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PubMed1994

Target cell-directed degradation of perforin mRNA in CTL: lack of correlation with loss of protein and lytic ability.

We have previously shown that CTL and NK cells rapidly down regulate perforin mRNA and become functionally inactive within 4-6 hr after exposure to sensitive target cells (TC). We report here for the first time that CTL also down regulate perforin mRNA upon exposure to resistant, but binding, TC. When three separate human MHC-restricted CTL lines were exposed to resistant TC, perforin mRNA was rapidly degraded. Removal of both extracellular Ca++ and Mg++ prevented perforin message down regulation, whereas removal of Ca++ alone did not, indicating that CTL:TC binding was required. Unlike the response of CTL exposed to sensitive TC, resistant TC did not trigger serine esterase (SE) release, suggesting distinct signalling pathways for perforin mRNA down regulation and granule exocytosis. Moreover, using western analysis, we showed that there was limited (< 10%) perforin protein release after CTL:TC interaction, suggesting that CTL loss of lytic activity after exposure to sensitive TC is not due to massive depletion of perforin. Treatment of CTL with mAb to CD2, CD3, CD2 + CD3, CD8, Class I and LFA-1 did not induce perforin mRNA down regulation. Furthermore, mAb to CD2, CD3, CD8, Class I, Class II, CD54 and LFA-1 did not block TC-mediated perforin mRNA down regulation although lysis of TC was inhibited.

Cell Line↗
PubMed1996

The effects of adjuvants on CTL induction by V3:Ty-virus-like particles (V3-VLPs) in mice.

We have previously described the generation of HIV-1 V3-specific cytotoxic T-lymphocytes (CTL) responses in BALB/c (H-2d) mice following immunization with Ty-virus-like particles carrying the V3 loop of gp120 (V3-VLPs) without adjuvant. In this study the effects of various adjuvants on CTL induction by V3-VLPs was examined. Mice immunized with V3-VLPs formulated in aqueous-based adjuvants, Detox, gamma-inulin, galactosaminylmuramyl dipeptide and Chemivax generated V3-specific CTL responses, although at reduced levels when compared to the no adjuvant group. V3-VLPs prepared in Alhydrogel, algamulin or as an oil emulsion in SAF-MF failed to generate V3-specific CTL responses. The mechanism whereby alum prevented the induction of a CTL response was investigated further. Immunization with V3-VLPs prepared in non-saturating doses of alum or alum plus EDTA primed for strong CTL responses, indicating that free VLPs do, but alum-bound VLPs do not enter the MHC class I processing pathway of antigen-presenting cells (APCs). Furthermore, V3-VLPs with very low doses of alum led to an enhancement of the CTL response. The formulation of hybrid Ty-VLPs in oil based or precipitating adjuvants, therefore, inhibits access to the MHC class I processing pathway of APCs. The intact particulate structure of hybrid VLPs is therefore strictly necessary for CTL induction.

Adjuvants, Immunologic↗
PubMed1997

Cytokines and impaired CD8+ CTL activity among elderly persons and the enhancing effect of IL-12.

We have previously demonstrated that about 70% of elderly persons exhibit deficient cytotoxic T lymphocyte (CD8+ CTL) responses against influenza viruses when compared to young persons. Since IFN-gamma, a Th1 cytokine and IL-4, a Th2 cytokine, stimulate and inhibit CD8+ CTL responses respectively, their role(s) in the age-related CTL deficiency was investigated. Lymphocytes from young adults (34 +/- 5 years old) and elderly subjects (71 +/- 1 years old) were stimulated in vitro with influenza A/H3N2, A/H1N1 or influenza B virus for 6-7 days. The CD8+ CTL activity against virus-infected autologous target cells was significantly lower among the elderly than the young subjects (P < 0.01). Following stimulation with influenza virus, IL-4 production in both age groups was similar on day 3 but significantly higher among elderly persons on day 6 (P < 0.05). In contrast, T cells from the elderly produced significantly lower IFN-gamma than did those from young persons on both days (P < 0.05). Treatment of T cells from young and elderly adults with recombinant human IL-12, a pivotal cytokine that stimulates Th1 cytokines, resulted in enhancement of CD8+ CTL activity and IFN-gamma production in a dose dependent manner (P < 0.01). IL-12-dependent enhancement of CTL activity was not always abrogated by anti-IFN-gamma antibody treatment. These results suggest that deficient influenza virus-specific CTL activity among the elderly is attributable to a Th1 to Th2 cytokine production switch. Immunotherapy with IL-12 could represent a useful approach to correct the CD8+ CTL deficiency and cytokine imbalance among elderly humans.

