Gammadelta T cell receptor repertoire in blood and colonic mucosa of rhesus macaques

Although their precise roles are not well defined, gammadelta T lymphocytes are recognized as regular components of immune responses. These cells express a limited T cell receptor repertoire and they can be stimulated by soluble ligands without conventional processing and presentation by major histocompatibility antigens. Progress in this area has been limited by the substantial differences between murine and human gammadelta T cells and the lack of knowledge about these cells in nonhuman primates. We used molecular analysis of T cell receptor diversity to characterize gammadelta T cell populations from peripheral blood and colon of rhesus macaques (Macaca mulatta). The gammadelta T cell receptor diversity was limited and distinct for these tissue compartments, particularly in the TCRGV2 family. Furthermore, the TCRDV1 + subset of peripheral blood gammadelta T cells showed signs of progressive oligoclonalization as a function of age. Similar observations have been reported for human tissue samples and our results validate rhesus macaques as an appropriate animal model for studying primate gammadelta T cell populations.     

Vγ9/Vδ2 T cell activation induced by bacterial low molecular mass compounds depends on the 1-deoxy-d-xylulose 5-phosphate pathway of isoprenoid biosynthesis

Isopentenyl diphosphate (IPP), an important precursor of isoprenoid biosynthesis in prokaryotic and eukaryotic organisms, has been shown to activate Vgamma9/Vdelta2 T cells, the major subset of human gammadelta T cells. The biosynthesis of IPP has been first described as the acetate/mevalonate pathway. Recently, 1-deoxy-D-xylulose 5-phosphate (DOXP) and 2-C-methyl-D-erythritol 4-phosphate have been shown to be key metabolites in the DOXP pathway also leading to the formation of IPP in some eubacteria such as Escherichia coli. Here we report that the low molecular mass fraction of extracts from bacteria using the DOXP pathway induces Vgamma9/Vdelta2 T cell activation, while analogous preparations from bacteria using the classical mevalonate pathway fail to do so. Addition of 1-deoxy-D-xylulose potentiates the ability of E. coli extracts to activate Vgamma9/Vdelta2 T cells. As the amounts of IPP present in the bacterial preparations are not sufficient to induce significant Vgamma9/Vdelta2 T cell activation, our data suggest that compounds other than IPP associated with the DOXP pathway are responsible for Vgamma9/Vdelta2 T cell activation.     

Low-intensity workers: lessons learned from supervising primary care mental health workers and dilemmas associated with such roles

The UK government's latest primary care mental health initiative, Improving Access to Psychological Therapies (IAPT), seeks to achieve its aims partly through the extensive use of low-intensity workers (LIWs). As clinical supervisors to teams of primary care mental health workers (PCMHWs) already offering brief, evidence-based interventions in primary care mental health services, we draw on the growing body of literature and our own experience to discuss dilemmas associated with the roles of such workers. These concern clinical governance, training, supervision and integration into existing services. We discuss how IAPT service characteristics may provide solutions to some of these dilemmas. We argue that it is unlikely they will be completely resolved and that IAPT services, in addressing future challenges, could benefit from lessons learned from the PCMHW role.     

An increased number of circulating γ/δ TCR + T cells in patients with chronic viral hepatitis

Abstract There is evidence that γ/δ TCR + T cells are specialized in recognizing different antigens, but their immunologic role as a second TCR is still unclear. The aim of this study was to investigate the percentage and absolute numbers of circulating γ/δ TCR + T cells in patients with chronic viral hepatitis (CVH) and to compare with HBsAg⁺, HCV healthy carriers and healthy subjects. Forty nine patients with CVH-24 with chronic active (CAH) and 25 with chronic persistent hepatitis (CPH)-, 21 HBsAg⁺, 20 HCV asymptomatic carriers and 20 healthy subjects were enrolled in the study. Lymphocyte subsets were determined after incubation with monoclonal antibodies to T total (CD5) and T γ/δ cells (γ/δ-1) using immunofluorescence microscopy. An increased number of circulating γ/δ TCR + T cells was found in patients with CVH in comparison with asymptomatic carriers and normal controls: this increase was more profound in patients with CAH, compared to CPH patients. These results indicate a correlation between circulating γ/δ TCR + T cells in CVH patients and activity and chronicity of the disease.     

