Thiol-mediated redox regulation of intestinal lamina propria T lymphocytes.

Intestinal lamina propria T lymphocytes (LP-Ts) have a markedly low proliferative potential both in vivo and in vitro. Here, we have identified that the capacity of antigen-presenting cells to release cysteine upon receptor-ligand interactions represents a critical parameter for proliferation of LP-Ts. The availability of cysteine is limiting for the intracellular production of glutathione, which in turn is essential for cell cycle progression. When cysteine is provided either directly or by addition of the reducing agent 2-mercaptoethanol to cystine-containing culture medium, proliferation of LP-T is fully restored. Importantly, coculture with peripheral blood monocytes that easily take up cystine, reduce cystine, and secrete cysteine also restores reactivity of LP-Ts to T cell receptor/CD3 stimulation. In marked contrast, lamina propria macrophages lack this capacity to elaborate cysteine, and thereby secure physiological unresponsiveness to antigen exposure in the intestinal microenvironment. The well-documented local recruitment of blood monocytes in inflammatory bowel disease (IBD) may thus represent an important parameter underlying hyperresponsiveness of T cells, an essential component of the pathogenesis of IBD.

Delineation of an immunoregulatory amplifier population recognizing autologous Ia molecules. Analysis with human T cell clones.

Autoreactive T lymphocytes were generated by culturing human peripheral blood mononuclear cells with an antigen-specific major histocompatibility complex (MHC)-restricted autologous inducer T cell, termed RW17C and subsequently cloned in soft agar. The majority of such clones (AC1-13) expressed the T3+T4+T8-T11+Ia+ phenotype and were directed at autologous class II MHC gene products found on B cells, macrophages, and B lymphoblastoid cells as judged by their proliferative response to the latter. For this recognition, the clones employed a T3-Ti molecular complex and a T4 structure analogous to those found on allospecific T cells. Perhaps more importantly, it was observed that the same AC1-13 autoreactive clones (AC) induced autologous B cells to produce high levels of immunoglobulin in the absence of exogenous antigen and could synergize with the RW17C clone to effect maximal B cell Ig production. These results support the notion that such autoreactive cells can function in a physiologic amplifier role by facilitating induction via an internal set of signals (i.e. autologous MHC).

Peptide variability exists within alpha and beta subunits of the T cell receptor for antigen.

The T cell receptor for antigen (Ti) has recently been identified as a 90-kdalton T3-associated clonotypic structure composed of one 49-51-kdalton alpha and one 43-kdalton beta subunit, which are disulfide linked. Here, Ti molecules from two alloreactive CTL clones derived from the same donor but of differing specificities (CT8III and CT4II) are directly compared following isolation with anticlonotypic monoclonal antibodies. Isoelectric focusing shows that the alpha subunits (pI 4.4-4.7) are more acidic than the beta subunits (pI 6.0-6.2) but that each glycoprotein species is distinctive. More importantly, two-dimensional peptide maps of 125I-labeled surface receptors indicate that the beta chains of Ti1 and Ti2 appear unique and share only two peptides in common. In contrast, peptide maps of Ti1 and Ti2 alpha chains are more related although not identical. These results suggest that the human T cell receptor is composed of constant as well as variable regions and that at least one of the latter is located within the beta subunit.

Amino acid residues required for binding of lymphocyte function-associated antigen 3 (CD58) to its counter-receptor CD2.

Efficient activation and regulation of the cellular immune response requires engagement of T cell accessory molecules as well as the antigen-specific T cell receptor. The lymphocyte function-associated antigen (LFA) 3 (CD58)/CD2 accessory pathway, one of the first discovered, has been extensively characterized in terms of structure and function of the CD2 molecule, which is present on all T lymphocytes and natural killer cells of the human immune system. The binding site of human CD2 for LFA-3 has been localized to two epitopes on one face of the first immunoglobulin (Ig)-like domain of this two-domain, Ig superfamily molecule. Human LFA-3 is genetically linked and is 21% identical in amino acid sequence to CD2, suggesting that this adhesive pair may have evolved from a single ancestral molecule. We have aligned the amino acid sequences of LFA-3 and CD2 and mutagenized selected amino acids in the first domain of LFA-3 that are analogous to those implicated in the binding site of CD2. The data show that K30 and K34, in the predicted C-C' loop, and D84, in the predicted F-G loop of LFA-3, are involved in binding to CD2, suggesting that two complementary sites on one face of the first domain of each molecule bind to each other.

