Comparison of immune responses in patients infected with Vibrio cholerae O139 and O1.

Vibrio cholerae O139 has recently emerged as the second etiologic agent of cholera in Asia. A study was carried out to evaluate the induction of specific immune responses to the organism in V. cholerae O139-infected patients. The immune responses to V. cholerae O139 Bengal were studied in patients by measuring antibody-secreting cells (ASC), as well as vibriocidal and antitoxic antibodies in the circulation. These responses were compared with those in patients with V. cholerae O1 disease. Strong immunoglobulin A (IgA) and IgM ASC responses were seen against the homologous lipopolysaccharide or serogroup of V. cholerae. The magnitude and isotype of the responses were similar in O139- and O1-infected patients. Vibriocidal antibody responses were seen against bacteria of the homologous but not heterologous serogroup, and these responses reflect the lack of cross-protection between the infections caused by the two serogroups. The two groups of patients showed comparable cholera toxin-specific ASC responses, with the IgG isotype dominating over the IgA isotype, as well as comparable antitoxic immune responses in plasma. These results suggest that despite having a polysaccharide capsule, V. cholerae O139 induces systemic and intestine-derived ASC responses in peripheral blood comparable to those seen in patients with V. cholerae O1 disease.     

CD4+ T-cell subsets in intestinal inflammation

Intestinal CD4⁺ T cells are essential mediators of immune homeostasis and inflammation. Multiple subsets of CD4⁺ T cells have been described in the intestine, which represents an important site for the generation and regulation of cells involved in immune responses both within and outside of the gastrointestinal tract. Recent advances have furthered our understanding of the biology of such cells in the intestine. Appreciation of the functional roles for effector and regulatory populations in health and disease has revealed potential translational targets for the treatment of intestinal diseases, including inflammatory bowel disease. Furthermore, the role of dietary and microbiota-derived factors in shaping the intestinal CD4⁺ T-cell compartment is becoming increasingly understood. Here, we review recent advances in understanding the multifaceted roles of CD4⁺ T cells in intestinal immunity.     

Laboratory-acquired Vibrio cholerae O1 infection in Austria, 2008

Vibrio cholerae infection is a rare but well-documented cause of laboratory-associated illness. We report on the first case of indigenous cholera documented in Austria after more than fifty years. In April 2008, the National Reference Centre for V. cholerae received an isolate of V. cholerae O1, serotype Ogawa, cultured from the stool specimen of a patient consulting a general practitioner because of watery diarrhea. The 23 year old microbiology student had been working with viable V. cholerae for 4 weeks in a practical laboratory course. Two days before onset of symptoms an open 300 mL Erlenmeyer flask with approx. 30 mL of overnight V. cholerae culture tipped over and spilled into a laboratory shaker near the student's working place. Wearing gloves and protective gowns, the student and her supervisor immediately cleaned and decontaminated the shaker. As a consequence of this laboratory incident, the institution in question replaced the clamp-less shaker plate by a traditional shaker plate with mechanical clamps.     

T-cell responses to CD1-presented lipid antigens in humans with Mycobacterium tuberculosis infection

CD1-restricted presentation of lipid or glycolipid antigens derived from Mycobacterium tuberculosis has been demonstrated by in vitro experiments using cultured T-cell lines. In the present work, the frequency of T-cell responses to natural mycobacterial lipids was analyzed in ex vivo studies of peripheral blood lymphocytes from human patients with pulmonary tuberculosis, from asymptomatic individuals with known contact with M. tuberculosis documented by conversion of their tuberculin skin tests, and from healthy tuberculin skin test-negative individuals or individuals vaccinated with Mycobacterium bovis BCG. Proliferation and gamma interferon enzyme-linked immunospot assays using peripheral blood lymphocytes and autologous CD1(+) immature dendritic cells revealed that T cells from asymptomatic M. tuberculosis-infected donors responded with significantly greater magnitude and frequency to mycobacterial lipid antigen preparations than lymphocytes from uninfected healthy donors. By use of these methods, lipid-antigen-specific proliferative responses were minimally detectable or absent in blood samples from patients with active tuberculosis prior to chemotherapy but became detectable in blood samples drawn 2 weeks after the start of treatment. Lipid antigen-reactive T cells were detected predominantly in the CD4-enriched T-cell fractions of circulating lymphocytes, and anti-CD1 antibody blocking experiments confirmed the CD1 restriction of these T-cell responses. Our results provide further support for the hypothesis that lipid antigens serve as targets of the recall response to M. tuberculosis, and they indicate that CD1-restricted T cells responding to these antigens comprise a significant portion of the circulating pool of M. tuberculosis-reactive T cells in healthy individuals with previous exposure to M. tuberculosis.     

