High Avidity CTLs Research Articles

Abstract 473: CD4+ T cell help differentially influences anti-tumor immunity as a function of T cell avidity

Abstract One of the major hurdles for cancer immunotherapy is that most tumors express self-antigens to which the immune system is unresponsive due to self-tolerance. Our lab and others have demonstrated that high-avidity tumor-specific CD8+ T cells are superior in anti-tumor activity to low avidity T cells. However, adoptive transfer of high-avidity, tumor-specific CTL populations may fail due to loss of CTL effector functions upon tumor infiltration. Therefore, strategies to maintain effector functions of high avidity CTL, or enhancement of low avidity CTL are desirable. To investigate whether provision of CD4+ T cell help will enhance the effectiveness of high-avidity CTLs in anti-tumor immunity as compared to lower avidity T cells, we utilized two transgenic mouse lines developed in our lab that bear different TCR transgenes specific for a common melanoma differentiation antigen, tyrosinase-related protein 2 (TRP-2(180-188)). T cells from clone 24 TCR Tg mice (TCRhi) show higher avidity than those from clone 37 (TCRlo) mice based on tetramer dissociation and in vitro effector function assays. Here we report that co-activation of CD4+ T cells (OVA323-339 specific, CD4-OVA) with TRP-2-specific CD8+ T cells can boost the in vitro function of TCRhi cells, including proliferation, IFN-γ secretion, Gr-B secretion as well as cell-mediated cytotoxicity, but had minimal effect on TCRlo cells. We observed that only adoptive co-transfer of CD4-OVA cells with TCRhi cells suppressed tumor growth of (5 day) established B16 melanoma tumors. Furthermore, those mice receiving co-transferred CD4-OVA cells with TCRhi cells developed autoimmune depigmentation at the sites of vaccination and tumor challenge. Our finding may have important implications for enhancement of cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 473. doi:10.1158/1538-7445.AM2011-473

HLA‐A2‐restricted glypican‐3 peptide‐specific CTL clones induced by peptide vaccine show high avidity and antigen‐specific killing activity against tumor cells

Glypican-3 (GPC3) is an onco-fetal antigen that is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we identified an HLA-A2-restricted GPC3(144-152) (FVGEFFTDV) peptide that can induce GPC3-reactive CTLs without inducing autoimmunity in HLA-A2 transgenic mice. In this study, we carried out a phase I clinical trial of HLA-A2-restricted GPC3(144-152) peptide vaccine in 14 patients with advanced HCC. Immunological responses were analyzed by ex vivo γ-interferon enzyme-linked immunospot assay. The frequency of GPC3(144-152) peptide-specific CTLs after vaccination (mean, 96; range, 5-441) was significantly larger than that before vaccination (mean, 6.5; range, 0-43) (P < 0.01). An increase in the GPC3(144-152) peptide-specific CTL frequency was observed in 12 (86%) of 14 patients after vaccination. Additionally, there was a significant correlation between the maximum value of GPC3(144-152) peptide-specific CTLs after vaccination and the dose of the peptide injected (P = 0.0166, r = 0.665). Moreover, we established several GPC3(144-152) peptide-specific CTL clones from PBMCs of patients vaccinated with GPC3(144-152) peptide by single cell sorting using Dextramer and CD107a antibody. These CTL clones had high avidity (the recognition efficiency showing 50% cytotoxicity was 10(-10) or 10(-11) M) and could recognize HCC cell lines expressing GPC3 in an HLA-class I-restricted manner. These results suggest that GPC3(144-152) peptide vaccine can induce high avidity CTLs capable of killing HCC cells expressing GPC3. This trial was registered with University Hospital Medical Information Network number 000001395.

