技术资料

Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules,we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization,lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization,dependent on dynein motor function,occurred before MTOC polarization,and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion,one that may prepare,but not commit cells for precision secretory function. View Publication

T cell development requires a period of postthymic maturation. Why this is the case has remained a mystery,particularly given the rigors of intrathymic developmental checkpoints,successfully traversed by only ∼5% of thymocytes. We now show that the first few weeks of T cell residence in the lymphoid periphery define a period of heightened susceptibility to tolerance induction to tissue-restricted antigens (TRAs),the outcome of which depends on the context in which recent thymic emigrants (RTEs) encounter antigen. After encounter with TRAs in the absence of inflammation,RTEs exhibited defects in proliferation,diminished cytokine production,elevated expression of anergy-associated genes,and diminished diabetogenicity. These properties were mirrored in vitro by enhanced RTE susceptibility to regulatory T cell-mediated suppression. In the presence of inflammation,RTEs and mature T cells were,in contrast,equally capable of inducing diabetes,proliferating,and producing cytokines. Thus,recirculating RTEs encounter TRAs during a transitional developmental stage that facilitates tolerance induction,but inflammation converts antigen-exposed,tolerance-prone RTEs into competent effector cells. View Publication

IL-12 is an immunoregulatory cytokine,which promotes Th1 cell differentiation and is a major inducer of IFN-gamma. IFN-beta,a Type I IFN used in the treatment of multiple sclerosis,has been shown to significantly increase the expression of the anti-inflammatory cytokine IL-10,a major suppressor of Th1 cytokines. The beneficial immunomodulatory effects of IFN-beta may in part be a result of its ability to suppress IL-12. However,IL-12 and IFN-beta signal via the STAT4 pathway. Our aim was to investigate the relationship between IL-12 and IFN-beta by observing the effect of prior exposure to IL-12 or IFN-beta on the ability of T cells to subsequently respond to the other cytokine. We report that IFN-beta increases IL-12-induced STAT4 phosphorylation and up-regulates IL-12 receptor beta1 and beta2 expression. However,despite this up-regulation,IFN-beta suppressed IL-12-induced IFN-gamma expression. Our results suggest that this may be a result of the parallel induction of IL-10 by IFN-beta. View Publication

BACKGROUND/AIM Live attenuated vaccines confer partial protection in pigs before the appearance of neutralizing antibodies,suggesting the contribution of cell-mediated immunity (CMI). However,PRRSV-specific T-lymphocyte responses and protective mechanisms need to be further defined. To this end,the hypothesis was tested that PRRSV-specific T-lymphocytes induced by exposure to type-2 PRRSV can recognize diverse isolates. METHODS An IFN-gamma ELISpot assay was used to enumerate PRRSV-specific T-lymphocytes from PRRSVSD23983-infected gilts and piglets born after in utero infection against 12 serologically and genetically distinct type-1 and -2 PRRSV isolates. The IFN-gamma ELISpot assay using synthetic peptides spanning all open reading frames of PRRSVSD23983 was utilized to localize epitopes recognized by T-lymphocytes. Virus neutralization tests were carried out using the challenge strain (type-2 PRRSVSD23983) and another strain (type-2 PRRSVVR2332) with high genetic similarity to evaluate cross-reactivity of neutralizing antibodies in gilts after PRRSVSD23983 infection. RESULTS At 72 days post infection,T-lymphocytes from one of three PRRSVSD23983-infected gilts recognized all 12 diverse PRRSV isolates,while T-lymphocytes from the other two gilts recognized all but one isolate. Furthermore,five of nine 14-day-old piglets infected in utero with PRRSVSD23983 had broadly reactive T-lymphocytes,including one piglet that recognized all 12 isolates. Overlapping peptides encompassing all open reading frames of PRRSVSD23983 were used to identify ≥28 peptides with T-lymphocyte epitopes from 10 viral proteins. This included one peptide from the M protein that was recognized by T-lymphocytes from all three gilts representing two completely mismatched MHC haplotypes. In contrast to the broadly reactive T-lymphocytes,neutralizing antibody responses were specific to the infecting PRRSVSD23983 isolate. CONCLUSION These results demonstrated that T-lymphocytes recognizing antigenically and genetically diverse isolates were induced by infection with a type 2 PRRSV strain (SD23983). If these reponses have cytotoxic or other protective functions,they may help overcome the suboptimal heterologous protection conferred by conventional vaccines. View Publication

