A novel 80-kD cell surface structure identifies human circulating lymphocytes with natural killer activity

Human lymphocytes with natural killer (NK) activity, including most activated gamma/delta+ T lymphocytes, recognize and lyse tumor target cells without requiring recognition of major histocompatibility complex antigen. However, unlike gamma/delta+ T lymphocytes, NK cells do not express CD3/T cell receptor (TCR) molecules, and the receptors involved in cell-mediated cytotoxicity are unknown. To further delineate circulating NK cells, we developed monoclonal antibodies (mAbs) against the human NK leukemia YT2C2. We report the isolation of a mAb termed BY55, recognizing at the cell surface a novel 80-kD protein with restricted expression. In addition to the immunizing cell line, this mAb binds to circulating NK cells, gamma/delta+ cells, and a minor subset of alpha/beta+ T lymphocytes. Expression of the BY55 mAb- reactive epitope/molecule is regulated by activation, as short-term culture of peripheral blood lymphocytes (PBL) with phorbol ester induced its downmodulation. Furthermore, BY55 mAb reactivity was found neither with the NK nor with the TCR alpha/beta+ gamma/delta+ clones tested. Biochemical studies as well as phenotypic analysis revealed that this structure is different from all previously identified molecules on the lymphocyte cell surface. Interestingly, we found that BY55+ cells exert most NK activity obtained with fresh circulating lymphocytes. We report that within fresh E rosette-positive PBL only a subset of the CD16+, CD56+, and CD57+ cells coexpressed BY55 molecule, indicating that BY55 mAb defines a unique subset mediating NK activity of circulating PBL.     

CD4-CD8- T cell receptor alpha beta T cells: generation of an in vitro major histocompatibility complex class I specific cytotoxic T lymphocyte response and allogeneic tumor rejection

The generation of an in vitro major histocompatibility complex class I specific response of CD4-CD8- T cell receptor (TCR) alpha beta cytotoxic T lymphocytes (CTL) and their allogeneic tumor rejection were investigated. Inocula of BALBRL male 1 were rejected in C57BL/6 (B6) mice treated with minimum essential medium (MEM) (control), anti-L3T4 (CD4) monoclonal antibody (mAb) or anti-Lyt-2.2 (CD8) mAb and CTL against the tumor were generated in vitro. No rejection and no induction of CTL were observed in B6 mice treated with anti-L3T4 (CD4) plus anti-Lyt-2.2 (CD8) mAb. CTL with the classical Thy-1+ CD3+CD4-CD8+ TCR alpha beta phenotype were generated in mixed lymphocyte tumor cell culture (MLTC) spleen cells from B6 mice treated with MEM (control) or anti-L3T4 (CD4) mAb, whereas CTL with an unusual Thy-1+CD3+CD4-CD8- TCR alpha beta phenotype were generated in MLTC spleen cells from anti-Lyt-2.2 (CD8) mAb-treated B6 mice. Both types of CTL were reactive with both H-2Kd and Dd (Ld) class I antigen. These findings suggest that when CD4+ cells were blocked by anti-L3T4 (CD4) mAb, CD8+ CTL mediated rejection, and when CD8+ cells were blocked by anti-Lyt-2.2 (CD8) mAb, CD4+ cells were capable of mediating rejection, although less efficiently than CD8+ cells, by inducing CD4-CD8- TCR alpha beta CTL. The finding that adoptive transfer of CD4 and CD8-depleted MLTC spleen cells, obtained from anti-Lyt-2.2 (CD8) mAb-treated B6 mice that had rejected BALBRL male 1, resulted in rejection of BALBRL male 1 inoculated into B6 nu/nu mice confirmed the above notion. CTL clones with the CD4-CD8- TCR alpha beta phenotype specific for Ld were established.     

Melanoma cells and normal melanocytes share antigens recognized by HLA- A2-restricted cytotoxic T cell clones from melanoma patients

HLA-A2-restricted, CD3+, CD8+, alpha/beta+ cytotoxic T cell (CTL) clones were isolated from peripheral blood (PBL) or tumor infiltrating lymphocytes (TIL) of two HLA-A2+ melanoma patients (9742 and 5810), to evaluate the possible recognition of autologous melanoma and of allogeneic HLA-A2-matched normal melanocytes. These CTL clones lysed not only fresh and cultured autologous melanoma cells, but also allogeneic HLA-A2+, but not HLA-A2-, normal melanocytes. The lysis of autologous neoplastic cells and of melanocytes could be inhibited by an anti-HLA-A2 monoclonal antibody (mAb). Lysis of the normal melanocytes was not dependent on the presence of human or fetal calf serum in the culture medium. HLA-A2-restricted CTL clones recognized not only proliferating melanocytes cultured in complete melanocyte medium, but also melanocytes made quiescent by culture for up to 6 d in a basal medium devoid of exogenous factors such as phorbol ester (O- tetradecanoyl phorbol 13-acetate [TPA]), epidermal growth factor, insulin, and pituitary extracts. Analysis of specificity of four CTL clones (A75, A83, A94, and 119) from patient 9742, performed on a panel of 39 targets, indicated that the three HLA-A2-restricted CTL (A75, A83, and A94) lysed all but one of nine allogeneic melanomas expressing the HLA-A2 molecule with no reactivity on nine HLA-A2- allogeneic melanomas. Only a few instances of borderline reactivity were seen by the same effectors on 21 targets of nonmelanocyte lineage, including 12 carcinomas of different histology, four Epstein-Barr virus-transformed B cells (lymphoblastoid cell lines [LCL]), including the autologous LCL, four lines of normal fibroblasts, and normal kidney cells. Lack of reactivity on allogeneic targets of nonmelanocyte lineage occurred in spite of expression of HLA-A2 on 14 of these targets as determined by conventional tissue typing and cytofluorimetric analysis with four different anti-HLA-A2 mAb. These data indicate that tissue-related antigens can be expressed on normal and neoplastic cells of the melanocyte lineage and can be recognized in association with HLA-A2 by CTL clones from melanoma patients.     

Induction of cytotoxic T lymphocytes with peptides in vitro: identification of candidate T-cell epitopes in hepatitis B virus X antigen.

Cytotoxic T lymphocytes (CTL) have been suggested to contribute to viral clearance during hepatitis B virus (HBV) infection. To induce effective CTL against viral infection by peptide vaccination, it is essential to identify the epitope peptides recognized by CTL. Here, 15 peptide sequences that contain HLA-A2.1-restricted CTL binding consensus motif were identified on hepatitis B virus X (HBx) protein and synthesized for further characterization. In the binding assay, 8 of 15 synthetic peptides enhanced the expression of HLA-A2.1 molecules on the surface of T2 cells, a human transport-associated antigen processing-deficient cell line. This result implies that these eight peptides are able to bind to the HLA-A2.1 molecules. These peptides were further tested for their ability to activate CTL from peripheral blood mononuclear cells (PBMCs) isolated from HBV chronic carriers. Five of eight tested peptides activated PBMC-derived T cells, resulting in the lysis of the target T2 cells pulsed with the same peptide. Furthermore, the CTL responses to HBx antigen in HBV chronic carriers were shown to be polyclonal, multispecific, and mediated mainly by CD8+ T cells. In contrast, these responses were not detected in uninfected healthy blood donors. Although the five CTL epitope peptides identified in this study have not been proven to be the naturally processed epitopes in HBV-infected hepatocytes, they could be candidates for peptide-based immunotherapy against HBV infection.     

A novel beta 4, alpha 6 integrin-associated epithelial cell antigen involved in natural killer cell and antigen-specific cytotoxic T lymphocyte cytotoxicity

Efficient immune responses require interactions between cell adhesion molecules on lymphocytes and counter-receptors on antigen presenting cells or target cells. While target-specific receptors or ligands have not been identified for natural killer (NK) cells, cell adhesion molecules have been implicated in the interaction between NK cell effectors and tumor cell targets. Herein, we describe monoclonal antibodies (mAbs) against a carcinoma cell line that efficiently block the cytolytic activity of interleukin 2-activated NK cell lines and clones. L280 mAb reacts with secretory epithelial cells in normal human tissues, but does not react with hematopoietic cells or other tissue types. Biochemical analysis revealed that L280 mAb immunoprecipitates the beta 4, alpha 6 integrin, as well as a novel 98-kD glycoprotein, and probably reacts with a carbohydrate epitope on these molecules. Involvement of the L280 antigen in cellular immunity is not restricted to NK cell-mediated cytotoxicity. L280 mAb also efficiently inhibits alloantigen-specific cytotoxicity against Colo-205 cells mediated by human histocompatibility leukocyte antigen (HLA)-A2 alloantigen specific alpha beta-TCR+ and gamma delta-TCR+ cytotoxic T lymphocyte (CTL) clones. Additionally, we demonstrate that L280 mAb blocks cytotoxicity mediated by influenza peptide-specific HLA-restricted CTL clones. These data indicate that the antigen recognized by L280 mAb is important in both NK and CTL function, and that an as yet unidentified receptor for this epithelial antigen is present on both NK and T lymphocytes. The restricted expression of L280 antigen indicates that this molecule may be important in immune reactions in epithelial tissues.     

Clearance of influenza virus respiratory infection in mice lacking class I major histocompatibility complex-restricted CD8+ T cells

Transgenic mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption and normal mice that had been treated with a CD8-specific mAb were infected intranasally with an H3N2 influenza A virus. Both groups of CD8T cell-deficient mice eliminated the virus from the infected respiratory tract. Potent CTL activity was detected in lung lavage populations taken from mice with intact CD8+ T cell function, with minimal levels of cytotoxicity being found for inflammatory cells obtained from the antibody-treated and beta 2-m mutant mice. We therefore conclude that cells infected with an influenza A virus can be cleared from the respiratory tract of mice lacking both functional class I major histocompatibility complex (MHC) glycoproteins and class I MHC-restricted, CD8+ effector T cells.     

CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta

The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells.     

Selective activation of gamma/delta + T cell clones by single anti-CD2 antibodies

The CD2 antigen is the target for an "alternative" T cell activation pathway. Numerous studies have demonstrated that pairs of monoclonal antibodies (mAbs) directed toward two different epitopes are required for activation of T cell receptor (TCR)-alpha/beta + T cells via CD2. We have now explored the activation of human TCR-gamma/delta + T cell clones by a panel of anti-CD2 mAbs directed against the sheep erythrocyte-binding (T11.1) epitope of CD2. Seven of seven gamma/delta + clones expressing different molecular forms of the TCR-gamma/delta responded to stimulation by a single anti-CD2 mAb (OKT11, 9E8, BW0110, M-T910) with IL-2 secretion and/or proliferation. Immobilization of anti-CD2 mAbs in microculture plates was essential for activation of gamma/delta + clones, which occurred in the absence of feeder cells. In addition to interleukin 2 (IL-2) production and proliferation, anti-CD2 mAbs also triggered cytotoxic effector activity in gamma/delta + clones as measured against FcR+ P815 target cells. In contrast to gamma/delta + clones (but in line with established data), none of five CD4+ or CD8+ TCR-alpha/beta + clones were activated by any of the tested individual anti-CD2 mAbs. Taken together, our results reveal a striking difference between cloned gamma/delta + and alpha/beta + T cells in that gamma/delta + T cells are selectively activated by a single anti-CD2 (T11.1) mAb, without need for the simultaneous signal of a second anti-CD2 mAb directed against another (T11.2 or T11.3) CD2 epitope.     

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope.

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202-221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site.     

