Immunohistochemical Study of the β-Chemokine Receptors CCR3 and CCR5 and Their Ligands in Normal and Alzheimer’s Disease Brains

Chemokines belong to an expanding family of cytokines the primary function of which is recruitment of leukocytes to inflammatory sites. Recent evidence has shown their presence in the central nervous system. Because inflammatory responses have been implicated in the pathogenesis of Alzheimer’s disease (AD), we studied the expression of CCR3, CCR5, and their ligands in normal and AD brains by immunohistochemistry. CCR3 and CCR5 are present on microglia of both control and AD brains, with increased expression on some reactive microglia in AD. Immunohistochemistry for MIP-1β, MIP-1α, RANTES, eotaxin, and MCP-3 (ligands for CCR5 and/or CCR3) revealed the presence of MIP-1β predominantly in a subpopulation of reactive astrocytes, which were more widespread in AD than control brains, and MIP-1α predominantly in neurons and weakly in some microglia in both AD and controls. Many of the CCR3⁺ or CCR5⁺ reactive microglia and MIP-1β⁺ reactive astrocytes were found associated with amyloid deposits. Immunoreactivity for eotaxin, RANTES, and MCP-3 were not detected. Detection of these β-chemokine receptors on microglia and some of their ligands in reactive astrocytes and neurons as well as microglia suggests a role for this system in glial-glial and glial-neuronal interactions, potentially influencing the progression of AD.     

M-Cell Surface β1 Integrin Expression and Invasin-Mediated Targeting of Yersinia pseudotuberculosis to Mouse Peyer’s Patch M Cells

Quantitative analysis of Yersinia pseudotuberculosis infection of murine gut loops revealed that significantly more wild-type bacteria associated with Peyer’s patch M cells than with dome enterocytes or goblet cells. An invasin-deficient mutant was significantly attenuated for M-cell invasion, while β1 integrin expression was demonstrated in the apical membranes of M cells but not enterocytes. M-cell targeting by Yersinia pseudotuberculosis in vivo may, therefore, be mediated primarily by the interaction of invasin with cell surface β1 integrins.     

Plasma elafin,cathelicidin, and α-defensins are increased in paediatric inflammatory Crohn’s disease and reflect disease location

IntroductionThe aim of our study was to assess antimicrobial peptides in children with Crohn’s disease (CD).MethodsPlasma elafin, cathelicidin, and α- and β-defensins were assessed in 35 children with CD using immunoassays. Phenotype and location of CD were assessed based on the results of endoscopic and radiological studies.ResultsWe found increased elafin, cathelicidin, and α-defensins in children with inflammatory phenotype as compared to stricturing and penetrating phenotypes of CD. Additionally, we found increased elafin and cathelicidin in colonic location and α-defensins in ileal CD locations.ConclusionsAssessing antimicrobial peptides may be helpful in estimating of phenotype and location of CD lesions.     

Influence of Acute-Phase Parasite Load on Pathology, Parasitism, and Activation of the Immune System at the Late Chronic Phase of Chagas’ Disease

To obtain low and high parasite loads in the acute phase of Chagas’ disease, A/J mice were infected with 10³ or 10⁵ Trypanosoma cruzi trypomastigotes of the Y strain and treated on day 6 with benznidazol. One year later, chronically infected mice were screened for subpatent parasitemias, tissue pathology, and immune response. Mice infected with the high parasite inoculum showed higher levels of chronic parasitemias, heart and striated muscle inflammation, and activation of the immune system than did mice infected with the low inoculum. Concerning the activation of the immune system, the main findings for high-dose-infected mice were (i) increased numbers of splenocytes, with preferential expansion of CD8⁺ and B220⁻ CD5⁻ cells, many of them bearing a macrophage phenotype; (ii) higher frequencies of B (B220⁺), CD4⁺, and CD8⁺ large lymphocytes; (iii) a shift of CD4⁺ cells towards a CD45RB^Low phenotype; (iv) increased frequencies of both CD45RB^Low and CD45RB^High large CD4⁺ cells; (v) augmented numbers of total immunoglobulin (Ig)-secreting cells, with predominance of IgG2a-producing cells; and (vi) increased production of gamma interferon and interleukin 4. In addition, these mice presented lower IgM and higher IgG2a and IgG1 parasite-specific serum antibody levels. Our results indicate that the parasite load at the acute phase of T. cruzi infection influences the activation of the immune system and development of Chagas’ disease pathology at the late chronic phase of the disease.     

