Development and ontogeny of hamster T cell subpopulations

The Syrian hamster is unique among laboratory animals because products of class I MHC genes are monomorphic. Thus, this species may be a model in which to test the relationship between MHC polymorphism and the T cell antigen receptor repertoire. Recently, cytotoxic and helper T cell subpopulations have been distinguished on the basis of cell surface phenotype detected with monoclonal antibodies (mAb). We used these reagents (mAb 110 detects all peripheral T cells and mAb 38 detects cytotoxic T cells) to dissect and categorize thymic populations according to relative maturational status. The two mAb divide thymocytes into four subpopulations in the young adult. Two (110+ 38+, 110+ 38-) were peripheral-like and were housed in the medulla, exclusively; another subset (110- 38+) consisted almost entirely of TdT+ cortical thymocytes. The fourth subset (110- 38-), bearing neither marker, was heterogeneous and consisted mostly of medium-large-size thymocytes, including cells with an early phenotype (nuclear TdT+). Cells with the cortical phenotype proved to be the most sensitive to cortisone treatment, whereas those which expressed the medullary marker, 110, were most resistant. To ascertain the relationship between 110- and 110+ T lineage cells, we followed the appearance of the four thymic subpopulations during ontogeny of the hamster thymus. Adult-like thymic architecture (delineation of cortex and medulla) as well as the two 110- subsets were established before expression of 110 antigen was apparent in the thymus. However, lymphocytes bearing the 110 antigen were found in lymph nodes prior to thymus during ontogeny, concomitant with developing T cell function in peripheral tissue. This finding implies that cells lacking 110 antigen were exported from the thymus and subsequently acquired expression of the molecule in the periphery, and we suggest that acquisition of 110 antigen may be a stage of postthymic maturation. Although 110+ cells appeared to be the most mature subset by several criteria, all functional thymocytes of adults or neonates were not 110+. Thus, we conclude that the 110 marker is acquired after T cells reach functional maturity. Moreover, the response profile of isolated 38+ thymocytes was analogous to peripheral 38+ T cells, suggesting that the dichotomy of function detected with our mAb also occurs before acquisition of 110 antigen. We have modeled what is known about hamster T cell development into a hypothetical scheme.     

Antigen presentation in Syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.     

Syrian hamsters express diverse MHC class I gene products

MHC class I glycoproteins are highly diverse in most species. The Syrian hamster has long been thought to express monomorphic MHC class I molecules and thus be an exception to this rule. Here we show that Syrian hamsters express diverse MHC class I gene products. The nucleotide sequences of the alpha 1 and alpha-2 domains of classical Syrian hamster MHC class I molecules are highly variable and show evidence of having been under selective pressures at their Ag recognition sites. Interestingly, none of the Syrian hamster class I genes was closely related to their counterparts in the mouse. These observations suggest that Syrian hamsters in the wild may express diverse MHC class I molecules.     

A prominent role for invariant T cells in the amphibian Xenopus laevis tadpoles

Invariant T (iT) cells expressing an invariant or semi-invariant T cell receptor (TCR) repertoire have gained attention in recent years because of their potential as specialized regulators of immune function. These iT cells are typically restricted by nonclassical MHC class I molecules (e.g., CD1d and MR1) and undergo differentiation pathways distinct from conventional T cells. While the benefit of a limited TCR repertoire may appear counterintuitive in regard to the advantage of the diversified repertoire of conventional T cells allowing for exquisite specificity to antigens, the full biological importance and evolutionary conservation of iT cells are just starting to emerge. It is generally considered that iT cells are specialized to recognize conserved antigens equivalent to pathogen-associated molecular pattern. Until recently, little was known about the evolution of iT cells. The identification of class Ib and class I-like genes in nonmammalian vertebrates, despite the heterogeneity and variable numbers of these genes among species, suggests that iT cells are also present in ectothermic vertebrates. Indeed, recent studies in the amphibian Xenopus have revealed a drastic overrepresentation of several invariant TCRs in tadpoles and identified a prominent nonclassical MHC class I-restricted iT cell subset critical for tadpole antiviral immunity. This suggests an important and perhaps even dominant role of multiple nonclassical MHC class I-restricted iT cell populations in tadpoles and, by extension, other aquatic vertebrates with rapid external development that are under pressure to produce a functional lymphocyte repertoire with small numbers of cells.     

