Search

Clostridioides difficile is an enteric pathogen that can cause significant clinical disease in both humans and animals. However, clinical disease arises most commonly after treatment with broad-spectrum antibiotics. The organism's ability to cause naturally occurring disease in mice is rare, and little is known about its clinical significance in highly immunocompromised mice. We report on 2 outbreaks of diarrhea associated with C. difficile in mice. In outbreak 1, 182 of approximately 2, 400 NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) and related strains of mice became clinically ill after cessation of a 14-d course of 0.12% amoxicillin feed to control an increase in clinical signs associated with Corynebacterium bovis infection. Most mice had been engrafted with human tumors; the remainder were experimentally naïve. Affected animals exhibited 1 of 3 clinical syndromes: 1) peracute death; 2) severe diarrhea leading to euthanasia or death; or 3) mild to moderate diarrhea followed by recovery. A given cage could contain both affected and unaffected mice. Outbreak 2 involved a small breeding colony (approximately 50 mice) of NOD. CB17- Prkdc scid /NCrCrl (NOD- scid ) mice that had not received antibiotics or experimental manipulations. In both outbreaks, C. difficile was isolated, and toxins A and B were detected in intestinal content or feces. Histopathologic lesions highly suggestive of C. difficile enterotoxemia included fibrinonecrotizing and neutrophilic typhlocolitis with characteristic 'volcano' erosions or pseudomembrane formation. Genomic analysis of 4 isolates (3 from outbreak 1 and 1 from outbreak 2) revealed that these isolates were closely related to a pathogenic human isolate, CD 196. To our knowledge, this report is the first to describe naturally occurring outbreaks of C. difficile -associated typhlocolitis with significant morbidity and mortality in highly immunocompromised strains of mice.

Background : The immune response to Listeria monocytogenes (LM) is characterized by formation of leukocyte rich foci of infection in liver and spleen.  Although much has been gained in our understanding of immune response through the study of LM, little is known about spatio-temporal regulation of immune response to Listeria in liver. Methods: We utilize a combination of molecular, genetic and intravital microscopic approaches to gain insight into the dynamics of foci and leukocyte behavior during hepatic Listeriosis.  Results : LM foci efficiently exclude blood flow, indicating the presence of a barrier separating the foci and healthy tissue.  Despite this barrier, sinusoidal myelomonocytic cells readily enter or transiently interact with cells at the edge of foci of infection.  Next, utilizing L9.6 transgenic CD8 + T cells specific for an endogenously processed LM antigen, p60 217-225, along with LM deficient in this epitope, we define the role of TCR in T cell migratory behavior in infected liver.  Surprisingly, T cell behavior varies with micro-anatomic locale.  Near foci, non-specific adhesion mechanisms dominate lymphocyte behavior.  Antigen specific effects on motility became detectable only distal to foci.  Conclusions: These data suggest that LM antigens act in a paracrine manner to mediate protection from Listeriosis in the liver., Competing Interests: No competing interests were disclosed.

Clostridium difficile is a spore-forming anaerobic bacterium that causes colitis in patients with disrupted colonic microbiota. While some individuals are asymptomatic C. difficile carriers, symptomatic disease ranges from mild diarrhea to potentially lethal toxic megacolon. The wide disease spectrum has been attributed to the infected host's age, underlying diseases, immune status, and microbiome composition. However, strain-specific differences in C. difficile virulence have also been implicated in determining colitis severity. Because patients infected with C. difficile are unique in terms of medical history, microbiome composition, and immune competence, determining the relative contribution of C. difficile virulence to disease severity has been challenging, and conclusions regarding the virulence of specific strains have been inconsistent. To address this, we used a mouse model to test 33 clinical C. difficile strains isolated from patients with disease severities ranging from asymptomatic carriage to severe colitis, and we determined their relative in vivo virulence in genetically identical, antibiotic-pretreated mice. We found that murine infections with C. difficile clade 2 strains (including multilocus sequence type 1/ribotype 027) were associated with higher lethality and that C. difficile strains associated with greater human disease severity caused more severe disease in mice. While toxin production was not strongly correlated with in vivo colonic pathology, the ability of C. difficile strains to grow in the presence of secondary bile acids was associated with greater disease severity. Whole-genome sequencing and identification of core and accessory genes identified a subset of accessory genes that distinguish high-virulence from lower-virulence C. difficile strains. IMPORTANCE Clostridium difficile is an important cause of hospital-associated intestinal infections, and recent years have seen an increase in the number and severity of cases in the United States. A patient's antibiotic history, immune status, and medical comorbidities determine, in part, the severity of C. difficile infection. The relative virulence of different clinical C. difficile strains, although postulated to determine disease severity in patients, has been more difficult to consistently associate with mild versus severe colitis. We tested 33 distinct clinical C. difficile isolates for their ability to cause disease in genetically identical mice and found that C. difficile strains belonging to clade 2 were associated with higher mortality. Differences in survival were not attributed to differences in toxin production but likely resulted from the distinct gene content in the various clinical isolates., (Copyright © 2017 Lewis et al.)