Identification of three new bovine T-cell receptor delta variable gene subgroups expressed by peripheral blood T cells

To understand the biology of γδ T cells in ruminants, it is necessary to have a comprehensive picture of γδ T-cell receptor gene diversity and expression. In this study, three new subgroups of bovine T-cell receptor δ (TRD) variable genes were identified by RT-PCR and sequencing and homology with TRDV genes from other mammals determined. Previously unidentified TRDV subgroup genes described in this study include the bovine homologues of ovine TRDV2, TRDV3, and TRDV4 which were named accordingly. TRDV2 subgroup has two genes (TRDV2-1 and TRDV2-2) while we found the previously identified TRDV1 has at least eight genes corresponding to separate genomic sequences. Nucleotide and amino acid sequences for particular gene subgroups between cattle and sheep were more than 87% identical but identities among TRDV subgroups within a species were much less, with bovine TRDV4 having <45% identity to the other three bovine TRDV gene subgroups. Analysis of circulating bovine γδ T cells revealed that genes from all four TRDV subgroups were expressed in combination with TRDJ1, TRDJ3, and TRDC, although TRDV4 was the least represented, and all displayed a variety of CDR3 junctional lengths. Finally, some genes within the TRDV1, TRDV2, and TRDV3 subgroups recombined with TRAV incorporating TRAJs, suggesting dual use.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database under the accession numbers DQ275147, DQ275148, DQ275149, and DQ280318.     

Identification of a domain in the delta subunit (S238-V264) of the alpha4beta3delta GABAA receptor that confers high agonist sensitivity

We have expressed the alpha4beta3delta and alpha4beta3gamma2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the alpha4beta3delta receptor (EC50 approximately 1.4 micromol/L) than of the alpha4beta3gamma2L subtype (EC50 approximately 27.6 micromol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric delta/gamma2L subunits with alpha4 and beta3 subunits. A stretch of amino acids in the delta subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the delta and gamma2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.     

T cell up-regulation of CD127 is associated with reductions in the homeostatic set point of the peripheral T cell pool during malnourishment

The following study was undertaken to better understand the mechanisms that relate the homeostatic set point of the peripheral T cell population to energy availability in mice. We report that the total number of peripheral naïve and memory CD4+ and CD8+T cells notably declined after one week of malnourishment, a time period too short to be entirely due to malnutrition-induced thymic involution. Peripheral malnourished T cells expressed higher levels of the IL-7 receptor component, CD127, and were less sensitive to death-by-neglect as compared to control T cells. Overall levels of IL-7 were similar in malnourished and control mice. Adoptive transfer studies revealed that CD127 expression did not correlate with increased survival in vivo and that all naïve CD8+T cells upregulated CD127, regardless of initial expression levels. Corticosterone levels were elevated in malnourished mice and this correlated in time with peripheral T cell up-regulation of CD127 and the diminishment of the peripheral T cell pool. Overall, these data suggest a model in which CD127 levels are up-regulated quickly during malnourishment, thereby increasing the scavenge rate of IL-7, and providing a mechanism to quickly adjust the total number of T cells during malnutrition.     

Perinatal supplementation with thymoquinone improves diabetic complications and T cell immune responses in rat offspring

Background

Epidemiological studies have shown that the offspring of mothers who experience diabetes mellitus during pregnancy are seven times more likely to develop health complications later in life compared to offspring born to nondiabetic mothers.

Aim of the study

We investigated whether supplementation with a natural antioxidant (thymoquinone; TQ) in female rats with streptozotocin (STZ)-induced gestational diabetes (GD) improved diabetic complications and T cell immune responses in their offspring.

