The technical limitations of isolating neutrophils without contaminating leukocytes, while concurrently minimizing neutrophil activation, is a barrier to determining specific neutrophil functions. We aimed to assess the use of FACS for generating highly pure quiescent neutrophil populations in an antibody-free environment. Peripheral blood human granulocytes and murine bone marrow-derived neutrophils were isolated by discontinuous Percoll gradient and flow-sorted using FSC/SSC profiles and differences in autofluorescence. Postsort purity was assessed by morphological analysis and flow cytometry. Neutrophil activation was measured in unstimulated-unsorted and sorted cells and in response to fMLF, LTB4, and PAF by measuring shape change, CD62L, and CD11b expression; intracellular calcium flux; and chemotaxis. Cytokine production by human neutrophils was also determined. Postsort human neutrophil purity was 99.95% (sem=0.03; n=11; morphological analysis), and 99.68% were CD16(+ve) (sem=0.06; n=11), with similar results achieved for murine neutrophils. Flow sorting did not alter neutrophil activation or chemotaxis, relative to presorted cells, and no differences in response to agonists were observed. Stimulated neutrophils produced IL-1β, although to a lesser degree than CXCL8/IL-8. The exploitation of the difference in autofluorescence between neutrophils and eosinophils by FACS is a quick and effective method for generating highly purified populations for subsequent in vitro study.

Natalizumab treatment is associated with progressive multifocal leukoencephalopathy (PML) development. Treatment duration, prior immunosuppressant use, and JCV serostatus are currently used for risk stratification, but PML incidence stays high. Anti-JCV antibody index and L-selectin (CD62L) have been proposed as additional risk stratification parameters.

This study aimed at verifying and integrating both parameters into one algorithm for risk stratification.

Multicentric, international cohorts of natalizumab-treated MS patients were assessed for JCV index (1921 control patients and nine pre-PML patients) and CD62L (1410 control patients and 17 pre-PML patients).

CD62L values correlate with JCV serostatus, as well as JCV index values. Low CD62L in natalizumab-treated patients was confirmed and validated as a biomarker for PML risk with the risk factor "CD62L low" increasing a patient's relative risk 55-fold (p < 0.0001). Validation efforts established 86% sensitivity/91% specificity for CD62L and 100% sensitivity/59% specificity for JCV index as predictors of PML. Using both parameters identified 1.9% of natalizumab-treated patients in the reference center as the risk group.

Both JCV index and CD62L have merit for risk stratification and share a potential biological relationship with implications for general PML etiology. A risk algorithm incorporating both biomarkers could strongly reduce PML incidence.

Heterogeneity of lymphocyte populations demonstrates the diversity of cellular immune responses and provide a better understanding of the immune system. CD3+ CD8+ T cells exhibit a low CD8 expressing (CD8low) population in flow cytometric analysis of peripheral blood T cells. In healthy donors, this population consists of 0.2-7.0% of all CD8 T cells. The majority of the CD8low T cell population showed an elevated expression of CD25, CD45RA, and CD95L, and low levels of CD28, CD62L and CD45RO. Circulating CD8low T cells resemble cytotoxic effector cells because they express cytolytic mediators and are able to execute cytotoxicity. A restricted T cell receptor profile with increased Vbeta9, Vbeta14 and Vbeta23 expression was observed and the CD8low T cell population contain Epstein-Barr virus-specific T cells. Therefore, the CD8low population represent a subset of activated CD8 effector T cells, resulting most probably from a continuous and/or balanced immune response to intracellular pathogens.

