疟原虫红前期适应性免疫相关机制的研究进展

红前期是疟原虫进入宿主的第一个时期,阻断红前期疟原虫的发育,就能够从源头上阻断疟疾的发生和传播,有效的红前期疟疾疫苗对于控制疟疾的发病及阻断疟疾的传播有着重要意义。对红前期免疫机制的深入研究对于合理的红前期亚单位疫苗的设计具有重要的指导意义,本文仅就目前疟原虫红前期适应性免疫相关机制的研究做一综述。     

Protective and pathogenic roles of CD8+ T cells during malaria infection

CD8+ T cells play a key role in protection against pre-erythrocytic stages of malaria infection. Many vaccine strategies are based on the idea of inducing a strong infection-blocking CD8+ T cell response. Here, we summarize what is known about the development, specificity and protective effect of malaria-specific CD8+ T cells and report on recent developments in the field. Although work in mouse models continues to make progress in our understanding of the basic biology of these cells, many questions remain to be answered - particularly on the roles of these cells in human infections. Increasing evidence is also emerging of a harmful role for CD8+ T cells in the pathology of cerebral malaria in rodent systems. Once again, the relevance of these results to human disease is one of the primary questions facing workers in this field.     

Evidence of cross‐stage CD8+ T cell epitopes in malaria pre‐erythrocytic and blood stage infections

Malaria parasites have a complex, multistage life cycle and there is a widely held view that each stage displays a distinct set of antigens presented to the immune system. Yet, molecular analysis of malaria parasites suggests that many putative antigenic targets are shared amongst the different stages. The specificities of these cross‐stage antigens and the functions of the immune responses they elicit are poorly characterized. It is well‐known that CD8+ T cells play opposing immune functions following Plasmodium berghei (Pb) infection of C57BL/6 mice. Whilst these cells play a crucial role in protective immunity against pre‐erythrocytic stages, they are implicated in the development of severe disease during blood stages. Recently, CD8+ T cell epitopes derived from proteins supposedly specific for either pre‐erythrocytic or blood stages have been described. In this brief report, we have compiled and confirmed data that the majority of the mRNAs and/or proteins from which these epitopes are derived display expression across pre‐erythrocytic and blood stages. Importantly, we provide evidence of cross‐stage immune recognition of the majority of these CD8+ T cell epitopes. Hence, our findings provide a resource to further examine the relevance of antigen‐specific cross‐stage responses during malaria infections.     

Comprehensive longitudinal analysis of hepatitis C virus (HCV)‐specific T cell responses during acute HCV infection in the presence of existing HIV‐1 infection

Summary. The aim of this study was to study the development of HCV‐specific T cell immunity during acute HCV infection in the presence of an existing HIV‐1 infection in four HIV‐1 infected men having sex with men. A comprehensive analysis of HCV‐specific T cell responses was performed at two time points during acute HCV infection using a T cell expansion assay with overlapping peptide pools spanning the entire HCV genome Three patients with (near) normal CD4+ T cell counts (range 400–970 × 10⁶/L) either resolved (n = 1) or temporary suppressed HCV RNA. In contrast, one patient with low CD4+ T cell counts (330 × 10⁶/L), had sustained high HCV RNA levels. All four patients had low HCV‐specific CD8+ T cell responses, and similar magnitudes of CD4+ T cell responses. Interestingly, individuals with resolved infection or temporary suppression of HCV‐RNA had HCV‐specific CD4+ T cell responses predominantly against nonstructural (NS) proteins. While the individual with high HCV RNA plasma concentrations had CD4+ T cell responses predominantly directed against Core. Our data show that an acute HCV infection in an HIV‐1 infected person can be suppressed in the presence of HCV‐specific CD4+ T cell response targeting non‐structural proteins. However further research is needed in a larger group of patients to evaluate the role of HIV‐1 on HCV‐specific T cell responses in relation to outcome of acute HCV infection.     

