Early Effector Research Articles

Tumor-Reactive CD8+ Early Effector T Cells Identified at Tumor Site in Primary and Metastatic Melanoma

CD8(+) T cells at the earliest stage of effector generation have not been identified at tumor site of melanoma patients. Such early effectors, if present, should be characterized by a specific phenotype, distinct from that expressed at later stages of the antigen-induced differentiation program, by short-lived effector cells, memory precursors, and terminal effectors. Here, we show that neoplastic tissues from primary and metastatic lesions of melanoma patients contain a subset of CD8(+) T cells expressing FOXP3. CD8(+) FOXP3(+) CD25(+) T lymphocytes were found in tumor-invaded lymph nodes (TILN), s.c. metastases, and advanced primary lesions. Their frequency was significantly higher in TILN compared with tumor-free lymph nodes or with peripheral blood and in primary tumors compared with TILN. CD8(+) FOXP3(+) T cells did not express markers of regulatory [CTLA-4, CCL4, interleukin-10 (IL-10), transforming growth factor-β1], exhausted (PD-1), or senescent (CD57) CD8(+) T lymphocytes. Instead, this subset showed an antigen-experienced "EM1" phenotype (CCR7(-) CD45RA(-) CD28(+) CD27(+)) and exhibited a CD127(-), KLRG1(-), HLA-DR(+), CD38(+), T-bet(+), perforin(+) "early effector" profile predicted by current models. CD8(+) FOXP3(+) T cells produced IFN-γ on short in vitro activation, recognized autologous tumor by CD107a mobilization, and expressed Ki-67 on ex vivo analysis. In response to autologous tumor plus IL-2/IL-15, the CD8(+) FOXP3(+) T cells proliferated promptly and showed competence for differentiation (downregulation of CD27 and upregulation of T-bet). These results suggest development of early phases of antitumor immunity even in advanced melanoma. Moreover, the CD8(+) FOXP3(+) "early effector" subset may be an invaluable tool for monitoring immunity at tumor site.

Regional Selectivity of rab5 and rab7 Protein Upregulation in Mild Cognitive Impairment and Alzheimer's Disease

Endocytic alterations are one of the earliest changes to occur in Alzheimer's disease (AD), and are hypothesized to be involved in the selective vulnerability of specific neuronal populations during the progression of AD. Previous microarray and real-time quantitative PCR experiments revealed an upregulation of the early endosomal effector rab5 and the late endosome constituent rab7 in the hippocampus of people with mild cognitive impairment (MCI) and AD. To assess whether these select rab GTPase gene expression changes are reflected in protein levels within selectively vulnerable brain regions (basal forebrain, frontal cortex, and hippocampus) and relatively spared areas (cerebellum and striatum), we performed immunoblot analysis using antibodies directed against rab5 and rab7 on postmortem human brain tissue harvested from cases with a premortem clinical diagnosis of no cognitive impairment (NCI), MCI, and AD. Results indicate selective upregulation of both rab5 and rab7 levels within basal forebrain, frontal cortex, and hippocampus in MCI and AD, which also correlated with Braak staging. In contrast, no differences in protein levels were found in the less vulnerable cerebellum and striatum. These regional immunoblot assays are consistent with single cell gene expression data, and provide protein-based evidence for endosomal markers contributing to the vulnerability of cell types within selective brain regions during the progression of AD.

Tarp regulates early Chlamydia-induced host cell survival through interactions with the human adaptor protein SHC1.

Many bacterial pathogens translocate effector proteins into host cells to manipulate host cell functions. Here, we used a protein microarray comprising virtually all human SRC homology 2 (SH2) and phosphotyrosine binding domains to comprehensively and quantitatively assess interactions between host cell proteins and the early phase Chlamydia trachomatis effector protein translocated actin-recruiting phosphoprotein (Tarp), which is rapidly tyrosine phosphorylated upon host cell entry. We discovered numerous novel interactions between human SH2 domains and phosphopeptides derived from Tarp. The adaptor protein SHC1 was among Tarp's strongest interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to tumor necrosis factor-induced apoptosis. Collectively, our findings reveal a critical role for SHC1 in early C. trachomatis-induced cell survival and suggest that Tarp functions as a multivalent phosphorylation-dependent signaling hub that is important during the early phase of chlamydial infection.

