穿孔素介导的细胞凋亡机制在抗流感病毒感染免疫防御中作用的研究

目的：穿孔素介导的细胞凋亡机制在流感病毒初次感染中作用的研究。方法：用流感病毒A／PR／8／34经鼻感染穿孔素基因敲除鼠和同源对照C57BL／6小鼠，采用PFU方法测定肺内流感病毒增殖状况；免疫组织化学染色方法观察小鼠病毒感染后感染细胞的凋亡情况；利用乳酸脱氢酶释放法检测感染鼠脾淋巴细胞NK活性及CTL杀伤活性。结果：穿孔素基因缺乏导致流感病毒在小鼠肺内大量增殖；小鼠清除感染病毒所需时间延长；病毒感染细胞发生凋亡的时间亦因穿孔素的缺乏而延迟；感染小鼠脾淋巴细胞NK活性及CTL杀伤活性均显著降低。结论：穿孔素依赖的细胞介导的细胞毒效应在控制流感病毒初次感染，快速清除感染病毒方面起重要作用。     

Tumor necrosis factor alpha production from CD8+ T cells mediates oviduct pathological sequelae following primary genital Chlamydia muridarum infection

The immunopathogenesis of Chlamydia trachomatis-induced oviduct pathological sequelae is not well understood. Mice genetically deficient in perforin (perforin(-/-) mice) or tumor necrosis factor alpha (TNF-α) production (TNF-α(-/-) mice) displayed comparable vaginal chlamydial clearance rates but significantly reduced oviduct pathology (hydrosalpinx) compared to that of wild-type mice. Since both perforin and TNF-α are effector mechanisms of CD8(+) T cells, we evaluated the role of CD8(+) T cells during genital Chlamydia muridarum infection and oviduct sequelae. Following vaginal chlamydial challenge, (i) mice deficient in TAP I (and therefore the major histocompatibility complex [MHC] I pathway and CD8(+) T cells), (ii) wild-type mice depleted of CD8(+) T cells, and (iii) mice genetically deficient in CD8 (CD8(-/-) mice) all displayed similar levels of vaginal chlamydial clearance but significantly reduced hydrosalpinx, compared to those of wild-type C57BL/6 mice, suggesting a role for CD8(+) T cells in chlamydial pathogenesis. Repletion of CD8(-/-) mice with wild-type or perforin(-/-), but not TNF-α(-/-), CD8(+) T cells at the time of challenge restored hydrosalpinx to levels observed in wild-type C57BL/6 mice, suggesting that TNF-α production from CD8(+) T cells is important for pathogenesis. Additionally, repletion of TNF-α(-/-) mice with TNF-α(+/+) CD8(+) T cells significantly enhanced the incidence of hydrosalpinx and oviduct dilatation compared to those of TNF-α(-/-) mice but not to the levels found in wild-type mice, suggesting that TNF-α production from CD8(+) T cells and non-CD8(+) cells cooperates to induce optimal oviduct pathology following genital chlamydial infection. These results provide compelling new evidence supporting the contribution of CD8(+) T cells and TNF-α production to Chlamydia-induced reproductive tract sequelae.     

Enhanced resistancy of thioredoxin-transgenic mice against influenza virus-induced pneumonia

Thioredoxin (TRX) is a small redox-active protein with anti-oxidant effect and redox-regulating functions. Using TRX transgenic (Tg) mice in which human TRX is overexpressed systemically under the control of beta-actin promoter, the effects of influenza virus infection were examined in TRX Tg mice and wild type C57BL/6 mice. (1) Median lethal dose (LD50) against influenza virus infection in wild-type C57BL/6 mice was 10(-5.3) dilution, while that of TRX Tg mice was 10(-4.2) dilution. Thus, TRX Tg mice were more resistant against the virus infection than wild-type mice. (2) The body weights of wild-type mice 7 days after infection with a sublethal dose of the virus (10(-6) dilution) decreased significantly, whereas those of TRX Tg mice increased slightly. (3) Histopathology of the lung at 3 weeks after sublethal infection of influenza virus showed that severe alveolar or bronchiolar destruction was observed in wild-type mice, while mild viral pneumonia was seen in the TRX Tg mice. (4) Local (IgA) and systemic (IgG) antibody productions against influenza virus hemagglutinin in mice surviving 3 weeks after infection were similar between wild-type and TRX Tg mice. These results indicate that overexpression of TRX in Tg mice suppresses the inflammatory overshoot of viral pneumonia caused by influenza virus infection, resulting in the reduction of mortality without affecting the host's systemic immune responses to the infection. TRX may play some important roles in regulating the inflammatory process in the primary host defense against infection.     

