Lysis of CD4+ T cells expressing HIV-1 gag peptides by gag-specific CD8+ cytotoxic T cells

The vast majority of in vitro experiments testing the cytotoxic T lymphocytes (CTL) activity in HIV infection has been performed with target cells consisting of autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) expressing Human immunodeficiency virus type I (HIV-1) proteins. However data concerning the lysis of primary CD4+ T lymphocytes expressing HIV-1 antigens by CTLs is still lacking. To study the CTL activity against such primary targets, we used a system involving PBMCs of an HIV+ asymptomatic patient (PT) as effector cells and the CD4+ lymphocytes or B-LCLs of his healthy HLA-identical twin brother (HTW) as target cells. These syngeneic targets were either infected with recombinant vaccinia virus containing HIV-1 gag gene (gag-vac), or coated with HIV-1 gag peptides. We demonstrate in this study that PT CTLs (which were CD3+, CD4-, CD8+, TCRalphabeta+, TCRgammadelta-, CD56-) specifically lysed both types of syngeneic target cells expressing gag-vac; however, CD4+ T cells expressing HIV gag proteins were lysed less efficiently than B-LCLs expressing the same HIV epitopes. On the other hand, no specific lysis was detected when the target cells were uninfected or infected by wild-type vaccinia virus.     

Evaluation of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte responses utilizing B-lymphoblastoid cell lines transduced with the CD4 gene and infected with HIV-1

Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4HIV-1) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HIV-1 were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env- and gag-specific CTL effectors lysed LCL-CD4HIV-1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HIV also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of LCL-CD4HIV-1 should facilitate the analysis of the range of HIV-1 gene products recognized by CTL in seropositive persons.     

TAP1-independent loading of class I molecules by exogenous viral proteins

Presentation of peptides derived from endogenous proteins on class I molecules needs functional TAP peptide transporters. To reveal whether class I-associated presentation of exogenous proteins also required the presence of TAP transporters, we assessed in vitro the ability of spleen cells and macrophages from TAP1-deficient mice (TAP1-/-) to present peptides derived from exogenous recombinant viral proteins on their class I molecules. We found that recombinant glyco- and nucleoprotein from lymphocytic choriomeningitis virus and nucleoprotein of vesicular stomatitis virus were presented as efficiently by TAP1-/- cells as by control cells. Peptide regurgitation was not involved. Since particulate, non-replicating antigens can efficiently prime anti-viral cytotoxic T cells in vivo, this new, TAP-independent pathway of class I-associated antigen presentation may be applicable for vaccine strategies.     

P-glycoprotein plays an insignificant role in the presentation of antigenic peptides to CD8+ T cells

Most antigenic peptides presented to CD8+ T cells are generated from cytosolic precursors and are translocated by TAP into the endoplasmic reticulum, where they associate with MHC class I molecules. TAP-deficient cells exhibit a limited capacity to deliver peptides from cytosolic proteins to class I molecules. One candidate for an alternative peptide transporter is P-glycoprotein, which transports numerous substances, including peptides, across membranes. Elevation of P-glycoprotein expression is partially responsible for the resistance developed by neoplasias to chemotherapeutic drugs. Overexpression of P-glycoprotein has been reported to enhance the expression of class I molecules. Here, we investigated the role of P-glycoprotein in the generation of peptide-MHC complexes. We were unable to detect P-glycoprotein-mediated transport of synthetic peptides into the endoplasmic reticulum of either T2 cells (TAP-deficient) infected with a recombinant vaccinia virus (rVV) expressing P-glycoprotein or drug-resistant cells in which TAP is inactivated by a peptide from the herpes simplex virus ICP47 protein. Expression of rVV-encoded P-glycoprotein in T2 cells was unable to enhance cell surface expression of any of three MHC class I allomorphs tested. rVV-mediated expression of P-glycoprotein enabled T2 cells to produce limited amounts of class I-peptide complexes from cytosolic antigens, but this was not blocked by a drug that inhibits its transporter function, and a similar degree of presentation was mediated by functionally inactive mutated forms of P-glycoprotein. Thus, this was a nonspecific effect that we attributed to diminished membrane integrity resulting from P-glycoprotein overexpression. Taken together, our findings cast serious doubts that P-glycoprotein is a biologically significant transporter of cytosolic peptides.     

Generation of CD8+ and CD4+ T-cell response to dendritic cells genetically engineered to express the MART-1/Melan-A gene

Both CD8+ and CD4+ T cells have demonstrated roles in antitumor immune response in many animal tumor systems. In many human tumor systems, although abundant literature exists on the evidence of tumor antigen-specific CD8+ CTL response, only limited information is available on tumor antigen-specific CD4+ T-cell response. Using the MART-1/Melan-A (MART-1) antigen system as a prototype human tumor-associated antigen (TAA)- and dendritic cell (DC)-based MART-1 antigen presentation system (i.e., DCs transduced with an adenoviral vector-based construct carrying the MART-1 gene), we explored, in vitro, the feasibility of generating both CD8+ and CD4+ T-cell responses in the same individual. Here, we show that autologous DCs from both HLA-A2-positive melanoma patients and normal healthy individuals that are transduced with an adenoviral vector containing the MART-1 antigen are capable of inducing both MART-1-specific CD8+ and CD4+ T cells in in vitro coculture. After several rounds of stimulation, both the CD4+ and CD8+ T cells synthesized IFN-gamma when they were specifically stimulated. The CD8+ T cells generated in such cocultures also recognized the MART-1(27-35) peptide, AAGIGILTV, in 4-h cytotoxicity assays. These observations, therefore, suggest that Th1-type responses can be generated, in vitro, by stimulation with DCs that are genetically modified to express a TAA. Although the outcome of this type of genetically engineered DC-based stimulation may vary from system to system, this type of in vitro antigen presentation may be very useful in more comprehensive analyses of CD4+ T-cell response to defined TAAs, and such genetically engineered autologous DCs might be better candidates to serve as surrogate cancer vaccines.     

Histocompatibility leukocyte antigen-A2-restricted and tumor-specific cytotoxic T lymphocytes from tumor-infiltrating lymphocytes of patient with testicular embryonal cancer

T lymphocytes play an important role in tumor rejection. To understand T cell-mediated specific immunity at the tumor site of testicular embryonal cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) of a patient with testicular embryonal cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted and tumor-specific cytotoxicity. We established a CD3+CD4-CD8+ cytotoxic T lymphocyte (CTL) line from the IL-2-activated TIL of a 37-year-old patient with testicular embryonal cancer. A 6 h 51Cr-release assay was performed to measure the cytotoxicity of the CTL. The CD3+CD4-CD8+ CTL line showed cytotoxicity against HLA-A2+ tumor cells, including freshly isolated autologous tumor cells, adenocarcinoma cell lines from various organs (lung, breast, pancreas, colon and kidney) and squamous cell carcinomas (esophagus and oral cavity). No other cell lines examined, including an autologous tumor cell line and HLA-A2- tumor cell lines, were lysed by this CTL line. These results suggest the existence of HLA-A2-restricted and tumor-specific CTL at the tumor site of testicular embryonal cancer.     

Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.     

Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor zeta chain (universal T cell receptor). CD8+ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.     

Immunity to porcine rubulavirus infection in adult swine

The immune response against the porcine rubulavirus was analyzed in experimentally infected adult pigs. High titers of virus neutralizing and hemagglutinating inhibitory antibodies were identified in infected animals. The antibody specificity was directed towards HN, M, and NP rubula virion proteins; immunodominance of HN proteins was demonstrated. Peripheral blood mononuclear cells from infected, but not from non-infected pigs proliferated in vitro in response to virus antigenic stimuli, showing a bell-shaped plot with the highest peak at 5 weeks post-infection. Virus-induced lymphoblasts expressed CD4+ CD8+ phenotype, whereas lectin-induced lymphoblasts were mainly identified as CD4+ CD8- cells. Phenotype analysis of freshly prepared PBMC revealed increased number of both monocytes (PoM1+) and total T lymphocytes (CD2+) early during infection, with reduced values of B lymphocytes at 4 weeks post-infection. Decrease in CD4+ CD8- blood cells was observed at 3 weeks post-infection, whereas both CD4- CD8+ and CD4+ CD8+ cells increased 1 and 4 weeks post-infection, respectively. This work discusses the relevance of CD4+ CD8+ T cells in the control of porcine rubulavirus infection.     