The role of Tyrosine kinase 2 on Th17 development in a murine model of allergic asthma (141.7)

Abstract Mutations of the Janus Kinase Tyrosine Kinase 2 (Tyk2) in humans are associated with hypereosinophilia, Hyper-IgE and susceptibility to viral and mycobacterial infections. This syndrome was found to be the result of defective signaling of both IL 12 and IFNalpha as well as abnormal responses to IL 6, IL-10 and IL-23. Here we describe asthmatic Tyk2 deficient mice suffered from extreme airway eosinophilic inflammation and hyper IgE production compared to wild type mice. As these symptoms are associated with an increase in Th2 cytokines IL-4 and IL-5 in the airways, the major goal of the project is to investigate the role of Tyk2 in CD4+ T cell subset differentiation in the lung in a murine model of acute asthma. Th2 and early Th17 effector T cells are important in establishing the inflammation seen in the airways in this disease. In contrast, regulatory T cells and late developed Th17 T cells have a protective function on it. Under Th17 skewing conditions naïve CD4+ T cells from Tyk2 deficient mice showed an IL-6-dependent defect on Th17 cell development. Consistently, we found hyper-proliferation of Th2 cells associated with a constitutive inhibition of the Th1 and Th17 pathway in the lung of asthmatic Tyk2 deficient mice compared to wild type mice. In summary these data suggest that overexpression of Tyk2 could lead to amelioration of the asthmatic symptoms because this would suppress the pathogenic disease-specific CD4+ T effector cells in the lung.

HVEM-LIGHT interactions promote generation of memory Th2 cells that mediate lung inflammation (91.8)

Abstract Memory Th2 cells are important in the pathogenesis of allergic responses including asthma, orchestrating recruitment of eosinophils and other inflammatory cells. Members of the tumor necrosis factor (TNF) receptor superfamily have emerged as key contributors to T cell activity, and therefore we investigated whether the engagement of the herpesvirus entry mediator (HVEM) on T cells by the TNF family molecule LIGHT controls Th2 mediated lung inflammation. Using systems where either HVEM or LIGHT are only defective on responding antigen-specific T cells, we demonstrated that the presence of HVEM on T cells is indispensible for the generation and persistence of Th2 memory cells and for allergen-induced Th2 lung inflammation, despite having a limited effect on early Th2 effector responses. Using adoptive co-transfer experiments we demonstrate that HVEM-LIGHT interaction on CD4 T cells can support longevity of T cells and promote memory generation. We also observed reduced activity of protein kinase B (PKB/Akt) and NF-κB in HVEM-deficient effector CD4 T cells, correlating with their poor survival. Furthermore, expression of an active form of PKB in HVEM-deficient T cells rescued Th2 cell survival in vivo, and restored lung inflammation in recall responses. Collectively, our results demonstrate a critical role of HVEM signals in the pathogenesis of Th2 derived lung inflammation and suggest that blocking of HVEM-LIGHT interactions could be a novel therapeutic target for asthma

CD4+ T cell regulation of CD25 expression controls the development of effector CD8+ T cells in acute and chronic virus infections. (137.14)

Abstract Both CD4+ T cell help and IL-2 have been postulated to ‘program’ activated CD8+ T cells for memory cell development. However, the link between these two signals has not been well elucidated. We have studied effector and memory CD8+ T cell differentiation following infection with vaccinia virus and murine gammaherpes virus in the absence of both CD4+ T cells and CD25. Expression of CD25 on antigen-specific CD8+ T cells peaked 3-4 days after infection and was dependent on CD4+ T cell help. CD4+ T cell or CD25-deficiency led to normal early effector CD8+ T cell differentiation, but a subsequent lack of accumulation of CD8+ T cells resulting in overall decreased memory cell generation. Interestingly, KLRG1high CD127low short-lived effector cells were diminished in the absence of IL-2 signaling, while memory precursors remained intact. Interestingly, the mononucleosis-like expansion of Vβ4+ T cells seen in mice infected with MHV-68 was impaired in a similar fashion to antigen specific responses. Upon secondary antigen encounter, CD25-deficient CD8+ T cells were capable of undergoing robust expansion, but short-lived effector development was again impaired. These results demonstrate that CD4+ help and IL-2 signaling are linked via CD25 upregulation, which controls the expansion and differentiation of antigen-specific effector CD8+ T cells.

