Localization Of CD8 Research Articles

Hybrid Immuno-RT for Bulky Tumors: Standard Fractionation with Partial Tumor SBRT

Bulky tumors remain challenging to be treated. Stereotactic body radiation therapy (SBRT) is effective against radioresistant tumor cells and can induce immunogenic cell death (ICD) that leads to T-cell-mediated antitumor effects. Low-dose radiation (LDRT) can inflame the tumor microenvironment (TME) by recruiting T cells. We designed a novel radiotherapy technique (RT, ERT) whose dose distribution map resembles the "eclipse" by concurrently delivering LDRT to the whole tumor, meanwhile SBRT to only a part of the same tumor. This study examined the safety and efficacy of ERT to bulky lesions with PD-1 inhibitors in mice and patients. In mice with CT26 colon or LLC1 lung bulky tumors (400 - 500 cm3), the whole tumor was irradiated by LDRT (2 Gy x 3), meanwhile the tumor center was irradiated by SBRT (10 Gy x 3); αPD-1 was given weekly. The dependence of therapeutic effects on CD8+ T cells was determined using depleting antibodies. Frequencies of CD8+ T cells and M1 macrophages (Mφ) were determined by flow cytometry. Multiplex Immunohistochemistry (mIHC) was applied to analyze the number and the location of CD8+ T cells and their subpopulations, as well as the phospho-eIF2α level (the ICD marker) of tumor cells in TME. Patients with advanced lung or liver bulky tumors who failed standard treatment or with oncologic emergencies were treated. Kaplan-Meier method was applied to estimate patients' progression-free survival (PFS) and overall survival (OS). ERT/αPD-1 is superior to SBRT/αPD-1 or LDRT/αPD-1 in controlling bulky tumors in both mouse models in a CD8+ T-cell dependent manner. In the CT26 model, ERT/αPD-1 resulted in complete tumor regression in 3/11 mice and induced more CD8+ T cells and M1 Mφ in TME compared to other groups. mIHC analysis showed that ERT/αPD-1 induced higher bulk, stem-like (TCF1+ TIM3- PD-1+), and more differentiated (TCF1- TIM3+ PD-1+) CD8+ T cells infiltration into the tumor center and periphery compared to other groups. Compared to untreated or LDRT-treated tumor centers, tumor centers irradiated with ERT or SBRT showed elevated phospho-eIF2α accompanied by higher dendritic cell infiltration. In total, 39 advanced cancer patients were treated with ERT/αPD-1 or plus chemotherapy. Radiation-induced pneumonitis occurred in 1 of 26 patients receiving thoracic ERT. There were two cases of grade III toxicity associated with PD-1 inhibitors. No toxicity above grade III was observed. The objective response rate was 38.5%. The median PFS was 5.6 months and median OS was not reached at a median follow-up of 11.7 months. ERT/αPD-1 showed superior efficacy in controlling bulky tumor in two mouse models. The hybrid immuno-RT (ERT) combing PD-1 inhibitors was safe and effective in patients with bulky tumors. Further clinical trials in combination with bioimaging to identify the optimal SBRT target region for the bulky tumor are warranted.

IFN-γ-STAT1-mediated CD8+ T-cell-neural stem cell cross talk controls astrogliogenesis after spinal cord injury

BackgroundSpinal cord injury (SCI) causes nearly all patients to suffer from protracted disabilities. An emerging therapeutic strategy involving the recruitment of endogenous neural stem cells (NSCs) has been developed. However, endogenous NSCs in the adult spinal cord differentiate into mostly astrocytes after traumatic injury, forming glial scars, which is a major cause of regeneration failure in SCI. Thus, understanding which factors drive the activation and differentiation of endogenous NSCs after SCI is critical for developing therapeutic drugs.MethodsThe infiltration, state, and location of CD8+ T cells in spinal cord after traumatic injury were analyzed by flow cytometry and immunofluorescence (IF) staining. The Basso Mouse Scale (BMS) scores and rotarod testing were used for motor behavioral analysis. NSCs were co-cultured with CD8+ T cells. EdU assay was used to detect proliferative cells. Western blotting was used to analyze the expression levels of STAT1, p-STAT1, and p27. ChIP-seq and ChIP-qRT-PCR analyses were used to detect the downstream of STAT1. Nestin-CreERT2::Ai9 transgenic mice were used to genetic lineage tracing of Nestin+ NSCs after SCI in vivo.ResultsA prolonged increase of activated CD8+ T cells occurs in the injured spinal cords. The behavioral analysis demonstrated that the administration of an anti-CD8 antibody promotes the recovery of locomotor function. Then, we discovered that CD8+ T cells suppressed the proliferation of NSCs and promoted the differentiation of NSCs into astrocytes by the IFN-γ-STAT1 pathway in vitro. ChIP-seq and ChIP-qRT-PCR analysis revealed that STAT1 could directly bind to the promoters of astrocyte marker genes GFAP and Aldh1l1. Genetic lineage tracing of Nestin+ NSCs demonstrated that most NSCs differentiated into astrocytes following SCI. Depleting CD8+ T cells reduced the differentiation of NSCs into astrocytes and instead promoted the differentiation of NSCs into oligodendrocytes.ConclusionIn conclusion, CD8+ T cells suppressed the proliferation of NSCs and promoted the differentiation of NSCs into astrocytes by the IFN-γ-STAT1-GFAP/Aldhl1l axis. Our study identifies INF-γ as a critical mediator of CD8+ T-cell-NSC cross talk and a potential node for therapeutic intervention in SCI.