Clinical and immunopathologic alterations in rhesus macaques affected with globoid cell leukodystrophy

Globoid cell leukodystrophy, or Krabbe’s disease, is a severe disorder of the central and peripheral nervous system caused by the absence of galactocerebrosidase (GALC) activity. Herein, we describe the clinical, neuropathological, histochemical, and immunohistological features observed in rhesus macaques affected with Krabbe’s disease. Clinical signs included pronounced muscle tremors of head and limbs, difficulty ambulating, ataxia, hypermetria, proprioceptive deficits, and respiratory abnormalities. Histopathologically, all animals presented with evidence of demyelination in the peripheral and central nervous systems and accumulation of mononuclear and multinuclear globoid cells in the cerebral and cerebellar white matter associated with severe gliosis. Using immunohistochemistry and multi-label confocal microscopy, it was determined that globoid cells were CD68⁺, HAM56⁺, LN5⁺, CD163⁺, IBA-1⁺, and Glut-5⁺, suggesting that both peripheral blood-derived monocytes/macrophages and resident parenchymal microglia gave rise to globoid cells. Interestingly, many of the globoid cells and parenchymal microglia with a more ameboid morphology expressed HLA-DR, indicating immune activation. Increased expression of iNOS, TNF-α, and IL-1β were observed in the affected white matter, colocalizing with globoid cells, activated microglia, and astrocytes. Cytokine mRNA levels revealed markedly increased gene expression of CCL2 in the brain of affected macaques. CCL2-expressing cells were detected throughout the affected white matter, colocalizing with GFAP⁺ cells and astrocytes. Collectively, these data suggest that dysregulation of monocyte/macrophage/microglia and up-regulation of certain cytokines may contribute to the pathogenesis of Krabbe’s disease.     

CD4+ CD25+ T-Cell Production in Healthy Humans and in Patients with Thymic Hypoplasia

Regulatory T cells are found primarily in the CD4⁺ CD25⁺ fraction of T cells and play an important role in the prevention of autoimmunity. We examined CD4⁺ CD25⁺ T cells in 33 healthy children and adults and compared them to a population with an inherited form of thymic hypoplasia and a predisposition to autoimmune disease. Absolute numbers of CD4⁺ CD25⁺ T cells were markedly higher in healthy infants than in infants with chromosome 22q11.2 deletion syndrome.     

CD34+ Cord Blood Cell-Transplanted Rag2−/− γc−/− Mice as a Model for Epstein-Barr Virus Infection

Recent studies suggest that Epstein-Barr virus (EBV) can infect naïve B cells, driving them to differentiate into resting memory B cells via the germinal center reaction. This hypothesis has been inferred from parallels with the biology of normal B cells but has never been proven experimentally. Rag2^−/− γ_c^−/− mice that were transplanted with human CD34⁺ cord blood cells as newborns were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we used this model to better define the strategy of EBV infection of human B cells in vivo and to compare this model system with different conditions of EBV infection in humans. Our results support the model of EBV persistence in vivo in cases that were characterized by follicular hyperplasia and a relatively normal CD4⁺ and CD8⁺ T-cell distribution. Intriguingly, in cases that were characterized by nodular and diffuse proliferation with a preponderance of CD8⁺ T cells, similar to infectious mononucleosis, EBV still infects naïve B cells but also induces clonal expansion and ongoing somatic mutations without germinal center reactions. Our results reveal different strategies of EBV infection in B cells that possibly result from variations in the host immune response. Future experiments might allow understanding of the mechanisms responsible for persistent EBV infection and provide targets for more highly tailored therapeutic interventions.     

