Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes.

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.

Effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo.

Mycophenolic acid, a selective inhibitor of the de novo synthesis of guanosine nucleotides in T and B lymphocytes, has been proposed to inhibit human immunodeficiency virus (HIV) replication in vitro by depleting the substrate (guanosine nucleotides) for reverse transcriptase. Here we show that mycophenolic acid induced apoptosis and cell death in a large proportion of activated CD4+ T cells, thus indicating that it may inhibit HIV infection in vitro by both virological mechanisms and immunological mechanisms (depletion of the pool of activated CD4+ T lymphocytes). Administration of mycophenolate mophetil, the ester derivate of mycophenolic acid, to HIV-infected subjects treated with anti-retroviral therapy and with undetectable viremia resulted in the reduction of the number of dividing CD4 + and CD8+ T cells and in the inhibition of virus isolation from purified CD4+ T-cell populations. Based on these results, the potential use of mycophenolate mophetil in the treatment of HIV infection deserves further investigation in controlled clinical trials.

AA-amyloidosis caused by visceral leishmaniasis in a human immunodeficiency virus-infected patient.

AA-amyloidosis in the setting of chronic visceral leishmaniasis (VL) has been reported in animal models but documentation in humans is unavailable. Here, we report on a Portuguese man who in 1996 was diagnosed with both human immunodeficiency virus (HIV)-infection and VL. Antiretroviral treatment led to sustained suppression of HIV viremia but CD4+ lymphocytes rose from 8 to only 160 cells/mL. Several courses of antimony treatment did not prevent VL relapses. Renal failure developed in 2006 and renal biopsy revealed AA-amyloidosis. The patient had cryoglobulinemia and serum immune complexes containing antibodies directed against seven leishmanial antigens. Antimony plus amphotericin B, followed by oral miltefosine resulted in a sustained VL treatment response with elimination of circulating Leishmania infantum DNA and CD4+ recovery. The concomitant reduction of serum AA levels and disappearance of circulating leishmanial immune complexes suggests that prolonged VL may lead to AA-amyloidosis in immunocompromised humans.

Assessing ageing of individual T lymphocytes: mission impossible?

Effector T lymphocytes are the progeny of a limited number of antigen-specific precursor cells and it has been estimated that clonotypic human T cells may expand million fold on their way reaching high cell numbers that are sufficient for immune protection. Moreover, memory T cell responses are characterized by repetitive expansion of antigen-specific T cell clonotypes, and limitations in the proliferative capacity could lead to immune senescence. Because telomeres progressively shorten as a function of cell division, telomere length is a powerful indicator of the replicative in vivo history of human T lymphocytes. In this review, we summarize observations made over the last decade on telomere length dynamics of well-defined T cell populations derived from healthy donors and patients with infectious disease or cancer. We focus on T cell differentiation, T cell ageing, and natural and vaccine induced immune responses. We also discuss the scientific evidence for in vivo replicative senescence of antigen-specific T cells, and evaluate the available methods for measuring telomere lengths and telomerase activity, and their potential and limitations to increase our understanding of T cell physiology.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Tuberculin Skin Test and CD4+/CD8+ T Cell Counts in Adults Infected with the Human Immunodeficiency Virus in Medellín, Colombia

To evaluate the effect of BCG vaccination and T lymphocyte subpopulations on the reactivity to the tuberculin skin test, 113 asymtomatic HIV+ individuals were tuberculin tested by intradermal injection of 5TU of purified protein derivative and the levels of circulating lymphocyte (CD3, CD4 and CD8) subpopulations determined by indirect immunofluorescence. Ninety-two percent of the subjects included in the study were males. The mean age of the group was 32.1±7.4 years. Sixty-two percent presented a BCG scar. However, only 22% exhibited positive tuberculin reactions (³5mm) irrespective of the presence of the BCG scar. Tuberculin positive individuals exhibited higher CD4+ cell counts (p=0.004) and CD4+/CD8+ ratios (p=0.006) than tuberculin negative (<5mm) HIV+ individuals. The number of individuals with positive tuberculin reactions was significantly higher in subjects with more than 500 CD4+ lymphocytes/ml (p=0.02) or CD4+/CD8+ ratios ³1.12 (p=0.002). These results suggest that a prior BCG vaccination does not influence the reactivity to the tuberculin skin test in HIV+ asymptomatic individuals and that the number of CD4+ lymphocytes and the CD4+/CD8+ ratio positively correlate with the tuberculin reactivity

Circulating soluble CR1 (CD35). Serum levels in diseases and evidence for its release by human leukocytes.

