In this paper we describe a number of immunological parameters for dogs with a chronic Leishmania infantum infection which exhibit patterns of progressive disease or apparent resistance. The outcome of infection was assessed by isolation of parasites, serum antibody titers to Leishmania antigen, and development of clinical signs of leishmaniasis. Our studies show that 3 years after experimental infection, asymptomatic or resistant dogs responded to L. infantum antigen both in lymphocyte proliferation assays in vitro and in delayed-type hypersensitivity reaction, whereas no serum antibodies to parasite antigen were shown. In

Standard in vitro methods for assessing T-cell activation have typically measured either the proliferative responses of peripheral blood mononuclear cell (PBMC) cultures to various provocative stimuli employing tritiated thymidine incorporation or the secretion of specific cytokines from activated cells. These bulk assay methods suffer the drawback of being lengthy assays, and, in addition, they do not provide information about functional responses of individual lymphocyte subsets. This report describes a three-color flow cytometric method for the rapid analysis (4 hours) of individual T-cell subsets in whole blood responding to various provocative stimuli, including pokeweed mitogen, the comitogenic monoclonal antibodies (mAbs) CD2/CD2R, the superantigen staphylococcal enterotoxin B (SEB), and the speciac antigen Candlda albicans. After 4 hours, CD69 expression in response to CD2/ CDZR paralleled thymidine incorporation measured after 72 hours. Variations in the proportions of CD4+ and CD4-T cells expressing CD69 were observed with Merent stimuli. These observations demonstrate the potential of multiparameter flow cytometry for the investigation of functional responses of individual T-cell subsets to a variety of stimuli in whole blood. o 1995 wiley-Liss, Inc.

IL-2-dependent cell lines were established from normal peripheral blood T lymphocytes that express neither CD4 nor CD8 differentiation antigens. CD3+,4-,8- cell lines from 15 different donors failed to react with WT31, an mAb directed against the T cell antigen receptor alpha/beta heterodimer. Anti-Leu-4 mAb was used to isolate the CD3/T cell antigen receptor complex from 125I-labeled CD3+,4-,8- (WT31-) T cells. Using detergent conditions that preserved the CD3/T cell antigen receptor complex, an approximately 90 kD disulfide-linked heterodimer, composed of approximately 45- and approximately 40- (or approximately 37-) kD subunits, was coimmunoprecipitated with the invariant 20-29-kD CD3 complex. Analysis of these components by nonequilibrium pH gradient electrophoresis indicated that the approximately 40-kD and approximately 37-kD subunits were similar, and quite distinct from the more basic approximately 45-kD subunit. None of these three subunits reacted with an antibody directed against a beta chain framework epitope. Heteroantiserum against a T cell receptor gamma chain peptide specifically reacted with both the approximately 37- and approximately 40-kD CD3-associated proteins, but not with the approximately 45-kD subunit. CD3+,4-,8- cells failed to transcribe substantial amounts of functional 1.3-kb beta or 1.6-kb alpha mRNA, but produced abundant 1.6-kb gamma mRNA. Southern blot analysis revealed that these CD3+,4-,8- cell lines rearranged both gamma and beta genes, and indicated that the populations were polyclonal. The expression of a CD3-associated disulfide-linked heterodimer on CD3+,4-,8- T cell lines established from normal, adult peripheral blood contrasts with prior reports describing a CD3-associated non-disulfide-linked heterodimer on CD3+/WT31- cell lines established from thymus and peripheral blood obtained from patients with immunodeficiency diseases. We propose that this discrepancy may be explained by preferential usage of the two C gamma genes in T lymphocytes.

Recent studies have demonstrated that therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms; however, the potential of enhancing the efficacy of such treatments using nano-scale delivery platforms requires more in depth evaluation. As such, we employed a nanolipoprotein (NLP) platform to enhance the efficacy of innate immune agonists. Here, we demonstrate that the synthetic Toll-like receptor (TLR) agonists monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG) can be readily incorporated into NLPs. Conjugation of MPLA and CpG to NLPs (MPLA:NLP and CpG:NLP, respectively) significantly enhanced their immunostimulatory profiles both in vitro and in vivo compared to administration of agonists alone, as evidenced by significant increases in cytokine production, cell surface expression of activation markers, and upregulation of immunoregulatory genes. Importantly, enhancement of cytokine production by agonist conjugation to NLPs was also observed in primary human dendritic cells. Furthermore, BALB/c mice pretreated with CpG:NLP constructs survived a lethal influenza challenge whereas pretreatment with CpG alone had no effect on survival.

The gamma-TCR is encoded by genes composed of V, J, and C elements that demonstrate a limited potential for recombinational diversity. These genes are rearranged, transcribed, and translated into proteins early during thymic ontogeny. Lymphocytes express gamma-TCR proteins on the plasma membrane only in association with the CD3 complex. gamma-TCR glycoproteins usually associate with another non-gamma glycoprotein, designated delta-TCR, to form a heterodimer receptor. Both non-disulfide-bonded and disulfide-bonded gamma/delta-TCR heterodimers have been identified on the plasma membrane of human T lymphocytes. On certain gamma-TCR-bearing T cell lines, a delta-TCR protein cannot be visualized by autoradiography. It is possible that delta-TCR proteins are associated with gamma-TCR glycoproteins on these cell lines but are not efficiently radiolabeled. Alternatively, it has been suggested that homodimers of gamma-TCR proteins can assemble with CD3 and be expressed on the plasma membrane of these cells. In adult lymphoid tissues, the majority of T lymphocytes expresses a CD3, alpha/beta antigen receptor, whereas only a minor subset (less than 5% of peripheral blood lymphocytes, lymph node, spleen, and thymocytes) express a CD3, gamma/delta antigen receptor. IL-2-dependent cell lines of both murine and human CD3, gamma/delta T cells have been established. Most CD3, gamma/delta T cell lines mediate cytotoxicity against a broad spectrum of tumor-cell targets, although the functional significance of this observation remains unclear. Cytotoxicity is apparently not restricted by or directed against MHC antigens. Antibodies against CD3 or gamma-TCR can induce proliferation and IL-2 secretion and can either augment or inhibit cytotoxicity, demonstrating that the gamma/delta-TCR is a functional receptor. The ligand recognized by this receptor has not been identified. The physiological role of T lymphocytes expressing gamma/delta-TCR, the molecular and structural properties of delta-TCR, and the relationship between CD3, alpha/beta T lymphocytes and CD3, gamma/delta T lymphocytes are the major unresolved questions that will be the primary focus of further experimentation.