Antibiotic administration can disrupt the intestinal microbiota and down-regulate innate immune defenses, compromising colonization resistance against orally acquired bacterial pathogens. Vancomycin-resistant Enterococcus faecium (VRE), a major cause of antibiotic-resistant infections in hospitalized patients, thrives in the intestine when colonization resistance is compromised, achieving extremely high densities that can lead to bloodstream invasion and sepsis. Viral infections, by mechanisms that remain incompletely defined, can stimulate resistance against invading bacterial pathogens. We report that murine norovirus infection correlates with reduced density of VRE in the intestinal tract of mice with antibiotic-induced loss of colonization resistance. Resiquimod (R848), a synthetic ligand for Toll-like receptor 7 (TLR-7) that stimulates antiviral innate immune defenses, restores expression of the antimicrobial peptide Reg3γ and reestablishes colonization resistance against VRE in antibiotic-treated mice. Orally administered R848 triggers TLR-7 on CD11c(+) dendritic cells, inducing interleukin-23 (IL-23) expression followed by a burst of IL-22 secretion by innate lymphoid cells, leading to Reg3γ expression and restoration of colonization resistance against VRE. Our findings reveal that an orally bioavailable TLR-7 ligand that stimulates innate antiviral immune pathways in the intestine restores colonization resistance against a highly antibiotic-resistant bacterial pathogen., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2016, American Association for the Advancement of Science.)

Antibiotic administration disrupts the intestinal microbiota, increasing susceptibility to pathogens such as Clostridium difficile. Metronidazole or oral vancomycin can cure C. difficile infection, and administration of these agents to prevent C. difficile infection in high-risk patients, although not sanctioned by Infectious Disease Society of America guidelines, has been considered. The relative impacts of metronidazole and vancomycin on the intestinal microbiota and colonization resistance are unknown. We investigated the effect of brief treatment with metronidazole and/or oral vancomycin on susceptibility to C. difficile, vancomycin-resistant Enterococcus, carbapenem-resistant Klebsiella pneumoniae, and Escherichia coli infection in mice. Although metronidazole resulted in transient loss of colonization resistance, oral vancomycin markedly disrupted the microbiota, leading to prolonged loss of colonization resistance to C. difficile infection and dense colonization by vancomycin-resistant Enterococcus, K. pneumoniae, and E. coli. Our results demonstrate that vancomycin, and to a lesser extent metronidazole, are associated with marked intestinal microbiota destruction and greater risk of colonization by nosocomial pathogens., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Infection with the opportunistic enteric pathogen Clostridium difficile is an increasingly common clinical complication that follows antibiotic treatment-induced gut microbiota perturbation. Innate lymphoid cells (ILCs) are early responders to enteric pathogens; however, their role during C. difficile infection is undefined. To identify immune pathways that mediate recovery from C. difficile infection, we challenged C57BL/6, Rag1(-/-) (which lack T and B cells), and Rag2(-/-)Il2rg(-/-) (Ragγc(-/-)) mice (which additionally lack ILCs) with C. difficile. In contrast to Rag1(-/-) mice, ILC-deficient Ragγc(-/-) mice rapidly succumbed to infection. Rag1(-/-) but not Ragγc(-/-) mice upregulate expression of ILC1- or ILC3-associated proteins following C. difficile infection. Protection against infection was restored by transferring ILCs into Ragγc(-/-) mice. While ILC3s made a minor contribution to resistance, loss of IFN-γ or T-bet-expressing ILC1s in Rag1(-/-) mice increased susceptibility to C. difficile. These data demonstrate a critical role for ILC1s in defense against C. difficile., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2⁺Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2⁺Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2⁺Mo and Mo-DCs exert innate antifungal activity. First, CCR2⁺Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2⁺Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2⁺Mo and their derivatives in innate antifungal immunity in the lung.

