CCR2+ Inflammatory Dendritic Cells and Translocation of Antigen by Type III Secretion Are Required for the Exceptionally Large CD8+ T Cell Response to the Protective YopE69-77 Epitope during Yersinia Infection

During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8⁺ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8⁺ T cells recognize the epitope YopE_69-77. The features of the interaction between pathogen and host that result in this large CD8⁺ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE_69-77-specific CD8⁺ T cells generated during the large response are polyclonal and are produced by a “translocation-dependent” pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE_69-77-specific CD8⁺ T cell response (~10% of the large expansion) can be generated in a “translocation-independent” pathway in which CD8α⁺ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE_69-77-specific CD8⁺ T cell expansion because this response was significantly reduced in Ccr2^-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE_69-77-specific CD8⁺ T cells ex vivo and promoted the expansion of YopE_69-77-specific CD8⁺ T cells in infected Ccr2^-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8⁺ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE_69-77-specific CD8⁺ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective adaptive immune response.     

Memory-Like Antigen-Specific Human NK Cells from TB Pleural Fluids Produced IL-22 in Response to IL-15 or Mycobacterium tuberculosis Antigens

Our previous result indicated that memory-like human natural killer (NK) cells from TB pleural fluid cells (PFCs) produced large amounts of IFN-γ in response to Bacille Calmette Guerin (BCG). Furthermore, recent studies have shown that human lymphoid tissues harbored a unique NK cell subset that specialized in production of interleukin (IL)-22, a proinflammatory cytokine that mediates host defense against pathogens. Yet little information was available with regard to the properties of IL-22 production by memory-like human NK cells. In the present study, we found that cytokines IL-15 induced and IL-12 enhanced the levels of IL-22 by NK cells from TB PFCs. In addition, IL-22 but not IL-17 was produced by NK cells from PFCs in response to BCG and M.tb-related Ags. More importantly, the subset of specific IL-22-producing NK cells were distinct from IFN-γ-producing NK cells in PFCs. CD45RO⁺ or CD45RO^- NK cells were sorted, co-cultured with autologous monocytes and stimulated with BCG for the production of IL-22. The result demonstrated that CD45RO⁺ but not CD45RO^- NK cells produced significantly higher level of IL-22. Anti-IL-12Rβ1 mAbs (2B10) partially inhibit the expression of IL-22 by NK cells under the culture with BCG. Consistently, BCG specific IL-22-producing NK cells from PFCs expressed CD45RO^highNKG2D^highgranzyme B^high. In conclusion, our data demonstrated that memory-like antigen-specific CD45RO⁺ NK cells might participate in the recall immune response for M. tb infection via producing IL-22, which display a critical role to fight against M. tb.     

Pathogenic Yersinia Promotes Its Survival by Creating an Acidic Fluid-Accessible Compartment on the Macrophage Surface

Microbial pathogens and host immune cells each initiate events following their interaction in an attempt to drive the outcome to their respective advantage. Here we show that the bacterial pathogen Yersinia pseudotuberculosis sustains itself on the surface of a macrophage by forming acidic fluid-accessible compartments that are partially bounded by the host cell plasma membrane. These Yersinia-containing acidic compartments (YACs) are bereft of the early endosomal marker EEA1 and the lysosomal antigen LAMP1 and readily form on primary macrophages as well as macrophage-like cell lines. YAC formation requires the presence of the Yersinia virulence plasmid which encodes a type III secretion system. Unexpectedly, we found that the initial formation of YACs did not require translocation of the type III effectors into the host cell cytosol; however, the duration of YACs was markedly greater in infections using translocation-competent Y. pseudotuberculosis strains as well as strains expressing the effector YopJ. Furthermore, it was in this translocation- and YopJ-dependent phase of infection that the acidic environment was critical for Y. pseudotuberculosis survival during its interaction with macrophages. Our findings indicate that during its extracellular phase of infection Y. pseudotuberculosis initiates and then, by a separate mechanism, stabilizes the formation of a highly intricate structure on the surface of the macrophage that is disengaged from the endocytic pathway.     