CD4+ T-cell responses and distribution at the colonic mucosa during Brachyspira hyodysenteriae-induced colitis in pigs

The spirochaete Brachyspira hyodysenteriae causes swine dysentery, a severe colitis characterized by mucosal enlargement as a result of crypt elongation and epithelial necrosis. Most efforts to understand the pathogenesis of this disease have focused on the aetiological agent and its virulence factors. However, the host immune response has been considered an important factor in disease development. Previous research has shown that B. hyodysenteriae induces systemic CD4(+) and gammadelta T-cell responses after intramuscular immunization. Here, we have evaluated changes in the CD4(+) and gammadelta T-cell composition and distribution the different compartments of the colonic mucosa of pigs challenged with B. hyodysenteriae. We report that, in infected pigs, gammadelta T cells were significantly depleted from the epithelial layer, although their numbers were maintained in the lamina propria. In addition, CD4(+) T cells aggregated in clusters located in the lamina propria and submucosa. Ex vivo analyses of CD4(+) T-cell responses to B. hyodysenteriae antigens correlated with the changes in the mucosal CD4(+) T-cell distribution observed in infected pigs; CD4(+) T cells recovered from peripheral blood and colonic lymph nodes of infected pigs proliferated to B. hyodysenteriae antigens, whereas no differences were found in the gammadelta T-cell responses between challenged and control groups. In addition, colonic lymph node CD4(+) T cells had a predominant memory/activated phenotype. These results indicate that infection with B. hyodysenteriae induces a mucosal CD4(+) T-cell response and points to CD4(+) T cells being important contributors to the immunopathogenesis of swine dysentery.     

Direct measurement of CD4+ and CD8+ T-cell responses to CMV in HIV-1-infected subjects

Data from murine models of chronic viral infection suggest that CD4+ T-cell responses to viral pathogens are important in sustaining the number and/or function of CD8+ cytotoxic T-cell (CTL) effectors. In this study, we used cytokine flow cytometry (CFC), staining with HLA-A*0201-peptide tetramers, and peptide stimulation with epitopic peptides to study functional CD4+ and CD8+ T-cell responses to cytomegalovirus (CMV) in human subjects coinfected with CMV and the human immunodeficiency virus, type 1 (HIV-1). We show that strong CD4+ and CD8+ T-cell responses to CMV antigens are sustained over time in HIV-1-infected individuals. Those who maintain a strong CD4+ T-cell response to CMV are also likely to maintain higher frequencies of CD8+ T cells capable of binding to HLA-A*0201-CMV pp65 (A2-pp65) tetramers as well as responses to pp65 peptide stimulation with effector cytokine production. These data support the hypothesis that declines in frequencies of CD4+ T-cell responses to CMV are associated with an inability to sustain high levels of CMV-specific CD8+ T-cell responses in HIV-1-infected subjects. These declines may precede the onset of CMV-associated end organ disease.     

Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses

Whereas the definition of B-cell memory is based on well-known cellular properties and differentiation steps, the process of T-cell memory generation was, until recently, less well understood. A series of recent reports, however, have drastically modified our notion of CD8(+) memory T cells. They show that, in addition to division, the generation of efficient memory cells requires a previously unknown differentiation process. As a whole, the generation of CD8(+) memory T cells appears to mimic the generation of memory B cells. Both processes depend on the help of CD4(+) T cells, they are irreversible, they have the same mechanism, and they occur progressively during the late expansion phase of the primary immune response.     

