Low Avidity CTL Research Articles

Increased PRAME antigen-specific killing of malignant cell lines by low avidity CTL clones, following treatment with 5-Aza-2'-Deoxycytidine.

The cancer testis antigen Preferentially Expressed Antigen of Melanoma (PRAME) is overexpressed in many solid tumours and haematological malignancies whilst showing minimal expression in normal tissues and is therefore a promising target for immunotherapy. HLA-A0201-restricted peptide epitopes from PRAME have previously been identified as potential immunogens to drive antigen-specific autologous CTL responses, capable of lysing PRAME expressing tumour cells. CTL lines, from 13 normal donors and 10 melanoma patients, all of whom were HLA-A0201 positive, were generated against the PRAME peptide epitope PRA(100-108). Specific killing activity against PRA(100-108) peptide-pulsed targets was weak compared with CTL lines directed against known immunodominant peptides. Moreover, limiting dilution cloning from selected PRAME-specific CTL lines resulted in the generation of a clone of only low to intermediate avidity. Addition of the demethylating agent 5-aza-2'-Deoxycytidine (DAC) increased PRAME expression in 7 out of 11 malignant cell lines including several B lineage leukaemia lines and also increased class I expression. Pre-treatment of target cells was associated with increased sensitivity to antigen-specific killing by the low avidity CTL. When CTL, as well as of the target cells, were treated, the antigen-specific killing was further augmented. Interestingly, one HLA-A0201-negative DAC-treated line (RAJI) showed increased sensitivity to killing by clones despite a failure of expression of PRAME or HLA-A0201. Together these data point to a general increased augmentation of cancer immunogenocity by DAC involving both antigen-specific and non-specific mechanisms.

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

The shared tumor associated antigen cyclin‐A2 is recognized by high‐avidity T‐cells

Cyclin-A2, a key cell cycle regulator, has been shown to be overexpressed in various types of malignancies with little expression in normal tissue. Such tumor-associated genes potentially are useful targets for cancer immunotherapy. However, high-avidity cyclin-specific T cells are considered to be thymically deleted. We identified at least one nonameric HLA-A*0201 binding cyclin-A2 epitope by a reverse immunology approach. Using a highly efficient T-cell expansion system that is based on CD40-activated B (CD40-B) cells as sole antigen-presenting cells we successfully generated cyclin-A2 specific CTL from HLA-A*0201(+) donors. Interestingly, high-avidity cyclin-A2 specific CTL lines, which recognized peptide-pulsed and antigen expressing target cells, were indeed generated by stimulation with CD40-B cells when pulsed with low concentrations of peptide, whereas CD40-B cells pulsed at saturating concentrations could only induce low-avidity CTL, which recognized peptide-pulsed target cells only. One high-avidity CTL line was subcloned and CTL clones, whose peptide concentration required for half-maximal lysis were less than 1 nM, could lyse cyclin-A2 expressing tumor cells. Taken together, cyclin A2 is an attractive candidate for immune intervention in a significant number of cancer patients and high-avidity T cells can be readily generated using CD40-B cells as antigen-presenting cells.

Role of 3 domain of class I MHC molecules in the activation of high- and low-avidity CD8+ CTLs

CD8 can serve as a co-receptor or accessory molecule on the surface of CTL. As a co-receptor, CD8 can bind to the alpha3 domain of the same MHC class I molecules as the TCR to facilitate TCR signaling. To evaluate the role of the MHC class I molecule alpha3 domain in the activation of CD8(+) CTL, we have produced a soluble 227 mutant of H-2D(d), with a point mutation in the alpha3 domain (Glu227 --> Lys). 227 mutant class I-peptide complexes were not able to effectively activate H-2D(d)-restricted CD8 T cells in vitro, as measured by IFN-gamma production by an epitope-specific CD8(+) CTL line. However, the 227 mutant class I-peptide complexes in the presence of another MHC class I molecule (H-2K(b)) (that cannot present the peptide) with a normal alpha3 domain can induce the activation of CD8(+) CTL. Therefore, in order to activate CD8(+) CTL, the alpha3 domain of MHC class I does not have to be located on the same molecule with the alpha1 and alpha2 domains of MHC class I. A low-avidity CD8(+) CTL line was significantly less sensitive to stimulation by the 227 mutant class I-peptide complexes in the presence of the H-2K(b) molecule. Thus, low-avidity CTL may not be able to take advantage of the interaction between CD8 and the alpha3 domain of non-presenting class I MHC molecules, perhaps because of a shorter dwell time for the TCR-MHC interaction.