Protection against polyoma virus-induced tumors is perforin-independent

CD8 T cells are necessary for controlling tumors induced by mouse polyoma virus (PyV), but the effector mechanism(s) responsible have not been determined. We examined the PyV tumorigenicity in C57BL/6 mice mutated in Fas or carrying targeted disruptions in the perforin gene or in both TNF receptor type I and type II genes. Surprisingly, none of these mice developed tumors. Perforin/Fas double-deficient radiation bone marrow chimeric mice were also resistant to PyV-induced tumors. Anti-PyV CD8 T cells in perforin-deficient mice were found not to differ from wild type mice with respect to phenotype, capacity to produce cytokines or maintenance of memory T cells, indicating that perforin does not modulate the PyV-specific CD8 T cell response. In addition, virus was cleared and persisted to similar extents in wild type and perforin-deficient mice. In summary, perforin/granzyme exocytosis is not an essential effector pathway for protection against PyV infection or tumorigenesis.     

Perforin pathway is essential for protection of mice against lethal ocular HSV-1 challenge but not corneal scarring

Perforin (cytolysin; pore-forming protein) is expressed in both CD8(+) cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, and is a major factor responsible for the cytolytic activities of these cells. Both CD8(+) T-cells and NK cells are important in eliminating cells infected with certain viruses. We examined the role of perforin in a mouse model of HSV-1 infection using perforin-deficient mice. Na?ve perforin knockout (perforin(0/0)) mice were more susceptible to lethal HSV-1 ocular challenge (60% survival), than na?ve parental C57BL/6 (100% survival). In contrast, both C57BL/6 and perforin(0/0) mice had similar levels of HSV-1 induced corneal scarring. Vaccination of perforin(0/0) mice induced a significantly higher HSV-1 neutralizing antibody titer than vaccination of C57BL/6 mice, and the mice were completely protected against lethal ocular challenge. These results suggest that in na?ve mice ocularly challenged with HSV-1, the perforin pathway was involved in protection against death, but not in protection against corneal scarring.     

Animal model of Mycobacterium abscessus lung infection

Chronic lung disease as a result of Mycobacterium abscessus is an emerging infection in the United States. We characterized the lung immune responses in mice and guinea pigs infected with M. abscessus. C57BL/6 and leptin-deficient ob/ob mice challenged with a low-dose aerosol (LDA) of M. abscessus did not develop an infection. However, when challenged with a high-dose aerosol (HDA), C57BL/6 and ob/ob mice developed an established infection and a pulmonary immune response consisting of an early influx of IFN-gamma+ CD4+ T cells; this immune response preceded the successful clearance of M. abscessus in both strains of mice, although mycobacterial elimination was delayed in the ob/ob mice. Infected guinea pigs showed an increased influx of lymphocytes into the lungs with bacterial clearance by Day 60. In contrast to the C57BL/6 and ob/ob mice and guinea pigs, IFN-gamma knockout (GKO) mice challenged with a LDA or HDA of M. abscessus showed a progressive lung infection despite a robust influx of T cells, macrophages, and dendritic cells, culminating in extensive lung consolidation. Furthermore, with HDA challenge of the GKO mice, emergence of IL-4- and IL-10-producing CD4+ and CD8+ T cells was seen in the lungs. In conclusion, IFN-gamma is critically important in the host defense against M. abscessus. As the number of effective drugs against M. abscessus is limited, the GKO mice provide a model for in vivo testing of novel drugs.     