The intestinal microbiota contributes to the development of the immune system, and conversely, the immune system influences the composition of the microbiota. Toll-like receptors (TLRs) in the gut recognize bacterial ligands. Although TLR signaling represents a major arm of the innate immune system, the extent to which TLRs influence the composition of the intestinal microbiota remains unclear. We performed deep 16S ribosomal RNA sequencing to characterize the complex bacterial populations inhabiting the ileum and cecum of TLR- and MyD88-deficient mice. The microbiota of MyD88- and TLR-deficient mouse colonies differed markedly, with each colony harboring distinct and distinguishable bacterial populations in the small and large intestine. Comparison of MyD88-, TLR2-, TLR4-, TLR5-, and TLR9-deficient mice and their respective wild-type (WT) littermates demonstrated that the impact of TLR deficiency on the composition of the intestinal microbiota is minimal under homeostatic conditions and after recovery from antibiotic treatment. Thus, differences between TLR-deficient mouse colonies reflected long-term divergence of the microbiota after extended husbandry in isolation from each other. Long-term breeding of isolated mouse colonies results in changes of the intestinal microbiota that are communicated to offspring by maternal transmission, which account for marked compositional differences between WT and mutant mouse strains.

Microbial penetration of the intestinal epithelial barrier triggers inflammatory responses that include induction of the bactericidal C-type lectin RegIIIγ. Systemic administration of flagellin, a bacterial protein that stimulates Toll-like receptor 5 (TLR5), induces epithelial expression of RegIIIγ and protects mice from intestinal colonization with antibiotic-resistant bacteria. Flagellin-induced RegIIIγ expression is IL-22 dependent, but how TLR signaling leads to IL-22 expression is incompletely defined. By using conditional depletion of lamina propria dendritic cell (LPDC) subsets, we demonstrated that CD103(+)CD11b(+) LPDCs, but not monocyte-derived CD103(-)CD11b(+) LPDCs, expressed high amounts of IL-23 after bacterial flagellin administration and drove IL-22-dependent RegIIIγ production. Maximal expression of IL-23 subunits IL-23p19 and IL-12p40 occurred within 60 min of exposure to flagellin. IL-23 subsequently induced a burst of IL-22 followed by sustained RegIIIγ expression. Thus, CD103(+)CD11b(+) LPDCs, in addition to promoting long-term tolerance to ingested antigens, also rapidly produce IL-23 in response to detection of flagellin in the lamina propria., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Listeria monocytogenes is a facultative intracellular bacterium that causes systemic infections in immunocompromised hosts. Early recruitment of myeloid cells, including inflammatory monocytes and neutrophils, to sites of L. monocytogenes infection is essential for the control of infection and host survival. Because previous experimental studies used depleting or blocking Abs that affected both inflammatory monocytes and neutrophils, the relative contributions of these cell populations to defense against L. monocytogenes infection remain incompletely defined. In this article, we used highly selective depletion strategies to either deplete inflammatory monocytes or neutrophils from L. monocytogenes-infected mice and demonstrate that neutrophils are dispensable for early and late control of infection. In contrast, inflammatory monocytes are essential for bacterial clearance during the innate and adaptive phases of the immune response to L. monocytogenes infection.

