Due to advances in sequencing technology, somatically mutated cancer antigens, or neoantigens, are now readily identifiable and have become compelling targets for immunotherapy. In particular, ...neoantigen-targeted vaccines have shown promise in several pre-clinical and clinical studies. However, to date, neoantigen-targeted vaccine studies have involved tumors with exceptionally high mutation burdens. It remains unclear whether neoantigen-targeted vaccines will be broadly applicable to cancers with intermediate to low mutation burdens, such as ovarian cancer. To address this, we assessed whether a derivative of the murine ovarian tumor model ID8 could be targeted with neoantigen vaccines. We performed whole exome and transcriptome sequencing on ID8-G7 cells. We identified 92 somatic mutations, 39 of which were transcribed, missense mutations. For the 17 top predicted MHC class I binding mutations, we immunized mice subcutaneously with synthetic long peptide vaccines encoding the relevant mutation. Seven of 17 vaccines induced robust mutation-specific CD4 and/or CD8 T cell responses. However, none of the vaccines prolonged survival of tumor-bearing mice in either the prophylactic or therapeutic setting. Moreover, none of the neoantigen-specific T cell lines recognized ID8-G7 tumor cells in vitro, indicating that the corresponding mutations did not give rise to bonafide MHC-presented epitopes. Additionally, bioinformatic analysis of The Cancer Genome Atlas data revealed that only 12% (26/220) of HGSC cases had a ≥90% likelihood of harboring at least one authentic, naturally processed and presented neoantigen versus 51% (80/158) of lung cancers. Our findings highlight the limitations of applying neoantigen-targeted vaccines to tumor types with intermediate/low mutation burdens.
Cancers accumulate mutations over time, each of which brings the potential for recognition by the immune system. We evaluated T-cell recognition of the tumor mutanome in patients with ovarian cancer ...undergoing standard treatment.
Tumor-associated T cells from 3 patients with ovarian cancer were assessed by ELISPOT for recognition of nonsynonymous mutations identified by whole exome sequencing of autologous tumor. The relative levels of mutations and responding T cells were monitored in serial tumor samples collected at primary surgery and first and second recurrence.
The vast majority of mutations (78/79) were not recognized by tumor-associated T cells; however, a highly specific CD8(+) T-cell response to the mutation hydroxysteroid dehydrogenase-like protein 1 (HSDL1)(L25V) was detected in one patient. In the primary tumor, the HSDL1(L25V) mutation had low prevalence and expression, and a corresponding T-cell response was undetectable. At first recurrence, there was a striking increase in the abundance of the mutation and corresponding MHC class I epitope, and this was accompanied by the emergence of the HSDL1(L25V)-specific CD8(+) T-cell response. At second recurrence, the HSDL1(L25V) mutation and epitope continued to be expressed; however, the corresponding T-cell response was no longer detectable.
The immune system can respond to the evolving ovarian cancer genome. However, the T-cell response detected here was rare, was transient, and ultimately failed to prevent disease progression. These findings reveal the limitations of spontaneous tumor immunity in the setting of standard treatments and suggest a high degree of ignorance of tumor mutations that could potentially be reversed by immunotherapy.
Mutated cancer antigens, or neoantigens, represent compelling immunological targets and appear to underlie the success of several forms of immunotherapy. While there are anecdotal reports of ...neoantigen-specific T cells being present in the peripheral blood and/or tumors of cancer patients, effective adoptive cell therapy (ACT) against neoantigens will require reliable methods to isolate and expand rare, neoantigen-specific T cells from clinically available biospecimens, ideally prior to clinical relapse. Here, we addressed this need using "mini-lines", large libraries of parallel T cell cultures, each originating from only 2,000 T cells. Using small quantities of peripheral blood from multiple time points in an ovarian cancer patient, we screened over 3.3 × 10
6
CD8
+
T cells by ELISPOT for recognition of peptides corresponding to the full complement of somatic mutations (n = 37) from the patient's tumor. We identified ten T cell lines which collectively recognized peptides encoding five distinct mutations. Six of the ten T cell lines recognized a previously described neoantigen from this patient (HSDL1
L25V
), whereas the remaining four lines recognized peptides corresponding to four other mutations. Only the HSDL1
L25V
-specific T cell lines recognized autologous tumor. HSDL1
L25V
-specific T cells comprised at least three distinct clonotypes and could be identified and expanded from peripheral blood 3-9 months prior to the first tumor recurrence. These T cells became undetectable at later time points, underscoring the dynamic nature of the response. Thus, neoantigen-specific T cells can be expanded from small volumes of blood during tumor remission, making pre-emptive ACT a plausible clinical strategy.
Highlights ► HPV infection is associated with cervical and a number of other cancers. ► CD8 T cells specific to HPV E7 can cause tumors to regress in preclinical models. ► Pentarix is a therapeutic ...vaccine directed against 5 strains of HPV. ► Pentarix elicits strong CD8 immunity against HPV.
