CD4+ proliferative T-cell clones reactive to autologous Epstein-Barr virus-transformed B cells (EBV-LCL) can be restricted by HLA class I molecules

In this study, we show that an HLA-loss variant, EBV-LCL .180, which lacks HLA-DR, DP, DQ and B determinants but expresses HLA-A and C molecules, can activate autologous proliferative T cells which recognize EBV-LCL antigens (or LYDMA). T-cell clones isolated from bulk culture (A.180) of T cell primed with autologous variant .180 when tested for their proliferative reactivity to .180 and three autologous class I-loss variants, each lacking specific class I determinants, showed requirement for class I HLA molecules. Clones A.180.Cl and A.180.F3D respond to the A2+, B-null, C+ mutant .53, but not to the A-null, B5+, C+ mutant .144, or the A-null, B-null, C+ mutant .184, indicating that these clones are restricted by an HLA-A2 determinant. These proliferative T-cell clones express the CD4 marker and are noncytotoxic, even in the presence of concanavalin A (Con A) lectin. In coculture experiments, A.180.Cl was further shown to provide lymphokine "help" for EBV-LCL-specific, autologous cytotoxic T-cell clones. These results suggest the repertoire of HLA determinants employed by EBV-LCL-specific proliferative T cells, in addition to the previously shown HLA-DR, DP, and DQ determinants, also includes class I molecules.     

Self-reactive, T cell receptor-gamma delta+, lymphocytes from the intestinal epithelium of weanling mice.

Intraepithelial T lymphocytes (IEL) are dispersed throughout the intestinal epithelial lining but their role in cellular immune defense is unknown. Their location suggests that their highly activated state may be due to constant exposure to bacterial Ag. To study IEL specificity and function we have prepared a panel of IEL-T cell hybridomas from both adult and weanling C57B1/6 mice. Many of these expressed TCR-gamma delta, a cell type rare in peripheral lymph nodes and spleen but predominant at epithelial surfaces. We have identified a subset of gamma delta T cells from weanling mice which is self reactive, i.e., these hybrids secrete IL-2 spontaneously, without antigenic stimulation or a requirement for APC. Self-reactive TCR-gamma delta+ hybrids and lines, all of which bear a particular TCR (V gamma 1.1C gamma 4V delta 6), have previously been derived from neonatal thymus and the skin. Northern blot and immunoprecipitation analyses suggest that the self-reactive IEL hybrids also bear a C gamma 4/V delta 6 TCR. Antibody inhibition experiments showed that the self-reactivity of the IEL hybrids is TCR mediated. Spontaneous IL-2 production was blocked by soluble anti-CD3 and anti-TCR-gamma delta antibodies but not by antibodies to the TCR-alpha beta. The self-reactive IEL hybrids lack class II MHC and the class I-like proteins CD1 and TLA but express class I MHC. IEL hybrids may also require the vitronectin receptor as an accessory molecule for their activation because spontaneous IL-2 production is blocked by antibody to the vitronectin receptor as well as by the extracellular matrix protein active site peptide RGDS, but not the control peptide RGES. V gamma 1.1C gamma 4V delta 6 T cells in the thymus, skin, and intestine may represent a small and unique subpopulation of lymphocytes with a potential for autoimmune reactivity at peripheral sites.     

Human V gamma 9-V delta 2 T cell receptor-gamma delta lymphocytes show specificity to Daudi Burkitt's lymphoma cells

Peripheral blood TCR-gamma delta cells with different functional V gamma or V delta gene rearrangements represent two nonoverlapping subsets. The major subset uses the V gamma 9 and the V delta 2 gene segments and the minor subset the V delta 1 gene segments in its functional TCR rearrangement. Upon in vitro activation, these TCR-gamma delta lymphocytes display MHC-unrestricted lytic activity, against a wide variety of tumor cells of distinct histologic origin. Here we show that fresh TCR-gamma delta lymphocytes that express a V gamma 9-V delta 2 encoded TCR display a specific proliferative response to Daudi, Burkitt's lymphoma cells. Moreover, cloned V gamma 9-V delta 2 lymphocytes show the capacity to lyse Daudi cells, whereas none of the cloned V gamma 1 TCR-gamma delta lymphocytes shows such specificity. Nucleotide diversity at the V-D-J junction of the TCR-V delta 2 gene did not contribute to this Daudi cell specificity. Comparison of the MHC-unrestricted cytolytic capacities of the V gamma 9-V delta 2 and the V delta 1 clones using a panel of distinct types of tumor target cells showed that on average, the level of MHC unrestricted lysis of V gamma 9-V delta 2 clones against these tumor cells exceeded that of V delta 1 clones. However, in contrast to all these tumor cell lines, only the Daudi cells showed such an absolute distinction in susceptibility to lysis by V gamma 9-V delta 2 and V delta 1 clones. V gamma 9-V delta 2 clones that were generated with a stimulator cell other than Daudi did not lyse their stimulator cells but nevertheless showed specific cytolysis of Daudi cells. The specific proliferation to and cytolysis of Daudi cells of the entire V gamma 9-V delta 2 subpopulation of TCR-gamma delta lymphocytes is reminiscent of a superantigen response.     

A murine CD4-, CD8- T cell receptor-gamma delta T lymphocyte clone specific for herpes simplex virus glycoprotein I.

The role of TCR-gamma delta T lymphocytes in immune responses is currently not well understood. TCR-gamma delta cells have a limited repertoire suggesting that TCR-gamma delta T a limited number of evolutionarily conserved Ag such as nonpolymorphic MHC and heat shock proteins. TCR-gamma delta T lymphocytes appear in enhanced numbers in skin lesions produced by Mycobacterium leprae and in the synovial fluid of joints affected by rheumatoid arthritis, raising the possibility that this subset of T lymphocytes may play a role in control of infectious processes and in autoimmune diseases. We report the identification of a TCR-gamma delta T cell clone isolated from a HSV-infected mouse that recognizes glycoprotein I of HSV type 1. Clone recognition of glycoprotein I does not appear to require the expression of MHC class I or class II gene products. These data suggest that TCR-gamma delta lymphocytes may play an important role in the immune response to viral infections.     

Recognition of the product of a novel MHC TL region gene (27b) by a mouse gamma delta T cell receptor

The gamma delta T cell receptor (TCR) derived from the mouse KN6 T cell hybridoma recognizes an autologous determinant encoded by a broadly expressed gene mapping in the TL region of the major histocompatibility complex (MHC). We have cloned the gene and demonstrated that it is a novel class I gene (designated 27b) belonging to a hitherto undescribed TL region gene cluster in strain C57BL/6. The BALB/c allele of 27b, gene T17c, is defective because it lacks an appropriate splice acceptor site, which explains the lack of recognition of BALB/c stimulator cells by the KN6 cells. We propose that gamma delta TCR and nonclassical MHC and MHC-related class I molecules have coevolved to recognize a conserved set of endogenous and foreign determinants.     

Staphylococcal enterotoxin-mediated human T-T cell interactions.

Staphylococcal enterotoxins (SE) are known to stimulate a large proportion of T cells. SE bind to MHC-class II molecules on APC and a particular segment of certain TCR V beta and V gamma gene products. Resting human T cells do not express HLA class II Ag and therefore cannot present SE to T cells. Activated human T cells, however, do express HLA-DR, -DP, and -DQ Ag and could consequently serve as APC for SE. As such, local immune responses to SE might be regulated and/or abrogated by SE-mediated T-T cell interactions leading to T cell destruction. We have investigated if such SE-mediated T-T cell interactions can occur in vitro using human cytolytic TCR-alpha beta+ and TCR-gamma delta+ T cell clones. We demonstrate that the TCR-alpha beta+ T cell clones can efficiently present staphylococcal enterotoxin A (SEA) to each other: T cell clones coated with SEA are lysed by SEA-reactive T cell clones but not by a SEA-nonreactive T cell clone. Furthermore, the SEA-reactive TCR-alpha beta+ clones (but not the SEA-nonreactive clone) destruct themselves in the presence of SEA at low concentrations of SEA (less than 0.01 microgram/ml). Also, SEA-coated T cell clones can induce proliferative responses although such responses are much weaker than those induced when B cells are used as stimulator cells. In contrast, the SEA-reactive TCR-gamma delta+ T cell clones are resistant to autokilling in the presence of SEA and they do not lyse SEA-coated TCR-gamma delta+ targets. However, such targets can be lysed by TCR-alpha beta+ effector cells. These results indicate that TCR-gamma delta+ cells are relatively resistant to lysis and that during local nonspecific immune responses triggered by SE, which induces HLA-class II expression by the responding T cells, SE-mediated T-T cell interactions may play a role in the regulation and/or abrogation of these immune responses.     

Comparison of antigen specificity, class II major histocompatibility complex restriction, and in vivo behavior of myelin basic protein-specific T cell lines and clones derived from (BALB/c x SJL/J) mice

Immunization with myelin basic protein (BP) causes experimental allergic encephalomyelitis (EAE) in certain strains of mice. SJL/J (H-2s) is the prototype sensitive strain. Although BALB/c (H-2d) is resistant to EAE through use of an identical immunization protocol, (BALB/c x SJL/J)F1 hybrid mice develop EAE after immunization with BP. T cell clones specific for BP have been isolated from a highly encephalitogenic line of (BALB/c x SJL/J)F1 hybrid T cells raised against bovine BP. The clones were examined for their H-2 restriction and specificity for heterologous forms of BP (mouse, rat, and bovine BP). The results revealed the clones cross-reacting with mouse (self) BP were almost always restricted to F1 hybrid class II major histocompatibility complex (MHC) elements. In contrast, mouse cross-reactive clones derived from a nonencephalitogenic (BALB/c x SJL/J) T cell line raised against rat BP were largely restricted to H-2d elements. These clones did not cross-react with bovine BP. Four additional lines were generated by carrying the original rat and bovine F1 T cell lines on parental antigen-presenting cells thus generating lines biased toward homozygous (SJL/J, H-2s, or BALB/c, H-2d) restriction elements. These "parentally restricted" T cell lines did not induce EAE when injected in vivo. These results suggest that in this F1 strain sensitivity to T cell-induced EAE is associated with epitopes on murine BP that associate with F1 class II MHC restricting elements. In contrast, nonencephalitogenic T cell lines contain a high proportion of murine cross-reactive clones restricted to H-2d, the haplotype of the classically resistant BALB/c mouse. This work illustrates the use of T cell lines and clones in a model system to further analyze the role of MHC restriction elements in autoimmune disease occurring in heterozygous individuals.     

Two better cell lines for making hybridomas expressing specific T cell receptors

Two variants of the AKR thymoma BW5147 have been isolated which can no longer express functional TCR alpha- and beta-chains. By generating hybridomas with these variant fusion lines, TCR of any normal T lymphocyte, including TCR-gamma/delta, can be studied at a clonal level, without interference of the BW5147-derived receptor chains. In this study one of the variants has been useful in identifying the reactivity to allogeneic MHC Ag of BW5147 itself.     

Recognition of naturally processed and ovarian cancer reactive CD8<Superscript>+</Superscript> T cell epitopes within a promiscuous HLA class II T-helper region of NY-ESO-1

NY-ESO-1 is frequently expressed in epithelial ovarian cancer (EOC) and elicits spontaneous humoral and cellular immune responses in a proportion of EOC patients. The identification of NY-ESO-1 peptide epitopes with dual HLA-class I and class II specificities might be useful in vaccination strategies for generating cognate CD4+ T cell help to augment CD8+ T cell responses. Here, we describe two novel NY-ESO-1-derived MHC class I epitopes from EOC patients with spontaneous humoral immune response to NY-ESO-1. CD8+ T cells derived from NY-ESO-1 seropositive EOC patients were presensitized with a recombinant adenovirus encoding NY-ESO-1or pooled overlapping peptides. These epitopes, ESO127-136 presented by HLA-A68 molecule, and ESO127-135 restricted by HLA-Cw15 allele, are located within ESO119-143, a promiscuous HLA-class II region containing epitopes that bind to multiple HLA-DR alleles. The novel epitopes were naturally processed by APC or naturally presented by tumor cell lines. In addition, these epitopes induced NY-ESO-1-specific CTL in NY-ESO-1 seropositive EOC patients. Together, the results indicate that ESO119-143 epitope has dual HLA classes I and II specificities, and represents a potential vaccine candidate in a large number of cancer patients.     