The C1.7 Ag is a surface marker previously shown to be expressed on all NK cells and on a subset of CD8+ T cells. We report in this study that C1.7 Ag expression on peripheral blood-derived CD8+ T cells overlaps with activation markers S6F1high and CD29high and is reciprocally expressed with CD62L. C1.7 Ag expression can be induced in vitro on CD8+ T cells by anti-CD3 cross-linking, suggesting that C1.7 Ag is activation dependent. In contrast to NK cells, C1.7 Ag does not signal on CD8+ T cells, nor does it induce redirected lysis upon ligation. The proportion of C1.7 Ag+CD8+ T cells is increased in HIV-infected patients compared with healthy donors. In 69 HIV-infected patients, we observed a significant inverse correlation between the percentage of C1.7 Ag-expressing CD8+ T cells and the absolute CD4+ T cell count. Two-year clinical follow-up of patients with initial CD4+ T cell count of >400 cells/mm3 and a normal proportion of C1.7 Ag+CD8+ T cells revealed that these patients were clinically stable with minimal HIV-associated symptoms. In contrast, 10 of 12 patients with CD4+ T cell counts of >400 cells/mm3 and an elevated proportion of C1.7 Ag+CD8+ T cells were symptomatic. ANOVA analysis of patients indicates that C1.7 Ag is a better predictor of disease progression than CD4 count. Overall, our findings indicate that C1.7 Ag is the first described marker for activated/memory CD8+ T cells and a useful parameter for evaluating the level of CD8+ T cell activation in vivo.

Human MM is a haematologic disorder characterized by the accumulation of malignant plasma cells (PC), primarily in the bone marrow (BM). Although these cells characteristically home to the BM, in recent years several groups have detected the presence of related malignant B cells in the peripheral blood (PB) which could be implicated in the progression and spread of the disease. However, the proportion and origin of these clonotypic circulating B cells is still controversial. In this study, using a triple-staining flow cytometric procedure and a whole blood lysis method, PB B lineage cells could be divided into two populations according to their distinct repertoires of cell adhesion molecules and B cell antigens in untreated MM patients. The results show that: (i) the percentage and the absolute number of PB CD19+ B cells were decreased in MM patients compared with controls; (ii) the quantity and percentage of B cell antigens (CD20, CD22, CD24, DR, CD138) and adhesion molecules (beta1- and beta2-integrins, CD44, CD54, CD56, CD61 and CD62L) expressed by these PB CD19+ cells of MM patients and healthy subjects were similar and all of them were virtually polyclonal cells; (iii) a very minor circulating CD19-CD38++CD45-/dim subset was also detected which expressed CD138 (B-B4) (high intensity), monoclonal cytoplasmic immunoglobulin (cIg), and was negative for pan-B antigens (CD19, CD20, CD24, DR), surface immunoglobulin (sIg) and several adhesion molecules such as CD62L, CD18 and CD11a; this CD19-CD38++CD45-/dim CD138++ subset was not found in normal blood and exhibited a phenotypic profile which was closely related to that of malignant BM plasma cells, with the exception of the CD56 antigen. Polymerase chain reaction (PCR) analysis of IgH clonotypic rearrangements confirmed these results. We postulate that, in MM patients, circulating B lineage cells may be divided into two different categories: polyclonal CD19+ B cells and a very minor proportion of clonal CD138++ PC that escape from the BM.

L-selectin mediates tethering and rolling of lymphocytes in high endothelial venules (HEV) of lymph nodes (LN) and of leukocytes at inflammatory sites. We used transgenic mice expressing varying levels of wild-type or a non-cleavable mutant form of L-selectin on T cells to determine the relationship between L-selectin density, tethering and rolling, and migration into LN. T cells expressing supraphysiological levels of either wild-type or non-cleavable L-selectin showed rolling parameters similar to C57BL/6 T cells in hydrodynamic flow assays and during rolling in Peyer's patch HEV. In contrast, PMA- or antigen-activated T cells and L-selectin(+/-) T cells expressing subphysiological levels of L-selectin showed reduced numbers of rolling cells with increased rolling velocity. Short-term homing studies showed that elevated expression of L-selectin above physiological levels had no effect on T cell migration to LN; however, low L-selectin expression resulted in reduced T cell homing to LN. Thus, T lymphocyte migration into LN is regulated by the density of cell surface L-selectin. In addition, there is a saturable density of L-selectin required for optimal homing to PLN in C57BL/6 mice, the L-selectin level on circulating naive T cells promotes optimal homing, and increased expression above saturating levels promotes no further increase in T cell recruitment.

OBJECTIVE

Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation.

METHODS

We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8(+) T cells with human leukocyte antigen (HLA) class I-matched AML blasts in microtiter plates. Before culture, CD8(+) T cells were separated into CD62L((high)+) and CD62L((low)+/neg) subsets enriched for naive/central memory and effector memory cells, respectively.