Expression of Ley antigen in human immunodeficiency virus-infected human T cell lines and in peripheral lymphocytes of patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC)

Ley determinant (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----R) defined by mAb BM-1 is highly expressed in human immunodeficiency virus (HIV)-infected T cell lines and in CD3+ peripheral mature T cells of patients with acquired immune deficiency syndrome (AIDS) or with AIDS-related complex (ARC). Ley expression increased greatly in the CD3+ population in the advanced stage of AIDS when the CD4+ population decreased greatly. Six other carbohydrate antigens tested by their respective mAbs were not detected in these same cells. None of the carbohydrate antigens tested by the seven mAbs used in this study were found in noninfected T cell lines and in normal peripheral blood lymphocytes.     

Species-restricted interactions between CD8 and the alpha 3 domain of class I influence the magnitude of the xenogeneic response

As compared with the vigorous T cell response normally observed against allogeneic MHC molecules, T cells recognize xenogeneic MHC molecules poorly. To define structural features of the MHC molecule important for such species-specific recognition, HLA-A2(A2)-specific murine CTL were examined for their recognition of transfected cell lines expressing the class I molecules A2 or A2/H-2Kb(A2/Kb). A2/Kb is a chimeric molecule consisting of the alpha 1 and alpha 2 domains of A2 and the alpha 3, transmembrane, and cytoplasmic regions of Kb. The majority of CTL clones showed enhanced recognition of transfected cell lines expressing this chimeric molecule. Enhanced recognition was shown to correlate with sensitivity of the CTL clones to inhibition by anti-CD8 antibody. These results suggested that CD8 may interact with class I in a species-specific manner, and that suboptimal CD8 interaction with the alpha 3 domain of xenogeneic molecules may be an important contribution to poor xenoreactivity. This conclusion was supported by the capacity of A2/Kb, but not A2 human cell transfectants, to induce a primary in vitro CTL xenoresponse specific for A2.     

Autologous tumor-specific cytotoxic T lymphocytes in the infiltrate of human metastatic melanomas. Activation by interleukin 2 and autologous tumor cells, and involvement of the T cell receptor

TIL from metastatic melanoma proliferated by greater than 1,000-fold (840-3,675, mean 1,543) after 6 wk in culture of mixtures of TIL and tumor cells with rIL-2 alone. Cytolysis was restricted to autologous tumor cells. CD8+ T cells were the predominant population of TIL before and after expansion, and were primarily responsible for autologous tumor-specific CTL activity. No other rIL-2-activated lymphocytes from peripheral blood, lymph nodes with melanoma metastasis, or TIL from sarcoma or renal cell carcinoma had autologous tumor-specific CTL activity. There were few or no CD16+ NK cells in TIL from metastatic melanoma before or after incubation with rIL-2, respectively. However, TIL from sarcoma or renal cell carcinoma contained a substantial proportion of CD3-CD16+ NK cells, which increased in number in culture with rIL-2. Purified CD16+ NK cells as well as CD3+CD16- T cells from rIL-2-activated TIL of renal cell carcinoma displayed MHC-nonrestricted cytotoxicity. At the clonal level as determined by limiting dilution, 8 of 10 clones from melanoma TIL displayed cytotoxicity restricted to autologous tumor cells, while all 13 clones from renal cancer TIL equally lysed autologous and allogeneic tumor cells. Anti-T cell receptor (TCR)-alpha/beta(WT31) mAb as well as anti-CD3 mAb inhibited autologous melanoma cell-specific CTL activity mediated by rIL-2-activated TIL at the effector phase. These two mAbs also inhibited rIL-2-dependent proliferation of these TIL when added to the culture. Pretreatment of fresh melanoma cells with mAb to MHC antigens followed by washing inhibited specific CTL activity. These results suggest that both TCR-alpha/beta on effector TIL and MHC antigens on fresh tumor cells are involved in the specific immune-recognition. After reaching maximum propagation, TIL from metastatic melanoma responded poorly to rIL-2 alone. However, stimulation with fresh autologous melanoma cells restored both CTL activity and proliferation in response to rIL-2. The latter is associated with IL-2 receptor (Tac antigen) expression on the surface. These results indicate that TIL from metastatic melanomas may have unique characteristics different from lymphocytes obtained from the other sources, and may contain precursor CTL sensitized in vivo to autologous tumor cells, and thus can be propagated in larger numbers with rIL-2 alone while retaining autologous tumor-specific CTL activity.     