T cell antigen receptor engagement and specificity in the recognition of stress-inducible MHC class I-related chains by human epithelial gamma delta T cells

Human gamma delta T cells with the TCR variable region V(delta)1 occur mainly in epithelia and respond to stress-induced expression of the MHC class I-related chains A and B, which have no function in Ag presentation. MIC function as ligands for NKG2D-DAP10, an activating receptor complex that triggers NK cells, costimulates CD8 alpha beta and V(gamma)9V(delta)2 gamma delta T cells, and is required for stimulation of V(delta)1 gamma delta T cells. It is unresolved, however, whether triggering of V(delta)1 gamma delta TCRs is also mediated by MIC or by unidentified cell surface components. Soluble MICA tetramers were used as a binding reagent to demonstrate specific interactions with various V(delta)1 gamma delta TCRs expressed on transfectants of a T cell line selected for lack of NKG2D. Tetramer binding was restricted to TCRs derived from responder T cell clones classified as reactive against a broad range of MIC-expressing target cells and was abrogated when TCRs were composed of mismatched gamma- and delta-chains. These results and the inability of V(delta)1 gamma delta T cells to respond to target cells expressing the ULBP/N2DL ligands of NKG2D, which are highly divergent from MIC, indicate that MIC delivers both the TCR-dependent signal 1 and the NKG2D-dependent costimulatory signal 2. This dual function may serve to prevent erroneous gamma delta T cell activation by cross-reactive cell surface determinants.     

Edmonston measles virus prevents increased cell surface expression of peptide-loaded major histocompatibility complex class II proteins in human peripheral monocytes

Gamma interferon (IFN-gamma) induces expression of the gene products of the major histocompatibility complex (MHC), whereas IFN-alpha/beta can interfere with or suppress class II protein expression. In separate studies, measles virus (MV) was reported to induce IFN-alpha/beta and to up-regulate MHC class II proteins. In an attempt to resolve this paradox, we examined the surface expression of MHC class I and class II proteins in MV-infected peripheral monocytes in the presence and absence of IFN-alpha/beta. Infection of purified monocytes with Edmonston B MV resulted in an apparent increase in cell surface expression of HLA-A, -B, and -C class I proteins, but it had no effect on the expression of HLA-DR class II proteins. MV-infected purified monocytes expressed IFN-alpha/beta, but no measurable IFN-gamma expression was detected in supernatant fluids. Class II protein expression could be enhanced by coculture of purified monocytes with uninfected peripheral blood mononuclear cell (PBMC) supernatant. MV infection of PBMCs also did not affect expression of class II proteins, but the expression of HLA-A, -B, and -C class I proteins was increased two- to threefold in most donor cells. A direct role for IFN-alpha/beta suppression of MHC class II protein expression was not evident in monocytes since MV suppressed class II protein expression in the absence of IFN-alpha/beta. Taken together, these data suggest that MV interferes with the expression of peptide-loaded class II complexes, an effect that may potentially alter CD4(+)-T-cell proliferation and the cell-mediated immune responses that they help to regulate.     

Functional and phenotypic differences between CD4+ and CD4- T cell receptor-gamma delta clones from peripheral blood.

CD4+ TCR-gamma delta+ T cells comprise a very small subset of TCR-gamma delta+ T cells. CD4+ TCR gamma delta+ T cell clones were established to study the phenotypical and functional characteristics of these cells. Thirty-four CD4+ TCR-gamma delta+ T cell clones were established after sorting CD4+ T cells from a pre-expanded TCR-gamma delta+ T cell population. These clones as well as the CD4- TCR-gamma delta+ T cells from the same donor used V gamma 2 and V delta 2. In a second cloning experiment CD4+ TCR-gamma delta+ T cells were cloned directly from freshly isolated TCR-gamma delta+ T cells using a cloning device coupled to a FACS sorter. Forty-three clones were obtained, which all expressed CD4 and TCR-gamma delta. Eleven of these clones used V delta 1 and three of them coexpressed V gamma 2. The other CD4+ TCR-gamma delta+ T cell clones used both V delta 2 and V gamma 2. CD4+ TCR-gamma delta+ T cell clones expressed CD28 irrespective of the V gamma or V delta usage, and were CD11b negative. Three CD4-CD8+ TCR-gamma delta+ clones expressed CD8 alpha but not CD8 beta and were CD11b positive. CD28 expression among CD4-CD8+ and CD4-CD8- was variable but lower than on CD4+ T cell clones. CD4- TCR-gamma delta+ T cell clones using V gamma 2 and V delta 2 specifically lyse the Burkitt lymphoma cell line Daudi and secrete low levels of IFN-gamma and granulocyte-macrophage-CSF upon stimulation with Daudi. In contrast, most CD4+ T cell clones that use V gamma 2 and V delta 2 had a very low lytic activity against Daudi cells and secrete high levels of IFN-gamma and granulocyte-macrophage-CSF after stimulation with Daudi cells. The NK-sensitive cell line K562 was killed efficiently by the CD4- TCR-gamma delta+ T cell clones, but not by CD4+ TCR-gamma delta+ T cell clones, and could not induce cytokine secretion in CD4+ or CD4- T cell clones. CD4+ TCR-gamma delta+ T cell clones, but not the CD4- clones, could provide bystander cognate T cell help for production of IgG, IgM, and IgA in the presence of IL-2 and IgE in the presence of IL-4. Thus, CD4+ TCR-gamma delta+ T cells are similar to CD4+ TCR-alpha beta+ T cells in their abilities to secrete high levels of cytokines and to provide T cell help in antibody production.(ABSTRACT TRUNCATED AT 400 WORDS)     

