Human effector memory T cells express CD86: a functional role in naive T cell priming

The glycoprotein CD86 expressed on APCs provides a costimulatory signal necessary for an efficient activation of naive T cells. In contrast, there is controversy about the condition of expression and the function of CD86 on T cells. In this study, we have analyzed the phenotype and the biological activity of CD86+ T cells generated from human PBMC. Results show that CD86 expression on T cells is induced by long term stimulation via CD3 and IL-2R and is down-regulated as the cells become quiescent. The CD86-expressing cells are memory effector T cells: 1) they express CD45RO and high levels of the activation markers CD25, CD54, and HLA-Dr; 2) they selectively express CD30, CD40-ligand, and CD70; and 3) in response to stimulation, most of them produce IFN-gamma before dying by apoptosis. We then analyzed whether CD86 expressed on T cells is functional. Results show that paraformaldehyde-fixed CD86+ T cells enhance the proliferation and production of IFN-gamma by anti-CD3 mAb-stimulated naive T cells and induce proliferation of resting allogenic T cells. All these effects are prevented by neutralizing anti-CD86 mAbs. In contrast, we report no autocrine effect of CD86 in CD86+ T cell activation. In conclusion, these data show that human memory effector T cells express a functional form of CD86 that can costimulate naive T cell responses.     

The recirculation of naive and memory lymphocytes

It has been clearly shown that continuous recirculation of lymphocytes is crucial for the development of primary immune responses and that naive CD4 cells are distinguished from memory CD4 cells by differences in expression of several adhesion molecules. These findings suggest that changes in migratory behavior accompany the naive to memory cell transition. This area is first reviewed and then to evaluate this hypothesis, we compare the tissue distributions of highly purified naive and memory CD4 cells after transfer to syngeneic recipients. Naive cells which express high levels of L-selectin, and low levels of alpha 4 and beta 2 integrins, and CD44 localized in secondary lymphoid organs and were detectable in these tissues and in the blood for several weeks after transfer. Memory cells, which have a reciprocal phenotype, showed a markedly different distribution, particularly with respect to tissues where entry is controlled through high endothelial venules.     

Kinetics of the response of naive and memory CD8 T cells to antigen: similarities and differences

We have studied the kinetics of the antigen induced response of naive and memory CD8 T cells expressing a transgenic T cell receptor (TCR) specific for the glycoprotein peptide amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV). Memory T cells were generated in vivo by adoptive transfer of LCMV TCR transgenic T cells into normal recipient mice, followed by LCMV infection. The results demonstrated that the cell cycle progression and kinetics of TCR down-modulation, CD25 and CD69 up-regulation were identical in naive and memory T cells after antigen recognition. Moreover, the two T cell populations did not differ in respect of activation thresholds and in their proliferative capacities neither in vitro nor in vivo. However, memory CD8 T cells could be more rapidly induced to become cytolytic and to secrete high levels of interleukin-2 and interferon-gamma than naive T cells. LCMV GP33-specific CD8 memory T cells were only slightly more efficient in reducing LCMV titers in the spleen but were far more effective than naive LCMV GP33-specific T cells in controlling subcutaneous tumor growth of B16.F10 melanoma cells which expressed the LCMV GP33 epitope as tumor-associated antigen. Thus, in our experiments the main difference between CD8 memory T cells and naive cells is the ability of the former to rapidly acquire effector cell functions.     

Cytoskeletal proteins: the evolution of cell division

The prokaryotic cell division protein FtsZ and eukaryotic tubulin have been shown to have very similar structures and are most likely homologs. The evolutionary transition from FtsZ to tubulin could provide a window into the transition from prokaryotic cells to eukaryotic cells.     

Localization and function of early cell division proteins in filamentous Escherichia coli cells lacking phosphatidylethanolamine

Escherichia coli cells that contain the pss-93 null mutation are completely deficient in the major membrane phospholipid phosphatidylethanolamine (PE). Such cells are defective in cell division. To gain insight into how a phospholipid defect could block cytokinesis, we used fluorescence techniques on whole cells to investigate which step of the cell division cycle was affected. Several proteins essential for early steps in cytokinesis, such as FtsZ, ZipA, and FtsA, were able to localize as bands to potential division sites in pss-93 filaments, indicating that the generation and localization of potential division sites was not grossly affected by the absence of PE. However, there was no evidence of constriction at most of these potential division sites. FtsZ and green fluorescent protein (GFP) fusions to FtsZ and ZipA often formed spiral structures in these mutant filaments. This is the first report of spirals formed by wild-type FtsZ expressed at normal levels and by ZipA-GFP. The results suggest that the lack of PE may affect the correct interaction of FtsZ with membrane nucleation sites and alter FtsZ ring structure so as to prevent or delay its constriction.     

