Biology of murine gamma delta T cells.

Murine lymphocytes express either a T-cell receptor alpha beta or a gamma delta heterodimer. The function of alpha beta cells are well characterized, while gamma delta cells remain an enigmatic population. In the mouse, gamma delta cells appear in significant proportions in the epithelia of various nonlymphoid tissues such as the skin, intestine, tongue, lung, and reproductive organs. While gamma delta T-cell subsets with distinct antigen receptor repertoires are associated with certain organs, diversified populations of gamma delta cells showing heterogeneous TCR phenotypes, as a result of junctional region diversification and usage of different V chains, can be found in the lymphoid organs and in the intestinal epithelia. Recent evidence has shown that gamma delta cells might recognize heat shock proteins, possibly in association with classical and nonpolymorphic MHC molecules. Together with their tissue distribution, gamma delta cells may represent the first line of defense of the immune system. gamma delta Cells are the first T cells to colonize the thymus. Intriguingly, there is more evidence to support the hypothesis that they might also affect the development of alpha beta cells and other hematopoietic stem cells.

Synthesis of some quaternary ammonium alkylating agents and their effects on soman-inhibited acetylcholinesterase.

A number of compounds were synthesized and tested for their ability to realkylate the phosphonate anion of "aged", soman-inhibited acetylcholinesterase. None were found able to do so, but two of the compounds in particular, [2-(4-pyridyl)ethyl]diethylmethylammonium iodide (6) and its 2-isomer 7, proved able to slow the rate of aging significantly.

Tolerance and self-reactivity in V gamma 1.1C gamma 4 transgenic mice.

Immunological tolerance is the process of inhibiting or eliminating lymphocytes that recognize self-derived antigens. By removing potentially harmful self-reactive clones, this mechanism allows for the random generation of a diverse repertoire of T-cells capable of responding to foreign pathogens. Although all self-reactive T-cells should be removed from the repertoire, it is quite clear from many recent studies that a significant fraction of T-cells bearing gamma delta T-cell receptors (TCR) recognize self-derived antigens in normal healthy mice. The presence of self-reactive T-cells in healthy animals presents a paradox which may be explained by understanding the transient expression of the antigens (e.g., MHC class Ib, Heat Shock Proteins) that have been identified for gamma delta T-cells thus far. Data from experiments with V gamma 1.1C gamma 4 transgenic mice demonstrating the presence of self-reactive gamma delta T-cells and their influence on lymphoid development and immune surveillance will be examined in this review.

Cytokine transcription in lymph nodes of cattle in different stages of bovine leukemia virus infection.

Bovine leukemia virus (BLV) is a transforming oncovirus that contains no oncogenes or preferred site of proviral integration. The role of cytokines in the disease process of BLV is potentially important due to the similarity of BLV with other retroviruses in which cytokines play a role, such as HTLV-I and -II. Mesenteric and supra-mammary lymph nodes were obtained from a panel of nine cattle. Three were non-infected controls, three were BLV-positive aleukemic (AL), and three were BLV-positive persistent lymphocytotic (PL). Mononuclear cells were perfused from the organs and total RNA extracted from either 1 x 10(8) unseparated cells or 1 x 10(7) purified CD4/CD8 T-cells. cDNA was generated and subjected to RT-PCR to analyze cytokine transcription during disease progression. cDNA levels were normalized using beta-actin PCR at sub-plateau cycle number, enabling a semi-quantitative assessment of cytokine gene transcripts. Using this approach, IL-2, IL-10 and IFN-gamma message was detected in the T-cell fractions of all of the BLV-infected animals, but not in the non-infected controls.

Bovine cytokine expression during different phases of bovine leukemia virus infection.

The potential role of aberrant cytokine production in the pathogenesis of bovine leukemia virus (BLV) was studied by analyzing cytokine mRNA expression in pokeweed-stimulated PBMLs of cows in different phases of disease progression. To analyze the mRNA, a semi-quantitative RT-PCR assay was developed. The RT-PCR assay was developed for detection of IL-2, -4, -6, -10, -12, IFN-gamma and actin using cDNA derived from phorbol-stimulated peripheral blood mononuclear leukocytes. Using a PCR specific for BLV tax, agar gel immunodiffusion and white blood cell counts, BLV-negative, BLV-positive aleukemic (AL), and BLV-positive persistently lymphocytotic (PL) cattle were identified. Peripheral blood lymphocytes cultured in vitro for 24 h in pokeweed mitogen were analyzed for cytokine production using the RT-PCR assay. Consistently elevated levels of IL-2 and IL-12 in AL and PL cattle in pokeweed mitogen-stimulated cells was detected, while IFN-gamma was elevated in the AL but not the PL cattle.

