Humanized mice as a model to study human immunity

Mice with human immune systems have evolved over the last three decades. Over the years, these humanized mouse models have provided us with valuable information on human immunity. Recent developments in recipient mouse strains and engraftment protocols have resulted in models with high level of de novo formation of human immune cells. Here we describe the development and improvements of humanized BALB/c/Rag2 -/- /IL2Rγ -/- and NOD/SCID/IL2Rγ -/- mice and employ such mice or studies on various aspects of human immunity. We show that engraftment with human cells depends on the recipient strain and conditioning regiment. While we confirm that T cells that developed in the mouse are functional and respond to allogeneic cells and mitogens in vitro, no rejection was observed in vivo after transplantation of human β-islets under the kidney capsule of humanized mice (Paper I). In a second study, we showed that human CD56 dim NK cells in humanized mice and in recipients of a bone marrow transplant are subject to further differentiation. We observed that CD57 and killer cell immunoglobulin-like receptors (KIRs) are acquired during differentiation of CD56 dim cells. (Paper II) Infection studies in humanized mice have thus far been almost exclusively limited to infections with pathogens specifically targeting human immune cells. We explored the use of humanized mice for mycobacterial infections, which are not restricted to infection of human cells. We found that humanized mice contained higher bacterial titers in comparison to controls. While this finding could be attributed to dysfunctional T cell responses and impaired anti-mycobactericidal responses by human macrophages, we found that humanized mice infected with Mycobacterium bovis BCG or Mycobacterium tuberculosis developed organized granulomas similar to those found in humans. Furthermore, we demonstrated that human CD4 + cells impair mycobacterial control but are essential for the development or maintenance of granulomas (Paper III). Finally, we used humanized mice to shed light on Epstein Barr Virus induced latency. Infections with this human specific virus resulted in a CD4/CD8 T cell ratio skewed towards CD8 and the differentiation of T cells from naïve to effector memory cells. A variable number of infected mice showed tumors and B-cell proliferation ex vivo. In vivo depletion of CD8 + cells increased the frequency of tumors and ex vivo proliferation of transformed B cells. Surprisingly, ex vivo proliferation of B cells from CD8 + cell depleted or non-depleted mice was inhibited in presence of cyclosporine A, suggesting that CD4 + T cells exerted a supporting effect on cells displaying a latency type which otherwise would not proliferate in vitro. This finding was confirmed by analysis of viral promoters in CD4 + , CD8 + and non-depleted infected animals (Paper IV). In conclusion, our studies show that current humanized mouse models can be used to improve our knowledge of various aspects of human immunity, such as alloreactivity, the ontogeny of hematopoietic cells, the immunobiology of human-lymphoid specific as well as non-species specific microorganisms and the regulation of granuloma formation in infectious and non-infectious diseases