Serum inflammatory markers in the diagnosis and assessment of Crohn’s disease activity

IntroductionThe aim of our study was to evaluate the diagnostic characteristics of selected inflammatory markers and the results of multiplication of their concentrations in the diagnosis and assessment of Crohn’s disease (CD) activity.MethodsWe studied 49 patients with CD and 31 healthy controls. The CD patients were assigned to subgroups with active and inactive disease based on the Crohn’s Disease Activity Index score. Serum interleukins and C-reactive protein (CRP) were measured using immunoassays.ResultsSerum CRP and interleukins: IL-6, IL-17A, IL-23 were significantly higher in the CD group than in controls, with the best diagnostic performance for IL-23. Only serum IL-6 and CRP were significantly higher in active than in inactive disease, with the better performance of CRP. Multiplication results did not perform better than individual multipliers.ConclusionsSerum CRP may be useful in the assessment of CD activity and there is a need for introduction of IL-23 for the CD diagnosis.     

Expansion of a Novel Pulmonary CD3− CD4+ CD8+ Cell Population in Mice during Chlamydia pneumoniae Infection

A new pulmonary T-cell-like lymphocyte population with the phenotype CD3⁻ CD4⁺ CD8⁺ was discovered in mice. CD4⁺ CD8⁺ but CD3⁺ cells among murine intestinal intraepithelial lymphocytes have previously been described. We describe herein a dramatic expansion of the CD3⁻ CD4⁺ CD8⁺ cell population in response to experimental respiratory infection. After intranasal Chlamydia pneumoniae infection, CD4⁺ CD8⁺ cells became transiently the dominant lymphocyte type (maximum of 87% of all lymphocytes) in the lungs of NIH/S mice but remained virtually undetectable in spleen and blood. The enrichment of these cells was not a C. pneumoniae-specific event, since infection of NIH/S mice with influenza A virus also resulted in an increase in the number of CD4⁺ CD8⁺ cells (maximum of 42% of all lymphocytes). In addition to outbred NIH/S mice, two other mouse strains were studied: BALB/c (H-2^d) and C57BL/6 (H-2^b). C. pneumoniae-infected BALB/c mice responded with an intermediate increase in the number of CD4⁺ CD8⁺ cells in lungs, whereas C57BL/6 mice did not respond. The double-positive CD4⁺ CD8⁺ cells lacked a major part of the T-cell receptor complex, being both CD3⁻ and TCR αβ⁻. However, when they were stimulated in vitro with a T-cell mitogen, they responded by proliferation but did not secrete gamma interferon. The dramatic expansion of this cell population at the infection site suggests an active role for them in respiratory infection, but the specification of this requires further study.     

Low CD4+ T-Lymphocyte Values in Human Immunodeficiency Virus-Negative Adults in Botswana

CD4⁺-lymphocyte counts (LCs) play a crucial role in the management and monitoring of HIV infection. Variability in CD4⁺ LCs has been reported to occur as a result of measurement techniques and/or biological variations. We report on the CD4⁺ LCs of healthy human immunodeficiency virus (HIV)-seronegative adults in Botswana. Samples were obtained from HIV-seronegative blood donors. The median CD4⁺ LC was 726 cells/mm³ (for females, 782 cells/mm³; for males, 698 cells/mm³). The median CD8⁺ LC was 488 cells/mm³ (for females, 494 cells/mm³; for males, 485 cells/mm³). The median CD4⁺-to-CD8⁺ ratio was 1.57 (for females, 1.66; for males, 1.51). Our findings of low CD4⁺ LCs among HIV-negative adults in Botswana are significant and have important implications for the management of HIV disease in the population of this sub-Saharan African country.     

Association of CD4+ CD25+ T Cells with Prevention of Severe Destructive Arthritis in Borrelia burgdorferi-Vaccinated and Challenged Gamma Interferon-Deficient Mice Treated with Anti-Interleukin-17 Antibody

CD4⁺ CD25⁺ T cells are a population of regulatory T cells responsible for active suppression of autoimmunity. Specifically, CD4⁺ CD25⁺ T cells have been shown to prevent insulin-dependent diabetes mellitus, inflammatory bowel disease, and pancreatitis. Here, we present evidence that CD4⁺ CD25⁺ T cells also play a major role in controlling the severity of arthritis detected in Borrelia burgdorferi-vaccinated gamma interferon-deficient (IFN-γ°) C57BL/6 mice challenged with the Lyme spirochete. When B. burgdorferi-vaccinated and challenged IFN-γ° mice were treated with anti-interleukin-17 (IL-17) antibody, the number of CD4⁺ CD25⁺ T cells increased in the local lymph nodes. Furthermore, histopathologic examination showed the mice to be free of destructive arthritis. When these anti-IL-17-treated B. burgdorferi-vaccinated and challenged mice were also administered anti-CD25 antibody, the number of CD4⁺ CD25⁺ T cells in the local lymph nodes decreased. More importantly, severe destructive arthropathy was induced. In addition, delayed administration of anti-CD25 antibody decreased the severity of the arthritis. These results suggest that CD4⁺ CD25⁺ T cells are involved in regulation of a severe destructive arthritis induced with an experimental model of vaccination and challenge with B. burgdorferi.     