Sleep Deprivation Induces Changes in Immunity in Trichinella spiralis-Infected Rats

Sleep is considered an important predictor of immunity. A lack of sleep may reduce immunity, which increases susceptibility to any type of infection. Moreover, sleep deprivation in humans produces changes in both, the percent of circulating immune cells (T cells and NK cells) and cytokine levels (IL-1, IFNγ, TNΦ-αα, IL-6 and IL-17). The aim of our study was to investigate whether sleep deprivation produces deregulation on immune variables during the immune response generated against the helminth parasite Trichinella spiralis. Because sleep deprivation is stressful per se, we designed another experiments to compared stress alone (consisting in movement restriction and single housing) with sleep deprivation, in both control (uninfected) and experimental (infected) rats. Our results demonstrate that the sleep deprivation and stress have a differential effect in mesenteric lymph nodes (MLN) and spleen. In uninfected rats sleep deprivation alone produces an increase in natural killer cells (NK+) and B cells (CD45+), accompanied by a decrease in cytotoxic T cells (CD3+CD8+) in spleen; while, in MLN, produces only an increase in natural killer cells (NK+). Both, SD and stress, produce an increased percentage of total T cells (CD3+) in spleen. In the MLN both are also associated to an increase in cytotoxic T cells (CD3+CD8+) and B cells (CD45+). In the spleens of parasitized rats, cell populations did not change. In spleens of both, sleep-deprived and stressed infected rats, we observed an increase in B cells (CD45+). In infected rats, sleep deprivation alone produced an increase in NK cells (NK+). In mesenteric node cell populations of parasitized rats, we observed a decrease in NK cells and an increase in T helper (CD4+) cells in both SD and stressed rats. Rats that were only subjected to stress showed a decrease in B cells (CD45+). These findings suggest that the immune response generated against infection caused by T. spiralis is affected when the sleep pattern is disrupted. These results support the notion that sleep is a fundamental process for an adequate and strong immune response generated against this parasite.     

The presence of professional phagocytes dictates the number of host cells targeted for Yop translocation during infection

Type III secretion systems deliver effector proteins from Gram‐negative bacterial pathogens into host cells, where they disarm host defences, allowing the pathogens to establish infection. Although Yersinia pseudotuberculosis delivers its effector proteins, called Yops, into numerous cell types grown in culture, we show that during infection Y. pseudotuberculosis selectively targets Yops to professional phagocytes in Peyer's patches, mesenteric lymph nodes and spleen, although it colocalizes with B and T cells as well as professional phagocytes. Strikingly, in the absence of neutrophils, the number of cells with translocated Yops was significantly reduced although the bacterial loads were similar, indicating that Y. pseudotuberculosis did not arbitrarily deliver Yops to the available cells. Using isolated splenocytes, selective binding and selective targeting to professional phagocytes when bacteria were limiting was also observed, indicating that tissue architecture was not required for the tropism for professional phagocytes. In isolated splenocytes, YadA and Invasin increased the number of all cells types with translocated Yops, but professional phagocytes were still preferentially translocated with Yops in the absence of these adhesins. Together these results indicate that Y. pseudotuberculosis discriminates among cells it encounters during infection and selectively delivers Yops to phagocytes while refraining from translocation to other cell types.     

Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague

Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host.     

CD8+ T Cells Restrict Yersinia pseudotuberculosis Infection: Bypass of Anti-Phagocytosis by Targeting Antigen-Presenting Cells

All Yersinia species target and bind to phagocytic cells, but uptake and destruction of bacteria are prevented by injection of anti-phagocytic Yop proteins into the host cell. Here we provide evidence that CD8⁺ T cells, which canonically eliminate intracellular pathogens, are important for restricting Yersinia, even though bacteria are primarily found in an extracellular locale during the course of disease. In a model of infection with attenuated Y. pseudotuberculosis, mice deficient for CD8⁺ T cells were more susceptible to infection than immunocompetent mice. Although exposure to attenuated Y. pseudotuberculosis generated T_H1-type antibody responses and conferred protection against challenge with fully virulent bacteria, depletion of CD8⁺ T cells during challenge severely compromised protective immunity. Strikingly, mice lacking the T cell effector molecule perforin also succumbed to Y. pseudotuberculosis infection. Given that the function of perforin is to kill antigen-presenting cells, we reasoned that cell death marks bacteria-associated host cells for internalization by neighboring phagocytes, thus allowing ingestion and clearance of the attached bacteria. Supportive of this model, cytolytic T cell killing of Y. pseudotuberculosis–associated host cells results in engulfment by neighboring phagocytes of both bacteria and target cells, bypassing anti-phagocytosis. Our findings are consistent with a novel function for cell-mediated immune responses protecting against extracellular pathogens like Yersinia: perforin and CD8⁺ T cells are critical for hosts to overcome the anti-phagocytic action of Yops.     

NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*^/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*^/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8⁺ T cells in lungs and draining lymph nodes of Nox1*^/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*^/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.     

Monkeypox Virus Infection of Rhesus Macaques Induces Massive Expansion of Natural Killer Cells but Suppresses Natural Killer Cell Functions

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques.     

Bioluminescent tracing of a Yersinia pestis pCD1+-mutant and Yersinia pseudotuberculosis in subcutaneously infected mice

Yersinia pestis has evolved from Yersinia pseudotuberculosis serotype O:1b. A typical Y. pestis contains three plasmids: pCD1, pMT1 and pPCP1. However, some isolates only harbor pCD1 (pCD1⁺-mutant). Y. pestis and Y. pseudotuberculosis share a common plasmid (pCD1 or pYV), but little is known about whether Y. pseudotuberculosis exhibited plague-inducing potential before it was evolved into Y. pestis. Here, the luxCDABE::Tn5::kan was integrated into the chromosome of the pCD1⁺-mutant, Y. pseudotuberculosis or Escherichia coli K12 to construct stable bioluminescent strains for investigation of their dissemination in mice by bioluminescence imaging technology. After subcutaneous infection, the pCD1⁺-mutant entered the lymph nodes, followed by the liver and spleen, and, subsequently, the lungs, causing pathological changes in these organs. Y. pseudotuberculosis entered the lymph nodes, but not the liver, spleen and lungs. It also resided in the lymph nodes for several days, but did not cause lymphadenitis or pathological lesions. By contrast, E. coli K12-lux was not isolatable from mouse lymph nodes, liver, spleen and lungs. These results indicate that the pCD1⁺-mutant can cause typical bubonic and pneumonic plague-like diseases, and Y. pestis has inherited lymphoid tissue tropism from its ancestor rather than acquiring these properties independently.     

Innate Immune Recognition of Yersinia pseudotuberculosis Type III Secretion

Specialized protein translocation systems are used by many bacterial pathogens to deliver effector proteins into host cells that interfere with normal cellular functions. How the host immune system recognizes and responds to this intrusive event is not understood. To address these questions, we determined the mammalian cellular response to the virulence-associated type III secretion system (T3SS) of the human pathogen Yersinia pseudotuberculosis. We found that macrophages devoid of Toll-like receptor (TLR) signaling regulate expression of 266 genes following recognition of the Y. pseudotuberculosis T3SS. This analysis revealed two temporally distinct responses that could be separated into activation of NFκB- and type I IFN-regulated genes. Extracellular bacteria were capable of triggering these signaling events, as inhibition of bacterial uptake had no effect on the ensuing innate immune response. The cytosolic peptidoglycan sensors Nod1 and Nod2 and the inflammasome component caspase-1 were not involved in NFκB activation following recognition of the Y. pseudotuberculosis T3SS. However, caspase-1 was required for secretion of the inflammatory cytokine IL-1β in response to T3SS-positive Y. pseudotuberculosis. In order to characterize the bacterial requirements for induction of this novel TLR-, Nod1/2-, and caspase-1-independent response, we used Y. pseudotuberculosis strains lacking specific components of the T3SS. Formation of a functional T3SS pore was required, as bacteria expressing a secretion needle, but lacking the pore-forming proteins YopB or YopD, did not trigger these signaling events. However, nonspecific membrane disruption could not recapitulate the NFκB signaling triggered by Y. pseudotuberculosis expressing a functional T3SS pore. Although host cell recognition of the T3SS did not require known translocated substrates, the ensuing response could be modulated by effectors such as YopJ and YopT, as YopT amplified the response, while YopJ dampened it. Collectively, these data suggest that combined recognition of the T3SS pore and YopBD-mediated delivery of immune activating ligands into the host cytosol informs the host cell of pathogenic challenge. This leads to a unique, multifactorial response distinct from the canonical immune response to a bacterium lacking a T3SS.     