Influenza Epitope-Specific CD8+ T Cell Avidity, but Not Cytokine Polyfunctionality, Can Be Determined by TCRβ Clonotype

Cytokine polyfunctionality has recently emerged as a correlate of effective CTL immunity to viruses and tumors. Although the determinants of polyfunctionality remain unclear, there are published instances of a link between the production of multiple effector molecules and the peptide plus MHC class I molecule avidity of T cell populations. Influenza A virus infection of C57BL/6J mice induces CTL populations specific for multiple viral epitopes, each with varying proportions of monofunctional (IFN-γ(+) only) or polyfunctional (IFN-γ(+)TNF-α(+)IL-2(+)) CTLs. In this study, we probe the link between TCR avidity and polyfunctionality for two dominant influenza epitopes (D(b)NP(366) and D(b)PA(224)) by sequencing the TCR CDR3β regions of influenza-specific IFN-γ(+) versus IFN-γ(+)IL-2(+) cells, or total tetramer(+) versus high-avidity CTLs (as defined by the peptide plus MHC class I molecule-TCR dissociation rate). Preferential selection for particular clonotypes was evident for the high-avidity D(b)PA(224)-specific set but not for any of the other subsets examined. These data suggest that factors other than TCRβ sequence influence cytokine profiles and demonstrate no link between differential avidity and polyfunctionality.

Provision of CD4+ T cell help preferentially enhances anti-tumor immunity of high avidity T cells (131.19)

Abstract It has been shown that high avidity tumor-specific CD8+ T cells are superior to low-avidity T cells in anti-tumor activity. However, adoptive transfer of high-avidity CTL populations could still fail as a therapy due to the fact that CTL effector function may be lost following tumor infiltration. To investigate whether provision of CD4+ T cell help will enhance the effectiveness of high avidity CTLs in anti-tumor immunity, we utilized transgenic mice developed in our lab which bear different TCR transgenes specific for a common melanoma differentiation antigen, tyrosinase-related protein 2 (TRP-2(180-188)). T cells from clone 24 Tg mice (TCRhi) show higher avidity than those from clone 37 Tg mice (TCRlo) based on tetramer dissociation and in vitro effector function assays. Here we report that co-activation of CD4+ T cells with TCRhi cells can boost the in vitro function of TCRhi cells, including proliferation, IFN-gamma secretion, Gr-B secretion, and antigen-specific cytotoxicity, but had minimal effects on TCRlo cells. In the treatment of subcutaneous B16 melanoma, we observed that adoptive co-transfer of CD4+ T cells with TCRhi cells (not with TCRlo cells) markedly suppressed tumor growth and induced autoimmune depigmentation at the sites of vaccination and tumor challenge. These findings suggest that provision of CD4+ T cell help preferentially enhances anti-tumor immunity of high-avidity T cells, which may have critical implications for cancer therapy.

From TCR Gene Transfer to TCR Gene Editing of Central Memory T Lymphocytes for Immunotherapy of Leukemia.