γδ+ T Cells Preferentially Respond to Live Rather than Killed Malaria Parasites

We have compared the in vitro responses of peripheral blood T cells from malaria-unexposed donors to live Plasmodium falciparum schizonts, freeze-thawed schizont extracts (P. falciparum schizont extracts [PfSE]), and parasite culture supernatants. We show that the cells responding to PfSE and parasite culture supernatants are predominantly CD4⁺ TCRαβ⁺ while in the presence of live schizonts there is an additional activation of TCRγδ⁺ cells. Activation of TCRγδ⁺ cells in response to PfSE was seen only when irradiated autologous feeder cells or recombinant interleukin-2 (IL-2) was added to the cultures. Live schizonts but not PfSE induced significant IL-2 production in vitro in the first 5 days after stimulation, suggesting that induction of early IL-2 by live parasites may contribute to the marked activation of the TCRγδ⁺ population.     

Foxp3+ T-regulatory cells in Sjogren's syndrome: correlation with the grade of the autoimmune lesion and certain adverse prognostic factors

Sjögren’s syndrome (SS) is a chronic autoimmune exocrinopathy associated with variable lymphocytic infiltration of the affected organs (primarily salivary and lacrimal glands) and broad clinical manifestations, including lymphoma development. To investigate the potential implication of Foxp3⁺ T-regulatory cells in the regulation of SS inflammatory responses, we studied their incidence in the minor salivary glands (MSGs) and their relationship with histopathological and clinical disease parameters. Similar percentages of infiltrating Foxp3⁺ cells were observed in the MSG lesions of all SS patients (n = 30) and non-SS sialadenitis controls (n = 7). Foxp3⁺ cells were not detected in sicca-complaining controls with negative biopsy (n = 6). In SS patients, Foxp3⁺ cell frequency varied according to lesion severity, with the highest and lowest frequencies obtained in intermediate and mild MSG lesions, respectively. In the peripheral blood of these patients, reverse distribution of Foxp3⁺ cells was observed. Furthermore, the frequency of Foxp3⁺ cells in the MSG lesions and peripheral blood was negatively associated (r = −0.6679, P = 0.0065). MSG-infiltrating Foxp3⁺ cells were found to positively correlate with biopsy focus score (P = 0.05), infiltrating mononuclear cells, dendritic cells, and macrophages (P ≤ 0.024 each), and serum C4 levels (P = 0.0328), whereas lower Foxp3⁺ cell incidence correlated with adverse predictors for lymphoma development, such as the presence of C4 hypocomplementemia (P = 0.012) and SG enlargement (tendency, P = 0.067). Our findings suggest that the Foxp3⁺ T-regulatory cell frequency in the MSG lesions of SS patients correlates with inflammation grade and certain risk factors for lymphoma development.     

Most CD56+ Intestinal Lymphomas Are CD8+CD5− T-Cell Lymphomas of Monomorphic Small to Medium Size Histology

