Human B Cell Receptor Repertoire Sequencing.

Next-generation sequencing has the potential to uncover the complex nature of B cell immunity by revealing the full complexity of B cell receptor (BCR) repertoires in health and disease. However, there are drawbacks which can compromise the validity of the repertoire analysis caused by quantitative bias and accumulation of sequencing errors during the library preparation and sequencing. Here, we provide an optimized protocol designed to minimize bias for reproducible and accurate preparation of human BCR repertoire libraries for high-throughput sequencing.

Cytomegalovirus drives Vδ1+ γδ T cell expansion and clonality in common variable immunodeficiency.

The function and phenotype of γδ T cells in the context of common variable immunodeficiency (CVID) has not been explored. CVID is a primary immunodeficiency disorder characterized by impaired antibody responses resulting in increased susceptibility to infections. γδ T cells are a subset of unconventional T cells that play crucial roles in host defence against infections. In this study, we aim to determine the roles and functions of γδ T cells in CVID. We observe a higher frequency of Vδ1+ γδ T cells compared to healthy controls, particularly in older patients. We also find a higher proportion of effector-memory Vδ1+ γδ T cells and a more clonal T cell receptor (TCR) repertoire in CVID. The most significant driver of the Vδ1+ γδ T cell expansion and phenotype in CVID patients is persistent cytomegalovirus (CMV) viremia. These findings provide valuable insights into γδ T cell biology and their contribution to immune defence in CVID.

Circulating effector γδ T cell populations are associated with acute coronavirus disease 19 in unvaccinated individuals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe coronavirus disease 2019 (COVID-19) in a small proportion of infected individuals. The immune system plays an important role in the defense against SARS-CoV-2, but our understanding of the cellular immune parameters that contribute to severe COVID-19 disease is incomplete. Here, we show that populations of effector γδ T cells are associated with COVID-19 in unvaccinated patients with acute disease. We found that circulating CD27neg CD45RA+ CX3CR1+ Vδ1effector cells expressing Granzymes (Gzms) were enriched in COVID-19 patients with acute disease. Moreover, higher frequencies of GzmB+ Vδ2+ T cells were observed in acute COVID-19 patients. SARS-CoV-2 infection did not alter the γδ T cell receptor repertoire of either Vδ1+ or Vδ2+ subsets. Our work demonstrates an association between effector populations of γδ T cells and acute COVID-19 in unvaccinated individuals.

Venetoclax treatment in patients with cancer has limited impact on circulating T and NK cells.

Venetoclax is an effective treatment for certain blood cancers, such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). However, most patients relapse while on venetoclax and further treatment options are limited. Combining venetoclax with immunotherapies is an attractive approach; however, a detailed understanding of how venetoclax treatment impacts normal immune cells in patients is lacking. In this study, we performed deep profiling of peripheral blood (PB) cells from patients with CLL and AML before and after short-term treatment with venetoclax using mass cytometry (cytometry by time of flight) and found no impact on the concentrations of key T-cell subsets or their expression of checkpoint molecules. We also analyzed PB from patients with breast cancer receiving venetoclax long-term using a single-cell multiomics approach (cellular indexing of transcriptomes and epitopes by sequencing) and functional assays. We found significant depletion of B-cell populations with low expression of MCL-1 relative to other immune cells, attended by extensive transcriptomic changes. By contrast, there was less impact on circulating T cells and natural killer (NK) cells, with no changes in their subset composition, transcriptome, or function following venetoclax treatment. Our data indicate that venetoclax has minimal impact on circulating T or NK cells, supporting the rationale of combining this BH3 mimetic drug with cancer immunotherapies for more durable antitumor responses.

Case Report: Cytomegalovirus Disease Is an Under-Recognized Contributor to Morbidity and Mortality in Common Variable Immunodeficiency.

BACKGROUND: Common Variable Immunodeficiency (CVID) is classified as a 'Predominantly Antibody Deficiency' (PAD), but there is emerging evidence of cellular immunodeficiency in a subset of patients. This evidence includes CVID patients diagnosed with cytomegalovirus (CMV) infection, a hallmark of 'combined immunodeficiency'. CMV infection also has the potential to drive immune dysregulation contributing to significant morbidity and mortality in CVID. We aim to determine the extent of cellular immune dysfunction in CVID patients, and whether this correlates with CMV infection status. METHODS: We conducted a single-center retrospective cohort study of individuals with CVID at the Royal Melbourne Hospital, and identified patients with and without CMV disease or viraemia. We then isolated T-cells from patient and healthy donor blood samples and examined T-cell proliferation and function. RESULTS: Six patients (7.6%, 6/79) had either CMV disease (pneumonitis or gastrointestinal disease), or symptomatic CMV viraemia. A high mortality rate in the cohort of patients with CVID and CMV disease was observed, with 4 deaths in the period of analysis (66.6%, 4/6). Individuals with CMV infection showed reduced T-cell division in response to T-cell receptor (TCR) stimulation when compared with CMV-negative patients. DISCUSSION: This study demonstrates the morbidity and mortality associated with CMV in CVID, and highlights the need for focused interventions for patients with CVID at risk of CMV disease.

Repeated Plasmodium falciparum infection in humans drives the clonal expansion of an adaptive γδ T cell repertoire.

Repeated Plasmodium falciparum infections drive the development of clinical immunity to malaria in humans; however, the immunological mechanisms that underpin this response are only partially understood. We investigated the impact of repeated P. falciparum infections on human γδ T cells in the context of natural infection in Malian children and adults, as well as serial controlled human malaria infection (CHMI) of U.S. adults, some of whom became clinically immune to malaria. In contrast to the predominant Vδ2+ T cell population in malaria-naïve Australian individuals, clonally expanded cytotoxic Vδ1effector T cells were enriched in the γδ T cell compartment of Malian subjects. Malaria-naïve U.S. adults exposed to four sequential CHMIs defined the precise impact of P. falciparum on the γδ T cell repertoire. Specifically, innate-like Vδ2+ T cells exhibited an initial robust polyclonal response to P. falciparum infection that was not sustained with repeated infections, whereas Vδ1+ T cells increased in frequency with repeated infections. Moreover, repeated P. falciparum infection drove waves of clonal selection in the Vδ1+ T cell receptor repertoire that coincided with the differentiation of Vδ1naïve T cells into cytotoxic Vδ1effector T cells. Vδ1+ T cells of malaria-exposed Malian and U.S. individuals were licensed for reactivity to P. falciparum parasites in vitro. Together, our study indicates that repeated P. falciparum infection drives the clonal expansion of an adaptive γδ T cell repertoire and establishes a role for Vδ1+ T cells in the human immune response to malaria.

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor.

Unlike conventional αß T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1+ and Vδ2+ γδ TCR-mediated ligand recognition, the mode of Vδ3+ TCR ligand engagement is unknown. MHC class I-related protein, MR1, presents vitamin B metabolites to αß T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2- γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3+ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3+ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Mucosal-Associated Invariant T Cell Effector Function Is an Intrinsic Cell Property That Can Be Augmented by the Metabolic Cofactor α-Ketoglutarate.

Mucosal-associated invariant T (MAIT) cells are an innate-like population of unconventional T cells that respond rapidly to microbial metabolite Ags or cytokine stimulation. Because of this reactivity and surface expression of CD45RO+, CD45RA-, and CD127+, they are described as effector memory cells. Yet, there is heterogeneity in MAIT cell effector response. It is unclear what factors control MAIT cell effector capacity, whether it is fixed or can be modified and if this differs based on whether activation is TCR dependent or independent. To address this, we have taken a systematic approach to examine human MAIT cell effector capacity across healthy individuals in response to ligand and cytokine stimulation. We demonstrate the heterogenous nature of MAIT cell effector capacity and that the ability to produce an effector response is not directly attributable to TCR clonotype or coreceptor expression. Global gene transcription analysis revealed that the MAIT cell effector capacity produced in response to TCR stimulation is associated with increased expression of the epigenetic regulator lysine demethylase 6B (KDM6B). Addition of a KDM6B inhibitor did not alter MAIT cell effector function to Ag or cytokine stimulation. However, addition of the KDM6B cofactor α-ketoglutarate greatly enhanced MAIT cell effector capacity to TCR-dependent stimulation in a partially KDM6B-dependent manner. These results demonstrate that the TCR-dependent effector response of MAIT cells is epigenetically regulated and dependent on the availability of metabolic cofactors.

Absence of mucosal-associated invariant T cells in a person with a homozygous point mutation in MR1.

The role unconventional T cells play in protective immunity in humans is unclear. Mucosal-associated invariant T (MAIT) cells are an unconventional T cell subset restricted to the antigen-presenting molecule MR1. Here, we report the discovery of a patient homozygous for a rare Arg31His (R9H in the mature protein) mutation in MR1 who has a history of difficult-to-treat viral and bacterial infections. MR1R9H was unable to present the potent microbially derived MAIT cell stimulatory ligand. The MR1R9H crystal structure revealed that the stimulatory ligand cannot bind due to the mutation lying within, and causing structural perturbation to, the ligand-binding domain of MR1. While MR1R9H could bind and be up-regulated by a MAIT cell inhibitory ligand, the patient lacked circulating MAIT cells. This shows the importance of the stimulatory ligand for MAIT cell selection in humans. The patient had an expanded γδ T cell population, indicating a compensatory interplay between these unconventional T cell subsets.

MAIT cell clonal expansion and TCR repertoire shaping in human volunteers challenged with Salmonella Paratyphi A.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can detect bacteria-derived metabolites presented on MR1. Here we show, using a controlled infection of humans with live Salmonella enterica serovar Paratyphi A, that MAIT cells are activated during infection, an effect maintained even after antibiotic treatment. At the peak of infection MAIT cell T-cell receptor (TCR)ß clonotypes that are over-represented prior to infection transiently contract. Select MAIT cell TCRß clonotypes that expand after infection have stronger TCR-dependent activation than do contracted clonotypes. Our results demonstrate that host exposure to antigen may drive clonal expansion of MAIT cells with increased functional avidity, suggesting a role for specific vaccination strategies to increase the frequency and potency of MAIT cells to optimize effector function.

Diverse Streptococcus pneumoniae Strains Drive a Mucosal-Associated Invariant T-Cell Response Through Major Histocompatibility Complex class I-Related Molecule-Dependent and Cytokine-Driven Pathways.

Mucosal-associated invariant T (MAIT) cells represent an innate T-cell population that can recognize ligands generated by the microbial riboflavin synthesis pathway, presented via the major histocompatibility complex class I-related molecule (MR1). Streptococcus pneumoniae is a major human pathogen that is also associated with commensal carriage; thus, host control at the mucosal interface is critical. The recognition of pneumococci by MAIT cells has not been defined nor have the genomics and transcriptomics of the riboflavin operon. We observed robust recognition of pneumococci by MAIT cells, using both MR1-dependent and MR1-independent pathways. The pathway used was dependent on the antigen-presenting cell. The riboflavin operon was highly conserved across a range of 571 pneumococci from 39 countries, dating back to 1916, and different versions of the riboflavin operon were also identified in related Streptococcus species. These data indicate an important functional relationship between MAIT cells and pneumococci.

Nutritional Stress Induced by Tryptophan-Degrading Enzymes Results in ATF4-Dependent Reprogramming of the Amino Acid Transporter Profile in Tumor Cells.