Methods

Three groups of female rats were tested: nondiabetics, diabetics treated with TQ during pregnancy and lactation periods and diabetics that were not treated with TQ (n = 10 female rats in each group).

Results

Our data demonstrated a significant decrease in the numbers of neonates born to diabetic rats compared with those born to control rats. GD led to macrosomic pups with several postpartum complications, such as a significant increase in plasma levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α (but not of IL-10); a marked decrease in the plasma level of IL-2; a marked reduction in the proliferative capacity of superantigen (SEB)-stimulated T-lymphocytes; and an obvious reduction in the number of circulating and thymus homing T cells. TQ supplementation of diabetic mothers during pregnancy and lactation periods had an obvious and significant effect on the number and mean body weight of neonates. Furthermore, TQ significantly restored the IL-2 level and T cell proliferation and subsequently rescued both circulating and thymus homing T cells in the offspring.

Conclusions

Our data suggest that nutritional supplementation of GD mothers with the natural antioxidant TQ during pregnancy and lactation periods improves diabetic complications and maintains an efficient T cell immune response in their offspring, providing a protective effect in later life.     

Therapeutic efficacy of antitumor dendritic cell vaccinations correlates with persistent Th1 responses,high intratumor CD8+ T cell recruitment and low relative regulatory T cell infiltration

Despite the increasing number of immunotherapeutic strategies for the treatment of cancer, most approaches have failed to correlate the induction of an anti-tumor immune response with therapeutic efficacy. We therefore took advantage of a successful vaccination strategy-combining dendritic cells and irradiated GM-CSF secreting tumor cells-to compare the immune response induced against 9L gliosarcoma tumors in cured rats versus those with progressively growing tumors. At the systemic level, the tumor specific cytotoxic responses were quite heterogeneous in uncured vaccinated rats, and were surprisingly often high in animals with rapidly-growing tumors. IFN-gamma secretion by activated splenic T cells was more discriminative as the CD4+ T cell-mediated production was weak in uncured rats whereas high in cured ones. At the tumor level, regressing tumors were strongly infiltrated by CD8+ T cells, which demonstrated lytic capacities as high as their splenic counterparts. In contrast, progressing tumors were weakly infiltrated by T cells showing impaired cytotoxic activities. Proportionately to the T cell infiltrate, the expression of Foxp3 was increased in progressive tumors suggesting inhibition by regulatory T cells. In conclusion, the main difference between cured and uncured vaccinated animals does not depend directly upon the induction of systemic cytotoxic responses. Rather the persistence of higher CD4+ Th1 responses, a high intratumoral recruitment of functional CD8+ T cells, and a low proportion of regulatory T cells correlate with tumor rejection.     

Molecular typing of Candida albicans isolates from patients and health care workers in a neonatal intensive care unit

Aims: The aim of this study was to investigate the genetic relatedness between Candida albicans isolates and to assess their nosocomial origin and the likeliness of cross‐transmission between health care workers (HCWs) and hospitalized neonates in a neonatal intensive care unit (NICU). Methods: We retrospectively analysed 82 isolates obtained from 40 neonates and seven isolates from onychomycosis of the fingers of five HCWs in a Tunisian NICU by using pulsed‐field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis with CA1 and CA2 as primers. Results: In RAPD analysis, the discriminatory power (DP) of CA1 and CA2 primers was 0·86 and 0·81, respectively. A higher DP was achieved by combining patterns generated by both primers (0·92), while PFGE karyotyping exhibited the lowest DP (0·62). The RAPD‐CA1/CA2 analysis revealed that 65·8% of isolates obtained from neonates derived from a limited number (6) of groups of genetically identical strains, that five temporal clusterings occurred during the study period and that three HCWs’ isolates and 11 isolates obtained from six neonates were identical. Conclusions: These findings argue for the nosocomial transmission of C. albicans in our NICU and for the transfer of strains from HCWs to patients. Significance and Impact of the Study: Identification of relatedness between Candida species obtained from neonates and health care workers by using molecular techniques with high discriminatory power is essential for setting up specific control measures in order to reduce the incidence of nosocomial candidiasis.     

