TCRγδ Cells Research Articles

Beneficial Effects of CpG-Oligodeoxynucleotide Treatment on Trauma and Secondary Lung Infection.

Although Streptococcus pneumoniae is usually found as a commensal in healthy individuals, it can act as a pathogen in trauma patients, causing such complications as early-onset pneumonia and sepsis. We discovered that treating mice with an A-class CpG-oligodeoxynucleotide (ODN) at 2 h after traumatic injury significantly improved mouse survival following early-onset secondary lung infection with S. pneumoniae. This study used mass cytometry (cytometry by time-of-flight) and Luminex technologies to characterize the cellular immune response to secondary S. pneumoniae lung infection at 1 and 3 d postinfection. We found increased expression of CD14, CD64, and PD-L1 on F4-80(+) and F4-80(+)CD11c(+) macrophages, CD11c(+) dendritic cells, and CD14(+)CD172a(+) cells after burn-injury and infection, supporting previous reports of innate immune cell activation in sepsis. CpG-ODN treatment at 2 h after burn-injury reversed these effects; improved pathogen clearance; and led to an increased expression of CD25, CD27, MHCII, and IL-17 on or in TCRγδ cells at 1 d postinfection. At 3 d postinfection, CpG-ODN treatment increased the expression of PD-L1 on innate cell subsets. Furthermore, we analyzed cytokine levels in lung-washout samples of TCRγδ cell-depleted (TCRγδ(-)) mice to demonstrate that the effects of CpG-ODN on cytokine expression after burn-injury and S. pneumoniae infection rely on functional TCRγδ cells. In summary, we demonstrate that cytometry by time-of-flight provides an effective strategy to systematically identify specific cellular phenotypic responses to trauma and bacterial pneumonia and to discover changes in immune system phenotypes associated with beneficial immunotherapy.

Inhibitory effect of antidepressant drugs on contact hypersensitivity reaction is connected with their suppressive effect on NKT and CD8+ T cells but not on TCR delta T cells

BackgroundContact hypersensitivity (CHS) reaction induced by a topical application of hapten is a cell-mediated antigen-specific type of skin inflammation mediated by interaction of several subtypes of T cell subpopulations. Recently, it has been shown that antidepressant drugs inhibit CHS reaction, although the mechanism of this effect remains unknown. The aim of the present study was to investigate the effect of 2-week desipramine or fluoxetine administration on the CHS reaction induced by picryl chloride (PCL) application in B10.PL mice and in knock-out mice established on B10.PL background: TCRδ−/− mice lacking TCRγδ T lymphocytes; β2m−/− mice lacking CD8+ T lymphocytes and CD1d−/− mice lacking CD1d dependent natural killer T (NKT) lymphocytes. MethodsB10.PL, TCRδ−/−, β2m−/− and CD1d−/− mice were divided into six groups: 1) vehicle-treated negative control group; 2) desipramine-treated negative control group; 3) fluoxetine-treated negative control group; 4) vehicle and PCL-treated group (positive control group); 5) desipramine and PCL-treated group; and 6) fluoxetine and PCL-treated group. CHS to PCL was tested by evaluation of ear swelling. Metabolic activity of spleen and lymph node cells were estimated by MTT test. ResultsThe antidepressants significantly suppressed the CHS reaction in B10.PL mice: desipramine by 55% and fluoxetine by 42% compared to the positive control. This effect was even stronger in TCRδ−/− mice, in which fluoxetine reduced the ear swelling by 73% in comparison with the vehicle-treated positive control group. On the other hand, desipramine and fluoxetine did not inhibit CHS reaction in β2m−/− and CD1d−/− mice. Moreover, PCL increased metabolic and/or proliferative activity of splenocytes in all four strains of mice whereas the antidepressants decreased this activity of splenocytes in B10.PL, TCRδ−/− and CD1d−/− mice. ConclusionThe results of the present study show that lack of CD8+ T cells or NKT cells abolishes the immunosuppressive effect of antidepressant drugs on PCL-induced CHS reaction in mice. These results suggest that antidepressant drug-induced inhibition of CHS reaction is connected with their inhibitory effect on ability of CD8+ T cells and NKT cells to induce and/or escalate CHS reaction. TCRγδ cells seem not to be involved in antidepressant-induced suppression of CHS.

Alpha-bisabolol, not a matter for cancer therapy. Commentary: "Research on the immunosuppressive activity of ingredients contained in sunscreens".

Alpha-bisabolol, not a matter for cancer therapy. Commentary: "Research on the immunosuppressive activity of ingredients contained in sunscreens".

The influence of enrofloxacin, florfenicol, ceftiofur and E. coli LPS interaction on T and B cells subset in chicks.