Tryptophan degradation is an immune escape strategy shared by many tumors. However, cancer cells' compensatory mechanisms remain unclear. We demonstrate here that a shortage of tryptophan caused by expression of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) resulted in ATF4-dependent upregulation of several amino acid transporters, including SLC1A5 and its truncated isoforms, which in turn enhanced tryptophan and glutamine uptake. Importantly, SLC1A5 failed to be upregulated in resting human T cells kept under low tryptophan conditions but was enhanced upon cognate antigen T-cell receptor engagement. Our results highlight key differences in the ability of tumor and T cells to adapt to tryptophan starvation and provide important insights into the poor prognosis of tumors coexpressing IDO and SLC1A5. Cancer Res; 76(21); 6193-204. ©2016 AACR.

MR1-Restricted Mucosal-Associated Invariant T Cells and Their Activation during Infectious Diseases.

MR1-restricted mucosal-associated invariant T (MAIT) cells recognize vitamin B metabolites, which are generated by a broad range of bacteria, from Escherichia coli to Mycobacterium tuberculosis and BCG. MAIT cells have been described as innate sensors of infection as they accumulate early in infected tissues. MAIT cells maintain an activated phenotype throughout the course of infections, secrete inflammatory cytokines, and have the potential to directly kill infected cells, playing an important role in shaping the host response. In this review, we will discuss the current knowledge regarding the molecular mechanisms that underline MAIT cells activation in sterile and non-sterile inflammatory conditions.

Immunology of a Transmissible Cancer Spreading among Tasmanian Devils.

Devil facial tumor disease (DFTD) is a transmissible cancer that has killed most of the Tasmanian devil (Sarcophilus harrissii) population. Since the first case appeared in the mid-1990s, it has spread relentlessly across the Tasmanian devil's geographic range. As Tasmanian devils only exist in Tasmania, Australia, DFTD has the potential to cause extinction of this species. The origin of DFTD was a Schwann cell from a female devil. The disease is transmitted when devils bite each other around the facial areas, a behavior synonymous with this species. Every devil that is 'infected' with DFTD dies from the cancer. Once the DFTD cells have been transmitted, they appear to develop into a cancer without inducing an immune response. The DFTD cancer cells avoid allogeneic recognition because they do not express MHC class I molecules on the cell surface. A reduced genetic diversity and the production of immunosuppressive cytokines may also contribute.

Identification of dendritic cells, B cell and T cell subsets in Tasmanian devil lymphoid tissue; evidence for poor immune cell infiltration into devil facial tumors.

The Tasmanian devil is under threat of extinction due to the transmissible devil facial tumor disease (DFTD). This fatal tumor is an allograft that does not induce an immune response, raising questions about the activity of Tasmanian devil immune cells. T and B cell analysis has been limited by a lack of antibodies, hence the need to produce such reagents. Amino acid sequence analysis revealed that CD4, CD8, IgM, and IgG were closely related to other marsupials. Monoclonal antibodies were produced against CD4, CD8, IgM, and IgG by generating bacterial fusion proteins. These, and commercial antibodies against CD1a and CD83, identified T cells, B cells and dendritic cells by immunohistochemistry. CD4(+) and CD8(+) T cells were identified in pouch young thymus, adult lymph nodes, spleen, bronchus- and gut-associated lymphoid tissue. Their anatomical distribution was characteristic of mammalian lymphoid tissues with more CD4(+) than CD8(+) cells in lymph nodes and splenic white pulp. IgM(+) and IgG(+) B cells were identified in adult lymph nodes, spleen, bronchus-associated lymphoid tissue and gut-associated lymphoid tissue, with more IgM(+) than IgG(+) cells. Dendritic cells were identified in lymph node, spleen and skin. This distribution is consistent with eutherian mammals and other marsupials, indicating they have the immune cell subsets for an anti-tumor immunity. Devil facial tumor disease tumors contained more CD8(+) than CD4(+) cells, but in low numbers. There were also low numbers of CD1a(+) and MHC class II(+) cells, but no CD83(+) IgM(+) or IgG(+) B cells, consistent with poor immune cell infiltration.