Expression of the DX5 antigen on CD8+ T cells is associated with activation and subsequent cell death or memory during influenza virus infection

The antigen recognized by the DX5 antibody (DX5 antigen) is expressed on all murine NK cells. In the present study we found that a proportion of CD8+ T cells (approximately 5%) also express the DX5 antigen in uninfected mice, and that numbers of CD8+ T cells expressing DX5 are significantly higher in the lungs of influenza virus-infected mice representing up to 50% of all CD8+ T cells on day 10 post infection. The expression of the DX5 antigen on CD8+ T cells was associated with a memory phenotype in uninfected C57BL/6 mice and with an activation phenotype during influenza virus infection. Interestingly, when lymphocytes were isolated from lungs of influenza virus-infected mice on day 10 post infection and adoptively transferred into recombination activating gene-1 (RAG1)-deficient mice, CD8+DX5+ cells could not be recovered from the recipient mice 2 days later. Moreover, CD8+DX5+ cells were not detected when lung cells were removed from day 10 influenza virus-infected mice and cultured in vitro for 2 days. However, CD8+DX5+ cells could be detected when apoptosis inhibitors were added to these cultures, suggesting that the CD8+DX5+ cells underwent apoptosis during cell culture. Furthermore, almost all DX5 expressing CD8+ cells from lungs of mice on day 10 post influenza virus infection stained positively with Annexin-V. Taken together, the data suggest that CD8+ T cells expressing DX5 are associated with an activation/memory phenotype and are biased towards apoptosis.     

Strain-dependent requirement for IFN-γ for respiratory control and immunotherapy in murine gammaherpesvirus infection

Interferon-γ (IFN-γ) and perforin (pfp) are important effector mechanisms used by CD8 T cells to clear virus-infected cells. In this study, we used IFN-γ/pfp double knockout mice to address if these two effector molecules play redundant roles in the control of acute infection with murine gammaherpesvirus-68 (MHV-68) in BALB/C mice. Perforin knockout (KO) mice and wild-type mice cleared infectious virus from the lungs, even following high-dose infection. However, the IFN-γ KO and IFN-γ/pfp double knockout (DKO) groups had higher virus titers in the lungs at day 10 post-infection, and both groups had higher mortality rates. In IFN-γ/pfp DKO mice, the virus titer and mortality rate were significant higher than in IFN-γ KO mice, indicating a role for perforin in protection from disease. WT mice given IFN-γ blocking antibody also showed significantly higher viral titers. In contrast, IFN-γ KO mice on a C57BL/6 background controlled respiratory infection comparably to wild-type mice. These data show that perforin plays a redundant role in the control of virus replication, but IFN-γ plays an essential role in BALB/C mice infected with MHV-68. We conclude that there is a marked strain-dependent difference in the effector mechanisms needed to control acute MHV-68 infection between C57BL/6 and BALB/C mice. In addition we show that immune therapy that re-establishes viral control after spontaneous reactivation in CD4-deficient mice depends upon perforin in C57BL/6 mice but IFN-γ in BALB/C mice.     

Increased host resistance against Pneumocystis carinii pneumonia in gammadelta T-cell-deficient mice: protective role of gamma interferon and CD8(+) T cells

Although a clear relationship between alphabeta T-cell receptor-positive (alphabeta-TCR(+)) CD4(+) T cells and susceptibility to Pneumocystis carinii infection exists, the role of other T-cell subsets is less clearly defined. Previous studies have shown that gammadelta-TCR(+) T cells infiltrate into the lung during P. carinii pneumonia. Therefore, the present study examined the role of gammadelta-TCR(+) T cells in host defense against P. carinii pneumonia. C57BL/6 (control) and B6.129P2-Tcrd(tm1Mom) (gammadelta-TCR(+) T-cell-deficient) mice were inoculated intratracheally with P. carinii. At specific time points, mice were sacrificed and analyzed for P. carinii burden, T-cell subsets, and cytokine levels in lung tissue. Analysis of P. carinii burden showed a more rapid and complete resolution of infection in gammadelta-TCR(+) T-cell-deficient mice than in C57BL/6 controls. This augmented resolution was associated with elevated gamma interferon (IFN-gamma) levels in bronchoalveolar lavage fluid predominantly produced by CD8(+) T cells, as well as an increased recruitment of CD8(+) T cells in general. In separate experiments, neutralization of IFN-gamma or depletion of CD8(+) T cells early during infection abolished the augmented resolution previously observed in gammadelta-TCR(+) T-cell-deficient mice. These results show that the presence of gammadelta-TCR(+) T cells modulates host susceptibility to P. carinii pneumonia through interactions with pulmonary CD8(+) T cells and tissue production of IFN-gamma.     

