Variant peptide vaccines are used clinically to induce T cell responses against tumor antigens (4
). However, in many patients these vaccines are ineffective at inducing clinical tumor regression or T cell responses with high TAA recognition efficiency (4
). Although the amount of TAA expressed by similar tumors in different patients varies (49
), this factor has been given little attention, as it relates to variant peptide vaccinations. Previous studies have not determined if the amount of TAA expression affects the quality of the TAA-crossreactive response induced by variant peptide vaccinations. We examined this issue by taking advantage of an increase in expression of the TAA GP70 that occurs with age in BALB/c mice. Young mice demonstrate T cell tolerance to GP70423–431
(AH1). However, GP70 expression increases with age, and AH1-specific T cell responses are further diminished in aging mice (19
). Vaccination of young mice with either of two peptide variants of AH1, A5 or 39, has previously been shown to elicit robust AH1-crossreactive T cell responses and AH1-specific tumor protection (5
). In this study, we determined if the increase in GP70 expression that occurs with age alters the AH1-crossreactive responses elicited by these two peptide variants.
Interestingly, the responses induced by variants A5 or 39 were affected differently by increased AH1-specific tolerance. Although immunization with variant A5 induced a robust response of A5-specific and AH1-crossreactive T cells in mice with undetectable splenic GP70 expression, they are absent in nearly all aging mice that have elevated splenic GP70 ( and ). Conversely, immunization with variant 39 induced a response containing 39-specific and AH1-crossreactive T cells in all aging mice regardless of the presence of detectable GP70 expression ( and data not shown). However, the AH1-crossreactive T cells elicited by variant 39 immunization of aging mice do not respond to stimulation with the AH1 epitope ().
The extent and quality of AH1 cross-reactivity in the T cell populations induced by each of these variants suggests a mechanism by which they might be affected differently by AH1-specific tolerance. First, nearly all of the variant A5-elicited T cells bind AH1-tet (), whereas only half of the variant 39-elicited T cells bind AH1-tet (). The 39-specific cells that do not cross-react with the AH1 peptide should not be susceptible to tolerance induced by the increased expression of GP70. Further, the AH1-tet+ T cells expanded by variant A5 in young mice respond significantly better to AH1 stimulation than the AH1-tet+ population expanded by variant 39 (). Thus, responses with greater AH1-reactivity, as detected by AH1-tet staining and AH1 stimulation, are more susceptible to the induction of tolerance by AH1 presentation.
These data suggest two reasons that T cell responses to variant 39 are maintained in AH1-tolerant mice. First, part of the 39-elicited response does not cross-react with the AH1 antigen ( and ). The lack of AH1-recognition by these T cells suggests precursors of these cells would be ignorant to AH1-specific tolerance. Second, a portion of the 39-elicited population recognizes AH1-tet, but does not respond to stimulation with AH1 peptide (). These cells do, however, respond to stimulation with 39 peptide (). The short half-life of AH1-tet binding with TCR suggests that insufficient TCR recognition may be responsible for poor AH1 functional recognition (33
). Tetramer binding, but failure to respond to peptide stimulation, has been observed in T cells previously (54
). Thus, it seems unlikely that precursors of either of these 39-elicited T cell populations, both lacking functional recognition of AH1 (), would be susceptible to AH1-specific tolerance. We propose that in aging GP70hi
mice, T cells with no functional recognition of AH1 are the only cells available to respond to variant 39 vaccination, as the 39-reactive cells with functional AH1 recognition have been tolerized by anergy or deletion. The absence of functionally AH1-reactive cells in the response of GP70hi
mice, present in the responses of both young BALB/c and aging gp70
-deficient mice (), demonstrates that variant-induced responses to TAA are increasingly suppressed by escalating TAA expression. The loss of A5-induced responses in GP70hi
mice also supports this conclusion (, ).
To determine why immunization with variant A5 does not elicit cognate responses in GP70hi
mice, we assessed the precursor frequency of T cells that bind A5-tet in naïve GP70hi
mice (). Similar numbers of A5-tet+
T cells were found in GP70hi
, young BALB/c and aging gp70−/−
mice, suggesting that in GP70hi
mice these cells must be unresponsive to variant A5 immunization. Analysis of PD-1 and IL-7Rα surface expression suggests that some of the A5-tet+
T cells in GP70hi
may be anergic, exhausted or undergoing deletion ()(3
). However, not all of the A5-tet+
cells in GP70hi
mice display a PD-1+
phenotype. Immunoregulatory cells may suppress the response of these PD-1−
cells. Others have shown that depletion or inhibition of Treg prior to immunization with the native AH1 antigen induces long-lasting and tumor-protective AH1-specific T cell responses, unlike immunization without Treg deletion or inhibition (57
). Thus, Treg may suppress AH1-specific cells. Another group showed that Treg depletion enhanced the functional avidity of a TAA-specific response, suggesting that T cells with greater avidity for tolerizing antigen may be preferentially suppressed by Treg (60
). Perhaps the increased GP70 expression in GP70hi
mice makes A5-specific cells more susceptible to Treg-mediated suppression than those same cells in mice with less GP70 expression. Alternatively, the increased number and frequency of Treg in aging mice (61
) may result in the enhanced suppression of AH1-specific T cells in aging mice. Further studies are needed to determine the mechanism and extent of suppression induced by Treg and anergy in this model.
Although variant peptide immunizations often induce robust responses from TAA-crossreactive T cells, the functional avidity of these cells for the TAA may be relatively low (35
). Data presented here suggest that as endogenous TAA expression increases, these variant-elicited responses may become further biased towards a T cell repertoire with poor functional recognition of the TAA. This bias may result from the peripheral suppression of precursor T cells with functional TAA-recognition. In the GP70 TAA model, Treg-mediated suppression has previously been demonstrated in young mice with low TAA expression (57
). The data presented here suggest that in mice with higher GP70 expression, anergy or deletion may also play a role in suppressing high-avidity T cells. We propose that T cells with poor TAA-recognition escape peripheral tolerance due to this poor recognition and remain available for variant peptide vaccine-elicited expansion. However, these data also suggest that precursor T cells with high functional avidity for the TAA may remain in individuals with high TAA expression. The presence of these T cells suggests that treatments that block suppressive mechanisms, such as PD-1 (63
) and Treg (60
), may allow their expansion during vaccination. Thus, variant peptide vaccination in conjunction with one or more of these treatments may induce the proliferation of T cells with high TAA-specific avidity in patients bearing normal or transformed tissues with high TAA expression.