A novel means of immunoregulation: CD46 attenuates effector responses of human conventional and unconventional T cells by distinct mechanisms (138.8)

Abstract Many studies including human genetics have implicated IL10-producing cells as key in limiting immune pathology during infection and in preventing autoimmunity. Here we show that the complement regulator CD46 regulates IL10 production in an IL2-dependent fashion. More specifically, CD3/CD46-activation of human CD4+ T cells in low IL2 induces strong IFNγ secretion, whereas increasing IL2 induces a ‘switch’ to immunosuppressive Tr1-like cells that are initially IFN-γ+/IL10+, and then IL-10+. We propose that CD46 activation initially promotes antimicrobial Th1 effector responses, while expansion of this response increases local IL2, promoting a CD46-dependent switch to IL10-mediated immunoregulation. The significance of this in vivo is supported by the failure of CD3/CD46-activated CD4+ T cells from rheumatoid arthritis patients to switch to the IL10+ state, producing ≥20-fold more IFNγ than IL10. IL10-switching of CD4+ T cells is mediated by the CYT-1 isoform of the alternatively spliced cytoplasmic tail of CD46. Interestingly, human γδ T cells, whose importance to the early effector phase of immune responses is increasingly apparent, express predominantly the CYT-2 isoform of CD46. Thus, they do not produce IL10 in response to TCR/CD46 co-engagement, but rather significantly decrease their IFNγ and TNFα production. Thus, we propose that CD46 uses distinct molecular mechanisms to regulate unconventional and conventional T cells across the span of the immune response.

Collapsin Response Mediator Protein 4a (CRMP4a) Is Upregulated in Motoneurons of Mutant SOD1 Mice and Can Trigger Motoneuron Axonal Degeneration and Cell Death

Embryonic motoneurons from mutant SOD1 (mSOD1) mouse models of amyotrophic lateral sclerosis (ALS), but not wild-type motoneurons, can be triggered to die by exposure to nitric oxide (NO), leading to activation of a motoneuron-specific signaling pathway downstream of the death receptor Fas/CD95. To identify effectors of mSOD1-dependent cell death, we performed a proteomic analysis. Treatment of cultured mSOD1 motoneurons with NO led to a 2.5-fold increase in levels of collapsin response mediator protein 4a (CRMP4a). In vivo, the percentage of mSOD1 lumbar motoneurons expressing CRMP4 in mSOD1 mice increased progressively from presymptomatic to early-onset stages, reaching a maximum of 25%. Forced adeno-associated virus (AAV)-mediated expression of CRMP4a in wild-type motoneurons in vitro triggered a process of axonal degeneration and cell death affecting 60% of motoneurons, whereas silencing of CRMP4a in mSOD1 motoneurons protected them from NO-induced death. In vivo, AAV-mediated overexpression of CRMP4a but not CRMP2 led to the death of 30% of the lumbar motoneurons and an 18% increase in denervation of neuromuscular junctions in the gastrocnemius muscle. Our data identify CRMP4a as a potential early effector in the neurodegenerative process in ALS.

CD4 + T cell regulation of CD25 expression controls development of short-lived effector CD8 + T cells in primary and secondary responses

Both CD4(+) T cell help and IL-2 have been postulated to "program" activated CD8(+) T cells for memory cell development. However, the linkage between these two signals has not been well elucidated. Here we have studied effector and memory CD8(+) T cell differentiation following infection with three pathogens (Listeria monocytogenes, vesicular stomatitis virus, and vaccinia virus) in the absence of both CD4(+) T cells and IL-2 signaling. We found that expression of CD25 on antigen-specific CD8(+) T cells peaked 3-4 days after initial priming and was dependent on CD4(+) T cell help, likely through a CD28:CD80/86 mediated pathway. CD4(+) T cell or CD25-deficiency led to normal early effector CD8(+) T cell differentiation, but a subsequent lack of accumulation of CD8(+) T cells resulting in overall decreased memory cell generation. Interestingly, in both primary and recall responses KLRG1(high) CD127(low) short-lived effector cells were drastically diminished in the absence of IL-2 signaling, although memory precursors remained intact. In contrast to previous reports, upon secondary antigen encounter CD25-deficient CD8(+) T cells were capable of undergoing robust expansion, but short-lived effector development was again impaired. Thus, these results demonstrated that CD4(+) T cell help and IL-2 signaling were linked via CD25 up-regulation, which controls the expansion and differentiation of antigen-specific effector CD8(+) T cells, rather than "programming" memory cell traits.