Neutrophil-dendritic cell interaction plays an important role in live attenuated Leishmania vaccine induced immunity

BackgroundNeutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen^-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen^-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo.Methodology/findingsLdCen^-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen^-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen^-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4⁺T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen^-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen^-/- parasites by DCs. Neutrophil depletion in LdCen^-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4⁺Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4⁺ T cells in vivo.ConclusionsCollectively, LdCen^-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.     

In Vivo IFN-γ Secretion by NK Cells in Response to Salmonella Typhimurium Requires NLRC4 Inflammasomes

Natural killer (NK) cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ) contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c⁺ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18), independently of Toll-like receptor (TLR)-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.     

Lymphocyte subpopulations in lymph nodes and peripheral blood: a comparison between patients with stable angina and acute coronary syndrome

Objective

Atherosclerosis is characterized by a chronic inflammatory response involving activated T cells and impairment of natural killer (NK) cells. An increased T cell activity has been associated with plaque instability and risk of acute cardiac events. Lymphocyte analyses in blood are widely used to evaluate the immune status. However, peripheral blood contains only a minor proportion of lymphocytes. In this study, we hypothesized that thoracic lymph nodes from patients with stable angina (SA) and acute coronary syndrome (ACS) might add information to peripheral blood analyses.

Methods

Peripheral blood and lymph nodes were collected during coronary by-pass surgery in 13 patients with SA and 13 patients with ACS. Lymphocyte subpopulations were assessed by flow cytometry using antibodies against CD3, CD4, CD8, CD19, CD16/56, CD25, Foxp3, CD69, HLA-DR, IL-18 receptor (R) and CCR4.

Results

Lymph nodes revealed a lymphocyte subpopulation profile substantially differing from that in blood including a higher proportion of B cells, lower proportions of CD8⁺ T cells and NK cells and a 2-fold higher CD4/CD8 ratio. CD4⁺CD69⁺ cells as well as Foxp3⁺ regulatory T cells were markedly enriched in lymph nodes (p<0.001) while T helper 1-like (CD4⁺IL-18R+) cells were more frequent in blood (p<0.001). The only significant differences between ACS and SA patients involved NK cells that were reduced in the ACS group. However, despite being reduced, the NK cell fraction in ACS patients contained a significantly higher proportion of IL-18R⁺ cells compared with SA patients (p<0.05).

Conclusion

There were several differences in lymphocyte subpopulations between blood and lymph nodes. However, the lymphocyte perturbations in peripheral blood of ACS patients compared with SA patients were not mirrored in lymph nodes. The findings indicate that lymph node analyses in multivessel coronary artery disease may not reveal any major changes in the immune response that are not detectable in blood.     

NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens

Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8–10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4⁺ and CD8⁺ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D⁺ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2^-/-Il2rg^-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia.     

scFv from Antibody That Mimics gp43 Modulates the Cellular and Humoral Immune Responses during Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv) that mimics the main antigen of P. brasiliensis (gp43) confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4⁺ and CD8⁺ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4⁺ and CD8⁺ T cells.     

Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus colubriformis: tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions, and in vitro lymphocyte transformations during primary infections

The kinetics of the lymphocyte responses of Trichostrongylus colubriformis-infected and normal guinea pigs were measured by the in vivo uptake of tritiated thymidine either as dpm ³H/mg tissue or as the percentage change in [³H] -labeled lymphoblasts in autoradiographs of tissue impression smears and sections. The lymphoid response was predominantly a local one centering on the infected area of the small intestine. The greatest lymphocyte reactions as assessed by counts of labeled lymphoblasts occurred in the Peyer's patches and mesenteric lymph nodes where the peak responses took place 11 and 6 days after infection, respectively. The local nature of the responses was exemplified by the fact that the mesenteric lymph nodes of the anterior small intestine showed a peak response on the sixth day but the response from the posterior small intestine peaked 7 days later. A similar but less dramatic relationship existed among the Peyer's patches. In addition no labeled lymphoblast response was elicited in the inguinal lymph nodes or cecal lymphoid patches throughout the infection and the first increased responsiveness of the spleen did not take place until after Day 13, by which time the lymphoid proliferations associated with the infected intestine had subsided. Initially, the spleen showed a marked depletion of labeled blast cells during the first 7 days of the infection. This was taken as indicating at the time the infection was being established the export of cells capable of transformation in response to parasite antigen. This was supported by the observation that large numbers of phytohemagglutinin responsive lymphocytes were found in the peripheral circulation at this time. The in vitro responsiveness of peripheral lymphocytes to T. colubriformis antigen was also studied. Positive lymphocyte transformations first occurred 6 days after infection but thereafter declined to the normal level by Day 13; the peak transformation ratio was found 25 days after infection but by Day 38 it had declined to a low but persistently positive level. There was a correlation between the circulation of specifically sensitized cells, probably of thymic origin, IgE antibody titers, and the development of positive dermal delayed hypersensitivity reactions in infected guinea pigs, suggesting a close relationship among these three immunological phenomena.All lymphoblast responses in Peyer's patches, mesenteric lymph nodes, and lamina propria of the intestine were completed before the immune elimination of the parasite commenced 10 days after infection. During the first 10 days of infection specifically sensitized lymphocytes appeared and disappeared from the circulation. The loss of circulating sensitized lymphocytes at the time immune elimination of the parasite was taking place in the gut suggested that the sensitized cells were “homing-in” on the local area of infection. After the immune elimination of the parasite had commenced, the level of sensitized lymphocytes and IgE antibodies then increased rapidly in the blood. Evidence from the kinetics of the hemagglutinating antibodies indicated that stage specific antigens occur in T. colubriformis.     