Lack of correlation between agglutinability, the surface distribution of con A and post-confluence inhibition of cell division in ten cell lines

The neoplastic cells from seven patients with childhood lymphoblastic lymphoma were studied for cell surface markers and surface morphology in the scanning electron microscope (SEM). The cells were studied for surface immunoglobulin (Slg), complement receptors (EAC), receptors for cytophilic antibody (IgGEA), and nonimmune rosette formation with sheep red blood cells (E). In one patient the cells exclusively bound E, suggesting a T-lymphocytic origin. In two patients the cells bound EAC, but demonstrated no other B-lymphocytic markers. In two patients no markers were detected, and in two patients receptors for both E and EAC were demonstrated. Additional studies in one of these patients permitted simultaneous demonstration of both markers on the same neoplastic cells. The neoplastic cells were also examined by SEM after fixation and critical point dehydration. No consistent surface morphology was observed. In four patients the cells were predominately smooth, whereas in two patients variable numbers of surface microvilli were present. A correlation of the surface features with membrane markers could not be established. A comparison of the surface markers with clinical and cytologic features revealed clinical homogeneity in spite of the heterogeneous immunologic markers. This heterogeneity was most likely a reflection of neoplastic alteration and disordered differentiation of the cells. The observation of complement receptors on the cells of four cases is a feature not previously reported in this disease and should be investigated in other presumed T-cell malignancies.     

Diaschisis in right putaminal hemorrhage: correlation with motor function

Local cerebral lesions may cause depression of function in remote areas of the brain presumably by a transneural mechanism; it has been called "diaschisis". In the present study, to investigate the relationship between motor function and "diaschisis" regional brain blood flow in hypertensive right putaminal hemorrhage was studied in 33 patients (mean age, 55 years; 22 men, 11 women). The hematoma was treated conservatively in 8 patients, aspirated stereotaxically in 9 patients, and evacuated through a craniotomy in 16 patients. The regional blood flow in the right motor cortex and the left cerebellar hemisphere was measured by a single photon emission CT with N-isopropyl-p- (123 I) iodo-amphetamine intravenous injection method, and was evaluated by the RI count on early image/the decay-corrected RI count on delayed image (E/D) value. The time during which regional brain blood flow was measured ranged from 29 to 35 days from the onset. There was a positive correlation between the grade in the motor function in the subacute stage and the regional blood flow in the right motor cortex and the left cerebellar hemisphere. There was also a positive correlation between the Barthel index in the chronic stage (mean follow-up periods: 40 months) and the regional blood flow in the right motor cortex and the left cerebellar hemisphere. The results of the present study suggest that in right putaminal hemorrhage the flow reduction in areas remote from the primary lesion, i.e., "diaschisis", may reflect not only the degree of functional abnormalities of the internal capsule, but also the possibility of functional recovery.     

Establishment of oral mucosa phenotype in vitro in correlation to epithelial anchorage

We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother-bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein-tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.     

A homozygous mutation in the luteinizing hormone receptor causes partial Leydig cell hypoplasia: correlation between receptor activity and phenotype

Leydig cell hypoplasia (LCH) is characterized by a decreased response of the Leydig cells to LH. As a result, patients with this syndrome display aberrant male development ranging from complete pseudohermaphroditism to males with micropenis but with otherwise normal sex characteristics. We have evaluated three brothers with a mild form of LCH. Analysis of their LH receptor (LHR) gene revealed a homozygous missense mutation resulting in a substitution of a lysine residue for a isoleucine residue at position 625 of the receptor. In vitro analysis of this mutant LHR, LHR(I625K), in HEK293 cells indicated that the signaling efficiency was significantly impaired, which explains the partial phenotype. We have compared this mutant LHR to two other mutant LHRs, LHR(A593P) and LHR(S616Y), identified in a complete and partial LCH patient, respectively. Although the ligand-binding affinity for all three mutant receptors was normal, the hormonal response of LHR(A593P) was completely absent and that of LHR(S616Y) and LHR(I625K) was severely impaired. Low cell surface expression explained the reduced response of LHR(S616Y), while for LHR(I625K) this diminished response was due to a combination of low cell surface expression and decreased coupling efficiency. For LHR(A593P), the absence of a reduced response resulted from both poor cell surface expression and a complete deficiency in coupling. Our experiments further show a clear correlation between the severity of the clinical phenotype of patients and overall receptor signal capacity, which is a combination of cell surface expression and coupling efficiency.     