Adolescent↗
PubMed1999

CTL epitopes identified with a defective recombinant adenovirus expressing measles virus nucleoprotein and evaluation of their protective capacity in mice.

Cytotoxic T-lymphocyte (CTL) responses to measles virus (MV) play an important role in recovery from infection, with one of the major target proteins for CTL activity being the nucleoprotein (Np). In this report, a replication-deficient adenovirus-5 recombinant, expressing for MV Np (Rad68) was tested for in vivo priming of MV Np-specific CTL responses in BALB/c and CBA mice. In both strains of mice strong Np-specific CTL responses were induced and these responses were shown to be MHC class I restricted. Using overlapping 15mer peptides spanning residues 1-505 of MV Np a single epitope comprising residues 281-295 was identified in BALB/c mice whereas, in CBA mice two epitopes comprising residues 51-65 and 81-95, were identified. These epitopes were found to contain class I motifs for H-2L(d) and H-2K(k) MHC molecules, respectively. Immunization of BALB/c and CBA mice with the respective CTL epitopes resulted in the in vivo induction of peptide-and MV Np-specific CTL responses. In addition, the identified H-2K(k) restricted CTL epitopes conferred some protection against encephalitis induced following intracerebral challenge with a lethal dose of canine distemper virus (the Np of which shares 70% sequence homology with MV Np). These findings highlight the potential of using well-defined CTL epitopes to control virus infection.

Adenoviridae↗
PubMed2000

Bone marrow cells inhibit the generation of autologous EBV-specific CTL.

Recently, we reported that human bone marrow cells (BMC) inhibited the proliferative (recall) response of lymphocytes to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) protein antigens [12]. To clarify further the effect of BMC on the immune response to viral antigens, we obtained PBL from EBV IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) 1 year after renal transplantation and generated EBV-specific CTL in vitro in the presence or absence of autologous BMC. The addition of freshly aspirated autologous iliac crest BMC from either R or LRD caused a significant inhibitory effect on the generation of EBV-specific CTL from CTL precursors, in contrast to the addition of autologous PBL used as controls (62.29 +/- 10.85% inhibition using BMC from the kidney transplant recipients; 74.47 +/- 15.21% inhibition using BMC from the living-related donors). This inhibitory effect was only exerted during the CTL generation phase; but not in the effector CTL killing phase. The expression of CD94, a component of the killer inhibitory receptor (KIR) on CD3(+) cells was elevated in the cultures with BMC, in contrast to the cultures without BMC. The BMC inhibitory effect was partially abrogated by pre-incubation of the CTL effectors with anti-CD94 monoclonal antibody, in contrast with its isotype control. In addition, supernatants obtained from the CTL generating cultures with BMC contained high levels of prostaglandin E(2) (PGE(2)), and EBV-specific CTL activity was inhibited by the addition of exogenous PGE(2) in the absence of BMC. The induction of CD40L cell surface expression by anti-CD3 was also decreased on the effector T cell population when BMC were added. There was a concomitant reduction in protein kinase C (PKC) activity. These studies demonstrate that BMC exert an inhibitory effect on T cell-mediated immunity to viral antigens in humans by regulating autologous effector T cell generation and early T cell activation signaling pathways.

Antigens, CD↗
PubMed2000

Bone marrow cells promote TH2 polarization and inhibit virus-specific CTL generation.