T cell responses in melanoma patients after vaccination with tumor-mRNA transfected dendritic cells

We have developed an individualized melanoma vaccine based on autologous dendritic cells (DCs) transfected with autologous tumor-mRNA. The vaccine targets the unique spectrum of tumor antigens in each patient and may recruit multiple T cell clones. In a recent phase I/II trial, we demonstrated T cell responses against vaccine antigens in 9/19 patients evaluable by T cell assays. Here, we report a follow-up study that was conducted to characterize interesting T cell responses and to investigate the effects of long-term booster vaccination. Two patients were selected for continued vaccine therapy. The clinical follow-up suggested a favorable clinical development in both patients. The immunological data (T cell proliferation/IFNgamma ELISPOT/Bioplex cytokine assays) indicated sustained T cell responses and suggested an enhancing effect of booster vaccinations. Both CD4(+) and CD8(+) T cell responses were demonstrated. From post-vaccination samples, we generated 39 T cell clones that responded specifically to stimulation by mRNA-transfected DCs and 12 clones that responded to mock-transfected DCs. These data clearly indicate a two-component vaccine response, against transfected and non-transfected antigens. T cell receptor (TCR) clonotype mapping, performed on 11 tDC-specific clones, demonstrated that 10/11 clones had different TCRs. The results thus indicate a broad spectrum T cell response against antigens encoded by the transfected tumor-mRNA. We generally observed mixed Th1/Th2 cytokine profiles, even in T cell clones that were confirmed to be derived from a single cell. This finding suggests that cytokine patterns after cancer vaccination may be more complex than indicated by the classic Th1/Th2 dichotomy.     

Cytotoxic T cell responses to Streptococcus are associated with improved prognosis of oral squamous cell carcinoma

Several species of Streptococcus, such as S. salivarius, S. mitis, and S. anginosus, are found to extensively colonize the oral cavity and the upper respiratory tract, and have been shown to increase in patients with oral squamous cell carcinoma (OSCC). Accumulating evidence have revealed that commensal bacteria are involved in antitumor immunity via T cell-mediated mechanisms, but the role of Streptococcus enrichment in OSCC is yet unclear. In this study, we stimulated peripheral blood mononuclear cells from non-cancer controls (NCs) and OSCC patients with S. salivarius, S. mitis, and S. anginosus. We observed that compared to NC subjects, OSCC patients at earlier stages had higher frequencies of granzyme B-expressing CD8 T cells for all Streptococcus species tested, while OSCC patients at more advanced stages had higher frequencies of granzyme B-expressing CD8 T cells for S. anginosus but not other Streptococcus species. In OSCC patients, the Streptococcus-reactive CD8 T cells presented significantly lower levels of PD-1 and TIM-3 expression than Streptococcus-nonreactive CD8 T cells. The clinical outcomes of OSCC patients in our cohort were tracked for 24 months after the resection of the primary tumor. In patients that did not present tumor recurrence, the frequencies of S. salivarius-reactive and S. mitis-reactive CD8 T cells were significantly higher than that in patients that developed recurrent tumor. Furthermore, in patients with tumor recurrence, the duration between primary tumor resection and tumor recurrence was positively associated with the frequencies of S. salivarius-reactive and S. anginosus-reactive CD8 T cells. Together, we demonstrated that Streptococcus-reactive CD8 T cell responses might contribute to antitumor immunity in OSCC patients.     

Induction of immunity against experimental tuberculosis with mycobacterial mannophosphoinositides encapsulated in liposomes containing lipid A

Abstract Mycobacterial mannophosphoinositides (PIMs) encapsulated in liposomes made of egg phosphatidylcholine (EPC) and cholesterol (CH) (2:1.5 molar ratio) were able to induce humoral and delayed type hypersensitivity (DTH, foot-pad swelling reaction) responses in mice without the help of any carrier protein. Animals immunized with this glycophospholipid antigen demonstrated enhanced percent survival on intravenous challenge with virulent M. tuberculosis . On fractionation of PIMs, pentamannophosphoinositide (PIM₅) was found to induce higher antibody and DTH reaction and proved to be more immunoprotective than other fractions. Inclusion of lipid A as immunomodulator in liposomes containing PIMs or PIM₅ resulted in a significantly increased immune response. Further, mice immunized with PIMs or PIM₅ in lipid A-containing liposomes exhibited decreased mortality on challenge with M. tuberculosis H₃₇Rv, which was comparable to BCG vaccinated animals.     

