Phenotyping renal leukocyte subsets by four-color flow cytometry: Characterization of chemokine receptor expression

To investigate mechanisms of cell-mediated injury in renal inflammatory disease it is critical to determine the surface phenotype of infiltrating renal leukocyte subsets. However, the cell-specific expression of many leukocyte receptors is difficult to characterize in vivo. Here, we report a protocol based on flow cytometry that allows simultaneous characterization of surface receptor expression on different subsets of infiltrating renal leukocytes. The described technique combines an adapted method to prepare single cell suspensions from whole kidneys with subsequent four-color flow cytometry. We recently applied this technique to determine the differential expression of murine chemokine receptors CCR2 and CCR5 on infiltrating renal leukocyte subsets. In this article, we summarize our current findings on the validity of the method as compared with immunohistology and in situ hybridization in two murine models of nonimmune ( obstructive nephropathy) and immune-mediated ( lupus nephritis) inflammatory renal disease. Flow cytometry analysis revealed an accumulation of CCR5-, but not CCR2-positive lymphocytes in inflamed kidneys, compared to the peripheral blood. Particularly renal CD8(+) cells expressed CCR5 (79% in obstructed kidneys, 90% in lupus nephritis). In both models, infiltrating renal macrophages were positive for CCR2 and CCR5. These data corresponded to immunohistological and in situ hybridization results. They demonstrate that flow cytometric analysis of single cell suspensions prepared from inflamed kidneys is a rapid and reliable technique to characterize and quantify surface receptor expression on infiltrating renal leukocyte subsets

Can the plasma PD-1 levels predict the presence and efficiency of tumor-infiltrating lymphocytes in patients with metastatic melanoma?

Background: The immune response in melanoma patients is locally affected by presence of tumor-infiltrating lymphocytes (TILs), generally divided into brisk, nonbrisk, and absent. Several studies have shown that a greater presence of TILs, especially brisk, in primary melanoma is associated with a better prognosis and higher survival rate. Patients and Methods: We investigated by enzyme-linked immunosorbent assay (ELISA) the correlation between PD-1 levels in plasma and the presence/absence of TILs in 28 patients with metastatic melanoma. Results: Low plasma PD-1 levels were correlated with brisk TILs in primary melanoma, whereas intermediate values correlated with the nonbrisk TILs, and high PD-1 levels with absent TILs. Although the low number of samples did not allow us to obtain a statistically significant correlation between the plasma PD-1 levels and the patients' overall survival depending on the absence/presence of TILs, the median survival of patients having brisk type TILs was 5 months higher than that of patients with absent and nonbrisk TILs. Conclusions: This work highlights the ability of measuring the plasma PD-1 levels in order to predict the prognosis of patients with untreated metastatic melanoma without a BRAF mutation at the time of diagnosis

Antigen receptor variable region repertoires expressed by T cells infiltrating thyroid, retroorbital, and pretibial tissue in Graves' disease

To date, it has remained unclear whether T cells infiltrating thyroid, retroorbital, and pretibial tissue of patients with Graves' ophthalmopathy and pretibial dermopathy represent a primary immune response that is directed against certain antigenic determinants shared among these involved tissues. To characterize these T cells at the molecular level, we compared the T cell antigen receptor (TcR) variable (V) region gene usage in thyroid, retroorbital, pretibial tissue, and peripheral blood mononuclear cells of two patients with Graves' disease, ophthalmopathy, and pretibial dermopathy. Ribonucleic acid was extracted, reverse transcribed, and amplified using the PCR and 22 V alpha and 23 V beta gene-specific oligonucleotide primers. The resulting TcR V alpha and V beta transcripts were verified by Southern hybridization analysis using TcR C region-specific, digoxigenin-labeled oligonucleotide probes. In addition, complementarity determining regions 3 and junctional regions of TcR V beta genes were sequenced. Marked similarities of intrathyroidal, retroorbital, and pretibial TcR V alpha and V beta gene repertoires were noted with respect to the degree of TcR V gene restriction and the patterns of individual V genes expressed. Sequence analysis of junctional domains of V beta families revealed oligoclonality of intrahyroidal, retroorbital, and pretibial T cells. In addition, certain conserved junctional motifs were shared by T cells derived the thyroid gland and the extrathyroidal sites. Our results suggest that in the two patients with Graves' disease and extrathyroidal manifestations studied, similar antigenic determinants may have contributed to the recruitment and oligoclonal expansion of T cells both within the thyroid gland and at the involved extrathyroidal sites

18F-FAC PET Visualizes Brain-Infiltrating Leukocytes in a Mouse Model of Multiple Sclerosis.

