BL/6 mice infected with HSV-1 via the oral mucosa.     

Nature and nurture: T-cell receptor-dependent and T-cell receptor-independent differentiation cues in the selection of the memory T-cell pool

The initiation of a T‐cell response begins with the interaction of an individual T‐cell clone with its cognate antigen presented by MHC. Although the strength of the T‐cell receptor (TCR) –antigen–MHC (TCR‐pMHC) interaction plays an important and obvious role in the recruitment of T cells into the immune response, evidence in recent years has suggested that the strength of this initial interaction can influence various other aspects of the fate of an individual T‐cell clone and its daughter cells. In this review, we will describe differences in the way CD4⁺ and CD8⁺ T cells incorporate antigen‐driven differentiation and survival signals during the response to acute infection. Furthermore, we will discuss increasing evidence that the quality and/or quantity of the initial TCR‐pMHC interaction can drive the differentiation and long‐term survival of T helper type 1 memory populations.     

Methoxychlor metabolite HPTE alters viability and differentiation of embryonic thymocytes from C57BL/6 mice

Endocrine-disrupting chemicals (EDC) are widespread in the built and natural environments. Heightened public awareness of their potential danger has led to concern about whether EDC and their metabolites have significant negative biological effects. Studies have shown that EDC like DDT and other organochlorine pesticides, such as methoxychlor (MXC), have adverse effects on immune cells, but no studies have addressed the impact of HPTE, the primary metabolite of MXC. To elucidate the presence and significance of HPTE adverse effects, this study explored the impact of HPTE on a critical window and component of immune system development, embryonic T-cell development. Lesions at this phase of development can lead to lifelong immune dysfunction and increased incidence of immune disease, such as autoimmunity. Embry-onic thymocytes (GD 16-18) from C57BL/6 mice were subjected to an in vitro differentiation culture that mimicked early steps in thymocyte development in the presence of 0.005, 0.05, 0.5, 5, or 50?μM HPTE, or a model endocrine disruptor, DES. The results indicated that compared to the vehicle control, HPTE- and DES-induced death of thymocytes. Annexin-V staining and Caspase 8, markers of programed cell death, revealed that the loss of cells was due at least in part to induction of apoptosis. Moreover, HPTE-induced cell death not only resulted in selective loss of double positive thymocytes, but also loss of developing CD4 intermediate cells (post-double positive partially differentiated thymocyte population). Phenotypic analysis of thymocyte maturation (T-cell receptor, TCR) and TCR ligation (CD5) surface markers revealed that surviving embryonic thymocytes expressed low levels of both. Taken together these data demonstrate that immature embryonic thymocytes are sensitive to HPTE exposure and that HPTE exposure targets thymocyte populations undergoing critical differentiation steps. These findings suggest HPTE may play a pivotal role in MXC exposure-induced immune dysfunction.     

Different subsets of natural killer T cells may vary in their roles in health and disease

Natural killer T cells (NKT) can regulate innate and adaptive immune responses. Type I and type II NKT cell subsets recognize different lipid antigens presented by CD1d, an MHC class‐I‐like molecule. Most type I NKT cells express a semi‐invariant T‐cell receptor (TCR), but a major subset of type II NKT cells reactive to a self antigen sulphatide use an oligoclonal TCR. Whereas TCR‐α dominates CD1d‐lipid recognition by type I NKT cells, TCR‐α and TCR‐β contribute equally to CD1d‐lipid recognition by type II NKT cells. These variable modes of NKT cell recognition of lipid–CD1d complexes activate a host of cytokine‐dependent responses that can either exacerbate or protect from disease. Recent studies of chronic inflammatory and autoimmune diseases have led to a hypothesis that: (i) although type I NKT cells can promote pathogenic and regulatory responses, they are more frequently pathogenic, and (ii) type II NKT cells are predominantly inhibitory and protective from such responses and diseases. This review focuses on a further test of this hypothesis by the use of recently developed techniques, intravital imaging and mass cytometry, to analyse the molecular and cellular dynamics of type I and type II NKT cell antigen‐presenting cell motility, interaction, activation and immunoregulation that promote immune responses leading to health versus disease outcomes.     

Characteristics of IL‐17 induction by Schistosoma japonicum infection in C57BL/6 mouse liver

Schistosomiasis japonica is a severe tropical disease caused by the parasitic worm Schistosoma japonicum. Among the most serious pathological effects of S. japonicum infection are hepatic lesions (cirrhosis and fibrosis) and portal hypertension. Interleukin‐17 (IL‐17) is a pro‐inflammatory cytokine involved in the pathogenesis of many inflammatory and infectious conditions, including schistosomiasis. We infected C57BL/6 mice with S. japonicum and isolated lymphocytes from the liver to identify cell subsets with high IL‐17 expression and release using flow cytometry and ELISA. Expression and release of IL‐17 was significantly higher in hepatic lymphocytes from infected mice compared with control mice in response to both non‐specific stimulation with anti‐CD3 monoclonal antibody plus/anti‐CD28 monoclonal antibody and PMA plus ionomycin. We then compared IL‐17 expression in three hepatic T‐cell subsets, T helper, natural killer T and γδT cells, to determine the major source of IL‐17 during infection. Interleukin‐17 was induced in all three subsets by PMA + ionomycin, but γδT lymphocytes exhibited the largest increase in expression. We then established a mouse model to further investigate the role of IL‐17 in granulomatous and fibrosing inflammation against parasite eggs. Reducing IL‐17 activity using anti‐IL‐17A antibodies decreased infiltration of inflammatory cells and collagen deposition in the livers of infected C57BL/6 mice. The serum levels of soluble egg antigen (IL) ‐specific IgGs were enhanced by anti‐IL‐17A monoclonal antibody blockade, suggesting that IL‐17 normally serves to suppress this humoral response. These findings suggest that γδT cells are the most IL‐17‐producing cells and that IL‐17 contributes to granulomatous inflammatory and fibrosing reactions in S. japonicum‐infected C57BL/6 mouse liver.     