The expression of the natural killer (NK) cell marker CD56 has been reported to occur in NK cell lymphomas/leukemias and a small group of peripheral T-cell lymphomas but has not been studied extensively in primary intestinal non-B-cell lymphomas. Normal human jejunal intraepithelial lymphocytes (IELs) are mainly T-cell receptor (TCR)-αβ⁺CD3⁺CD8⁺CD5^low and include an ~15% fraction of CD56⁺ cells that could be the cells of origin for CD56⁺ intestinal T-cell lymphoma (ITL). To test this hypothesis, 70 cases diagnosed as ITL were immunophenotyped, and 15 CD56⁺ cases (21%) were identified. The majority of the CD56⁺ lymphomas was of monomorphic small to medium-sized histology, shared the common phenotype βF1^±CD3ε/cyt⁺CD8⁺CD4⁻CD5⁻CD57⁻TIA-1⁺ and had clonally rearranged TCR γ-chain genes. In contrast, the CD56⁻ lymphomas were mainly composed of pleomorphic medium and large cells or had a morphology most consistent with anaplastic large-cell lymphoma and were mostly CD8⁻. These findings suggest that the majority of CD56⁺ intestinal lymphomas are morphologically and phenotypically distinct T-cell lymphomas most likely derived from activated cytotoxic CD56⁺CD8⁺ IELs. Some overlapping histological and clinical features between CD56⁺ and CD56⁻ ITLs indicate that the former belong to the clinicopathological entity of ITL. The consistent expression of cytotoxic-granule-associated proteins introduces ITL (both CD56⁺ and CD56⁻) into the growing family of usually aggressive extranodal lymphomas of cytotoxic T-cell and NK-cell derivation. In contrast to putative NK-cell lymphoma of the sinonasal region, intestinal NK-cell lymphoma seems to be very rare.     

Functional and Morphological Development of Lymphoid Tissues and Immune Regulatory and Effector Function in Rhesus Monkeys: Cytokine-Secreting Cells, Immunoglobulin-Secreting Cells, and CD5+ B-1 Cells Appear Early in Fetal Development

Little is known regarding the timing of immune ontogeny and effector function in fetal humans and nonhuman primates. We studied the organization of lymphocyte and antigen-presenting cell populations in developing lymphoid tissues of rhesus monkey fetuses during the second and third trimesters (65 to 145 days of gestation; term = 165 days). Immunoglobulin-secreting and cytokine-secreting cells were detected at day 80. The thymus, spleen, lymph nodes, and intestinal mucosa were examined for cells expressing CD3, CD5, CD20, CD68, p55, and HLA-DR. In the spleens of 65-day-old fetuses (early second trimester), the overwhelming majority of total lymphocytes were CD5⁺ CD20⁺ B-1 cells. The remaining lymphocytes were CD3⁺ T cells. By day 80, splenic B and T cells were equal in number. Intraepithelial CD3⁺ CD5⁻ T cells and lamina propria CD20⁺ CD5⁺ B cells were present in the intestines of 65-day-old fetuses. By day 80, numerous CD20⁺ CD5⁺ B cells were present in the jejunums and colons and early lymphocyte aggregate formation was evident. The spleens of 80- to 145-day-old fetuses contained immunoglobulin M (IgM)-secreting cells, while IgA-, IgG-, interleukin-6-, and gamma interferon-secreting cells were numerous in the spleens and colons. Thus, by the second trimester, the lymphoid tissues of the rhesus monkey fetus have a complete repertoire of properly organized antigen-presenting cells, T cells, and B cells.     

Processing of Mycobacterium tuberculosis Bacilli by Human Monocytes for CD4+ αβ and γδ T Cells: Role of Particulate Antigen