Oral squamous cell carcinoma (OSCC) tumors from heavy alcohol consumers are associated with higher levels of TLR9 and a particular immunophenotype: Impact on patient survival.

Oral squamous cell carcinoma (OSCC) is one of the most frequent types of oral cancer in developing countries and its burden correlates with exposure to tobacco and excessive alcohol consumption. Toll like receptors (TLRs) are major sensors of inflammatory stimuli, from both microbial and sterile causes and as such, they have been related to tumor progression and metastasis. Here, we evaluated the expression of TLR2, 4 and 9 as well as CD3+, CD8+ and Granzyme B+ cell infiltration by immunohistochemistry in oral samples of 30 patients with OSCC, classified according to their consumption of alcohol. Our findings indicate that there is a significant association between heavy alcohol consumption and tumors with higher expression levels of TLR9. Moreover, patients with TLR9high tumors, as well as those who indicated high consumption of alcohol exhibited a diminished overall survival. TCGA data analysis indicated that TLR9high tumors express a significant increase in some genes related with the oral cavity itself, inflammation and tumor promotion. Our analysis of tumor infiltrating leukocytes demonstrated that the major differences perceived in heavy alcohol consumers was the location of CD8+ T cells infiltrating the tumor, which showed lower numbers intratumorally. Our data suggest the existence of a pathogenic loop that involves alcohol consumption, high TLR9 expression and the immunophenotype, which might have a profound impact on the progression of the disease.

Distinct Immune Gene Programs Associated with Host Tumor Immunity, Neoadjuvant Chemotherapy, and Chemoimmunotherapy in Resectable NSCLC.

Our understanding of the immunopathology of resectable non-small cell lung cancer (NSCLC) is still limited. Here, we explore immune programs that inform of tumor immunity and response to neoadjuvant chemotherapy and chemoimmunotherapy in localized NSCLC. Targeted immune gene sequencing using the HTG Precision Immuno-Oncology panel was performed in localized NSCLCs from three cohorts based on treatment: naïve (n = 190), neoadjuvant chemotherapy (n = 38), and neoadjuvant chemoimmunotherapy (n = 21). Tumor immune microenvironment (TIME) phenotypes were based on the location of CD8+ T cells (inflamed, cold, excluded), tumoral PD-L1 expression (<1% and ≥1%), and tumor-infiltrating lymphocytes (TIL). Immune programs and signatures were statistically analyzed on the basis of tumoral PD-L1 expression, immune phenotypes, and pathologic response and were cross-compared across the three cohorts. PD-L1-positive tumors exhibited increased signature scores for various lymphoid and myeloid cell subsets (P < 0.05). TIME phenotypes exhibited disparate frequencies by stage, PD-L1 expression, and mutational burden. Inflamed and PD-L1+/TILs+ NSCLCs displayed overall significantly heightened levels of immune signatures, with the excluded group representing an intermediate state. A cytotoxic T-cell signature was associated with favorable survival in neoadjuvant chemotherapy-treated NSCLCs (P < 0.05). Pathologic response to chemoimmunotherapy was positively associated with higher expression of genes involved in immune activation, chemotaxis, as well as T and natural killer cells (P < 0.05 for all). Among the three cohorts, chemoimmunotherapy-treated NSCLCs exhibited the highest scores for various immune cell subsets including T effector and B cells (P < 0.05). Our findings highlight immune gene programs that may underlie host tumor immunity and response to neoadjuvant chemotherapy and chemoimmunotherapy in resectable NSCLC.

The Clinical Significance of Hepatic CD69+ CD103+ CD8+ Resident-Memory T Cells in Autoimmune Hepatitis.