Inflammatory (Ly6C(hi) CCR2+) monocytes provide defense against infections but also contribute to autoimmune diseases and atherosclerosis. Monocytes originate from bone marrow and their entry into the bloodstream requires stimulation of CCR2 chemokine receptor by monocyte chemotactic protein-1 (MCP1). How monocyte emigration from bone marrow is triggered by remote infections remains unclear. We demonstrated that low concentrations of Toll-like receptor (TLR) ligands in the bloodstream drive CCR2-dependent emigration of monocytes from bone marrow. Bone marrow mesenchymal stem cells (MSCs) and their progeny, including CXC chemokine ligand (CXCL)12-abundant reticular (CAR) cells, rapidly expressed MCP1 in response to circulating TLR ligands or bacterial infection and induced monocyte trafficking into the bloodstream. Targeted deletion of MCP1 from MSCs impaired monocyte emigration from bone marrow. Our findings suggest that bone marrow MSCs and CAR cells respond to circulating microbial molecules and regulate bloodstream monocyte frequencies by secreting MCP1 in proximity to bone marrow vascular sinuses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30-1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ T cells in effector sites.

Pulmonary infection of mice with Aspergillus fumigatus induces concurrent T helper type 1 (Th1) and Th17 responses that depend on Toll-like receptor/MyD88 and Dectin-1, respectively. However, the mechanisms balancing Th1 and Th17 CD4 T cell populations during infection remain incompletely defined. In this study, we show that Dectin-1 deficiency disproportionally increases Th1 responses and decreases Th17 differentiation after A. fumigatus infection. Dectin-1 signaling in A. fumigatus-infected wild-type mice reduces IFN-γ and IL-12p40 expression in the lung, thereby decreasing T-bet expression in responding CD4 T cells and enhancing Th17 responses. Absence of IFN-γ or IL-12p35 in infected mice or T-bet in responding CD4 T cells enhances Th17 differentiation, independent of Dectin-1 expression, in A. fumigatus-infected mice. Transient deletion of monocyte-derived dendritic cells also reduces Th1 and boosts Th17 differentiation of A. fumigatus-specific CD4 T cells. Our findings indicate that Dectin-1-mediated signals alter CD4 T cell responses to fungal infection by decreasing the production of IL-12 and IFN-γ in innate cells, thereby decreasing T-bet expression in A. fumigatus-specific CD4 T cells and enabling Th17 differentiation.

Monocytes play a central role in defense against infection, but the mechanisms promoting monocyte recruitment and activation remain incompletely defined. Defense against Listeria monocytogenes, an intracellular bacterial pathogen, requires in vivo MCP-1 induction and CCR2-dependent recruitment of Ly6C(high) monocytes from bone marrow to sites of infection. Herein, we demonstrate that infection of bone marrow-derived macrophages with virulent L. monocytogenes induces MCP-1 expression in two phases. The first phase is rapid, induces low-level production of MCP-1, and is dependent on TLR/MyD88 signaling. The second phase promotes prolonged, higher level MCP-1 secretion and is dependent on signaling via the type I IFN receptor (IFNAR). Although attenuated L. monocytogenes strains that remain confined to the phagosome trigger TLR/MyD88-mediated signals and induce low-level MCP-1 expression, only cytosol-invasive bacteria promote IFNAR-dependent MCP-1 expression. In vivo, deficiency of either MyD88 or IFNAR signaling does not impair early monocyte emigration from bone marrow and recruitment to infected spleen. Loss of both MyD88 and IFNAR-mediated MCP-1 induction, however, results in deficient Ly6C(high) monocyte recruitment and increased susceptibility to L. monocytogenes infection. Our studies demonstrate that distinct but partially overlapping signal transduction pathways provide redundancy that ensures optimal monocyte recruitment to sites of microbial infection.