Mycobacterium tuberculosis readily activates both CD4⁺ and Vδ2⁺ γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and γδ T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2⁺ γδ T cells. For γδ T cells, recognition of M. tuberculosis-infected monocytes was dependent on Vδ2⁺ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4⁺ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vδ2⁺ γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4⁺ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4⁺ and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4⁺ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4⁺ and γδ T cells.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is spread readily from person to person by inhalation of aerosolized mycobacteria (8). A hallmark of M. tuberculosis infection is the ability of most healthy individuals to control the infection by mounting an acquired immune response, in which antigen-specific T cells and mononuclear phagocytes arrest the growth of M. tuberculosis bacilli and maintain control over dormant bacilli within granulomas (reviewed in reference 25). This protective cellular immune response results in conversion of the tuberculin skin test from negative to positive and probably in increased resistance to reinfection with tubercle bacilli.CD4⁺ αβ-T-cell-receptor (αβ TCR)-bearing T cells (CD4⁺ T cells) are readily activated by mycobacterial antigens and have a dominant role in the protective immune response to M. tuberculosis in humans (2, 34). These CD4⁺ T cells not only secrete cytokines but also serve directly as cytotoxic effector cells against M. tuberculosis-infected macrophages (6). In addition to CD4⁺ T cells, M. tuberculosis antigens activate other human T-cell subsets such as γδ TCR⁺ T cells (γδ T cells) (15, 16, 18). Vδ2⁺ and Vγ9⁺ γδ T cells are particularly responsive to live M. tuberculosis (15). A role for both γδ and CD4⁺ T cells in protective immunity to acute M. tuberculosis infection has been demonstrated in murine models (20, 21, 26, 27). A recent study of humans suggests that Vγ9⁺ and Vδ2⁺ γδ T-cell numbers and function are reduced in tuberculosis patients (23).Functional comparisons of human CD4⁺ and γδ T-cell responses of healthy tuberculin-positive persons demonstrate that both T-cell subsets have similar cytotoxic effector functions for M. tuberculosis-infected monocytes and produce large amounts of gamma interferon (IFN-γ), with γδ T cells being slightly more efficient producers of IFN-γ than CD4⁺ T cells (37). Despite similarities in function, these two T-cell subsets differ in the mycobacterial antigens recognized by their TCRs and the manners in which antigens are presented to them by M. tuberculosis-infected mononuclear phagocytes. CD4⁺ T cells recognize a wide diversity of mycobacterial peptides in the context of class II major histocompatibility complex (MHC) molecules, which include secreted as well as somatic antigens (6, 13, 33, 37). In contrast, Vγ9⁺ and Vδ2⁺ γδ T cells, the dominant γδ TCR subsets activated by M. tuberculosis, recognize mycobacterial antigens in a non-MHC-restricted manner and the repertoire of antigens includes small phosphate-containing antigens such as TUBag’s (5, 9, 19, 22, 29, 36).Both blood monocytes and alveolar macrophages infected with M. tuberculosis are efficient antigen-presenting cells for mycobacterial antigen-specific CD4⁺ and γδ T cells (1, 5). However, little is known about how M. tuberculosis-infected mononuclear phagocytes process antigens for these two T-cell subsets. M. tuberculosis bacilli are taken up by mononuclear phagocytes through a variety of surface receptors, including complement receptor 4, mannose receptor, and complement receptor 3 (17, 31, 32). Within mononuclear phagocytes, the mycobacteria reside within phagosomes and modulate the phagosome by preventing fusion with acidic lysosomal compartments (7). Although the vacuolar membranes surrounding the phagosome acquire endosomal markers, the vesicular proton ATPase is actively excluded, resulting in an elevated pH of 6.3 to 6.5 compared to the normal lysosomal pH of 4.5 (7, 35). The elevated pH in the phagosome does not appear to inhibit the ability of mycobacterial antigens to be processed and presented to CD4⁺ and Vδ2⁺ γδ T cells. This study was undertaken to gain insight into the mechanisms used by monocytes infected with live M. tuberculosis bacilli to process mycobacterial antigens for presentation to both CD4⁺ and γδ T cells.     

Regulatory T Cells Modulate Staphylococcal Enterotoxin B-Induced Effector T-Cell Activation and Acceleration of Colitis

Oral administration of bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) activates mucosal T cells but does not cause mucosal inflammation. We examined the effect of oral SEB on the development of mucosal inflammation in mice in the absence of regulatory T (Treg) cells. SCID mice were fed SEB 3 and 7 days after reconstitution with CD4⁺ CD45RB^high or CD4⁺ CD45RB^high plus CD4⁺ CD45RB^low T cells. Mice were sacrificed at different time points to examine changes in tissue damage and in T-cell phenotypes. Feeding SEB failed to produce any clinical effect on SCID mice reconstituted with CD4⁺ CD45RB^high and CD4⁺ CD45RB^low T cells, but feeding SEB accelerated the development of colitis in SCID mice reconstituted with CD4⁺ CD45RB^high T cells alone. The latter was associated with an increase in the number of CD4⁺ Vβ8⁺ T cells expressing CD69 and a significantly lower number of CD4⁺ CD25⁺ Foxp3⁺ T cells. These changes were not observed in SCID mice reconstituted with both CD45RB^high and CD45RB^low T cells. In addition, SEB impaired the development of Treg cells in the SCID mice reconstituted with CD4⁺ CD45RB^high T cells alone but had no direct effect on Treg cells. In the absence of Treg cells, feeding SEB induced activation of mucosal T cells and accelerated the development of colitis. This suggests that Treg cells prevent SEB-induced mucosal inflammation through modulation of SEB-induced T-cell activation.     