Abstract C238: Nov-002, a cellular redox modulator, enhances the antitumor effect of adoptively transferred T cells in a murine melanoma model

Abstract Oxidative signaling involving the glutathione system of redox control has been implicated in the regulation of T cell function. NOV-002, a glutathione disulfide mimetic, added to standard chemotherapy increased anti-tumor activity and survival in advanced non-small cell lung cancer patients compared to chemotherapy alone. Similarly, NOV-002 treatment significantly increased circulating T cell levels [CD4+, CD8+] in these patients. Here, we investigated whether the addition of NOV-002 to our previously established murine adoptive therapy (AT) regimen could enhance the anti-tumor activity of the transferred T cells. Adoptive transfer of early effector Pmel CD8+ cells activated in the presence of IL-12 (PmelIL12) into lymphopenic hosts is effective in regressing previously established B16 tumors. Daily administration of NOV-002 (25 mg/kg, i.p.) for 7 days following AT in C57BL/6 mice resulted in a significant delay in tumor progression when compared to mice subjected to AT alone, consistent with a marked enhancement of PmelIL12 anti-tumor immune activity. This enhanced anti-tumor effect was accompanied by a significantly longer median overall survival in mice treated with AT + NOV-002 compared to those treated with AT + saline [45 vs 28 days; 95% CI 34–56 days vs 23–32 days, respectively; p=0.02]. We then investigated, whether NOV-002 creates a more favorable micro-environment for expansion of transferred Pmel cells in a lymphopenic host. To this end, non-tumor bearing mice were treated with cyclophosphamide (CTX, 200mg/kg; to induce lymphopenia) or saline, and 7 days later 5×106 Pmel cells were adoptively transferred into all mice. In addition, mice were treated with either NOV-002 (25 mg/kg, i.p.) or saline daily for 7 days following AT. All mice were vaccinated with gp100 (the melanoma tumor antigen towards which the Pmel T cells are specifically reactive) 24 hours after AT to elicit in vivo activation and proliferation of transferred Pmel cells. On day 3 after vaccination, peripheral blood was collected, and analyzed by flow cytometry for the presence of donor Pmel cells (= Ly5.1+ cells) which represents T cell priming in response to vaccination. A significantly lower frequency of donor Pmel cells were seen in the animals treated with CTX relative to saline controls (9% vs. 20%; p=0.01). The addition of NOV-002 to CTX treatment, however, resulted in significantly higher frequencies of activated Pmel cells than CTX alone (18% vs 9%; p=0.03). Taken together, these results indicate that NOV-002 enhances the effect of AT thereby generating a significantly greater antitumor T cell response, resulting in decreased tumor growth, and improved survival. Moreover, NOV-002 enhances the expansion of CD8+ T cells in a lymphopenic host. These findings are consistent with the hypothesis that enhanced immune responsiveness may contribute to the clinical profile of NOV-002 in oncology trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C238.

Chk2 down-regulation by promoter hypermethylation in human bulk gliomas

Aims Gliomas account for 80% of malignant brain tumors. DNA damage response and subsequent checkpoint control pathways could maintain the integrity of the genome and thus defend tumorigenesis. Four kinases, ATM, ATR, ChK1 and Chk2 are the damage sensors and the early effectors in DNA damage responses. Given their importance, we investigated the transcriptional regulation of these four genes. Main methods Tissues from ten normal brains and thirty human gliomas were utilized for mRNA analysis via real-time PCR. Another twelve normal brain tissues and forty gliomas were used for confirmation. Methylation-specific PCR (MSP) was used to determine the methylation status of the Chk2 promoter. Quantitative chromatin immunoprecipitation (ChIP) was used to measure the influence of methylation on Sp1 binding. Key findings We found that the expression of ATR, ChK1 and Chk2 in gliomas was significantly down-regulated relative to the normal brain tissues. The most significant reduction of expression was of the Chk2 gene, whose expression was ∼ 10-fold decreased in gliomas ( P < 0.0001). Down-regulation of Chk2 was validated in the second real-time PCR analysis. This reduction in expression was partially due to promoter methylation. The Chk2 proximal promoter recruited Sp1 for transcriptional activation. We found that hypermethylation of the Chk2 promoter undermined the binding of the transcriptional factor Sp1. Significance Our results indicate that Chk2 methylation could be involved in glioma carcinogenesis and Chk2 expression may potentially be used for the diagnosis of glioma.