This study aimed to investigate the influence of enrofloxacin, florfenicol, ceftiofur and E. coli LPS interaction on T and B subsets in thymus and spleen of newly-hatched chicks. A 126, 1-day-old chicks were administered enrofloxacin, florfenicol or ceftiofur in recommended doses according to the currently treatment schedule advises. E .coli LPS was given intravenously once at the dose of 200 μg kg−1 BW on the 2nd day of experiment (d. e.). On the 6th and the 14th d. e. thymus and spleens were subjected to flow cytometry investigation.The most significant changes were demonstrated in spleen. The antibiotics administration decreased the percentage of B and T cells subset. Moreover, this suppressive effect was enhanced by E. coli LPS administration. On the 6th d. e. the percentage of CD3+TCRγδ−, CD3+TCRγδ+, CD4+CD8−, CD4−CD8+ decreased significantly after ceftiofur and LPS treatment. A lower percentage of CD3+TCRγδ−, CD4−CD8+ and CD3+TCRγδ+ was observed in enrofloxacine and LPS treated group. The decrease percentage of CD3+TCRγδ+cells and Bu-1+ was found after florfenicol and LPS treatment. On the 14th d. e. a decreased percentage of CD4+CD8− and increased percentage of CD4−CD8+ cells was shown in ceftiofur or enrofloxacine and LPS treated groups. In addition decreased percentage of CD3+TCRγδ+ was found in all antibiotic and LPS treated groups.In this study, it was shown that enrofloxacine, florfenicol, ceftiofur treatment may change the proportions among lymphocytes subset and might have an impact on the immune response to bacterial endotoxins in chicks.

Phosphoantigen Presentation to TCR γδ Cells, a Conundrum Getting Less Gray Zones.

The mechanistic requirements of antigen recognition by T cells expressing a γδ TCR has revealed important differences with those of αβ TCR cells and, despite impressive new data generated in the very recent years, they remain poorly understood. Based on the structure of the TCR chains and the tissue distribution, γδ cells are represented in a variety of populations. The major subset of human peripheral blood γδ cells express Vγ9Vδ2 TCR heterodimers and are all stimulated by phosphorylated metabolites (commonly called phosphoantigens). Phosphoantigens are molecules with a very small mass and only stimulate Vγ9Vδ2 cells in the presence of antigen-presenting cells, suggesting a strict requirement for dedicated antigen-presenting molecules. Recent studies have identified butyrophilin (BTN) 3A1 as the molecule necessary to stimulate Vγ9Vδ2 cells. BTN3A1 extracellular, transmembrane, and cytoplasmic domains have different functions, including cognate interaction with the Vγ9Vδ2 TCR, binding of the phosphoantigens, and interaction with cytoplasmic proteins. This review mainly discusses the known molecular mechanisms of BTN3A1-mediated antigen presentation to γδ cells and proposes a model of phosphoantigen presentation, which integrates past and recent studies.

Lymphocyte subpopulations of bone marrow of breast cancer patients

We have studied bone marrow lymphocytes subpopulations in 67 patients with breast cancer and 13 patients with benign processes in the mammary gland. Morphological study of the cellular composition of the bone marrow and immunological analysis of lymphocyte subpopulations have been done. Comparative characterization of lymphocyte subpopulations in the bone marrow of patients with breast cancer and benign breast diseases was done by flow cytometry. The gate of Mature lymphocytes (CD45++) studied subpopulations of T cells (CD3), b cells (CD19), NK cells (CD56+CD3–). Among T lymphocytes (CD3+) was studied TCRγδ and TCRαβ cells, a subpopulation of CD4, CD8, CD4+, CD25+ T-regulatory cells. Within В-lymphocytes (CD19+) was studied subpopulations CD10, CD38, CD5 lymphocytes. We revealed a significant increase of CD5+, CD38+ B-cells in patients with breast cancer compared to benign tumors. Index of maturation of erythrocytes was significantly higher in BC 0.96 in comparison with benign processes 0.92. When comparing immunological data with histological structure of the tumor in the group of breast cancer patients, it was noted that the percent of CD10+ B-lymphocytes was significantly higher in infiltrative ductal cancer in comparison to infiltrative lobular carcinoma (19.3 and 8.0 % accordingly; p = 0.024).