Brain-infiltrating leukocytes contribute to multiple sclerosis (MS) and autoimmune encephalomyelitis and likely play a role in traumatic brain injury, seizure, and stroke. Brain-infiltrating leukocytes are also primary targets for MS disease-modifying therapies. However, no method exists for noninvasively visualizing these cells in a living organism. 1-(2'-deoxy-2'-18F-fluoroarabinofuranosyl) cytosine (18F-FAC) is a PET radiotracer that measures deoxyribonucleoside salvage and accumulates preferentially in immune cells. We hypothesized that 18F-FAC PET could noninvasively image brain-infiltrating leukocytes. Methods: Healthy mice were imaged with 18F-FAC PET to quantify if this radiotracer crosses the blood-brain barrier (BBB). Experimental autoimmune encephalomyelitis (EAE) is a mouse disease model with brain-infiltrating leukocytes. To determine whether 18F-FAC accumulates in brain-infiltrating leukocytes, EAE mice were analyzed with 18F-FAC PET, digital autoradiography, and immunohistochemistry, and deoxyribonucleoside salvage activity in brain-infiltrating leukocytes was analyzed ex vivo. Fingolimod-treated EAE mice were imaged with 18F-FAC PET to assess if this approach can monitor the effect of an immunomodulatory drug on brain-infiltrating leukocytes. PET scans of individuals injected with 2-chloro-2'-deoxy-2'-18F-fluoro-9-β-d-arabinofuranosyl-adenine (18F-CFA), a PET radiotracer that measures deoxyribonucleoside salvage in humans, were analyzed to evaluate whether 18F-CFA crosses the human BBB. Results: 18F-FAC accumulates in the healthy mouse brain at levels similar to 18F-FAC in the blood (2.54 ± 0.2 and 3.04 ± 0.3 percentage injected dose per gram, respectively) indicating that 18F-FAC crosses the BBB. EAE mice accumulate 18F-FAC in the brain at 180% of the levels of control mice. Brain 18F-FAC accumulation localizes to periventricular regions with significant leukocyte infiltration, and deoxyribonucleoside salvage activity is present at similar levels in brain-infiltrating T and innate immune cells. These data suggest that 18F-FAC accumulates in brain-infiltrating leukocytes in this model. Fingolimod-treated EAE mice accumulate 18F-FAC in the brain at 37% lower levels than control-treated EAE mice, demonstrating that 18F-FAC PET can monitor therapeutic interventions in this mouse model. 18F-CFA accumulates in the human brain at 15% of blood levels (0.08 ± 0.01 and 0.54 ± 0.07 SUV, respectively), indicating that 18F-CFA does not cross the BBB in humans. Conclusion: 18F-FAC PET can visualize brain-infiltrating leukocytes in a mouse MS model and can monitor the response of these cells to an immunomodulatory drug. Translating this strategy into humans will require exploring additional radiotracers

Immune DNA signature of T-cell infiltration in breast tumor exomes.

Tumor infiltrating lymphocytes (TILs) have been associated with favorable prognosis in multiple tumor types. The Cancer Genome Atlas (TCGA) represents the largest collection of cancer molecular data, but lacks detailed information about the immune environment. Here, we show that exome reads mapping to the complementarity-determining-region 3 (CDR3) of mature T-cell receptor beta (TCRB) can be used as an immune DNA (iDNA) signature. Specifically, we propose a method to identify CDR3 reads in a breast tumor exome and validate it using deep TCRB sequencing. In 1,078 TCGA breast cancer exomes, the fraction of CDR3 reads was associated with TILs fraction, tumor purity, adaptive immunity gene expression signatures and improved survival in Her2+ patients. Only 2/839 TCRB clonotypes were shared between patients and none associated with a specific HLA allele or somatic driver mutations. The iDNA biomarker enriches the comprehensive dataset collected through TCGA, revealing associations with other molecular features and clinical outcomes

Role of Rip2 in development of tumor-infiltrating MDSCs and bladder cancer metastasis.

Tumor invasion and metastases represent a complex series of molecular events that portends a poor prognosis. The contribution of inflammatory pathways mediating this process is not well understood. Nod-like receptors (NLRs) of innate immunity function as intracellular sensors of pathogen motifs and danger molecules. We propose a role of NLRs in tumor surveillance and in programming tumor-infiltrating lymphocytes (TILs). In this study, we examined the downstream serine/threonine and tyrosine kinase Rip2 in a murine model of bladder cancer. In Rip2-deficient C57Bl6 mice, larger orthotopic MB49 tumors developed with more numerous and higher incidence of metastases compared to wild-type controls. As such, increased tumor infiltration of CD11b+ Gr1hi myeloid-derived suppressor cells (MDSCs) with concomitant decrease in T cells and NK cells were observed in Rip2-deficient tumor bearing animals using orthotopic and subcutaneous tumor models. Rip2-deficient tumors showed enhanced epithelial-to-mesenchymal transition, with elevated expression of zeb1, zeb2, twist, and snail in the tumor microenvironment. We found that the absence of Rip2 plays an intrinsic role in fostering the development of granulocytic MDSCs by an autocrine and paracrine effect of granulocytic colony stimulating factor (G-CSF) expression. Our findings suggest that NLR pathways may be a novel modality to program TILs and influence tumor metastases

The Prognostic Importance of CD20(+) B lymphocytes in Colorectal Cancer and the Relation to Other Immune Cell subsets

The anti-tumour immune response is critical to patient prognosis in colorectal cancer (CRC). The aim of this study was to investigate infiltration of B lymphocytes into CRC tumours, and their clinical relevance, prognostic value and relation to other immune cell subsets. We used multiplexed immunohistochemistry and multispectral imaging to assay the amount of infiltrating CD20(+) B lymphocytes along with infiltration of CD8(+) cytotoxic T cells, FOXP3(+) T regulatory cells, CD68(+) macrophages and CD66b(+) neutrophils, in 316 archival CRC tissue specimens. A higher density of infiltrating CD20(+) B lymphocytes was associated with tumours of the right colon (P = 0.025) and of lower stages (P = 0.009). Furthermore, patients whose tumours were highly infiltrated by CD20(+) B lymphocytes had a significantly improved disease-specific survival (HR = 0.45, 95% CI 0.28-0.73, P = 0.001), which remained significant in multivariable analysis. CD20(+) B lymphocytes were highly and positively associated with CD8(+) T lymphocytes (P <0.001), and part of the prognostic role was found to be a cooperative effect between these lymphocyte subsets. Our results support a favourable prognostic value of tumour-infiltrating CD20(+) B lymphocytes in CRC. Furthermore, a cooperative prognostic effect between CD20(+) B lymphocytes and CD8(+) T lymphocytes is suggested.Peer reviewe

Frequency over function : raised levels of CD127low/- regulatory T cells in the tumour microenvironment compared with the periphery of head and neck cancer patients

Objective: Regulatory T cells (Tregs) are known to infiltrate the tumour microenvironment of many cancers, including head and neck malignancies, and are thought to contribute to the host's impaired anti-tumour immune response. However, their immunosuppressive function remains poorly understood within the tumour microenvironment and this study aimed to address this. Methods: The frequency and suppressive capacity of two CD4?CD127low/- Treg populations, separated on the basis of different levels of CD25 expression (CD25inter and CD25high), from the tumour/node microenvironment and peripheral circulation of newly-presenting head and neck squamous cell carcinoma patients (n=19), were assessed using multicolour flow cytometry. Results: The proportion of Tregs (CD4?CD25high/?interCD127low/-) in the tumour/node microenvironment was significantly elevated compared with the peripheral circulation (p&lt;0.001) and similar percentages were present in both the primary tumour and metastatic lymph node. The percentage of suppression induced by Tregs isolated from tumour associated nodes on the proliferation of nodal effector T cells was similar to that of peripheral Tregs on peripheral effector T cells. However, when the suppressive activity of both nodal and peripheral Tregs was compared on the same peripheral effectors, peripheral Tregs suppressed proliferation to a greater extent. Conclusion: This work shows that the recruitment and percentages of tumour infiltrating Tregs are key factors in modulating the immune environment of head and neck tumours