What Is a Good Tomato? A Case of Valuing in Practice

As a contribution to the field of valuation studies this article lays out a number of lessons that follow from an exploratory inquiry into ‘good tomatoes’. We held interviews with tomato experts (developers, growers, sellers, processors, professional cooks and so-called consumers) in the Netherlands and analysed the transcriptions carefully. Grouping our informants’ concerns with tomatoes into clusters, we differentiate between five  registers of valuing. These have to do with money, handling, historical time, what it is to be natural, and sensual appeal. There are tensions between and within these registers, that lead to clashes and compromises. Accordingly, valuing tomatoes does not fit into inclusive formal schemes. Neither is it simply a matter of making judgements. Our informants told us how they know whether a tomato is good, but also revealed what they do to make tomatoes good. Their valuing includes activities such as pruning tomato plants and preparing tomato dishes. But if such activities are meant to make tomatoes good, success is never guaranteed. This prompts us to import the notion of care. Care does not offer control, but involves sustained and respectful tinkering towards improvement. Which is not to say in the end the tomatoes our informants care for are good. In the end these tomatoes get eaten. And while eating performs tomatoes as ‘good to eat’, it also finishes them off. Valuing may lead on to destruction. An important lesson for valuation studies indeed.As a contribution to the field of valuation studies this article lays out a number of lessons that follow from an exploratory inquiry into ‘good tomatoes’. We held interviews with tomato experts (developers, growers, sellers, processors, professional cooks and so-called consumers) in the Netherlands and analysed the transcriptions carefully. Grouping our informants’ concerns with tomatoes into clusters, we differentiate between five  registers of valuing. These have to do with money, handling, historical time, what it is to be natural, and sensual appeal. There are tensions between and within these registers, that lead to clashes and compromises. Accordingly, valuing tomatoes does not fit into inclusive formal schemes. Neither is it simply a matter of making judgements. Our informants told us how they know whether a tomato is good, but also revealed what they do to make tomatoes good. Their valuing includes activities such as pruning tomato plants and preparing tomato dishes. But if such activities are meant to make tomatoes good, success is never guaranteed. This prompts us to import the notion of care. Care does not offer control, but involves sustained and respectful tinkering towards improvement. Which is not to say in the end the tomatoes our informants care for are good. In the end these tomatoes get eaten. And while eating performs tomatoes as ‘good to eat’, it also finishes them off. Valuing may lead on to destruction. An important lesson for valuation studies indeed

Stratification of PD-1 blockade response in melanoma using pre- and post-treatment immunophenotyping of peripheral blood

Efficacy of checkpoint inhibitor therapies in cancer varies greatly, with some patients showing complete responses while others do not respond and experience progressive disease. We aimed to identify correlates of response and progression following PD-1-directed therapy by immunophenotyping peripheral blood samples from 20 patients with advanced malignant melanoma before and after treatment with the PD-1 blocking antibody pembrolizumab. Our data reveal that individuals responding to PD-1 blockade were characterised by increased CD8 T cell proliferation following treatment, while progression was associated with an increase in CTLA-4-expressing Treg. Remarkably, unsupervised clustering analysis of pre-treatment T cell subsets revealed differences in individuals that went on to respond to PD-1 blockade compared to individuals that did not. These differences mapped to expression of the proliferation marker Ki67 and the costimulatory receptor CD28 as well as the inhibitory molecules 2B4 and KLRG1. While these results require validation in larger patient cohorts, they suggest that flow cytometric analysis of a relatively small number of T cell markers in peripheral blood could potentially allow stratification of PD-1 blockade treatment response prior to therapy initiation

IL-21 shapes germinal center polarization via light zone B cell selection and cyclin D3 upregulation

Germinal center (GC) dysregulation has been widely reported in the context of autoimmunity. Here, we show that interleukin 21 (IL-21), the archetypal follicular helper T cell (Tfh) cytokine, shapes the scale and polarization of spontaneous chronic autoimmune as well as transient immunization-induced GC. We find that IL-21 receptor deficiency results in smaller GC that are profoundly skewed toward a light zone GC B cell phenotype and that IL-21 plays a key role in selection of light zone GC B cells for entry to the dark zone. Light zone skewing has been previously reported in mice lacking the cell cycle regulator cyclin D3. We demonstrate that IL-21 triggers cyclin D3 upregulation in GC B cells, thereby tuning dark zone inertial cell cycling. Lastly, we identify Foxo1 regulation as a link between IL-21 signaling and GC dark zone formation. These findings reveal new biological roles for IL-21 within GC and have implications for autoimmune settings where IL-21 is overproduced