RESULTS

In eight different related and unrelated donor/AML pairs, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs expressed T-cell receptors of single Vbeta-chain families, indicating their clonal origin. The majority of CTL clones were obtained from mini-MLLCs initiated with CD62L((high)+) cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10(8)-10(10) cells within 6 to 8 weeks. Three of four representative CTL clones were capable of completely preventing engraftment of human primary AML blasts in nonobese diabetic/severe combined immune deficient IL2Rgamma(null) mice.

CONCLUSIONS

The mini-MLLC approach allows the efficient in vitro expansion of AML-reactive CTL clones from CD8(+)CD62L((high)+) precursors of healthy donors. These CTLs can inhibit leukemia engraftment in immunodeficient mice, suggesting their potential biological relevance.

Previous studies have demonstrated the utility of S100B as a surrogate marker of brain-related pathologies, e.g. neuropsychiatric disorders, and melanoma progression, which have an inflammatory component. This study addresses the relevance of S100B(+) lymphocytes in mediating such responses. S100B expression was determined in human peripheral blood leukocytes isolated from healthy volunteers using flow cytometry. S100B(+) lymphocytes were characterised for phenotype, cytokine production and S100B secretion. In addition, we investigated whether S100B activates monocytes and neutrophils. S100B(+) cells comprised 2-4% of all lymphocytes and the majority displayed a CD3(+) CD8(+) phenotype; fewer cells were CD3(-) CD56(+) NK lymphocytes. Comparison of S100B(+) and S100B(-) CD3(+) CD8(+) cells revealed no differences in production of interferon gamma (IFNγ) and interleukin-2 (IL-2). Stimulation of S100B(+) CD3(+) CD8(+) lymphocytes with anti-CD3 or phytohaemagglutinin resulted in release of S100B. High concentrations of recombinant human S100B triggered upregulation of CD11b and membrane shedding of CD62L in granulocytes and monocytes. These findings set the stage for a new field of research addressing a S100B-mediated crosstalk between the innate and adaptive immune systems if close proximity of effector and responder cells accomplishes sufficient local S100B levels. In various physiological and pathological conditions S100B might function as an interface to immunological processes, distinct from known cytokine- and chemokine-mediated pathways.

To examine mechanisms of mobilization and homing of hematopoietic progenitor cells, coexpression of CD34 and the adhesion molecules L-selectin (CD62L), VLA-4 (alpha 4 beta 1-integrin, CD49d/CD29), and LFA-1 (alpha L beta 2-integrin, CD11a/CD18) was evaluated. Samples from leukapheresis (LP) products and bone marrow (BM) were obtained on the same day from patients who received granulocyte colony-stimulating factor (G-CSF) after cytotoxic chemotherapy. The proportion of CD34+ cells expressing L-selectin tended to be greater in LP products compared with BM. In samples from both sources, the mean fluorescence intensity of CD34 was significantly greater on CD34+/L-selectin-positive cells compared with the CD34+/L-selectin-negative cell subset. Three-color immunofluorescence showed that early CD34+/HLA-DRdim or CD34+/HLA-DR- progenitor cells were strongly positive for L-selectin, whereas L-selectin-negative cells were only found in the CD34+HLA-DRbright subset. The mean fluoresence intensity of VLA-4 and LFA-1 was significantly greater on CD34+ cells from BM compared with LP products. Moreover, a distinct population of CD34dim/VLA-4bright and CD34dim/LFA-1bright cells was found only in samples from BM. This subset may be enriched for myeloid progenitor cells, since the cloning efficiency of CD34+ cells for CFU-GM was significantly greater in BM samples than in LP products. Binding of CD34+ cells to endothelial cells was partially inhibited by a blocking antibody to beta 2-integrin. In conclusion, L-selectin is expressed in significant amounts on more primitive CD34+ cells which circulate in considerable numbers in the peripheral blood. This suggests that L-selectin plays a role in redistribution and homing of hematopoietic progenitor cells to the bone marrow following cytotoxic damage. Conversely, strong expression of VLA-4 and LFA-1 was mainly found on lineage-committed progenitor cells of the bone marrow.

