Cell-mediated suppression of HIV-specific cytotoxic T lymphocytes

CTL specific for HIV have been described in lungs of infected patients at early stages of HIV disease. In order to characterize the evolution over time of HIV-specific CTL, we have analyzed the cytotoxic function and the cell surface phenotype of the alveolar lymphocytes from 41 patients at various stages of HIV disease. We demonstrated a progressive decline of alveolar anti-HIV CTL activity and detected Ts cells from the lungs of patients with advanced HIV disease. These alveolar T cells strongly suppressed the effector phase of anti-HIV CTL lysis. They lacked a marked specificity of function because they also block anti-HLA CTL response and were not restricted by the HLA-class-I transplantation Ag. They displayed the CD3, CD8, and HNK1 markers, were CD4 and CD16 negative, and lacked NK activity. The presence of Ts cells at late stages of HIV disease could thus partly explain the inefficiency of host defenses against HIV.     

The presence of NK alloantigens on cloned cytotoxic T lymphocytes

A panel of sera raised against NK-1.1 and NK-2.1 alloantigens was tested for reactivity against a panel of cloned antigen-dependent CTL lines. By using indirect immunofluorescence and flow cytofluorimetry, weak, but clear and consistent, reactivity was found on all CTL. Concordant with the genetics of NK alloantigens, C57BL/6-derived clones were reactive with anti-NK-1.1 and anti-NK-2.1 sera, whereas CBA-derived clones were reactive with anti-NK-2.1 sera but not with anti-NK-1.1 sera. Cloned CTL lines were also able to partially and specifically absorb the antibodies from NK alloantiserum that reacted with splenic NK cells. These results indicate that cloned CTL lines express at least some of the NK alloantigen determinants present on splenic NK cells and have important implications regarding the relationship of CTL and NK cells.     

Studies of cloned simian immunodeficiency virus-specific T lymphocytes. gag-specific cytotoxic T lymphocytes exhibit a restricted epitope specificity.

CD8+ CTL inhibit the replication of HIV and simian immunodeficiency virus of macaques (SIVmac) in PBL and, therefore, are likely to play an important role in containing the spread of the AIDS virus in infected individuals. We have generated a series of gag-specific lytic T lymphocyte clones from PBL: of an SIVmac-infected rhesus monkey. These T cell clones are CD3+CD8+ and are MHC class I-restricted in their target specificity. They are, therefore, CTL. Interestingly, all gag-specific CTL clones, as well as the gag-specific lytic activity of PBL of this monkey, demonstrated specificity for a single 25 amino acid fragment of the SIVmac gag protein. Moreover, they were restricted in their lytic function by a single MHC class I allele. These findings illustrate a powerful method for cloning AIDS virus-specific T lymphocytes and demonstrate a remarkably restricted epitope specificity of this AIDS virus-specific CTL response.     

Cell surface molecules involved in NK recognition by cloned cytotoxic T lymphocytes

Short-term treatment of cloned mouse cytotoxic T lymphocytes (CTL) with interferon (IFN) induces lytic activity for natural killer- (NK) sensitive targets. Extended culture of CTL in high concentrations of interleukin 2 induces promiscuous lytic activity in which state both NK-sensitive and NK-resistant target cells are lysed. Cold-target competition analysis showed that the development of NK activity was associated with the acquisition of binding activity for NK-sensitive but not for NK-resistant targets, whereas the development of promiscuous lytic activity was associated with the acquisition of binding activity for both types of target. Antigen-specific cytolysis was inhibited by antibodies to Ly-2, Ly-5, LFA-1 and to the V region of the T cell antigen receptor (TCR), whereas NK and promiscuous lytic activity in the same cells was resistant to inhibition by anti-Ly-2 and anti-TCR. NK activity was expressed normally against a variant NK-sensitive cell line lacking all MHC antigens. These results show that, in contrast to antigen-specific recognition, the NK and promiscuous lytic activities of CTL are expressed without participation of effector cell Ly-2 and TCR molecules or target cell MHC molecules, and are most likely mediated through novel and distinct receptor systems.     

Recognition of cloned influenza virus hemagglutinin gene products by cytotoxic T lymphocytes.

The influenza A virus hemagglutinin (HA) is an integral membrane glycoprotein expressed in large quantities on infected cell surfaces and is known to serve as a target antigen for influenza virus-specific cytotoxic T lymphocytes (CTL). Despite the fact that HAs derived from different influenza A virus subtypes are serologically non-cross-reactive, the HA has been implicated by previous experiments to be a target antigen for the subset of T cells capable of lysing cells infected with any human influenza A subtype (cross-reactive CTL). To directly determine whether the HA is recognized by cross-reactive CTL, we used vaccinia virus recombinants containing DNA copies of the PR8 (A/Puerto Rico/8/34) (H1N1) or JAP (A/JAP/305) (H2N2) HA genes. When these viruses were used to stimulate HA-specific CTL and to sensitize target cells for lysis by HA-specific CTL, we found no evidence for HA recognition by cross-reactive CTL aside from a relatively small degree of cross-reactivity between H1 and H2 HAs. Results of unlabeled target inhibition studies were consistent with the conclusion that the HA is, at most, only a minor target antigen for cross-reactive CTL.     