Aspergillus fumigatus, a common environmental fungus, can cause lethal invasive infections in immunocompromised hosts. In immunocompetent individuals, however, inhaled A. fumigatus spores prime CD4(+) T cells and activate immune responses that prevent invasive infection. Calibration of inflammatory responses to levels that prevent fungal invasion without inducing collateral tissue damage is essential for host survival, but the underlying regulatory mechanisms remain undefined. Although IL-10 is a validated regulatory cytokine that suppresses immune responses, and IL-10 deficiency or blockade generally enhances immune responses, we find that A. fumigatus-specific T cell frequencies are markedly reduced in airways of IL-10-deficient mice. T cell priming, proliferation, and survival were unaffected by IL-10 deficiency and did not account for decreased frequencies of A. fumigatus-specific T cells in the airways of IL-10-deficient mice. Instead, IL-10 deficiency results in redistribution of A. fumigatus-specific T cells from infected lungs to the gut, a process that is reversed by antibiotic-mediated depletion of intestinal microbes. Our studies demonstrate that disregulated immune responses in the gut can result in dramatic redistribution of pathogen-specific T cells within the host.

Regulation of CD8 T cell expansion and contraction is essential for successful immune defense against intracellular pathogens. IL-10 is a regulatory cytokine that can restrict T cell responses by inhibiting APC functions. IL-10, however, can also have direct effects on T cells. Although blockade or genetic deletion of IL-10 enhances T cell-mediated resistance to infections, the extent to which IL-10 limits in vivo APC function or T cell activation/proliferation remains unknown. Herein, we demonstrate that primary and memory CD8 T cell responses following Listeria monocytogenes infection are enhanced by the absence of IL-10. Surface expression of the IL-10R is transiently up-regulated on CD8 T cells following activation, suggesting that activated T cells can respond to IL-10 directly. Consistent with this notion, CD8 T cells lacking IL-10R2 underwent greater expansion than wild-type T cells upon L. monocytogenes infection. The absence of IL-10R2 on APCs, in contrast, did not enhance T cell responses following infection. Our studies demonstrate that IL-10 produced during bacterial infection directly limits expansion of pathogen-specific CD8 T cells and reveal an extrinsic regulatory mechanism that modulates the magnitude of memory T cell responses.

Aspergillus fumigatus is a mold that causes a spectrum of diseases, including lethal lung infections in immunocompromised humans and allergic asthma in atopic individuals. T helper 1 (Th1) CD4(+) T cells protect against invasive A. fumigatus infections whereas Th2 CD4(+) T cells exacerbate asthma upon inhalation of A. fumigatus spores. Herein, we demonstrate that A. fumigatus-specific T cells were rapidly primed in lymph nodes draining the lung and fully differentiated into interferon-gamma (IFN-gamma)-producing Th1 CD4(+) T cells upon arrival in the airways. T-bet induction in A. fumigatus-specific CD4(+) T cells was enhanced by MyD88-mediated signals in draining lymph nodes, but T cell proliferation, trafficking, and Th1 differentiation in the airways were Toll-like receptor (TLR) and MyD88 independent. Our studies demonstrate that CD4(+) T cell differentiation during respiratory fungal infection occurs incrementally, with TLR-mediated signals in the lymph node enhancing the potential for IFN-gamma production whereas MyD88-independent signals promote Th1 differentiation in the lung.

Major histocompatibility complex (MHC) molecules carrying selected peptides will bind specifically to their cognate T-cell receptor on individual clones of reactive T cells. Fluorescently labeled, tetrameric MHC-peptide complexes have been widely used to detect and quantitate antigen-specific T-cell populations via flow cytometry. We hypothesized that such MHC-peptide tetramers could also be used to selectively deplete unique reactive T-cell populations, while leaving the remaining T-cell repertoire and immune response intact. In this report, we successfully demonstrate that a tetramer-based depletion of T cells can be achieved in a murine model of allogeneic bone marrow transplantation. Depletion of a specific alloreactive population of donor splenocytes (< 0.5% of CD8+ T cells) prior to transplantation significantly decreased morbidity and mortality from graft-versus-host disease. There was no early regrowth of the antigen-specific T cells in the recipient and in vivo T-cell proliferation was greatly reduced as well. Survival was increased more than 3-fold over controls, yet the inherent antitumor activity of the transplant was retained. This method also provides the proof-of-concept for similar strategies to selectively remove other unwanted T-cell clones, which could result in novel therapies for certain autoimmune disorders, T-cell malignancies, and solid organ graft rejection.