C receptor type 1 (CR1, CD35) is present in a soluble form in plasma (sCR1). Soluble CR1 was measured with a specific ELISA assay in normal individuals and in patients with different diseases. The mean serum concentration of sCR1 in 31 normal donors was 31.4 +/- 7.8 ng/ml, and was identical in plasma. An increase in sCR1 was observed in 36 patients with end-stage renal failure on dialysis (54.8 +/- 11.7 ng/ml, p &lt; 0.0001), and in 22 patients with liver cirrhosis (158.3 +/- 49.9 ng/ml, p &lt; 0.0001). The mean sCR1 levels dropped from 181 +/- 62.7 to 52.1 +/- 24.0 ng/ml (p &lt; 0.001) in nine patients who underwent liver transplantation, and was 33.5 +/- 7.3 in 10 patients with functioning renal grafts, indicating that the increase in sCR1 was reversible. Soluble CR1 was elevated in some hematologic malignancies (&gt; 47 ng/ml), which included B cell lymphoma (12/19 patients), Hodgkin's lymphoma (4/4), and chronic myeloproliferative syndromes (4/5). By contrast, no increase was observed in acute myeloid or lymphoblastic leukemia (10) or myeloma (5). In two patients with chronic myeloproliferative syndromes, sCR1 decreased rapidly after chemotherapy. The mean concentration of sCR1 was not significantly modified in 181 HIV-infected patients at various stages of the disease (34.8 +/- 14.4 ng/ml), and in 13 patients with active SLE (38.3 +/- 19.6 ng/ml), although in both groups the number of CR1 was diminished on E. There was a weak but significant correlation between sCR1 and CR1 per E in HIV infection and SLE (r = 0.39, p &lt; 0.0001, and r = 0.60, p &lt; 0.03 respectively). In vitro, monocytes, lymphocytes, and neutrophils were found to release sCR1 into culture supernatants. In vivo, sCR1 was detected in the serum of SCID mice populated with human peripheral blood leukocytes. The sCR1 levels correlated with those of human IgG (r = 0.97, p &lt; 0.0001), suggesting synthesis of sCR1 by the transferred lymphocytes. The mechanisms underlining the increased levels of sCR1 and its biologic consequences remain to be defined.

Optimum in vitro expansion of human antigen-specific CD8 T cells for adoptive transfer therapy.

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

Eokines: synthesis, storage and release from human eosinophils

Eosinophils are prominent inflammatory cells in asthma and other allergic disorders, as well as in helminthic parasite infections. Recently, eosinophils have been reported to synthesize and store a range of regulatory proteins within their secretory granules (eokines). Eokines comprise a group of cytokines, chemokines, and growth factors which are elaborated by eosinophils. These proteins, and the messages which encode them, appear to be identical to those produced by lymphocytes and other tissues. Interestingly, immunoreactivity to many of these eokines has been found to co-localize to the eosinophil´s secretory granules. In this review, we have discussed the repertoire of 18 eokines so far identified in eosinophils, and focused on four of these, namely, interleukin-2 (IL-2), IL-4, granulocyte/macrophage colony-stimulating factor (GM-CSF), and RANTES. These four eokines co-localize to the crystalloid granules in eosinophils, as shown in studies using subcellular fractionation and immunogold labeling in electron microscopy. During stimulation by physiological triggers, for example, with serum-coated particles, eosinophils release these mediators into the surrounding supernatant. In addition, eokines are likely to be synthesized within eosinophils rather than taken up by endocytosis, as show in detection of mRNA for each of these proteins using in situ hybridization, RT-PCR, and in the case of RANTES, in situ RT-PCR. Eokines synthesis and release from eosinophils challenges the commonly held notion that these cells act downstream of key elements in immune system, and indicate that they may instead belong to the afferent arm of immunity.

