Low-avidity Cytotoxic T Lymphocytes Research Articles

Optimisation of anti-cancer peptide vaccines to preferentially elicit high-avidity T cells

Therapeutic cancer vaccines often do not substantially reduce tumour burden, despite stimulating anti-tumour cytotoxic T lymphocytes (CTLs). Recent experiments have shown that the majority of vaccine-elicited CTLs may be of low-avidity. Moreover, low-avidity CTLs, which are abundant, do not kill cancer cells and potentially inhibit the ability of high-avidity T cells to kill cancer cells.By modelling CTL selection using a system of ordinary differential equations, we show that the efficacy of the peptide vaccine may be improved by controlling its delivery and dosage to preferentially elicit high-avidity CTLs. Our simulations predict that weekly, reduced doses of a vaccine may result in a greater than 90% reduction in cancer concentration. By contrast, a standard vaccine protocol such as a high-dose injection given every 2 weeks induces only a 65% reduction. Our model demonstrates a proof-of-concept approach to targeting immune responses for CTL selection, thereby offering a technique to potentially improve existing therapies.

Antigen-Specific Inhibition of High-Avidity T Cell Target Lysis by Low-Avidity T Cells via Trogocytosis

Current vaccine conditions predominantly elicit low-avidity cytotoxic T lymphocytes (CTLs), which are non-tumor-cytolytic but indistinguishable by tetramer staining or enzyme-linked immunospot from high-avidity CTLs. Using CTL clones of high or low avidity for melanoma antigens, we show that low-avidity CTLs can inhibit tumor lysis by high-avidity CTLs in an antigen-specific manner. This phenomenon operates invivo: high-avidity CTLs control tumor growth in animals but not in combination with low-avidity CTLs specific for the same antigen. The mechanism involves stripping of specific peptide-major histocompatibility complexes (pMHCs) via trogocytosis by low-avidity melanoma-specific CTLs without degranulation, leading to insufficient levels of specific pMHC on target cellsurface to trigger lysis by high-avidity CTLs. As such, peptide repertoire on the cell surface is dynamic and continually shaped by interactions with Tcells. These results describe immune regulation by low-avidity Tcells and have implications for vaccine design.

Increased sensitivity to antigen in high avidity CD8+ T cells results from augmented membrane proximal T‐cell receptor signal transduction

The functional avidity of a cytotoxic T lymphocyte (CTL) is known to be a critical determinant of the efficacy with which it clears pathogens. High avidity cells, which are by definition highly sensitive to peptide antigen, are superior for elimination of viruses and tumours. Our studies have established the ability of T cells to undergo avidity modulation as a result of antigen encounter. High and low avidity cells established in this manner exhibit significant differences in the amount of peptide required to elicit effector function. However, how signalling is regulated in these cells as it relates to the control of peptide sensitivity remains to be defined. To address this question, we compared T-cell receptor (TCR) signal transduction events in high and low avidity CTL generated from OT-I(rag2-) TCR transgenic mice. Our data suggest that divergent signalling is initiated at the TCR-associated CD3ζ, with low avidity CTL requiring higher amounts of pMHC to achieve threshold levels of phosphorylated CD3ζ compared with high avidity CTL. Further, this difference is transduced further downstream to mitogen-activated protein kinase and Ca(2+) signalling pathways. These results suggest that regulated control of the initiation of TCR signalling in high versus low avidity cells determines the amount of peptide required for T-cell activation.

In vivo administration of artificial Antigen Presenting Cells licenses low avidity T cells for treatment of cancer (131.13)

Abstract The development of effective anti-tumor immune responses is normally constrained by low avidity, tumor-specific cytotoxic T lymphocytes (CTL) which are unable to eradicate the tumor. Therefore, new strategies to rescue anti-tumor activity of low avidity melanoma-specific CTL in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low avidity CTL we immunized mice bearing metastatic lung melanoma with artificial Antigen Presenting Cells (aAPC), made by covalently coupling pepMHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system and complete tumor eradication in a mouse TRP-2 antigen system, when low avidity CTL specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTL and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release and TCR down-regulation in low avidity CTL. Therefore, in vivo aAPC administration represents a potentially novel approach to improve adoptive immunotherapy of cancer.

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 (≤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.

Identification of a potential human telomerase reverse transcriptase-derived, HLA-A1-restricted cytotoxic T-lymphocyte epitope.

The catalytic subunit of human telomerase reverse transcriptase (hTERT) is expressed in the majority of tumor cells of different histological origins as opposed to most normal somatic cells. This implicates hTERT as a widely expressed tumor-associated antigen and an attractive candidate for antigen-specific tumor immunotherapy. T lymphocytes specific for hTERT-derived epitopes have been isolated and shown reactive with hTERT-expressing tumor cells. To further increase the applicability of hTERT as a target antigen for immunotherapy, we set out to identify potential hTERT-derived, HLA-A1-restricted cytotoxic T-lymphocyte (CTL) epitopes. The "reverse immunology" approach, involving computer-assisted epitope prediction, in vitro CTL induction, and tetramer-guided CTL isolation, resulted in specific CTLs against hTERT-derived, HLA-A1-binding peptides. Intermediate- to low-avidity CTLs were induced against the hTERT325-333 peptide and recognized endogenously processed hTERT. Recognition of endogenous hTERT depended on an increase of hTERT expression above normal levels in tumor cells through hTERT transduction, most probably as a result of limited CTL avidity. The altered peptide ligand hTERT699T-707 was designed to increase HLA-A1-binding affinity of the hTERT699-707 peptide and was used to induce CTLs. However, these CTLs poorly cross-recognized native hTERT699-707 and failed to recognize endogenously processed hTERT. In conclusion, our study has identified the hTERT325-333 peptide as a potential hTERT-derived epitope that may prove useful for induction and monitoring of hTERT-specific, HLA-A1-restricted CTL responses.

Changes to peptide structure, not concentration, contribute to expansion of the lowest avidity cytotoxic T lymphocytes.

The efficient in vitro expansion of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) for use in adoptive immunotherapy represents an important clinical goal. Furthermore, the avidity of expanded CTL populations often correlates closely with clinical outcome. In our study, high-avidity CTL lines could be expanded ex vivo from an antigen-primed animal using low peptide concentration, and intermediate peptide concentrations favored the generation of lower avidity CTL. Further increases in peptide concentration during culture inhibited the expansion of all peptide-specific CD8+ cells. In contrast, a single amino acid variant peptide efficiently generated functional CTL populations at high or low peptide concentration, which responded to wild-type epitope with the lowest average avidity seen in this study. We propose that for some peptides, the efficient generation of low-avidity CTL responses will be favored by stimulation with altered peptide rather than high concentrations of wild-type epitope. In addition, some variant peptides designed to have improved binding to major histocompatibility complex class I may reduce rather than enhance the functional avidity for the wild-type peptide of ex vivo-expanded CTL. These observations are relevant to in vitro expansion of CTL for immunotherapy and strategies to elicit regulatory or therapeutic immunity to neo-self-antigen when central tolerance has eliminated high-avidity, cognate T cells.

The avidity of allospecific cytotoxic T lymphocytes (CTL) determines their cytokine production profile.