Immune reactivity against a novel HLA-A3-restricted influenza virus peptide identified by predictive algorithms and interferon-gamma quantitative PCR

The use of appropriate antigenic peptides for the most common human major histocompatibility complex (MHC) alleles is required for the amplification of the autologous cytotoxic compartment and the development of cytotoxic T cell-mediated immunity. The human A2 allele of the MHC plays an important role for the identification of peptide-specific cytotoxic T cells (CTL) against tumor and viral epitopes. Computer-based prediction algorithms, which are available on the Internet, have already proved to be applicable for the identification of novel CTL epitopes. Using the bioinformatics approach, the authors have identified the novel influenza matrix protein-derived and HLA-A3-restricted 9-mer peptide RLEDVFAGK capable of inducing peptide specific CTL reactivity. Peripheral blood mononuclear cells (PBMC) from healthy individuals and patients with lung cancer were pulsed with this peptide and with the well-characterized HLA-A2-restricted influenza A virus matrix peptide GILGFVFTL. Using quantitative PCR (TaqMan; Applied Biosystems, Foster City, CA, U.S.A), reactivity for both peptides was determined by measuring the change in type 1 cytokine (IFN-gamma) expression upon in vitro stimulation. Peptide-specific reactivity matched well with the subsequently determined MHC-class I alleles of the tested individuals. Results from this study indicate that the use of bioinformatics and the PCR-based screening system for the monitoring of T cell reactivity may allow for the identification of novel CTL epitopes.     

Characterization of CD3+, CD4-, CD8- clones expressing the putative T cell receptor gamma gene product. Analysis of the activation pathways leading to interleukin 2 production and triggering of the lytic machinery

Four clones were derived from human peripheral blood T lymphocytes from which CD4+ and CD8+ cells had been removed by treatment with specific mAbs and complement. All expressed the CD2+, 3+, 4-, 8-, T44- phenotype, and did not react with the WT31 mAb, which is specific for a framework determinant of the CD3-associated alpha/beta heterodimer which serves as receptor for antigen on most human T lymphocytes. Surface iodination followed by crosslinking with dithiobis-succinimidyl propionate (DSP) and immunoprecipitation with anti-CD3 mAbs indicated that, in all four clones, the CD3-associated molecules consisted of a major 45 kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNA for the alpha chain was missing; beta chain mRNA was present in a defective form (1 kb instead of 1.3 kb). These data support the concept that these clones may express, in association with CD3, the molecular product of the T cell receptor gamma genes instead of the typical alpha/beta heterodimer. CD3+, WT31- clones lysed the NK-sensitive K562 target cells and produced IL-2 upon stimulation with PHA. In addition, they released IL-2 after triggering with soluble anti-CD3 mAbs or with an appropriate combination of anti-CD2 mAbs (in the presence of adherent cells). When CD3+, WT31- clones were incubated with an anti-CD3 producing hybridoma as triggering target, the latter was efficiently lysed. Target cell lysis also occurred when a suitable combination of anti-CD2 mAbs-producing hybridomas was used. Therefore, CD3+, WT31- cells appear to use two pathways of cell activation that function also in conventional CD3+, WT31+ T cells, but they lack a third putative pathway initiated by T44 surface molecules.     