Generating AML-Specific Peripheral Blood Autologous Cytotoxic T-Lymphocytes (CTLs)

Background: Ex-vivo expansion of CBT-cells using CD3/CD28 co-stimulatory beads, IL-2 + IL-7 and subsequent priming against leukemia cell lines using IL-15 generated specific CTLs. [1, 2]Hypothesis: We hypothesized that (a) patient-derived AML-specific PB auto CTLs could be generated with immune-stimulatory culture condition (b) Resistant AML samples would possess gene expression profiles similar to MDSCs (myeloid-derived suppressor cells) (c) Frequency of Tregs (CD4+CD25brightFoxP3+) and T-cell co-signaling molecules gene expression will be different between effective and ineffective CTLs.Methods: AML & auto T-cells were purified from cryopreserved PBMC of AML patients admitted with acute blast crisis (n=8). AML blasts were sustained in StemSpan™ Serum-Free media [STEMCELL Technologies] with MSC support + cytokine cocktail (IL-3, SCF, FLT3L, GMCSF, IL-4). T-cells were expanded in culture for 2 weeks as reported [1, 2] and subsequently primed with γ-irradiated auto AML weekly X 3 with IL15 + CD28ab [BD Biosciences]. At the end of week 3 (EOW3), cytotoxicity was assessed against AML and irrelevant targets - IM9 (lymphoid) and U937 (myeloid) cell lines, loaded with BATDA at an E:T ratio of 40:1, 20:1, 10:1 and 5:1 using DELFIA® EuTDA assay.[2] IFN-γ ELISPOT assay against same targets was also done.[2] RT-qPCR analysis was performed on AML & T-cells before and after priming, using Power SYBR® Green master mix (Thermo Fisher Scientific) and StepOne Plus system [Life Technologies]. Two-tailed student t-testcompared experimental groups.Results· T-cells expanded in all samples (n=8) with a median expansion of 155-fold (range 11-489), at EOW3.· ELISPOT assay was positive in 4/8 samples. [Fig 1]· CTL assay was difficult to standardize for primary AML blasts due to high degree of spontaneous apoptosis (>30% spontaneous release [SR]).· 2/8 samples were deemed evaluable (SR<30%).· Both samples showed AML-specific lysis. [Fig 2]· Overall, AML-specific autologous CTL could be generated from 5 of 8 samples based on ELISPOT & CTL assays, regardless of original FAB immunophenotype, not shown.· Tregs proportion declined significantly in effective CTLs post-priming as compared to pre-priming (56% to 24%, p-value 0.046, n=4). [Fig 3]· T-cell gene expression profiling showed significant differences in effective vs ineffective CTLs. [Table 1]· Resistant AML (n=3) had up-regulated downstream markers associated with MDSC generation compared to “non-resistant” AML (n=5). [Table 2]Conclusions (a) AML-specific auto CTLs can be generated (b) Tregs decreased with priming in effective CTLs (c) differential T-cell gene expression profile exists between effective and ineffective CTLs (d) AML gene expression suggests MDSC-like profile in resistant samples.Abstract 5806. TABLE 1:T-CELL GENE EXPRESSION PROFILE (POST VS PRE-PRIMING)Effective CTLs (n=5)Ineffective CTLs (n=3)GeneΔΔ Ct(Post - Pre) (mean, SEM)P-valueFold change (mean, SEM)ΔΔ Ct(Post - Pre) (mean, SEM)P-valueFold change (mean, SEM)4-1BB-3.17 (0.76)0.02514 (7.7)1.98 (1.04)0.190.39 (0.22)HVEM-2.43 (0.61)0.0287.3 (3.7)0.14 (1.65)0.951.57 (1.28)LIGHT-3.62 (0.73)0.01617.3 (7.3)1.78 (1.84)0.441.1 (0.98)PRKC-α-2.03 (0.47)0.0234.6 (1.1)1.89 (0.36)0.0340.29 (0.08)PRKC-θ-3.36 (0.59)0.0113.7 (6.7)0.25 (0.59)0.710.99 (0.41)LAIR1-3.81 (0.42)0.00316.2 (5.6)-1.35 (2.20)0.6017.15 (16.5)PP2A-2.40 (0.57)0.0256.7 (2.6)0.49 (1.57)0.791.89 (1.52)2B4-1.53 (1.14)0.274.98 (1.82)-3.48 (0.11)0.0211.2 (0.9)LTA-α-1.18 (0.78)0.233.61 (2.11)2.69 (0.18)0.0430.16 (0.02)LTA-β-0.93 (0.63)0.242.49 (0.99)2.24 (0.47)0.0420.23 (0.08)TABLE 2:GENE EXPRESSION PROFILE RESISTANT VS NON-RESISTANT AMLGeneΔΔ Ct(mean, SEM)95% CIP-valueRelative fold changeJAK1-4.63 (1.98)-9.480.210.057924.83JAK2-5.38 (0.94)-7.67-3.080.001241.52JAK3-5.90 (2.17)-12.811.010.072659.77S100A8-7.16 (2.66)-14.01-0.320.0432143.27S100A9-8.31 (2.75)-15.04-1.590.0233318.37c-myc-2.78 (0.59)-4.24-1.330.00346.89Refs:1.Davis et al. Cancer Research 2010;70(13):52492.Jeyaraj A, Chen X, Szabolcs P. IL-15 Induced Polyclonal CTL Generated From Expanded CBT Cells Against Leukemia Cell Lines Constitutes IFN-γ Producing Cells and TCRγδ Cells. ASH 2012 Annual Meeting [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Distribution of immune response cells in the pelvic urethra and the prepuce of rams