Abstract 374▪▪This icon denotes an abstract that is clinically relevant.The transfer of a T cell receptor (TCR) from a high-avidity tumor-specific lymphocyte to polyclonal lymphocytes as been proposed as an attractive approach to overcome the major limitations of adoptive immunotherapy of cancer, such as the difficulty in expanding rare, high-avidity tumor-specific CTLs from cancer patients, in conditions that can preserve their function and prevent exhaustion. However, complete exploitation of this approach is limited by technical and safety issues, like the low and transient transgene expression, unpredictable pairing of the exogenous and endogenous TCR chains and poor survival and expansion potential of gene-modified effector lymphocytes. We developed a strategy aimed at overcoming these limitations. First, to increase TCR expression and facilitate proper TCR pairing we used a codon-optimized high-avidity TCR, specific for an HLA-A2-restricted peptide from the oncogenic Wilms tumor antigen 1 (WT1), modified with point mutations to introduce cysteines into the constant regions of the α and β chains. We cloned genes encoding the TCR in third generation lentiviral vectors under the control of a bi-directional PGK or a bi-directional EF1α promoter. Second, to increase the expected life span and expansion potential of gene modified cells, we transduced lymphocytes upon activation with anti-CD3 and anti-CD28 antibody-conjugated beads (bCD3/CD28) and culture with low doses of IL-7/IL-15. Human T lymphocytes were efficiently transduced by both vectors. However, PGK promoter was superior to EF1α in sustaining stochiometric expression of WT1-specific TCR chains, at levels appropriate for efficient HLA-A2/WT1 pentamer binding (16%), for up to 70 days, in the absence of further T cell stimulation. The phenotype of transduced cells was consistent with early T cell differentiation (CD62L+, CD28+CD27+, IL7Rα+, IL-2+ γIFN±), a phenotype associated with long-term persistence of adoptively transferred lymphocytes. Most importantly, TCR transduced cells were able to specifically produce γIFN and exhibited cytotoxic activity against WT1+HLA-A2+ primary leukemic blasts from AML patients. Third, to further improve the safety of the strategy and ensure predictable levels of transgene expression, we developed, for the first time, a protocol for site-specific integration of transgenes into primary T lymphocytes by exploiting zinc finger nucleases (ZFNs). Delivery in primary T lymphocytes of ZFNs targeting the CCR5 gene, in association with a GFP expression cassette flanked by CCR5 homology arms, resulted in efficient (6%) site-specific integration of GFP into the CCR5 locus. Extensive molecular analysis on sorted gene-modified cells and clones confirmed site-specific integration and showed a biallelic CCR5 disruption in 87% of GFP+ clones, due to a combined site-specific integration in one CCR5 allele, and non-homologous end joining (NHEJ) repair in the other allele. Finally, we exploited ZFNs to address the major limitation of TCR gene transfer, represented by the co-expression in the same cell of the endogenous and tumor-specific TCR. Such co-expression results in a dilution of tumor-specific TCR, thus reducing the efficacy of gene-modified cells, and might result in autoreactive specificities due to misparing between endogenous and exogenous α and β TCR chains. To this purpose, we used a new set of ZFNs specific for the constant region of the TCR β chain and knock down the expression of endogenous TCR in 20% of Jurkat cell line and 3.5% of primary human lymphocytes. These results show that a complete editing of T cell specificities can be achieved in human lymphocytes. (E.P. and P.G. equally contributed to this work). Disclosures:Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment.

Differential TCR signal transduction pathways involved in high and low avidity CD8+ T-cell responsiveness (35.26)

Abstract CTLs that display high functional avidity are known to be optimal for pathogen clearance. However, the molecular mechanism that allows high avidity CTLs to respond at very low levels of peptide antigen remains to be elucidated. To investigate this question, we compared TCR signal transduction events in high and low avidity CD8+ T-cells generated from OT-I TCR transgenic mice. Upon stimulation with APCs preloaded with a range of Ova257-264 peptide concentrations (10^-6, 10^-9 and 10^-12 M), we found that high avidity CD8+ T-cells induced higher levels of MAPK ERK-1/2 phosphorylation as well as higher levels of the dephosphorylated form of NFAT at each peptide concentration used for stimulation. We next determined whether the observed divergence in NFAT activation was reflected in differences in calcium mobilization. We found efficient mobilization of Ca2+ in high avidity cells even when the lowest concentration of peptide was used for stimulation, a level of peptide at which no increase in calcium was observed in low avidity cells. The decreased peptide requirement for calcium mobilization in high avidity cells correlated with a decreased peptide requirement for ZAP-70 activation and CD3ζ phosphorylation, the latter of which was higher and prolonged in high versus low avidity cells. Thus the functional avidity of the CD8+ effector cells studies here is controlled by differential regulation of the most membrane proximal event, phosphorylation of CD3ζ. Studies are currently underway to determine the mechanism by which high avidity cells acheive CD3ζ phosphorylation at limiting peptide levels.

Circulating Leukemic Myeloid Dendritic Cells from Patient with Leukemia Elicit CDK2-Specific CTLs from Allogeneic HLA-A24+ Naive CD8+ T Cells.