CD8 T cell populations restricted by H2-M3 MHC class Ib molecules expand rapidly during primary Listeria monocytogenes infection but only minimally upon reinfection. In contrast, CD8 T cells restricted by MHC class Ia molecules undergo more delayed expansion during primary infection but rapid and robust expansion following reinfection. In this study we demonstrate that primary H2-M3-restricted CD8 T cell responses are unaffected by the frequency of naive MHC class Ia-restricted T cells during L. monocytogenes infection. The magnitude of H2-M3-restricted memory responses, in contrast, is down-modulated by increasing frequencies of MHC class Ia-restricted effector T cells following secondary systemic infection. Suppression by MHC class Ia-restricted T cells, however, is not a universal feature of MHC class Ib-restricted memory responses. Primary systemic L. monocytogenes infection followed by secondary tissue infection, for example, results in robust expansion of H2-M3-restricted memory T cells in draining lymph nodes, despite the activation of MHC class Ia-restricted memory T cell responses. Thus, whereas MHC class Ia-restricted memory T cell populations predominate in spleens following systemic reinfection, H2-M3-restricted memory T cell responses remain prominent in lymph nodes draining localized infections. Our studies demonstrate that interactions between CD8 T cell populations can differ, depending on the status of the responding T cells (naive vs memory) and the route of reinfection. These results may have important implications for prime-boost vaccination strategies.

H2-M3-restricted CD8+ T cells can exhibit cross-reactivity to different bacterially derived N-formylmethionine peptides. The extent of this promiscuity is unclear. We deleted the nonredundant fMIVTLF epitope and found that Listeria monocytogenes still primed fMIVTLF-specific T cells. Thus, cross-reactivity appears to be a more general characteristic of H2-M3-restricted T cells.

Immunization of mice with live or heat-killed Listeria monocytogenes (HKLM) efficiently primes pathogen-specific CD8(+) T cells. T lymphocytes primed by HKLM, however, undergo attenuated proliferation and do not fully differentiate. Thus, only infection with live bacteria induces long-term, CD8(+) T cell-mediated protective immunity. In this study we demonstrate that live and heat-killed bacteria, while both associating with Mac-3(+)CD11b(hi) cells, localize to distinct splenic areas following intravenous inoculation. While HKLM localize to the marginal zone and the splenic red pulp, live L. monocytogenes are carried to the T cell zone of splenic white pulp. Despite these differences, in vivo depletion of CD11c-expressing cells prevents priming of naive T cells by either HKLM or live L. monocytogenes. Analysis of CD11c(hi) dendritic cells (DC) reveals that infection with live L. monocytogenes induces higher levels of CD40, CD80 and CD86 expression than immunization with HKLM. Our results suggest that CD8(+) T cell priming following HKLM immunization or live infection is mediated by DC and that the disparate outcomes of priming can be attributed to suboptimal conditioning of DC in the absence of live, cytosol-invasive bacteria.

Immunosuppressive agents in current use are nonspecific. The capacity to delete specific CD8 T-cell clones of unique specificity could prove to be a powerful tool for dissecting the precise role of CD8(+) T cells in human disease and could form the basis for a safe, highly selective therapy of autoimmune disorders. Major histocompatibility complex (MHC) tetramers (multimeric complexes capable of binding to specific CD8 T-cell clones) were conjugated to (225)Ac (an alpha-emitting atomic nanogenerator, capable of single-hit killing from the cell surface) to create an agent for CD8 T-cell clonal deletion. The "suicide" tetramers specifically bound to, killed, and reduced the function of their cognate CD8 T cells (either human anti-Epstein-Barr virus (EBV) or mouse anti-Listeria in 2 model systems) while leaving the nonspecific control CD8 T-cell populations unharmed. Such an approach may allow a pathway to selective ablation of pathogenic T-cell clones ex vivo or in vivo without disturbing general immune function.