This laboratory recently reported that human bone marrow cells (BMC) inhibit the generation of virus-specific CTL in culture. The culture supernatants contained increased levels of prostaglandin E(2) (PGE(2)) (shown to favor TH2 cell development) and also inhibited EBV-CTL effector cell development. In this study, we obtained PBL from Epstein-Barr virus (EBV) IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) one year after renal transplantation. EBV-specific CTL were then generated in vitro by stimulating PBL with autologous EBV-transformed B cells (EBV-B) in the presence or absence of autologous BMC. The addition of BMC to the EBV-CTL generation cultures increased the intracellular expression in CD3+ cells of IL-4,-5,-6,-10, and -13. These CD3+ cells also expressed increased levels of the TH2 associated receptor CCR3. Inhibition was even observed by preparing EBV-CTL generating cultures in trans-wells that separated the autologous BMC from the PBL + EBV-B. It was then observed that CD3+ cells obtained after 7 days of culture in the presence of autologous BMC could be used as inhibitors of EBV-CTL generation. Protein Kinase A (PKA), a cAMP kinase that is involved in the upregulation of TH2 cytokine activity, was increased in EBV-CTL cultures by the presence of BMC. Additionally, IL-4-mediated signal transduction and activation of transcription (STAT-6) phosphorylation was slightly increased. These results show that the BMC inhibition is mediated by soluble factors (cytokines) and that cell-cell contact in this autologous system is not required, so that BMC (at least partially, via cytokine production) promote TH2 polarization in culture. Moreover, TH2 cells induced by culturing with autologous BMC directly inhibit EBV-CTL generation, and TH2 associated PKA, CCR3, and STAT-6 phosphorylation are enhanced by BMC.

Bone Marrow Cells↗
PubMed1998

Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens.

DNA vaccines have been shown to be an effective means of inducing cytotoxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccines are processed and presented to CTL may allow for improvements in CTL responses in older animals. Since CTL recognize short peptides presented by MHC class I molecules, and since ubiquitin-dependent proteolysis is widely believed to be responsible for degradation of endogenously synthesized antigens and generation of these peptide ligands, we sought to use ubiquitin (Ub) conjugation to target influenza virus nucleoprotein (NP) antigen into the Ub-proteasome degradation pathway for MHC class I-restricted antigen processing and presentation. However, the addition of the Ub moiety did not affect the half-life of Ub-NP protein in transiently transfected human rhabdomyosarcoma (RD) cells. Moreover, the modifications of NP DNA vaccine with Ub conjugation did not affect their ability to induce a CTL response specific for the H-2Kd-restricted NP147-155 epitope, as assessed by both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting dilution analysis (LDA). In contrast, the anti-NP antibody (Ab) responses were dramatically reduced in mice immunized with low doses (1 microgram) of Ub-NP constructs, compared with mice immunized with wild-type NP DNA. These results demonstrate that Ub conjugation alone does not guarantee targeting of endogenously synthesized antigens for rapid degradation by proteasomes. Furthermore, the ability of ubiquintination to reduce Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP from transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed.

Animals↗
PubMed1999

Priming of CD8+ CTL effector cells in mice by immunization with a stress protein-influenza virus nucleoprotein fusion molecule.

Literature is accumulating which suggests the potential for stress proteins to form the basis of a novel vaccine technology. Immunization with mammalian tumor-derived stress proteins and their associated peptides promote anti-tumor immunity. Vaccination with HIV-1 p24 antigen fused to mycobacterial heat shock protein (Hsp) Hsp71 enhances p24-specific immunity, as measured by p24-specific antibody production and in vitro cell proliferation and cytokine induction. An ovalbumin-Hsp71 fusion protein primes ovalbumin-specific CTL activity and resistance to challenge with an ovalbumin-expressing tumor. We have extended these observations by using a mycobacterial Hsp65 fusion molecule to prime CTL specific for a viral antigen. Gene fusion constructs were generated from DNA encoding Mycobacterium bovis strain BCG Hsp65 and individual fragments of influenza virus nucleoprotein (NP) encompassing H-2Kd- and H-2Db-restricted CTL epitopes. The ability of these purified recombinant fusion proteins to prime NP-specific CTL was assessed in mice of appropriate H-2 haplotypes. We observed that adjuvant-free immunization with either fusion protein elicited significant CTL activity when administered at doses of 10-100 micrograms per mouse. An NP fusion protein made with glutathione-S-transferase failed to elicit NP-specific CTL, indicating that the phenomenon requires Hsp65 sequences. A single immunization with the Hsp65-NP fusion protein elicited CTL activity which persisted for a minimum of 4 months post-immunization, at which time it could be boosted by a second immunization. To our knowledge, this is the first report of a member of the Hsp60 family priming for antigen-specific CTL activity when employed as a fusion protein partner.