CD8α/α homodimers fail to function as co‐receptor for a CD8‐dependent TCR

In this study, we have started to dissect the molecular basis of CD8 dependence of a high and low avidity CTL clone specific for the same peptide epitope. Using anti-CD8alpha and anti-CD8beta antibodies, we found that cytotoxicity and IFN-gamma production by high but not by low avidity CTL was strongly CD8 dependent. We isolated the TCR genes of both types of CTL clones and used retroviral gene transfer to analyse the function of these TCR in primary T cells of wild-type and CD8beta-deficient mice. Both TCR triggered antigen-specific killing in wild-type T cells, and blocking experiments showed that CD8 dependence/independence co-transferred with the TCR into primary T cells, indicating that it was dictated by the TCR itself. Gene transfer experiments into CD8beta-deficient T cells revealed that only the TCR derived from the CD8-independent CTL clone elicited antigen-specific cytotoxicity, while the CD8-dependent TCR was non-functional in the absence of the CD8beta-chain. These data indicate a striking difference between CD8alpha/beta heterodimers and CD8alpha/alpha homodimers as only the former were able to provide co-receptor function for the CD8-dependent TCR.

CD8+ T Cells Possess the Potential to Differentiate into Both a High or Low Avidity Effector Cell (87.7)

Abstract During an immune response, CTL clones are present that exhibit a wide spectrum of peptide sensitivities. While the property of functional avidity has been well documented, the range of mechanism(s) by which this T cell property can be regulated is unknown. For example, an unresolved question is whether distinct naïve T cell subsets exist that display “inherent” differences in their peptide requirements for activation, i.e. functional avidity. In this scenario distinct naïve T cell clones would selectively respond upon encountering a high vs. low antigen concentration. Alternatively, differences in peptide sensitivity could be an “induced” property within the responding CTL population following encounter with an APC. Although the latter model is consistent with data from previous studies, formal demonstration that a single clone can give rise to both high and low avidity daughter cells is lacking. Using a TCR transgenic model in which both high and low avidity CTL can be generated, we now demonstrate that naïve T cells do not exhibit differences in the amount of peptide required for conjugate formation and subsequent proliferation/activation. Furthermore, we show that individual T cell clones are capable of differentiating into either a high or low avidity CTL. These results provide the first formal demonstration that an individual cell can give rise to both high and low avidity progeny, suggesting that avidity modulation at the level of an individual cell may be an important contributor to the functional avidity of the CD8+ T cell response generated in vivo. This work was supported by National Institutes of Health grant R01 AI43591 to M.A.A-M.

High Avidity Cyclin E1-Derived Peptide-Specific CTL Kill Lymphoid Leukemia Cells and Cross-Recognize a Homologous Cyclin E2-Derived Peptide.

Using a similar strategy that successfully identified PR1 as a leukemia-associated antigen (LAA), we identified two homologous HLA-A2-restricted peptides from cyclin E1 (CCNE1) and cyclin E2 (CCNE2) that could be used to elicit peptide-specific CTL from healthy donors in vitro. Two homologous nonameric peptides from CCNE1 (CCNE1144–152) and CCNE2 (CCNE2144–152), which differ by a single amino acid at position 7, have equal binding affinity for HLA-A2 and each elicited peptide-specific CTL with equal efficiency, as measured by specific lysis of T2 cells pulsed with either peptide (CCNE1 59.7% vs CCNE2 72.6% specific lysis, respectively, at E: T 10:1). TCR-Vβ spectratype analysis showed CCNE1-CTL clones to be derived from 3 Vβ families, while CCNE2-CTL clones were derived from a single Vβ family. The CCNE1-CTL and the CCNE2-CTL bound to each of the CCNE1/A2 and CCNE2/A2 tetramers, but staining intensity was greater for the CCNE1-CTL, suggesting greater TCR avidity of the CCNE1-CTL for both peptides. Because each clone cross-recognized the other homologous peptide, we hypothesized that each clone would efficiently kill leukemia that over-expressed either or both CCNE1 and CCNE2 proteins. FACsorted high avidity CTL showed higher specific lysis of peptide-pulsed T2 than did low avidity CTL (38.8% vs 31.9% specific lysis, respectively, at E: T 10:1, p = 0.02). The fluorescence decay of tetramer dissociation (ln (peptide/HLA-A2 tetramer)) over time was linear for each clone, suggesting that avidity was proportional to TCR affinity and tetramer dissociation t1/2 was determined based on first order kinetics. CCNE1-CTL had higher affinity for CCNE1144–152/HLA-A2 (CCNE1/A2, t1/2=84.5min; CCNE2/A2, t1/2=25.3min) and preferentially killed CCNE1144–152-pulsed T2 cells (CCNE1, 56.9% vs CCNE2, 38%, respectively, at E: T 10:1, p = 0.004). Interestingly, CCNE2-CTL also had higher TCR affinity for CCNE1144–152/HLA-A2 (CCNE1/A2, t1/2=29.5min; CCNE2/A2, t1/2=10.7min), but showed only slightly higher specific lysis of CCNE1144–152-pulsed T2 cells (CCNE1 = 49.3% vs CCNE2 = 44.2% specific lysis, respectively, at E: T 10:1, p = 0.33). Each clone specifically lysed HLA-A2+ T-ALL leukemia cells in proportion to both CCNE1 and CCNE2 protein overexpression assessed by Western blot (CCNE1-CTL, R2=0.89; CCNE2-CTL, R2=0.88). In contrast, healthy HLA-A2+ BM cells, which do not overexpress CCNE1 or CCNE2, and control HLA-A2− CML cells that overexpress both proteins, were not lysed. Both the high and low affinity clones showed equal lysis of T-ALL cells that expressed large amounts of each protein (specific lysis = 24.3% by CCNE1-CTL, vs lysis = 23.8% by CCNE2-CTL, at E: T 10:1). However, high affinity CCNE1-CTL killed T-ALL cells significantly better than low affinity CCNE2-CTL (16.8% vs 6.6% lysis, respectively, at E: T 10:1, p =0.02) when the T-ALL expressed a 2.5-fold lower amount of both CCNE1 and CCNE2 proteins. We conclude that the CCNE1 and CCNE2 homologous self-peptides are lymphoid leukemia-associated antigens. Furthermore, while the higher TCR affinity of CCNE1-CTL suggests that the CCNE1 peptide is the more dominant epitope, ultimate target susceptibility is enhanced due to degeneracy of the resulting CTL clones against homologous peptide epitopes.

Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways.

Several factors may influence sensitivity of melanoma cells to CTL lysis. One is the avidity of the CTL TCR. A second is that certain cytotoxic drugs have been reported to sensitize cancer cells to CTL lysis through Fas-mediated apoptosis. In this study, we examined whether antineoplastic agents 5-fluorouracil (5-FU) and dacarbazine (DTIC) sensitize melanoma cells to lysis of G209 peptide-specific CTL. Our results show that CTL generated from PBMC are HLA-A2 restricted and gp100 specific. Treatment with 5-FU or DTIC sensitized melanoma cells to lysis of G209-specific CTL. Most importantly, 5-FU- or DTIC-treated melanoma cells also became sensitive to low-avidity CTL, which per se are less cytolytic to melanomas. We sought to identify apoptotic pathways mediating this effect. The enhanced cytolysis was mediated through the perforin/granzyme pathway. Although 5-FU up-regulated FasR expression on melanoma cells, sensitization was not blocked by anti-Fas Ab, and the G209-specific CTL was Fas ligand (FasL) negative. However, when G209-specific CTL were stimulated to express FasL, FasL signaling also contributed to enhanced cytolysis. DTIC treatment, which did not increase FasR expression, also sensitized FasL-mediated killing induced by neutralizing anti-Fas Ab. For CD95L-positive G209-specific CTL, the sensitization was primarily mediated through the perforin/granzyme pathway regardless of up-regulation of FasR. The findings demonstrate that cytotoxic drug-mediated sensitization primes both perforin/granzyme and Fas-mediated killing by melanoma-specific CTL. Considering that most of autoreactive antitumor CTL are low avidity, the findings provide experimental basis for understanding cytotoxic and immunologic therapeutic synergy in melanoma.

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.

Lessons learned from HIV treatment interruption: safety, correlates of immune control, and drug sparing.

Therapeutic approaches seeking to limit the exposure to antiretroviral drugs while retaining the benefits of continuous therapy have become an active area of investigation in HIV therapy research. Although early attempts to use interruptions of therapy as auto-vaccination strategies have shown little success, much has been discovered in regards to immunological correlates of viral control in acute and chronic infection, viral evolution, and the safety of single or multiple therapy interruptions in different patient sub-groups (acutely infected, chronically infected, and multi-drug resistant). Here we review safety data and candidate factors that may contribute to the striking differences observed between patients that undergo similar treatment interruption strategies but achieve different outcomes in controlling HIV replication. Differences between acute and chronic infection in the viral component (e.g. diversity of the viral pool) and the host immune system (e.g. low avidity CTL memory response), which may not be reversed by ART, may determine the potential for suppressive immune response upon therapy interruption. Consistent with goals of limiting toxicity and cost of antiretroviral drug regimens, safety outcomes to date indicate that intermittent therapy strategies may safely continue to be investigated in early and chronically infected patients. Based on ongoing research, we identify the topics to be targeted in future studies.