Lipid Rafts and Caveolin-1 Coordinate Interleukin-1β (IL-1β)-dependent Activation of NFκB by Controlling Endocytosis of Nox2 and IL-1β Receptor 1 from the Plasma Membrane

Recent evidence suggests that signaling by the proinflammatory cytokine interleukin-1beta (IL-1beta) is dependent on reactive oxygen species derived from NADPH oxidase. Redox signaling in response to IL-1beta is known to require endocytosis of its cognate receptor (IL-1R1) following ligand binding and the formation of redox-active signaling endosomes that contain Nox2 (also called redoxosomes). The consequent generation of reactive oxygen species by redoxosomes is responsible for the downstream recruitment of IL-1R1 effectors (IRAK, TRAF6, and IkappaB kinase kinases) and ultimately for activation of the transcription factor NFkappaB. Despite this knowledge of the signaling events that occur downstream of redoxosome formation, an understanding of the mechanisms that coordinate the genesis of redoxosomes following IL-1beta stimulation has been lacking. Here, we demonstrate that lipid rafts play an important role in this process. We show that Nox2 and IL-1R1 localize to plasma membrane lipid rafts in the unstimulated state and that IL-1beta signals caveolin-1-dependent endocytosis of both proteins into the redoxosome. We also show that inhibiting lipid raft-mediated endocytosis prevents NFkappaB activation. Finally, we demonstrate that Vav1, a Rac1 guanine exchange factor and activator of Nox2, is recruited to lipid rafts following IL-1beta stimulation and that it is required for NFkappaB activation. Our results fill in an important mechanistic gap in the understanding of early IL-1R1 and Nox2 signaling events that control NFkappaB activation, a redox-dependent process important in inflammation.

Immune Reconstitution: How It Should Work, What's Broken, and Why It Matters

Of the multiple cell lineages undergoing reconstitution in the setting of hematopoietic stem cell transplantation (HSCT), this overview will focus on reconstitution of the T cell arm of the immune system. In clinical HSCT, the autologous setting is free of constraints imposed by reactions to allogeneic antigens, but markers are lacking to distinguish reconstitution from transferred cells versus stem or progenitor cells left residual in the host. In allogeneic transplants, the converse is true – the stem cell origins of the reconstituted populations can be determined, but the biology of such reconstitution is confounded by concomitant allogeneic responses. Because of these constraints, the basic biology of T cell immune reconstitution was first delineated in mouse models in which the availability of congenic strains allowed stem cell origins of reconstituted cell populations to be investigated without the presence of confounding allogenic reactions. Two pathways of T cell regeneration were identified: the thymic-dependent maturation of new T cells from marrow progenitors, and thymic-independent expansion of mature peripheral T cells. 1 These initial studies also determined that no other pathways existed by which substantial peripheral populations of T cells were generated, and also established a means for investigating T cell reconstitution in humans by analyzing the expression of isoforms of the leukocyte common antigen CD45 as markers of naive and memory populations which correlated with the pathway of regeneration. They also demonstrated that homeostatic expansion of mature T cells occurred in lymphopenic hosts. Studies of such expansion have formed central concepts of homeostasis – that T cell levels are maintained by a balance between cytokine-consuming cells and homeostatic cytokines that support the maturation, proliferative expansion and survival of T cells.2 In translating these murine findings to humans, the recovery of CD4+ T cells in pediatric patients who were severely lymphopenic following chemotherapy treatment were initially tracked (Figure 1). There was a strong correlation between the recovery of total CD4+ T cell numbers and the recovery of naive, CD45RA+CD45RO- CD4+ T cells after as few as six months. 3 Furthermore, recovery of naive CD4+ cells was associated with a marked expansion of thymus volume, consistent with a dynamic regulation of the thymus.

Evidence that CT694 is a novel Chlamydia trachomatis T3S substrate capable of functioning during invasion or early cycle development

Chlamydia trachomatis is an obligate intracellular parasite, occupies a membrane-bound vacuole throughout development and is capable of manipulating the eukaryotic host by translocating effector molecules via a type III secretion system (T3SS). The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early cycle events. We present evidence here that the C. trachomatis protein CT694 represents an early cycle-associated effector protein. CT694 is secreted by the Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two-hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. Additionally, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibres that correlated with the ability of CT694 to interact with AHNAK. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis, and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development particular to this species.