Prime-boost immunization is a promising strategy for inducing and amplifying pathogen- or tumor-specific memory CD8 T cell responses. Although expansion of CD8 T cell populations following the second Ag dose is integral to the prime-boost strategy, it remains unclear when, after priming, memory T cells become competent to proliferate. In this study, we show that Ag-specific CD8 T cells with the capacity to undergo extensive expansion are already present at the peak of the primary immune response in mice. These early memory T cells represent a small fraction of the primary immune response and, at early time points, their potential to proliferate is obscured by large effector T cell populations that rapidly clear Ag upon reimmunization. With sufficient Ag boosting, however, secondary expansion of these memory cells can be induced as early as 5-7 days following primary immunization. Importantly, both early and delayed boosting result in similar levels of protective immunity to subsequent pathogen challenge. Early commitment and differentiation of memory T cells during primary immunization suggest that a short duration between priming and boosting is feasible, providing potential logistic advantages for large-scale prime-boost vaccination of human populations.

Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8(+) T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules. Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L. monocytogenes-specific CD8(+) T cells. To investigate epitope cross-recognition by H2-M3-restricted CD8(+) T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L. monocytogenes. Infection with fMIGWII-deficient L. monocytogenes unexpectedly primed CD8(+) T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo. Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses. HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L. monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells. Our results demonstrate that the dominant H2-M3-restricted CD8(+) T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L. monocytogenes. Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.

Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes are considered pivotal to prevent lymphoproliferative disease (LPD) in allogeneic stem cell transplantation (SCT) recipients. We evaluated the recovery of EBV-specific CD8+ T cells after partially T-cell-depleted SCT and studied the interaction between EBV-specific CD8+ T cells, EBV reactivation, and EBV-LPD. EBV-specific CD8+ T cells were enumerated using 12 class I HLA tetramers presenting peptides derived from 7 EBV proteins. Blood samples were taken at regular intervals after SCT in 61 patients, and EBV DNA levels were assessed by real-time polymerase chain reaction. Forty-five patients showed EBV reactivation, including 25 with high-level reactivation (ie, more than 1000 genome equivalents [geq] per milliliter). Nine of these 25 patients progressed to EBV-LPD. CD8+ T cells specific for latent or lytic EBV epitopes repopulated the peripheral blood at largely similar rates. In most patients, EBV-specific CD8+ T-cell counts had returned to normal levels within 6 months after SCT. Concurrently, the incidence of EBV reactivations clearly decreased. Patients with insufficient EBV-specific CD8+ T-cell recovery were at high risk for EBV reactivation in the first 6 months after SCT. Failure to detect EBV-specific CD8+ T cells in patients with high-level reactivation was associated with the subsequent development of EBV-LPD (P =.048). Consequently, the earlier defined positive predictive value of approximately 40%, based on high-level EBV reactivation only, increased to 100% in patients without detectable EBV-specific CD8+ T cells. Thus, impaired recovery of EBV-specific CD8+ T cells in patients with high-level EBV reactivation may identify a subgroup at very high risk for EBV-LPD and supports that EBV-specific CD8+ T cells protect SCT recipients from progressive EBV reactivation and EBV-LPD.

H2-M3-restricted T cells respond more rapidly to primary Listeria monocytogenes infection than conventional MHC class Ia-restricted T cells. Reinfection with L. monocytogenes, while inducing explosive proliferation of H2-K(d)-restricted T cells, does not stimulate significant expansion of H2-M3-restricted CTL. These disparate responses to reinfection are apparent within 5 days of primary L. monocytogenes infection. However, H2-M3-restricted memory T cells are generated, and are indistinguishable from classically restricted T cells in terms of cell surface memory markers and longevity. Early responses of H2-M3- and H2-K(d)-restricted memory T cells to reinfection are similar, with increases in size and expression of activation markers. Interestingly, priming of H2-M3-restricted T cells with an L. monocytogenes-derived N-formyl peptide plus anti-CD40 generates memory T cells that expand upon re-exposure to Ag during L. monocytogenes infection. Our data indicate that disparate H2-M3- and MHC class Ia-restricted memory T cell responses reflect intrinsic differences between these T cell populations. Although distinct proliferative programs appear to be hardwired in these populations during primary L. monocytogenes infection, under different inflammatory circumstances M3-restricted T cell populations can maintain the ability to expand upon re-exposure to Ag.

