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PubMed2001

Human vascular endothelial cells stimulate a lower frequency of alloreactive CD8+ pre-CTL and induce less clonal expansion than matching B lymphoblastoid cells: development of a novel limiting dilution analysis method based on CFSE labeling of lymphocytes.

We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8(+) T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8(+) T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8(+) T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8(+) T cells by either BLC or HUVEC derived from the same donor. CD8(+) T cells expanded by allostimulation were identified as CD8(+), CFSE(low) cells and were categorized as CTL by the expression of intracellular perforin and IFN-gamma. Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p < 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8(+) T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8(+) T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.

Antigens, CD↗

PubMed2002

Characterization of the Fas ligand/Fas-dependent apoptosis of antiretroviral, class I MHC tetramer-defined, CD8+ CTL by in vivo retrovirus-infected cells.

C57BL/6 (B6; H-2(b)) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8(+) CTL responses to the immunodominant K(b)-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2(b) congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2(b) spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2(b) cells function as "veto" cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2(b) veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8(+) CTL were identified by a labeled K(b)/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2(b) veto cell-mediated depletion of these CTL. A 65-93% reduction in the number of B6 K(b)/KSPWFTTL tetramer(+) CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2(b) veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2(b) veto cells) to block inhibition demonstrated that AKR.H-2(b) veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer(+) CTL cocultured with AKR.H-2(b) veto cells was annexin V positive and Fas(high), indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2(b) cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of Vbeta4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.

AKR murine leukemia virus↗

PubMed2003

Impacts of avidity and specificity on the antiviral efficiency of HIV-1-specific CTL.

Although CD8(+) CTLs are presumed to be an important mediator of protective immunity in HIV-1 infection, the factors that determine CTL antiviral efficiency are poorly understood. Two factors that have been proposed to influence CTL antiviral function are antigenic avidity and epitope specificity. In this study we evaluate these by examining the activity of HIV-1-specific CTL against acutely infected cells. The ability of CTL to kill infected cells is variable and depends more on epitope specificity than functional avidity within the range for the tested clones (50% of maximal killing, 50 pg/ml to 100 ng/ml); killing efficiency is similar for different clones recognizing the same epitope, despite their variation in avidity. When CTL clones are tested for their ability to suppress viral replication, similar results are observed. Inhibition is more dependent on epitope specificity than functional avidity among the tested clones (50% of maximal killing, 20 pg/ml to 20 ng/ml). Thus, CTL specificity can be an overriding factor in the ability of CTL to interact with HIV-1-infected cells, indicating that factors determining the process of epitope presentation on infected cells have a key influence on CTL efficiency. These results suggest that CTL specificity may have a pivotal role in the immunopathogenesis of infection, and that simple quantitative measures of CTL may be insufficient indicators of the CTL response to HIV-1.

Antiviral Agents↗

PubMed1999

Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication.

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.

Animals↗

PubMed1999

The antiviral activity of HIV-specific CD8+ CTL clones is limited by elimination due to encounter with HIV-infected targets.

Adoptive immunotherapy of virus infection with viral-specific CTL has shown promise in animal models and human virus infections and is being evaluated as a therapy for established HIV-1 infection. Defining the individual obstacles for success is difficult in human trials. We have therefore examined the localization, persistence, and antiviral activity of HIV-1 gag-specific CTL clones in both HIV-1-infected and uninfected haplotype-matched human (hu)-PBL-SCID mice. Injection of gag-specific clones but not control CTL into HIV-1-infected hosts reduced plasma viremia by >10-fold but failed to eliminate the virus infection from most treated animals. The failure to eradicate virus did not reflect selection of escape variants because the gag epitope remained unmutated in virus isolates obtained after CTL therapy. Injection of carboxyfluorescein diacetate succinimide ester-labeled CTL demonstrated markedly different fates for gag-specific CTL in the presence or absence of HIV-1 infection. HIV-1-specific CTL rapidly disappeared in infected recipients, whereas they were maintained at high numbers in uninfected mice. By contrast, control CTL were long lived in both infected and uninfected recipients. Thus, interaction of CTL with virus-infected target cells in vivo leads not only to target destruction but also to the rapid disappearance of the infused CTL, and it limits the capacity of CTL therapy to eliminate HIV-1 infection.