MicroRNA in the immune system, microRNA as an immune system

The advent of microRNA has potentially uncovered a new level of complexity to be considered for every biological process. Through the modulation of transcription and translation, microRNA alter the basal state of cells and the outcome of stimulatory events. The exact effect of the microRNA network and individual microRNA on cellular processes is only just starting to be dissected. In the immune system, microRNA appear to have a key role in the early differentiation and effector differentiation of B cells. In T cells, microRNA have been shown to be key regulators of the lineage induction pathways, and to have a strong role in the induction, function and maintenance of the regulatory T-cell lineage. MicroRNA are also important for regulating the differentiation of dendritic cells and macrophages via toll-like receptors, with responsibilities in suppressing effector function before activation and enhancing function after stimulation. In addition to regulating key processes in the immune system, microRNA may also represent an archaic immune system themselves. Small interfering RNA of viral origin has been shown to function as an intracellular mediator in the suppression of viral infection in eukaryotes as diverse as plants, insects, nematodes and fungi, and there is growing evidence that endogenous mammalian microRNA can have similar impacts. In this article we speculate that the anti-viral function of microRNA drove the expression of different subsets of microRNA in different cellular lineages, which may have, in turn, led to the myriad of roles microRNA play in lineage differentiation and stability.

Ex vivo activity of early cytokine conditioned effector CD8+ T cells inversely correlates with their anti-tumor activity in vivo (41.1)

Abstract Enhanced effector activity such as cytokine secretion, cell proliferation , cytolytic activity, etc. in response to in vitro stimulation has been widely used as a means to predict in vivo activity after immune mediated interventions, such as adoptive transfer or systemic vaccination. Contrary to this, here we show that the enhanced in vivo survival and anti-tumor activity of cytokine conditioned early activated CD8+ T cells inversely correlates with their effector functions in vitro. When compared with late effector PmelIL-12, early effector showed decreased production of IFNγ and TNFα, and decreased expression of the degranulation marker CD107a in response to in vitro reactivation. However, after transfer into lymphodepleted hosts, early effector PmelIL-12 survived for at least 20 days and were able to effectively control the progression of B16 tumors in a superior manner than late effector PmelIL-12. Thus, effector activity in response to ex vivo antigenic restimulation does not necessarily predict in vivo activity. Other parameters such as surface expression of functional markers (CCR7, CD62L) and /or survival (CD127) could serve as better predictors of in vivo activity. Developing and validating accurate predictors of in vivo lymphocyte function would greatly facilitate our ability to rationally design novel immune based therapies for clinical trials that have significant anti-tumor activity.

Human memory CCR4+CD8+ T cell subset has the ability to produce multiple cytokines

The CC chemokine receptor (CCR)4 is associated with trafficking of specialized cutaneous memory type 2 T(h) cells in the skin. However, a CD8(+) T cell population expressing CCR4 still remains uncharacterized. In the present study, we investigated the expression and function of CCR4 on human CD8(+) T cells and characterized CCR4(+)CD8(+) human T cells. Multi-color flow cytometric analysis revealed that CCR4(+)CD8(+) T cells were predominantly found in the CD27(+)CD28(+)CD45RA(-) memory subset and expressed the CCR7(+/-)CCR5(-) phenotype. CCR4(+)CD8(+) T cells expressed neither perforin (Per) nor granzymes (Gra) A/B, suggesting that they were more immature memory T cells than the CCR6(+)CD8(+) early effector memory T cells that express GraA and Per. CCR4(+)CD8(+) T cells effectively produced IL-4, IFN-gamma, IL-2 and tumor necrosis factor-alpha, indicating that they are memory T cells having the ability to secrete type 1 and type 2 cytokines. These cells also showed chemotaxic activity in response to CC chemokine receptor ligand (CCL)17/thymus and activation-regulated chemokine and CCL22/macrophage-derived chemokine. These results suggest that CCR4(+)CD8(+) T cells are in an immature memory T cell subset in the differentiation pathway of human CD8(+) T cells and that they migrate to inflammatory sites in the skin where they are involved in cutaneous immunity.