Aberrantly expressed self-antigens in leukemic cells are a kind of leukemia-associated-antigens (LAAs). Although such self-antigen-derived LAAs potentially induce high avidity CTLs in patients with leukemia, these CTLs usually do not persist in the patients because they undergo apoptosis upon encountering with leukemic cells. In allogeneic stem cell transplant (allo-SCT) recipients with leukemia, LAAs-specific high avidity CTL may be elicited from donor-derived naive T cells that are sensitized by residual leukemic cells. Cyclin-dependent kinase 2 (CDK2) is a cell cycle regulator protein that is aberrantly expressed by leukemia cells. We previously reported that two CDK2-derived peptides (CDK2 158-166, CDK2 178-186) avidly bound to HLA-A24 molecule to elicit each peptide-specific CTLs from HLA identical donor of a AML patient, and CDK2-specific CD8 + T cells preferentially killed the recipient AML cells which aberrantly expressed CDK2 protein (Blood. 108 (11): a3173. 2006). When we assessed cryopreserved PBMCs obtained before and after allo-SCT from 16 HLA-A24+ patients (6 AML, 1 MDS, 1 CML, 2 ALL, 4 NHL and 2 RCC) using CDK2 158/A24 and CDK2 178/A24 pentamers, small populations (0.1–1.0%) of CDK2 158 and CDK2 178-specific CTL were detectable in 6 patients after SCT but not before SCT. All of the 6 patients had MRD at the time of SCT and achieved molecular CR after SCT. None of the 3 patients relapsed after SCT did not show CDK2 immunity. There is no relationship between the appearance of CDK2-specific CTLs and the development of GvHD (Figure). Leukemic myeloid dendritic cells (mDC) are known to present in vivo in patients with leukemia, and be able to trigger a protective antileukemic immune response by allogeneic T-cells. We hypothesized that residual circulating leukemic mDCs may sensitize donor-derived T cells by cross-presenting CDK2-peptides early after allo-SCT and elicit high avidity CTLs specific to leukemic cells. mDCs subset was identified with by three-color staining using mAbs against CD85k, CD33, CD14 and CD16. Circulating mDCs represented 5.4% of PBMCs in a patient with CML-CP at diagnosis. Leukemic mDCs from the patient were purified with CD1c mAb-conjugated maginetic beads and were assessed for their ability to stimulate allogeneic T cells to acquire specific cytotoxicity against CDK2-peptides. The purity of the mDCs as determined by flow cytometry was 92% of living isolated cells. Naive CD8 + T cells isolated from healthy individual were cultured with the leukemic mDCs for 12 days and subjected to pentamers staining. After the coculturing, the proportion of CDK2 158/A24 and CDK2 178/A24 pentamers + CD8 T cells increased 0.6% to 2.2% and 0.6% to 2.6%, respectively. These data suggest that CDK2-specific CTLs can be induced from donor-derived T cells due to in vivo sensitization of donor T cells by residual leukemic mDCs and this may be a mechanism responsible for the generation of CDK2-specific CTLs in allo-SCT recipients with MRD without vaccination of CDK2-peptides. Vaccination with CDK2-peptides after allo-SCT may be useful in both enhancing CDK2-specific immunity in patients with MRD and in generating CDK2 immunity in those without MRD. [Display omitted]

A Novel Functional CTL Avidity/Activity Compartmentalization to the Site of Mucosal Immunization Contributes to Protection of Macaques against Simian/Human Immunodeficiency Viral Depletion of Mucosal CD4+ T Cells