Detection of intracellular IFN‐γ and IL‐4 cytokines in CD4+ and CD8+ T cells in the peripheral blood of dogs naturally infected with Leishmania infantum

Canine leishmaniosis (CanL) is a systemic zoonotic disease the clinical manifestations of which can range from self‐healing cutaneous lesions to disseminated visceral disease. Effective activation of cellular immunity is the cornerstone of resistance against Leishmania infantum in infected dogs. The aim of this cross‐sectional, controlled study was the intracellular detection of interleukin 4 (IL‐4) and interferon‐γ (IFN‐γ) in CD4+ and CD8+ lymphocytes in the peripheral blood of 40 dogs naturally infected with L. infantum by applying flow cytometry. The percentage of CD4+IL‐4+ and CD8+IL‐4+ lymphocytes (with or without immunostimulation) was low in the clinically healthy and subclinically infected dogs in contrast to clinically affected ones. In the same groups of dogs, the percentage of CD4+IFN‐γ+ and CD8+IFN‐γ+ T cells in their resting phase and following specific immunostimulation with Leishmania soluble antigen (LSA) was also low. CD4+IL‐4+ and CD8+IL‐4+ T cell percentage was higher in sick compared to clinically healthy and subclinically infected dogs, after immunostimulation. The corresponding figure of CD8+IL‐4+ cells in sick dogs after LSA immunostimulation was also increased thus underlining the important role these cells may play in humoral immunity and perhaps the progression of CanL.     

A whole‐killed,blood‐stage lysate vaccine protects against the malaria liver stage

Although the attenuated sporozoite is the most efficient vaccine to prevent infection with the malaria parasite, the limitation of a source of sterile sporozoites greatly hampers its application. In this study, we found that the whole‐killed, blood‐stage lysate vaccine could confer protection against the blood stage as well as the liver stage. Although the protective immunity induced by the whole‐organism vaccine against the blood stage is dependent on parasite‐specific CD4⁺ T‐cell responses and antibodies, in mice immunized with the whole‐killed, blood‐stage lysate vaccine, CD8⁺, but not CD4⁺ effector T‐cell responses greatly contributed to protection against the liver stage. Thus, our data suggested that the whole‐killed, blood‐stage lysate vaccine could be an alternative promising strategy to prevent malaria infection and to reduce the morbidity and mortality of patients with malaria.     

Interferon-gamma-independent CD8+ T cell-mediated protective anti-malaria immunity elicited by recombinant adenovirus

Recombinant adenovirus, expressing the CS protein of Plasmodium yoelii, AdPyCS, was shown to induce a comparable degree of T cell-mediated protection against malaria as a single dose of irradiated P. yoelii sporozoites, causing inhibition of liver stage development. We now report that differently from sporozoite-induced immunity, interferon (IFN)-gamma does not mediate the protective immunity induced by AdPyCS, since a similar degree of protection was observed in AdPyCS immunized mice lacking IFN-gamma-/- and the IFN-gamma receptor (IFN-gammaR-/-) compared to that in wild-type mice. Depletion of CD8+ T cells from these immunized mice almost completely abolished the AdPyCS-induced immunity, indicating that the immunization with AdPyCS induces CD8+ T cell-mediated protective anti-malaria immunity, which is independent of IFN-gamma.     

Batf3 deficiency proves the pivotal role of CD8α+ dendritic cells in protection induced by vaccination with attenuated Plasmodium sporozoites

Increasing evidence indicates that hepatic CD8α⁺ dendritic cells (DCs) are important antigen cross‐presenting cells (APC) involved in the priming of protective CD8⁺ T‐cell responses induced by live‐attenuated Plasmodium sporozoites. Experimental proof for a critical role of CD8α⁺ DCs in protective pre‐erythrocytic malaria immunizations has pivotal implications for vaccine development, including improved vectored subunit vaccines. Employing Batf3^?/? mice, which lack functional CD8α⁺ DCs, we demonstrate that deficiency of these particular APCs completely abolishes protection and corresponding signatures of vaccine‐induced immunity. We show that in wild‐type, but not in Batf3^?/?, mice CD8α⁺ DCs accumulate in the liver after immunization with live irradiation‐attenuated P. berghei sporozoites. IFN‐γ production by Plasmodium antigen‐specific CD8⁺ T cells is dependent on functional Batf3. In addition, our results demonstrate that the dysfunctional cDC‐CD8+ T‐cell axis correlates with MHC class II upregulation on splenic CD8α^? DCs. Collectively, these findings underscore the essential role of CD8α⁺ DCs in robust protection induced by experimental live‐attenuated malaria vaccines.     

