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Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy.

Supplemental Figure 1. Sequence of pIRS-21097-1105 TCR chains. Supplemental Figure 2. Sequence of pCDC25b38-46 TCR chains. Supplemental Figure 3. Electroporation of IVT RNA encoding murine TCR chains results in functional cell surface expression of phosphopeptide-specific TCR. Supplemental Figure 4. pSer1100-IRS-2 staining of colon melanoma metastasis

Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy.

Lymphatic and blood vessels are formed by specialized lymphatic endothelial cells (LEC) and blood endothelial cells (BEC), respectively. These endothelial populations not only form peripheral tissue vessels, but also critical supporting structures in secondary lymphoid organs, particularly the lymph node (LN). Lymph node LEC (LN-LEC) also have been shown to have important immunological functions that are not observed in LEC from tissue lymphatics. LN-LEC can maintain peripheral tolerance through direct presentation of self-antigen via MHC-I, leading to CD8 T cell deletion; and through transfer of self-antigen to dendritic cells for presentation via MHC-II, resulting in CD4 T cell anergy. LN-LEC also can capture and archive foreign antigens, transferring them to dendritic cells for maintenance of memory CD8 T cells. The molecular basis for these functional elaborations in LN-LEC remain largely unexplored, and it is also unclear whether blood endothelial cells in LN (LN-BEC) might express similar enhanced immunologic functionality. Here, we used RNA-Seq to compare the transcriptomic profiles of freshly isolated murine LEC and BEC from LN with one another and with freshly isolated LEC from the periphery (diaphragm). We show that LN-LEC, LN-BEC, and diaphragm LEC (D-LEC) are transcriptionally distinct from one another, demonstrating both lineage and tissue-specific functional specializations. Surprisingly, tissue microenvironment differences in gene expression profiles were more numerous than those determined by endothelial cell lineage specification. In this regard, both LN-localized endothelial cell populations show a variety of functional elaborations that suggest how they may function as antigen presenting cells, and also point to as yet unexplored roles in both positive and negative regulation of innate and adaptive immune responses. The present work has defined in depth gene expression differences that point to functional specializations of endothelial cell populations in different anatomical locations, but especially the LN. Beyond the analyses provided here, these data are a resource for future work to uncover mechanisms of endothelial cell functionality.

BackgroundPhosphorylated peptides presented by MHC molecules represent a new class of neoantigens expressed on cancer cells and recognized by CD8 T-cells. These peptides are promising targets for cancer immunotherapy. Previous work identified an HLA-A*0201-restricted phosphopeptide from insulin receptor substrate 2 (pIRS2) as one such target. The purpose of this study was to characterize a second phosphopeptide, from breast cancer antiestrogen resistance 3 (BCAR3), and to evaluate safety and immunogenicity of a novel immunotherapic vaccine comprising either or both of these phosphorylated peptides.MethodsPhosphorylated BCAR3 protein was evaluated in melanoma and breast cancer cell lines by Western blot, and recognition by T-cells specific for HLA-A*0201-restricted phosphorylated BCAR3 peptide (pBCAR3126-134) was determined by51Cr release assay and intracellular cytokine staining. Human tumor explants were also evaluated by mass spectrometry for presentation of pIRS2 and pBCAR3 peptides. For the clinical trial, participants with resected stage IIA–IV melanoma were vaccinated 6 times over 12 weeks with one or both peptides in incomplete Freund’s adjuvant and Hiltonol (poly-ICLC). Adverse events (AEs) were coded based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V.4.03, with provision for early study termination if dose-limiting toxicity (DLT) rates exceeded 33%. The enrollment target was 12 participants evaluable for immune response to each peptide. T-cell responses were assessed by interferon-γ ELISpot assay.ResultspBCAR3 peptides were immunogenic in vivo in mice, and in vitro in normal human donors, and T-cells specific for pBCAR3126-134controlled outgrowth of a tumor xenograft. The pIRS21097-1105peptide was identified by mass spectrometry from human hepatocellular carcinoma tumors. In the clinical trial, 15 participants were enrolled. All had grade 1 or 2 treatment-related AEs, but there were no grade 3–4 AEs, DLTs or deaths on study. T-cell responses were induced to the pIRS21097-1105peptide in 5/12 patients (42%, 90% CI 18% to 68%) and to the pBCAR3126-134peptide in 2/12 patients (17%, 90% CI 3% to 44%).ConclusionThis study supports the safety and immunogenicity of vaccines containing the cancer-associated phosphopeptides pBCAR3126-134and pIRS21097-1105, and the data support continued development of immune therapy targeting phosphopeptides. Future studies will define ways to further enhance the magnitude and durability of phosphopeptide-specific immune responses.Trial registration numberNCT01846143