Increased levels of inflammatory mediators in children and adults infected with Vibrio cholerae O1 and O139

Investigations were carried out to study the production of factors associated with the innate immune response in the systemic and mucosal compartments in adults and children infected with Vibrio cholerae O1 and V. cholerae O139. The levels of nonspecific mediators of the innate defense system, i.e., prostaglandin E(2) (PGE(2)), leukotriene B(4) (LTB(4)), and lactoferrin (Lf), as well as myeloperoxidase (MPO), were elevated at the acute stage of the disease in stools obtained from both O1- and O139-infected adults and children. In the systemic compartment, the levels of Lf were increased after onset of disease, which in children remained elevated up to convalescence compared to the healthy controls. Increased concentrations of C-reactive protein were seen in the sera of adult cholera patients at the acute stage of infection. Elevated levels of the nitric oxide (NO*) metabolites (nitrite and nitrate [NO(2)(-) and NO(3)(-)]) were detected in plasma but not in urine. The activity of the scavenger of reactive oxygen species, superoxide dismutase, was higher in the plasma of adults immediately after the onset of disease, suggesting that an active scavenging of reactive oxygen species was taking place. The concentration of 8-iso-prostaglandin F(2 alpha) remained unchanged in the systemic and mucosal compartments in the study subjects. After the recovery of patients from cholera, the concentration of the majority of the metabolites decreased to baseline levels by day 30 after the onset of infection. Immunohistochemical staining showed increased tissue expression of MPO, Lf, and inducible nitric oxide synthase at the acute stage in the duodenal biopsies of adults and rectal biopsies obtained from children with cholera. Very little difference was seen in the levels of the different inflammatory mediators in patients infected with V. cholerae O1 or the encapsulated V. cholerae O139. In summary, these results suggest that elevated concentrations of Lf, MPO, PGE(2), LTB(4), and NO*, as well as other metabolites, during the acute stage of the disease indicate that the innate defense system, as well as the inflammatory process, is activated in both adults and pediatric patients infected with V. cholerae O1 and O139.     

Virus-specific effector CD4+ T-cell responses in hemodialysis patients with hepatitis C virus infection

Patients with chronic renal failure undergoing hemodialysis who are infected with hepatitis C virus (HCV) may test consistently anti-HCV negative. Because CD4(+) T-cells provide help for antibody production virus-specific effector CD4(+) T-cell responses were investigated in relation to anti-HCV positivity in 15 hemodialysis patients grouped according to HCV antibody and viremia. CD4(+) T-cell reactivity was studied in peripheral blood mononuclear cells by standard lymphocyte proliferation assay and phenotypic/functional characterization (cell-surface staining/cytokine secretion) by flow cytometry. HCV-specific CD4(+) T-cell proliferation in viremic hemodialysis patients was weak or absent independently of their anti-HCV status. Virus-specific CD4(+) T-cells displayed a memory phenotype and showed low to undetectable capacity to secrete effector interferon (IFN)-gamma. Impaired activation-induced cytokine secretion appeared to be Th1 (IFN-gamma) but not Th2 (interleukin-4)-directed and was virus-specific as cytomegalovirus responses were preserved. The frequency ex vivo of CD3(+)CD4(+)IFN-gamma(+) T-cells was independent of the HCV antibody status and comparable between viremic (range: 0.08-1.54%) or non-viremic (0.11-3.2%) hemodialysis patients and healthy donors (0.13-1.10%; P = 0.58). The numbers of CD3(+)CD4(+)IFN-gamma(+) T-cells augmented slightly (P = 0.047) in HCV-infected hemodialysis patients but markedly in only one (greater than ninefold) after HCV stimulation. In conclusion, hemodialysis patients show limited HCV-specific effector CD4(+) Th1-cell responses which nonetheless seem unrelated to the anti-HCV status and are not more impaired due to the ongoing hemodialysis.     

Presentation of exogenous whole inactivated simian immunodeficiency virus by mature dendritic cells induces CD4+ and CD8+ T-cell responses

Interactions between HIV-1 and dendritic cells (DCs) play an important role in the initial establishment and spread of infection and development of antiviral immunity. We used chemically inactivated aldrithiol-2 (AT-2) simian immunodeficiency virus (SIV) with functional envelope glycoproteins to study virus interactions with DCs and developed an in vitro system to evaluate the quality of SIV antigen (Ag) presentation by DCs to T cells. AT-2 SIV interacts authentically with T cells and DCs and thus allows assessment of natural SIV-specific responses. CD4+ and CD8+ T cells from blood or lymph nodes of SIV-infected macaques released interferon-gamma (IFN gamma) and proliferated in response to a variety of AT-2 SIV isolates. Responses did not vary significantly as a function of the quantitative envelope glycoprotein content of the virions. Presentation of Ags derived from AT-2 SIV by DCs was more potent than presentation by comparably Ag-loaded monocytes. Interestingly, SIV-pulsed mature DCs stimulated both CD4+ and CD8+ T-cell responses, whereas immature DCs primarily stimulated CD4+ T cells. Further studies using AT-2 inactivated virus may help to define better the details of the virus-DC interactions critical for infection versus induction of antiviral immune responses.     

