Tumor Infiltrating Lymphocytes Target HLA-I Phosphopeptides Derived From Cancer Signaling in Colorectal Cancer.

There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients' tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients' tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.

A cross sectional assessment of nutrient intake and the association of the inflammatory properties of nutrients and foods with symptom severity in a large cohort from the UK Multiple Sclerosis Registry.

To assess the intake of nutrients in people with multiple sclerosis (pwMS) compared to a control population, and to assess the pro/ anti-inflammatory properties of nutrients/ foods and their relationships with fatigue and quality of life. This was a cross sectional study in which 2410 pwMS (686 men; 1721 women, 3 n/a, mean age 53 (11 years)) provided dietary data using a food frequency questionnaire that was hosted on the MS Register for a period of 3 months and this was compared to a cohort of 24,852 controls (11,250 male, 13,602 female, mean age 59 years). Consent was implied by anonymously filling out the questionnaire. A Wilcoxon test was used to compare intake between pwMS and controls, and a bivariate analyses followed by chi2 test were undertaken to identify significance and the strength of the relationship between pro/anti-inflammatory dietary factors and fatigue and EQ-5D. Compared to controls, all nutrients were significantly lower in the MS group (P < .05). Bivariate associations showed a significant correlation between consuming fish and lower clinical fatigue (χ2(1) = 4.221, P< .05), with a very low association (φ (phi) = -0.051, P = .04. Positive health outcomes on the EQ-5D measures were associated with higher carotene, magnesium oily fish and fruits and vegetable and sodium consumption (P < .05). Fiber, red meat, and saturated fat (women only) consumption was associated with worse outcomes on the EQ-5D measures (P < .05). pwMS have different dietary intakes compared to controls, and this may be associated with worse symptoms.

Isolation of Major Histocompatibility Complex (MHC)-Associated Peptides by Immunoaffinity Purification.

MHC molecules on the surface of cells are responsible for the presentation of antigenic peptides to CD8+ and CD4+ T cells. Downstream analysis of these peptides can offer insight into various disease processes with an immune component, as in autoimmune diseases and cancer. A critical step in studying MHC-associated peptides is their isolation from MHC molecules on the surface of cells. In this chapter, we detail anti-MHC antibody conjugation to beads, immunoaffinity purification of the MHC molecules, and peptide elution. This method can be used for analysis of unmodified and/or posttranslationally modified peptides by mass spectrometry.

Aberrant X chromosome skewing and acquired clonal hematopoiesis in adult-onset common variable immunodeficiency.

Advances in genomic medicine have elucidated an increasing number of genetic etiologies for patients with common variable immunodeficiency (CVID). However, there is heterogeneity in clinical and immunophenotypic presentations and a limited understanding of the underlying pathophysiology of many cases. The primary defects in CVID may extend beyond the adaptive immune system, and the combined defect in both the myeloid and lymphoid compartments suggests the mechanism may involve bone marrow output and earlier progenitors. Using the methylation profile of the human androgen receptor (AR) gene as a surrogate epigenetic marker for bone marrow clonality, we examined the hematopoietic compartments of patients with CVID. Our data show that clonal hematopoiesis is common among patients with adult-onset CVID who do not have associated noninfectious complications. Nonblood tissues did not show a skewed AR methylation status, supporting a model of an acquired clonal hematopoietic event. Attenuation of memory B cell differentiation into long-lived plasma cells (CD20-CD27+CD38+CD138+) was associated with marked changes in the postdifferentiation methylation profile, demonstrating the functional consequence of clonal hematopoiesis on humoral immunity in these patients. This study sheds light on a potential etiology of a subset of patients with CVID, and the findings suggest that it is a stage of an acquired lymphocyte maturation disorder.

A Clinical Review of Surgical vs Transcatheter Aortic Valve Replacement in Geriatric Patients.