Initial viral load determines the magnitude of the human CD8 T cell response to yellow fever vaccination

CD8 T cells are a potent tool for eliminating intracellular pathogens and tumor cells. Thus, eliciting robust CD8 T-cell immunity is the basis for many vaccines under development. However, the relationship between antigen load and the magnitude of the CD8 T-cell response is not well-described in a human immune response. Here we address this issue by quantifying viral load and the CD8 T-cell response in a cohort of 80 individuals immunized with the live attenuated yellow fever vaccine (YFV-17D) by sampling peripheral blood at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90. When the virus load was below a threshold (peak virus load < 225 genomes per mL, or integrated virus load < 400 genome days per mL), the magnitude of the CD8 T-cell response correlated strongly with the virus load (R² ∼ 0.63). As the virus load increased above this threshold, the magnitude of the CD8 T-cell responses saturated. Recent advances in CD8 T-cell–based vaccines have focused on replication-incompetent or single-cycle vectors. However, these approaches deliver relatively limited amounts of antigen after immunization. Our results highlight the requirement that T-cell–based vaccines should deliver sufficient antigen during the initial period of the immune response to elicit a large number of CD8 T cells that may be needed for protection.CD8 T cells provide a powerful mechanism for elimination of intracellular pathogens and tumor cells. Accordingly, a major thrust of current vaccine research focuses on stimulating robust T-cell immunity for defense against infections such as HIV, malaria, tuberculosis, Ebola virus, herpes viruses, and hepatitis C virus (HCV) (1–8). Inducing effective CD8 T-cell immunity is also an important goal for cancer vaccines (9, 10). However, how antigen load affects the CD8 T-cell response has not been quantified in a detailed manner during a human immune response. In this study we address this question using the human live attenuated yellow fever vaccine (YFV-17D) vaccine.The dynamics of CD8 T-cell responses to intracellular infection have been extensively studied in model systems. Infection typically stimulates a rapid burst of proliferation in antigen-specific CD8 T cells with division occurring as quickly as once in 4–6 h (11). This expansion results in a large population of effector CD8 T cells that aid in clearance of infected cells. Although most (90–95%) of the effector CD8 T cells die, a small fraction differentiate to form long-term memory CD8 T cells (12). Detailed quantitative measurements of the dynamics of virus and the CD8 T-cell response to the YFV-17D vaccine allow us to characterize these basic features of the CD8 T-cell responses in humans. Additionally, tracking the dynamics of both virus and CD8 T cells over time in a large cohort allows us to explore the relationship between amount of antigen and the magnitude of expansion and answer the following questions: Is there a threshold amount of virus required to generate a response? Does the magnitude of the response increase proportionally, or does it saturate with viral load? Although a number of studies have considered the complex relationship between numbers of specific CD8 T cells and virus loads during the chronic phase of HIV and HCV infections (3, 13–23), very few studies (24–27) have investigated these questions in the context of the generation of immune response following acute infections and vaccination.We addressed these questions by measuring the dynamics of both virus and virus-specific CD8 T cells following immunization with the YFV-17D vaccine. The YFV-17D vaccine comprises a highly efficacious, live attenuated virus that causes an acute infection and stimulates a robust immune response conferring lifelong protection against the yellow fever virus (YFV) (28, 29). Because yellow fever is not endemic to the United States, immunization with YFV-17D induces a primary immune response (30, 31). Previous work with YFV-17D has identified CD8 T cells specific for some of the YFV epitopes and defined the stages of expansion, contraction, and memory maintenance (32–38). We now know that YFV stimulates a polyfunctional, broadly targeting, and long-lasting CD8 T-cell response. Of particular note, we have previously demonstrated that the magnitude of the total effector CD8 T-cell response against YFV can be measured using the Ki-67⁺ Bcl-2^lo HLA-DR⁺ CD38⁺ phenotype of activated T cells seen early after vaccination (38). In the current study, we followed a large cohort of 80 individuals with intensive sampling at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90 postvaccination to quantify viral load in plasma (39). Additionally, we quantified the magnitude of the YFV-specific effector CD8 T-cell response at days 0, 3, 7, 14, 30, and 90 postvaccination using the Ki-67⁺Bcl-2^lo phenotype. We find that different individuals have different virus loads following infection and generate CD8 T-cell responses of different sizes. This allows us to determine the relationship between virus load and magnitude of the CD8 T-cell response.The majority of vaccines that are currently under development use replication-incompetent or single-cycle vectors such as Modified Vaccinia Ankara, adenovirus, and DNA. Although these approaches are inherently safe, they may express and deliver relatively limited amounts of antigen. Our results emphasize the requirement that T-cell–based vaccines deliver sufficient antigen to elicit a large CD8 T-cell response that may be needed for protection.     