Random Mutagenesis Identifies a C-Terminal Region of YopD Important for Yersinia Type III Secretion Function

A common virulence mechanism among bacterial pathogens is the use of specialized secretion systems that deliver virulence proteins through a translocation channel inserted in the host cell membrane. During Yersinia infection, the host recognizes the type III secretion system mounting a pro-inflammatory response. However, soon after they are translocated, the effectors efficiently counteract that response. In this study we sought to identify YopD residues responsible for type III secretion system function. Through random mutagenesis, we identified eight Y. pseudotuberculosis yopD mutants with single amino acid changes affecting various type III secretion functions. Three severely defective mutants had substitutions in residues encompassing a 35 amino acid region (residues 168–203) located between the transmembrane domain and the C-terminal putative coiled-coil region of YopD. These mutations did not affect regulation of the low calcium response or YopB-YopD interaction but markedly inhibited MAPK and NFκB activation. When some of these mutations were introduced into the native yopD gene, defects in effector translocation and pore formation were also observed. We conclude that this newly identified region is important for YopD translocon function. The role of this domain in vivo remains elusive, as amino acid substitutions in that region did not significantly affect virulence of Y. pseudotuberculosis in orogastrically-infected mice.     

YopP-Expressing Variant of Y. pestis Activates a Potent Innate Immune Response Affording Cross-Protection against Yersiniosis and Tularemia

Plague, initiated by Yersinia pestis infection, is a rapidly progressing disease with a high mortality rate if not quickly treated. The existence of antibiotic-resistant Y. pestis strains emphasizes the need for the development of novel countermeasures against plague. We previously reported the generation of a recombinant Y. pestis strain (Kim53ΔJ+P) that over-expresses Y. enterocolitica YopP. When this strain was administered subcutaneously to mice, it elicited a fast and effective protective immune response in models of bubonic, pneumonic and septicemic plague. In the present study, we further characterized the immune response induced by the Kim53ΔJ+P recombinant strain. Using a panel of mouse strains defective in specific immune functions, we observed the induction of a prompt protective innate immune response that was interferon-γ dependent. Moreover, inoculation of mice with Y. pestis Kim53ΔJ+P elicited a rapid protective response against secondary infection by other bacterial pathogens, including the enteropathogen Y. enterocolitica and the respiratory pathogen Francisella tularensis. Thus, the development of new therapies to enhance the innate immune response may provide an initial critical delay in disease progression following the exposure to highly virulent bacterial pathogens, extending the time window for successful treatment.     

Respiratory Dendritic Cell Subsets Differ in Their Capacity to Support the Induction of Virus-Specific Cytotoxic CD8+ T Cell Responses

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC - exhibiting the CD103⁺ and CD11b^hi phenotype, respectively - are the primary DC presenting antigen to naïve CD4⁺ and CD8⁺ T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103⁺ RDC subset preferentially drives efficient proliferation and differentiation of naive CD8⁺ T cells responding to infection into effector cells, and only the CD103⁺ RDC subset can present to naïve CD8⁺ T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103⁺ and CD11b^hi RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines.