Humanized mice: are we there yet?

Animal models have been instrumental in increasing the understanding of human physiology, particularly immunity. However, these animal models have been limited by practical considerations and genetic diversity. The creation of humanized mice that carry partial or complete human physiological systems may help overcome these obstacles. The National Institute of Allergy and Infectious Diseases convened a workshop on humanized mouse models for immunity in Bethesda, MD, on June 13–14, 2005, during which researchers discussed the benefits and limitations of existing animal models and offered insights into the development of future humanized mouse models

Expansion of Melanoma-specific Cytolytic CD8+ T Cell Precursors in Patients with Metastatic Melanoma Vaccinated with CD34+ Progenitor-derived Dendritic Cells

Cancer vaccines aim at inducing (a) tumor-specific effector T cells able to reduce/eliminate the tumor mass, and (b) long-lasting tumor-specific memory T cells able to control tumor relapse. We have shown earlier, in 18 human histocompatibility leukocyte antigen (HLA)-A*0201 patients with metastatic melanoma, that vaccination with peptide-loaded CD34–dendritic cells (DCs) leads to expansion of melanoma-specific interferon γ–producing CD8+ T cells in the blood. Here, we show in 9 out of 12 analyzed patients the expansion of cytolytic CD8+ T cell precursors specific for melanoma differentiation antigens. These precursors yield, upon single restimulation with melanoma peptide–pulsed DCs, cytotoxic T lymphocytes (CTLs) able to kill melanoma cells. Melanoma-specific CTLs can be grown in vitro and can be detected in three assays: (a) melanoma tetramer binding, (b) killing of melanoma peptide–pulsed T2 cells, and (c) killing of HLA-A*0201 melanoma cells. The cytolytic activity of expanded CTLs correlates with the frequency of melanoma tetramer binding CD8+ T cells. Thus, CD34-DC vaccines can expand melanoma-specific CTL precursors that can kill melanoma antigen–expressing targets. These results justify the design of larger follow-up studies to assess the immunological and clinical response to peptide-pulsed CD34-DC vaccines

Interferon and Granulopoiesis Signatures in Systemic Lupus Erythematosus Blood

Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease characterized by flares of high morbidity. Using oligonucleotide microarrays, we now show that active SLE can be distinguished by a remarkably homogeneous gene expression pattern with overexpression of granulopoiesis-related and interferon (IFN)-induced genes. Using the most stringent statistical analysis (Bonferroni correction), 15 genes were found highly up-regulated in SLE patients, 14 of which are targets of IFN and one, defensin DEFA-3, a major product of immature granulocytes. A more liberal correction (Benjamini and Hochberg correction) yielded 18 additional genes, 12 of which are IFN-regulated and 4 granulocyte-specific. Indeed immature neutrophils were identified in a large fraction of SLE patients white blood cells. High dose glucocorticoids, a standard treatment of disease flares, shuts down the interferon signature, further supporting the role of this cytokine in SLE. The expression of 10 genes correlated with disease activity according to the SLEDAI. The most striking correlation (P < 0.001, r = 0.55) was found with the formyl peptide receptor-like 1 protein that mediates chemotactic activities of defensins. Therefore, while the IFN signature confirms the central role of this cytokine in SLE, microarray analysis of blood cells reveals that immature granulocytes may be involved in SLE pathogenesis

Breast cancer instructs dendritic cells to prime interleukin 13–secreting CD4+ T cells that facilitate tumor development

We previously reported (Bell, D., P. Chomarat, D. Broyles, G. Netto, G.M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K.A. Palucka, and J. Banchereau. 1999. J. Exp. Med. 190: 1417–1426) that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which cluster with CD4+ T cells. We now show that CD4+ T cells infiltrating breast cancer tumors secrete type 1 (interferon γ) as well as high levels of type 2 (interleukin [IL] 4 and IL-13) cytokines. Immunofluorescence staining of tissue sections revealed intense IL-13 staining on breast cancer cells. The expression of phosphorylated signal transducer and activator of transcription 6 in breast cancer cells suggests that IL-13 actually delivers signals to cancer cells. To determine the link between breast cancer, DCs, and CD4+ T cells, we implanted human breast cancer cell lines in nonobese diabetic/LtSz-scid/scid β2 microglobulin–deficient mice engrafted with human CD34+ hematopoietic progenitor cells and autologous T cells. There, CD4+ T cells promote early tumor development. This is dependent on DCs and can be partially prevented by administration of IL-13 antagonists. Thus, breast cancer targets DCs to facilitate its development