Role of Vibrio cholerae O139 surface polysaccharides in intestinal colonization

Since the first occurrence of O139 Vibrio cholerae as a cause of cholera epidemics, this serogroup has been investigated intensively, and it has been found that its pathogenicity is comparable to that of O1 El Tor strains. O139 isolates express a thin capsule, composed of a polymer of repeating units structurally identical to the lipopolysaccharide (LPS) O side chain. In this study, we investigated the role of LPS O side chain and capsular polysaccharide (CPS) in intestinal colonization by with genetically engineered mutants. We constructed CPS-negative, CPS/LPS O side chain-negative, and CPS-positive/LPS O side chain-negative mutants. Furthermore, we constructed two mutants with defects in LPS core oligosaccharide (OS) assembly. Loss of LPS O side chain or CPS resulted in a approximately 30-fold reduction in colonization of the infant mouse small intestine, indicating that the presence of both LPS O side chain and CPS is important during the colonization process. The strain lacking both CPS and LPS O side chain and a CPS-positive, LPS O side chain-negative core OS mutant were both essentially unable to colonize. To characterize the role of surface polysaccharides in survival in the host intestine, resistance to several antimicrobial substances was investigated in vitro. These investigations revealed that the presence of CPS protects the cell against attack of the complement system and that an intact core OS is necessary for survival in the presence of bile.     

Neisserial immunoglobulin A1 protease induces specific T-cell responses in humans.

We have previously shown that immunoglobulin A1 (IgA1) protease, an exoenzyme of pathogenic neisseriae, can trigger the release of proinflammatory cytokines from human monocytic subpopulations. Here, we demonstrate a dose-dependent T-cell response to recombinant gonococcal IgA1 protease (strain MS11) in healthy human blood donors. This response was delayed in comparison to the immune response against tetanus toxoid. Stimulation with IgA1 protease led to the activation of CD4(+) and CD8(+) T cells, as well as CD19(+) B cells and CD56(+) NK cells, indicated by de novo expression of CD69. Only CD4(+) T cells proliferated and stained positive for intracellular gamma interferon (IFN-gamma). Both proliferation and IFN-gamma production were dependent on antigen presentation via major histocompatibility complex class II. Peripheral blood mononuclear cells stimulated with IgA1 protease produce IFN-gamma and tumor necrosis factor alpha but no, or very low amounts of, interleukin-10 (IL-10) or IL-4, indicating a Th1-based proinflammatory immune response. These findings support the significance of IgA1 protease as a virulence determinant of bacterial meningitis and its function as a dominant proinflammatory T-cell antigen.     

Quantitative studies of CD8+ T-cell responses during microbial infection

MHC class I tetramer staining, intracellular cytokine staining and ELISPOT assays have made it possible to quantify CD8(+) T-cell responses precisely during and following viral and bacterial infection. Although these quantitative methods are by now familiar and trusted components of the immunologist's toolbox, their application to models of microbial infection continues to provide surprising insights into mammalian adaptive immunity. In the past year there have been many exciting new findings on CD8(+) T-cell priming, expansion and memory formation in response to microbial infection.     

Concomitant augmentation of type 1 CD4+ and CD8+ T-cell responses during successful interferon-alpha and ribavirin treatment for chronic hepatitis C virus infection

The combined interferon-alpha (IFN-alpha) and ribavirin (IFN-alpha/ribavirin) therapy for chronic hepatitis C virus (HCV) infection results in sustained viral eradication in 31%-64% of the patients. Previous studies have strongly suggested that HCV-specific T-cell responses maybe modulated during this therapy. The objective of this study was to further define the effect of IFN-alpha/ribavirin therapy on type 1 and type 2 HCV-specific CD4(+) and CD8(+) T-cell responses during IFN-alpha/ribavirin therapy. Toward this, serial CD8(+) T-cell responses to HCV-derived epitopes and CD4(+) T-cell responses to the HCV core antigen were analyzed in four patients before (baseline), during (at 24 weeks), and at the end (at 48 weeks) of IFN-alpha/ribavirin therapy. Therapy-induced viral clearance in three patients was associated with a significant augmentation of HCV-specific type 1 CD4(+) and CD8(+) T-cell responses. In contrast, in a patient who did not respond to therapy, a significant HCV-specific CD4(+) Th2 cell reactivity was observed accompanied by a lack of augmentation of the HCV-specific CD8(+) T-cell reactivity. These results indicate that enhancement of HCV-specific CD4(+) and CD8(+) T-cell responses is an important factor in determining the response to the IFN-alpha/ribavirin therapy and the outcome of the HCV infection.     