Characterization of F21.A, a monoclonal antibody that recognize a leucocyte surface antigen for killer whale homologue to beta-2 integrin.

The specificity of F21.A, a monoclonal antibody raised against bottlenose dolphin leucocytes, was characterized in killer whale on the basis of immunoprecipitation of a protein of 94 kDa, as well as flow cytometric analysis. While minimally expressed on resting cells, F21.A labeled a homologue to beta-2 integrin in 89-97% of PMA-activated neutrophils, 53-66% of activated monocytes, and activated B cells but not T cells. Activation of neutrophils reached its maximum 10 min after PMA stimulation. F21.A did not label intracellular stores as did both cross-reacting anti-canine CD11b and CD18, suggesting that an activation-induced conformational change would expose a neoepitope recognized by F21.A. F21.A labeling was largely inhibited by pre-incubation with plasma, suggesting a binding site closely related to that for fibrinogen. In vitro phagocytosis and respiratory burst were almost fully inhibited upon pre-incubation with F21.A, demonstrating its functional importance. This antibody is foreseen as a possible valuable diagnostic and research tool in cetacean immunology.

Stable, stoichiometric delivery of diverse protein functions.

As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.

First wave fetal thymocytes expressing V3J gamma 1C gamma 1-V delta 1J delta 2C delta T cell receptors are not required for alpha beta T cell receptor rearrangement and expression.

We have determined the fetal expression of V gamma 3, delta and beta TcRs in mice transgenic for the V gamma 1.1J4C gamma 4 TcR chain. The first wave thymocytes appearing at day 14 and disappearing by day 17 in normal mice was absent from the transgenic mice. However, both mice had an almost identical number of gamma delta-bearing thymocytes throughout gestation. Therefore, it is most likely that the V gamma 3J gamma 1C gamma 1 chain was replaced in the transgenic mice by the V gamma 1.1J gamma 4C gamma 4 transgene. The appearance, although slightly earlier for the transgenic mice, of alpha beta-bearing thymocytes was also very similar between transgenic and control mice during gestation. These data suggest that whatever the role of the first wave thymocytes expressing V3-V delta 1 TcRs is, it most likely is not required for the rearrangement, expression and maturation of the alpha beta TcR repertoire. We are currently analyzing a series of gamma delta transgenic mice to determine whether other restricted populations of gamma delta-bearing T cells are involved in specific aspects of immune development or function.

Role of T cells and cytokines in fatal and resolving experimental babesiosis: protection in TNFRp55-/- mice infected with the human Babesia WA1 parasite.

We characterized the cytokine response and T-cell requirements of mice infected with the intraerythrocytic parasites Babesia microti and WA1. WA1 infections were fatal, whereas B. microti infections were resolved. We measured production of tumor necrosis factor (TNF)-alpha, interferon-gamma, interleukin (IL)-10, and IL-4 by splenic CD4+, CD8+, and gammadelta+ T cells using flow cytometry. WA1 inoculation stimulated TNF-alpha production, whereas resolving B. microti infections were characterized by increased IL-10 and IL-4. The role of TNF-alpha in WA1 infections was further investigated by inoculating TNFRp55-/- mice with a lethal dose of WA1. A survival rate of 90% in the TNFRp55-/- mice indicated that a disruption in the TNF-alpha pathway abrogated the pathologic mechanism of WA1. Inoculation of WA1 into CD4-/- and CD8-/- mice resulted in survival rates of 60% and 78%, respectively, whereas WA1 infection in gammadelta-/- and control mice was fatal. These results suggest that CD8+ T cells may contribute to the WA1-associated disease. Babesia-infected CD4-/- mice experienced a longer duration of parasitemia, indicating that CD4+ T cells participate in parasite elimination. These studies demonstrate differences in immune responses during fatal or resolving Babesia infections, and they identify TNF-alpha as an important mediator of the WA1-associated pathogenesis.