Antigen-like effects of monoclonal antibodies directed at receptors on human T cell clones.

Recent studies suggested that the clonally unique Ti epitopes defined by non-cross-reactive monoclonal antibodies might represent the variable regions of the antigen receptor. Here we determine whether such anti-Ti antibodies could trigger clonal T cell activation. Anticlonotypic monoclonal antibodies to the 49/43-kdalton heterodimer of a given clone or antibodies to the 20/25-kdalton membrane associated monomorphic T3 molecule selectively induce proliferation and IL-2 secretion when linked to a solid support. In contrast, anti-T4 and anti-T8 antibodies under the same conditions have no effect. In conclusion, these results imply that anticlonotypic antibody functions in a fashion analogous to antigen and further support the notion that the T3-Ti molecular complex represents the antigen receptor on human T lymphocytes.

Identification of a novel dimeric phosphoprotein (PP29/30) associated with signaling receptors in human T lymphocytes and natural killer cells.

Two-dimensional gel electrophoresis of in vitro phosphorylated proteins coprecipitated by CD2 monoclonal antibody (mAb) from Brij58 lysates of resting human T lymphocytes and natural killer (NK) cells resulted in the identification of a novel 29/30-kD disulfide-linked dimer (pp29/30). Comparative two-dimensional analysis of CD2, CD3, CD4, CD5, and CD8 immunoprecipitates revealed that pp29/30 associates with these signaling receptor complexes but not with CD18, CD27, and CD29 in human T lymphocytes. Analysis of CD2 immunoprecipitates prepared from T cell antigen receptor/CD3-modulated T lymphocytes indicated that pp29/30 preferentially associates and comodulates with the human T cell antigen receptor (TCR). Since tyrosine phosphorylated pp29/30 selectively interacts with the Src homology type 2 domains (SHZ) of the protein tyrosine kinases p56lck and p59fyn but not ZAP70 the present data suggest that pp29/30 represents a novel signaling receptor associated phosphoprotein likely involved in the activation of human T lymphocytes and NK cells.

Clonotypic structures involved in antigen-specific human T cell function. Relationship to the T3 molecular complex.

Monoclonal antibodies were produced against a human cytotoxic T cell clone, CT8III (specificity: HLA-A3), with the view of defining clonally restricted (clonotypic) surface molecules involved in its antigen recognition function. Two individual antibodies, termed anti-Ti1A and anti-Ti1B, reacted exclusively with the CT8III clone when tested on a panel of 80 additional clones from the same donor, resting or activated T cells, B cells, macrophages, thymocytes, or other hematopoietic cells. More importantly, the two antibodies inhibited cell-mediated killing and antigen-specific proliferation of the CT8III clone but did not affect the functions of any other clone tested. This inhibition was not secondary to generalized abrogation of the CT8III clone's function, because interleukin 2 responsiveness was enhanced. To examine the relationship of the structures defined by anti-clonotypic antibodies with known T cell surface molecules, antibody-induced modulation studies and competitive binding assays were performed. The results indicated that the clonotypic structures were associated with, but distinct from, the 20,000-mol wt T3 molecule expressed on all mature T lymphocytes. Moreover, in contrast to anti-T3, anti-Ti1A and anti-Ti1B each immunoprecipitated two molecules of 49,000 and 43,000-mol wt from 131I-labeled CT8III cells under reducing conditions. The development of monoclonal antibodies to such polymorphic T cell surface structures should provide important probes to further define the surface receptor for antigen.

Functional characterisation of decoy receptor 3 in Crohn's disease.

AIMS: Both epithelial barrier dysfunction and apoptosis resistance of immune cells contribute to the pathogenesis of Crohn's disease. The soluble decoy receptor 3 (DcR3) acts in an anti-apoptotic manner by neutralising the death ligand CD95L. Here, we investigated the possible involvement of DcR3 in Crohn's disease. METHODS: The epithelial fraction of human small intestinal mucosa samples was obtained by laser microdissection. Expression of DcR3 was examined by global gene expression profiling, quantitative reverse transcription polymerase chain reaction, immunoblot analysis, and immunohistochemistry. DcR3 concentrations in the serum of patients with Crohn's disease were measured by enzyme-linked immunosorbent assay. Apoptosis assays were performed to study the effects of DcR3 in intestinal epithelial cells and lamina propria T cells. RESULTS: DcR3 is over-expressed in the epithelial layer of ileum specimens in patients with Crohn's disease, both at actively inflamed and non-active sites. DcR3 serum levels are significantly elevated in patients with active and non-active Crohn's disease as compared to healthy controls. The expression of DcR3 in intestinal epithelial cells is induced by tumour necrosis factor alpha. Increased DcR3 expression is associated with activation of nuclear factor kappa B (NF-kappaB) and results in protection of intestinal epithelial cells and lamina propria T cells from CD95L-induced apoptosis. CONCLUSIONS: DcR3 may promote inflammation in Crohn's disease by inhibiting CD95L-induced apoptosis of epithelial and immune cells as well as by inducing NF-kappaB activation.

Surface structures involved in target recognition by human cytotoxic T lymphocytes.

Cloned human cytotoxic T lymphocytes and monoclonal antibodies inhibiting their function (anti-T3A, anti-T4A, and anti-T8A) were used to elucidate the role of T cell surface glycoproteins in cell-mediated lympholysis involving individual classes of gene products of the major histocompatibility complex on target cells. The results indicate that several surface molecules are required for specific target recognition: T3 and T4 on T4+ cytotoxic T lymphocytes and T3 and T8 on T8+ cytotoxic T lymphocytes.

Identification of the receptor for antigen and major histocompatibility complex on human inducer T lymphocytes.

Human T cell clones and monoclonal antibodies directed at their surface structures were used to define the receptor for the antigen and major histocompatibility complex on inducer T lymphocytes. The results indicated that the receptor is a single complex consisting of the monomorphic T3 molecule with a molecular weight of 20,000 to 25,000 and a clonotypic disulfide linked heterodimer Ti with a molecular weight of 90,000. Sepharose-bound monoclonal antibodies (anti-Ti4 or anti-T3) to the receptor could activate clonal proliferation and inducer function for B cell immunoglobulin secretion and thus substitute for the appropriate combination of major histocompatibility complex gene product and specific antigen.

Up-regulation of the phosphoinositide 3-kinase pathway in human lamina propria T lymphocytes.

Human intestinal lamina propria T lymphocytes (LPT), when investigated ex vivo, exhibit functional properties profoundly different from those of peripheral blood T lymphocytes (PBT). One prominent feature represents their enhanced sensitivity to CD2 stimulation when compared to PBT. Given that LPT are hyporesponsive to T cell receptor (TCR)/CD3 stimulation, an alternative activation mode, as mimicked by CD2 triggering in vitro, may be functional in mucosal inflammation in vivo. This study provides insight into signalling events associated with the high CD2 responsiveness of LPT. When compared to PBT, LPT show an increased activation of the phosphoinositide 3/protein kinase B/glycogen synthase kinase 3beta (PI3-kinase/AKT/GSK-3beta) pathway in response to CD2 stimulation. Evidence is provided that up-regulation of this pathway contributes to the enhanced CD2-induced cytokine production in LPT. Given the importance of TCR-independent stimulation for the initiation of intestinal immune responses analysis of signalling pathways induced by 'co-stimulatory' receptors may provide valuable information for therapeutic drug design.