The diverse inflammatory response found in the liver of patients with autoimmune hepatitis (AIH) is well established, but identification of potentially pathogenic subpopulations has proven enigmatic. We report herein that CD69+ CD103+ CD8+ tissue-resident memory T cells (TRM ) are significantly increased in the liver of patients with AIH compared to chronic hepatitis B, NAFLD, and healthy control tissues. In addition, there was a significant statistical correlation between elevation of CD8+ TRM cells and AIH disease severity. Indeed, in patients with successful responses to immunosuppression, the frequencies of such hepatic CD8+ TRM cells decreased significantly. CD69+ CD8+ and CD69+ CD103+ CD8+ T cells, also known as CD8+ TRM cells, reflect tissue residency and are well known to provide intense immune antigenic responses. Hence, it was particularly interesting that patients with AIH also manifest an elevated expression of IL-15 and TGF-β on inflammatory cells, and extensive hepatic expression of E-cadherin; these factors likely contribute to the development and localization of CD8+ TRM cells. Based on these data and, in particular, the relationships between disease severity and CD8+ TRM cells, we studied the mechanisms involved with glucocorticoid (GC) modulation of CD8+ TRM cell expansion. Our data reflect that GCs in vitro inhibit the expansion of CD8+ TRM cells induced by IL-15 and TGF-β and with direct down-regulation of the nuclear factor Blimp1 of CD8+ TRM cells. Our data suggest that CD8+ TRM cells play a critical role in the pathogenesis of AIH, and GCs attenuate hepatic inflammation through direct inhibition of CD8+ TRM cell expansion.

CD8+ T-cell lymphocyte infiltration predict clinical outcomes Wilms’ tumor.

e22001 Background: The abundance and location of CD8+ tumor-infiltrating lymphocytes demonstrate important facets of the anticancer immune response. CD8-expressing lymphocytes have been used in immunotherapy for multiple cancers. This study aims to determine the association between the abundance and localization of CD8+ tumor-infiltrating lymphocytes and clinical outcomes Wilms’ tumor. Methods: This retrospective study employed 42 pediatric patients diagnosed with Wilms’ tumor. CD8+ tumor-infiltrating lymphocyte counts were calculated based on the mean percentage of stroma occupied by CD8+ lymphocytes at the center and the invasive border of the tumor using immunohistochemistry. Results: CD8+ tumor-infiltrating lymphocyte counts were significantly higher in the center and the invasive border of the early-stage tumor samples. CD8+ tumor-infiltrating lymphocytes in the invasive border and tumor center positively correlated with tumor invasion, regional lymph node invasion, histological type, metastasis, and stage of the tumor. A high CD8+ tumor-infiltrating lymphocyte scores at the invasive margin of the tumor correlated with low tumor recurrence. Low CD8+ tumor-infiltrating lymphocyte scores in the two tumor regions correlated with poor prognosis and shorter disease-free survival. Conclusions: Overall, these findings show that patients with high CD8+ tumor-infiltrating lymphocytes are associated with better clinical outcomes. Therefore, measuring the abundance of CD8+ tumor-infiltrating lymphocytes may be useful in predicting response to cancer immunotherapies.

CD8+ T-cell lymphocytes infiltration predict clinical outcomes in Wilms' tumor.

The abundance and location of CD8+ tumor-infiltrating lymphocytes demonstrate important facets of the anticancer immune response. CD8-expressing lymphocytes have been used in immunotherapy for multiple cancers. This study aims to determine the association between the abundance and localization of CD8+ tumor-infiltrating lymphocytes and clinical outcomes of Wilms' tumor. This retrospective study employed 42 pediatric patients diagnosed with Wilms' tumor. CD8+ tumor-infiltrating lymphocyte counts were calculated based on the mean percentage of stroma occupied by CD8+ lymphocytes at the center and the invasive border of the tumor using immunohistochemistry. CD8+ tumor-infiltrating lymphocyte counts were significantly higher in the center and the invasive border of the early-stage tumor samples. CD8+ tumor-infiltrating lymphocytes in the invasive border and tumor center positively correlated with tumor invasion, regional lymph node invasion, histological type, metastasis, and stage of the tumor. A high CD8+ tumor-infiltrating lymphocyte scores at the invasive margin of the tumor correlated with low tumor recurrence. Low CD8+ tumor-infiltrating lymphocyte scores in the two tumor regions correlated with poor prognosis and shorter disease-free survival. Overall, these findings show that patients with high CD8+ tumor-infiltrating lymphocytes are associated with better clinical outcomes. Therefore, measuring the abundance of CD8+ tumor-infiltrating lymphocytes may be useful in predicting response to cancer immunotherapies.

Tissue-resident memory T cells invade the brain parenchyma in multiple sclerosis white matter lesions.