Development of an interleukin-2 receptor expression assay and its use in evaluation of cellular immune responses in bottlenose dolphin (Tursiops truncatus).

We describe optimization of a peripheral blood mononuclear leukocyte proliferation assay and development of an interleukin-2 receptor (IL-2R) expression assay for bottlenose dolphins (Tursiops truncatus). Peripheral blood mononuclear leukocytes obtained from both Sea World (February 1993) and the Naval Command Control and Ocean Surveillance Center (March 1993) (San Diego, California, USA) were stimulated with the mitogens concanavalin A (ConA) and phytohemagglutinin (PHA) and evaluated for optimum proliferation and IL-2R expression. Based on these optimization assays, standard conditions were established and used to assess immune function in a population of apparently healthy, free-ranging bottlenose dolphins from Sarasota Bay, Florida (USA) in June 1993. A positive correlation was observed between proliferation assays using ConA and PHA as the stimulants. However, IL-2R expression induced by both mitogens differed significantly.

Blastogenesis and interleukin-2 receptor expression assays in the harbor seal (Phoca vitulina).

Two in vitro functional assays were developed to evaluate mitogen-induced responses of peripheral blood mononuclear leukocytes (PBML) from free-ranging harbor seals, Phoca vitulina. Lymphocyte proliferation was measured by a standard blastogenesis assay following optimization of culture conditions including mitogen concentration, cell density, and incubation time. These optimized parameters, with the exception of incubation time, were subsequently employed to measure lymphocyte activation by analytical flow cytometry using fluorochrome-based identification of cell surface interleukin-2 receptor (IL-2r) expression. Baseline values established for free-ranging harbor seals had extensive animal variability; there was evidence that the samples were derived from a group of animals with a normal distribution. Positive correlations were observed between blastogenesis assays, and between blastogenesis and activation assays, when using pokeweed or concanavalin A as the stimulus. However, no relationship was found in the expression of the IL-2r induced by these mitogens. This result supports the contention that the two mitogens stimulate different lymphocyte subpopulations. This was observed only with the IL-2r expression assay because of its unique ability to measure the number of T lymphocytes initially activated rather than the ultimate number of progeny cells identified by blastogenesis. Both assays, used concurrently, should provide a more comprehensive representation of lymphocyte competence and serve as a measure of animal health.

Cell interactions in alveolar macrophage-mediated suppression of the immune response: an unusual suppressor pathway involving a population of T-cells that express Lyt-1, L3T4, and I-J.

Studies from this laboratory have demonstrated that incubation of murine alveolar macrophages (AM) with SRBC-primed spleen cells (SC) results in suppression of the in vitro plaque-forming cell (PFC) response and that suppression is mediated by a soluble factor contained in supernatants obtained from cultures of AM and SC. In the present study, immunological techniques employing monoclonal antibody (MoAb) were used to isolate various T-cell subsets in order to determine the phenotype of the cells which interact with AM to produce suppression. Spleen cell populations depleted of Thy-1+-, Lyt-1+-, L3T4+-, or I-J+-bearing cells failed to generate suppressive supernatants when cultured with AM. Depletion of Lyt-2+ T-cells (the classical suppressor/effector subset) did not alter the ability of the remaining cell population to cooperate with AM for generation of suppressive supernatants. Direct suppression of the PFC response in cultures containing AM was abrogated after treatment of the spleen cells with anti-I-J, but not anti-Lyt-2 MoAbs. Reconstitution of the AM-mediated suppressive response with enriched populations of SC required the presence of T-cells which expressed Lyt-1, L3T4, and I-J. These results suggest the existence of an unusual suppressor pathway involving I-J restriction but which appears to be mediated by the interaction of AM with a population of T-cells that expresses surface markers characteristic of T-helper cells.

Horse (Equus caballus) T-cell receptor alpha, gamma, and delta chain genes: nucleotide sequences and tissue-specific gene expression.