Immunoreactivity of five antigens of Mycobacterium tuberculosis in patients attending a public health care facility in an area with high endemicity for TB

The objective of this study was to evaluate people attending a primary health clinic in Rio de Janeiro, Brazil for immunoreactivity to five Mycobacterium tuberculosis antigens, as these antigens are markers of immune response and factors associated with active TB. The serum antibody titers of different categories of patients (defined by microbiological and radiological characteristics and by response to therapy on follow‐up) to 38 kDa, 16 kDa, MPT64, ESAT‐6 and MT10.3 antigens were determined blind with ELISA. Positive tests to each antigen were defined with ROC analysis. OR were calculated for factors associated with humoral response in patients with active TB. A total of 201 patients underwent serological testing. Patients with confirmed active TB responded more frequently to MPT64 (44%), 16 kDa (37.7%) and 38 kDa (36.1%). ESAT‐6 and MT10.3 were also able to distinguish people in TB groups from controls. TB infected subjects responded less frequently to ESAT‐6 and MT10.3 (3.7% and 11%, respectively). Sensitivity and specificity to all antigens combined were 58.4% and 60.7%, respectively. Reactivity to 38 kDa and to MPT64 was more likely among alcohol users OR 2.61 (95%CI;1.05–6.94) and OR 3.27 (95%CI;1.33–8.15), respectively. 16 kDa antigen elicited a more protective response among smokers, OR 0.29 (95%CI; 0.10–0.83). It was concluded that reactivity to all antigens tested represented markers of active disease. ESAT‐6 and MT10.3 could not be identified as markers of TB infection in this community. Sensitivity was higher to all antigens combined, but at a cost of lower specificity. Interestingly, among factors associated with positive immunoreactivity, alcohol use and smoking seem to polarize the humoral response in different directions. This finding deserves further investigation.     

Cognate T and B cell interaction and association of follicular helper T cells with B cell responses in Vibrio cholerae O1 infected Bangladeshi adults

Vibrio cholerae O1 can cause life threatening diarrheal disease if left untreated. T cells can play critical roles in inducing B cell mediated immunity. As the mechanism of T cell dependent B cell maturation is not well established, we hypothesized that a specific population of T (follicular helper T, Tfh) cells, are involved in B cell maturation following cholera. We found flowcytometrically that V. cholerae infection induces significant increases in circulating Tfh cells expressing B cell maturation associated protein CD40L early in disease. The increased Tfh cells expressing CD40L recognize cholera toxin most prominently, with lessened responses to V. cholerae membrane preparation (MP) and V. cholerae cytolysin (VCC). We further showed that early induction of Tfh cells and CD40L was associated with later memory B cell responses to same antigens. Lastly, we demonstrated in vitro that Tfh cells isolated after cholera can stimulate class switching of co-cultured, isolated B cells from patients with cholera, leading to production of the more durable IgG antibody isotype colorimetrically. These studies were conducted on circulating Tfh cells; future studies will be directed at examining role of Tfh cells during cholera directly in gut mucosa of biopsied samples, at the single cell level if feasible.     

Induction and regulation of CD8+ cytolytic T cells in human tuberculosis and HIV infection

Tuberculosis and HIV continue to be the world-leading killers among infectious diseases, primarily affecting poor people in many developing countries. Despite differences in the immunopathogenesis of human infection with tuberculosis and HIV, experimental evidence from clinical studies and relevant animal models can be used to reflect on the cellular mechanisms responsible for an increased risk of active tuberculosis among HIV-infected individuals. In this review, we will discuss the molecular features and regulation of cytolytic T cells and how deficient cytolytic T cell responses contribute to the pathogenesis of TB and HIV infection as well as TB/HIV co-infection.     