Expanded CD14+ CD16+ Monocyte Subpopulation in Patients with Acute and Chronic Infections Undergoing Hemodialysis

Infections are frequent complications in end-stage renal failure patients undergoing hemodialysis (HD), and peripheral blood monocytes are important cells in host defense against infections. The majority of circulating monocytes express high levels of lipopolysaccharide receptor antigen CD14 and are negative for the immunoglobulin Fcγ receptor type III (CD16). We studied the occurrence of a minor subpopulation coexpressing low levels of CD14 together with CD16 in HD patients. In healthy controls CD14⁺ CD16⁺ monocytes account for 8% ± 4% of CD14⁺ monocytes, with an absolute number of 29 ± 14 cells/μl. In stable HD patients the CD14⁺ CD16⁺ subpopulation was significantly elevated (14% ± 3%, or 66 ± 28 cells/μl), while the number of CD14⁺⁺ monocytes (monocytes strongly positive for CD14) remained constant. In HD patients suffering from chronic infections a further rise in CD14⁺ CD16⁺ monocytes was observed (128 ± 71 cells/μl; P < 0.01) such that this subpopulation constituted 24% of all blood monocytes. In contrast, numbers of CD14⁺⁺ cells did not change compared to those for stable HD patients, indicating that the CD14⁺ CD16⁺ monocyte subpopulation was selectively expanded. During acute infections the CD14⁺ CD16⁺ cell subpopulation always expanded. A whole-blood assay revealed that CD14⁺ CD16⁺ monocytes exhibited a higher phagocytosis rate for Escherichia coli bacteria than CD14⁺⁺ monocytes, underlining their role during host defense. In addition, CD14⁺ CD16⁺ monocytes expressed higher levels of major histocompatibility complex (MHC) class II antigens (HLA-DR, -DP, and -DQ) and equal amounts of MHC class I antigens (HLA-ABC). Thus, CD14⁺ CD16⁺ cells constitute a potent phagocytosing and antigen-presenting monocyte subpopulation, which is expanded during acute and chronic infections commonly observed in chronic HD patients.

Peripheral blood monocytes are members of the mononuclear phagocytic system, which plays a central role in immunoregulation and host defense against immunopathogenic organisms (7). Monocytes are activated through molecular signals provided by structures of the infective organisms (8, 27, 28, 34, 35) or inflammatory mediators and chemotactic factors released by other cells during the infective challenge (22, 44, 47). However, blood monocytes represent a heterogeneous cell population and can be distinguished by variations in morphology (38, 58), membrane antigen expression (39), and release of inflammatory mediators (12, 25, 41).While the lipopolysaccharide (LPS) receptor antigen CD14 is expressed by nearly all circulating peripheral blood monocytes, monocytes differ markedly in cell surface CD14 density as well as in the expression of immunoglobulin Fcγ receptors (53, 67). The majority of monocytes strongly positive for CD14 (CD14⁺⁺) express Fcγ receptor I (CD64) and Fcγ receptor II (CD32) and are negative for Fcγ receptor III (CD16) (18). Only a small population was identified by the absence of Fcγ receptors (63). Nevertheless, a subset of monocytes characterized by low-level expression of CD14 and expression of the CD16 antigen has also been described (40). In healthy subjects these CD14⁺ CD16⁺ cells account for about 10% of all monocytes and are thought to be more mature cells than the regular CD14⁺⁺ monocytes, as they exhibit features of tissue macrophages (66). In various infectious or inflammatory diseases such as AIDS and asthma the CD14⁺ CD16⁺ monocyte subpopulation is markedly expanded (36, 43, 50). A more than 10-fold increase of these cells during septicemia was demonstrated, and CD14⁺ CD16⁺ cells become the predominant type of monocytes in some septic patients (14).Patients with end-stage renal failure undergoing chronic hemodialysis (HD) show an impaired immune response (10) with a high prevalence of infectious complications (17). Most of these infections are of bacterial origin, representing a major cause of morbidity and mortality in chronic HD patients (24). Furthermore, acute or chronic inflammatory processes, among them pneumonia and vascular access site infections, are common hazards in uremic patients undergoing chronic regular HD. Despite some data on the functional abnormalities of polymorphonuclear leukocytes in uremia (19), little information exists on the level of monocytes and their subsets in maintenance dialysis patients.In an effort to further understand the importance of the distinct monocyte population expressing Fcγ receptor type III, we determined the levels of these cells in patients with end-stage renal failure undergoing chronic HD. This allowed the level of CD14⁺ CD16⁺ cells to be compared to that of CD14⁺⁺ cells and the total monocyte count in whole blood. To investigate the proinflammatory role of CD14⁺ CD16⁺ monocytes, stable patients as well as patients with acute or chronic signs of infections or inflammatory processes were studied. Furthermore, we analyzed cell surface HLA expression of CD14⁺ CD16⁺ monocytes by immunophenotyping and compared their phagocytic competence with that of regular CD14⁺⁺ blood monocytes.     