The pathogens of the reproductive system in the male can penetrate and establish by ascending route, from to the prepuce to the urethra, accessory glands, epididymis and testicles. The aim of this paper is determine the distribution and number of cells involved in the immune response in prepuce and pelvic urethra of rams, without apparent clinical alterations in testicle, epididymis and prepuce. The distribution of some of the cells involved in the immune response at the level of the prepuce and the pelvic urethra was quantified in four one-year-old rams seronegative for B. ovis and A. seminis and without apparent lesions in the testicles, the epididymis, and the prepuce. At the moment of slaughter, samples were taken from the preputial fornix and the pelvic urethra and placed in 10% formalin and under freezing conditions. CD4, CD8, WC1, CD45RO, CD14 and CD1b cells were demonstrated by immunohistochemistry, and immunoglobulin-containing cells (ICC) of the IgA, IgG and IgM classes were demonstrated by immunofluorescence. The labeled cells present in the mucosa of both organs were counted with an image analyzer. The total number of cells was compared between both tissues and differentially between the epithelium and the connective tissue of the mucosa. Significant differences were found in the total number of CD4, CD45RO, and WC1 lymphocytes, in CD14 macrophages, and CD1b dendritic cells, with mean values being greater in the fornix than in the urethra (p&lt;0.05) in all cases. Only dendritic cells were found in the prepuce. No differences were found in the number of CD8 lymphocytes between both organs. The ratio between each cell type in the connective and the intraepithelial tissues and between organs was 10/1 for CD4 in the fornix (p&lt;0.05), against 7/1 in the urethra (p&lt;0.05), while CD8 had a 1/1 distribution in both mucosae. The WC1 ratio was 5/1 in both mucosae (p&lt;0.05). CD45RO labeling was 19/1 in the prepuce (p&lt;0.05) and 1/1 in the urethra. IgA-containing cells did not show differences in the total number of cells in both tissues. In the urethra, no IgG-containing cells were observed and IgM-containing cells were scarce; in contrast, both cell types were present in the prepuce, in amounts greater than in the urethra (p&lt;0.05). IgA-, IgG-, and IgM-containing cells were located in both organs in the mucosal connective tissue. The presence of antigen-presenting cells, macrophages, and dendritic cells, as well as of lymphocytes CD4, CD8 TCR γδ (WC1), IgA-, IgG and IgM positive cells, and CD45RO cells suggests that both mucosae may behave as inductive and effector sites for the mucosal immune response.

IL-15 Induced Polyclonal CTL Generated From Expanded CBT Cells Against Leukemia Cell Lines Constitutes IFN-γ Producing Cells and TCRγδ Cells

