Suppressed cytotoxic T lymphocyte responses in experimental cysticercosis.

Mitogen-induced T cell responses are suppressed in mice infected with larvae of Taenia crassiceps. The effects of experimental infection on specific T cell responses, however, have not been examined. In the present study, we demonstrate that larval-infected mice exhibit suppressed ability to develop anti-virus specific cytotoxic T lymphocyte (CTL) responses while maintaining apparently normal natural killer (NK) cell responsiveness.

A novel CD8-independent high-avidity cytotoxic T-lymphocyte response directed against an epitope in the phosphoprotein of the paramyxovirus simian virus 5.

Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo.

An abrupt and concordant initiation of apoptosis: antigen-dependent death of CD8+ CTL.

The ability of CD8+ cytotoxic T lymphocytes (CTL) to clear viral infections may be limited when high avidity CTL encounter supra-optimal antigen density on antigen-presenting cells (APC) and undergo antigen-dependent apoptosis of CTL (ADAC). Previously, we have shown ADAC in CD8+ populations to be Fas independent, TNF-alpha receptor 2 (TNFR2) mediated, caspase dependent, and accompanied by a decrease in Bcl-2. We now employ flow cytometry to follow ADAC within individual CD8+ cells to demonstrate that the intense TCR signal induced in high avidity CTL by supra-optimal antigen density results 8 - 16 h later in a caspase-independent TNFR2 down-modulation that is directly related to the stimulating APC antigen density and concludes in a rapid onset of apoptosis by 18 - 24 h. Individual CTL undergoing apoptosis exhibit a dramatic and concurrent: (1) positive staining with Annexin V and propidium iodide; (2) transformation to a smaller cell size characteristic of apoptosis; and (3) a nearly complete loss of Bcl-2, c-IAP1, and TRAF2. We conclude that the antigen-dependent apoptosis of CD8+ CTL occurs when a tandem TCR/TNFR2 signal initiates an abrupt and concordant onset of multiple apoptotic events.

Peptide requirement for CTL activation reflects the sensitivity to CD3 engagement: correlation with CD8alphabeta versus CD8alphaalpha expression.

In our previous studies, CTL that were sensitive to low concentrations of peptide Ag were found to be far superior to those requiring high concentrations of Ag for reducing viral burden when adoptively transferred into SCID mice. Thus it is important that we understand the mechanisms that control the requirement for peptide Ag with the long-term goal of selectively expanding these exquisitely sensitive cells in vivo. Although TCR affinity is one parameter that can affect the CTL sensitivity for Ag, we investigated whether additional mechanisms may also be involved. In studies using a TCR transgenic mouse model, we successfully generated CTL with identical TCR affinity that possess distinctly different activation requirements. Using both peptide Ag and anti-CD3 Ab to activate the CTL lines of high vs low avidity, we found that the variations in activation threshold are the result of differences in the required number of engaged TCR. Additionally, we have observed that the ratio of CD8alphabeta to CD8alphaalpha is significantly greater in CTL lines that are more sensitive to TCR engagement, which may contribute to the lower activation threshold of these CTL following CD3 engagement. These studies identify a novel mechanism by which the activation requirements of Ag-specific CTL are determined by demonstrating a direct correlation between the sensitivity to TCR engagement, the expression of levels CD8alphabeta vs alphaalpha, and the amount of peptide Ag required to reach the threshold for activation.

High-avidity CTL exploit two complementary mechanisms to provide better protection against viral infection than low-avidity CTL.

Previously, we observed that high-avidity CTL are much more effective in vivo than low-avidity CTL in elimination of infected cells, but the mechanisms behind their superior activity remained unclear. In this study, we identify two complementary mechanisms: 1) high-avidity CTL lyse infected cells earlier in the course of a viral infection by recognizing lower Ag densities than those distinguished by low-avidity CTL and 2) they initiate lysis of target cells more rapidly at any given Ag density. Alternative mechanisms were excluded, including: 1) the possibility that low-avidity CTL might control virus given more time (virus levels remained as high at 6 days following transfer as at 3 days) and 2) that differences in efficacy might be correlated with homing ability. Furthermore, adoptive transfer of high- and low-avidity CTL into SCID mice demonstrated that transfer of a 10-fold greater amount of low-avidity CTL could only partially compensate for their decreased ability to eliminate infected cells. Thus, we conclude that high-avidity CTL exploit two complementary mechanisms that combine to prevent the spread of virus within the animal: earlier recognition of infected cells when little viral protein has been made and more rapid lysis of infected cells.

Differential expansion and survival of high and low avidity cytotoxic T cell populations during the immune response to a viral infection.

The importance of high avidity CTL for the effective clearance of viral infections is now well established. Thus one would predict that the preferential activation and expansion of high avidity CTL following viral challenge and retention of these cells in the memory pool would be optimal for the immune response. However, whether this actually occurs during the immune response to viral infection is unknown. In this report I have analyzed the avidity of the CTL specific for the OVA(257-264) peptide during acute infection with a recombinant vaccinia expressing ovalbumin and in the memory population. I have found that the relative ratio of high and low avidity CTL varies over the course of an immune response. Thus CTL avidity is an important factor in the expansion and survival of CTL in vivo.

Supraoptimal peptide-major histocompatibility complex causes a decrease in bc1-2 levels and allows tumor necrosis factor alpha receptor II-mediated apoptosis of cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) are primary mediators of viral clearance, but high viral burden can result in deletion of antigen-specific CTLs. We previously reported a potential mechanism for this deletion: tumor necrosis factor (TNF)-alpha-mediated apoptosis resulting from stimulation with supraoptimal peptide-major histocompatibility complex. Here, we show that although death is mediated by TNF-alpha and its receptor (TNF-RII), surprisingly neither the antigen dose dependence of TNF-alpha production nor that of TNF-RII expression can account for the dose dependence of apoptosis. Rather, a previously unrecognized effect of supraoptimal antigen in markedly decreasing levels of the antiapoptotic protein Bc1-2 was discovered and is likely to account for the gain in susceptibility or competence to sustain the death signal through TNF-RII. This decrease requires a signal through the TCR, not just through TNF-RII. Although death mediated by TNF-RII is not as widely studied as that mediated by TNF-RI, we show here that it is also dependent on proteolytic cleavage by caspases and triggered by a brief initial encounter with antigen. These results suggest that determinant density can regulate the immune response by altering the sensitivity of CTLs to the apoptotic effects of TNF-alpha by decreasing Bc1-2 levels.

Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases.

Activation of ICE/Ced-3 family proteases (caspases) has been proposed to mediate both the granule exocytosis and Fas-Fas ligand pathways of rapid target cell death by cytotoxic T lymphocytes. In agreement with this model, two peptide fluoromethyl ketone caspase inhibitors and baculovirus p35 blocked apoptotic nuclear damage and target cell lysis by the CTL-mediated Fas-Fas ligand pathway. The peptide caspase inhibitors also blocked drug-induced apoptotic cell death in tumor cells. In contrast, the caspase inhibitors blocked CTL granule exocytosis-induced target apoptotic nuclear damage, but did not inhibit target lysis. These results are consistent with recent demonstrations that granzyme B can activate caspases leading to apoptotic nuclear damage, but show that target cell lysis by CTL granule exocytosis occurs by a caspase-independent pathway.

Cytotoxic T lymphocyte (CTL) adherence assay (CAA): a non-radioactive assay for murine CTL recognition of peptide-MHC class I complexes.

Cytotoxic T lymphocytes (CTL) form an important immune surveillance system against intracellular pathogens. Here we describe a simple, visual assay for identifying peptides specifically recognized by CTL, based on the discovery that CTL develop increased adhesive properties upon TCR triggering. Several CTL lines were shown to pellet to the bottom of a round bottom 96-well plate in the absence of peptide. In contrast, these same CTL lines incubated with their cognate peptide, allowing them to present peptide to each other, adhered to the sides of the well and were readily distinguished by macroscopic visual examination of the plate after 4-5 h or overnight incubation. This CTL adherence assay (CAA) demonstrated peptide specificity and MHC restriction, and was titratable with peptide concentration. With this technique, a minimal-sized, malaria CTL epitope was correctly identified from a panel of overlapping nonamers, although the adherence pattern of two mono-substituted, variant peptides was less predictive of lytic activity. Also, substitutions in an HIV-1 envelope CTL epitope that reduced lytic activity were correctly predicted. Inhibitors of RNA and protein synthesis, upon preincubation, abrogated the adherence, indicating, at minimum, a need for live cells. Wortmannin, a PI-3 kinase inhibitor, inhibited the peptide specific adherence, consistent with a role for TCR or integrin signal transduction in CAA. Other cytoskeletal and metabolic inhibitors had no effect. Adherence of the T cells may involve low affinity, nonspecific interactions since wells coated with FCS, BSA or milk powder all produced an effective CAA in the presence of peptide under serum free conditions. Consequently, CAA may represent a rapid, simple method for screening large numbers of peptides to find cytolytic epitopes for a given CTL line and may identify additional epitopes causing T cell activation and adherence but not cytolytic activity.

Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL.

Experimental data suggest that negative selection of thymocytes can occur as a result of supraoptimal antigenic stimulation. It is unknown, however, whether such mechanisms are at work in mature CD8+ T lymphocytes. Here, we show that CD8+ effector cytotoxic T lymphocytes (CTL) are susceptible to proliferative inhibition by high dose peptide antigen, leading to apoptotic death mediated by TNF-alpha release. Such inhibition is not reflected in the cytolytic potential of the CTL, since concentrations of antigen that are inhibitory for proliferation promote efficient lysis of target cells. Thus, although CTL have committed to the apoptotic pathway, the kinetics of this process are such that CTL function can occur before death of the CTL. The concentration of antigen required for inhibition is a function of the CTL avidity, in that concentrations of antigen capable of completely inhibiting high avidity CTL maximally stimulate low avidity CTL. Importantly, the inhibition can be detected in both activated and resting CTL. Blocking studies demonstrate that the CD8 molecule contributes significantly to the inhibitory signal as the addition of anti-CD8 antibody restores the proliferative response. Thus, our data support the model that mature CD8+ CTL can accommodate an activation signal of restricted intensity, which, if surpassed, results in deletion of that cell.

Expression of HLA class I molecules and the transporter associated with antigen processing in hepatocellular carcinoma.

The expression of the HLA class I molecules on the cell surface was investigated in hepatocellular carcinoma (HCC) cell lines using complement-mediated cytotoxicity (CMC) and flow cytometric analysis. Although HLA-A antigens were detected by CMC in all cell lines tested, HLA-B and -C antigens were not detectable in six of seven HCC cell lines. These results were also confirmed by flow cytometric analysis focusing on HLA-Bw4 and Bw6 public antigens. Furthermore, complementary DNA (cDNA) from each cell line was tested for the expression of HLA-A, -B, -C and the transporter associated with antigen processing genes (TAP1 and TAP2). Two cell lines showed a reduced level of one or both of the TAP messenger RNAs (mRNAs), and one of these showed a reduction of HLA-B and -C gene expression as well, but the others had detectable mRNA levels. These results demonstrate that hepatocellular carcinoma cell lines tested in the current study lose or decrease the expression of HLA-B and -C alleles on the cell surface, even though mRNA encoding these alleles is present, suggesting that the loss of the HLA molecules might be caused by posttranscriptional events or failure to transport and load peptides necessary for HLA expression. The selective loss of HLA-B and -C, but not -A, molecules (which also excludes a beta 2-microglobulin defect) is intriguing, and may be attributable to the ability of some of the HLA-A molecules to load signal peptides not requiring TAP transport, or to natural selection of HLA-B or -C locus-specific immune surveillance.

Molecular analysis of presentation by HLA-A2.1 of a promiscuously binding V3 loop peptide from the HIV-envelope protein to human cytotoxic T lymphocytes.

P18(IIIB) is a highly immunogenic peptide from the V3 loop of the HIV-1 gp160 envelope protein that is presented promiscuously by multiple class I MHC molecules. Understanding the molecular basis for promiscuous presentation may have many practical applications. As the highly prevalent HLA-A2.1 class I molecule is known to present P18(IIIB) for recognition by cytotoxic T lymphocytes (CTL) found in peripheral blood mononuclear cells of HIV+ donors, a P18(IIIB)-specific CTL line was generated from and HLA-A2(+), HIV- donor in order to define the molecular basis for, and ultimately improve upon the binding of, this peptide to HLA-A2.1. The minimal epitope recognized by the line was a decamer, I10, with the sequence RGPGRAFVTI. Interestingly, this decamer is identical to the minimal epitope from P18(IIIB) seen by murine CTL restricted by H-2Dd. A panel of Ala-substituted peptides was employed in MHC-binding and T cell response studies to identify MHC- and TCR-binding residues. Notably, many of the agretopic and epitopic residues identified were identical to those involved in the corresponding interactions of I10 with the H-2Dd MHC molecule and murine I10-specific CTL. The I10 peptide does not contain the described HLA-A2.1 binding motif. Instead a Pro at P3, a Phe at P7 and an Ile at P10 are utilized for MHC binding. Agretopic residue similarities with the hepatitis B nucleocapsid decamer suggest that these residues may comprise an alternative motif of anchors utilized by decamers for binding to HLA-A2.1.

Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy.

The conventional approach to cytotoxic T-lymphocyte (CTL) induction uses maximal antigen concentration with the intent of eliciting more CTL. However, the efficacy of this approach has not been systematically explored with regard to the quality of the CTLs elicited or their in vivo functionality. Here, we show that a diametrically opposite approach elicits CTLs that are much more effective at clearing virus. CTLs specific for a defined peptide epitope were selectively expanded with various concentrations of peptide antigen. CTLs generated with exceedingly low-dose peptide lysed targets sensitized with > 100-fold less peptide than CTLs generated with high-dose peptide. Differences in expression of T-cell antigen receptors or a number of other accessory molecules did not account for the functional differences. Further, high-avidity CTLs adoptively transferred into severe combined immunodeficient mice were 100- to 1000-fold more effective at viral clearance than the low-avidity CTLs, despite the fact that all CTL lines lysed virus-infected targets in vitro. Thus, the quality of CTLs is as important as the quantity of CTLs for adoptive immunotherapy, and the ability to kill virally infected targets in vitro is not predictive of in vivo efficacy, whereas the determinant density requirement described here is predictive. Application of these principles may be critical in developing effective adoptive cellular immunotherapy for viral infections and cancer.

Definition of TCR recognition sites on Ld-tum- complexes.

The P911 variant of the P815 mastocytoma was shown by Lurquin et al. (Cell 58:293, 1989) to elicit rapid tumor rejection in a syngeneic host. This rejection was mediated by Ld-restricted cytotoxic T lymphocytes (CTL) for which targets could be sensitized by the synthetic peptide designated tum- (P91A-.12-24). In a previous study, T cell clones specific for Ld-tum- complexes displayed very restricted TCR usage and a characteristic TCR motif in the V alpha CDR3 region, predicted to interact with peptide. However, in contrast to the majority of Ld peptide ligands that are nonamers, the tum- peptide is a 13-mer and its sequence does not fit the Ld binding motif. Thus, to define shorter versions of the tum- 13-mer and residues involved in TCR recognition, nonamer derivatives were synthesized and compared in several different binding and functional assays. From these comparisons, the peptide TQNHRALDL was found to be the optimal nonamer. CTL recognition of Ala-substituted analogues of this peptide indicated that the His and Arg residues at positions 4 and 5 are important for TCR contact. We propose that these basic residues of the tum- peptide interact with the previously defined acidic residues in the CDR3 region of several TCR known to recognize Ld-tum- complexes.