Multiple sclerosis is a chronic inflammatory, demyelinating disease, although it has been suggested that in the progressive late phase, inflammatory lesion activity declines. We recently showed in the Netherlands Brain Bank multiple sclerosis-autopsy cohort considerable ongoing inflammatory lesion activity also at the end stage of the disease, based on microglia/macrophage activity. We have now studied the role of T cells in this ongoing inflammatory lesion activity in chronic multiple sclerosis autopsy cases. We quantified T cells and perivascular T-cell cuffing at a standardized location in the medulla oblongata in 146 multiple sclerosis, 20 neurodegenerative control and 20 non-neurological control brain donors. In addition, we quantified CD3+, CD4+, and CD8+ T cells in 140 subcortical white matter lesions. The location of CD8+ T cells in either the perivascular space or the brain parenchyma was determined using CD8/laminin staining and confocal imaging. Finally, we analysed CD8+ T cells, isolated from fresh autopsy tissues from subcortical multiple sclerosis white matter lesions (n = 8), multiple sclerosis normal-appearing white matter (n = 7), and control white matter (n = 10), by flow cytometry. In normal-appearing white matter, the number of T cells was increased compared to control white matter. In active and mixed active/inactive lesions, the number of T cells was further augmented compared to normal-appearing white matter. Active and mixed active/inactive lesions were enriched for both CD4+ and CD8+ T cells, the latter being more abundant in all lesion types. Perivascular clustering of T cells in the medulla oblongata was only found in cases with a progressive disease course and correlated with a higher percentage of mixed active/inactive lesions and a higher lesion load compared to cases without perivascular clusters in the medulla oblongata. In all white matter samples, CD8+ T cells were located mostly in the perivascular space, whereas in mixed active/inactive lesions, 16.3% of the CD8+ T cells were encountered in the brain parenchyma. CD8+ T cells from mixed active/inactive lesions showed a tissue-resident memory phenotype with expression of CD69, CD103, CD44, CD49a, and PD-1 and absence of S1P1. They upregulated markers for homing (CXCR6), reactivation (Ki-67), and cytotoxicity (GPR56), yet lacked the cytolytic enzyme granzyme B. These data show that in chronic progressive multiple sclerosis cases, inflammatory lesion activity and demyelinated lesion load is associated with an increased number of T cells clustering in the perivascular space. Inflammatory active multiple sclerosis lesions are populated by CD8+ tissue-resident memory T cells, which show signs of reactivation and infiltration of the brain parenchyma.

Differential phenotypes and localization of CD8 T cell responses to acute and chronic viral infections

Abstract Norovirus is the most common viral cause of gastroenteritis globally. Most infections are either acute, symptomatic, and rapidly cleared or persist asymptomatically. However, the mechanisms underlying these outcomes are unknown. MNV-CW3 and -CR6 are murine noroviruses which demonstrate immune correlates of acute and chronic infection respectively. This research interrogates the mechanisms underlying strain-specific differential antiviral CD8 T cell responses to acute or chronic MNV. At days 3, 4, 5 and 8 post-infection, the phenotype and quantity of adoptively transferred MNV specific CD8 T cells in the spleen, mesenteric lymph node (MLN), and intestines are analyzed by flow cytometry. Concurrently, fluorescent microscopy is used to determine the biogeographical distribution of antiviral CD8 T cells throughout the intestine. Flow cytometry shows activated MNV-specific CD8 T cells first accumulate in the MLN following oral infection with either virus suggesting this is the site of immune activation though CR6 infection results in a delayed, reduced CD8 T cell response as shown by CFSE dilution. Supporting the MLN as a primary initiation site of the antiviral CD8 T cell response, prevention of T cell egress from activation sites by FTY720 treatment enriches activated CD8 T cells in the MLN in CW3 infections with a concurrent decrease in the spleen. Furthermore, by day 8 post-infection CD8 T cell populations are skewed to memory precursors or short-lived effectors in CW3 and CR6, respectively. These data suggest these highly related viral strains may induce activation of distinct populations of, or pathways in, APC populations with long-lasting effects on CD8 T cell function and the outcome of infection.

Macrophages/Microglia Represent the Major Source of Indolamine 2,3-Dioxygenase Expression in Melanoma Metastases of the Brain.

The manifestation of brain metastases in patients with advanced melanoma is a common event that limits patient's survival and quality of life. The immunosuppressive properties of the brain parenchyma are very different compared to the rest of the body, making it plausible that the current success of cancer immunotherapies is specifically limited here. In melanoma brain metastases, the reciprocal interplay between immunosuppressive mediators such as indoleamine 2, 3-dioxygenase (IDO) or programmed cell death-ligand 1 (PD-L1) in the context of neoplastic transformation are far from being understood. Therefore, we analyzed the immunoreactive infiltrate (CD45, CD3, CD8, Forkhead box P3 [FoxP3], CD11c, CD23, CD123, CD68, Allograft Inflammatory factor 1[AIF-1]) and PD-L1 with respect to IDO expression and localization in melanoma brain metastases but also in matched metastases at extracranial sites to correlate intra- and interpatient data with therapy response and survival. Comparative tissue analysis identified macrophages/microglia as the major source of IDO expression in melanoma brain metastases. In contrast to the tumor infiltrating lymphocytes, melanoma cells per se exhibited low IDO expression levels paralleled by cell surface presentation of PD-L1 in intracranial metastases. Absolute numbers and pattern of IDO-expressing cells in metastases of the brain correlated with recruitment and localization of CD8+ T cells, implicating dynamic impact on the regulation of T cell function in the brain parenchyma. However, paired analysis of matched intra- and extracranial metastases identified significantly lower fractions of cytotoxic CD8+ T cells in intracranial metastases while all other immune cell populations remain unchanged. In line with the already established clinical benefit for PD-L1 expression in extracranial melanoma metastases, Kaplan-Meier analyses correlated PD-L1 expression in brain metastases with favorable outcome in advanced melanoma patients undergoing immune checkpoint therapy. In summary, our data provide new insights into the landscape of immunosuppressive factors in melanoma brain metastases that may be useful in the implication of novel therapeutic strategies for patients undergoing cancer immunotherapy.