Adoptive Transfer↗

PubMed1984

Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation, and analysis to phenotype and specificity.

Activation of peripheral blood lymphocytes (PBL) from a melanoma patient either in secondary MLC in which EBV-transformed B cells from the cell line JY were used as stimulator cells, or by co-cultivation with the autologous melanoma cells in a mixed leukocyte tumor cell culture (MLTC) resulted in the generation of cytotoxic activity against the autologous melanoma (O-mel) cells. From these activated bulk cultures four cloned cytotoxic T lymphocyte (CTL) lines were isolated. The CTL clone O-1 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+), and O-36 (T3+, T4-, T8+, OKM-, HNK-, and HLA-DR+) were obtained from MLC, whereas the CTLC clones O-C7 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+) and O-D5 (T3+, T4-, T8+, OKM-1-, HNK, and HLA-DR+) were isolated from autologous MLTC. All four CTL clones were strongly cytotoxic for O-mel cells but failed to lyse autologous fibroblasts and autologous T lymphoblasts. Moreover, the CTL clones lacked NK activity as measured against K562 and Daudi cells. Panel studies indicated that the CTL clones also killed approximately 50% of the allogeneic melanoma cells preferentially, whereas the corresponding T lymphoblasts were not lysed. Monoclonal antibodies against class I (W6/32) and class II (279) MHC antigens failed to block the reactivity of the CTL clones against O-mel and allogeneic melanoma cells, indicating that a proportion of human melanoma cells share determinants that are different from HLA antigens and that are recognized by CTL clones. In contrast to the CTL clones isolated from MLTC, the clones obtained from MLC also lysed JY cells, which initially were used as stimulator cells. The reactivity of O-36 against JY could be inhibited with W6/32, demonstrating that this reactivity was directed against class I MHC antigens. These results suggest that the lysis of O-mel and JY cells by O-36 has to be attributed to two independent specificities of this CTL clone. The specificity of the other cross-reactive CTL clone (O-1) could not be determined. The notion that individual CTL clones can have two specificities was supported by the following observations. The cytotoxic reactivity of both O-1 (T4+) and O-36 (T8+) against JY was blocked by monoclonal antibodies directed against T3 and human LFA-1, and against T3, T8, and human LFA-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Cellular and molecular mechanisms of the IL-12-induced increase in allospecific murine cytolytic T cell activity. Implications for the age-related decline in CTL.

The mechanism(s) by which IL-12 augments CTL generation and the potential for IL-12 to alter the age-related decline in CTL activity were assessed using primary CTL stimulated by allogeneic cells. IL-12 at 2 U/ml enhanced the generation of allospecific CTL response in young adult mice by 1.5- to 5-fold, and optimal augmentation was achieved and maintained with as low as 0.1 U/ml IL-12. In contrast to the enhancement of MHC-unrestricted lytic activity, which was most efficient when IL-12 was added near the end of the 5 day culture period, nearly equivalent augmentation of Ag-specific CTL was induced when IL-12 was added from 1 to 5 days before harvest. The augmentation of CTL by IL-12 was not associated with increased incorporation of [3H]TdR. Increased IFN-gamma production was not required for IL-12 to augment CTL activity because when three different Abs against mouse IFN-gamma were each included in the MLC at concentrations abolishing recoverable IFN-gamma protein, they did not inhibit the IL-12 effect but, under some conditions, further increased CTL activity. Ab to IL-2 also did not alter the IL-12 effect. Depletion of ASGM1+ cells from the responding population before stimulation did not significantly alter the response to IL-12. However, depletion of CD8+ T cells from effector cells obtained after stimulation substantially reduced the IL-12-induced augmentation of CTL. Thus, the results suggested that IL-12 augments CTL activity primarily through an effect on T cells, and the augmentation was associated with an approximate doubling of perforin levels, which was not caused by a significantly increased proportion of CD8+ cells. Finally, IL-12 was effective for moderating the age-related decrease in CTL activity, suggesting a potential role for immunomodulation by IL-12.

Aging↗

PubMed1994

Association of HER2/neu expression with sensitivity to tumor-specific CTL in human ovarian cancer.

To study potential sources of tumor-associated Ags in human ovarian cancer, we have established two ovarian tumor cell lines (OvS1 and OvA2) from two ovarian cancer patients, which express the cellular oncogene HER2/neu. Corresponding tumor infiltrating lymphocyte cultures have also been established and display an autologous tumor-specific pattern of cytotoxicity that is HLA-A2 restricted. To determine the potential relationship between HER2/neu expression and CTL-mediated cytolysis, we first established tumor cell clones from OvS1. These were categorized as high or low expressors of HER2/neu (cOvS1+ or cOvS1-, respectively), and cOvS1+ clones displayed a significantly higher sensitivity to CTL killing as compared with cOvS1- clones. To modulate the expression of HER2/neu, ovarian cancer cells were treated with IFN-gamma. After this exposure, HER2/neu expression was significantly decreased, whereas the expression of HLA Class I was significantly increased. Despite the increase in HLA Class I molecules on the cell surface, CTL-mediated cytolysis of both OvS1 and OvA2 was significantly decreased. IFN-gamma treated cOvS1+ clones displayed a similar decrease in sensitivity to CTL killing, whereas IFN-gamma treated cOvS1- clones displayed an increase or no change in sensitivity to CTL. To confirm this apparent association between HER2/neu expression and CTL recognition, melanoma tumor cell lines that were insensitive to ovarian tumor-specific CTL were transfected with the HER2/neu gene. An HLA-A2+ HER2/neu-transfected melanoma cell line was made sensitive to HLA-A2 restricted ovarian tumor-specific CTL but not to HLA-A2 unrestricted CTL, whereas an HLA-A2- HER2/neu-transfected melanoma remained insensitive to HLA-A2 restricted CTL. These results demonstrate that the sensitivity of ovarian epithelial tumor cells to CTL-mediated lysis is associated with the level of expression of HER2/neu, suggesting that this oncogene product may serve as a source of tumor-associated Ags or as an inducer of such peptides. This is the first time in a human tumor system that oncogene expression has been related to the induction of antigenicity. These results prompt us to approach new strategies for immunotherapy of cancer.

Antibodies, Monoclonal↗

PubMed1997

The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection.

We have previously described the development of a lipopeptide-based vaccine, Theradigm-HBV, capable of inducing CTL responses in humans. This vaccine incorporates the HLA-A2.1-restricted CTL epitope hepatitis B core Ag 18-27, linked to the universal helper T lymphocyte (HTL) epitope tetanus toxoid (TT) 830-843. Herein, we report the results of a phase I trial designed to examine the effects of Theradigm-HBV dose and regimen on the magnitude and duration of the memory CTL response. A total of four injections of up to 5 mg/dose were found to be a safe and effective means of generating substantial memory CTL responses. Precursor frequency analysis demonstrated CTL responses of similar magnitude to those previously observed in patients who successfully cleared hepatitis B virus infection and to influenza-specific memory CTL responses induced by natural exposure to influenza virus. Theradigm-HBV induced CTL responses that persisted for more than 9 months after the last injection. HTL responses were associated with significant CTL responses, and sustained HTL activity was necessary for development of persistent memory CTL activity. These results represent the first demonstration of the importance of HTL activity for development of long-lived memory CTL responses in humans. In conclusion, our results show that lipopeptides safely induce specific CTL activity in humans of such magnitude and persistence as to be of potential therapeutic significance.

Acute Disease↗

PubMed1997

HLA class-I-restricted and tumor-specific CTL in tumor-infiltrating lymphocytes of patients with gastric cancer.

Immune recognition of human cancers except melanoma is not well understood at either the cellular or the molecular level. We demonstrate in this study the existence of HLA class-I-restricted and tumor-specific CTL in IL-2-activated TIL (tumor-infiltrating lymphocytes) of all 4 gastric cancer patients tested. We established HLA A2-restricted and adenocarcinoma-specific CTL in 2 HLA A0201+ patients, and HLA A2402-restricted CTL recognizing both adenocarcinoma and squamous-cell carcinomas (SCC) in the 2 remaining HLA A2402+ patients. Further, HLA A3101-restricted and adenocarcinoma-specific CTL were established in 1 of the 2 HLA A2402+ patients who had HLA A3101 allele. HLA A2-, A2402- and A3101-restricted CD8+ CTL clones were established from these parental CTL lines. The 2 HLA A2-restricted CTL lines lysed 8 of 13 HLA A2+ adenocarcinoma cell lines established from different organs (stomach, colon, lung and breast) with different subtypes (HLA A0201, A0206 and A0207). The HLA A2-restricted CTL line recognized 9 and 6 different HPLC fractions of peptides eluted from the HLA A0201+ breast and HLA A0201+ colon adenocarcinoma cell lines, respectively. Allele-specific deletion of HLA A2 or A24 molecules was observed in some tumor lines that were not susceptible to lysis by the CTL lines. These results suggest that TIL of gastric cancer possess CTL recognizing different peptide antigens binding to different HLA-A alleles that are widely expressed on adenocarcinomas and also, to some extent, on SCC from different organs.

Adenocarcinoma↗

PubMed1989

Different class I antigen oligosaccharides on a murine tumor and a lectin-resistant variant are not responsible for the differential recognition of the tumors by CTL.

Previous studies have shown that whereas a highly malignant mouse cell line termed MDAY-D2 (d haplotype) does not elicit a detectable response by cytotoxic T lymphocytes (CTL) in DBA/2 mice, strong anti-tumor CTL are generated against a wheat-germ-agglutinin-resistant variant, designated MDW3. Additional evidence suggests these anti-MDW3 CTL may not be a consequence of a unique antigenic determinant on the variant cells. Because MDW3 cells are expected to differ from MDAY-D2 cells in their surface carbohydrate structures (due to their lectin resistance) and Class I major histocompatibility molecules play a crucial role in CTL-mediated responses, we speculated that the Asn-linked oligosaccharides present on Class I molecules of MDAY-D2 and MDW3 might be different and could potentially influence recognition analyses and Con A-Sepharose affinity chromatography clearly demonstrated that the oligosaccharides isolated from the H-2Dd molecule of MDAY-D2 cells are larger and more highly branched than those of the MDW3 variant. Taken together with the finding that anti-MDW3 CTL are restricted by H-2Dd, these results suggested that the larger H-2Dd oligosaccharides on MDAY-D2 cells could potentially mask or perturb determinants required for recognition by these CTL. To test this postulate, the surface Class I oligosaccharides of both MDAY-D2 and MDW3 cells were converted to simpler hybrid structures by treatment with the oligosaccharide processing inhibitor, swainsonine. However, no effect was observed on the lysis or binding of either MDAY-D2 or MDW3 cells by anti-MDW3 CTL. Thus, the results do not support the possibility that the larger H-2Dd oligosaccharides on MDAY-D2 cells are, in themselves, responsible for the poor recognition of the parent tumor by anti-MDW3 CTL. Our data do indicate, however, that CTL target binding and effector functions are not dependent on the fine structure of complex Asn-linked carbohydrates present on Class I molecules and possibly on other, accessory molecules at the target cell surface, since MDW3 cells maintained their sensitivity to lysis by CTL following swainsonine treatment.

Alkaloids↗

PubMed2005

Evaluation of high functional avidity CTL to Gag epitope clusters in EIAV carrier horses.

Cytotoxic T lymphocytes (CTL) are critical for lentivirus control including EIAV. Since CTL from most EIAV carrier horses recognize Gag epitope clusters (EC), the hypothesis that carrier horses would have high functional avidity CTL to optimal epitopes in Gag EC was tested. Twenty-two optimal EC epitopes were identified; two in EC1, six in EC2, and seven each in EC3 and 4. However, only five of nine horses had high functional avidity CTL (<or=11 nM) recognizing six epitopes in EC; four in relatively conserved EC3; and one each in EC1 and 2. Horses with high functional avidity CTL had significantly more days since the last clinical episode than horses with low avidity CTL, and this was not explained by analyzing duration of infection. Furthermore, there was a significant inverse correlation between the CTL functional avidity of the nine horses and the days since the last clinical episode. Gag CTL epitope escape variants were found in three horses, but only one of these was recognized by high functional avidity CTL. Thus, not all carrier horses had high functional avidity CTL to Gag EC, but those that did had longer periods without disease episodes.

Amino Acid Sequence↗

PubMed1998

OK-432 develops CTL and LAK activity in mononuclear cells from regional lymph nodes of lung cancer patients.

We examined the effect of OK-432 on induction of cytotoxic T lymphocytes (CTL) directed against autologous tumor cells (ATC) and lymphokine-activated killer (LAK) cells from mononuclear cells separated from regional lymph node cells (RLMNCs) of 49 lung cancer patients. We also examined the phenotypic changes of RLMNCs during incubation with or without OK-432. Significant CTL activity and LAK activity against ATC developed from RLMNCs after stimulation with OK-432 or IL-2. Sequential treatment with OK-432 plus IL-2 or IL-2 plus OK-432 also developed significant CTL activity and LAK activity from RLMNCs. The CTL activity produced by OK-432 alone was as high as the CTL activity developed by IL-2 alone, OK-432 plus IL-2, or IL-2 plus OK-432. There was no significant difference in the CTL activities achieved by these four treatments. The proportion of CD25+ cells in RLMNCs after incubation with OK-432 was twice that before incubation. Although OK-432 increased IL-2 receptor expression on RLMNCs, it showed no synergistic effect with IL-2 in developing CTL and LAK activity. After incubation with OK-432, the proportion of HLA-DR + cells was also increased significantly. Moreover, the proportions of HLA-ABC+ and HLA-DR+ (class I and class II major histocompatibility complex antigens) cells in ATC were significantly larger than in Daudi cells. OK-432 alone could develop CTL activity against ATC from the RLMNCs of lung cancer patients that was as high as that developed by IL-2 alone or by sequential treatment with OK-432 plus IL-2 or IL-2 plus OK-432. The CTL developed from the RLMNCs of lung cancer patients may recognize class I and/or II antigens on the surface of ATC. These results indicated that treatment with OK-432 might be therapeutically useful for lung cancer patients as a CTL inducer rather than a LAK inducer.

Aged↗

PubMed2002

Functionally distinct helper T-cell epitopes of HCV and their role in modulation of NS3-specific, CD8+/tetramer positive CTL.