Circulating CD20dim T-lymphocytes increase with age: evidence for a memory cytotoxic phenotype

The CD20 antigen has been regarded as a B lineage specific, 35 kDa, non-glycosylated membrane phosphoprotein, which functions as either a Ca2+ ion channel or as a regulatory protein of such a channel. Weak expression of CD20 (CD20dim), however, has recently been reported on a sub-population of T lymphocytes. We present results which confirm the existence of a CD20dim T lymphocyte population and show that such cells have a reduced antibody-binding capacity, when compared to CD20bright B-cells (10337 +/- 642 and 346311 +/- 24264 respectively). In addition, CD20dim cell counts vary with age, with the highest levels occurring in octogenarians: cord blood 0.3 +/- 0.1% (n = 13), 20-60 year-old group 2.1 +/- 1.1% (n = 18) and individuals > or = 61 years of age 6.9 +/- 3.2% (n = 10) (P < 0.001). Further characterization of CD20dim T cells, using three colour flow cytometry, demonstrated a predominantly memory cytotoxic phenotype, in that the cells were CD8+CD28+CD45RO+T-CR alpha beta +CD38-HLA-DR-.     

CD66: role in the regulation of neutrophil effector function

Neutrophils express several heavily glycosylated carcinoembryonic antigen (CEA)-related glycoproteins (CD66 antigens) which have been implicated in adhesion to E-selectin and as receptors for the lectins galectin 3 and bacterial type-1 fimbriae. The role of the CD66 antigens in neutrophil effector function was examined using non-cross-reacting and cross-reacting domain-mapped CD66 monoclonal antibody (mAb), which recognize epitopes on biliary glycoprotein (BGP; CD66a), CEA gene family member 6 (CGM6; CD66b), nonspecific cross-reacting antigen 90 (NCA90; CD66c) or CGM1 (CD66d). We show that BGP-specific mAb which recognize an AB-domain epitope strongly augment adhesion to fibrinogen by an Fc receptor- and beta2 integrin-dependent mechanism. Co-ligation of BGP with the glycophosphatidylinositol (GPI)-anchored CGM6 and NCA90 also caused increased beta2 integrin-mediated adhesion, receptor clustering and priming of formyl-Met-Leu-Phe (fMLP)-induced oxidant production by neutrophils, but only a small change in expression of L-selectin and CR3 compared to the chemotactic peptide fMLP. Ligation of CGM6 or NCA90 alone did not cause activation of the neutrophil in any of the assays used and did not cause priming of fMLP-induced oxidant production even when a secondary cross-linking reagent was used. We propose that specific cross-linking of neutrophil BGP with CGM6 and NCA90 contributes significantly to the regulation of neutrophil function during neutrophil recruitment.     

Priming of CD8+ CTL effector cells in mice by immunization with a stress protein-influenza virus nucleoprotein fusion molecule

Literature is accumulating which suggests the potential for stress proteins to form the basis of a novel vaccine technology. Immunization with mammalian tumor-derived stress proteins and their associated peptides promote anti-tumor immunity. Vaccination with HIV-1 p24 antigen fused to mycobacterial heat shock protein (Hsp) Hsp71 enhances p24-specific immunity, as measured by p24-specific antibody production and in vitro cell proliferation and cytokine induction. An ovalbumin-Hsp71 fusion protein primes ovalbumin-specific CTL activity and resistance to challenge with an ovalbumin-expressing tumor. We have extended these observations by using a mycobacterial Hsp65 fusion molecule to prime CTL specific for a viral antigen. Gene fusion constructs were generated from DNA encoding Mycobacterium bovis strain BCG Hsp65 and individual fragments of influenza virus nucleoprotein (NP) encompassing H-2Kd- and H-2Db-restricted CTL epitopes. The ability of these purified recombinant fusion proteins to prime NP-specific CTL was assessed in mice of appropriate H-2 haplotypes. We observed that adjuvant-free immunization with either fusion protein elicited significant CTL activity when administered at doses of 10-100 micrograms per mouse. An NP fusion protein made with glutathione-S-transferase failed to elicit NP-specific CTL, indicating that the phenomenon requires Hsp65 sequences. A single immunization with the Hsp65-NP fusion protein elicited CTL activity which persisted for a minimum of 4 months post-immunization, at which time it could be boosted by a second immunization. To our knowledge, this is the first report of a member of the Hsp60 family priming for antigen-specific CTL activity when employed as a fusion protein partner.     