Graft-versus-host disease in recipients of grafts from natural killer T cell-deficient (Jalpha281(-/-)) donors

Valpha14i natural killer T cells (NKT cells) are a CD1-restricted subset of NKT cells that express an invariable Valpha14+ Jalpha281+ alphabeta T-cell receptor. To determine whether the absence of Valpha14i NKT cells from the graft affects the development of acute GVHD, we induced GVH reactions using Jalpha281(-/-) mice as donors in the C57BL/6-->(C57BL/6 x DBA/2)F1-hybrid strain combination. Recipients of grafts from Jalpha281(-/-) donors were not protected from either the morbidity or the severe wasting syndrome associated with the development of acute GVHD, but the concentrations of some T helper 1 (Th1) and Th2 cytokines were different from those seen in recipients of grafts from wild-type donors. Interferon-gamma was seen earlier (day 4) in recipients of grafts from Jalpha281(-/-) donors but did not reach the concentrations seen in recipients of grafts from wild-type donors on day 8 (P < 0.02). On day 8, the amount of tumour necrosis factor-alpha released into the serum following the injection of a small amount of lipopolysaccharide was lower in recipients of grafts from Jalpha281(-/-) donors (P < 0.02). The amount of interleukin (IL)-5 was also lower in recipients of grafts from Jalpha281(-/-) donors, when compared to the concentration seen in recipients of grafts from wild-type donors (P < 0.002). IL-13 was seen in recipients of grafts from Jalpha281(-/-) donors but not in recipients of grafts from wild-type donors. Our findings show that the absence of donor Valpha14i NKT cells is associated with lower concentrations of some Th1 cytokines. We also observed higher IL-13 concentrations and lower IL-5 concentrations in recipients of grafts from Jalpha281(-/-) donors indicating a variable effect on Th2 cytokine production.     

Chronic exposure to stress predisposes to higher autoimmune susceptibility in C57BL/6 mice: Glucocorticoids as a double‐edged sword

Stress activates the hypothalamic‐pituitary‐adrenocortical axis to promote the release of corticosterone (CORT), which consequently suppresses pathogenic stimulation of the immune system. Paradoxically, however, stress often promotes autoimmunity through yet unknown mechanisms. Here we investigated how chronic variable stress (CVS), and the associated alterations in CORT levels, affect the susceptibility to experimental autoimmune encephalomyelitis (EAE) in female and male C57BL/6 mice. Under baseline (nonstressed) conditions, females exhibited substantially higher CORT levels and an attenuated EAE with less mortality than males. However, CVS induced a significantly worsened EAE in females, which was prevented if CORT signaling was blocked. In addition, females under CVS conditions showed a shift toward proinflammatory Th1/Th17 versus Th2 responses and a decreased proportion of CD4⁺CD25⁺ Treg cells. This demonstrates that whereas C57BL/6 female mice generally exhibit higher CORT levels and an attenuated form of EAE than males, they become less responsive to the immunosuppressive effects of CORT under chronic stress and thereby prone to a higher risk of destructive autoimmunity.     

Dynamics of immune effector mechanisms during infection with Mycobacterium avium in C57BL/6 mice

Opportunistic infections with non‐tuberculous mycobacteria such as Mycobacterium avium are receiving renewed attention because of increased incidence and difficulties in treatment. As for other mycobacterial infections, a still poorly understood collaboration of different immune effector mechanisms is required to confer protective immunity. Here we have characterized the interplay of innate and adaptive immune effector mechanisms contributing to containment in a mouse infection model using virulent M. avium strain 104 in C57BL/6 mice. M. avium caused chronic infection in mice, as shown by sustained organ bacterial load. In the liver, bacteria were contained in granuloma‐like structures that could be defined morphologically by expression of the antibacterial innate effector protein Lipocalin 2 in the adjoining hepatocytes and infiltrating neutrophils, possibly contributing to containment. Circulatory anti‐mycobacterial antibodies steadily increased throughout infection and were primarily of the IgM isotype. Highest levels of interferon‐γ were found in infected liver, spleen and serum of mice approximately 2 weeks post infection and coincided with a halt in organ bacterial growth. In contrast, expression of tumour necrosis factor was surprisingly low in spleen compared with liver. We did not detect interleukin‐17 in infected organs or M. avium‐specific T helper 17 cells, suggesting a minor role for T helper 17 cells in this model. A transient and relative decrease in regulatory T cell numbers was seen in spleens. This detailed characterization of M. avium infection in C57BL/6 mice may provide a basis for future studies aimed at gaining better insight into mechanisms leading to containment of infections with non‐tuberculous mycobacteria.     

牛MBP诱导BALB/c和C57BL/6小鼠实验性变态性脑脊髓炎样反应的研究

为了进一步研究实验性变态反应性脑脊髓炎(EAE)动物模型,采用从新鲜牛脊髓中提取的MBP免疫诱导非敏感品系BALB/c小鼠和C57BL/6小鼠,通过对实验动物的症状、中枢神经系统的病理改变及细胞因子水平变化的检测,较成功地建立了C57BL/6、BALB/c小鼠的EAE模型,实验方法稳定可靠。