AbstractAbstract 1910 Background:We have previously demonstrated that ex vivo expanded CBT cells expanded with CD3/CD28 co-stimulatory beads + IL-2 and IL-7 were receptive to subsequent in vitro priming against killed lymphoid and myeloid leukemia cells in the presence of IL-7, IL-12, and IL-15, (Davis et al. Cancer Research 2010;70(13):5249). Hypothesis and Objectives:Here, we wanted to test 1) the minimum prerequisite cytokine requirement, hypothesizing that higher specificity may be obtained if less exogenous cytokine is employed. 2) Determine the mechanism of CTL’s kill by employing blocking antibodies against HLA-ABC, HLA-DR and TCRγδ recognition sites. 3) Characterize the critical cellular phenotype of the CTL to identify whether the cytotoxicity is specific to CD4, CD8, TCRαβ, or TCRγδcell populations or the observed cytotoxicity is the contribution from multiple cellular phenotypes? Methods:CTL was generated from already expanded >98% pure T cells either with a combination of cytokines (IL-7, -12 & -15) versus IL-15, versus IL-7 alone and were compared from the same cultures by matched pair Student T-test. After 3 weeks of CTL cultures, the cytotoxicity (specific lysis) was tested against fresh IM9 leukemia cells (loaded with BATDA) at an Effector: Target ratio of 40:1, 20:1 and 10:1. The blocking with HLA class I, class II and TCRγδ antibodies were done before performing the CTL assay. ELISPOT assay tested the specificity (non-specific target recognition) and magnitude of activity as measured by IFN-γ spot forming cells (SFC). Results:From the data shown in Figure 1, it is evident that IL-15 alone is sufficient to support both priming and expansion of IM-9 specific CTL. After 3 weeks of CTL cultures, the observed specific lysis of fresh IM9 leukemia with IL-15 alone versus in combination was 73% and 75% respectively at an Effector: Target ratio of 40:1. The generated CTL exhibit no cytotoxicity against non-specific targets against a myeloid leukemia cell line (U937). While cytotoxicity by Europium release assay was comparable, CTL from IL-15 alone cultures displayed higher specificity in ELISPOT assays with much less non-specific target recognition as measured by IFN-γ spot forming cells (SFC) (Figure 2A). Though the relative (fold) expansion of CTL observed in the group with all cytokines is significantly higher, the numbers are comparable between the combination of cytokines and IL-15 alone towards the later weeks of priming and expansion, data not shown. [Display omitted] [Display omitted] By blocking experiments, it is evident that, even though no single MoAb completely abolished cytotoxicity, the most significant diminution was identified when the effectors were blocked with TCRγδ (Figure 2B). Multicolor FACS analysis indicates that after exposing the CTLs to specific targets, ∼7–8% of the cells are secreting IFN- γ and ∼ 20–25% of the generated CTLs are TCRγδ positive. Immunoscope analysis of TCRγδ spectratype identifies oligoclonal restriction of CTL cultures with lytic activity compared to identical cytokine expanded T cells lacking leukemia–specific CTL activity (Figure 3). [Display omitted] Conclusion:A polyclonal leukemia specific CTL can be generated with IL-15 alone from previously expanded and partially mature CB T cells. While the cytotoxicity is preserved compared to those primed/expanded by multiple cytokines the specificity is higher in IL-15 alone. Notably, the CTL activity is not confined to a single cellular subset and possibly the most significant activity resides in the TCRγδ expressing T cell population. Disclosures:No relevant conflicts of interest to declare.

Human TCRγδ+T cells represent a novel target for IL‐27 activity

IL-27 and TCRγδ(+) T lymphocytes play critical roles in both innate and adaptive immune responses in health and disease, including infection and tumors. Although the activity of IL-27 is well characterized in different human immune cells, no information is available on the role of IL-27 in human TCRγδ(+) T lymphocytes. Here, we provide the first evidence that TCRγδ(+) T lymphocytes express both gp130 and WSX-1 chains of IL-27R, and that IL-27 may function in TCRγδ(+) T cells by (i) inducing STAT1 and STAT3 phosphorylation, (ii) stimulating cytotoxicity against tumor cells through upregulation of cytotoxic granules production, (iii) reducing the release of Th2-related cytokines, such as IL-5 and IL-13, and inducing IFN-γ production, and (iv) upregulating the expression of CD62L. These results highlighted a novel immunoregulatory property of human IL-27 that may be relevant in the immune response against tumors. Our results may offer new perspectives for the development of future clinical trials using IL-27 and TCRγδ(+) cells for cancer immunotherapy.

Mechanisms underlying T cell guided innate immunity after inhalation of Staphylococcus enterotoxin A (SEA) (173.1)

Abstract Inhalation of Staphylococcus aureus enterotoxin A (SEA) in mice induces strong pulmonary inflammation marked by vascular permeability and alveolitis, thereby modeling aspects of lung injury in humans. After SEA inhalation CD8+Vβ3+ T cells robustly expand but the early events of this response in the lung are unclear. Our new data show that Vβ3+ T cells are activated quickly after SEA inhalation since CD69 and the high affinity IL-2 receptor α-chain (CD25) were upregulated by 3 hours. This coincided with several changes in the innate immune system including recruitment of neutrophils, monocytes and NK cells into the lung, and imprinting an activation phenotype on macrophages. Importantly, in the absence of T cells all of these effects were abrogated. As a way to understand how T cells condition innate immunity, we analyzed cytokine production and found an unexpected role for TCRαβ T cells promoting IL-17 secretion by TCRγδ T cells. We demonstrated that TCRγδ cells did not produce IL-17 when tested in TCRβ-/- mice. Currently, we are defining how TCRγδ cells are induced to synthesize IL-17 by the TCRαβ cells. In sum, TCRαβ T cells mediate pro-inflammatory responses, not only as a direct source of cytokines, but also by guiding the recruitment and activation of innate cells. Thus, our data show role reversal between the adaptive immune and innate immune system.