The IL-17F sequence variant is associated with susceptibility to tuberculosis

The interleukin (IL)-17 gene plays a key role in host defence against infections from microbes, including Mycobacterium tuberculosis. Genetic factors contribute to host defence. However, whether genetic variation in IL-17 is associated with altered susceptibility to tuberculosis is unknown. A total of 596 pulmonary tuberculosis (PTB) patients, 176 extra-pulmonary tuberculosis (EPTB) patients, and 622 control patients from a Chinese Han population were recruited. Two single-nucleotide polymorphisms (SNPs) in IL-17F (rs1889570 and rs763780) and one SNP in IL-17A (rs2275913) were genotyped using the SNaPshot technique. Of the three SNPs in the IL-17 gene tested, there was an increased frequency of the rs1889570 G allele and the rs763780 C allele in the PTB patients and an increased frequency of the rs763780 C allele in the EPTB patients compared with the control patients. There were also significant differences in the distribution of the rs763780 genotype between the PTB and EPTB patients and the controls. The patients who had the CT/TT genotype of the rs763780 SNP were more susceptible to tuberculosis, compared to the CC genotype. There was no significant difference observed between the IL-17 SNPs when the PTB and EPTB patients were compared. Genetic variation in IL-17F is associated with altered susceptibility to tuberculosis and may provide valuable information in the development of tuberculosis.     

哮喘患者外周血Treg和Th1/Th2的变化及其与哮喘病情的关系

目的：探讨哮喘患者外周血调节性T细胞（Treg）以及辅助性T细胞（Th1/Th2）的比例的变化,探讨其在哮喘的临床治疗中的作用。方法：80例哮喘患者（哮喘组）按临床表现分为急性发作期组（54例）和缓解期组（26例）,同时选择50例健康体检者。应用流式细胞仪检测上述各组外周血CD4＋CD25＋Foxp3＋Treg、CD4＋IFN-γ＋Th1和CD4＋IL-4＋Th2细胞水平,并进行统计学分析。结果：哮喘组CD4＋CD25＋Foxp3＋Treg水平亦明显低于正常对照组（P〈0.05。其中急性发作期组Treg水平明显低于缓解期组和正常对照组（P〈0.05）。而哮喘组Th1/Th2比值显著低于对照组（P〈0.05）,且在哮喘急性发作组中Th1/Th2比值显著低于缓解期组和正常对照组（P〈0.05）。结论：提示Treg和Th在哮喘的发生和发展中起着重要的作用。     

Down-regulation of macaque ΓΔ+ T cells in lymphoid compartments after rectal infection with SIVsmmPBj14

The dynamics of T cells expressing the ΓΔ T-cell receptor in mucosae and other compartments during the course of human immunodeficiency virus (HIV)-1 infection are poorly understood. To examine the impact of an acquired immunodeficiency syndrome virus on the ΓΔ+ T-cell population, rectal inoculation of macaques with simian immunodeficiency virus (SIV)-PBj14 was used as a model. After rectal inoculation, five macaques were sacrificed on days 4, 5, or 7 and then assessed for changes in the ΓΔ T-cell receptor repertoire in different lymphoid compartments. There was decreased representation of ΓΔ+ T cells in the intestinal mucosae, blood, and spleens. Overall, the reduced number of total ΓΔ+ T cells was consistent with decreases in the VΓ or VΔ T-cell sub-populations. Nevertheless, there was no consistent deletion or expansion of a selected VΓ+ or VΔ+ cell sub-population. These results demonstrate that SIV-PBj14 replication and dissemination after mucosal inoculation resulted in a decline of detectable ΓΔ+ T cells, suggesting that macaque ΓΔ+ T cells are susceptible to down-regulation or destruction during acute SIV-PBj14 infection.