Animals↗
PubMed1999

CTL-defined cancer vaccines: perspectives for active immunotherapeutic interventions in minimal residual disease.

The characterization of tumor-associated antigens recognized by cellular or humoral effectors of the immune system has opened new perspectives for cancer therapy. Several categories of cancer-associated antigens have been described as targets for cytotoxic T lymphocytes (CTL) in vitro and in vivo: (1) 'Cancer-Testis' (CT) antigens expressed in different tumors and normal testis, (2) melanocyte differentiation antigens, (3) point mutations of normal genes, (4) antigens that are overexpressed in malignant tissues, and (5) viral antigens. Clinical studies with peptides derived from these antigens have been initiated to induce specific CTL responses in vivo. Immunological and clinical parameters for the assessment of peptide-specific reactions have been defined, i.e. induction of DTH-, CTL-, autoimmune-, and tumor-regression responses. Preliminary results demonstrate that tumor-associated peptides alone elicit specific DTH- and CTL-responses leading to tumor regression after intradermal injection. GM-CSF was proven effective to enhance peptide-specific immune reactions by amplification of dermal peptide-presenting dendritic cells. Long lasting complete tumor regressions have been observed after induction of CTL by peptide immunization. Based on these results, active immunotherapy with tumor-associated antigens may be a promising approach for patients with minimal residual disease, who are at high risk for tumor recurrence. However, in single cases with disease progression after an initial tumor response either a loss of the respective tumor antigen targeted by CTL or of the presenting MHC class I molecule was detected as mechanisms of immune escape under immunization in vivo. Based on these observations, cytokines to enhance antigen- and MHC-class I expression in vivo are being evaluated to prevent immunoselection. Recently, a strategy utilizing spontaneous antibody responses to tumor-associated antigens (SEREX) has led to the identification of a new CT antigen, NY-ESO-1. In a melanoma patient with high titer antibody against NY-ESO-1 also a strong HLA-A2 restricted CTL reactivity against the same antigen was found. Clinical studies involving tumor antigens that induce both antibody- and CTL-responses will show whether these are better candidates for immunotherapy of cancer.

Cancer Vaccines↗
PubMed2000

HLA-G has a concentration-dependent effect on the generation of an allo-CTL response.

Human leucocyte antigen (HLA) -G is expressed on trophoblast cells during pregnancy, suggesting a role in protection of the semiallogeneic fetus. Published data suggest that HLA-G protects a cell against natural killer cell lysis. It has been hypothesized that HLA-G may also protect the fetus by preventing allo-cytotoxic T lymphocyte (CTL) responses. To test this hypothesis, we assayed the effects of various concentrations of purified HLA-G on CTL response in a mixed lymphocyte culture (MLC) system. We found that concentrations > or =0.1 microg/ml of HLA-G suppressed the allo-CTL response by 30-100% over the control, but, paradoxically, concentrations of 0.01-0.05 microg/ml of HLA-G augmented the allo-CTL response by 25-50% over the control. Concentrations < or = 0.001 microg/ml HLA-G had no effect. Addition of HLA-G to preprimed allo-CTL effector cells did not affect their killing ability. Allo-CTL suppressive doses of HLA-G induced a T helper type 2 (Th2) cytokine response, whereas allo-CTL-enhancing doses of HLA-G induced a Th1-type cytokine response. HLA-G purified from first-trimester placenta does not affect allo-proliferative responses nor does it alter the percentage of CD4+ or CD8+ T cells in MLCs. These findings support a potential role for HLA-G-mediated suppression of allo-CTL formation in normal pregnancies. In addition, the effects observed at lower concentrations of HLA-G may have interesting implications for the condition of pre-eclampsia in which concentrations of this HLA class I molecule are reduced.