Dysregulated protein phosphorylation plays a major role in cancer cell malignancy. We showed that phosphopeptides are presented by MHC-I molecules, representing a new class of cancer neoantigens. We found that healthy donors with no evident prior cancer had robust CD8+ T cell responses to a cohort of HLA-B7+ leukemia-associated phosphopeptides. Here we report that CD8+CD45RO+ T cells purified from blood of different healthy donors showed responses to 2–25% of HLA-A2+ and 0–23% of HLA-B7+ cancer-associated phosphopeptides, confirming that individuals with no evident cancer history have pre-existing phosphopeptide specific immune memory. Six of 59 HLA-A2+ phosphopeptides were “immunodominant,” with memory responses evident in the majority of analyzed donors. This suggests that these phosphopeptides result from processes common in cellular transformation or viral infection. In contrast, no immunodominant HLA-B7+ phosphopeptides were identified among the 168 analyzed, suggesting that mechanisms driving their expression in healthy donors are less common. Robust day 14 responses were observed against a small cohort of HLA-B7+ phosphopeptides in 2 donors. Responses to some of these phosphopeptides could be detected ex vivo and were evident over 3 years, suggesting they reflected chronic antigen exposures. In contrast, responses to 2 phosphopeptides evident ex vivo diminished or disappeared over 14 days in culture, suggesting a recent acute immune response composed of terminal effectors unable to persist long term. These results provide evidence of ongoing immune surveillance in healthy individuals to phosphopeptides known to be expressed on cancers.

Identifying appropriate antigens is essential to developing effective cancer vaccines. Protein phosphorylation plays a major role in signal transduction pathways that are dysregulated and contribute to the malignant phenotype of cancer cells. Our lab has shown that phosphopeptides derived from these proteins can be presented by major histocompatibility complex Class-I molecules and represent a new class of cancer-associated neoantigens. We previously reported that healthy donors without evident prior cancer had unusually robust CD8 T-cell responses to a cohort of HLA-B7+ leukemia-associated phosphopeptides, suggesting that they might be due to pre-existing phosphopeptide specific memory T cells. To test this, we evaluated CD8+CD45RO+ memory T cells purified from the blood of healthy donors. These cells showed robust responses to 2-25% of HLA-A2+ and 0-23% of HLA-B7+ cancer-associated phosphopeptides. This demonstrates that healthy individuals with no evident cancer history have pre-existing immune memory to some cancer-associated phosphopeptides. We identified 3 “immunodominant” HLA-A2 associated phosphopeptides, in that memory responses were evident to them in the majority of analyzed donors. Such immunodominance suggests that these phosphopeptides result from signaling processes that occur commonly in transformation- or viral infection-related processes. In contrast, immunodominant phosphopeptides were not evident in the context of HLA-B7. While 2 donors showed pre-existing immune memory to a large number of shared targets, other donors showed memory to only small numbers of nonoverlapping phosphopeptides. This suggests that the mechanisms driving expression of these phosphopeptide antigens among healthy donors are much less common. We also found that responses to some phosphopeptides could be detected directly ex vivo. In some cases, the phosphopeptide targets of these ex vivo responses did not stimulate responses after 2 weeks in culture. These results suggest that some phosphopeptides in some donors may be the targets of ongoing immune responses composed of effector or effector memory cells that do not persist long-term in culture. These results provide evidence of regular, ongoing immune surveillance in healthy individuals to cancer-associated phosphopeptides. Citation Format: Amanda M. Lulu, Kara L. Cummings, Victor H. Engelhard. Pre-existing immune memory to cancer-associated phosphopeptides in healthy donors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B68.