Clonal analysis of CD4+/CD8+ T cells in a patient with aplastic anemia.

T cell clones were established from peripheral blood of a patient with severe aplastic anemia. 8 of 18 individual clonal T cell populations stably coexpressed CD4 and CD8 molecules, a phenotype characteristic for thymocytes and a minor subpopulation of circulating T lymphocytes. Analysis of T cell receptor genes revealed identical rearrangements of T cell receptor beta chain genes, suggesting clonality of these T cells. CD4+/CD8+ T cells clones were found to be efficiently cytotoxic towards autologous lymphoblasts. Autocytotoxicity could be blocked by a CD3 MAb, a MAb specific for monomorphic MHC class II determinants, and particularly, by an MHC-DP-specific MAb, suggesting specificity for autologous DP molecules. Perhaps more important, CD4+/CD8+ T cell clones inhibited differentiation of autologous progenitor enriched bone marrow cells in vitro by a direct cell-mediated mechanism. These data suggest that circulating cytotoxic CD4+/CD8+ T cell clones specific for autologous MHC-DP determinants may be involved in hematopoietic failure in some cases of aplastic anemia.

Interleukin 1 stimulates T lymphocytes to produce granulocyte-monocyte colony-stimulating factor.

T lymphocytes are thought to cooperatively interact with monocytes to produce colony-stimulating factors (CSF). However, little is known about monocyte-mediated signals leading to CSF-secretion by T lymphocytes, although soluble monocyte products have been implicated. We have employed monoclonal antibody anti-T3B covalently coupled to CnBr-activated Sepharose 4B beads, to show that multimeric ligation of T cell antigen receptor leads to T cell receptiveness to interleukin 1 (IL-1), as indicated by T cell production of CSF, which induces growth of myeloid progenitor cells into neutrophil, eosinophil, and monocyte colonies. To investigate the molecular basis of these findings, total RNA was extracted from T3B Sepharose-primed and IL-1-stimulated T lymphocytes and probed for granulocyte-monocyte-CSF (GM-CSF), granulocyte-CSF (G-CSF), and monocyte-CSF (M-CSF) mRNA. GM-CSF, but not G-CSF or M-CSF, messages were detected. Nuclear "run on" assays revealed that IL-1 action is effective primarily at the level of GM-CSF gene transcription. These results suggest a previously unrecognized role of IL-1 in the regulation of GM-CSF secretion by T cells.

Selective blockade of the antigen-receptor-mediated pathway of T cell activation in patients with impaired primary immune responses.

We investigated impaired cellular immune responses of individuals on chronic hemodialysis by using monoclonal antibodies that trigger differential pathways of T cell activation. Reduced cellular reactivity, which exists in a high proportion of such patients, can be attributed to a failure of the monocyte population to support the process of primary T cell activation in vitro. This defect results in a lack of interleukin 2 production, which is critically dependent on a monocyte-derived signal. In contrast, T lymphocyte function was found to be physiologic. Perhaps more important, the degree of monocyte dysfunction in vitro correlated with the same patients' in vivo responses to hepatitis B vaccination. Addition of recombinant human interleukin 2 fully reconstituted their deficient immune response in vitro.

Spontaneous responsiveness to cytokines by human T-cell leukemias.

The molecular basis for autonomous growth of malignant forms of human T lymphocytes is not known. It can be investigated by functional responses of malignant cells in comparison with untransformed counterparts. At least two pathways (the CD2 and CD3 pathways) of human T-cell activation have been recently defined on the basis of monoclonal antibody activities in vitro, an experimental model exists which can be used to investigate which pathway of T-cell triggering might be involved in malignant growth. In untransformed T lymphocytes, responses to addition of cytokines are strictly controlled by signals which are mediated through triggering molecules including CD2 and CD3 and we therefore investigated 20 freshly recovered human T leukemias with regard to spontaneous growth in response to interleukins. The majority of cases (16 out of 20) investigated displayed spontaneous responsiveness to cytokines (interleukins 1, 4, and 6), which might be related to activation signals mediated through the CD2 pathway. The functional repertoire of T leukemias did not correlate with expression of differentiation antigens conventionally employed for leukemia typing.