2007P - Spatio-temporal separation of tumor infiltrating CD8+ T-cells and HER2/neu+ tumor cells in tumor-immune milieu of infiltrating ductal carcinoma of the breast

BackgroundDesignated tumor infiltrating lymphocytes (TIL) within tumor microenvironment (TME) comprised of both regulatory cells and effector cells, and their interaction with tumor cells account for the immune escape and immunosurveillance mechanisms respectively. The number and location of CD8+ tumor infiltrating lymphocytes (TILs) are the measures of immune response and carries prognostic significance and predictive potential in the era of immuno-oncology. The present study was an attempt to find the relation between the treatment response and the spatio-temporal separation of tumor infiltrating CD8+ T-cells and HER2/neu+ tumor cells in breast cancer TME. MethodsIn this study, a total of 7 breast cancer patients were enrolled and surgically removed fresh tumor tissues were evaluated for the baseline HER2/neu and CD8 expression by immunohistochemistry (IHC) using dual stain (duplex IHC). The tumor tissues were also tested for the response to HER2 blocker in a novel humanized ex vivo platform that preserves tumour microenvironment using tumour explants, maintained in defined tumour grade-matched matrix support and autologous patient serum. ResultsTumors responded to HER2 blockade had a majority of the CD8+ T cells (60-80%) concentrated around the HER2/neu+ tumor area (within 50-100µm). While, the non-responding tumors had significantly lesser number of the CD8+T cells (20-40%) proximal to HER2/neu+ zone compared to the responders. The present study indeed delineated a preferential spatial relationship of tumor infiltrating CD8+ cells in HER2/neu+ tumor region especially for the recognition of tumor antigen, cross presentation and tumor cell killing. ConclusionsAssessment of distribution pattern of effector cells surrounding tumor area had a correlation on the efficacy of HER2 inhibition. It warrants validation in the larger cohort of patient tumors to underpin its clinical relevance for possible immunotherapies like combination of check point inhibitors with HER2/neu blocker and others. Identification of other co-stimulatory and pathway molecules on the tumors not responded to HER2 blocker is being studied to understand the mechanism of resistance. Legal entity responsible for the studyThe authors. FundingMitra RxDx Inc. DisclosureAll authors have declared no conflicts of interest.

PD-1 high expression predicts lower local disease control in stage IV M0 nasopharyngeal carcinoma

BackgroundTumor-infiltrating lymphocytes (TILs) play a critical role in tumor immune surveillance and immune suppression. Understanding tumor infiltrating T cell subset density, location and PD-1/PD-L1 expression might provide insight for the prediction of tumor therapeutic response and clinical outcome. The purpose of this study was to evaluate the expression and localization of CD8, FoxP3, PD-1, and PD-L1 in primary tumor tissues and their effects on prognosis of stage IV M0 locally advanced nasopharyngeal carcinoma (NPC) patients.MethodsSixty NPC patients with stage IV M0 locally advanced disease were treated with definitive chemoradiation. Tumor biopsies from primary lesion were analyzed for the expression and localization of CD8, FoxP3, PD-1, and PD-L1 by immunohistochemistry. Their associations with local disease control and survival of NPC were analyzed.ResultsThe average follow-up time was 43 months (range from 14 to 61 months). High expression of CD8+, FoxP3+, PD-1+ and PD-L1+ was observed in 60, 86.7, 56.7, and 91.7% of patients, respectively. There was no correlation between clinicopathological features and the expression of these immune markers. High PD-1 expression was found to be associated with lower local disease control (5-year LRFS 23.2% vs 96.8%, p < 0.001) and unfavorable clinical outcome (5-year OS 47.4% vs 73.3%, p = 0.014). In multivariate analysis, PD-1 expression was also an adverse prognostic factor for 5-year OS (HR: 3.68, P = 0.023) and LRFS (HR: 16.89, 1.27–11.84, P = 0.007). Those with PD-1 distribution in both stroma and tumor region had the poorest prognosis. However, PD-1 expression has no significant correlation with 5-year RRFS (p = 0.980) and DMFS (p = 0.865). Patients with both PD-1 and PD-L1 high expression had significant poor local disease control (5-year LRFS 96.0% vs 43.0%, p < 0.001) and overall survival (5-year OS 80.8% vs 45.1%, p < 0.001) compared with the others. Other immune markers were not found having corrections with disease control and survival.ConclusionsPD-1 high expression, especially with PD-L1 co-expression, is associated with high local recurrence and unfavorable clinical outcome for stage IV M0 NPC patients, and might be a potential target for immunotherapy.