CD4+ T cell responses are impaired in chronic HCV infection. To determine factor(s) involved in CD4+ T cell dysregulation, we examined the effect of extracellular core on the alteration of CD4+ T cell responses and the cell surface level of core-binding protein, gC1qR on CD4+ T cells from acute HCV patients with resolved and chronic infection. During the acute phase of infection, the frequency of gC1qR+CD4+ T cells increased in both resolved and chronic HCV infection compared to healthy controls. Notably, 6 months later, the frequency of gC1qR+CD4+ T cells maintained elevated in chronic patients compared to that in resolved patients. In addition, TCR stimulation increased the frequency of gC1qR+CD4+ T cells, resulting in core-induced inhibition of T cell responses in both resolved and chronic patients. These results suggest that HCV infection expands gC1qR+CD4+ T cells, which increase the susceptibility to core-mediated immune dysregulation and facilitate the establishment of HCV persistency.

Complement is activated during the early phase of viral infection and promotes destruction of virus particles as well as the initiation of inflammatory responses. Recently, complement and complement receptors have been reported to play an important role in the regulation of innate as well as adaptive immune responses during infection. The regulation of host immune responses by complement involves modulation of dendritic cell activity in addition to direct effects on T-cell function. Intriguingly, many viruses encode homologs of complement regulatory molecules or proteins that interact with complement receptors on antigen-presenting cells and lymphocytes. The evolution of viral mechanisms to alter complement function may augment pathogen persistence and limit immune-mediated tissue destruction. These observations suggest that complement may play an important role in both innate and adaptive immune responses to infection as well as virus-mediated modulation of host immunity.

Immune control of many intracellular pathogens, including Trypanosoma cruzi , is reported to be dependent on the production of nitric oxide. In this study, we show that mice deficient in inducible nitric oxide synthase (iNOS or NOS2) exhibit resistance to T. cruzi infection that is comparable to that of wild-type mice. This is the case for two iNOS-deficient mouse strains, Nos2 tm1Lau and Nos2 N5, infected with the Brazil or Tulahuen strain of T. cruzi . In all cases, blood parasitemia, tissue parasite load, and survival rates are similar between wild-type and iNOS-deficient mice. In contrast, both wild-type and Nos2 tm1Lau mice died within 32 days postinfection when treated with the nitric oxide synthase inhibitor aminoguanidine. Increased transcription of NOS1 or NOS3 is not found in iNOS-knockout (KO) mice, indicating that the absence of nitric oxide production through iNOS is not compensated for by increased production of other NOS isoforms. However, Nos2 tm1Lau mice exhibit enhanced expression of tumor necrosis factor alpha, interleukin-1, and macrophage inflammatory protein 1α compared to that of wild-type mice, and these alterations may in part compensate for the lack of iNOS. These results clearly show that iNOS is not required for control of T. cruzi infection in mice.

Cancer cells display novel phosphopeptides in association with MHC class I and II molecules. In this study, we evaluated two HLA-A2–restricted phosphopeptides derived from the insulin receptor substrate (IRS)-2 and the cell-cycle regulator CDC25b. These proteins are both broadly expressed in multiple malignancies and linked to cancer cell survival. Two phosphopeptides, termed pIRS-21097–1105 and pCDC25b38–46, served as targets of strong and specific CD8 T-cell memory responses in normal human donors. We cloned T-cell receptor (TCR) cDNAs from murine CD8 T-cell lines specific for either pIRS-21097–1105 or pCDC25b38–46. Expression of these TCRs in human CD8 T cells imparted high-avidity phosphopeptide-specific recognition and cytotoxic and cytokine-secreting effector activities. Using these cells, we found that endogenously processed pIRS-21097–1105 was presented on HLA-A2+ melanomas and breast, ovarian, and colorectal carcinomas. Presentation was correlated with the level of the Ser1100-phosphorylated IRS-2 protein in metastatic melanoma tissues. The highest expression of this protein was evident on dividing malignant cells. Presentation of endogenously processed pCDC25b38–46 was narrower, but still evident on HLA-A2+ melanoma, breast carcinoma, and lymphoblastoid cells. Notably, pIRS-21097–1105–specific and pCDC25b38–46–specific TCR-expressing human CD8 T cells markedly slowed tumor outgrowth in vivo. Our results define two new antigens that may be developed as immunotherapeutic agents for a broad range of HLA-A2+ cancers. Cancer Res; 74(23); 6784–95. ©2014 AACR.