EBV-specific CD4+ T cell clones exhibit vigorous allogeneic responses

Alloreactive T cells play a key role in mediating graft-vs-host disease and allograft rejection, and recent data suggest that most T cell alloreactivity resides within the CD4 T cell subset. Particularly, T cell responses to herpesvirus can shape the alloreactive repertoire and influence transplantation outcomes. In this study, we describe six distinct EBV-specific CD4(+) T cell clones that cross-reacted with EBV-transformed lymphoblastoid cell lines (LCLs), dendritic cells, and endothelial cells expressing MHC class II alleles commonly found in the population. Allorecognition showed exquisite MHC specificity. These CD4(+) T cell clones efficiently killed dendritic cells or LCLs expressing the cross-reactive allogeneic MHC class II molecules, whereas they did not kill autologous LCLs. Endothelial cells expressing the proper allogeneic MHC molecules were poorly killed, but they induced high-level TNF-alpha production by the EBV-specific CD4(+) T cell clones. As already proposed, the strong alloreactivity toward LCLs suggest that these cells could be used for selective depletion of alloreactive T cells.     

Increased CD8+ cytotoxic T cell responses to myelin basic protein in multiple sclerosis

Autoreactive T cells of CD4 and CD8 subsets recognizing myelin basic protein (MBP), a candidate myelin autoantigen, are thought to contribute to and play distinct roles in the pathogenesis of multiple sclerosis (MS). In this study we identified four MBP-derived peptides that had high binding affinity to HLA-A2 and HLA-A24 and characterized the CD8(+) T cell responses and their functional properties in patients with MS. There were significantly increased CD8(+) T cell responses to 9-mer MBP peptides, in particular MBP(111-119) and MBP(87-95) peptides that had high binding affinity to HLA-A2, in patients with MS compared with healthy individuals. The resulting CD8(+) T cell lines were of the Th1 phenotype, producing TNF-alpha and IFN-gamma and belonged to a CD45RA(-)/CD45RO(+) memory T cell subset. Further characterization indicated that the CD8(+) T cell lines obtained were stained with MHC class I tetramer (HLA-A2/MBP(111-119)) and exhibited specific cytotoxicity toward autologous target cells pulsed with MBP-derived peptides in the context of MHC class I molecules. These cytotoxic CD8(+) T cell lines derived from MS patients recognized endogenously processed MBP and lysed COS cells transfected with genes encoding MBP and HLA-A2. These findings support the potential role of CD8(+) CTLs recognizing MBP in the injury of oligodendrocytes expressing both MHC class I molecules and MBP.     

Presentation of Candida albicans and purified protein derivative soluble antigens by Epstein-Barr virus-transformed human lymphoblastoid B-cell lines

Cells other than the macrophage can function as antigen-presenting cells (APCs). These class II-bearing accessory cells include dendritic cells, epidermal Langerhans cells, B cells, murine B-cell tumors, and human Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL). We investigated the ability of EBV-LCL to present two soluble antigens, Candida albicans and purified protein derivative of tuberculin (PPD). The EBV-LCL derived from B cells of two different individuals can present both antigens to bulk cultures of autologous antigen-primed peripheral blood lymphocytes. The responses of PPD-reactive T-cell clones were weaker to PPD when presented by EBV-LCL than by PBL-APCs, with some clones responding only to PPD presented by PBL-APCs. This suggests that EBV-LCL are not equivalent to PBL monocytes in APC function, and that expression of class II major histocompatibility complex antigen is not sufficient in enabling antigen-presenting capability.