Differential phenotypes and localization of CD8 T cell responses to acute and chronic viral infection

Abstract Norovirus represents the major viral source of gastroenteritis worldwide and the leading cause of foodborne illness in North America. Most infections are acute, symptomatic, and rapidly cleared but some cases persist asymptomatically or induce post-infectious irritable bowel syndrome. Despite the global economic burden of these infections no vaccine to prevent disease exists. Using immune correlates of acute vs chronic infection in mouse models following infection with related strains of murine norovirus, MNV-CW3 and MNV-CR6 respectively, this research interrogates the mechanisms underlying strain-specific differential antiviral CD8 T cell responses. The phenotype and quantity of MNV specific CD8 T cells are analyzed by flow cytometry. Since MNV-CW3 and -CR6 replication is higher in the small and large intestine, respectively, immunofluorescent microscopy is used to determine whether CD8 T cells are preferentially recruited to either tissue. Combining these complementary techniques provides novel insight into mechanisms governing intestinal antiviral T cell responses. Preliminary data shows activated MNV-specific CD8 T cells first accumulate in the MLN, suggesting this is the site of immune activation following oral infection with either virus. However, the magnitude and kinetics of T cell activation are reduced in CR6-infected mice compared to CW3-infected mice. This data suggests that these highly related viral strains induce activation of distinct populations of, or pathways in, APC populations with long-lasting effects on the outcome of infection. These results may have broad impacts on our understanding of how non-latent, chronic viral infections persist within a host.

Relationship of infiltrating intraepithelial T lymphocytes in the diagnosis of oral lichen planus versus oral epithelial dysplasia: a pilot study

The aim of this study was to identify the type and distribution of CD4+ and CD8+ T lymphocytes in oral mucosal specimens to potentially distinguish between underlying alterations or patterns in oral epithelial dysplasia and oral lichen planus. This pilot study included 10 archived tissue samples that were received at the University of North Carolina Oral and Maxillofacial Pathology Laboratory and were diagnosed as oral lichen planus and moderate to severe epithelial dysplasia. Dual staining with CD4 and CD8 antibodies was carried out on each case. Slides were scanned in the Aperio ScanScope FL (Leica Biosystems, Wetzlar, Germany) and archived. Histomorphometric analysis was performed to detect inflammatory cells expressing CD4 and CD8 biomarkers in the epithelial and connective tissue regions. No differences were found in the amount and ratio of CD4+/CD8+ lymphocytes among the 3 groups analyzed; however, the intraepithelial CD8+ lymphocyte distribution was strikingly different between lichen planus and moderate to severe epithelial dysplasia. The localization of CD8+ cells can be potentially useful as an adjunctive diagnostic procedure to distinguish oral epithelial dysplasia from other inflammatory entities, such as lichen planus.

The effect of feeding mice during gestation and nursing with Rhodiola kirilowii extracts or epigallocatechin on CD4 and CD8 cells number and distribution in the spleen of their progeny

Rhodiola kirilowii, a member of Crassulaceae family, grows wildly in Asiatic mountains and is also cultivated in some European countries. Its underground parts traditionally are used for enhance physical and mental performance of the body.In our previous papers we reported immuno- and angio-modulatory effects of aqueous and hydro-alcoholic extracts of radix and rhizome of this plant in mice. In the present work we evaluated the effect of Rhodiola kirilowii water- (RKW) or hydro-alcoholic (RKW-A) extracts and epigallocatechin (one of the polyphenols present in these extracts) given to mice, during pregnancy and nursing period, on the number and localization of CD4+ and CD8+ cells in spleens of adult progeny mice. Previously, we observed several abnormalities in functionality of spleen cells collected from these mice. No differences in CD4+ T cells localisation or numbers were found between all tested mice groups. In contrast, CD8+ T cells localisation and staining were altered in progeny of water or alcohol extract-fed mice. CD8+ T cells were found not only in the PALS but also in the B cell follicle and in the red pulp. Furthermore, CD8+ T cells from T cell zones in the progeny of extract-fed mice showed much intensive staining for CD8 antigen and significantly higher numbers per area in comparison to control mice.