Notch Induces T Cell Receptor γδ Thymocytes to Differentiate along a Bipotent CD4 CD8 Double Positive Pathway (111.6)

Abstract It is well documented that in wildtype mice TCRγδ+ cells differentiate along a double negative (DN) pathway whereas TCRαβ+ cells differentiate along the double positive (DP) pathway, suggesting that the TCR itself induces lineage differentiation. Under experimental conditions and in genetically modified mice, however, evidence was presented suggesting that rather than TCR itself, TCR signal strength and notch determine lineage choice. In the human thymus, a “chimeric” DP TCRγδ+ cell population is present and constitutes a sizeable fraction of the γδ population. We asked the question whether these cells belong to the αβ or γδ lineage or whether these cells are bipotent. We found that TCRγδ DP cells are bipotent cells since strong TCR signals induces differentiation to TCRγδ cells, whereas Notch activation induces TCRαβ lineage differentiation. We furthermore could show that Notch signaling diverts TCRγδ DN cells to the DP pathway and induces strong proliferation. In line with these findings, TCRγδ+ acute lymphoblastic leukemias (ALL) with activating Notch1 mutations follow the DP differentiation pathway, whereas the DN ALL cells are devoid of these activating Notch1 mutations. We were able to confirm that also in vivo TCRγδ DP have rearranged TCRβ chains, actively rearrange the TCRα locus and delete the TCRδ locus (αβ lineage). Using TCRα rearrangements as a lineage marker, we could show that a subpopulation of mature TCRγδ cells is derived from DP cells.

Notch induces human T-cell receptor γδ+ thymocytes to differentiate along a parallel, highly proliferative and bipotent CD4 CD8 double-positive pathway

In wild-type mice, T-cell receptor (TCR) γδ(+) cells differentiate along a CD4 CD8 double-negative (DN) pathway whereas TCRαβ(+) cells differentiate along the double-positive (DP) pathway. In the human postnatal thymus (PNT), DN, DP and single-positive (SP) TCRγδ(+) populations are present. Here, the precursor-progeny relationship of the various PNT TCRγδ(+) populations was studied and the role of the DP TCRγδ(+) population during T-cell differentiation was elucidated. We demonstrate that human TCRγδ(+) cells differentiate along two pathways downstream from an immature CD1(+) DN TCRγδ(+) precursor: a Notch-independent DN pathway generating mature DN and CD8αα SP TCRγδ(+) cells, and a Notch-dependent, highly proliferative DP pathway generating immature CD4 SP and subsequently DP TCRγδ(+) populations. DP TCRγδ(+) cells are actively rearranging the TCRα locus, and differentiate to TCR(-) DP cells, to CD8αβ SP TCRγδ(+) cells and to TCRαβ(+) cells. Finally, we show that the γδ subset of T-cell acute lymphoblastic leukemias (T-ALL) consists mainly of CD4 SP or DP phenotypes carrying significantly more activating Notch mutations than DN T-ALL. The latter suggests that activating Notch mutations in TCRγδ(+) thymocytes induce proliferation and differentiation along the DP pathway in vivo.

Murine and Bovine γδ T Cells Enhance Innate Immunity against Brucella abortus Infections

γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα−/−, and GMCSF−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.

CD4 and CD8 TCRαβ Cells Are selected On MHC Expressed On Thymocyte Precursors in OP9-DL1 Cultures.

Abstract 3670Poster Board III-606Human CD34+ haematopoietic precursor cells cultured on delta-like-ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells (TEC) which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4+ and cytotoxic CD8+ TCRαβ cells is not expected.Phenotypically mature CD27+ CD1− TCRγδ as well as TCRαβ cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single positive (SP) TCRαβ cells were observed. Mature CD8 SP cells consisted of two subpopulations: one expressing mainly CD8αβ and one expressing CD8αα dimers. TCRαβ CD8αα and TCRγδ cells both expressed the IL2Rβ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8αβ+ and CD4+ TCRαβ+ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8αβ+ and CD4+ T cells. These T cells had the characteristics of conventional T cells: CD4+ cells expressed ThPOK, CD40L and high levels of IL-2 and IL-4; CD8+ cells expressed EOMES, RUNX3 and high levels of granzyme, perforin and IFN-γ.Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8+ cells, whereas blocking MHC class I antibodies and CD8 antibodies inhibited generation of mature CD27+ CD8+ SP cells. The presence of dendritic cells was not required for the generation of mature CD4+ or CD8+ T cells.In conclusion, we have demonstrated positive selection of TCRαβ+ cells in the absence of TEC. The data suggest that positive selection is induced by interaction between T precursor cells without requirement for other haematologic or stromal elements. Disclosures:No relevant conflicts of interest to declare.