Cellular localization of perforin 1 in murine cloned cytotoxic T lymphocytes

The localization of perforin 1 (P1) in cytotoxic cells was studied by immuno-electron microscopy by using a monospecific rabbit antiserum against highly purified mouse P1 and protein A gold as a second ligand. P1 was found in specific granules of cloned cytotoxic T lymphocytes (CTL). Within the granules, P1 antigen was localized in the fine granular matrix, whereas the vesicular compartment remained free of gold particles. The amount of P1 antigen detectable by immuno-electron microscopy varied between different CTL clones. CTL with NK-like activity had the highest level of P1 antigen. A cytotoxicity loss CTL mutant had no detectable P1 antigen, suggesting an important role of P1 during cell-mediated cytolysis. P1 antigen was undetectable also in bone marrow macrophages, indicating a different cytolytic mechanism of these cells.     

Efficiency and effectiveness of cloned virus-specific cytotoxic T lymphocytes in vivo

The efficiency of cloned class I MHC restricted CTL specific for the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus in either mediating virus clearance or immunopathologic disease in mice during acute infection was quantitated. Cloned CTL specific for either an internal (nucleoprotein) or surface (glycoprotein) protein of lymphocytic choriomeningitis virus, when administered intracerebrally 5 days after the initiation of infection induced fatal immunopathology, indicating that both internal and surface viral Ag play a role in immune mediated disease in vivo. Dose-response analysis indicated that only 10(2) to 10(3) cloned CTL injected intracerebrally were required to induce mortality in 50% of inoculated syngeneic mice. Thus relatively few virus-specific CTL are required to induce an acute immunopathologic disease in the central nervous system. In contrast, if cloned CTL are adoptively transferred at the time of initiation of viral infection they provide protection as demonstrated by their ability to eliminate virus from the brain and thus terminate the acute infection.     

Direct cross-priming by th lymphocytes generates memory cytotoxic T cell responses

Under optimal Ag stimulation, CTL become functional effector and memory T cells. Professional APCs (pAPC) are considered essential for the activation of CTL, due to their unique capacity to provide costimulation and present exogenous Ags through MHC class I molecules. In this study, we report a novel means by which Th lymphocytes acquire and present MHC class I determinants to naive CTL. Although previous studies have looked at T cell Ag presentation to activated T cells, this study presents the first example of Ag presentation by Th cells to naive CTL. We report that activated Th cells can function as effective pAPC for CTL. Our results show that: 1) In addition to acquisition of cell surface molecules, including MHC class I/peptide complexes, from pAPC, Th cells can acquire and present MHC class I-binding peptides through TCR-MHC class II interactions with pAPC; 2) the acquired Ag can be functionally presented to CTL; and 3) Ag presentation by Th cells induces naive CTL to proliferate and preferentially differentiate into cells that phenotypically and functionally resemble central memory T cells. These findings suggest a novel role of Th cells as pAPC for the development of memory immune responses.     

The site of action of N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK) on cloned cytotoxic T lymphocytes

N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK), an irreversible inhibitor of trypsin-like serine proteases, is a potent, nontoxic inhibitor of cytotoxic T lymphocyte (CTL) activity with half-maximal inhibition of an alloreactive CTL clone occurring at [TLCK] = 30 microM. We have utilized TLCK as an affinity probe for functionally important CTL surface molecules by raising rabbit antibodies specific for the tosyl group and employing them as immunoprecipitating reagents. When 125I-labeled cloned CTL were treated with TLCK, immunoprecipitation with rabbit anti-tosyl antibodies and analysis by polyacrylamide gel electrophoresis revealed a small number of TLCK-binding proteins. Prior alkylation of radiolabeled CTL with iodoacetamide inhibited TLCK binding only slightly, suggesting that TLCK binding did not occur via free sulfhydryl groups. Thymocytes and a second CTL clone both had very similar patterns of TLCK-binding proteins; in contrast the TLCK-binding proteins of B cells differed greatly. Sequential immunoprecipitation experiments identified the predominant CTL TLCK-binding protein as T200. Lymphocyte function-associated antigen-1 also reacted with TLCK but to a lesser extent. The inhibitory role of cell-surface bound TLCK (vs intracellular TLCK) was demonstrated by protection experiments using Concanavalin A, a reversible ligand of the CTL cell surface. These experiments suggest that T200 may be required for cytotoxic activity of CTL.     