Gag-specific CD4 proliferative responses correlate inversely with HIV-1 RNA levels in infected adults,and robust responses are characteristic of long-term nonprogressive infection. However,strong responses are seldom detected in adult subjects with progressive infection and are not generally reconstituted on highly active antiretroviral therapy (HAART). To date,the role of HIV-1-specific Th responses in children has not been thoroughly examined. We characterized Gag-specific CD4 responses among 35 perinatally infected subjects,including 2 children who spontaneously control viremia without antiretroviral therapy,21 children with viral loads (VL) of textless400 on HAART,and 12 viremic children. Gag-specific Th activity was assessed by lymphoproliferative assay,and responses were mapped using overlapping Gag peptides in an IFN-gamma ELISPOT. Robust proliferative responses were detected in the children exhibiting spontaneous control of viremia,and mapping of targeted Gag regions in one such subject identified multiple epitopes. Among children textgreateror=5 years old,14 of 17 subjects with VL of textless400 on HAART demonstrated a significant p24 proliferative response (median p24 stimulation index,20),in contrast with only 1 of 9 viremic children (median p24 stimulation index,2.0; p = 0.0008). However,no subject younger than 5 years of age possessed a significant response,even when viremia was fully suppressed. When compared with adults with VL of textless400 on HAART,Th responses among children with VL of textless400 were both more frequent (p = 0.009) and of greater magnitude (p = 0.002). These data suggest that children may have a greater intrinsic capacity to reconstitute HIV-1-specific immunity than adults,and may be excellent candidates for immune-based therapies. View Publication

BACKGROUND: Patients with type 2 diabetes mellitus are at increased risk for the development of atherosclerosis. A pivotal event in the development of atherosclerosis is macrophage foam cell formation. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 regulate macrophage cholesterol efflux and hence play a vital role in macrophage foam cell formation. We have previously found that chronic elevated glucose reduces ABCG1 expression. In the present study,we examined whether patients with type 2 diabetes mellitus had decreased ABCG1 and/or ABCA1,impaired cholesterol efflux,and increased macrophage foam cell formation. METHODS AND RESULTS: Blood was collected from patients with and without type 2 diabetes mellitus. Peripheral blood monocytes were differentiated into macrophages,and cholesterol efflux assays,immunoblots,histological analysis,and intracellular cholesteryl ester measurements were performed. Macrophages from patients with type 2 diabetes mellitus had a 30% reduction in cholesterol efflux with a corresponding 60% increase in cholesterol accumulation relative to control subjects. ABCG1 was present in macrophages from control subjects but was undetectable in macrophages from patients with type 2 diabetes mellitus. In contrast,ABCA1 expression in macrophages was similar in both control subjects and patients with type 2 diabetes mellitus. Macrophage expression of ABCG1 in both patients and control subjects was induced by treatment with the liver X receptor agonist TO-901317. Upregulation of liver X receptor dramatically reduced foam cell formation in macrophages from patients with type 2 diabetes mellitus. CONCLUSIONS: ABCG1 expression and cholesterol efflux are reduced in patients with type 2 diabetes mellitus. This impaired ABCG1-mediated cholesterol efflux significantly correlates with increased intracellular cholesterol accumulation. Strategies to upregulate ABCG1 expression and function in type 2 diabetes mellitus could have therapeutic potential for limiting the accelerated vascular disease observed in patients with type 2 diabetes mellitus. View Publication

The multikinase inhibitors sunitinib,sorafenib,and axitinib have an impact not only on tumor growth and angiogenesis,but also on the activity and function of immune effector cells. In this study,a comparative analysis of the growth inhibitory properties and apoptosis induction potentials of tyrosine kinase inhibitors on T cells was performed. Tyrosine kinase inhibitor treatment resulted in a dramatic decrease in T cell proliferation along with distinct impacts on the cell cycle progression. This was at least partially associated with an enhanced induction of apoptosis although triggered by distinct apoptotic mechanisms. In contrast to sunitinib and sorafenib,axitinib did not affect the mitochondrial membrane potential but resulted in an induction or stabilization of the induced myeloid leukemia cell differentiation protein (Mcl-1),leading to an irreversible arrest in the G2/M cell cycle phase and delayed apoptosis. Furthermore,the sorafenib-mediated suppression of immune effector cells,in particular the reduction of the CD8(+) T cell subset along with the down-regulation of key immune cell markers such as chemokine CC motif receptor 7 (CCR7),CD26,CD69,CD25,and CXCR3,was not observed in axitinib-treated immune effector cells. Therefore,axitinib rather than sorafenib seems to be suitable for implementation in complex treatment regimens of cancer patients including immunotherapy. View Publication

BAFF plays a central role in B-lineage cell biology; however,the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study,we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation,as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs),excluding bone marrow PCs,express BAFF-R uniformly. In contrast,only tonsillar memory B cells (MB) and PCs,from both tonsil and bone marrow tissues,express BCMA. Furthermore,we show that TACI is expressed by MB cells and PCs,as well as a subpopulation of activated CD27(neg) B cells. In this regard,we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition,we found that TACI expression requires activation of the ERK1/2 pathway,since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC),since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively,our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells. View Publication