CD4+CD25+ regulatory T cells (T(reg)) play an important role in maintaining immunologic tolerance. Glucocorticoid-induced TNFR family-related gene (GITR) expressed preferentially at high levels on T(reg) has been shown to be a key player of regulating T(reg)-mediated suppression. A recent study reports that NF-kappaB-inducing kinase (NIK) expression in thymic stroma is important for the normal production of T(reg) but not for its suppression capacity. In this report, we have shown that T(reg) from NIK-deficient mice display hyperproliferative activities upon GITR stimulation through an IL-2-independent mechanism. Furthermore, high dose IL-2, anti-CD28 stimulation, or GITR ligand-transduced bone marrow-derived dendritic cells used as APC (culture conditions which drive T(reg) proliferation in vitro) could not ablate this difference in proliferative activity between NIK-deficient and wild-type T(reg). Additional experiments have shown NIK-deficient mice have a higher ratio of CD4+CD25+CD62L(low) T(reg) both in thymus and periphery than their wild-type littermates. This CD62(low) subset is responsible for the hyperproliferative activity upon GITR stimulation. These data suggest a novel role of NIK in controlling the development and expansion of CD4+CD25+ regulatory T cells.

BACKGROUND

Mechanisms underlying low-penetrance, common, non-protein coding variants in breast cancer risk loci are largely undefined. We showed previously that the non-protein coding mammary carcinoma susceptibility locus Mcs5a/MCS5A modulates breast cancer risk in rats and women. The Mcs5a allele from the Wistar-Kyoto (WKy) rat strain consists of two genetically interacting elements that have to be present on the same chromosome to confer mammary carcinoma resistance. We also found that the two interacting elements of the resistant allele are required for the downregulation of transcript levels of the Fbxo10 gene specifically in T-cells. Here we describe mechanisms through which Mcs5a may reduce mammary carcinoma susceptibility.

METHODS

We performed mammary carcinoma multiplicity studies with three mammary carcinoma-inducing treatments, namely 7,12-dimethylbenz(a)anthracene (DMBA) and N-nitroso-N-methylurea (NMU) carcinogenesis, and mammary ductal infusion of retrovirus expressing the activated HER2/neu oncogene. We used mammary gland and bone marrow transplantation assays to assess the target tissue of Mcs5a activity. We used immunophenotyping assays on well-defined congenic rat lines carrying susceptible and resistant Mcs5a alleles to identify changes in T-cell homeostasis and function associated with resistance.

RESULTS

We show that Mcs5a acts beyond the initial step of mammary epithelial cell transformation, during early cancer progression. We show that Mcs5a controls susceptibility in a non-mammary cell-autonomous manner through the immune system. The resistant Mcs5a allele was found to be associated with an overabundance of gd T-cell receptor (TCR)+ T-cells as well as a CD62L (L-selectin)-high population of all T-cell classes. In contrast to in mammary carcinoma, gdTCR+ T-cells are the predominant T-cell type in the mammary gland and were found to be overabundant in the mammary epithelium of Mcs5a resistant congenic rats. Most of them simultaneously expressed the CD4, CD8, and CD161α markers. In cultured T-cells of Mcs5a resistant congenic rats we found increased mitogen-induced proliferation and production of Th1 cytokines IFNg, IL-2, and Tumor Necrosis Factor (TNF), but not Th2 cytokines IL-4 and IL-6, or Th17 cytokine IL-17 when compared with susceptible control rats.

CONCLUSIONS

These data support a hypothesis that Mcs5a displays a non-mammary cell-autonomous mechanism of action to modulate breast cancer risk through the immune system. The resistant Mcs5a allele is associated with alterations in T-cell homeostasis and functions, and overabundance of γδTCR+ T-cells in carcinogen-exposed mammary epithelium.

A better understanding of protective immune memory against contagious bovine pleuropneumonia (CBPP) is needed in order to facilitate the development of safer vaccines based on selected components of the pathogen. For this purpose, cells collected from lymph nodes draining the lungs of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC)-infected cattle were stimulated with the pathogen in vitro and evaluated concurrently for proliferation (CFSE based method), expression of activation, memory markers and cytokine production. Direct evidence is presented for a major contribution of CD4+ T cells to the vigorous proliferative and T1 biased cytokine recall responses observed in cattle that have recovered from infection but not in animals developing the acute form of the disease. Two different phenotypes of MmmSC-specific memory CD4 were observed based on CD62L expression and proliferative capacities. Furthermore, recall proliferation of B cells also occurred but was strictly dependent on the presence of CD4. The information provided in this study will facilitate the search for MmmSC antigens that have potential for the development of subunit vaccines against CBPP.