Human eosinophil-lymphocyte interactions

While the eosinophil's effector functions clearly can contribute to the pathogenesis of allergic diseases, the evolutionary benefit to having eosinophils as a distinct class of leukocytes is not clear, especially if one must reconsider the nominally beneficial role of eosinophils in parasite host defense. Eosinophils are equipped to respond to lymphocytes and their cytokines (and not solely the eosinophil growth factor cytokines), but the functional consequences of such eosinophil responses need to be defined. Conversely, eosinophils, as antigen-presenting cells (APCs) or sources of lymphocyte-active cytokines, may stimulate and effect lymphocyte functioning. Eosinophils share with CD4+ lymphocytes expression of a number of receptors, including CD4 and IL-2R, and specific alpha4 integrins that may help in their common recruitment and activation. Further, elucidation of the interactions between lymphocytes and eosinophils will contribute to a broader understanding of the functioning of eosinophils in "normal" ongoing immune responses and in allergic disorders.

The spleen is an important site of T cell activation during human hepatosplenic schistosomiasis

We have undertaken a comparative immunephenotypic study of spleen cells from hepatosplenic patients (HS) and uninfected individuals (NOR) using flow cytometry. Our data did not show any significant differences in the mean percentage of T-cells and B-cells between the two groups. Analysis of activated T-cells demonstrated that HS present an increased percentage of CD3+HLA-DR+ splenocytes in comparison to NOR. Analysis of T-cell subsets demonstrated a significant increase on the percentage of both activated CD4+ T-splenocytes and CD8+ cells in HS. We did not find any difference in the mean percentage of CD28+ T-cells. Analysis of the B-cell compartment did not show any difference on the percentage of B1-splenocytes. However, the spleen seems to be an important reservoir/source for B1 lymphocytes during hepatosplenic disease, since after splenectomy we found a decreased the percentage of circulating B1-lymphocytes. We observed an increase on the percentage of CD2+CD3- lymphocytes in the spleen of HS suggesting that the loss of CD3 by activated T-cells or the expansion of NK-cells might play a role in the development/maintenance of splenomegaly.

Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909.

The induction of potent CD8+ T cell responses by vaccines to fight microbes or tumors remains a major challenge, as many candidates for human vaccines have proved to be poorly immunogenic. Deoxycytidyl-deoxyguanosin oligodeoxynucleotides (CpG ODNs) trigger Toll-like receptor 9, resulting in dendritic cell maturation that can enhance immunogenicity of peptide-based vaccines in mice. We tested whether a synthetic ODN, CpG 7909, could improve human tumor antigen-specific CD8+ T cell responses. Eight HLA-A2+ melanoma patients received 4 monthly vaccinations of low-dose CpG 7909 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and incomplete Freund's adjuvant. All patients exhibited rapid and strong antigen-specific T cell responses: the frequency of Melan-A-specific T cells reached over 3% of circulating CD8+ T cells. This was one order of magnitude higher than the frequency seen in 8 control patients treated similarly but without CpG and 1-3 orders of magnitude higher than that seen in previous studies with synthetic vaccines. The enhanced T cell populations consisted primarily of effector memory cells, which in part secreted IFN- and expressed granzyme B and perforin ex vivo. In vitro, T cell clones recognized and killed melanoma cells in an antigen-specific manner. Thus, CpG 7909 is an efficient vaccine adjuvant that promotes strong antigen-specific CD8+ T cell responses in humans.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Vertical human immunodeficiency virus type 1 - HIV-1 -transmission - a review

Several factors appear to affect vertical HIV-1 transmission, dependent mainly on characteristics of the mother (extent of immunodeficiency, co-infections, risk behaviour, nutritional status, immune response, genetical make-up), but also of the virus (phenotype, tropism) and, possibly, of the child (genetical make-up). This complex situation is compounded by the fact that the virus may have the whole gestation period, apart from variable periods between membrane rupture and birth and the breast-feeding period, to pass from the mother to the infant. It seems probable that an extensive interplay of all factors occurs, and that some factors may be more important during specific periods and other factors in other periods. Factors predominant in protection against in utero transmission may be less important for peri-natal transmission, and probably quite different from those that predominantly affect transmission by mothers milk. For instance, cytotoxic T lymphocytes will probably be unable to exert any effect during breast-feeding, while neutralizing antibodies will be unable to protect transmission by HIV transmitted through infected cells. Furthermore, some responses may be capable of controlling transmission of determined virus types, while being inadequate for controlling others. As occurence of mixed infections and recombination of HIV-1 types is a known fact, it does not appear possible to prevent vertical HIV-1 transmission by reinforcing just one of the factors, and probably a general strategy including all known factors must be used. Recent reports have brought information on vertical HIV-1 transmission in a variety of research fields, which will have to be considered in conjunction as background for specific studies.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.