beta2-microglobulin-associated regulation of interferon-gamma and virus-specific immunoglobulin G confer resistance against the development of chronic coxsackievirus myocarditis

To gain insight into the strategies of the immune system to confer resistance against the development of chronic coxsackievirus B3 (CVB3) myocarditis we compared the course of the disease in C57BL/6 mice, beta2-microglobulin knockout (beta2m(-/-)) mice, and perforin-deficient (perforin(-/-)) mice. We found that perforin(-/-) mice as well as immunocompetent C57BL/6 mice reveal a resistant phenotype with complete elimination of the virus from the heart in the course of acute myocarditis. In contrast, myocardial CVB3 infection of beta2m(-/-) mice was characterized by a significantly higher virus load associated with a fulminant acute inflammatory response and, as a consequence of virus persistence, by the development of chronic myocarditis. Interferon-gamma secretion of stimulated spleen cells was found to be significantly delayed in beta2m(-/-) mice compared to perforin(-/-) mice and C57BL/6 control mice during acute myocarditis. In addition, generation of virus-specific IgG and neutralizing antibodies were found to be significantly decreased in beta2m(-/-) mice during acute infection. From these results we conclude that protection against the development of chronic myocarditis strongly depends on the expression of beta2m, influencing the catabolism of IgG as well as the production of protective cytokines, such as interferon-gamma. Moreover, CVB3-induced cardiac injury and prevention of chronic myocarditis was found to be unrelated to perforin-mediated cytotoxicity in our model system.     

Properties of murine (CD8+)CD27- T cells

In humans, loss of CD27 expression is associated with the stable acquisition of effector functions by CD8+ T cells. We found that murine (CD8+)CD27- T cells were confined to the primed CD62L(dull/-)CD44(bright)CCR7- T cell population. (CD8+)CD27- T cells were absent from lymph nodes but could be found in blood, spleen and in non-lymphoid organs such as lung and liver. Late after primary influenza virus infection, low percentages of antigen-specific CD27- cells emerged in the lung and spleen. After recovery from secondary influenza virus infection, high percentages of influenza-specific CD27- T cells were found in the lung and the loss of CD27 on lung CD8+ T cells coincided with high granzyme B expression. After murine cytomegalovirus infection, loss of CD27 expression on virus-specific CD8+ T cell populations was sustained and especially marked in liver and lung. We suggest that in mice, CD27 is lost from CD8+ T cells only after repetitive antigenic stimulation. Moreover, the high expression of both granzyme B and perforin in the CD27- T cells suggests that the lack of CD27 on murine CD8+ T cells can be used to identify memory T cells with expression of cytotoxic effector molecules.     

Respiratory virus-induced regulation of asthma-like responses in mice depends upon CD8 T cells and interferon-gamma production

Respiratory virus infections can significantly influence the development of airway disease by both predisposing and exacerbating the developing lung immune environment. In contrast, the initiation of a more desirable anti-viral response may better prepare the local environment and protect it from developing an adverse long-term disease phenotype. BALB/c or C57BL/6 mice exposed to respiratory syncytial virus (RSV) infection at the same time as allergen sensitization were assessed for airway function, cytokine responses, and inflammatory parameters. Depending on the genetic strain of mouse used, BALB/c versus C57BL/6, RSV could differentially protect against the development of airway allergen responses. Although RSV was able to block allergen sensitization and induction of airway hyperresponsiveness and eosinophilic inflammation in C57BL/6 mice, the infection did not reduce the allergic responses in BALB/c mice. The alteration of airway responsiveness did not depend on the timing of RSV infection in C57BL/6 mice in conjunction to the allergen sensitization protocol. Neutralization experiments demonstrated that interferon-gamma contributed significantly to the RSV-induced airway attenuation of the allergic responses, whereas transfer of CD8 T cells from RSV-infected animals suggested that they were partially responsible for the altered environment. These data suggest that a respiratory viral infection impacts on the local lung environment and may reflect specific aspects of the hygiene hypothesis. However, the outcome of this interaction depends on the immunological response of the host.     