OBJECTIVE

Interferon (IFN)-alpha is an effective drug for treatment of uveitis in Behçet's disease. This study was undertaken to investigate the mechanism of action of IFN-alpha in the treatment of various types of noninfectious sight-threatening uveitis.

METHODS

Eleven patients with refractory uveitis, and 13 healthy individuals were enrolled. The number of circulating plasmacytoid dendritic cells (pDCs) and their capacity to produce IFN-alpha in culture on stimulation with synthetic oligodinucleotides containing the CpG-motif were studied. Peripheral blood CD4+ T-cell phenotype and activation status were evaluated by flow cytometry at 0, 2, and 8 weeks after treatment for expression of CD69, CD62L, chemokine receptors (CCR4, CXCR3, and CCR5), and intracellular cytokines (TNF-alpha, IFN-gamma, and IL-10).

RESULTS

All patients experienced a positive clinical response to IFN-alpha treatment. There was no significant difference between patients and control subjects in the number of circulating pDCs, but there was a significant decrease in the capability of patients' pDCs to produce IFN-alpha in response to CpG (P < 0.001). Peripheral blood CD4+ T cells expressed reduced levels of surface CD62L (P < 0.005) as a measure of activation and higher levels of chemokine receptors CXCR3, CCR4, and CCR5 (P < 0.005, P < 0.05, and P < 0.05, respectively); in addition, intracellular T-cell IL-10 levels were increased once the treatment was initiated (P < 0.01).

CONCLUSIONS

The data suggest that IFN-alpha may control uveitis by promoting induction of IL-10-producing T-cells, possibly T-regulatory cells. Dysregulation of the T-cell population in patients with uveitis may be associated with a defect in the pDCs' ability to produce IFN-alpha, which can be circumvented with administration of exogenous IFN-alpha.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autoimmune disease that is caused by mutations in the AIRE gene. Murine studies have linked AIRE to thymocyte selection and peripheral deletional tolerance, but the pathogenesis of the human disease remains unclear. In this study, we show that APECED patients have elevated IL-7 levels and a drastically decreased expression of IL-7R on CD8(+) T cells. This is associated with increased proliferation and a decreased expression of the negative TCR regulator CD5 in the CD45RO(-) subset. The CD45RO(-) cells also display oligoclonal expansions, decreased expression of the lymph node homing factors CCR7 and CD62L, and increased expression of perforin, consistent with the accumulation of highly differentiated effector cells. The CD45RO(-)CCR7(+)CD8(+) population of cells with markers characteristic of naive phenotype is also skewed, as shown by decreased expression of CD5 and increased expression of perforin. The putative CD31(+) recent thymic emigrant population is likewise affected. These data are consistent with IL-7 dysregulation inducing a decreased threshold of TCR signaling and self-antigen-driven proliferation, probably in synergy with the failed thymic selection. The resultant loss of CD8(+) T cell homeostasis is likely to play a significant role in the pathogenesis of APECED. Our findings may also hold lessons for other diseases in which the IL-7-IL-7R pathway has emerged as a risk factor.

Mycobacterium bovis infection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4(+) cells from infected animals. CD62L MFI on PPD- and PWM-stimulated gammadelta T-cell receptor-positive (TCR(+)) and CD8(+) cells was also reduced compared to that of nonstimulated gammadelta TCR(+) and CD8(+) cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4(+) cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.