Immune reactions induced by interleukin-2 transfected colorectal cancer cells in vitro: predominant induction of lymphokine-activated killer cells.

One aim of the genetic modification of tumor cells is the generation of immunogenic variants that can be used for the induction of immune responses against tumors. We engineered the human colorectal carcinoma cell line SW480 by means of plasmid transfection to secrete interleukin (IL)-2. Transfection of SW480 cells resulted in stable IL-2 secretion at 5-30 ng/ml per 10(5) cells in 24 h and, unexpectedly, in CD54 expression on the cell surface. SW480 variants expressing IL-2 and CD54 were tested for their capacity to induce T lymphocyte activation in vitro in comparison to untransfected and CD54 transfected cells. The cytolytic effector function of a class I MHC restricted CD8+, peptide antigen specific T cell clone was augmented following expression of CD54. IL-2 secreting SW480 variants did not further increase antigen-dependent cytolysis. Primary activation of resting T lymphocytes was assessed following allogeneic stimulation. When compared with unmodified SW480 cells, CD54 expressing variants did not initiate T cell proliferation. In contrast, IL-2 secreting SW480 cells strongly promoted primary T cell proliferation. Similarly, exogenous IL-2 and SW480 cells induced T cell proliferation which was not only due to IL-2 but was dependent on tumor cells. However, following the initial wave of cell growth in response to IL-2 secreting SW480 cells T lymphocytes could not be restimulated with SW480 or IL-2 secreting variants and could not be further expanded. T cells initially activated by IL-2 secreting SW480 cells exhibited cytolytic activity towards SW480 cells. This reactivity, however, was transient and completely blocked by K562 cells, suggesting MHC-unrestricted, nonspecific cytotoxicity. We conclude that endogenous IL-2 secretion by the colorectal carcinoma cell line SW480 does not result in the activation of MHC restricted specific T lymphocytes but predominantly induces lymphokine-activated killer cells. Considering that tumor cell vaccines are aimed at inducing tumor-specific immune responses, our in vitro observation would rather argue against the in vivo application of such a tumor cell modification in colorectal cancer.

Potential of CD80-transfected human breast carcinoma cells to induce peptide-specific T lymphocytes in an allogeneic human histocompatibility leukocyte antigens (HLA)-A2.1+-matched situation.

Allogeneic human histocompatibility leukocyte antigen (HLA)-matched tumor cell lines that have been made immunogenic by the transfer of genes encoding for costimulatory molecules such as CD80 are considered to be potential vaccines for the induction of systemic immune reactions in cancer patients. We used a human HLA-A2.1+ CD80-transfected breast carcinoma cell line (KS-CD80) and investigated in vitro the efficiency at which antigen (Ag)-specific responses were induced following the stimulation of allogeneic HLA-A2.1-matched T lymphocytes. The influenza matrix protein M1 was used as a model Ag. It was either endogenously expressed or exogenously loaded as a peptide (matrix protein), and the frequency of the generated specific T cells was determined. The expression of CD80 in KS cells was required for an effective activation and expansion of Ag-specific T cells. This response was augmented following the pretreatment of KS-CD80 cells with interferon-gamma and tumor necrosis factor-alpha. Interleukin-4 (IL-4), IL-7, and IL-12 further increased T-cell expansion. IL-7 was best at supporting the generation of T cells with Ag specificity. This investigation demonstrates that allogeneic CD80+ tumor cells can induce Ag-specific, HLA-restricted T lymphocytes at a high frequency. Our study supports the use of allogeneic cell lines for the induction of specific T-cell responses in tumor patients.

Interleukin-12 requires initial CD80-mediated T-cell activation to support immune responses toward human breast and ovarian carcinoma.