Abstract The development of vaccines that elicit robust CD8+ T cell immunity has long been a subject of intense investigation. Although whole exogenous protein has not historically been considered as ...useful for eliciting CD8+ T cell immunity, we report herein that whole, protein antigen is capable of eliciting profound levels of CD8+ T cell immunity if it is administered via repeated, daily subcutaneous immunization in combination with the TLR3 agonist poly(I:C). Mice immunized for four consecutive days with 100 μg of either whole exogenous OVA or whole HPV16 E7 protein combined with 10 μg of poly(I:C) mounted remarkable antigen-specific CD8+ T cell responses as measured by tetramer staining and ELISPOT analysis of splenocytes and peripheral blood, with up to 30% of peripheral CD8+ T cells being antigen specific within 7–8 days of vaccination. CD8+ T cell immunity elicited using this vaccination approach was critically dependent upon cross presentation, as either whole protein or long synthetic peptides were highly effective immunogens whereas minimal peptide epitopes were not. Vaccine-induced CD8+ T cells were also able to regress large, established tumors in vivo . Together these data suggest that ‘cluster’ vaccination with exogenous antigen combined with TLR3 agonist may constitute a profoundly important advancement in therapeutic vaccine design.
Oncogenic "driver" mutations are theoretically attractive targets for the immunotherapy of lymphoid cancers, yet the proportion that can be recognized by T cells remains poorly defined. To address ...this issue without any confounding effects of the patient's immune system, we assessed T cells from 19 healthy donors for recognition of three common driver mutations in lymphoma: MYD88
L265P
, EZH2
Y641F
, and EZH2
Y641N
. Donors collectively expressed the 10 most prevalent HLA class I alleles, including HLA-A*02:01. Peripheral blood T cells were primed with peptide-loaded dendritic cells (DC), and reactive T cells were assessed for recognition of naturally processed mutant versus wild type full-length proteins. After screening three driver mutations across 17-26 HLA class I alleles and 3 × 10
6
−3 × 10
7
T cells per donor, we identified CD4
+
T cells against EFISENCGEII from EZH2
Y641N
(presented by HLA-DRB1*13:02) and CD8
+
T cells against RPIPIKYKA from MYD88
L265P
(presented by HLA-B*07:02). We failed to detect RPIPIKYKA-specific T cells in seven other HLA-B*07:02-positive donors, including two lymphoma patients. Thus, healthy donors harbor T cells specific for common driver mutations in lymphoma. However, such responses appear to be rare due to the combined limitations of antigen processing, HLA restriction, and T cell repertoire size, highlighting the need for highly individualized approaches for selecting targets.
Immunohistochemical detection of increased levels of protein-associated nitrotyrosine has become widely used as a surrogate marker of in situ inflammation. However, the potential consequences of ...protein-associated nitrotyrosine formation in terms of cellular immune recognition has received surprisingly little attention. Using a well-defined I-E(K)-restricted epitope of pigeon cytochrome c, we previously demonstrated that conversion of a single tyrosine residue to nitrotyrosine can have a profound effect on recognition by CD4 T cells. In this study, we used the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein (gp33) to demonstrate that conversion of tyrosine to nitrotyrosine can also profoundly affect recognition of MHC class I-restricted epitopes. Conversion of the Y4 residue of the gp33 epitope to nitrotyrosine completely abrogated recognition by gp33-specific T cells from P14 TCR-transgenic mice. In contrast, CD8(+) T cells specific for "nitrated gp33" (NY-gp33) can be readily elicited in C57BL/6 mice after immunization with NY-gp33 peptide. Interestingly, T-T hybridomas specific for NY-gp33 peptide were found to fall into two distinct subsets, being specific for NY-gp33 presented in the context of either H-2D(b) or H-2K(b). This latter result is surprising in light of previous structural studies showing that Y4 comprises a critical TCR-contact residue when presented by H-2D(b) but that the same residue points downward into the peptide-binding groove of the MHC when presented by H-2K(b). Together, these results indicate that nitrotyrosine formation can impact T cell recognition both directly, through alteration of TCR-contact residues, or indirectly, through alterations in MHC-contact positions.
Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated ...a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.
Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.
The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.
Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody ...responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens.
We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-gamma ELISPOT and MHC class I pentamer staining.
We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy.
Abstract Introduction Tumor-infiltrating CD8+ T cells are strongly associated with survival in high-grade serous ovarian cancer, but their functional phenotype remains poorly defined. The mucosal ...integrin CD103 (αE /β7 ) facilitates the infiltration of T cells into epithelial tissues, including gut and lung mucosa, solid organ allografts, and various epithelial cancers. We reasoned that CD103 might also be expressed by tumor-reactive T cells in ovarian cancer. Methods Flow cytometry was used to assess the frequency and phenotype of CD103-expressing T cells in primary ascites fluid from 13 patients with high-grade serous ovarian cancer and 2 patients with recurrent disease. Results We report that a subset of patients with advanced serous ovarian cancer have profoundly elevated frequencies of CD103-expressing CD8+ cells in ascites (between 20% and 70% of CD8+ cells in ascites were CD103+ ) and that CD103 expression correlated with levels of TGF-β in ascitic fluid. Conversely, CD103 was not expressed on CD4+ cells, even in those patients with very high frequencies of CD8+ CD103+ cells. CD8+ CD103+ cells were antigen-experienced (CD45RA− CD45RO+ CD62Llo CCR7− ) and of an intermediate (EM2) effector memory phenotype (CD27+ CD28− ). TCR repertoire analysis indicated significant skewing between CD8+ CD103− and CD8+ CD103+ T cell subsets, suggesting the two populations contain distinct antigenic specificities. Lastly, HLA pentamer analysis revealed that one patient in the cohort harbored a high frequency of CD8+ T cells in ascites that were specific for the tumor antigen NY-ESO-1, and that ∼ 75% of these NY-ESO-1 specific CD8+ T cells were CD103+. Conclusions CD103+ may be a marker of activated and tumor-reactive CD8+ T cells in high-grade serous ovarian cancer.
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