Horse (Equus caballus) T-cell receptor alpha (TCRA), gamma (TCRG), and delta (TCRD) chain genes were isolated from a cDNA library and characterized. Five unique TCRAV families, including four full-length sequences, five distinct TCRAJ genes, and a single TCRAC gene were identified. TCRAV genes had closest homology with human sequences and least similarity to rat genes. Among eight horse TCRG genes, two distinct constant region genes with considerable variation in the connecting region were identified, but no variable or joining genes were present. Southern blot hybridization confirmed the presence of at least two TCRGC genes and indicated that the vast majority of horse alpha beta T cells rearrange either one or both TCRG alleles. Analysis of horse TCRD genes revealed the presence of eight unique TCRDV genes representing seven families, each having closest nucleotide homology with sheep sequences. Six unique TCRDJ genes were isolated; however, four of these sequences differed by only one base pair and thus likely represented alleles of a single gene. One horse TCRDC gene was present among fifteen clones analyzed and, based on Southern blot hybridizations, was deleted in polyclonal alpha beta T-cell populations, indicating that the TCRD locus is probably located within the TCRA locus as in other species. Polymerase chain reaction using horse-specific primers for the detection of TCRAC and TCRDC gene expression indicated that gamma delta T cells are located at numerous sites throughout the body, and with the exception of bone marrow where only TCRAC transcripts were detected, are closely associated with alpha beta T cells. This finding indicates that these two T-cell populations may be functionally interactive.

Differential production of intracellular gamma interferon in alpha beta and gamma delta T-cell subpopulations in response to peritonitis.

Flow cytometry was used to measure T-cell intracellular gamma interferon and surface interleukin 2 re-ceptor expression in response to peritonitis in rats. Interleukin 2 receptor expression levels were similar in the two T-cell subsets, but gamma interferon production was increased fivefold in gamma delta T cells compared with pro-duction in alpha beta T cells. Our results provide further evidence of an early and vigorous gamma delta T-cell response to bac-terial infection.

Structure and biological activity of the subgenomic Mtv-6 endogenous provirus.

The Mtv-6 provirus has an incomplete genome, but retains a functional superantigen gene (sag) which directs the thymic deletion of CD4+ T cells expressing T cell receptors containing the V beta 3 or V beta 5 chains. To better understand the Mtv-6 superantigen, the structure and biological activity of the Mtv-6 provirus was analyzed. First, the complete nucleotide sequence was determined, and the mutation producing the subgenomic provirus was identified. Second, the nucleotide sequence of the 5' end of the sag gene transcript (including the splice junction) was determined by sequence analysis of a cDNA clone. Third, the superantigen activity of Mtv-6 was analyzed in mice carrying the Mtv-6 provirus isolated by selective breeding on a genetic background free of endogenous and exogenous mouse mammary tumor virus (MMTV). These studies demonstrate that (i) the Mtv-6 provirus contains a 6.2-kb deletion between two 12-bp direct repeats encompassing the central portion of the provirus but not affecting sag gene splicing or translation, (ii) the sag gene transcript has the structure predicted from previous S1 nuclease mapping studies, and (iii) the Mtv-6 superantigen can direct thymic deletion of target V beta 3+ and V beta 5+ T cells in the absence of gene products from full-length MMTV proviruses.

Characterization of horse (Equus caballus) T-cell receptor beta chain genes.

Genes encoding the horse (Equus caballus) T-cell receptor beta chain (TCRB) were cloned and characterized. Of 33 cDNA clones isolated from the mesenteric lymph node, 30 had functionally rearranged gene segments, and three contained germline sequences. Sixteen unique variable segments (TCRBV), 14 joining genes (TCRBJ), and two constant region genes (TCRBC) were identified. Horse TCRBV were grouped into nine families based on similarity to human sequences. TCRBV2 and TCRBV12 were the most commonly represented horse families. Analysis of predicted protein structure revealed the presence of conserved regions similar to those seen in TCRB of other species. A decanucleotide promoter sequence homologous to those found in humans and mice was located in the 5' untranslated region of one horse gene. Germline sequences included the 5' region of the TCRBD2 gene with flanking heptamer/nonamer recombination signals and portions of the TCRBJ2-C2 intron. Southern blot hybridizations demonstrated restriction fragment length polymorphisms at the TCRBC locus among different horse breeds.

Up-regulation of tumor necrosis factor-alpha and interferon-gamma expression in the spleen and lungs of mice infected with the human Babesia isolate WA1.