Functionally Mature CD4 and CD8 TCRαβ Cells Are Generated in OP9-DL1 Cultures from Human CD34+ Hematopoietic Cells

Human CD34(+) hematopoietic precursor cells cultured on delta-like ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4(+) and cytotoxic CD8(+) TCRalphabeta cells is not expected. Phenotypically mature CD27(+)CD1(-) TCRgammadelta as well as TCRalphabeta cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCRalphabeta cells were observed. Mature CD8 single-positive cells consisted of two subpopulations: one expressing mainly CD8alphabeta and one expressing CD8alphaalpha dimers. TCRalphabeta CD8alphaalpha and TCRgammadelta cells both expressed the IL2Rbeta receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8alphabeta(+) and CD4(+) TCRalphabeta cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8alphabeta(+) and CD4(+) T cells. These T cells had the characteristics of conventional T cells: CD4(+) cells expressed ThPOK, CD40L, and high levels of IL-2 and IL-4; CD8(+) cells expressed Eomes, Runx3, and high levels of granzyme, perforin, and IFN-gamma. Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8(+) cells. Similarly, the presence of dendritic cells was not required for the generation of mature CD4(+) or CD8(+) T cells. These data suggest that positive selection of these cells is induced by interaction between T precursor cells.

Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis.

The commensal fungus Candida albicans causes oropharyngeal candidiasis (OPC; thrush) in settings of immunodeficiency. Although disseminated, vaginal, and oral candidiasis are all caused by C. albicans species, host defense against C. albicans varies by anatomical location. T helper 1 (Th1) cells have long been implicated in defense against candidiasis, whereas the role of Th17 cells remains controversial. IL-17 mediates inflammatory pathology in a gastric model of mucosal candidiasis, but is host protective in disseminated disease. Here, we directly compared Th1 and Th17 function in a model of OPC. Th17-deficient (IL-23p19−/−) and IL-17R–deficient (IL-17RA−/−) mice experienced severe OPC, whereas Th1-deficient (IL-12p35−/−) mice showed low fungal burdens and no overt disease. Neutrophil recruitment was impaired in IL-23p19−/− and IL-17RA−/−, but not IL-12−/−, mice, and TCR-αβ cells were more important than TCR-γδ cells. Surprisingly, mice deficient in the Th17 cytokine IL-22 were only mildly susceptible to OPC, indicating that IL-17 rather than IL-22 is vital in defense against oral candidiasis. Gene profiling of oral mucosal tissue showed strong induction of Th17 signature genes, including CXC chemokines and β defensin-3. Saliva from Th17-deficient, but not Th1-deficient, mice exhibited reduced candidacidal activity. Thus, the Th17 lineage, acting largely through IL-17, confers the dominant response to oral candidiasis through neutrophils and antimicrobial factors.

OP9-DL1 Cell Line Supports the Development of Phenotypically and Functionally Mature Tcrαβ And Tcrγδ T Cells, through Both Conventional and Aberrant Developmental Pathways