Murine cytomegalovirus replication in salivary glands is controlled by both perforin and granzymes during acute infection

The course of mouse cytomegalovirus (MCMV) infection was compared between wild-type and mutant C57BL / 6 (B6) mice deficient in either RAG-2, perforin, granzyme A, granzyme B or combinations thereof at two time points post infection (p. i.). At day 15 p. i., virus titers were similarly elevated in salivary glands of all mutant, but not wild-type B6 mice and undetectable in lung and spleen tissues of any of the mouse strains. Significant pathological alterations were only seen in salivary glands and spleen from RAG2(- / -), but not in those from other mice whereas few inflammatory foci were observed in lung tissues of all mice except B6. At day 30 p. i., elevated virus titers were observed only in salivary glands, lung and spleen from RAG2(- / -), but in none of the other mice, and were accompanied by extended pathological alterations in all three organs. The data extend previous reports on the critical role of NK / CD8(+) T cells in the early control of MCMV infection by showing that both perforin and granzymes A / B contribute to viral elimination in salivary glands; however, neither of the three molecules alone seem to be indispensable for the final control of infection.     

Characterization of age-related changes in natural killer cells during primary influenza infection in mice

The current investigation examined the importance of natural killer (NK) cells during the innate immune response to primary influenza infection in young and aged mice. Young (6-8 weeks) and aged (22 months) C57BL/6 mice were infected intranasally with influenza A virus, and NK cell-mediated cytotoxicity was determined in lung and spleen during the first 4 days of infection. Aged mice demonstrated both a decrease in influenza-inducible NK activity and a reduction in the percentage and number of NK1.1+ cells in response to primary influenza infection, relative to young mice. In order to further establish a role for NK cells in controlling influenza infection, young mice were depleted of NK cells in vivo by injecting rabbit anit-NK1.1 antibody 2 days and 1 day prior to influenza infection. Young mice depleted of NK cells exhibited increased weight loss and lung virus titers during the course of infection, compared to young mice infected with influenza virus. These data indicate that NK cell function is impaired in response to primary influenza infection in aged mice. More importantly, these results underscore the essential role of NK cells in controlling virus titers in lung during the early course of influenza infection, regardless of age.     

IL-18, but not IL-12, is required for optimal cytokine production by influenza virus-specific CD8+ T cells

The potent innate cytokines IL-12 and IL-18 are considered to be important antigen-independent mediators of IFN-gamma production by NK cells and T lymphocytes. The present analysis addresses the physiological role of IL-12 and IL-18 in the generation of virus-specific CD8+ T cells. Both wt C57BL/6J (B6) mice and mice with disrupted IL-12p40 (IL-12p40(-/-)) or IL-18 (IL-18(-/-)) genes were infected with an influenza A virus and the characteristics of the resultant epitope-specific CD8+ T cell responses were compared. While IL-12 appeared to have no notable effect on either virus growth or on CD8+ T cell response profiles, the absence of IL-18 was associated with delayed virus clearance from the lung and, despite normal numbers, a significantly reduced production of IFN-gamma, TNF-alpha, and IL-2 by epitope-specific CD8+ T cells. While this cytokine phenotype was broadly maintained in IL-12p40/IL-18 double-knockout mice, no evidence was seen for any additive effect. Together, our results suggest that IL-18, but not IL-12, induces optimal, antigen-specific production of key cytokines by CD8+ T cells for the efficient clearance of influenza virus from the lungs of infected mice.     