Mechanisms regulating expansion of CD8+ T cells during HIV‐1 infection

Abnormal immune activation and expansion of CD8+ T cells, especially of memory and effector phenotypes, take place during HIV‐1 infection, and these abnormal features persist during administration of antiretroviral therapy (ART) to infected patients. The molecular mechanisms for CD8+ T‐cell expansion remain poorly characterized. In this article, we review the literature addressing features of CD8+ T‐cell immune pathology and present an integrated view on the mechanisms leading to abnormal CD8+ T‐cell expansion during HIV‐1 infection. The expression of molecules important for directing the homing of CD8+ T cells between the circulation and lymphoid tissues, in particular CCR5 and CXCR3, is increased in CD8+ T cells in circulation and in inflamed tissues during HIV‐1 infection; these disturbances in the homing capacity of CD8+ T cells have been linked to increased CD8+ T‐cell proliferation. The production of IL‐15, a cytokine responsible for physiological proliferation of CD8+ T cells, is increased in lymphoid tissues during HIV‐1 infection as result of microbial translocation and severe inflammation. IL‐15, and additional inflammatory cytokines, may lead to deregulated proliferation of CD8+ T cells and explain the accumulation of CD8+ T cells in circulation. The decreased capacity of CD8+ T cells to localize to gut‐associated lymphoid tissue also contributes to the accumulation of these cells in blood. Control of inflammation, through ART administration during primary HIV‐1 infection or therapies aimed at controlling inflammation during HIV‐1 infection, is pivotal to prevent abnormal expansion of CD8+ T cells during HIV‐1 infection.     

CD4~+CD25~+调节性T细胞对啮齿类疟原虫感染过程及结局的影响

目的探讨CD4+CD25+调节性T细胞数量在约氏疟原虫(致死型)和夏氏疟原虫混合感染小鼠免疫应答中的动态变化。方法DBA/2和BALB/c小鼠分别经腹腔注射致死型约氏疟原虫(P.y17XL)、夏氏疟原虫(P.cAS)和P.y17XL+P.cAS(1∶1)混合感染的红细胞,计数红细胞感染率;采用流式细胞术动态检测脾细胞中Tregs细胞数量的变化。结果P.y17XL+P.cAS感染的DBA/2小鼠于感染后18d自愈;而BALB/c小鼠于感染过程中虽然出现死亡(8d),但与P.y17XL感染小鼠(5d)相比,死亡时间明显延迟;P.y17XL和P.y17XL+P.cAS感染的DBA/2鼠于感染后第5d CD4+CD25+调节性T细胞数量均达峰值,随后缓慢下降,相比P.cAS感染的DBA/2鼠于感染后第10d达峰值,是其它两种同天感染鼠的3倍和2倍,且小鼠全部死亡;而P.y17XL和P.y17XL+P.cAS感染的BALB/c小鼠CD4+CD25+调节性T细胞数量于感染后均迅速升高,分别于感染后第5d和第8d达30%左右,小鼠全部死亡,相比P.cAS感染的BALB/c小鼠CD4+CD25+调节性T细胞数量于感染后缓慢升高,于感染后第5d达14%,随后开始下降。结论①P.y17XL+P.cAS混合感染DBA/2鼠后CD4+CD25+调节性T细胞数量的动态变化与P.y17XL单独感染表现出完全相同的模式;BALB/c鼠:CD4+CD25+调节性T细胞数量的动态变化与P.y17XL单独感染也表现出完全相同的模式,但变化的时间明显被滞后;②CD4+CD25+调节性T细胞的异常升高与感染结局密切相关。     

T cell receptor Vβ chain restriction and preferred CDR3 motifs of liver‐kidney microsomal antigen (LKM‐1)‐reactive T cells from autoimmune hepatitis patients