Donor-specific CTL present within the cardiac allograft during a rejection episode are distinct from those that populate the cardiac allograft in the absence of rejection. Whereas the former generally have a high avidity for donor cells, the latter mainly have a low avidity for donor cells. This observation made us reason that high-avidity CTL are implicated in transplant rejection, whereas low-avidity CTL are not. In the present study, we analyse whether both CTL subsets were distinct with respect to their IL-2, IL-4, IL-6 and interferon-gamma (IFN-gamma) secretion pattern. CTL clones with either a high or a low avidity for donor antigens were stimulated with donor cells, third party cells, or immobilized anti-CD3 MoAb and the amount of cytokine released was measured. High- and low-avidity CTL clones were found to differ with respect to their IFN-gamma production profile. Stimulation with donor cells resulted in IFN-gamma secretion by high-avidity CTL clones, but not by low-avidity CTL clones. CD3 stimulation, in contrast, led to secretion of equivalent amounts of IFN-gamma by both CTL subsets. These observations indicate that low-avidity CTL are fully capable of producing IFN-gamma, but, in contrast to high avidity CTL, fail to do so when they encounter donor cells. As IFN-gamma favours the occurrence of transplant rejection, this observation emphasizes the relevance of high-avidity CTL in the rejection process. Additionally, the data show that the cytokine production profile of CTL depends on the nature of the stimulus.

Molecular and functional bases of self-antigen recognition in long-term persistent melanocyte-specific CD8+ T cells in one vitiligo patient.

Vitiligo patients possess high frequencies of circulating CD8+ T lymphocytes specific for the melanocyte differentiation antigen Melan-A/MART-1. These self-specific T cells exhibit intact functional properties and their T cell receptors are selected for a narrow range of high affinities of antigen recognition, suggesting their important role in the pathogenesis of vitiligo. In order to understand the molecular base for this unexpected, optimal T cell receptor recognition of a self-antigen, a tetramer-guided ex vivo analysis of the T cell receptor repertoire specific for the Melan-A antigen in a patient affected by vitiligo is reported. All T cell receptors sequenced corresponded to different clonotypes, excluding extensive clonal expansions and revealing a large repertoire of circulating Melan-A-specific T lymphocytes. A certain degree of T cell receptor structural conservation was noticed, however, as a single AV segment contributed to the alpha chain rearrangement in 100% of clones and a conserved amino acid sequence was found in the beta chain complementarity determining region 3 of various high affinity cells. We suggest that the conserved alpha chain confers self-antigen recognition, necessary for intrathymic selection and peripheral homeostasis, to many synonymous T cell receptors, whereas the beta chain fine tunes the T cell receptor affinity of the specific cells. In addition, we demonstrate that many high avidity T cell clones from this patient were capable of specifically lysing normal, HLA-matched melanocytes. These autoreactive clones persisted for more than 3 y in the patient's peripheral blood. These data, together with the skin-homing potential of the clones, directly point to the in vivo pathogenic role of melanocyte-specific cytotoxic T lymphocytes in vitiligo.

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

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

Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer.

We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1-restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1-restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon expression of wild-type and partially humanized high-affinity T cell antigen receptor (TCR) genes derived from the transgenic mice. These results demonstrate that TCR gene transfer can be used to circumvent self-tolerance of autologous T lymphocytes to universal tumor antigens and thus provide the basis for a TCR gene transfer-based broad-spectrum immunotherapy of malignant disease.

C1.7 monoclonal antibody designates high-avidity CD4+ cytotoxic T lymphocytes involved in clinical heart rejection.

It is assumed that not all donor-specific cytotoxic T lymphocytes (CTLs), but only those with a high avidity for donor antigens, can function as terminal effector cells in transplant rejection. In the present study, we searched for markers that would exclusively designate these high-avidity CTL. FACS analysis of donor-specific CTL clones obtained from heart transplant patients revealed that high- and low-avidity CTL varied in their expression of p38, a surface molecule involved in signal transduction, which is stained by the antibody C1.7. High- and low-avidity CD8+ CTL and high-avidity CD4+ CTL expressed p38, whereas low-avidity CD4+ CTL did not. Noncytotoxic and naive CD4+ lymphocytes also lacked p38 surface expression. Therefore, we conclude that p38 is a marker for CD4+ lymphocytes with the potency to damage the transplanted heart. Accordingly, p38 might be used to analyze the contribution of CD4+ CTL in immune responses, such as transplant rejection.

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

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