Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.

https://science.sciencemag.org/content/sci/283/5403/857.full.pdf

Control of Viremia in Simian Immunodeficiency Virus Infection by CD8+ Lymphocytes

In addition to CD4, the human immunodeficiency virus (HIV) requires a coreceptor for entry into target cells. The chemokine receptors CXCR4 and CCR5, members of the G protein-coupled receptor superfamily, have been identified as the principal coreceptors for T cell line-tropic and macrophage-tropic HIV-1 isolates, respectively. The updated coreceptor repertoire includes numerous members, mostly chemokine receptors and related orphans. These discoveries provide a new framework for understanding critical features of the basic biology of HIV-1, including the selective tropism of individual viral variants for different CD4 C target cells and the membrane fusion mechanism governing virus entry. The coreceptors also provide molecular perspectives on central puzzles of HIV-1 disease, including the selective transmission of macrophage-tropic variants, the appearance of T cell line-tropic variants in many infected persons during progression to AIDS, and differing susceptibilities of individuals to infection and disease progression. Genetic findings have yielded major insights into the in vivo roles of individual coreceptors and their ligands; of particular importance is the discovery of an inactivating mutation in the CCR5 gene which, in homozygous form, confers strong resistance to HIV-1 infection. Beyond providing new perspectives on fundamental aspects of HIV-1 transmission and pathogenesis, the coreceptors suggest new avenues for developing novel therapeutic and preventative strategies to combat the AIDS epidemic.

CHEMOKINE RECEPTORS AS HIV-1 CORECEPTORS: Roles in Viral Entry, Tropism, and Disease

We examined the regulation of virus-specific CD8 T cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. Our study shows that within the same persistently infected host, different mechanisms can operate to silence antiviral T cell responses; CD8 T cells specific to one dominant viral epitope were deleted, whereas CD8 T cells responding to another dominant epitope persisted indefinitely. These virus-specific CD8 T cells expressed activation markers (CD69hi, CD44hi, CD62Llo) and proliferated in vivo but were unable to elaborate any antiviral effector functions. This unresponsive phenotype was more pronounced under conditions of CD4 T cell deficiency, highlighting the importance of CD8– CD4 T cell collaboration in controlling persistent infections. Importantly, in the presence of CD4 T cell help, adequate CD8 effector activity was maintained and the chronic viral infection eventually resolved. The persistence of activated virus-specific CD8 T cells without effector function reveals a novel mechanism for silencing antiviral immune responses and also offers new possibilities for enhancing CD8 T cell immunity in chronically infected hosts.

https://rupress.org/jem/article-pdf/188/12/2205/1117308/98-1509.pdf

Viral Immune Evasion Due to Persistence of Activated T Cells Without Effector Function

Chemokine receptors comprise a large family of seven transmembrane domain G protein-coupled receptors differentially expressed in diverse cell types. Biological activities have been most clearly defined in leukocytes, where chemokines coordinate development, differentiation, anatomic distribution, trafficking, and effector functions and thereby regulate innate and adaptive immune responses. Pharmacological analysis of chemokine receptors is at an early stage of development. Disease indications have been established in human immunodeficiency virus/acquired immune deficiency syndrome and in Plasmodium vivax malaria, due to exploitation of CCR5 and Duffy, respectively, by the pathogen for cell entry. Additional indications are emerging among inflammatory and immunologically mediated diseases, but selection of targets in this area still remains somewhat speculative. Small molecule antagonists with nanomolar affinity have been reported for 7 of the 18 known chemokine receptors but have not yet been studied in clinical trials. Virally encoded chemokine receptors, as well as chemokine agonists and antagonists, and chemokine scavengers have been identified in medically important poxviruses and herpesviruses, again underscoring the importance of the chemokine system in microbial pathogenesis and possibly identifying specific strategies for modulating chemokine action therapeutically. The purpose of this review is to update current concepts of the biology and pharmacology of the chemokine system, to summarize key information about each chemokine receptor, and to describe a widely accepted receptor nomenclature system, ratified by the International Union of Pharmacology, that is facilitating clear communication in this area.