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.     

In vitro responses of human CD45R0brightRA- and CD45R0-RAbright T cell subsets and their relationship to memory and naive T cells

The cellular basis of immunological memory, particularly with respect to T cells is not understood. In humans, monoclonal antibodies to CD45 have been used to identify memory (CD45R0) and naive (CD45RA) T cells. However, this identification has been called into question by various studies which suggest that high molecular weight CD45 isoforms may be re-expressed by previously activated cells. In the present study, using cultures which supported responses of naive T cells, we examined the responses of purified CD45R0brightRA- or CD45R0(-)-RAbright T cell subsets. The former subset was found to respond preferentially to recall antigens with minimal responses apparent to neo-(or non-recall)-antigens. The inverse pattern was found for CD45R0-RAbright T cells, which converted to CD45R0brightRA- after stimulation with a neo-antigen. Moreover, the two populations of T cells exhibited distinct response kinetics with a faster response evident from the CD45R0brightRA- T cells compared to the CD45R0-RAbright subset. The poor responses of CD45R0-RAbright T cells to recall antigens compared to neo-antigens suggests that this putative naive population is specifically depleted of reactive T cells following an encounter with antigen. We propose that T cell priming results in the stimulation of many CD45R0-RAbright T cells with various T cell receptor specificities from which memory T cells are selected for survival. If re-expression of higher molecular weight isoforms does occur in humans in vivo, our results suggest that R0 expression would be retained (CD45R0+RA+). Alternatively, if primed CD45R0-RAbright T cells exist, they are not prevalent in peripheral blood and thus may be sequestered within lymphoid tissues. Our data support the view that in human peripheral blood, CD45R0bright and CD45RAbright expression identify memory and naive CD4+ T cells, respectively.     

The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division

The Bacillus subtilis divIVA gene, first defined by a mutation giving rise to anucleate minicells, has been cloned and characterized. Depletion of DivIVA leads to inhibition of the initiation of cell division. The residual divisions that do occur are abnormally placed and sometimes misorientated relative to the long axis of the cell. The DivIVA phenotype can be suppressed by disruption of the MinCD division inhibitor, suggesting that DivIVA controls the topological specificity of MinCD action and thus septum positioning. A DivIVA-GFP fusion targets to new and used sites of cell division, consistent with it having a direct role in topological specification.     

Selective inhibition of T cell proliferation but not expression of effector function by human alveolar macrophages

BACKGROUND: Alveolar macrophages are thought to play an important part in regulating lung immune responses. While it is clear that human alveolar macrophages suppress T cell proliferation in vitro, the mechanisms by which this is achieved are not clear, nor is it known whether alveolar macrophages also inhibit other aspects of T cell function. METHODS: Peripheral blood mononuclear cells were stimulated with phytohaemagglutinin or house dust mite allergen, and cultured with variable numbers of autologous alveolar macrophages obtained by bronchoalveolar lavage from 20 normal subjects. RESULTS: Alveolar macrophages induced a reversible inhibition of T cell proliferation in response to both mitogen and allergen stimulation, with the latter being considerably more susceptible to inhibition. This was achieved via heterogenous mechanisms, involving both soluble factors derived from alveolar macrophages and cell-cell contact. Despite inhibiting proliferation, alveolar macrophages had little or no effect on T cell calcium flux, the characteristic changes in CD3, CD2, CD28 and interleukin-2 (IL-2) receptor expression which accompany normal T cell activation, and IL-2 and interferon gamma secretion. In contrast, alveolar macrophages inhibited the tyrosine phosphorylation of proteins which may be involved in IL-2 receptor-associated signal transduction. CONCLUSIONS: The immunoregulatory properties of alveolar macrophages are relatively selective, allowing T cell activation and cytokine secretion while inhibiting T cell proliferation within the lung.