The presence of high-avidity CTLs in the right compartment can greatly affect clearance of a virus infection (for example, AIDS viral infection of and dissemination from mucosa). Comparing mucosal vs systemic immunization, we observed a novel compartmentalization of CTL avidity and proportion of functionally active Ag-specific CD8(+) T cells to tissues proximal to sites of immunization. Whereas both s.c. and intrarectal routes of immunization induced tetramer(+) cells in the spleen and gut, the mucosal vaccine induced a higher percentage of functioning IFN-gamma(+) Ag-specific CD8(+) T cells in the gut mucosa in mice. Translating to the CD8(+) CTL avidity distribution in rhesus macaques, intrarectal vaccination induced more high-avidity mucosal CTL than s.c. vaccination and protection of mucosal CD4(+) T cells from AIDS viral depletion, whereas systemic immunization induced higher avidity IFN-gamma-secreting cells in the draining lymph nodes but no protection of mucosal CD4(+) T cells, after mucosal challenge with pathogenic simian/human immunodeficiency virus. Mucosal CD4(+) T cell loss is an early critical step in AIDS pathogenesis. The preservation of CD4(+) T cells in colonic lamina propria and the reduction of virus in the intestine correlated better with high-avidity mucosal CTL induced by the mucosal AIDS vaccine. This preferential localization of high-avidity CTL may explain previous differences in vaccination results and may guide future vaccination strategy.

CDK2-Specific CTLs Can Be Generated In Vivo from Donor Derived T Cells in HLA-A24+ Patients with Leukemia in Remission after Allogeneic Stem Cell Transplantation.

Aberrantly expressed self-antigens in leukemic cells serve as leukemia-associated-antigens (LAAs) of leukemia reactive T cells. Although such self-antigen-derived LAAs potentially induce high avidity CTLs in patients with leukemia, these CTLs usually do not persist due to apoptosis upon encountering leukemic cells. In allogeneic stem cell transplant (allo-SCT) recipients with leukemia, residual leukemic cell may sensitize donor-derived T cells by LAA in vivo and induce high avidity CTLs specific to leukemic cells. Cyclin-dependent kinase 2 (CDK2) is a cell cycle regulator protein that is aberrantly expressed in AML, MDS, ALL and MCL cells. We previously reported that CDK2-derived nonamer peptides (CDK2158: TYTHEVVTL, CDK2167: WYRQPEILL, CDK2178: KYYSTAVDI) avidly bound to HLA-A24 molecule to elicit each peptide-specific CTL from peripheral blood mononuclear cells (PBMCs) of healthy individuals (Blood. 106 (11): a3103. 2005). When we generated CDK2158-specific CD8+ T cells from an HLA identical sibling donor of a patient with AML by stimulating donor PBMCs with CDK2158-coated HLA-A24-transfected T2, CDK2158-specific CD8+ T cells preferentially killed the recipient AML cells which aberrantly expressed CDK2 proteins. The percentages of specific lysis in the 51Cr-release assay at an E/T ratio of 40 were 36.8% for AML cells and 24.8% for autologous PBMCs. To determine if CDK2-specific CD8+ T cells are present in PBMCs from allo-SCT patients, we studied 14 patients possessing HLA-A24 [6 patients with AML (2 AML-M0, 3 AML-M2, 1 AML with multilinage dysplasia), 1 with MDS, 1 with CML, 2 with ALL, 2 with MCL and 2 with RCC] using CDK2158/A24 and CDK2178/A24 multimers. Cryopreserved PBMCs obtained before and 4–73 months after allo-SCT were assayed for multimer staining. The source of graft was unrelated BM in 4, related BM in 2, related PBMC in 3 and CB in 5 patients. All CB and 1 BM graft were HLA mismatched. Seven patients were in complete remission (CR) at the time of SCT while 7 were in non-CR. Ten patients remained in CR 4–79 months after SCT. Small populations (0.13–0.77 % of lymphocyte) of CDK2158 and CDK2178-specific CTL were detectable in 5 patients (3 with AML-M2, 1 with CML and 1 with ALL) 10–73 months after SCT. All of the 5 remained in CR 11–79 months after SCT. Only one of them had active cGvHD at sampling. On the other hand, among 7 patients who did not show an increase in the proportion of neither CDK2158 nor CDK2178-specific CTLs, 2 patients with AML-M0 relapsed 4 and 5 months after SCT, respectively. Both of them had active cGvHD at sampling. CDK2-specific CTLs were also undetectable in 2 patients with RCC after allo-SCT. These data provide evidence for the first time that CDK2-specific CTLs can be induced from donor-derived T cells without peptide immunization possibly due to in vivo sentitization of donor T cells by residual leukemic cells. Vaccination with CDK2-derived peptides after allo-SCT may therefore be useful in inducing CDK2-specific CTLs and thereby preventing relapse of leukemia. [Display omitted]