Characterization of the recognition and functional heterogeneity exhibited by cytokine‐induced killer cell subsets against acute myeloid leukaemia target cell

The polyclonal cytokine-induced killer (CIK) cells exhibit potent cytotoxicity against a variety of tumour cells including autologous and allogeneic acute myeloid leukaemic (AML) targets. At maturity, three lymphocyte subsets: CD3(-) CD56(+), CD3(+) CD56(-) and CD3(+) CD56(+), constitute the bulk of the CIK cell culture. The CD3(-) CD56(+) subset behaves like classical natural killer (NK) cells where cytotoxicity is potentiated by blocking the human leucocyte antigen Class I molecules in the AML targets. Both the CD3(+) CD56(+) and CD3(+) CD56(-) subsets, though known to kill autologous and allogeneic targets to a comparable degree and therefore non-major histocompatibility complex (MHC)-restricted, nevertheless require the presence of the MHC molecule on the target, which interacts with their CD3-T-cell receptor complex. Although CIK cells are often termed 'NK-like' T cells, we have demonstrated that the well-characterized NK receptors KIR, NKG2C/E, NKG2D and DNAM-1 are not involved in the process of AML recognition for the CD3(+) CD56(-) and CD3(+) CD56(+) subsets. The CD3(+) CD56(+) and CD3(+) CD56(-) subsets express a polyclonal and comparable TCRVbeta repertoire in a Gaussian distribution. The CD3(+) CD56(+) subset kills AML targets more efficiently than its CD3(+) CD56(-) counterpart because of the presence of a higher proportion of CD8(+) cells. The CD3(+) CD56(+) subset comprise more terminally differentiated late effector T cells that bear the CD27(+) CD28(-) or CD27(-) CD28(-) phenotype, with a higher granzyme A content. In comparison, the phenotype of the CD3(+) CD56(-) subset is consistent with early effector T cells that are CD27(+) CD28(+) and CD62L(+), known to be less cytotoxic but possess greater proliferative potential.

DNA and oligosaccharides stimulate oligomerization of human milk lactoferrin

Lactoferrin (LF), the glycoprotein transferring Fe+ ions, is contained in barrier liquids, human blood and milk. LF is an acute phase protein and one of the most important factors of nonspecific defense. The protein has unique set of biological functions. Using the methods of small-angle X-ray scattering and light-scattering it was shown for the first time that addition of DNA and oligosaccharides to LF with different level of initial oligomerization leads to an enhance in the oligomerization rate. 1M NaCl stimulates almost a complete dissociation of LF oligomeric complexes obtained in the pesence of DNA, oligosaccharides, or early founded oligomerization effectors (nucleotides). It was shown that LF oligomeric complexes obtained in the presence of different oligomerization effectors have different stability. Incubation with 50 mM MgCl2 leads to complete destruction of the protein complexes formed by ATP and oligosaccharide but partially dissociate the complexes with following formation of new in the case of AMP- and d(pT)10-dependent associates, which possess higher stability i n presence of the salt. A possible role of LF oligomerization for different biological functions of the protein is discussed.

Leukotrienes Are Potent Adjuvant during Fungal Infection: Effects on Memory T Cells

Leukotrienes (LTs) are potent lipid mediators involved in the control of host defense. LTB(4) induces leukocyte accumulation, enhances phagocytosis and bacterial clearance, and increases NO synthesis. LTB(4) is also important in early effector T cell recruitment that is mediated by LTB(4) receptor 1, the high-affinity receptor for LTB(4). The aims of this study were to evaluate whether LTs are involved in the secondary immune response to vaccination in a murine model of Histoplasma capsulatum infection. Our results demonstrate that protection of wild-type mice immunized with cell-free Ags from H. capsulatum against histoplasmosis was associated with increased LTB(4) and IFN-gamma production as well as recruitment of memory T cells into the lungs. In contrast, cell-free Ag-immunized mice lacking 5-lipoxygenase(-/-), a critical enzyme involved in LT synthesis, displayed a marked decrease on recruitment of memory T cells to the lungs associated with increased synthesis of TGF-beta as well as IL-10. Strikingly, these effects were associated with increased mortality to 5-lipoxygenase(-/-)-infected mice. These data establish an important immunomodulatory role of LTs, in both the primary and secondary immune responses to histoplasmosis.