Dendritic cell-based immunotherapy for colon cancer using an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope from tumor-associated antigen 90K

Tumor-associated antigen 90K is implicated in cell–cell and cell-extracellular matrix adhesion through its interaction with galectin-3 and integrin-β1 and is highly expressed in malignant tissues, making it a novel target for the development of new immunotherapies. We investigated a potential immunotherapy treatment for colon cancer using 90K-specific cytotoxic T lymphocytes induced by autologous dendritic cells and pulsed with 90K peptides. We selected three peptides (90K₃₅₁, 90K₅ and 90K₅₂₃) that bind to HLA-A*0201 molecules on the basis of their binding affinity, as determined by a peptide-T2 binding assay. Dendritic cells pulsed with 90K peptides resulted in the efficient generation of mature dendritic cells and exhibited enhanced T-cell stimulation and polarization of naive T cells toward Th1. Dendritic cells pulsed with 90K peptides generated potent cytotoxic T-lymphocytes that lysed T2 cells loaded with each 90K peptide, and 90K⁺/HLA-A2⁺ colon cancer cell lines, including HCT116 and SW480, in a dose-dependent and HLA-A*0201-restricted manner. No killing was observed in 90K⁺/HLA-A2⁻ DLD1 or 90K⁻/HLA-A2⁻ K562 cells. Therefore, we believe that cytotoxic T-lymphocytes stimulated by 90K peptide-pulsed dendritic cells naturally recognize the 90K peptide presented by colon cancer cells in the context of HLA-A2, and kill 90K-positive tumor cells. Dendritic cells pulsed with 90K peptides led to the induction of granzyme B and perforin positive CD8⁺ T cells against HCT116 and SW480 cells, but not DLD1 cells. In conclusion, 90K-specific cytotoxic T lymphocytes, generated by stimulating T cells with 90K peptide-pulsed dendritic cells, could be useful effector cells for the immunotherapy treatment of colon cancer.     

TLR4 inactivation protects from graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation. To clarify the role of Toll-like receptor 4 (TLR4), which is a major receptor for bacterial lipopolysaccharides (LPS), in the development of acute GVHD, we used a TLR4-knockout (TLR4^−/−) mouse GVHD model and analyzed the underlying immunological mechanisms. When TLR4^−/− mice were used as bone marrow and splenocyte cell graft donors or recipients, GVHD symptom occurrence and mortality were delayed compared to wild-type (TLR4^+/+) mice. In addition, histopathological analyses revealed that in TLR4^−/−→BALB/c chimeras, liver and small intestine tissue damage was reduced with minimal lymphocytic infiltration. In contrast to TLR4^+/+, TLR4^−/− mice dendritic cells did not express CD80, CD86, CD40, MHC-II or IL-12 during LPS induction and remained in an immature state. Furthermore, the ability of TLR4^−/− mice spleen dendritic cells to promote allogeneic T-cell proliferation and, in particular, T-helper cell 1 (Th1) development was obviously attenuated compared with TLR4^+/+ mice dendritic cells, and the levels of interferon-γ (IFN-γ) and IL-10, Th2-cell specific cytokines, were significantly higher in the serum of TLR4^−/−→BALB/c than in TLR4^+/+→BALB/c chimeric mice. Overall, our data revealed that TLR4 may play a role in the pathogenesis of GVHD and that targeted TLR4 gene therapy might provide a new treatment approach to reduce the risk of GVHD.