Dendritic cells (DCs) are professional antigen presenting cells with the ability to induce and regulate an immune response. DCs that capture and present antigen under noninflammatory conditions maintain an immature phenotype and acquire tolerogenic properties. These DCs generate regulatory T lymphocytes that potentiate tolerogenic responses. Here we developed a method for the generation of immature murine DCs able to process and present a specific antigen in a tolerogenic context. Immature DCs were prepared from bone marrow precursors after differentiation with granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence of vitamin D(3) and characterized by their low expression of major histocompatibility complex class (MHC) II and CD86 molecules. Purified phagosomes containing either MHC II molecules or ovalbumin were used to deliver antigens to immature DCs. More than 80% of the DCs captured the phagosomes, while maintaining a low expression of maturation markers and showing basal levels of secretion of activating cytokines such as interleukin (IL)-2 and IL-12. Treatment of the immature DCs with lipopolysaccharides (LPS) increased IL-10 secretion, in agreement with their anti-inflammatory and immune regulatory properties. Cocultures of transgenic OT-II T lymphocytes with the immature DCs carrying OVA-phagosomes succeeded in generating a subpopulation of regulatory T lymphocytes characterized by the expression of CD4, CD25, CD62L, and Foxp3. Taken together, our results suggest that vitamin D(3) generates immune tolerance through the modulation of DC phenotype and could be useful to induce tolerance to allotransplants.

Genetic modification of human T cells to express transgene-encoded polypeptides, such as tumor targeting chimeric antigen receptors, is an emerging therapeutic modality showing promise in clinical trials. The development of simple and efficient techniques for purifying transgene(+) T cells is needed to facilitate the derivation of cell products with uniform potency and purity. Unlike selection platforms that utilize physical methods (immunomagnetic or sorting) that are technically cumbersome and limited by the expense and availability of clinical-grade components, we focused on designing a selection system on the basis of the pharmaceutical drug methotrexate (MTX), a potent allosteric inhibitor of human dihydrofolate reductase (DHFR). Here, we describe the development of self inactivating (SIN) lentiviral vectors that direct the coordinated expression of a CD19-specific chimeric antigen receptor (CAR), the human EGFRt tracking/suicide construct, and a methotrexate-resistant human DHFR mutein (huDHFR(FS); L22F, F31S). Our results demonstrate that huDHFR(FS) expression renders lentivirally transduced primary human CD45RO(+)CD62L(+) central memory T cells resistant to lymphotoxic concentrations of MTX up to 0.1 μM. Our modular complementary DNA (cDNA) design insures that selected MTX-resistant T cells co-express functionally relevant levels of the CD19-specific CAR and EGFRt. This selection system on the basis of huDHFR(FS) and MTX has considerable potential utility in the manufacturing of clinical-grade T cell products.

Regulatory T cells (Treg) have been shown to play a role in the prevention of autoimmune diseases and transplant rejection. Based on an established protocol known to generate alloantigen reactive Treg in vivo, we have developed a strategy for the in vitro selection of Treg. Stimulation of unfractionated CD4(+) T cells from naive CBA.Ca (H2(k)) mice with C57BL/10 (H2(b)) splenocytes in the presence of an anti-CD4 antibody, YTS 177, resulted in the selection of Treg able to inhibit proliferation of naive T cells. In vivo, the cells were able to prevent rejection of 80% C57BL/10 skin grafts when co-transferred to CBA.Rag(-/-) mice together with naive CD45RB(high)CD4(+) cells. Purification of CD62L(+)CD25(+)CD4(+) cells from the cultures enriched for cells with regulatory activity; as now 100% survival of C57BL/10 skin grafts was achieved. Furthermore, differentiation of Treg could be also achieved when using purified CD25(-)CD4(+) naive T cells as a starting population. Interestingly, further in vitro expansion resulted in a partial loss of CD4(+) cells expressing both CD62L and CD25 and abrogation of their regulatory activity in vivo. This study shows that alloantigen stimulation in the presence of anti-CD4 in vitro provides a simple and effective strategy to generate alloreactive Treg.