https://pharmrev.aspetjournals.org/content/pharmrev/52/1/145.full.pdf

International Union of Pharmacology. XXII. Nomenclature for Chemokine Receptors

HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T-cells

MANY types of cancer and genetic disease are characterized by chromosome aberrations. Conventional cytogenetics, the analysis of banded metaphase chromosomes is time consuming and in many cases marker chromosomes cannot be identified by banding patterns alone. Flow cytometry offers an alternative approach where chromosomes can be labeled with one or more DNA binding dyes and passed through a flow cytometer. The resulting species-specific pattern is referred to as a flow karyotype. The first flow karyotypes were demonstrated in 1975 by Joe Gray and colleagues at the Lawrence Livermore Laboratories (1). They used a single Argon ion laser tuned to 488 nm and Ethidium Bromide as the DNA dye. In 1981, Sillar and Young described a new method for preparing chromosomes using polyamines to stabilize the chromosomes and digitonin to release them from the metaphase cells. This allowed much better resolution of the flow Karyotype (2). An improved strategy was then described exploiting the base pair binding preferences of Hoechst 33258 and Chromomycin A3 producing the bivariate flow karyotype which allowed better separation of the different chromosome types (3). Since then, several groups have analyzed and sorted human, animal, and plant chromosomes. The different human chromosome types have been sorted and chromosome specific libraries generated first in lambda phage (4) and then in cosmids (5,6). Sorting for library construction was a time consuming process usually taking days to accumulate enough chromosomes of each type. Individual chromosome types can also be sorted and amplified using polymerase chain reaction (PCR) and used to generate “chromosome paints,” fluorescently labeled probes that can be hybridized to metaphase chromosomes. This process requires sorting small numbers of chromosomes that can be achieved in a few minutes. Degenerate oligonucleotideprimed PCR (DOP PCR) can be used to generate such probes from any species, which will hybridize to the entire chromosome. Chromosomes paints have been generated against human chromosomes (7), mouse chromosomes (8), rat chromosomes (9), and many other species. It is possible to generate a chromosome paint from a single flow sorted chromosome although paints generated from a few hundred chromosomes seem to give more even “painting.” The human flow karyotype can often reveal marker chromosomes and other chromosome abnormalities and those chromosomes can be separated and used for downstream molecular studies or used to generate chromosome paints. Chromosome paints generated from marker chromosomes can be hybridized to normal metaphase spreads which will reveal the identity of the parent chromosomes from which the marker is derived (10). This process is referred to as reverse chromosome painting. The same approach has been used to study microchromosomes (11). More detailed mapping of chromosome breakpoints can be achieved using array painting where derivative chromosomes can be flow sorted, amplified using whole genome amplification and hybridization onto commercially available oligonucleotide microarrays (12). Flow cytometry seemed like it might offer a machine based semi-automated alternative to conventional cytogenetics. The flow karyotype generates a very conserved speciesspecific pattern. However, the instruments capable of giving high resolution flow karyotypes were bulky, expensive and difficult to use. They required two high powered water-cooled Argon lasers and needed a great deal of expertize to prepare, maintain and operate. The lasers were tuned to multiline ultra-violet (UV) and 457.9 nm and 300 mW of each was considered necessary which represented the maximum power available from 5 W Argon lasers. The system works better if the UV laser is the primary laser which would make it even more time consuming to transfer back to a configuration that is suitable for general cell sorting as usually the primary laser