Hepatitis C virus specific (HCV-specific) CD8+ cytotoxic T cells play a critical role in viral clearance. Low HCV-specific cytotoxic T lymphocyte (CTL) responses in chronic HCV infection may favor the persistence of virus, whereas stimulation and expansion of HCV-specific CTL activity may assist elimination of HCV infection. Helper T cells control the intensity of CD8+ T-cell responses and helper T-cell responses are known to be compromised in chronic carriers of HCV. In this study, we wanted to ascertain if strengthening the Th response could increase the intensity of CTL activity against HCV target antigens. We selected a synthetic CTL peptide NS3(1073-1081)), two Th1 epitopes, peptide NS3(358-375) and NS5B(155-172), and one Th2 epitope, peptide NS3(505-521). By using the four peptides alone or in combinations, we stimulated peripheral blood cells isolated from a chronic hepatitis C patient in vitro and then analyzed CD8 T cells specific for the NS3(1073-1081) CTL epitope in A2 tetramer staining and cytotoxicity assays. The results demonstrated that CTL responses could be augmented by helper T-cell epitopes NS3(358-375) and NS5B(155-172). Th2 epitope NS3(505-521) inhibited augmentation of CTL activity by Th1 epitopes. This inhibitory effect could be overcome by combining the two Th1 epitopes NS3(358-375) and NS5B(155-172) together with NS3(505-521). Under such conditions, CTL frequency was restored, but cytotoxic activity remained low suggesting that the help provided under these cultures was sufficient to drive proliferation of CTL, but not sufficient to drive differentiation into mature killer cells. These results may provide some insights into compromised CTL activity in HCV viral persistence.

Amino Acid Sequence↗

PubMed2002

Vaccine protection against functional CTL abnormalities in simian human immunodeficiency virus-infected rhesus monkeys.

Accumulating evidence suggests that HIV-specific CD8(+) CTL are dysfunctional in HIV-infected individuals with progressive clinical disease. In the present studies, cytokine production by virus-specific CTL was assessed in the rhesus monkey model for AIDS to determine its contribution to the functional impairment of CTL. CTL from monkeys infected with nonpathogenic isolates of simian and simian-human immunodeficiency virus expressed high levels of IFN-gamma, TNF-alpha, and IL-2 after in vitro exposure to a nonspecific mitogen or the optimal peptide representing a dominant virus-specific CTL epitope. However, similarly performed studies assessing these capabilities in CTL from monkeys infected with pathogenic immunodeficiency virus isolates demonstrated a significant dysfunction in the ability of the CTL to produce IL-2 and TNF-alpha. Importantly, CTL from vaccinated monkeys that effectively controlled the replication of a highly pathogenic simian-human immunodeficiency virus isolate following challenge demonstrated a preserved capacity to produce these cytokines. These experiments suggest that defects in cytokine production may contribute to CTL dysfunction in chronic HIV or SIV infection. Moreover, an AIDS vaccine that confers protection against clinical disease evolution in this experimental model also preserves the functional capacity of these CTL to produce both IL-2 and TNF-alpha.

Animals↗

PubMed2002

Tumor-specific CTL kill murine renal cancer cells using both perforin and Fas ligand-mediated lysis in vitro, but cause tumor regression in vivo in the absence of perforin.

Kidney cancer is a devastating disease; however, biological therapies have achieved some limited success. The murine renal cancer Renca has been used as a model for developing new preclinical approaches to the treatment of renal cell carcinoma. Successful cytokine-based approaches require CD8(+) T cells, but the exact mechanisms by which T cells mediate therapeutic benefit have not been completely identified. After successful biological therapy of Renca in BALB/c mice, we generated CTLs in vitro using mixed lymphocyte tumor cultures. These CTL mediated tumor-specific H-2K(d)-restricted lysis and production of IFN-gamma, TNF-alpha, and Fas ligand (FasL) in response to Renca. CTL used both granule- and FasL-mediated mechanisms to lyse Renca, although granule-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-gamma and TNF-alpha increased the sensitivity of Renca cells to CTL lysis by both granule- and FasL-mediated death pathways. Adoptive transfer of these anti-Renca CTL into tumor-bearing mice cured most mice of established experimental pulmonary metastases, and successfully treated mice were immune to tumor rechallenge. Interestingly, we were able to establish Renca-specific CTL from mice gene targeted for perforin (pfp(-/-)) mice. Although these pfp(-/-) CTL showed reduced cytotoxic activity against Renca, their IFN-gamma production in the presence of Renca targets was equivalent to that of wild-type CTL, and adoptive transfer of pfp(-/-) CTL was as efficient as wild-type CTL in causing regression of established Renca pulmonary metastases. Therefore, although granule-mediated killing is of paramount importance for CTL-mediated lysis in vitro, some major in vivo effector mechanisms clearly are independent of perforin.