OBJECTIVE: To provide an up-to-date review of the available evidence regarding management of elderly patients after transcatheter aortic valve replacement (TAVR). DATA SOURCES: A PubMed search of articles published (September 1969-December 2016) was done using a combination of the following words: aortic valve stenosis, geriatric, elderly, transcatheter aortic valve replacement, surgical aortic valve replacement, transcatheter aortic valve implantation (TAVI), and dual antiplatelet therapy. STUDY SELECTION/DATA EXTRACTION: Relevant original research, review articles, and guidelines were assessed for the management of elderly patients after TAVR. References from the above literature were also evaluated. Articles were selected for inclusion based on relevance to the topic, detailed methods, and complete results. DATA SYNTHESIS: Aortic valve stenosis is common in the geriatric population. While patients were historically treated with surgical aortic valve replacement (AVR), more patients are now undergoing TAVR. This article reviews the current literature regarding outcomes and pharmacotherapy between surgical and TAVR in the elderly population. CONCLUSION: Appropriate management of pharmacotherapy after surgical or TAVR can help improve outcomes in elderly patients, and pharmacists can provide guidance regarding evidence-based therapy.

Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia.

Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I-associated peptide antigens with O-linked ß-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376-84. ©2017 AACR.

Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency.

Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRß sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21(lo) B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease.

Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry.

Phosphorylation events within cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth. Phosphopeptides derived from aberrantly phosphorylated proteins that are presented on tumors and not on normal tissues by human leukocyte antigen (HLA) class I molecules are promising candidates for future cancer immunotherapies, because they are tumor specific and have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry-compatible, iron(III)-immobilized metal affinity chromatography (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a peptide esterification step to increase phosphopeptide specificity from these low-input samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian cancer, leukemia and colorectal cancer have been identified using these highly selective phosphopeptide enrichment protocols in combination with a program called 'CAD Neutral Loss Finder' that identifies all spectra containing the characteristic neutral loss of phosphoric acid from phosphorylated serine and threonine residues. This methodology enables the identification of HLA-associated phosphopeptides presented by human tissue samples containing as little as nanograms of peptide material in 2 d.

MHC class I-associated phosphopeptides are the targets of memory-like immunity in leukemia.

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.

Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer.

BACKGROUND: Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer. METHODS: We examined both in-house and publicly available breast cancer DNA microarray datasets relating to invasion and metastasis, thus identifying a cohort of candidate progression-associated biomarkers. Of these, 18 antibodies were released for extended analysis. Validated antibodies were screened against a tissue microarray (TMA) constructed from a cohort of consecutive breast cancer cases (n = 512) to test the immunohistochemical surrogate signature. RESULTS: Antibody screening revealed 3 candidate prognostic markers: the cell cycle regulator, Anillin (ANLN); the mitogen-activated protein kinase, PDZ-Binding Kinase (PBK); and the estrogen response gene, PDZ-Domain Containing 1 (PDZK1). Increased expression of ANLN and PBK was associated with poor prognosis, whilst increased expression of PDZK1 was associated with good prognosis. A 3-marker signature comprised of high PBK, high ANLN and low PDZK1 expression was associated with decreased recurrence-free survival (p < 0.001) and breast cancer-specific survival (BCSS) (p < 0.001). This novel signature was associated with high tumour grade (p < 0.001), positive nodal status (p = 0.029), ER-negativity (p = 0.006), Her2-positivity (p = 0.036) and high Ki67 status (p < 0.001). However, multivariate Cox regression demonstrated that the signature was not a significant predictor of BCSS (HR = 6.38; 95% CI = 0.79-51.26, p = 0.082). CONCLUSIONS: We have developed a comprehensive biomarker pathway that extends from discovery through to validation on a TMA platform. This proof-of-concept study has resulted in the identification of a novel 3-protein prognostic panel. Additional biochemical markers, interrogated using this high-throughput platform, may further augment the prognostic accuracy of this panel to a point that may allow implementation into routine clinical practice.