Antibodies, Monoclonal↗
PubMed1988

Human cytomegalovirus-specific cytotoxic T cells. Relative frequency of stage-specific CTL recognizing the 72-kD immediate early protein and glycoprotein B expressed by recombinant vaccinia viruses.

CTL are held to be an important host defense mechanism in persistent herpes-virus infections. We have therefore studied the nature and specificity of human cytomegalovirus (HCMV)-specific CTL in normal persistently infected individuals. This was achieved by using vaccinia recombinants encoding viral genes expressed at different stages of the virus replicative cycle, a structural glycoprotein gB (vac.gB) and the major 72-kD immediate early nonstructural protein (vac.IE) of HCMV, combined with limiting dilution analysis of the CTL response. In two subjects, 43 and 58% of HCMV CTL precursors (CTLp) lysed vac.IE-infected cells, in contrast to less than 6% lysing gB-infected cells. HCMV-specific CTL could also be generated by secondary in vitro stimulation with vac.gB- but not vac.IE-infected autologous fibroblasts. The high frequency of 72-kD IE protein-specific CTL suggests that this is at least a major recognition element for the HCMV-specific CTL response in asymptomatic persistently infected individuals, and CTL with this specificity may be important in maintaining the normal virus/host equilibrium.

Antigens, Viral↗
PubMed1992

How do CTL control virus infections? Evidence for prelytic halt of herpes simplex.

Cytotoxic T lymphocytes (CTL) induce in target cells a rapid, prelytic fragmentation of target cell DNA, accompanied by apoptosis. In contrast, complement and (with a few exceptions) chemical and physical means of inducing cytolysis induce necrosis, without DNA fragmentation. The function of the unusual DNA fragmentation induced by CTL remains to be elucidated. The major recognized function of CTL is in halting virus infections. Earlier, we proposed that CTL might halt virus infections prelytically, by fragmenting viral and cellular nucleic acids, and that in this case, cytolysis per se might be a less important function of CTL. We report here experiments designed to detect prelytic halt of virus replication. We employed in vivo-like conditions: fibroblast targets (difficult to lyse) were infected with herpes simplex virus (HSV), then incubated at low E/T cell ratios overnight. At the highest E/T ratios which produced less than 10% CTL-induced lysis, plaque-forming unit yield was reduced by about 50%. At higher E/T ratios which lysed 1/6 to 1/3 of the infected target cells, 3/4 to 9/10 of the virus production was prevented. The discrepancy between the level of lysis and the reduction in virus yield is evidence for significant CTL-induced prelytic halt of HSV replication. At present, it is unclear whether the antiviral effect observed involves an activity of CTL distinct from their lytic ability, such as their DNA fragmenting ability.

Animals↗
PubMed1994

Major and minor Kb-restricted epitopes encoded by the endogenous ecotropic murine leukemia virus AKR623 that are recognized by anti-AKR/Gross MuLV CTL.

C57BL/6 mice can generate a type-specific and class IH-2Kb-restricted CTL response against histocompatible AKR/Gross murine leukemia virus (MuLV) cell surface antigen positive (GCSA+) tumor cells. These anti-AKR/Gross MuLV CTL are also known to lyse SC.Kb/623 target cells expressing the molecular MuLV clone AKR623 (derived from the endogenous ecotropic MuLV provirus emv-11). To help identify AKR623 viral epitopes recognized by these CTL, four chimeric proviruses were constructed from two parental plasmids, pAKR623 and pAK7. It has been shown that SC.Kb/7 fibroblast targets expressing the emv-14-derived molecular clone AK7 are only poorly lysed by anti-AKR/Gross MuLV CTL. Data from experiments employing SC.Kb cells infected with the chimeras as targets against anti-AKR/Gross MuLV CTL supported the location of a previously identified immunodominant epitope located within the viral p15E transmembrane envelope protein, peptide TM134-141 (KSP-WFTTL). Furthermore, the use of Kb-motif-defined AKR623 encoded peptides together with data obtained using the chimeric viruses allowed the identification of three additional anti-AKR/Gross MuLV CTL epitopes. Peptides representing these epitopes, MA125-132 (RSALY-PAL), RT142-149 (SHRWYTVL), and RT456-463 (RMTHYQAM), are characterized herein with respect to their ability to confer lysis upon EMV- target cells and to stimulate tumor primed splenocytes in vitro. The identification and characterization of these additional epitopes allow for a better understanding of both the CTL response against GCSA+ tumor cells and the dysfunctional CTL response against EMV-14 and AK7.