We analyzed cytokine expression in mice infected with the intraerythrocytic parasites Babesia microti and WA1. In C3H/HeN mice, WA1 infections were fatal, whereas B. microti infections were resolved. We propose that the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) contribute to the WA1-associated disease. WA1 infection was characterized by up-regulation of TNFalpha and IFNgamma mRNA in the spleen. Previous studies in WA1-infected mice showed that pathologic lesions occurred primarily in the lungs, including pulmonary edema and intravascular margination of leukocytes. Analysis of cytokine expression in the lungs is important for an understanding of the disease process in WA1-infected mice. Expression of both TNFalpha and IFNgamma mRNA was increased in the lungs of WA1-infected mice. Immunohistochemical staining confirmed the upregulation of these proinflammatory cytokines in the lungs. Expression of TNFalpha and IFNgamma was not up-regulated in the lungs of B. microti-infected mice. The results implicate TNFalpha and IFNgamma in the pathogenesis of WA1-associated disease.

Characterization of horse (Equus caballus) immunoglobulin mu chain-encoding genes.

Horse (Equus caballus) immunoglobulin mu chain-encoding (IgM) variable, joining, and constant gene segments were cloned and characterized. Nucleotide sequence analyses of 15 cDNA clones from a mesenteric lymph node library identified 7 unique variable gene segments, 5 separate joining segments, and a single constant region. Based on comparison with human sequences, horse variable segments could be grouped into either family 1 of immunoglobulin (Ig) clan I or family 4 of Ig clan II subclan IV. All horse sequences had a relatively conserved 16 base pair (bp) segment in framework 3 which was recognized with high specificity in polymerase chain reaction by a degenerate oligonucleotide primer. Horse complementarity determining regions (CDR) had considerable variability in predicted amino acid content and length but also included the presence of relatively conserved residues and several canonical sequences that may be necessary in formation of the beta chain main structure and conformation of antigen-binding sites through interaction with light chain CDR. Sequence analysis of joining regions revealed the presence of nearly invariant 3' regions similar to those found in human and mouse genes. A single horse IgM constant region comprising 1472 bp and encoding 451 residues was also identified. Direct comparison of the horse constant region predicted amino acid sequence with those from eleven other species revealed the presence of 53 invariant residues with particularly conserved sequences within the third and fourth exons. Phylogenetic analysis using a neighbor-joining algorithm showed closest similarity of the horse mu chain-encoding constant region gene to human and dog sequences. Together, these findings provide insights into the comparative biology of IgM as well as data for additional detailed studies of the horse immune system and investigation of immune-related diseases.

Regulation of abortion by gamma delta T cells.

PROBLEM: T cells are present at the feto-maternal interface, but their function during pregnancy has not been fully elucidated. T cells bearing gamma delta T-cell receptor (TCR) may be particularly important, as some subsets can react to trophoblast cells by producing cytokines, such as interleukin-2 (IL-2). METHOD: We depleted T cells bearing the gamma delta receptor by injecting monoclonal antibodies (mAb) into females of the abortion-prone animal model CBA x DBA/2. We investigated the percentage and number of gamma delta T-cell receptor positive (TCR)+ cells in decidua and spleen during pregnancy in control and gamma delta-depleted female mice. Pregnant females were also exposed to ultrasonic sound stress to boost the abortion rate. RESULTS: Stress failed to increase the abortion rate in the gamma delta TCR-depleted mice. FACScan analysis show that the ratio of cells bearing the gamma delta TCR dramatically decreased after injection of mAB to the gamma delta TCR in spleen and decidua, these cells recovered six days after depletion, showing a change in cytokine pattern. Levels of TNF-alpha in decidual gamma delta T cells decreased; similar effects of decreasing Th1 cytokines could be observed in splenic gamma delta T cells. We further identified increased levels of intracellular TNF-alpha in the V delta 4 subset in the decidua, compared to spleen. CONCLUSIONS: Trophoblast recognition by the V delta 4 T-cell subset in the decidua may cause the release of abortogenic cytokines such as TNF-alpha. Depletion of such gamma delta TCR T cells during early pregnancy may promote successful pregnancy outcome in normal pregnancy and prevent stress-induced abortions.