An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells

The T cell receptor (TCR)-alpha/beta CD4-8- (double negative, DN) T cell subset is characterized by an oligoclonal repertoire and a restricted V gene usage. By immunizing mice with a DN T cell clone we generated two monoclonal antibodies (mAbs) against V alpha 24 and V beta 11, which have been reported to be preferentially expressed in DN T cells. Using these antibodies, we could investigate the expression and pairing of these V alpha and V beta gene products among different T cell subsets. V alpha 24 is rarely expressed among CD4+ and especially CD8+ T cells. In these cases it is rearranged to different J alpha segments, carries N nucleotides, and pairs with different V beta. Remarkably, V alpha 24 is frequently expressed among DN T cells and is always present as an invariant rearrangement with J alpha Q, without N region diversity. This invariant V alpha 24 chain is always paired to V beta 11. This unique V alpha 24-J alpha Q/V beta 11 TCR was found in expanded DN clones from all the individuals tested. These findings suggest that the frequent occurrence of cells carrying this invariant TCR is due to peripheral expansion of rare clones after recognition of a nonpolymorphic ligand.     

AIM-2: a novel tumor antigen is expressed and presented by human glioma cells

Antigen isolated from Immunoselected Melanoma-2 (AIM-2) was recently identified using melanoma-reactive CD8 T cells. AIM-2 antigen is expressed in a wide variety of tumor types, including neuroectodermal tumors, as well as breast, ovarian and colon carcinomas. In this study, we analyzed AIM-2 expression in glioblastoma multiforme (GBM) in primary cultured cells and established GBM cell lines. We found that the primary GBM cell lines expressed 88.4% and 93.0% of non-spliced and spliced AIM-2, respectively. Five out of seven of the established GBM cell lines expressed both non-spliced and spliced AIM-2. Furthermore, the C9 CTL clone, which is specific for AIM-2 peptide (RSDSGQQARY), efficiently recognized GBM tumor cells in an antigen-specific and HLA-A1 restricted manner. IFN-gamma treatment of the GBM tumor cells dramatically increased HLA-A1 expression levels and, consequently, increased CTL recognition of the treated tumor cells. More importantly, seven out of 12 HLA-A1 and AIM-2 positive patients from our dendritic cell clinical trial generated AIM-2 specific CTL activity in their PBMC after vaccinations. These data indicate that AIM-2 could be used as a tumor antigen target for monitoring vaccine trials or to develop antigen specific active immunotherapy for glioma patients.     

Viral escape by selection of cytotoxic T cell-resistant variants in influenza A virus pneumonia

Antigenic variation is a strategy exploited by influenza viruses to promote survival in the face of the host adaptive immune response and constitutes a major obstacle to efficient vaccine development. Thus, variation in the surface glycoproteins hemagglutinin and neuraminidase is reflected by changes in susceptibility to antibody neutralization. This has led to the current view that antibody-mediated selection of influenza A viruses constitutes the basis for annual influenza epidemics and periodic pandemics. However, infection with this virus elicits a vigorous protective CD8(+) cytotoxic T lymphocyte (CTL) response, suggesting that CD8(+) CTLs might exert selection pressure on the virus. Studies with influenza A virus-infected transgenic mice bearing a T cell receptor (TCR) specific for viral nucleoprotein reveal that virus reemergence and persistence occurs weeks after the acute infection has apparently been controlled. The persisting virus is no longer recognized by CTLs, indicating that amino acid changes in the major viral nucleoprotein CTL epitope can be rapidly accumulated in vivo. These mutations lead to a total or partial loss of recognition by polyclonal CTLs by affecting presentation of viral peptide by class I major histocompatibility complex (MHC) molecules, or by interfering with TCR recognition of the mutant peptide-MHC complex. These data illustrate the distinct features of pulmonary immunity in selection of CTL escape variants. The likelihood of emergence and the biological impact of CTL escape variants on the clinical outcome of influenza pneumonia in an immunocompetent host, which is relevant for the design of preventive vaccines against this and other respiratory viral infections, are discussed.     