Abstract IA23: Deconvolution of the triple-negative breast cancer microenvironment

Abstract Breast cancer heterogeneity is one of the principal obstacles both to predicting outcome and to determining an effective course of treatment for this disease. Although genomic technologies have been used to gain a better understanding, by identifying gene expression signatures associated with clinical outcome and breast cancer subtypes, relatively little is known about heterogeneity in the tumor microenvironment. It is now accepted that changes in the normal cells that constitute the tumor microenvironment (TME) play important roles in determining cancer progression and ultimate outcome. We and others have established that an immune gene expression signature correlates with good outcome in triple negative breast cancer (TNBC), yet fails to accurately predict outcome in all patients. Examining stromal heterogeneity in TNBC has identified four distinct stromal clusters, three of which are prognostic, contain distinct immune signatures and a signature of fibrosis. Lymphocytic infiltration and access to tumor parenchyma is not well understood due to high levels of spatial heterogeneity within tumors. We show that location of CD8+T cells is strongly influenced by TME subtypes and identify gene expression signatures predictive of distinct CD8+T cell localization and patient outcome. Since mounting evidence suggests that immune-checkpoint inhibitor immunotherapies may be promising for only a subset of TNBC patients, highlights the importance of understanding how the TME influences CD8+T cell location. Citation Format: Sadiq Saleh, Tina Gruosso, Mathieu Gigoux, Nicholas Bertos, Atilla Omeroglu, Dongmei Zuo, Sarkis Meterissian, Michael Hallett, Morag Park. Deconvolution of the triple-negative breast cancer microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA23.

Abstract PR07: Characterization of human effectors in the tumor microenvironment of NSCLC patients and lung tumor xenograft models

Abstract Non-small cell lung cancer (NSCLC) is one the most prevalent forms of lung cancer. Lung tumors consist of a heterogeneous population of tumor cells, immune cells and stromal cells. It is critical to assess the nature and localization of immune effector cells and their expression of immune checkpoint molecules to develop more effective therapeutic strategies. In this study, we used immunohistochemistry (IHC) to assess the localization of human immune cells and expression of immune checkpoint molecules to define the microenvironment composition. Furthermore, we developed a mouse xenograft model of human lung tumor using BLT-NSG mice with a reconstituted human immune system and A549 cells. Formalin-fixed paraffin embedded NSCLC lung tumor sections (n=28 adenocarcinoma, n=16 squamous) were processed by IHC to assess expression and localization of CD4, CD8, MPO, Foxp3, CD68 and cytokeratin, as well as immune checkpoint molecules (PD-1, Tim-3, LAG-3) and ligands (PD-L1 and VISTA). For the xenograft model, BLT-NSG mice (NSG mice transplanted with fetal liver CD34+ cells and autologous thymus) at 12-16 weeks post-transplant were injected with A549-luciferase (A549-Luc) cells via transpleural or intravenous injection. Weekly bioluminescence imaging was done to assess tumor growth. Lungs and spleens were harvested and enzymatically dissociated for flow cytometric analysis of human effectors and immune checkpoint molecules. We found that significantly more human effectors are localized to the stromal compartment (cytokeratin-) than within the cytokeratin+ tumor nests. The stromal compartment contained 12-fold more CD8+ T-cells, 23-fold more CD4+ T-cells, and 11-fold more regulatory T-cells (Foxp3+). Two-fold more neutrophils (MPO+) and macrophages (CD68+) were found in stroma outside of tumor nests. There were significantly more CD8+ T-cells (p&amp;lt;0.05) and MPO+ neutrophils (p&amp;lt;0.01) localized within the cytokeratin+ areas of squamous compared to adeno tumors. PD-1, Tim-3 or LAG-3 IHC showed that 100-fold more PD-1+ cells are found within the tertiary lymphoid structures in the stromal compartment. 19-fold more Tim-3+ cells, both lymphocytes and macrophages, are found in the stromal compartment. LAG-3+ cells are lymphocytes, and localized mostly to the stromal compartment (11-fold more than in cytokeratin+ areas). PD-L1 IHC showed that 12/16 (75%) of squamous tumors and 14/28 (50%) of adeno tumors express PD-L1 on tumor cells, and H-Scores of the PD-L1 staining from squamous tumors were significantly higher than the adeno tumors (p=0.019). A majority of macrophages expressed PD-L1, but the expression level varied from sample to sample (1+ to 3+ scoring). VISTA IHC showed that lymphocytes, neutrophils and macrophages expressed VISTA, and 26-fold more VISTA+ cells are found in the stromal compartment. In our A549 xenograft model study, NSG and BLT-NSG mice with IV-injected A549 cells showed tumor growth differences by bioluminescence imaging. Flow cytometric analyses showed an increased percentage of non-naïve CD8+ (88%) and non-naïve CD4+ (97%) cells compared to spleens (44% non-naïve CD8+ cells and 74% non-naïve CD4+ cells). There was an increased expression of LAG-3 in both human CD8+ (19-90%) and human CD4+ (9-61%) non-naïve T-cells in the lungs, but LAG-3+ cells in the spleen were minimal (&amp;lt;6%). PD-1 expression was high in non-naïve T-cell population in both lungs and spleen (&amp;gt;70%). In addition, there was an up-regulation of PD-L1+(4%) and VISTA+ (12%) in the human CD14+ population in the lungs. In conclusion, the expression of various immune checkpoint molecules in effector cells may limit their localization within the stromal compartment. Furthermore, our xenograft model was able to recapitulate our observations with human patient samples, and would be a valuable in vivo mouse tool to test new therapeutic agents. Citation Format: Hyun-Jeong Ra, Gisela Brake-Silla, Evgeniy Eruslanov, Michael Sharp, Charuhas Deshpande, Michael D. Feldman, Amy Ziober, Li-Ping Wang, Manuel A. Sepulveda, Linda A. Snyder, Gwenn Danet-Desnoyers. Characterization of human effectors in the tumor microenvironment of NSCLC patients and lung tumor xenograft models. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR07.