Abstract: Aims/Background: The liver‐kidney‐microsomal antigen (LKM‐1) has been recognized as a major CD4⁺ T cell antigen in autoimmune hepatitis (AIH). The aim of this study was to characterize the antigen recognition sites of the variable T cell receptor β‐chain (TCRBV) of T cells specific to LKM‐1. Methods: By repeated stimulation of T cells with a recombinant LKM‐1 antigen or an LKM‐derived peptide followed by limited dilution, we generated T cell clones. Usage of TCRBV was analyzed by RT‐PCR and CDR3 antigen recognition sites were sequenced. Results: The 18 LKM‐1 specific T cell clones isolated from six AIH patients preferentially expressed the TCR elements BV9, BV5S2+S3, BV6, and BV13S1. Four BV9+ T cell clones rearranged the joining element JB1S3 within their CDR3 regions. JB2S3 was detected in another four clones together with BV5S2+S3 or BV13S1. A conserved sequence motif, Q(N)G(X)N, was seen in the diversity regions of five clones (36%). In order to identify T cells expressing the preferred TCRBV molecules in situ, immunohistologic examination of liver biopsies was performed. In AIH patients an accumulation of T cells expressing TCRBV 13S1, BV8 and BV5S3 was observed. Conclusions: Our data define TCRBV restriction and preferred CDR3 features of LKM‐1 specific T cells. The in situ localization of T cells expressing these restricted TCR molecules may suggest a pathogenic relevance of LKM‐1 specific cellular immune responses.     

Increased Fas ligand expression of peripheral B‐1 cells correlated with CD4+ T‐cell apoptosis in filarial‐infected patients

Cellular hyporesponsiveness observed during helminth infections is attributed to factors such as antigen‐presenting cells (APC) dysfunction, increased interleukin‐10(IL‐10), regulatory T cells and induction of CD4⁺ T (Th)‐cell apoptosis. Increased Fas ligand (FasL) expression on the surface of B‐1 cells and induction of apoptosis of Th cells by FasL‐expressing B‐1 cells due to helminth infection were demonstrated in murine model of helminth infection where as profile of FasL expression, Th‐cell apoptosis and correlation between these two populations of cells in clinical filariasis remain unknown. In this study, we have scored the profile of apoptotic Th‐cell population and FasL‐expressing B‐1 cells in different clinical categories of filariasis. The peripheral apoptotic T‐helper cells were significantly increased in filarial patients compared to endemic controls. Expression of FasL on the surface of peripheral B‐1 cells increased in filarial patients and positively correlated with peripheral apoptotic T‐helper cells indicating FasL‐expressing B‐1 cells may be one of the important mediators of Th‐cell apoptosis and immune anergy during filarial pathology.     

Lipopeptide vaccines illustrate the potential role of subtype‐crossreactive T cells in the control of highly virulent influenza

Background The best form of protection against influenza is high‐titred virus‐neutralizing antibody specific for the challenge strain. However, this is not always possible to achieve by vaccination due to the need for predicting the emerging virus, whether it be a drift variant of existing human endemic influenza type A subtypes or the next pandemic virus, for incorporation into the vaccine. By activating additional arms of the immune system to provide heterosubtypic immunity, that is immunity active against all viruses of type A influenza regardless of subtype or strain, it should be possible to provide significant benefit in situations where appropriate antibody responses are not achieved. Although current inactivated vaccines are unable to induce heterosubtypic CD8⁺ T cell immunity, we have shown that lipopeptides are particularly efficient in this regard. Objectives To examine the role of vaccine‐induced CD8⁺ T cells in altering the course of disease due to highly virulent H1N1 influenza virus in the mouse model. Methods The induction of influenza‐specific CD8⁺ T cells following intranasal inoculation with lipopeptide vaccine was assessed by intracellular cytokine staining (ICS) and the capacity of these cells to reduce viral loads in the lungs and to protect against death after viral challenge was determined. Results and conclusions We show that CD8⁺ T cells are induced by a single intranasal vaccination with lipopeptide, they remain at substantial levels in the lungs and are efficiently boosted upon challenge with virulent virus to provide late control of pulmonary viral loads. Vaccinated mice are not only protected from death but remain active, indicative of less severe disease despite significant weight loss.