Characterization of determinants encoded by four Qa-1 genotypes and their recognition by cloned cytotoxic T lymphocytes

The alloantigens encoded by the four defined Qa-1 genotypes were characterized by cloned cytotoxic T lymphocyte (CTL) recognition. CTL clones specific for Qa-1a- and for Qa-1b-encoded antigens were generated. Examination of the reactivity of these clones with target cells from H-2r and H-2f strains provided the strongest evidence to date for the designation of the Qa-1c and Qa-1d genotypes, respectively, for these strains. Qa-1c-encoded antigens were recognized by most, but not all CTL clones that specifically lysed Qa-1b target cells, thus demonstrating that these antigens lack a Qa-1b-associated determinant. Similarly, Qa-1d encoded antigens were recognized by only half of the CTL clones that lysed Qa-1a target cells. In addition, one CTL clone that was cytotoxic for Qa-1b and Qa-1c target cells demonstrated low affinity, cross-reactive recognition of a Qa-1d encoded antigen. The reactivity patterns of the monoclonal CTL defined five Qa-1 determinants. Qa-1a, Qa-1b, and Qa-1d each encode multiple determinants. Two Qa-1d encoded determinants probably reside on different molecular species. Finally, large numbers of CTL clones tested on panels of target cells indicated that the Qa-1a strains expressed indistinguishable Qa-1.1 antigens and the Qa-1b strains expressed indistinguishable Qa-1.2 antigens. Therefore, additional polymorphism among these strains is improbable.     

Perforin mRNA in primary peritoneal exudate cytotoxic T lymphocytes

Considerable evidence indicates that cloned CTL cell lines kill target cells by releasing toxic granules that contain a cytolytic protein, called perforin, and several serine esterases (granzymes A to F). However, primary CTL, such as the highly cytolytic peritoneal exudate lymphocyte (PEL) cell population, have been found by a hemolytic assay to have no perforin, or perhaps only borderline levels of that protein, suggesting that these cells use a different lytic mechanism. To determine whether or not primary CTL express the perforin gene, we have here compared mRNA from PEL CTL and from a cloned CTL cell line, 2C, by Northern blot analysis using a perforin cDNA probe. CD8+ PEL CTL contain approximately 30% of the amount of perforin message present in 2C. Moreover, depletion of CD8+ T cells from the total peritoneal exudate cell population removes both cytolytic activity and perforin message. We have previously shown that PEL CTL elicit the same changes in target cells as cloned CTL cell lines and are resistant to lysis by the toxic granules purified from these cells lines. Taken together these results are consistent with the view that primary CTL, as well as long term cloned CTL cell lines, exercise their cytolytic activity by means of perforin.     

MIs locus recognition by a cloned line of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

This report demonstrates the expression of strong MIs locus MIsd) recognition by a cloned line of H-2-restricted influenza virus-specific CTL. This clone of F1 (H-2b/d; MIsb) origin was found to specifically proliferate in response to uninfected cells of CBA/J (H-2k, MIsd) origin but not to uninfected B10.BR or CBA/CaJ cells (H-2k, MIsb). In addition, proliferation by this cTL line was observed in response to histocompatible cells expressing cross-reactive MIsa determinants (DBA/2, NZB; H-2d, MIsa). This recognition was observed only at the level of CTL proliferation. The CTL line exhibited no cytotoxic activity for target cells of these MIs types. These observations are contrasted with the response of another cloned H-2-restricted influenza-specific CTL line that simultaneously exhibits alloreactivity for H-2k. The significance of these results for T lymphocyte recognition is discussed.     

The mechanism of anti-Lyt-2 inhibition of antibody-directed lysis by cytotoxic T lymphocytes

Bifunctional antibodies specific for a determinant within the T cell receptor (TcR) complex of cytotoxic T lymphocytes (CTL) and a determinant expressed on the surface of the target cell will effectively mediate cytolysis. In such a lytic system anti-Lyt-2 antibody can block cytolysis. We have observed that the amount of inhibition varies considerably from clone to clone and surprisingly correlates well with inhibition of conjugate formation as mediated by bifunctional antibody. This implies that inhibition of antibody-mediated killing occurs as the result of reduction of the avidity of the effector cell for its target, the same mechanism responsible for inhibition of receptor-mediated lysis by anti-Lyt-2. In light of the similarity between the mechanism of inhibition by anti-Lyt-2 of receptor-mediated and antibody-mediated cytolysis, we compared the ability of anti-Lyt-2 to inhibit cytolysis in these two different assay systems by using a number of different CTL clones. Whereas the majority of secondary CTL clones (presumed to have high affinity TcR) are inhibited equally in both assay systems, most primary CTL (presumed to have low affinity TcR) are more susceptible to inhibition by anti-Lyt-2 in their receptor-specific than their antibody-directed cytolysis. These results, taken together with an apparent correlation between the amount of Lyt-2 expressed on the cell surface and susceptibility to inhibition, suggest anti-Lyt-2 may block CTL function by sterically inhibiting mobility of the TcR complex.     