In vitro generation of mature T cells from human hematopoietic stem and progenitor cells (HSPC) could fulfill two existing needs. First, it could enhance and quicken T cell immune reconstitution after stem cell transplantation, which is very slow and generates a skewed TCR repertoire. Second, by generation of tumour antigen specific T cells it could provide an efficient therapy for numerous malignancies and could enhance GVT effect in the context of allogeneic SCT, without aggravating GVHD. T cells can be generated from human HSPC by culturing them on the murine stromal cell line OP9-transduced with the Notch ligand Delta-like-1 (OP9-DL1). Notch receptor activation is essential for T cell development. However, it is unclear whether Notch activation is sufficient for end maturation into functionally and phenotypically mature TCR positive cells. It was shown that human CD34+ cells cultured on OP9-DL1 differentiate to T cells which can proliferate and produce interferon-g upon polyclonal stimulation. The nature of the mature cells generated in these cultures, however, has not been well studied. CD34+ HSPC from postnatal thymus (PNT) or cord blood were cocultured with OP9-DL1, in the presence of the cytokines Flt-3L (5 ng/ml), SCF (2.5 ng/ml) and IL-7 (5 ng/ml). Every 3–5 days cells were harvested and transferred to fresh OP9-DL1 cells. At repetitive timepoints, an aliquot of the cells was analysed phenotypically. In some experiments, IL-15 was added to the culture. For some experiments, cells harvested from OP9-DL1 at the timepoint mature T cells were observed (usually about d 40 of culture), were transferred to feeder cells, consisting of JY cell line (5.104 cells/ml irradiated with 50 Gy and PBMC (5.105/ml irradiated with 40 Gy), in the presence of PHA (1 mg/ml). After 7 days, IL-2 (50 IU/ml) was added to the culture. Every 14 days, cells were restimulated with new feeders (irradiated JY and PBMC) and new addition of PHA. After 3 weeks of stimulation cells were stimulated overnight with 15 ng/ml PMA and 1500 ng/ml ionomycin, and 18 hours later cells were checked for intracellular presence of cytokines. We investigated whether the T cell population generated in these cultures contains mature cells with the characteristics of TCRγδ cells and of positively selected CD8 or CD4 single positive (SP) TCRαβ cells as observed in the human thymus. We found that under the described conditions, HSPC mature into CD1-CD27+ phenotypically mature T cells, with the TCRγδ fraction maturing faster and more efficiently compared to the TCRαβ fraction. Consistent with a mature phenotype, TCRγδ cells were mostly CD8αα or double negative (DN). No mature CD4 SP TCRαβ cells were observed and the mature CD8 SP cells co-expressed variable ratios of CD8αβ and CD8αα dimers, suggesting that these cells are not conventional positively selected TCRαβ cells. In support of this hypothesis, both mature CD1- TCRαβ and TCRγδ cells expressed the IL2Rβ receptor consitutively and both populations proliferated on IL-15 without prior antigen stimulation, CD8αα (TCRαβ and TCRγδ) cells being the most IL-15 responsive. Mature activated T cells secreted IFN-γ and TNFα, little or no IL-2 and IL-4, with no difference observed between TCRαβ and TCRγδ cells. These data suggest that CD8 TCRαβ cells generated in these cultures are unconventional CD8 cells possibly maturated through agonist selection. However, when cells harvested after 40 days of culture on OP9-DL1 were stimulated with PHA and IL-2 for 3 weeks, conventional appearing CD8αβ cells emerged, with a cytokine production profile similar to that of thymic CD8αβ TCRαβ T cells, with the majority of cells secreting IFN-γ and IL-2. We can conclude from these data that OP9-DL1 supports the development of both unconventional and conventional CD8+ TCRαβ cells, of which the generation and selection process are currently being investigated. Also the in vitro anti-tumor capacities of both populations need to be addressed.

Dysregulation of the host mevalonate pathway during early bacterial infection activates human TCR γδ cells

Primates, but not rodents, have T cell receptor Vgamma9-Vdelta2 T cells bridging innate and adaptive antimicrobial immunity. This T cell population is activated by prenyl pyrophosphates isolated from microbial or eukaryotic cells. Although the microbial metabolites are more active than the cellular ones, their involvement in TCR gammadelta activation during infection has not been studied. Here, we show that, during the initial phases of infections with Escherichia coli and Staphylococcus aureus, TCR gammadelta cells are activated by endogenous mevalonate metabolites. Infections with low bacteria inocula up-regulate the production and accumulation of host-derived TCR gammadelta stimulatory antigens within 1 h, which is followed by a peak of TCR gammadelta cell activation at 5 h. Infections induce the accumulation and dephosphorylation of the hydroxymethylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, resulting in increased activity of this enzyme and in increased synthesis of intermediate metabolites. Thus, primates have evolved the ability to readily respond to bacterial infection by sensing the dysregulation of the mevalonate pathway within infected cells, as a mechanism of immediate antimicrobial immunity.

Innate lymphocyte and dendritic cell cross-talk: a key factor in the regulation of the immune response

Dendritic cells (DC) are specialized in the presentation of antigens and the initiation of specific immune responses. They have been involved recently in supporting innate immunity by interacting with various innate lymphocytes, such as natural killer (NK), NK T or T cell receptor (TCR)-gammadelta cells. The functional links between innate lymphocytes and DC have been investigated widely and different studies demonstrated that reciprocal activations follow on from NK/DC interactions. The cross-talk between innate cells and DC which leads to innate lymphocyte activation and DC maturation was found to be multi-directional, involving not only cell-cell contacts but also soluble factors. The final outcome of these cellular interactions may have a dramatic impact on the quality and strength of the down-stream immune responses, mainly in the context of early responses to tumour cells and infectious agents. Interestingly, DC, NK and TCR-gammadelta cells also share similar functions, such as antigen uptake and presentation, as well as cytotoxic and tumoricidal activity. In addition, NK and NK T cells have the ability to kill DC. This review will focus upon the different aspects of the cross-talk between DC and innate lymphocytes and its key role in all the steps of the immune response. These cellular interactions may be particularly critical in situations where immune surveillance requires efficient early innate responses.