Expression of specific cytolytic activity by H-2I region-restricted, influenza virus-specific T lymphocyte clones

Among murine class II major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) clones specific for type A influenza virus, we have identified both noncytolytic clones and clones exhibiting H-2 I region-restricted cytolytic activity. After appropriate antigenic stimulation, both cytolytic and noncytolytic clones proliferated in the absence of exogenous interleukin 2. All of the clones possess the Thy-1.2+, Lyt-1+2-, L3T4+ phenotype. The class II MHC restriction of viral recognition by the CTL clones was mapped by proliferation using recombinant mouse strains and by inhibition of cytotoxic activity with monoclonal antibodies directed to class II MHC products and L3T4a. The restriction specificity of two CTL clones was unambiguously assigned to the E beta d chain by using L cell transfectant lines expressing E alpha kE beta d or E alpha kE beta k gene products. Analysis of the viral specificity of the cloned lines revealed subtype-specific and crossreactive patterns of viral antigen recognition; the pattern of viral antigen specificity exhibited by each clone in proliferation and cell-mediated cytotoxicity was identical. Each CTL clone also demonstrated antigen-dependent release of helper factor(s) that promoted in vitro primary anti-SRBC responses. Finally, the cytotoxic effector function of the class II MHC-restricted CTL clones was mediated by direct lysis of virus-infected cells, and not by secretion of a cytolytic lymphokine.     

Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines

Synthetic peptide analogues of sequences in the HER-2 protooncogene (HER-2) were selected based on the presence of HLA-A2.1 anchor motifs to identify the epitopes on HER-2 recognized by ovarian tumor-reactive CTL. 19 synthetic peptides were evaluated for recognition by four HLA- A2 ovarian-specific cytotoxic T lymphocyte (CTL) lines obtained from leukocytes associated with ovarian tumors. The nonapeptide E75 (HER-2, 369-377:KIFGSLAFL) was efficient in sensitizing T2 cells for lysis by all four CTL lines. This peptide was specifically recognized by cloned CD8+ CTL isolated from one of the ovarian-specific CTL lines. E75- pulsed T2 cells inhibited lysis by the same CTL clone of both an HLA- A2+ HER-2high ovarian tumor and a HER-2high cloned ovarian tumor line transfected with HLA-A2, suggesting that this or a structurally similar epitope may be specifically recognized by these CTL on ovarian tumors. Several other HER-2 peptides were recognized preferentially by one or two CTL lines, suggesting that both common and private HER-2 epitopes may be immunogenic in patients with ovarian tumors. Since HER-2 is a self-antigen, these peptides may be useful for understanding mechanisms of tumor recognition by T cells, immunological tolerance to tumor, and structural characterization of tumor antigens.     

Lysis of human melanoma cells by autologous cytolytic T cell clones. Identification of human histocompatibility leukocyte antigen A2 as a restriction element for three different antigens

From the peripheral blood of the melanoma patient (AV), we derived cytolytic T lymphocyte (CTL) clones that lysed the autologous tumor line SK-MEL-29, but not autologous EBV-B cells, K562, and other tumor targets. By immunoselection experiments it was shown that the CTL clones recognized at least three different antigens on the autologous tumor cells. We demonstrate here that these melanoma antigens are presented to the CTL in association with HLA-A2. First, HLA-A2-reactive pregnancy sera as well as an mAb against HLA-A2 inhibited the CTL lysis. Second, immunoselected melanoma subclones that were resistant to lysis by CTL clones against the three antigens described were found to lack expression of HLA-A2. By sensitizing the patient's lymphocytes against an HLA-A2- melanoma clone, we established a new series of CTL clones recognizing autologous AV melanoma cells. However, efficient lysis was only seen when target cells were pretreated with IFN-gamma. The lytic activity of these CTL was selectively inhibited by an mAb against a common HLA-B determinant. These results indicate that in addition to HLA-A2, other class I antigens are involved in the recognition of AV melanoma cells by autologous CTL.