Relationship among function, phenotype, and specificity in primary allospecific T cell populations: identification of phenotypically identical but functionally distinct primary T cell subsets that differ in their recognition of MHC class I and class II allodeterminants

The goal of the present study was to evaluate the relationship among function, Lyt phenotype, and MHC recognition specificity in primary allospecific T cell populations. By using Lyt-2+ and L3T4+ T cells obtained from the same responder populations, we assessed the ability of T cells of each phenotype to generate cytotoxic effector cells (CTL) and IL 2-secreting helper T cells in response to either class I or class II MHC allodeterminants. It was found that a discordance between Lyt phenotype and MHC recognition specificity does exist in primary allospecific T cells, but only in one T cell subpopulation with limited functional potential: namely, Lyt-2+ T cells with cytotoxic, but not helper, function that recognize class II MHC alloantigens. Target cell lysis by these Lyt-2+ class II-allospecific CTL was inhibited by anti-Ia monoclonal antibodies (mAb), but not anti-Lyt-2 mAb, indicating that they recognized class II MHC determinants as their "restriction" specificity and not as their "nominal" specificity even though they were Lyt-2+. A second allospecific T cell subset with limited functional potential was also identified but whose Lyt phenotype and MHC restriction specificity were not discordant: namely, an L3T4+ T cell subset with helper, but not cytotoxic, function specific for class I MHC allodeterminants presented in the context of self-Ia. Thus, the present study demonstrates that primary allospecific T cell populations contain phenotypically identical subpopulations of helper and effector cells that express fundamentally different MHC recognition specificities. Because the recognition specificities expressed by mature T cells reflect the selection pressures they encountered during their differentiation into functional competence, these findings suggest that functionally distinct but phenotypically identical T cell subsets may be selected independently of one another during ontogeny. Thus, the existence of Lyt-2+ CTL specific for class II allodeterminants can be explained by the hypothesis that the association of Lyt phenotype with MHC recognition specificity results from the process of thymic selection that these Lyt-2+ effector cells avoid.     

Existence of MHC class I-restricted alloreactive CD4+ T cells reacting with peptide transporter-deficient cells

It is generally accepted that as the result of positive thymic selection, CD8-expressing T cells recognize peptide antigens presented in the context of MHC class I molecules and CD4-expressing T cells interact with peptide antigens presented by MHC class II molecules. Here we report the generation of TCRalpha/beta(+), CD3(+), CD4(+), CD8(-), MHC class I-restricted alloreactive T-cell clones which were induced using peripheral blood mononuclear cells from healthy individuals following in vitro stimulation with transporter associated with antigen processing (TAP)-deficient cell lines T2. The CD4(+) T-cell clones showed an HLA-A2.1-specific proliferative response against T2 cells which was inhibited by anti-CD3 and anti-CD4 monoclonal antibodies. These results suggest that interaction of the TCR with peptide-bound HLA class I molecules contributes to antigen-specific activation of these co-receptor-mismatched T-cell clones. Antigen recognition by alloreactive MHC class I-restricted CD4(+) T cells was inhibited by removing peptides bound to HLA molecules on T2 cells suggesting that the alloreactive CD4(+) T cells recognize peptides that bind in a TAP-independent manner to HLA-A2 molecules. The existence of such MHC class I-restricted CD4(+) T cells which can recognize HLA-A2 molecules in the absence of TAP function may provide a basis for the development of immunotherapy against TAP-deficient tumor variants which would be tolerant to immunosurveillance by conventional MHC class I-restricted cytotoxic lymphocytes.     

The murine IL 2 receptor. III. Cellular requirements for the induction of IL 2 receptor expression on T cell subpopulations

The accessory cell requirements for the induction of the IL 2 receptor by the lectin Con A on murine T cell subsets were directly assayed with anti-IL 2 receptor monoclonal antibodies. Substantial levels of IL 2 receptor expression were induced on T lymphocytes of the MHC class I-restricted, suppressor/cytotoxic phenotype (L3T4-, Ly-2+) in the presence and absence of accessory cells. In contrast, high levels of IL 2 receptor expression could only be induced on T cells of the MHC class II-restricted, helper/inducer phenotype (L3T4+, LY-2-) in the presence, but not in the absence, of accessory cells. Ia- cells such as the P388D1 macrophage line or cultured fibroblasts (DAP X 3) were as efficient as the Ia+ B cell hybridoma LB in providing accessory cell function for the L3T4+, Ly-2- subset. PMA, but not purified human IL 1, could substitute for accessory cells for both IL 2 receptor expression and IL 2 secretion by the L3T4+, Ly-2- subset. These data suggest that IL 2 receptor induction on the L3T4+, Ly-2- subset is complex, possibly requiring a T cell-accessory cell interaction, whereas the lectin may directly trigger IL 2 receptor expression on L3T4-, Ly-2+ T cells.     