Mass Cytometry Defines Virus-Specific CD4 + T Cells in Influenza Vaccination

The antiviral response to influenza virus is complex and multifaceted, involving many immune cell subsets. There is an urgent need to understand the role of CD4+ T cells, which orchestrate an effective antiviral response, to improve vaccine design strategies. In this study, we analyzed PBMCs from human participants immunized with influenza vaccine, using high-dimensional single-cell proteomic immune profiling by mass cytometry. Data were analyzed using a novel clustering algorithm, denoised ragged pruning, to define possible influenza virus-specific clusters of CD4+ T cells. Denoised ragged pruning identified six clusters of cells. Among these, one cluster (Cluster 3) was found to increase in abundance following stimulation with influenza virus peptide ex vivo. A separate cluster (Cluster 4) was found to expand in abundance between days 0 and 7 postvaccination, indicating that it is vaccine responsive. We examined the expression profiles of all six clusters to characterize their lineage, functionality, and possible role in the response to influenza vaccine. Clusters 3 and 4 consisted of effector memory cells, with high CD154 expression. Cluster 3 expressed cytokines like IL-2, IFN-γ, and TNF-α, whereas Cluster 4 expressed IL-17. Interestingly, some participants had low abundance of Clusters 3 and 4, whereas others had higher abundance of one of these clusters compared with the other. Taken together, we present an approach for identifying novel influenza virus-reactive CD4+ T cell subsets, a method that could help advance understanding of the immune response to influenza, predict responsiveness to vaccines, and aid in better vaccine design

Transcriptional profiling of macrophages in situ in metastatic melanoma reveals localization-dependent phenotypes and function.

Modulation of immune function at the tumor site could improve patient outcomes. Here, we analyze patient samples of metastatic melanoma, a tumor responsive to T cell-based therapies, and find that tumor-infiltrating T cells are primarily juxtaposed to CD14+ monocytes/macrophages rather than melanoma cells. Using immunofluorescence-guided laser capture microdissection, we analyze transcriptomes of CD3+ T cells, CD14 + monocytes/macrophages, and melanoma cells in non-dissociated tissue. Stromal CD14+ cells display a specific transcriptional signature distinct from CD14+ cells within tumor nests. This signature contains LY75, a gene linked with antigen capture and regulation of tolerance and immunity in dendritic cells (DCs). When applied to TCGA cohorts, this gene set can distinguish patients with significantly prolonged survival in metastatic cutaneous melanoma and other cancers. Thus, the stromal CD14+ cell signature represents a candidate biomarker and suggests that reprogramming of stromal macrophages to acquire DC function may offer a therapeutic opportunity for metastatic cancers

Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.

Establishment of an in vivo small animal model of human tumor and human immune system interaction would enable preclinical investigations into the mechanisms underlying cancer immunotherapy. To this end, nonobese diabetic (NOD).Cg- PrkdcscidIL2rgtm1Wjl/Sz (null; NSG) mice were transplanted with human (h)CD34+ hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems [humanized NSG (HuNSG)]. HuNSG mice received human leukocyte antigen partially matched tumor implants from patient-derived xenografts [PDX; non-small cell lung cancer (NSCLC), sarcoma, bladder cancer, and triple-negative breast cancer (TNBC)] or from a TNBC cell line-derived xenograft (CDX). Tumor growth curves were similar in HuNSG compared with nonhuman immune-engrafted NSG mice. Treatment with pembrolizumab, which targets programmed cell death protein 1, produced significant growth inhibition in both CDX and PDX tumors in HuNSG but not in NSG mice. Finally, inhibition of tumor growth was dependent on hCD8+ T cells, as demonstrated by antibody-mediated depletion. Thus, tumor-bearing HuNSG mice may represent an important, new model for preclinical immunotherapy research. FASEB J 2018 Mar; 32(3):1537-1549

Cross-priming of cyclin B1, MUC-1 and survivin-specific CD8(+ )T cells by dendritic cells loaded with killed allogeneic breast cancer cells

INTRODUCTION: The ability of dendritic cells (DCs) to take up whole tumor cells and process their antigens for presentation to T cells ('cross-priming') is an important mechanism for induction of tumor specific immunity. METHODS: In vitro generated DCs were loaded with killed allogeneic breast cancer cells and offered to autologous naïve CD8(+ )T cells in 2-week and/or 3-week cultures. CD8(+ )T cell differentiation was measured by their capacity to secrete effector cytokines (interferon-γ) and kill breast cancer cells. Specificity was measured using peptides derived from defined breast cancer antigens. RESULTS: We found that DCs loaded with killed breast cancer cells can prime naïve CD8(+ )T cells to differentiate into effector cytotoxic T lymphocytes (CTLs). Importantly, these CTLs primed by DCs loaded with killed HLA-A*0201(- )breast cancer cells can kill HLA-A*0201(+ )breast cancer cells. Among the tumor specific CTLs, we found that CTLs specific for HLA-A2 restricted peptides derived from three well known shared breast tumor antigens, namely cyclin B1, MUC-1 and survivin. CONCLUSION: This ability of DCs loaded with killed allogeneic breast cancer cells to elicit multiantigen specific immunity supports their use as vaccines in patients with breast cancer