Studies on the mechanism of suppression by T cells in an adoptive secondary response.

Suppression of antibody synthesis by lymphocytes was studied using an adoptive secondary response model in which human serum albumin (HSA)-primed lymphocytes (memory cells) from the thoracic ducts of inbred rats were inhibited in irradiated recipients by nonimmune lymphocytes after mixed cell transfer. This investigation extended earlier work and formally showed that the suppressor cells were peripheral thymus-derived lymphocytes, which could rapidly recirculate from the blood to lymph, were present in spleen but not in bone marrow, and that primed T cells lacked this property to inhibit. The suppressive effect was independent of antigen dose but was markedly influenced by the form of antigen used for challenge in that suppression was significantly abrogated with aggregated HSA or with soluble HSA in the presence of specific antibody. Suppressor cells were found to exert their effect maximally at the time of antigen injection, but became ineffective by 40 hr following challenge. The results are considered within a larger framework of cellular regulation of antibody synthesis.     

Antigen-specific suppression of cytotoxic T cell responses: an idiotype-bearing factor regulates the cytotoxic T cell response to azobenzenearsonate-coupled cells

When A/J mice are injected subcutaneously with azobenzenearsonate- (ABA) coupled spleen cells, their splenocytes contain primed ABA-specific cytotoxic T lymphocyte (CTL) precursors. Animals that are not primed in vivo do not develop vigorous CTL activity when assessed after in vitro culture with ABA-coupled stimulators. Suppressor molecules derived from ligand-induced first-order ABA-specific suppressor T cells were evaluated for their ability to limit cytolytic T cell development. We have shown that an idiotype-bearing, hapten-specific suppressor factor suppresses priming for CTL in an H-2-unrestricted but allotype-restricted manner. The implication of these studies to regulatory networks is discussed.     

Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes

A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.     

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.     

Cyclosporine blocks the activation of antigen-dependent cytotoxic T lymphocytes directly by an IL-2-independent mechanism

The immunosuppressive drug Cyclosporin A (cyclosporine) inhibits the reactivation of quiescent Ag-dependent CTL in the presence of IL-2. Both proliferation and the regeneration of cytotoxicity are inhibited. The cytotoxic cells that are inhibited are Ag dependent for activation, whereas derived, Ag-independent, but still IL-2-dependent, cytotoxic cells are insensitive to cyclosporine. Cyclosporine also directly inhibits the effector phase of the cytotoxic cells, although not completely. The generation of primary CTL in mixed cultures was also blocked by cyclosporine in the presence of IL-2, in a time-dependent way that indicated that the sensitive time was early during the cultures. The CTL generated in primary cultures were significantly inhibited by cyclosporine in the assay, but this inhibition was less than for the cloned CTL lines.     

Allogeneic tumor-specific cytotoxic T lymphocytes

Summary We report the development of cytotoxic T lymphocytes specific for an allogeneic brain tumor in a rat model. DA strain cytotoxic T cell precursors stimulated by an allogeneic tumor (9L gliosarcoma) from the Fischer rat could generate a population of cytotoxic T lymphocytes that lysed the allogeneic 9L tumor but failed to lyse other targets, including Fischer concanavalin-A(ConA)-stimulated lymphoid blast targets. DA T cells depleted of reactivity to the Fischer haplotype (DA-f) retained reactivity to the 9L tumor, demonstrating that T cell precursors with specificity for normal Fischer alloantigens were not required for the generation of a response to the 9L Fischer tumor. The preferential lysis of the tumor target did not simply reflect a higher density of Fischer target antigens on the tumor than that found on normal Fischer ConA blast targets. First, the relative densities of class I antigen on the 9L tumor and normal Fischer ConA blasts were comparable. Second, cytotoxic T cells could not be generated from DA-f precursors when Fischer ConA blasts were used as stimulators. If DA-f T cells were simply responding to the higher density of Fischer antigen found on 9L tumor, it would have been expected that the ConA blasts expressing comparable levels of antigen to that found on the tumor would have generated cytotoxicity for both the 9L and ConA targets. We conclude that the cytotoxic T cells are specific for a determinant expressed only by the tumor. Such tumor-specific cytotoxic T cells could be useful in vivo for adoptive immunotherapy of brain tumors.