Modulation of human beta-defensin-1 (hBD-1) in plasmacytoid dendritic cells (PDC), monocytes, and epithelial cells by influenza virus, Herpes simplex virus, and Sendai virus and its possible role in innate immunity

hBD comprise a family of antimicrobial peptides that plays a role in bridging the innate and adaptive immune responses to infection. The expression of hBD-2 increases upon stimulation of numerous cell types with LPS and proinflammatory cytokines. In contrast, hBD-1 remains constitutively expressed in most cells in spite of cytokine or LPS stimulation; however, its presence in human PDC suggests it plays a role in viral host defense. To examine this, we characterized the expression of hBD-1 in innate immune cells in response to viral challenge. PDC and monocytes increased production of hBD-1 peptide and mRNA as early as 2 h following infection of purified cells and PBMCs with PR8, HSV-1, and Sendai virus. However, treatment of primary NHBE cells with influenza resulted in a 50% decrease in hBD-1 mRNA levels, as measured by qRT-PCR at 3 h following infection. A similar inhibition occurred with HSV-1 challenge of human gingival epithelial cells. Studies with HSV-1 showed that replication occurred in epithelial cells but not in PDC. Together, these results suggest that hBD-1 may play a role in preventing viral replication in immune cells. To test this, we infected C57BL/6 WT mice and mBD-1((-/-)) mice with mouse-adapted HK18 (300 PFU/mouse). mBD-1((-/-)) mice lost weight earlier and died sooner than WT mice (P=0.0276), suggesting that BD-1 plays a role in early innate immune responses against influenza in vivo. However, lung virus titers were equal between the two mouse strains. Histopathology showed a greater inflammatory influx in the lungs of mBD-1((-/-)) mice at Day 3 postinfection compared with WT C57BL/6 mice. The results suggest that BD-1 protects mice from influenza pathogenesis with a mechanism other than inhibition of viral replication.     

Role of T cells in resistance to Theiler's virus infection.

Intracerebral infection of C57BL/10SNJ mice with Theiler's virus results in acute encephalitis with subsequent virus clearance and absence of spinal cord demyelination. In contrast, infection of SJL/J mice results in acute encephalitis, virus persistence, and immune-mediated demyelination. These experiments examined the role of T-cell subsets in the in vivo immune response to Theiler's virus in resistant C57BL/10SNJ mice. Depletion of T-cell subsets with monoclonal antibodies (mAbs) directed at CD3 (pan-T-cell marker), CD4+ (class II-restricted) or CD8+ (class I-restricted) T cells resulted in increased frequency of paralysis and death as a result of acute encephalitis. Neuropathologic studies 10 days after infection demonstrated prominent necrosis, primarily in the pyramidal layer of hippocampus and in the thalamus of mice depleted of T-cell subsets. In immunosuppressed and infected C57BL/10SNJ mice, analysis of spinal cord sections 35 days after infection demonstrated small demyelinated lesions relatively devoid of inflammatory cells even though virus antigen could be detected by immunocytochemistry. Both CD4+ and CD8+ T cells are important in the resistance to infection with Theiler's virus in C57BL/10SNJ mice. However, subsequent spinal cord demyelination, to the extent observed in susceptible mice, depends on the presence of virus antigen persistence and a competent cellular immune response.     

Enhanced lung injury and delayed clearance of Pneumocystis carinii in surfactant protein A-deficient mice: attenuation of cytokine responses and reactive oxygen-nitrogen species

Surfactant protein A (SP-A), a member of the collectin family, selectively binds to Pneumocystis carinii and mediates interactions between pathogen and host alveolar macrophages in vitro. To test the hypothesis that mice lacking SP-A have delayed clearance of Pneumocystis organisms and enhanced lung injury, wild-type C57BL/6 (WT) and SP-A-deficient mice (SP-A(-/-)) with or without selective CD4(+)-T-cell depletion were intratracheally inoculated with Pneumocystis organisms. Four weeks later, CD4-depleted SP-A-deficient mice had developed a more severe Pneumocystis infection than CD4-depleted WT (P. carinii pneumonia [PCP] scores of 3 versus 2, respectively). Whereas all non-CD4-depleted WT mice were free of PCP, intact SP-A(-/-) mice also had evidence of increased organism burden. Pneumocystis infection in SP-A-deficient mice was associated histologically with enhanced peribronchial and/or perivascular cellularity (score of 4 versus 2, SP-A(-/-) versus C57BL/6 mice, respectively) and a corresponding increase in bronchoalveolar lavage (BAL) cell counts. Increases in SP-D content, gamma interferon, interleukin-4, interleukin-5, and tumor necrosis factor alpha in BAL fluid occurred but were attenuated in PCP-infected SP-A(-/-) mice compared to WT mice. There were increases in total BAL NO levels in both infected groups, but nitrite levels were higher in SP-A(-/-) mice, indicating a reduction in production of higher oxides of nitrogen that was also reflected in lower levels of 3-nitrotyrosine staining in the SP-A(-/-) group. We conclude that despite increases in inflammatory cells, SP-A-deficient mice infected with P. carinii exhibit an enhanced susceptibility to the organism and attenuated production of proinflammatory cytokines and reactive oxygen-nitrogen species. These data support the concept that SP-A is a local effector molecule in the lung host defense against P. carinii in vivo.     