Heat shock cognate protein 71-associated peptides function as an epitope for Toxoplasma gondii-specific CD4+ CTL

HLA-DR-restricted CD4+ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4+ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4+ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4+ CTL.     

CD4-mediated and CD8-mediated cytotoxic and proliferative immune responses to Toxoplasma gondii in seropositive humans

Both CD4+ and CD8+ cytotoxic T lymphocytes (CTL) are part of the human immune response to Toxoplasma gondii infection. To further our understanding of Toxoplasma immunity, we investigated factors influencing stimulation of CD4+ or CD8+ human T. gondii-specific immune cells. Both antigen-pulsed and Toxoplasma-infected antigen-presenting cells (APC) induced cell proliferation. Toxoplasma-infected APC elicited strong proliferation of CD4+ cells, but little or no proliferation of CD8+ cells, unless high antigen loads were used. Toxoplasma-infected APC stimulated specific cytotoxicity poorly or not at all, owing to death of stimulated cultures, whereas antigen-pulsed APC strongly elicited specific cytotoxicity. Cytotoxicity elicited by either type of APC resided exclusively in CD4+ T cells in polyclonal cultures. Thus, Toxoplasma-infected APC elicited stronger CD4-mediated than CD8-mediated cell proliferation and generated CD4+ CTL more readily than CD8+ CTL. Nonetheless, specific CD8+ memory cells were demonstrated, and rare CD8+ Toxoplasma-specific CTL were subcloned. Fixed Toxoplasma-infected APC (which induce CD8+ CTL) also elicited cell proliferation, but polyclonal cultures stimulated with these infected APC did not die. Unfixed Toxoplasma-infected APC strongly inhibited phytohemagglutinin-induced cell proliferation, whereas fixed APC did not. These data suggested that infected APC were inhibitory or lethal to some immune cells. Further investigations into interactions between immune cells and Toxoplasma-infected cells likely will help elucidate factors involved in the immunopathogenesis of Toxoplasma infection. As other intracellular parasites, including Plasmodium spp. and Leishmania spp., also elicit CD4+ CTL, such work may help establish paradigms governing immunity to intracellular parasites.     

An H2-T MHC class Ib molecule presents Listeria monocytogenes-derived antigen to immune CD8+ cytotoxic T cells

Mouse spleen T cells can adoptively transfer immunity to Listeria monocytogenes; this activity was markedly enhanced by stimulation with Con A in vitro before transfer. The enhanced and prolonged protection against L. monocytogenes in vivo was correlated with enhanced lysis in vitro of target cells infected with strains of L. monocytogenes that produce listeriolysin O (LLO). One of the targets of such cytotoxic cells from BALB/c (H2d) mice was a peptide that corresponded to amino acids 91 to 99 (p91-99) of the LLO molecule, which satisfies the binding motif of H2-Kd. Listeria-immune CD3+CD8+, but not CD3+CD8-, cells could also lyse H-2-incompatible, infected target cells. Immune cells from C57BL/6 (H2b) mice lysed allogeneic H-2d target cells infected with L. monocytogenes or a Bacillus subtilis transformant that secretes LLO, but did not lyse targets pulsed with p91-99. This H2-unrestricted cytolysis was therefore directed at a fragment of the LLO molecule other than p91-99. Listeria-infected bone marrow macrophages from congenic and recombinant strains of mice were lysed only when they shared the H2-T region or were Qa1-compatible with the immune cytotoxic cells; sharing of the H2-D, Q, or M region was insufficient. Thus, the immune response to L. monocytogenes included cytolytic CD8+ cells that recognized endogenously processed Listeria-derived Ags in the context of the class Ia H2-K molecule, as well as a class Ib H2-T molecule.     

Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein

Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.     

Herpes simplex virus inhibitor ICP47 destabilizes the transporter associated with antigen processing (TAP) heterodimer

Chemical cross-linking of the transporter associated with antigen processing (TAP) heterodimer was used to determine whether the herpes simplex virus inhibitor of TAP, ICP47, induces a conformational change in TAP. Cross-linking of TAP in cellular membranes produced a major species of approximately 220 kDa which was comprised solely of TAP.1 and TAP.2 and most likely represents the TAP heterodimer. Interestingly, prior treatment of TAP-containing membranes with TAP peptide substrates stimulated the formation of the cross-linked TAP heterodimer, whereas pretreatment of membranes with ICP47 completely blocked the formation of the cross-linked heterodimer. These data suggest that suitable substrates for TAP stabilize the TAP heterodimer, whereas ICP47 destabilizes the heterodimer. The results indicate that subtle conformational changes occur in the TAP heterodimer upon the binding of peptides and the inhibitor ICP47 and that ICP47 has a deleterious effect on TAP heterodimer structure, in addition to its role as a potent blocker of substrate binding to TAP.     

Human purified protein derivative-specific CD4+ T cells use both CD95-dependent and CD95-independent cytolytic mechanisms

CTL, both CD4+ and CD8+, are essential in the eradication of intracellular pathogens. Data generated using murine T cells have suggested a critical role for CD95 (Fas, Apo-1) in CD4+ T cell-induced apoptosis of target cells. In contrast, CD8+ CTL predominantly use the perforin/granzyme lytic pathway. At present little is known about the mechanism of CD4+ CTL lytic function during intracellular infection in humans. We have used human CD4+ T cells specific for purified protein derivative (PPD) of Mycobacterium tuberculosis to explore whether CD95 is the dominant cytolytic mechanism. PPD-reactive CD4+ clones efficiently lysed Ag-pulsed autologous monocytes, adherent macrophages, and EBV-transformed B cells. Addition of an antagonistic CD95 Ab had a minimal effect on cytolysis, whereas addition of MgEGTA to block perforin/granzyme resulted in complete inhibition of killing. In contrast, lysis of activated peripheral blood B cells could be partially blocked with the antagonistic CD95 Ab. Supporting these observations, monocytes, macrophages, and EBV-transformed B cells were not lysed by an agonistic CD95 Ab. Activated B cells were readily lysed by the agonistic CD95 Ab. T cell clones triggered through the TCR with anti-CD3 were capable of lysing the CD95-sensitive Jurkat T cell line in a CD95-dependent manner, but were also able to release granzymes. We conclude that human CD4+ T cells are capable of lysing PPD-pulsed targets using both perforin/granzyme and CD95 pathways. The contribution of CD95 is strictly dependent on target cell susceptibility to CD95-mediated killing.     

Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection

Analysis of antigen-induced negative selection of thymocytes in T cell receptor (TCR)-transgenic mice is complicated by the presence of an antigen-responsive peripheral T cell compartment. Our experiments address the question of whether and how peripheral T cell activation can affect immature thymocytes. Following three daily injections of peptide antigen into mice expressing a peptide-specific transgenic TCR and deficient for TAP1, we and others have found profound deletion of the CD4+CD8+ (DP) thymocyte subset. However, our work shows that even though mature CD8+ T cells are inefficiently selected in TAP1-deficient mice, there was a striking degree of peripheral expansion and activation of CD8+ peripheral T cells. Furthermore, when cells from TCR-transgenic mice were adoptively transferred, we found that deletion of nontransgenic DP thymocytes occurred in Thy-1-congenic and even more efficiently in TAP1-deficient recipients after repeated peptide injection resulting in peripheral T cell activation. In the adoptive transfer experiments the degree of deletion of immature bystander thymocytes was decreased upon blocking of TNF. These data show that deletion of DP thymocytes can result from excessive peripheral T cell activation and identify TNF as an important effector molecule for this process. When steps are taken to avoid peripheral T cell activation, peptide antigen can induce TCR-mediated thymocyte deletion, presumably in the thymus cortex, since injection of TAP1-deficient TCR-transgenic mice resulted in deletion of immature DP thymocytes prior to detectable peripheral T cell expansion and activation. This effect was not blocked by inhibiting tumor necrosis factor activity. In addition, DP depletion was seen in the absence of peripheral T cell activation when antibody-mediated depletion of CD8+ T cells was performed. Our work clearly shows that two mechanisms for deletion of DP thymocytes exist: deletion induced by antigen presentation in the thymus and deletion as a consequence of repeated stimulation of mature peripheral T cells.     

Cryopreservation of teeth. Organizational aspects of a tissue bank for tooth tissues

Squamous cell carcinoma (SCC) is one of the most common cancers in human. SCC, particularly, esophageal and lung SCC are relatively resistant to currently available regimens of chemotherapy or radiation therapy. Therefore, development of a specific immunotherapy using tumor specific cytotoxic T lymphocytes (CTL) would be important to offer other treatment modalities. However, generation of HLA class I-restricted CTL recognizing SCC has been rarely reported. We established the HLA A2601-restricted CTL cell line recognizing a peptide antigen expressed on SCC. This CD4- CD8+ cytotoxic T lymphocyte (KE-4 CTL) cell line was established in a patient with esophageal cancer. The KE-4 CTL recognized a peptide antigen on esohageal and lung SCC in an HLA A2601-restricted manner as evaluated by cytotoxity against a panel of tumor cells, transfection experiments with HLA A2601 cDNA, and reconstitution with eleted peptides. None of normal cells tested was lysed by this CTL. These results suggest the exstence of HLA A2601-restricted CTL precursors recognizing a peptide antigen on SCC in a patient with esophageal cancer.     

Progressive loss of IL-2-expandable HIV-1-specific cytotoxic T lymphocytes during asymptomatic HIV infection

In HIV-1 infection, circulating HIV-1-specific cytotoxic T lymphocytes (CTL) exist in different states of activation, including activated cytotoxic cells and memory cells. We report that a subpopulation of HIV-1-specific CTL is capable of clonal expansion upon culture with IL-2 without exogenous antigen. The IL-2-expandable HIV-1-specific CTL precursor frequency was reduced in patients with advancing infection, although HIV-1-specific memory CTL could still be detected by stimulation in vitro with allele-specific HIV-1 peptide. Longitudinal analysis during advancing infection showed a progressive decline in the IL-2-expandable HIV-1-specific CTL precursor (CTLp) frequency without a decline in Epstein-Barr virus (EBV)-specific or allo-specific CTLp frequencies. To address mechanisms that may contribute to the decline in the IL-2-expandable HIV-specific CTL response, the requirements for in vitro generation of HIV-1-specific and EBV-specific effector CTL were examined. In the absence of exogenous IL-2 in limiting dilution, generation of EBV-specific CD8+ effector CTL was dependent upon help from CD4+ cells. CD4+ help for EBV-specific CD8+ CTL was observed in asymptomatic HIV infection but not in advanced infection. In the presence of exogenous IL-2, CD4+ cells could also provide help for the optimal generation of HIV-1 peptide-specific CD8+ CTL, because in vitro depletion of CD4+ cells prior to culture using stimulation with an MHC class I-restricted HIV-1 peptide reduced the peptide-specific CD8+ CTL response. We conclude that there is a decline in the IL-2-expandable HIV-1-specific CTL response during advancing infection. There are a number of possible mechanisms for this decline, including a reduction in CD4+ T cell help for in vivo antigen-activated CD8+ T cells.