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.a.22895

Chromosome sorting for the masses

Fluorescent proteins (FPs) have become an essential tool for biological research. Since the isolation and description of GFP, hundreds of fluorescent proteins have been discovered and created with various characteristics. The excitation of these proteins ranges from ultra‐violet (UV) up to near infra‐RED (NIR). Using conventional cytometry with each detector assigned to each fluorochrome, great care must be taken when selecting the optimal bandpass filters to minimalize the spectral overlap. In the last 8 years, several companies have released full spectrum flow cytometers which eliminates the need to change optical filters for analyzing FPs. This addressed at least part of the problem however, the laser wavelengths in commercial instruments are generally not ideal for all fluorescent proteins yet do allow the separation of at least six FPs. Another technical challenge is to have convenient single color controls. If four different FPs are being used in an experiment, single color controls will be needed to compensate or unmix the data. In the case of cultured cells this will involve having each of the FPs expressed in cell lines separately with a parental cell line expressing none. In the case of in vivo experiments, colonies of animals may need to be maintained expressing each FP along with a wildtype animal. This represents a considerable expense and inconvenience. An appealing alternative is to produce and purify FPs and covalently couple to polystyrene microspheres. Such microspheres are ready to use and can be stored at 4°C for months or even years without any deterioration in fluorescence. The same procedure can be used to couple antibodies to these particles. Here we describe this procedure which can be executed in any lab without any special equipment or skills.

Building a spectral cytometry toolbox: Coupling fluorescent proteins and antibodies to microspheres

Functional marker chromosomes lacking detectable alpha satellite DNA: a tool for centromere study.

Fluorescent proteins (FPs) have become an essential tool for biological research. Since the isolation and description of green FP, hundreds of FPs have been discovered and created with various characteristics. The excitation of these proteins ranges from ultraviolet (UV) up to near infrared (NIR). Using conventional cytometry, with each detector assigned to a fluorochrome, great care must be taken when selecting the optimal bandpass filters to minimalize the spectral overlap as the emission spectra of FPs are broad. Full‐spectrum flow cytometers eliminate the need to change optical filters for analyzing FPs, which simplifies instrument setup. In experiments where more than one FP is used, single‐color controls are required. These can be cells expressing each of the proteins separately. In the case of the confetti system, for instance, when four FPs are used, all these proteins will need to be expressed separately so that compensation or spectral unmixing can be performed, and this can be inconvenient and expensive. An appealing alternative is to produce FPs in Escherichia coli, purify them, and covalently couple them to carboxylate polystyrene microspheres. Such microspheres are ready to use and can be stored at 4°C for months or even years without any deterioration in fluorescence. The same procedure can be used to couple antibodies or other proteins to these particles. Here, we describe how to express and purify FPs, how to couple them to microspheres, and how to evaluate the fluorescent properties of the particles. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/cpz1.745

Fluorescent Protein Production, Purification, and Coupling to Microspheres

BackgroundProstate cancer is caused by genomic aberrations in normal epithelial cells, however clinical translation of findings from analyses of cancer cells alone has been very limited. A deeper understanding of the tumour microenvironment is needed to identify the key drivers of disease progression and reveal novel therapeutic opportunities. ResultsIn this study, the experimental enrichment of selected cell-types and the development a Bayesian inference model for continuous differential transcript abundance analyses permitted definition of the transcriptional landscape of the prostate cancer microenvironment across the disease progression spectrum. An important role of monocytes and macrophages in prostate cancer progression and disease recurrence was reinforced by both our transcriptional landscape findings and by differential tissue composition analyses. ConclusionsThis study contributes to understanding of monocyte-derived recruitment in primary prostate cancer, and supports a clear direction for further investigation into mechanisms of the immune system that contribute to disease progression.

/pdf/dissection-of-prostate-tumour-stroma-and-immune-2lmav1aln9.pdf

Simon Monard

Papers

Chromosome sorting for the masses

Building a spectral cytometry toolbox: Coupling fluorescent proteins and antibodies to microspheres

Functional marker chromosomes lacking detectable alpha satellite DNA: a tool for centromere study.

Fluorescent Protein Production, Purification, and Coupling to Microspheres

Dissection of prostate tumour, stroma and immune transcription reveals a key contribution by the microenvironment to disease progression