Experimental Yersinia enterocolitica infection in euthymic and T-cell-deficient athymic nude C57BL/6 mice: comparison of time course, histomorphology, and immune response.

To elucidate the role of T lymphocytes in primary infection with Yersinia enterocolitica, we investigated the elimination rate of this pathogen, the histomorphology of tissue lesions, and the immune responses of athymic T-cell-deficient C57BL/6 nude mice and their euthymic littermates after parenteral infection with Y. enterocolitica of serotype O:8. While a low inoculum of 3 x 10(2) Y. enterocolitica cells (about 0.01 times the median lethal dose for normal C57BL/6 mice) was cleared by normal C57BL/6 mice within 7 to 10 days, athymic nude C57BL/6 mice developed progressive infections after this inoculum, leading to death on days 20 to 25 postinfection (p.i.). While normal C57BL/6 mice experienced short-term transient infections, nude mice exhibited a biphasic, progressive infectious process. Thus, in the early phase (days 1 to 7 p.i.), a rapid influx of CD11b/18-positive cells (Mac-1 antigen) and natural killer cells was evident in the spleens and livers of the nude mice. The late phase (from day 8 p.i. onward) was characterized by a rapid progression of the infection and a further influx of CD11b/18-positive cells into the liver accompanied by an increase in bacterial counts and development of tissue lesions particularly in the liver and spleen. In normal mice, granuloma-like lesions composed of CD11b/18-, CD4-, and CD8-positive cells could be observed. However, granulomata were not found in nude mice. Yersinia-specific immunoglobulin G antibodies appeared on day 15 p.i. in the sera of normal mice, while nude mice failed to develop significant antibody titers. Adoptive transfer of Yersinia-specific T cells into athymic nude mice mediated resistance to Y. enterocolitica infection and restored both the ability of granuloma formation and the production of specific antibodies. In summary, the data presented herein strongly suggest that T lymphocytes play an essential role in the defense of C57BL/6 mice against Y. enterocolitica.     

Critical role of cytotoxic T lymphocytes in immune clearance of rickettsial infection

Cytotoxic T-lymphocyte (CTL) activity developed against the major infected target cells of rickettsial infections, endothelial cells and macrophages. Spleen cells from mice immune to Rickettsia conorii exerted specific major histocompatibility complex (MHC) class I-matched CTL activity against R. conorii-infected SVEC-10 endothelial cells, with peak activity on day 10. Similarly, spleen cells from Rickettsia australis-immune mice exerted specific CTL activity against an R. australis-infected macrophage-like cell line. Gamma interferon (IFN-gamma) gene knockout mice were more than 100-fold more susceptible to R. australis infection than wild-type C57BL/6 mice. MHC class I gene knockout mice were the most susceptible, more than 50,000-fold more susceptible to a lethal outcome of R. australis infection than wild-type C57BL/6 mice. These results indicate that CTL activity was more critical to recovery from rickettsial infection than were the effects of IFN-gamma. The observation that perforin gene knockout mice were more than 100-fold more susceptible than wild-type C57BL/6 mice indicates that perforin-mediated activity accounts for a large component, but not all, of the CTL-mediated antirickettsial effect. CTL activity was expressed by immune CD8 T lymphocytes. Adoptive transfer of immune CD8 T lymphocytes from IFN-gamma gene knockout mice into R. australis-infected IFN-gamma gene knockout mice dramatically reduced the infectious rickettsial content in the organs, confirming that CD8 T lymphocytes provide immunity against rickettsiae besides that provided by the secretion of IFN-gamma. CTLs appear to be crucial to recovery from rickettsial infection.