IRF4-dependent dendritic cells regulate CD8+ T-cell differentiation and memory responses in influenza infection

Acute respiratory disease caused by influenza viruses is imperfectly mitigated by annual vaccination to select strains. Development of vaccines that elicit lung-resident memory CD8⁺ T cells (T_RM) would offer more universal protection to seasonal and emerging pandemic viruses. Understanding how lung-resident dendritic cells (DCs) regulate T_RM differentiation would be an important step in this process. Here, we used CD11c-cre-Irf4^f/f (KO) mice, which lack lung-resident IRF4-dependent CD11b⁺CD24^hi DCs and show IRF4 deficiency in other lung cDC subsets, to determine if IRF4-expressing DCs regulate CD8⁺ memory precursor cells and T_RM during influenza A virus (IAV) infection. KO mice showed defective CD8⁺ T-cell memory, stemming from a deficit of T regulatory cells and memory precursor cells with decreased Foxo1 expression. Transfer of wild-type CD11b⁺CD24^hi DCs into KO mice restored CD8⁺ memory precursor cell numbers to wild-type levels. KO mice recovered from a primary infection harbored reduced numbers of CD8⁺ T_RM and showed deficient expansion of IFNγ⁺CD8⁺ T cells and increased lung pathology upon challenge with heterosubtypic IAV. Thus, vaccination strategies that harness the function of IRF4-dependent DCs could promote the differentiation of CD8⁺ T_RM during IAV infection.     

Isolation of nontoxigenic Vibrio cholerae O1 from a human wound infection.

Vibrio cholerae serotype O1 organisms that do not produce cholera toxin and, in fact, lack the genetic material encoding the enterotoxin have recently been detected in coastal regions of the United States. Although these organisms have been assumed to be nonpathogenic, they have been considered a potential reservoir of toxigenic V. cholerae. In 1979, nontoxigenic V. cholerae O1 was isolated from a leg wound of an accident victim residing in New Orleans. The only known risk factors of the patient, besides his debilitated condition, were alcoholism and the consumption of raw oysters before recognition of his wound infection. Coincident with the identification of the isolate from the leg wound, an identical nontoxigenic V. cholerae O1 isolate was cultured from the sewage system serving the residence of this patient. Nontoxigenic V. cholerae O1 seems to be capable of multiplying in human tissue and may produce extraintestinal infection. This indigenous inhabitant of temperate coastal regions may not be avirulent and may be of public health significance.     

Low nadir CD4+ T-cell counts predict gut dysbiosis in HIV-1 infection

Human immunodeficiency virus (HIV)-1 infection causes severe gut and systemic immune damage, but its effects on the gut microbiome remain unclear. Previous shotgun metagenomic studies in HIV-negative subjects linked low-microbial gene counts (LGC) to gut dysbiosis in diseases featuring intestinal inflammation. Using a similar approach in 156 subjects with different HIV-1 phenotypes, we found a strong, independent, dose–effect association between nadir CD4+ T-cell counts and LGC. As in other diseases involving intestinal inflammation, the gut microbiomes of subjects with LGC were enriched in gram-negative Bacteroides, acetogenic bacteria and Proteobacteria, which are able to metabolize reactive oxygen and nitrogen species; and were depleted in oxygen-sensitive methanogenic archaea and sulfate-reducing bacteria. Interestingly, subjects with LGC also showed increased butyrate levels in direct fecal measurements, consistent with enrichment in Roseburia intestinalis despite reductions in other butyrate producers. The microbiomes of subjects with LGC were also enriched in bacterial virulence factors, as well as in genes associated with beta-lactam, lincosamide, tetracycline, and macrolide resistance. Thus, low nadir CD4+ T-cell counts, rather than HIV-1 serostatus per se, predict the presence of gut dysbiosis in HIV-1 infected subjects. Such dysbiosis does not display obvious HIV-specific features; instead, it shares many similarities with other diseases featuring gut inflammation.