Antimelanoma activity of CTL generated from peripheral blood mononuclear cells after stimulation with autologous dendritic cells pulsed with melanoma gp100 peptide G209-2M is correlated to TCR avidity.

Anchor residue-modified peptides derived from tumor-associated Ag have demonstrated success in engendering immune responses in clinical studies. However, tumor regression does not always correlate with immune responses. One hypothesis to explain this is that CTL resulting from such immunization approaches are variable in antitumor potency. In the present study, we evaluated this hypothesis by characterizing the activity of tumor-associated Ag-specific CTL. We chose an anchor residue-modified peptide from gp100, G209-2M, and used peptide-pulsed dendritic cells to generate CTL from PBMC of HLA-A2(+) normal donors. The specificities and avidities of the resulting CTL were evaluated. The results demonstrate that CTL generated by G209-2M can be classified into three categories: G209-2M-specific CTL which are cytotoxic only to G209-2M-pulsed targets; peptide-specific CTL which recognize both G209 and G209-2M peptides but not melanomas; and melanoma-reactive CTL which recognize peptide-pulsed targets as well as HLA-A2(+)gp100(+) melanomas. CTL that kill only peptide-pulsed targets require a higher peptide concentration to mediate target lysis, whereas CTL that lyse melanomas need a lower peptide concentration. Increasing peptide density on melanomas by loading exogenous G209 peptide enhances their sensitivity to peptide-specific CTL. High avidity CTL clones also demonstrate potent antimelanoma activity in melanoma model in nude mice. Injection of G209 peptide around transplanted tumors significantly enhances the antitumor activity of low avidity CTL. These results suggest that peptide stimulation causes expansion of T cell populations with a range of avidities. Successful immunotherapy may require selective expansion of the higher-avidity CTL and intratumor injection of the peptide may enhance the effect of peptide vaccines.

High-Avidity CTL Exploit Two Complementary Mechanisms to Provide Better Protection Against Viral Infection Than Low-Avidity CTL

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

Differential Expansion and Survival of High and Low Avidity Cytotoxic T Cell Populations during the Immune Response to a Viral Infection

The importance of high avidity CTL for the effective clearance of viral infections is now well established. Thus one would predict that the preferential activation and expansion of high avidity CTL following viral challenge and retention of these cells in the memory pool would be optimal for the immune response. However, whether this actually occurs during the immune response to viral infection is unknown. In this report I have analyzed the avidity of the CTL specific for the OVA257–264 peptide during acute infection with a recombinant vaccinia expressing ovalbumin and in the memory population. I have found that the relative ratio of high and low avidity CTL varies over the course of an immune response. Thus CTL avidity is an important factor in the expansion and survival of CTL in vivo.

Activation of Low Avidity CTL Specific for a Self Epitope Results in Tumor Rejection But Not Autoimmunity

To determine how self-tolerance can alter the ability of the immune system to respond against tumor-associated Ags that are also expressed by normal tissue, we designed experiments in which the same protein was expressed both as a tumor Ag and as a transgene product. Unlike conventional BALB/c mice that rejected renal carcinoma cells transfected with the influenza virus hemagglutinin (Renca-HA), transgenic mice that are tolerant of HA due to its expression as a self-Ag on pancreatic islet beta cells, (Ins-HA mice) supported progressive growth of these tumor cells. However, when Ins-HA mice were immunized with a recombinant strain of vaccinia virus expressing the dominant H-2Kd peptide epitope of HA before receiving Renca-HA cells, they too were able to reject the tumor cells. Rejection of Renca-HA cells by immunized Ins-HA mice was found to be associated with the generation of CTL having much lower avidity for target cells presenting the KdHA epitope than CTL from immunized conventional BALB/c mice. Significantly, we show that self-tolerance to the HA Ag is quantitative rather then absolute, and that vaccination of Ins-HA mice can activate low avidity KdHA-specific CD8+ T cells that are able to reject tumor cells expressing high levels of HA, yet these mice remain tolerant of pancreatic islet beta cells expressing HA.