Knutson, KL

Abstract Background: MHC class II molecules are normally restricted to professional antigen-presenting cells. Prior TNBC studies have focused on immune-compartment MHC-II. Using spatial transcriptomics, we recently identified aberrant tumor-cell MHC-II expression. Here, we characterized the immune architecture surrounding HLA-DRA-expressing tumor cells. Methods: High-plex single-cell spatial transcriptomics (CosMxTM SMI) was performed on treatment-naïve TNBC (Mayo TMA, n=65) and two neoadjuvant pembrolizumab cohorts (Mayo n=8; Emory n=4). Spatial neighborhoods were mapped relative to HLA-DRA-high tumor cells. Differential expression and adaptive immune gene-set scores were evaluated. Clinical relevance was assessed using FinXX (n=114), I-SPY2 (n=364), and Caris CODEai (n=3,662). Results: HLA-DRA-high tumor regions showed significant enrichment of adaptive immune subsets within ≤50 μm, including B cells, CD8+ T cells, NK cells, macrophages, and plasmacytoid dendritic cells. In pembrolizumab-treated cohorts, responders exhibited higher proportions of HLA-DRA-high tumor cells and greater numbers of B cells, plasmablasts, CD4+ and CD8+ T cells, and macrophages near tumor cells. Responders’ tumors showed upregulation of antigen-presentation machinery, B-cell/plasma-cell programs, CXCL13, and NOTCH3/DLL1. Across I-SPY2 and FinXX, higher HLA-DRA correlated with pathologic complete response and improved survival. In CODEai TNBC, high HLA-DRA expression correlated with higher CXCL13 (median TPM 7.5 vs. 1.1, q<0.05) and longer overall survival (24.2 vs. 18.5 months, HR 0.77, 95% CI 0.71-0.83, p<0.0001). This association was TNBC-specific. High CXCL13 also predicted improved survival (26.6 vs. 16.5 months, HR 0.64, 95% CI 0.59-0.69, p<0.0001). Conclusions: Tumor-cell HLA-DRA expression defines a highly organized adaptive immune niche enriched with B cells and activated T-cell populations in close proximity. Adaptive immune programs—including enhanced antigen presentation and CXCL13-mediated B-cell/plasma-cell pathways—consistently associate with improved outcomes across TNBC cohorts treated with chemotherapy and immune checkpoint blockade. Aberrant tumor-cell MHC-II expression may contribute to antitumor immunity and warrants further investigation as a potential therapeutic target. Citation Format: Yi Liu, Sachin Kumar Deshmukh, Thiti Susiriwatananont, Panuch Eiamprapaporn, Bogang Wu, Heikki Joensuu, Roberto A. Leon-Ferre, David Zahrieh, Judy C. Boughey, James Newell Ingle, Fergus J. Couch, Sharon Wu, Shipra Gandhi, Maryam Lustberg, George W. Sledge, Matthew P. Goetz, Keith L. Knutson, E Aubrey Thompson, Jodi Carter, Saranya Chumsri. Spatially resolved tumor-cell MHC class II shapes adaptive immunity and therapeutic response in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1200.

Background While SARS-CoV-2 vaccines are highly effective in healthy individuals, the magnitude and durability of humoral and cellular responses in patients with solid malignancies receiving chemotherapy remain understudied. Previous reports suggest that patients with cancer may not mount adequate immune response after SARS-CoV-2 vaccination. Additionally, most studies focused on humoral responses, while data regarding cellular immune responses are scarce. In this study, we evaluated humoral and cellular responses in patients with solid tumors receiving chemotherapy compared to healthy individuals up to one year after vaccinations. Methods Patients aged ≥18 who were willing to receive the SARS-CoV-2 vaccine were enrolled. Anti-SARS-CoV-2 immunoassays were used to detect antibodies against nucleocapsid and spike proteins. Human IFN-γ Fluorospot assay was used to determine antigen−specific T-cell responses. Data were compared between groups using Mann−Whitney test for continuous variables and Fisher’s exact test for categorical variables. Results A total of 67 subjects (47 patients with cancer and 20 healthy individuals) were included. 1–3 months following the second vaccine doses, 96% of patients with cancer and 100% of healthy individuals demonstrated a positive humoral response. While the positivity rate was not significantly different, patients with cancer had significantly lower spike IgG antibodies than healthy individuals. However, this difference diminished at 6 months when patients with cancer had increased antibodies compared to decreased antibodies in the healthy cohort and no difference was noticed at 12-month. Patients with cancer developed a similar antigen-specific T-cell response as healthy individuals at 1–3 months, 6 months and 12 months. There were no significant differences when comparing patients aged ≤ 55 years vs. >55 years, stages I-III vs. IV, single vs. multiple chemotherapy, and BNT162b2 vs. mRNA-1273 vaccines. There was a significant moderate correlation between neutralization and antibody levels at 12 months. However, despite patients with cancer having a significantly higher COVID risk score, there were no significant differences in COVID-19 infection and hospitalization between patients with cancer and healthy individuals. Conclusion Despite initial impaired antibody responses to SARS-CoV-2 vaccinations, patients with solid malignancies receiving chemotherapy effectively generated long-term cellular and humoral responses by 6 and 12 months, leading to similar infection and hospitalization rates compared to healthy individuals.

20043 Background: Studies have demonstrated that the generation of endogenous immunity to specific tumor antigens is associated with improved prognosis for many cancers. Thus, there has been increased efforts to identify tumor-associated antigens to which there is immunity, as these may be tumor rejection antigens. A candidate antigen is the folate receptor alpha (FRα) which is overexpressed in malignancies, especially breast and ovarian cancer. Prior studies involving ovarian and breast cancer patients have demonstrated the presence of FRα-specific cytotoxic T cells in the tumor-associated lymphocyte population. Methods: Using the RANKPEP CD4 T cell epitope prediction algorithm, we predicted promiscuous immunogenic regions of FRα, and tested for immunity in 30 breast or ovarian cancer patients and 18 healthy volunteer donors in order to attain a better understanding of the levels and extent of the endogenous FRα immune response. Immunity was examined using IFN-γ ELIspot analysis and IgG ELISAs. Results: Fourteen peptides were predicted as potential epitopes to which T cells may respond, seven each from the carboxy- and amino-terminus halves of the protein. Greater than 70% of patients demonstrated T cell immunity to at least one of the fourteen peptides. Patients responded to an average of 3 ± 0.7 peptides while healthy donors responded to only 1 ± 0.5 peptides (p = 0.02). Five peptides were recognized by >25% of patients. Responses to three of these peptides were higher (p > 0.05) in the patients than in healthy donors, suggesting that patients generated immunity upon cancer development. Compared to healthy individuals, patients developed higher immunity to the amino-terminus half of the receptor, suggesting natural epitope spreading (p = 0.03). There was no difference between patients and healthy donors in the immune responses to either non-specific stimuli (p = 0.2) or viral peptides (p = 0.5). Patients had detectable FRα-specific IgG antibody consistent with active FRα-specific helper T cell immunity. Conclusions: These findings demonstrate that the FRα is a target of the immune system in patients with breast or ovarian cancer. Understanding the antigens that are naturally targeted by the immune system may be important for diagnosis, prognosis, and immune-based therapies. No significant financial relationships to disclose.

Abstract While it remains unclear whether the immune system naturally evolved to prevent cancer, it is clear that many of its constituent cells and factors can greatly influence the clinical course of the disease in many different cancers, including breast cancer. Generally speaking, the immune system is a double edged sword and produces a broad array of both pro-tumor and anti-tumor factors. Of those which demonstrate anti-tumor activity, antigen-specific T cells (including both cytotoxic and helper T cells) are the most important as several studies have now shown that increased T cell infiltration is generally, and markedly, protective. More recent evidence also suggests that B cells can also be protective, particularly when producing tumor-specific antibodies or associated with intratumoral tertiary lymphoid structures. Thus, newer therapeutics being developed in academia and industry are being designed to directly augment adaptive immunity. The methods to manipulate the numbers of lymphocytes is growing and includes direct clonal expansion approaches such as vaccination and adoptive T cell therapy as well as more indirect methods that modulate cell intrinsic regulatory mechanisms (e.g. checkpoint and co-stimulatory pathways). In contrast, the pro-tumor activity of the immune system is largely mediated by the myeloid compartment of the immune system. While normal myeloid cells are not generally pro-tumorigenic, most tumors impair proper myeloid differentiation inducing intratumoral and systemic accumulation of defective myeloid cells which have profound tumor-promoting activity. This tumor-promoting activity of myeloid cells is mediated either through direct tumor growth-promoting activity (e.g. growth and angiogenic factors) or through blockade of adaptive immune response. Mechanisms by myeloid cells promote tumor growth appear to be diversified and are now only recently becoming understood, but nonetheless represent unique targets for therapeutic manipulation either alone in combination with other therapeutic modalities (e.g. checkpoint blockade). The basic underpinnings of tumor immunology are being rapidly identified and applied clinically to treat a variety of cancers and more recently breast cancer. This educational lecture will be a general overview of tumor immunology focusing on recent basic and therapeutic advances, highlighting those relevant to the field of breast cancer. Citation Format: Knutson KL. Basic science of immunotherapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr ES4-1.

Abstract Background: Recent studies have indicated that vaccination can protect against cancer development. One key aspect of developing vaccines is circumventing peripheral tolerance by identifying subdominant epitopes that are unique to the deregulated tumor microenvironment. While existing subdominant epitope vaccines are showing efficacy in preventing cancer, these vaccines are applicable only for subsets of patients with specific HLA subtypes. Therefore, we recently developed a degenerate HER2 subdominant epitope-based vaccine that should be useful in approximately 85% of all patients. The vaccine consists of a pool of four HLA-DR-restricted 15-amino acid epitopes (p59, p88, p422, and p885) that are naturally processed and are designed to elicit helper T cell immunity, the cornerstone of immune surveillance. Here we present Phase I trial results of this multi-peptide HER2 vaccine. Methods: Eligible women had HER2+ breast cancer (Stages II-III) and had completed standard treatment (i.e. surgery, chemotherapy, and trastuzumab) at least 90 days prior to enrollment and were rendered disease free. Vaccine included the above epitope pool along with adjuvant GM-CSF. Patients were vaccinated six times over six months and were monitored for toxicity at each visit. Peripheral blood samples were collected for immune responses evaluating for T cell and antibody immunity. Endpoints were safety and immunogenicity leading to the development CD4 helper T cells that recognized naturally-processed HER2. Results: Twenty-two subjects (age 33 to 69 years) were enrolled. At the present analysis, 21 have completed all 6 vaccination cycles; one patient withdrew after developing a grade 1 injection site reaction during the first vaccination cycle. Twenty patients have had LVEF measured after vaccination; only 2 patients had an LVEF drop of 10% or more but remained in the normal LEVF range. One severe toxicity was reported: a grade 3 INR increase considered unrelated to treatment. Mild to moderate (grade 1-2) toxicities included injection site reactions, fatigue, and white blood cell count decreases. All patients were alive at analysis and only one experienced a recurrence (median follow-up 507 days, range 22 – 844). Twenty patients have had immune response assessments. Vaccine induced T cell immunity was observed in 94% of patients to p59, in 94% of patients to p88, in 82% of patients to p422, and in 74% of patients to p885. Importantly, T cell immunity to naturally processed HER2 proteins occurred in 94% of patients. The mean number of T cells specific for each peptide, post vaccination, ranged from 349–528 T cells per million peripheral blood mononuclear cells (PBMCs). The mean number of T cells specific for whole HER2 protein was 783 T cells per million PBMCs compared to a mean of 898 T cells/million PBMCs specific for the foreign tetanus toxin. In contrast to T cell responses, modestly increased antibody immunity to HER2 occurred in 35% of patients, consistent with the T cell-inducing design of the vaccine. Conclusion: Our results show that it is possible to develop vaccines with broad HLA coverage that circumvent natural tolerance and induce tumor antigen-specific immunity in the vast majority of patients. Citation Format: Knutson KL, Kalli KR, Block MS, Hobday TJ, Padley DJ, Erskine CL, Dockter T, Suman VJ, Wilson G, Degnim AC. Robust generation of T cell immunity to HER2 in HER2+ breast cancer patients with a degenerate subdominant HLA-DR epitope vaccine. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-11-02.

Abstract PURPOSE: Hedgehog (HH) pathway has been implicated in maintenace and survival of breast cancer and cancer stem cells, EMT regulation, breast carcinogenesis and resistance to chemoradiation in solid tumors. Dysregulation of this pathway occurs in more than 50% of breast cancers. We have already demonstrated that immune-induced epithelial to mesenchymal transition (EMT) leads to the generation of breast cancer stem cells (BCSCs) from a parental epithelial breast cancer cell line in an in vivo murine model (Cancer Research 2009). The aim of our study is to demonstrate that resistance in the BCSCs is linked to HH hyperactivation and that the selective blockade of this pathway could eradicate this subpopulation of BCSCs in preclinical models. METHODS: One parental epithelial (E) and three mesenchymal BCSCs cell lines (M1, M2 and M3) were used for all the in vitro experiments. qRT-PCR was used to quantify the expression of the gene components of the pathway and gene targets. Measurement of Gli1, caspase 3 and Cyclin D1 protein expression was determined by Western blot. Cell viability assays were performed with Paclitaxel and the HH inhibitor GDC-0449. Apoptosis assays were performed when cell lines were treated with paclitaxel (10nM), GDC-0449 (25uM) or the combination of them. For the in vivo xenografted mouse model, 106 cells (E and M2) were inoculated sc respectively into the flank of 6–8 week-old syngeneic female FVB/N-TgN (MMTVneu) 202Mul/J mice. Treatment was started on daily GDC-0449 (ip), paclitaxel (ip on days 1, 4 and 8) or both drugs when tumors reached 0.5 cm2. Statistical analysis was performed using one-way ANOVA with the Bonferroni multiple comparison test. RESULTS: Activity of the HH measured by the expression of Gli1 was increased in BCSC cell lines as compared to E cell line (p < 0.001). This was antagonized as expected by treatment with GDC-0449. Reporter and expression assay showed that GDC-0449 decreases Gli1 transcriptional activity and levels of a HH downstream target gene, cyclin D1. In all BCSC lines (but not in the E cell line), a synergistic effect was showed with the combination treatment as compared to control (p ≤ 0.01) and to paclitaxel or GDC-0449 alone (p ≤ 0.05 respectively) as well as a highly increased apoptotic activity (p ≤ 0.001 with control and p ≤ 0.05 with monotherapy). Tumors derived from M2 injection regressed when the combination schedule was administered (p ≤ 0.05) at 2 weeks after the start of the treatment. CONCLUSION: The inhibition of the HH pathway with GDC-0449 plus paclitaxel demonstrates a synergistic therapeutic effect in comparison to monotherapy, as shown by increased apoptotic activity, cell cycle arrest and significant reduction of tumor size in vivo. Together these results provide the rationale for future clinical trials including the blockade HH and standard chemotherapy in order to eradicate the whole population of tumor cells (also BCSCs) within the tumors and avoid disease relapse and metastasis in breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-07.