AKR murine leukemia virus↗
PubMed1994

Lack of correlation between phenotype activation markers of CD8 lymphocytes and cytotoxic T lymphocyte (CTL) function in HIV-1 infection: evidence for rescue with rIL-2.

CTL activity against HIV-1 antigens expressed on HLA-A-matched EBV-transformed B target cells was detected in 33% (6/18) of freshly isolated PBMC (FPBMC) from patients in the early stages of HIV-1 infection (CDCII). No CTL activity was detected in FPMBC in patients with AIDS (CDCIV). However, the presence of CTL activity did not correlate with the expression of CTL activation markers. A dual-color flow cytometric examination revealed that the CD8+ lymphocytes bearing the memory (CD29) and activation (S6F1) surface molecules increased in number as the HIV-1 infection progressed. This functional and phenotypic discrepancy in memory CD8+ lymphocytes suggests that the memory CD8+ lymphocytes have lost cytotoxic function and become "paralyzed" as the HIV disease progresses. Incubation of PBMC of HIV(+) patients with rIL-2 reactivated predominantly HIV-specific CTL. However, rIL-2 stimulation also activated a "polyclonal or polyreactive" cytotoxic function. The reactivation of CTL function is rIL-2 dosage dependent and the amount of rIL-2 required for reactivation is associated with the severity of the disease. HIV antigen specific CTL in HIV(+) patients can be selectively expanded by HIV antigen stimulation in the presence of rIL-2. These results suggest that the in vivo IL-2 deficiency occurring in HIV-1 infection may be responsible in part for the "paralysis" of HIV specific CTL activity. Such activity can be rescued nonspecifically by exogenous rIL-2 stimulation and expanded specifically by HIV-1 antigen stimulation.

Antigens, Surface↗
PubMed1989

Development and evaluation of a limiting dilution analysis technique that can discriminate in vivo alloactivated cytotoxic T lymphocytes from their naive CTL precursors.

We describe a permutation of the conventional limiting dilution analysis (LDA) technique that allows, for the 1st time, the differential enumeration of alloantigen-specific CTL that have been activated by alloantigens in vivo. This technique does not detect nonactivated CTL precursors, even those with similar alloantigen specificity. Data are presented to validate this limiting dilution analysis technique. Using this LDA technique, we demonstrate that large numbers of the donor-reactive CTL arrive in sponge matrix allografts (f = 1/3,599 cells), most or all of which are in an activated state (f = 1/4,385 cells). In contrast, alloactivated CTL constitute only a small fraction (f = 1/57,208 cells) of the donor-reactive CTL in the regional lymph node (f = 1/1,873 cells) of the same sponge allograft recipients. As expected, regional lymph nodes from sponge isograft recipients contain DBA/2-reactive CTL precursors (f = 1/1,873 cells), but no activated DBA/2-reactive CTL (f less than 1/385,529 cells). This LDA technique should be useful in studies regarding activation and redistribution of alloreactive CTL caused by allograft implantation.

Animals↗
PubMed1991

Inhibition of anti-HLA-B7 alloreactive CTL by affinity-purified soluble HLA.