Specific prolongation of allograft survival by a T-cell-receptor-derived peptide.

Allograft rejection results from the specific recognition by host CD8+ T cells of allogeneic major histocompatibility complex (MHC) molecules on the tissue graft. The specificity of this cellular response is determined by the molecular interaction of the T-cell receptor (TCR) on host T cells with the MHC molecule and its bound ligand on the grafted tissue. To better understand the precise manner by which the TCR interacts with the MHC-peptide complex and how to therapeutically intervene, we have studied the allogeneic response to the mouse class I MHC molecule Ld. In this report, the therapeutic potential of a synthetic peptide derived from the TCR V beta 8 variable region that predominates in responses to Ld was tested. This V beta 8-derived peptide was found to dramatically and specifically block the in vivo and in vitro allogeneic response to Ld. Furthermore, this specific blocking is not dependent upon the presence of V beta 8+ effector cells nor does the V beta 8 peptide bind to the Ld ligand binding cleft. We propose that this peptide functions as an antagonist, competing with the native TCR for recognition of the Ld molecule.

Alloreactive cytotoxic T lymphocytes generated in the presence of viral-derived peptides show exquisite peptide and MHC specificity.

The nature of alloreactivity to MHC molecules has been enigmatic, primarily because of the observation that allogeneic responses are considerably stronger than syngeneic responses. To better determine the specificity potential of allogeneic responses, we have generated alloreactive CTL specific for exogenous, viral-derived peptide ligands. This approach allowed us to critically evaluate both the peptide- and MHC-specificity of these alloreactive T cells. Exploiting the accessibility of the H-2Ld class I molecule for exogenous peptide ligands, alloreactive CTL were generated that are specific for either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis virus (LCMV) peptides bound by Ld alloantigens. Peptide specificity was initially observed in bulk cultures of alloreactive CTL only when tested on peptide-sensitized T2.Ld target cells that have defective presentation of endogenous peptides. Subsequent cloning of bulk alloreactive CTL lines generated to MCMV yielded CTL clones that had exquisitely specific MCMV peptide recognition requirement. All of the MCMV/Ld alloreactive CTL clones were also exquisitely MHC-specific in that none of the CTL clones lysed targets expressing MCMV/Lq complexes, even though Lq differs from Ld by only six amino acid residues and Lq also binds the MCMV peptide. This observation clearly demonstrates that alloreactive CTL are capable of the same degree of specificity for target cell recognition as are syngeneic CTL in MHC-restricted responses.

Biased T cell receptor usage by Ld-restricted, tum- peptide-specific cytotoxic T lymphocyte clones.

We have investigated the TCR gene usage in a panel of H-2Ld-restricted, tum- peptide-specific CTL clones. These clones possess identical MHC restriction and peptide specificity, yet they vary dramatically in the amount of peptide required to sensitize targets for recognition. We previously demonstrated a precise quantitative correlation between the determinant density requirement of a given clone and the CD8 dependency. In this study we sequenced polymerase chain reaction copies of the TCR mRNA used by these clones, not only to correlate TCR structure with recognition of a specific class I/peptide complex, but also to determine if the functional affinity differences between these clones is reflected in the TCR gene products used. The number of TCR V beta, V alpha, and J alpha region gene segments expressed by these clones is very limited. Twelve of 17 clones express V beta 8 at comparable levels on the cell surface. Using PCR amplification of cDNA templates, cloning, and dideoxy sequencing, we have obtained the nucleotide sequence of the TCR V-(D)-J regions in seven of the V beta 8+ clones. Two of the clones use V beta 8.2 and identical J alpha gene segments. Three of the five V beta 8.3+ clones express identical V alpha and J alpha gene products and the other two use similar V alpha chains and J alpha chains with a shared motif in the predicted CDR3 region. Although no clear correlation between TCR gene usage and CD8 dependency was seen, the range of TCR gene usage in the tum- peptide-specific, Ld-restricted immune response is strikingly narrow and suggests a coselection of the alpha- and beta- chains for recognition of the class I/peptide complex.