Chromosomal distribution of the hamster intracisternal A-particle (IAP) retrotransposons

The retrotransposon-like elements of the intracisternal A-particle (IAP) sequences occur in about 900 copies per haploid hamster cell genome. By applying the fluorescent in situ hybridization (FISH) technique and four different, cloned segments of the IAP element as hybridization probes, these elements were found to be distributed in specific patterns over many of the 44 hamster chromosomes. The hybridization patterns were very similar regardless of whether all four probes or only the IAPI probe carrying the long terminal repeat (LTR) region were used. The IAP elements were found most abundantly, though not exclusively, on the short arms of at least 12 of the autosomes. Of the sex chromosomes, the shorter Y chromosome was stained on both arms, and the X chromosome on one arm by the IAP probes. Primary Syrian hamster cells, the established Syrian hamster cell line BHK21, and the adenovirus type 12 (Ad12)-transformed BHK21 cell line T637 yielded very similar results. In Chinese hamster ovary (CHO) or 3T3 mouse cells, signals could not be elicited by FISH using the Syrian hamster IAP probes. On Southern blots, the DNAs from these cell lines hybridized very weakly, if at all, to the IAP sequences. Thus, IAP sequences were retroposed after Syrian hamster and mouse or Syrian and Chinese hamsters had diverged in evolution.     

Structure of a class I gene from Syrian hamster

Syrian hamsters possess a multigene class I family yet fail to perform several associative immunologic functions. In an attempt to determine whether representative hamster genes are structurally functional, we have cloned two closely linked class I-like genes and determined the complete sequence of the 5' member. Its exon organization is similar to that seen in mouse and man, although only two intracytoplasmic domains are encoded instead of the usual three. Comparison of the predicted amino acid sequence and the 3' untranslated region to mouse and human genes suggest along with the linkage data that the hamster gene may be related to either or both K and Qa region genes but probably not to D and L region genes.     

Monoclonal antibodies to hamster class II MHC molecules distinguish T and B cells

Monoclonal antibodies were prepared against cell surface antigens present on Syrian hamster lymphocytes and a hamster B cell lymphoma line, GD-36. One of these antibodies, S11, precipitated glycoproteins of 29,000 and 39,000 m.w. These glycoproteins were shown to be identical to or a subset of la-like glycoproteins precipitated by hamster alloantisera; however, molecules identified by S11 differed from the predominant hamster la homologues detected with a cross-reactive monoclonal antibody to murine la.7. The immunofluorescence pattern of both anti-la reagents, S11 and anti-la.7, on hamster lymphoid cells is similar by fluorescence-activated cell sorter analysis. A subpopulation of spleen and lymph node cells stains brightly with these antibodies. By two-color fluorescence, this peripheral lymphocyte subpopulation, identified with monoclonal anti-hamster la, also bears surface immunoglobulin (IgM). These data strongly suggest that hamster resting peripheral B cells, and not T cells, express la antigens and can be identified and isolated differentially by using this marker.     

Isolation of cloned Syrian hamster insulinoma cell lines with limited capacity for insulin production.

Cell lines derived from a Syrian hamster insulinoma have been cloned in agar and maintained continuously in culture in vitro for 1 1/2 years. The cell lines have average doubling times of 48 h, they have modal chromosome numbers between 38 and 39, and they retain the ability to form tumors when injected into Syrian hamsters. The cells do not produce immunoreactive insulin when grown in vitro (less than 0.5 ng/mg protein), but do produce immunoreactive insulin when grown in vivo as tumors (mean value from six determinations = 7.1 +/- 1.5 ng/mg protein).     

T cells are not required for pathogenesis in the Syrian hamster model of hantavirus pulmonary syndrome

Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). In hamsters, ANDV causes a respiratory distress syndrome closely resembling human HPS. The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, T cell immunopathology has been implicated on the basis of circumstantial and corollary evidence. Here, we show that following ANDV challenge, hamster T cell activation corresponds with the onset of disease. However, treatment with cyclophosphamide or specific T cell depletion does not impact the course of disease or alter the number of surviving animals, despite significant reductions in T cell number. These data demonstrate, for the first time, that T cells are not required for hantavirus pathogenesis in the hamster model of human HPS. Depletion of T cells from Syrian hamsters did not significantly influence early events in disease progression. Moreover, these data argue for a mechanism of hantavirus-induced vascular permeability that does not involve T cell immunopathology.     

TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4-8+ T cells.

The transporter associated with the antigen processing 1 (TAP1) gene encodes a subunit for a transporter, presumed to be involved in the delivery of peptides across the endoplasmic reticulum membrane to class I molecules. We have generated mice with a disrupted TAP1 gene using embryonic stem cell technology. TAP1-deficient mice are defective in the stable assembly and intracellular transport of class I molecules and consequently show severely reduced levels of surface class I molecules. These properties are strikingly similar to those described for the TAP2 mutant cell line RMA-S. Cells from the TAP1-deficient mice are unable to present cytosolic antigens to class I-restricted cytotoxic T cells. As predicted from the near absence of class I surface expression, TAP1-deficient mice lack CD4-8+ T cells.     

Inhibitors of retrovirus infection are secreted by several hamster cell lines and are also present in hamster sera.