The objective of this study was to elucidate the interaction of naturally occurring soluble MHC class I molecules with alloreactive CTL and to discuss its possible relevance to graft acceptance. An anti-HLA-B7 specific CTL-line, BV.B7, was generated in vitro. On phenotyping the cells after 6 weeks, 80% were found to be CD8+, 14% CD4+ and 6% CD8+CD4+. CD4+ CTL were depleted using immunomagnetic beads precoated with an anti-CD4 antibody. Of the recovered CTL greater than 96% were CD8+. A total of 12 HLA-B7 target cell lines and PHA blasts tested were specifically lysed in a 51Cr-release assay. Soluble HLA class I molecules were isolated on affinity chromatography columns using the anti-HLA-B7 ME 1 and the anti-heavy chain W6/32 monoclonal antibodies. Antigen purity was confirmed by analysis on SDS-PAGE gels. CTL were preincubated with 0.1-1.8 micrograms/ml soluble HLA for 30 min at 37 degrees C and subsequently tested for cytotoxicity in the 51Cr-release assay; 1.1 micrograms/ml HLA-B7 molecules reduced CTL cytotoxicity by 50% whereas non-B7 HLA had no effect. Further, CTL cytotoxicity was reduced by preincubation with anti-CD8, anti-TcR, and anti-CD3 antibodies. We anticipate a possible down-regulatory role of soluble HLA on CTL in allogeneic transplantation.

Antibodies↗
PubMed2004

Epicutaneous application of CpG oligodeoxynucleotides with peptide or protein antigen promotes the generation of CTL.

Immunostimulatory oligodeoxynucleotides (ODN) are effective adjuvants in the induction of humoral and cellular immune responses when administered parenterally with antigen. The skin has recently become a target organ for the design of non-invasive vaccine technologies. Using ovalbumin (OVA) as a model antigen, we demonstrate that the application of ODN sequences to tape-stripped skin promotes the induction of potent cytotoxic T lymphocyte (CTL) responses to co-administered peptide. Induction of peptide-specific CTL required the presence of CpG motifs within the ODN. CTL afforded tumor protection against a tumor expressing an immunodominant OVA CTL epitope. CTL could also be induced to whole protein administered onto the skin. Differential CpG sequence activity was noted with respect to the induction of CTL to epicutaneous protein with an ODN sequence containing a poly-G motif having an optimal effect. Peptide-specific CTL could be detected in the peripheral blood as early as 6 d after a single immunization. These results highlight the potential of the bare skin as a route for vaccine development and indicate an important role for immunostimulatory ODN as adjuvants to generate functional CTL with the help of the skin immune system.

Adjuvants, Immunologic↗
PubMed2006

Detection of Wilms' tumor antigen--specific CTL in tumor-draining lymph nodes of patients with early breast cancer.

PURPOSE: The Wilms' tumor antigen (WT1) is overexpressed in approximately 90% of breast tumors and, thus, is a potential target antigen for the immunotherapy of breast cancer. We have tested the working hypotheses that WT1 can be immunogenic in patients with breast cancer and can stimulate CTL of sufficient avidity to kill tumor cells. EXPERIMENTAL DESIGN: Paired tumor-draining lymph node and peripheral blood samples were analyzed from five HLA-A2-positive patients with stage I/II breast cancer. Fluorescent HLA-A*0201/WT1 tetramers were used to quantify WT1-specific CTL and the functional capacity of the CTL was assessed using cytotoxicity assays and intracellular cytokine staining. RESULTS: WT1 tetramer-binding T cells expanded from all lymph node samples but none of the corresponding peripheral blood samples. Functional assays were carried out on T cells from the patient who had yielded the highest frequency of HLA-A*0201/WT1 tetramer-positive cells. The cytotoxicity assays showed WT1 peptide--specific killing activity of the CTL, whereas intracellular cytokine staining confirmed that the tetramer--positive T cells produced IFN-gamma after stimulation with WT1 peptide. These WT1-specific T cells killed HLA-A2-positive breast cancer cell lines treated with IFN-gamma but no killing was observed with untreated tumor cells. CONCLUSIONS: These results show that WT1-specific CTL can be expanded from the tumor-draining lymph nodes of breast cancer patients and that they can display peptide-specific effector function. However, the CTL only killed IFN-gamma-treated tumor targets expressing high levels of HLA-A2 and not tumor cells with low HLA expression. This suggests that induction of autologous WT1-specific CTL may offer only limited tumor protection and that strategies that allow a high level of peptide/MHC complex presentation and/or improve CTL avidity may be required.

Aged↗