Reconstitution of protective immunity by adoptive transfer of pathogen-specific T cells has been successful in patients with compromised cellular immunity. The in vivo effectiveness of in vitro-expanded CD8 CTLs is variable, however. For example, adoptively transferred Listeria monocytogenes-specific CD8 CTLs only confer protective immunity if challenge infection occurs within 48 hours of T cell infusion. Herein we show that transferred CTLs persist in lymphoid compartments for many weeks, but that their response to bacterial challenge decreases during the first week following transfer. While T cells transferred less than 48 hours before infection proliferate, those transferred 7 days before infection die. Remarkably, treatment of mice with anti-CD40 at the time of T cell infusion reprograms transferred T cells, allowing them to proliferate and confer protective immunity upon bacterial challenge 7 days later. Our study demonstrates, for the first time to our knowledge that CD40-mediated stimuli can influence CD8 T cell activation independent of concurrent antigen exposure. The ability to modulate long-term responsiveness of CD8 T cells with a transient, nonspecific inflammatory stimulus has importation implications for adoptive immunotherapy.

We investigated long-term memory and recall cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (VSVs) expressing Env and Gag. More than 7 months after a single vaccination with VSV-Env, approximately 6% of CD8(+) splenocytes stained with major histocompatibility complex class I tetramers containing the Env p18-I10 immunodominant peptide and showed a memory phenotype (CD44(Hi)). The level of tetramer-positive cells in memory was about 14% of the peak primary response. Recall responses elicited in these mice 5 days after boosting with a heterologous recombinant vaccinia virus expressing HIV-1 Env showed that 40 to 45% of CD8(+) splenocytes were tetramer positive and activated (CD62L(Lo)), and these cells produced gamma interferon after stimulation with Env peptide, indicating that they were functional. Five months after the boost, the long-term memory cell population (tetramer positive, CD44(Hi)) constituted 30% of the CD8(+) splenocytes. Recall responses to HIV-1 Gag were examined in mice primed with VSV recombinants expressing HIV-1 Gag protein and boosted with a vaccinia virus recombinant expressing Gag. Using this protocol, we found that approximately 40% of CD8(+) splenocytes were activated (CD62L(Lo)) and specific for a Gag immunodominant peptide (tetramer positive). The high-level Gag recall response elicited by the vaccinia virus-Gag was greater than that obtained by boosting with a VSV-Gag vector with a different VSV glycoprotein. The corresponding levels of CD44(Hi) memory cells were also higher long after boosting with vaccinia virus-Gag than after boosting with a glycoprotein exchange VSV-Gag. Our results show that VSV vectors elicit high-level memory CTL responses and that these can be amplified as much as six- to sevenfold using a heterologous boosting vector.

We investigated the primary cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (rVSVs). The primary response to Env peaked 5 to 7 days after intraperitoneal vaccination, at which time 40% of CD8(+) cells were Env tetramer positive and activated (CD62L(Lo)). These freshly isolated cells actively lysed target cells pulsed with the p18-I10 peptide and secreted gamma interferon and tumor necrosis factor alpha after stimulation with the Env p18-I10 peptide. The primary response to Env elicited by rVSVs was sixfold higher than that elicited by recombinant vaccinia viruses (rVVs) at 5 days postvaccination. An intranasal route of vaccination with VSV-Env also elicited a strong primary response to Env. The primary immune response to Gag elicited by rVSV peaked 7 days after vaccination, at which time 3% of CD8(+) cells were Gag tetramer positive and CD62L(Lo) and functional by intracellular cytokine staining. This response was eightfold higher than that elicited by rVV expressing Gag. VSV-GagEnv, which expresses both Gag and Env from a single recombinant, also induced strong cytotoxic T-lymphocyte (CTL) responses to both Env and Gag. Our quantitative analyses illustrate the potency of the VSV vector system in CTL induction.