One possible reason for the poor immunogenicity of tumors is the induction of peripheral tolerance by tumor cells that fail to deliver costimulatory signals. Furthermore, T cells stimulated with wild-type tumor cells often fail to secrete cytokines. The present study has been undertaken to identify cytokines that cooperate with CD80 in T-cell activation in vitro toward human breast and ovarian carcinoma cell lines. Tumor cell-mediated T-lymphocyte activation was analyzed directly in allogeneic mixed lymphocyte/tumor cell cultures as proliferation and effector functions were assessed in cytotoxic T-cell assays. Interleukin-7 (IL-7) amplified the proliferative response toward CD80-transfected breast and ovarian carcinomas and stimulated predominantly CD4+ T lymphocytes. IL-12 represses the proliferative response of naive T cells but cooperates with CD80-mediated activation during secondary stimulations. In long-term T-cell cultures, IL-12 synergizes with CD80 expression to stimulate cytolytic CD8+ T-cell lines, which recognize a breast carcinoma line in a human histocompatibility leukocyte antigen-restricted manner. These studies illustrate that costimulation is necessary for tumor cells to function as alloantigen-presenting cells. Furthermore, when added after the priming of T cells with CD80-transfected tumor cells, IL-12 could be helpful in propagating sufficient T-cell numbers to be used in adoptive transfers during cellular immunotherapy.

Definition of immunogenic determinants of the human papillomavirus type 16 nucleoprotein E7.

Specific T lymphocyte lines and T cell clones were established from peripheral blood mononuclear cells of asymptomatic seropositive individuals employing synthetic peptides which correspond to the sequence of the human papillomavirus (HPV) type 16 transforming protein E7. Specificity analysis of T cells as determined by means of [3H] thymidine incorporation after stimulation with individual peptides revealed three immunogenic determinants of E7 that are recognised in association with at least two different HLA haplotypes. One N-terminal region (aminoacids 5-18) was recognised by one T cell line. T cell clones and the corresponding T cell line established from another donor responded to a different N-terminal (17-38) and to a C-terminal region (69-86). The N-terminal sequence 5-18 and the C-terminal determinant contain a periodicity of hydrophilic and hydrophobic residues that have been found in many T cell epitopes. Phenotypic characterisation of T cell clones by indirect immunofluorescence revealed that the T cell clones expressed the CD4 surface glycoprotein suggesting that the specific E7 determinants were recognised in association with major histocompatibility complex (MHC) class II molecules. With regard to functional properties, at least three T cell clones exhibited specific cytotoxic activity towards autologous B lymphocytes transformed by Epstein-Barr virus in the presence of the relevant HPV16 E7 peptides. The implications of these results regarding the development of vaccination strategies and host-virus interaction are discussed.

Development of immunogenic colorectal cancer cell lines for vaccination: expression of CD80 (B7.1) is not sufficient to restore impaired primary T cell activation in vitro.

The capacity of colorectal carcinoma and melanoma cell lines to induce primary versus effector T lymphocyte activation in vitro was investigated. Established epithelial tumour cell lines derived from colorectal carcinoma and melanoma did not activate a primary proliferative response of resting T lymphocytes in allogeneic mixed lymphocyte tumour cell cultures (MLTCs). In contrast, the same tumour cells were effectively lysed by preactivated cytolytic T cell clones. This demonstrates that tumour cells are impaired in inducing a primary immune response but are susceptible to effector immune responses. Attempts at improving primary T cell activation revealed that exogenous cytokines, including interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2), were not effective. Expression of CD80 (B7.1), by transfecting a CD80 cDNA into the melanoma cell line SkMel63, improved T cell proliferation considerably. In contrast, CD80 expression in two colorectal carcinoma cell lines (SW480, SW707) did not result in T cell activation. This was not due to lack of class II MHC expression on SW480 since coexpression of a HLA-DR3 alloantigen and CD80 had no effect. Our data suggest that de novo CD80 expression is not, in general, sufficient to improve primary T cell activation by human tumour cells.