Deregulation of signaling pathways is a hallmark of malignant transformation. Signaling-associated phosphoproteins can be degraded to generate cancer-specific phosphopeptides that are presented by major histocompatibility complex (MHC) class I and II molecules and recognized by T cells; however, the contribution of these phosphoprotein-specific T cells to immune surveillance is unclear. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8(+) T cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and human leukocyte antigen-matched primary leukemia cells ex vivo. Notably, healthy individuals showed robust CD8(+) T cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients. This reduction correlated with clinical outcome; however, immunity was restored after allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T cell adoptive transfer immunotherapies.

A real-time PCR technique that allows for accurate and sensitive quantitation of tissue parasite burden in animals infected with the protozoan parasite Trypanosoma cruzi was developed. The utility of this method was demonstrated by confirmation of higher parasite load in mice with acute infections in comparison to chronically infected mice, detection of tissue-restricted parasite persistence in different parasite:host strain combinations, and the observation of increased tissue parasite burden with higher infective doses. This method should be a useful tool for monitoring parasite burden in hosts under various treatment regimens.

Identification of the antigens (Ags) recognized by CD8+ T cells specific for melanoma and other types of cancer cells has been motivated by the expectation that they will serve as the basis for a therapeutic vaccine or will be useful for the stimulation of more effective CD8+ CTL for adoptive immunotherapy. Few of the currently identified Ags are derived from source proteins directly associated with transformation or metastasis, and many are self-Ags which may be subject to tolerance mechanisms. We have defined a new array of phosphorylated peptides presented by HLA-A1, -A2, -A3, -B7, -B44 and HLA-B27 that are presented on melanomas from multiple patients at >1 copy per cell and derived from source proteins associated with tumorigenesis and metastasis. We evaluated the immunogenicity of several of the HLA-A2-restricted phosphopeptide Ags in an HLA-transgenic murine model system and in vitro human T cell cultures, evaluated the presence of the phosphorylated source protein or display of the phosphorylated epitope on cancers of different histological types, and investigated the ability of phosphopeptide-specific CD8+ T cells to prevent outgrowth of cancer cells in a xenogenic model. Evaluation of metastatic melanoma tissue microarrays and cancer cell lysates demonstrated that the phosphorylated source proteins for β-catenin (Ser33-phosphorylated), Breast cancer anti-estrogen resistance −3 (BCAR3; Thr130-phosphorylated), and Insulin receptor substrate-2 (IRS2; Ser1100-phosphorylated) were broadly expressed in melanomas and other cancers but not normal tissues. In addition, these epitopes can be utilized to elicit phosphopeptide-specific T cells in both murine HLA-A2 transgenic mice in vivo and human in vitro culture. The phospho-β-catenin, phospho-IRS2 and phospho-BCAR3-specific-T cells that were generated differentiate between the phosphorylated and non-modified forms of their respective peptides and recognize the endogenously processed and presented phosphopeptide on the surface of cancer cells. Finally, adoptive transfer of these phosphopeptide-specific T cells slowed outgrowth of melanoma tumors implanted in immunodeficient NOD.SCID.IL2Rα-/- mice. Thus, we have defined a new set of post-translationally modified epitopes derived from source proteins associated with tumorigenesis and metastasis that are immunogenic and capable of slowing tumor outgrowth in a xenograft model. Based on these results, we are currently planning a clinical trial utilizing these HLA-A2-restricted phosphopeptides in melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1584. doi:1538-7445.AM2012-1584