Recognition of Fresh Human Tumor by Human Peripheral Blood Lymphocytes Transduced with a Bicistronic Retroviral Vector Encoding a Murine Anti-p53 TCR

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the alpha- and beta-chain of a TCR from a highly avid murine CTL clone that recognized the human p53(264-272) epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.

Induction of Unresponsiveness Limits Tumor Protection by Adoptively Transferred MDM2-Specific Cytotoxic T Lymphocytes

There is evidence showing that high avidity CTLs can be more effective than low avidity CTLs for adoptive tumor immunotherapy. Because many T cell-recognized tumor antigens are nonmutated self-proteins, tolerance mechanisms are likely to render high avidity T cells unresponsive or cause T cell elimination by clonal deletion. We recently used the allo-restricted strategy to circumvent immunologic tolerance to a ubiquitously expressed tumor-associated protein, MDM2, and raised high avidity CTLs in humans and in mice. In this study, we investigated whether high avidity MDM2-specific CTLs can mediate tumor protection without causing damage to normal tissues in mice. Although the CTLs prolonged survival of tumor-bearing mice without causing damage to normal tissues, tumor protection was incomplete. We show that tumor growth occurred despite the continued presence of MDM2-specific CTLs and the continued susceptibility of tumor cells to CTL killing. However, analysis of the CTLs revealed that they had been rendered unresponsive in vivo because they did not produce interferon gamma in response to antigen-specific stimulation. These experiments suggest that induction of unresponsiveness may be an important mechanism limiting the efficacy of adoptive CTL therapy.

Cutting edge: predetermined avidity of human CD8 T cells expanded on calibrated MHC/anti-CD28-coated microspheres.

Cytotoxic CD8 T cells are key effectors in the immunotherapy of malignant and viral diseases. However, the lack of efficient methods for their in vitro priming and expansion has become a bottleneck to the development of vaccines and adoptive transfer strategies. Synthetic artificial APCs (aAPCs) are now emerging as an attractive tool for eliciting and expanding CTL responses. We show that, by controlling the MHC density on aAPCs, high- or low-avidity tumor-directed human CTL lines can be raised effectively in vitro if costimulation via CD28 and IL-12 is provided. Compared with low-avidity CTL lines, high-avidity CTLs need 100- to 1000-fold less peptide for activation, bind more MHC tetramers, and, as expected, are superior in recognizing tumor cell lines expressing Ag. We believe that the possibility to raise Ag-specific T cells with predetermined avidity will be crucial for the future use of aAPCs in immunotherapeutical settings.

Molecular mechanisms and biological significance of CTL avidity.