In vivo and in vitro analyses of α-galactosylceramide uptake by conventional dendritic cell subsets using its fluorescence-labeled derivative

Conventional dendritic cells (cDCs) present α-galactosylceramide (αGC) to invariant natural killer T (iNKT) cells through CD1d. Among cDC subsets, CD8+ DCs efficiently induce IFN-γ production in iNKT cells. Using fluorescence-labeled αGC, we showed that CD8+ DCs incorporated larger amounts of αGC and kept it intact longer than CD8– DCs. Histological analyses revealed that Langerin+CD8+ DCs in the splenic marginal zone, which was the unique equipment to capture blood-borne antigens, preferably incorporated αGC, and the depletion of Langerin+ cells decreased IFN-γ and IL-12 production in response to αGC. Furthermore, splenic Langerin+CD8+ DCs expressed more membrane-bound CXCL16, which possibly anchored iNKT cells in the marginal zone, than CD8− DCs. Collectively, it is suggested that the cellular properties and localization of CD8+ DCs are important for stimulation of iNKT cells by αGC.

Abstract A69: The immunological environment in triple negative breast cancer; impact on clinical outcomes towards a prognostic clinical test

Abstract It is now accepted that changes in the normal cells that constitute the tumor microenvironment (TME) play important roles in determining cancer progression and ultimate outcome. We and others have established that an immune signature, and more recently that the ability of CD8+ T cells to infiltrate the tumor bed, are correlated with good outcome in triple negative breast cancer. Importantly, since the cytotoxic effector functions of CD8+ T cells are mainly contact-dependent, the tumor microenvironment may act to inhibit access to the epithelial tumor bed, leading to uncontrolled tumor growth. Therefore, in order to determine the role that the TME plays on mediating the entry of CD8+ T cells into the epithelial tumor bed, our group performed laser-capture microdissection to separate the tumor epithelial compartment from the surrounding tumor-associated stroma from the primary tumor of 56 triple negative breast cancer patients and followed by gene expression profiling. Our aims are to provide clinical validation of predicted CD8+ T cell localisation based on our bioinformatic analysis, test our predictions that the identified stroma signature correlates with CD8+ T cell retention in other cohort of TN breast cancer patients and develop suitable markers of this stroma, and develop a muliplex based assay integrating CD8+ T cell localisation with stromal markers suitable for a retrospective study to validate predictions with outcome. Using gene expression profiling, our data have identified a canonical gene expression signature which correlates with retention of CD8+ T cells in the stroma and poor outcome. Thus, our results demonstrate that gene expression analysis of clinical triple negative breast cancer samples can predict the location of CD8+ T cells within the tumor and in turn the outcome. Citation Format: Mathieu Gigoux, Tina Gruosso, Nicholas Bertos, Sadiq Saleh, Atilla Omeroglu, Hong Zhao, Margarita Souleimanova, Sarkis Meterissian, Michael Hallett, Morag Park. The immunological environment in triple negative breast cancer; impact on clinical outcomes towards a prognostic clinical test. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A69.

Localization of CD8 T cell epitope within cardiac myosin heavy chain-α334–352 that induces autoimmune myocarditis in A/J mice

BackgroundCardiac myosin heavy chain-α (Myhc), an intracellular protein expressed in the cardiomyocytes, has been identified as a major autoantigen in cardiac autoimmunity. In our studies with Myhc334–352-induced experimental autoimmune myocarditis in A/J mice (H-2a), we discovered that Myhc334–352, supposedly a CD4 T cell epitope, also induced antigen-specific CD8 T cells that transfer disease to naive animals. Methods and resultsIn our efforts to identify the CD8 T cell determinants, we localized Myhc338–348 within the full length-Myhc334–352, leading to four key findings. (1) By acting as a dual epitope, Myhc338–348 induces both CD4 and CD8 T cell responses. (2) In a major histocompatibility complex (MHC) class I-stabilization assay, Myhc338–348 was found to bind H-2Dd—but not H-2Kk or H-2Ld—alleles. (3) The CD8 T cell response induced by Myhc338–348 was antigen-specific, as evaluated by MHC class I/H-2Dd dextramer staining. The antigen-sensitized T cells predominantly produced interferon-γ, the critical cytokine of effector cytotoxic T lymphocytes. (4) Myhc338–348 was found to induce myocarditis in immunized animals as determined by histology and magnetic resonance microscopy imaging. ConclusionsOur data provide new insights as to how different immune cells can recognize the same antigen and inflict damage through different mechanisms.