Expression of chemokine receptors and beta-chemokine production by peripheral blood mononuclear cells (PBMC) were determined in HIV-1-infected individuals before and after highly active anti-retroviral therapy (HAART) and their relationship to viral load, T cell phenotype and the expression of immunological activation markers was examined. We found that the expression of CCR5 is up-regulated in HIV-1-infected individuals while CXCR4 appears down-regulated on both CD4 and CD8 T cells compared with normal controls. These alterations are associated with the high levels of viral load. In addition, a relationship was observed between the degree of immune activation and chemokine receptor expression on T cells. However, after 3 months of combined anti-retroviral regimen, expression of CXCR4 significantly increased while CCR5 decreased when compared with pretherapy determinations. This was seen in strict association with a dramatic decrease of viral load and an increase of both CD45RA+/CD62L+ (naive) and CD45RA-/CD62L+ or CD45RA+/CD62L- (memory) T cells accompanied by a significant decrease of the expression of immune activation markers such as HLA-DR and CD38. At enrolment, both spontaneous and lectin-induced RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta production by PBMC were higher in HIV-1-infected individuals compared with normal controls, although differences for MIP-1beta were not statistically significant. However, RANTES and MIP-1alpha production decreased during HAART at levels closer to that determined with normal controls, while MIP-1beta production was less consistently modified. These data indicate that the expression of chemokine receptors CCR5 and CXCR4 and the production of beta-chemokines are altered in HIV-infected individuals, and suggest that their early modifications during HAART reflect both the peripheral redistribution of naive/memory T cell compartments and the decrease in levels of T cell activation. Such modifications in the expression of host determinants of viral tropism and the production of anti-viral molecules may play a role in the emergence of virus variants when a failure of HAART occurs.

Central memory T cells (Tcm) have not previously been characterized in cattle and any other ruminant species. Here we described two phenotypically and functionally different subsets of pathogen-specific memory CD4(+) T cells in cattle that survived infection with Mycoplasma mycoides subsp. mycoides small colony (MmmSC). The first subset is CD45RO(+)CD45R(-)CD62L(-) and comprises two thirds of IFN-gamma producing CD4(+) T cells after MmmSC recall stimulation. The second is CD45RO(+)CD45R(-)CD62L(+) and represents the majority of proliferating CD4(+) T cells after 7 days of stimulation. Cell sorting experiments confirmed that both CD4(+)CD62L(+) and CD4(+)CD62L(-) subsets are present in vivo and proliferate independently in recall responses to MmmSC. In addition, MmmSC stimulation strongly decreased CCR7 and increased CCR5 transcripts levels in CD4(+)CD62L(-) cells whereas CD4(+)CD62L(+) were only slightly affected. High levels of recall proliferation but low IFN-gamma production, together with the capacity to preferentially migrate through the lymph nodes (i.e., expression of CD62L and CCR7), are characteristics of Tcm, in humans and mice. Tcm are associated with long-term protective immunity and a privileged target for vaccine development. Our results demonstrate the existence of Tcm in cattle and suggest that CD62L may serve as a marker to monitor Tcm in infections and vaccine development studies in ruminant.

The pathogenesis of posttraumatic osteomyelitis, one of the major complications after orthopedic surgery, is not yet understood. Formation of bacterial biofilms on the implant is presumed, conferring resistance to antibiotic therapy and probably also to the host defense mechanisms. In that context, the polymorphonuclear neutrophils (PMN) having infiltrated the infected site were recovered and characterized phenotypically and functionally. Loss of CD62L and upregulation of CD14 were seen, as was expression of CD83. Expression of the latter is dependent on de novo protein synthesis and thus is indicative of an extended life span and a transdifferentiation of the PMN at the infected site. The infiltrated PMN had lost their chemotactic activity, whereas the capacity to produce superoxides was preserved and in some patients even enhanced. In vitro experiments done in parallel showed that long-term culture with interferon-gamma resulted in similar alterations of PMN: loss of chemotactic activity, whereas other functions of PMN, such generation of superoxides and phagocytosis of opsonized bacteria, were preserved or even enhanced. The loss of the migratory capacity of PMN having already emigrated from the blood vessel to the infected site is not expected to affect the host defense negatively. Assuming, however, that bacteria are organized as a biofilm and that infiltration into this biofilm is required for phagocytosis of the bacteria, our data could to some extent explain why despite being activated, the PMN are not able to control the infection. By releasing their cytotoxic, proteolytic, and collagenolytic potential, PMN might instead contribute to tissue destruction and eventually to osteolysis.