Follicular helper T cell signature in type 1 diabetes

The strong genetic association between particular HLA alleles and type 1 diabetes (T1D) indicates a key role for CD4+ T cells in disease; however, the differentiation state of the responsible T cells is unclear. T cell differentiation originally was considered a dichotomy between Th1 and Th2 cells, with Th1 cells deemed culpable for autoimmune islet destruction. Now, multiple additional T cell differentiation fates are recognized with distinct roles. Here, we used a transgenic mouse model of diabetes to probe the gene expression profile of islet-specific T cells by microarray and identified a clear follicular helper T (Tfh) cell differentiation signature. Introduction of T cells with a Tfh cell phenotype from diabetic animals efficiently transferred diabetes to recipient animals. Furthermore, memory T cells from patients with T1D expressed elevated levels of Tfh cell markers, including CXCR5, ICOS, PDCD1, BCL6, and IL21. Defects in the IL-2 pathway are associated with T1D, and IL-2 inhibits Tfh cell differentiation in mice. Consistent with these previous observations, we found that IL-2 inhibited human Tfh cell differentiation and identified a relationship between IL-2 sensitivity in T cells from patients with T1D and acquisition of a Tfh cell phenotype. Together, these findings identify a Tfh cell signature in autoimmune diabetes and suggest that this population could be used as a biomarker and potentially targeted for T1D interventions.This work was funded by an MRC Senior Fellowship (to L.S.K. Walker), a project grant from JDRF (to L.S.K. Walker and P. Narendran), and a studentship from Diabetes UK (to L.S.K. Walker and P. Narendran). L. Wardzinski and A. Kogimtzis were supported by a Wellcome Trust project grant (to L.S.K. Walker). M. Ono is a BBSRC David Philips fellow.Published versio

Coronaviruses as Vectors: Stability of Foreign Gene Expression

Coronaviruses are enveloped, positive-stranded RNA viruses considered to be promising vectors for vaccine development, as (i) genes can be deleted, resulting in attenuated viruses; (ii) their tropism can be modified by manipulation of their spike protein; and (iii) heterologous genes can be expressed by simply inserting them with appropriate coronaviral transcription signals into the genome. For any live vector, genetic stability is an essential requirement. However, little is known about the genetic stability of recombinant coronaviruses expressing foreign genes. In this study, the Renilla and the firefly luciferase genes were systematically analyzed for their stability after insertion at various genomic positions in the group 1 coronavirus feline infectious peritonitis virus and in the group 2 coronavirus mouse hepatitis virus. It appeared that the two genes exhibit intrinsic differences, the Renilla gene consistently being maintained more stably than the firefly gene. This difference was not caused by genome size restrictions, by different effects of the encoded proteins, or by different consequences of the synthesis of the additional subgenomic mRNAs. The loss of expression of the firefly luciferase was found to result from various, often large deletions of the gene, probably due to RNA recombination. The extent of this process appeared to depend strongly on the coronaviral genomic background, the luciferase gene being much more stable in the feline than in the mouse coronavirus genome. It also depended significantly on the particular genomic location at which the gene was inserted. The data indicate that foreign sequences are more stably maintained when replacing nonessential coronaviral genes

Direct contact between Plasmodium falciparum and human B-cells in a novel co-culture increases parasite growth and affects B-cell growth

Background: Plasmodium falciparum parasites cause malaria and co-exist in humans together with B-cells for long periods of time. Immunity is only achieved after repeated exposure. There has been a lack of methods to mimic the in vivo co-occurrence, where cells and parasites can be grown together for many days, and it has been difficult with long time in vitro studies. Methods and results: A new method for growing P. falciparum in 5% CO2 with a specially formulated culture medium is described. This knowledge was used to establish the co-culture of live P. falciparum together with human B-cells in vitro for 10 days. The presence of B-cells clearly enhanced parasite growth, but less so when Transwell inserts were used (not allowing passage of cells or merozoites), showing that direct contact is advantageous. B-cells also proliferated more in presence of parasites. Symbiotic parasitic growth was verified using CESS cell-line and it showed similar results, indicating that B-cells are indeed the cells responsible for the effect. In malaria endemic areas, people often have increased levels of atypical memory B-cells in the blood, and in this assay it was demonstrated that when parasites were present there was an increase in the proportion of CD19 + CD20 + CD27 − FCRL4 + B-cells, and a contraction of classical memory B-cells. This effect was most clearly seen when direct contact between B-cells and parasites was allowed. Conclusions: These results demonstrate that P. falciparum and B-cells undoubtedly can affect each other when allowed to multiply together, which is valuable information for future vaccine studies