CD8 CTLs are a major effector for protection against cancer as well as many infectious diseases, including HIV/AIDS. CD8 CTL recognize antigenic peptides in the context of class I MHC. CTL functional avidity has been shown to be an important determinant of in vivo efficacy. CTL that can recognize peptide/MHC only at high antigen density are termed low avidity CTL, while those that can recognize their cognate antigen at low densities are termed high avidity CTL. Recent studies have demonstrated that high avidity CTLs are essential for the effective clearance of viral infections and for the elimination of tumor cells. At this time, approaches that can target high avidity cells for expansion in vivo are not well defined; however, new insights are beginning to emerge. A recent study has shown that prime-boost immunization may be an effective method to generate high avidity CTLs that recognize HIV antigens. In addition, we recently found that high levels of costimulation (signal 2) can skew the CTL response toward higher avidity cells. Thus, vectors expressing a triad of costimulatory molecules (TRICOM) or dendritic cells expressing higher levels of costimulatory molecules, can be used to induce high avidity CTL. Finally a critical role for CD4+ T cell help in the generation of high avidity cells has recently been identified (Palmer, manuscript submitted). While high avidity CTLs are superior for viral and tumor clearance, they also have a greater sensitivity to antigen induced cell death. In some types of chronic infections, such as HIV and HCV, as well as in cancer, the host may lose, by clonal exhaustion or other apoptotic mechanisms, the effector cells that are most critical to viral or tumor clearance. In this review, we examine the current knowledge concerning CTL avidity. We discuss the factors that may distinguish high avidity CTLs from low avidity CTLs and describe some of the mechanisms these cells use to clear viral infections. In addition, we study possible immunization strategies that may be used to elicit higher avidity CTLs and describe what is known about the factors that render these cells more susceptible to apoptosis than low avidity CTLs. Finally, we will incorporate these various elements into a general discussion of possible approaches for induction and maintenance of an effective immune response that can result in clearance of tumors or chronic viral infections and the relevance to vaccine development.

Pregnancy can induce long-persisting primed CTLs specific for inherited paternal HLA antigens

Previous studies showed that pregnancy can prime the maternal cellular immune response directed against paternal HLA antigens. Primed CTLs specific for inherited paternal HLA antigens (IPA) were found in women who had formed HLA allo antibodies, whereas naive CTLs were present in women who did not form antibodies against the paternal HLA antigens. As HLA allo antibodies may disappear in time, it is not clear which women on the waiting list for transplantation have been sensitized to paternal HLA antigens and are at risk for graft rejection if paternal HLA antigens are shared by the donor organ. The presence of primed CTLs specific for a particular antigen is considered to be a reflection of sensitization. In the present study we investigated whether these primed CTLs persist in women who had been pregnant and had formed antibodies against the inherited paternal HLA class I antigens. For this purpose 14 women who had their last pregnancy 10 years ago were analyzed with respect to IPA-specific CTLp frequencies and the presence of high avidity CTLs directed against inherited paternal HLA class I antigens. Although primed CTLs specific for IPA’s were found more frequently in women with persisting alloantibodies, they still can be detected when the antibodies have disappeared. The current data show that primed CTLs directed against inherited paternal HLA antigens towards which antibodies have been formed in the past can persist for more than 10 years after pregnancy. The cellular test used in our study can be useful to detect presensitization in women with a history of pregnancy, who enter the waiting list for transplantation.

High Avidity CTLs for Two Self-Antigens Demonstrate Superior In Vitro and In Vivo Antitumor Efficacy

A majority of the human tumor-associated Ags characterized to date are derived from nonmutated "self"-proteins. Little is currently understood about the nature of the self-reactive lymphocytes that recognize these Ags. We recently characterized two nonmutated tumor-associated Ags for the B16 murine melanoma: tyrosinase-related protein-2 (TRP-2) and the endogenous retroviral envelope protein, p15E. We previously reported that both TRP-2 and p15E reactive CTL could be detected in the spleens of naive animals after a single in vitro stimulation using 10(-5)-10(-6) M of the appropriate Kb-binding 9-amino acid epitope. In this report we show that the CTL found in naive animals are low avidity lymphocytes, that respond only to high concentrations of peptide in vitro. We demonstrate that titration of in vitro-stimulating peptide to limiting concentrations distinguishes qualitative differences in the lymphocyte reactivity to these two Ags between vaccinated and unvaccinated animals. We further demonstrate that in vitro expansion of CTL in either high or low concentrations of stimulating peptide generated CTL cultures with different avidities for the relevant epitopes. CTL expanded in low concentrations demonstrated higher avidity for peptide-pulsed targets and better tumor recognition, when compared to CTL generated in the presence of high concentrations of Ag. More importantly, high avidity CTL demonstrated superior in vivo antitumor activity. These results demonstrate that qualitative differences in the CTL that recognize these two self-Ags are critically important to their in vitro and in vivo anti-tumor efficacy.