In the presence of TGF-β, CD4+CD62L+T cells can be induced to CD4+CD25+FoxP3+ regulatory T cells (iTregs). In our previous work, we have shown that adoptive transfer of iTregs promoted liver recovery from ischemia reperfusion injury (IRI). In this study, we examined the molecular mechanism underlying the liver IRI attenuation by iTregs in a mouse partial hepatic IRI model. We found that the population of hepatic Tregs decreased significantly at 24 h after reperfusion. Adoptive transfer of iTregs before IRI markedly increased the numbers of hepatic Tregs and attenuated liver IRI as indicated by reduced serum aminotransferases and proinflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). Ex vivo study indicated that iTregs suppressed IL-1β and TNF-α expression, promoted transcription of interleukin-10 (IL-10), and elevated phosphorylation of SMAD3 in Kupffer cells (KCs). Furthermore, inhibition of TGF-β signaling by anti-TGF-β abolished the effects on KCs. Treatment with TGF-β suppressed matrix metalloprotease (MMP9) production in KCs and protected liver from IRI. In conclusion, our results suggest that iTregs play a critical role in hepatic IRI by regulating pro-inflammatory and anti-inflammatory function of KCs through TGF-β.

This study explores whether previous failures on antiretroviral drug regimens preclude the possibility of immune restoration. This was assessed by evaluating T cell subset changes in individuals who received a salvage regimen of highly active antiretroviral therapy (HAART) after initially failing protease inhibitor monotherapy. Ten HIV-1-infected asymptomatic patients received a regimen of indinavir, zidovudine, and 3TC after failing saquinavir monotherapy. Changes in absolute numbers of naive, memory, and activated CD4+ and CD8+ T cells expressing a selection of CD45RA, CD62L, CD45RO, HLA-DR, and CD38 markers were monitored prospectively over 6 months. These measurements were correlated with plasma viral load along with alterations in a selected CD8+ V alpha/Vbeta T cell receptor (TCR) repertoire. Over 6 months there was a progressive increase in numbers of CD4+ memory (CD45RA-CD62L+) and naive (CD45RA+CD62L+) T cells, which displayed a modest inverse correlation with viral load. Two phases of CD8+ memory cell changes were identified, consisting of a transient increase in CD45RA+CD62L- numbers after 2 months and thereafter a progressive rise in CD45RA-CD62L+ cells until 6 months. A strong correlation existed between reduced viral load and loss of activated CD8+CD38+HLA-DR+ cell numbers. There was also a temporary broadening of the CD8+ V alpha/Vbeta TCR repertoire at 8 weeks, which became skewed after 6 months in parallel with reduced viral suppression. Closer analysis of naive and memory cell subset proportions in individual patients revealed that enlarged pools of naive subsets were evident in those patients with rebounds in viral load. Overall, drug-experienced patients responding to HAART displayed increased numbers of naive and memory CD4+ subsets, and reduced CD8+ cell activation with a loss of TCR skewing.

Type B gastritis in its active form is characterized by a dense infiltration of the lamina propria with granulocytes. Since the bacterium Helicobacter pylori does not invade the epithelial barrier, a signaling pathway chemoattractive for granulocytes must exist across this mucosal boarder. One possible mechanism tested was whether granulocytes are directly activated by water-soluble membrane proteins (WSP) from H. pylori. These findings were compared with the effects of WSP from other bacteria (Helicobacter felis, Campylobacter jejuni, Escherichia coli, and Staphylococcus aureus). A unique activation pattern by H. pylori WSP was found. Like all other WSP tested, they induced an upregulation of CD11b but had no influence on CD11c and, most strikingly, CD62L expression. In contrast, E. coli WSP, e.g., not only induce a strong CD11b and CD11c expression but also lead to a loss in surface CD62L. The lack of CD62L shedding conserves rolling of granulocytes along the endothelium, creating a favorable precondition for granulocytes to stick more readily to activated endothelium after H. pylori stimulation via CD11b-CD54 receptor-counterreceptor interaction. This may explain why H. pylori infection is a very strong stimulus for granulocyte infiltration. The active fraction for the induction of CD11b on granulocytes is a heat- and protease-sensitive protein with a molecular mass between 30 and 100 kDa. One activation step involved may be the binding of WSP to CD15 determinants on granulocytes with subsequent induction of CD11b.