Animals↗

PubMed2003

In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones.

Human papillomavirus (HPV) type 16 infection is strongly associated with the development of cervical carcinoma (CxCa) in women. The HPV16-derived oncoproteins E6 and E7, responsible for both onset and maintenance of malignant transformation, are expressed constitutively in CxCa cells and represent tumor-associated Ags. As a result, E6 and E7 constitute potential targets for adoptive CTL-mediated immunotherapy of CxCa. However, the availability to date of well-characterized HPV16-specific, CxCa-reactive human CTLs is extremely limited. The current study describes the in vitro generation and isolation of HPV16 E7-specific, CxCa-reactive human CTL clones from low-frequency healthy donor-derived CD8beta-positive precursors. For this purpose, an in vitro CTL induction protocol was used involving mature monocyte-derived dendritic cells as stimulator cells loaded with an HLA-A2.1-restricted, E7(11-20)-derived high-affinity altered peptide ligand. A double tetramer-guided isolation procedure and subsequent limiting-dilution cloning resulted in Ag-specific CTL clones. Stringent CTL characterization clearly indicated Ag-specific, HLA-A2.1-restricted reactivity against different HPV16-transformed CxCa cell lines. To allow expansion of E7(11-20)-specific CTL clones to numbers required for prolonged in vitro as well as in vivo application, their life span was significantly extended by ectopic expression of human telomerase reverse transcriptase. Collectively, our results show that optimized CTL induction and stringent CTL selection procedures, followed by human telomerase reverse transcriptase-mediated life span extension will allow continued availability of low-frequency HPV16-specific, CxCa-reactive human CTL clones. This may enhance the prospects of HPV16-specific adoptive CTL immunotherapy in CxCa patients.

Antigen Presentation↗

PubMed2004

Differential requirement for IFN-gamma in CTL maturation in acute murine graft-versus-host disease.

Although IFN-gamma is the archetypal Th1 cytokine, its role in CTL maturation is uncertain. We used an in vivo mouse model of CTL development, parent-into-F(1) acute graft-vs-host disease (AGVHD), to evaluate this issue. In AGVHD, transfer of naive parental T cells into F(1) hosts stimulates the development of allospecific CTL effectors that eliminate host lymphocytes, particularly B cells. Complete elimination of IFN-gamma, using IFN-gamma-deficient donors and administering anti-IFN-gamma mAb, suppressed B cell elimination, down-regulated TNF-alpha production, and enhanced Th2 cytokine production, but did not allow the B cell expansion characteristic of chronic GVHD (CGVHD). Because complete CTL inhibition results in full-blown CGVHD that is IFN-gamma independent, these observations indicate that IFN-gamma elimination only partially blocks CTL development. IFN-gamma elimination did not inhibit donor T cell engraftment or activation in the AGVHD model, but almost completely blocked Fas/Fas ligand (FasL) gene expression, protein up-regulation, and Fas/FasL-mediated CTL killing. In contrast, IFN-gamma elimination only partially inhibited perforin gene expression and perforin-mediated CTL activity. The contributions of IFN-gamma to CTL development were indirect, because IFN-gamma receptor-deficient donor cells differentiated normally into allospecific CTLs. Consistent with the view that the Fas/FasL and perforin pathways each mediate CTL killing in AGVHD, the absence of both perforin and IFN-gamma (perforin knockout donor cells and anti-IFN-gamma mAb) converted AGVHD to CGVHD. Thus, both IFN-gamma-dependent induction of Fas/FasL and IFN-gamma-independent induction of perforin contribute to CTL-mediated elimination of host B cells in AGVHD. Suppression of both pathways is required for typical CGVHD development.

Acute Disease↗