We have previously shown that Chinese hamster ovary (CHO) cells are resistant to infection by gibbon ape leukemia virus and amphotropic pseudotype retroviral vectors because of the secretion of factors that inhibit retrovirus infection. Such factors were not secreted by any mouse or human cell lines tested. Secretion of the inhibitors and resistance to infection are abrogated by treatment of CHO cells with the glycosylation inhibitor tunicamycin. Here we show that the inhibitory activities against gibbon ape leukemia virus and amphotropic viruses are partially separable and that glycosylation mutations in CHO cells mimic the effects of tunicamycin treatment. We find that several hamster cell lines derived from both Chinese and Syrian hamsters secrete inhibitors of retrovirus infection, showing that these inhibitors are not unique to the CHO cell line. Inhibitory factors are also present in the sera of Chinese and Syrian hamsters but were not detected in bovine serum. These results suggest the presence of specific factors that function to inhibit retrovirus infection in hamsters.     

Persistent infection of some standard cell lines by lymphocytic choriomeningitis virus: transmission of infection by an intracellular agent.

Cell-free cytoplasmic extracts of the Syrian hamster cell lines C13/SV28 and BHK-21F were immunogenic in Syrian hamsters. The resulting antisera cross-reacted completely with antisera against lymphocytic choriomeningitis virus (LCMV) in an immunoradiometric assay employing BHK-21F antigen. Several other Syrian hamster cell lines not previously known to be infected with LCMV were also strongly positive when assayed for viral antigens. Also, several mouse sera and antisera raised in Syrian hamsters against cells transformed by papovaviruses had high titers of anti-LCMV activity. No cytopathic effect was evident in any of the persistently infected cell lines. Culture media from these cells were not infectious and showed no evidence of defective interfering particles. However, cell-free extracts of all the persistently infected cells contained material capable of transmitting the persistent infection to uninfected cells of Syrian hamsters, rats, mice, green monkeys, and humans. The onset of infection is much slower than when LCMV virions are used. When 2 X 10(6) uninfected BHK cells were treated with an extract from 100 persistently infected cells, the new infection was apparent within about 12 days. When an extract from 10(6) cells was used, the new infection was apparent within about 5 days, but not sooner. The intracellular infectious material was sensitive to treatment with deoxycholate, Nonidet P-40, or ether but resistant to treatment with RNase or trypsin. It was also large (5,000S) and heterodisperse on sucrose gradients. The infectious material was probably contained in large lipid vesicles and their integrity was probably essential for infection. When a few persistently infected cells were cocultivated with many uninfected cells, a few discrete colonies positive for LCMV antigens were observed after about 5 days. Since the culture media were not infectious, the infection probably spread by cell-cell contact. Several different experiments indicated that interferon did not play a major role in mediating persistence in this case. Persistent infections by LCMV can be maintained without expression of extracellular virus particles and without appearance of large amounts of viral antigens on the cell surface. Cell-cell contact could still allow transmission of intracellular infectious material. In an animal, these properties could circumvent immune surveillance.     

Malignant transformation of hamster kidney cells by BK virus.

Primary hamster kidney cells were transformed by BK virus, a new human papovavirus. Transformed (HKBK) cell produced BK virus T antigen and induced tumors in hamsters that developed antibodies to BK virus T antigen. BK virus was rescued from HKBK cells by Sendai virus-assisted fusion with permissive cells. One out of six cell lines derived from HKBK cell-induced tumors showed the same characteristics as HKBK cells.     

Subpopulations of the T4+ inducer T cell subset in man: evidence for an amplifier population preferentially expressing Ia antigen upon activation

Prior studies demonstrated that Ia molecules were expressed on a fraction of human peripheral T4+ inducer cells upon stimulation by soluble antigen. In the present study, we utilized a fluorescence-activated cell sorter to separate antigen-activated T4+,Ia+ and T4+,Ia- populations and characterized their function. It was found that the T4+,Ia+ population contained the majority of proliferating T cells as assessed by tritiated thymidine incorporation. This proliferation largely appeared to be nonspecific. Despite macrophage repletion, elimination of the Ia+ subset of T cells with monoclonal anti-Ia antibody and complement treatment equally diminished subsequent proliferation to both the triggering antigen and an unrelated antigen. Moreover, the antigen-induced Ia+ subset of T cells alone produced a nonspecific helper factor, LMF. In contrast, the the T4+,Ia- population showed minimal proliferation to soluble antigen and did not generate LMF. Nevertheless, both T4+,Ia+ and T4+,Ia- inducer T cells were required to generate maximal immunoglobulin production by B cells in an antigen-driven system. We conclude that the human T4+ inducer T cell subset is comprised of at least 2 functionally distinct subpopulations, which are capable of acting in a synergistic fashion to provide help to B cells.     

Generation of T cell help through a MHC class I-restricted TCR

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8beta-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.