The role of HTLV-1 clonality, proviral structure, and genomic integration site in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) occurs in ∼5% of human T-lymphotropic virus type 1 (HTLV-1)-infected individuals and is conventionally thought to be a monoclonal disease in which a single HTLV-1(+) T-cell clone progressively outcompetes others and undergoes malignant transformation. Here, using a sensitive high-throughput method, we quantified clonality in 197 ATL cases, identified genomic characteristics of the proviral integration sites in malignant and nonmalignant clones, and investigated the proviral features (genomic structure and 5' long terminal repeat methylation) that determine its capacity to express the HTLV-1 oncoprotein Tax. Of the dominant, presumed malignant clones, 91% contained a single provirus. The genomic characteristics of the integration sites in the ATL clones resembled those of the frequent low-abundance clones (present in both ATL cases and carriers) and not those of the intermediate-abundance clones observed in 24% of ATL cases, suggesting that oligoclonal proliferation per se does not cause malignant transformation. Gene ontology analysis revealed an association in 6% of cases between ATL and integration near host genes in 3 functional categories, including genes previously implicated in hematologic malignancies. In all cases of HTLV-1 infection, regardless of ATL, there was evidence of preferential survival of the provirus in vivo in acrocentric chromosomes (13, 14, 15, 21, and 22).

Accurate interrogation of FCGR3A rs396991 in European and Asian populations using a widely available TaqMan genotyping method.

A polymorphism in the receptor for the Fc region of IgG, Fc γ-receptor IIIa (FcγRIIIa, FCGR3A rs396991), has been inconsistently shown in the literature to have an effect on response to monoclonal antibody therapy in several indications. The rs396991 (T/G) polymorphism leads to an F176V substitution and increased affinity for IgG. This variant has proven difficult to genotype accurately, primarily because of extensive homology between the FCGR3A and FCGR3B genes. We have shown that rs396991 can be genotyped by PCR amplification, followed by direct Sanger sequencing of the product, without coamplification of FCGR3B, and that the rs396991 TaqMan assay (C__25815666_10) agrees with Sanger sequencing results in 100% of European and Asian samples tested, but it has a small error rate in African and American populations. C__25815666_10 is therefore suitable to interrogate rs396991 in studies involving Europeans and Asians; however for other populations, the default genotyping method should be PCR followed by Sanger sequencing.

HTLV-1 proviral integration sites differ between asymptomatic carriers and patients with HAM/TSP.

BACKGROUND: HTLV-1 causes proliferation of clonal populations of infected T cells in vivo, each clone defined by a unique proviral integration site in the host genome. The proviral load is strongly correlated with odds of the inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is evidence that asymptomatic HTLV-1 carriers (ACs) have a more effective CD8 + T cell response, including a higher frequency of HLA class I alleles able to present peptides from a regulatory protein of HTLV-1, HBZ. We have previously shown that specific features of the host genome flanking the proviral integration site favour clone survival and spontaneous expression of the viral transactivator protein Tax in naturally infected PBMCs ex vivo. However, the previous studies were not designed or powered to detect differences in integration site characteristics between ACs and HAM/TSP patients. Here, we tested the hypothesis that the genomic environment of the provirus differs systematically between ACs and HAM/TSP patients, and between individuals with strong or weak HBZ presentation. METHODS: We used our recently described high-throughput protocol to map and quantify integration sites in 95 HAM/TSP patients and 68 ACs from Kagoshima, Japan, and 75 ACs from Kumamoto, Japan. Individuals with 2 or more HLA class I alleles predicted to bind HBZ peptides were classified 'strong' HBZ binders; the remainder were classified 'weak binders'. RESULTS: The abundance of HTLV-1-infected T cell clones in vivo was correlated with proviral integration in genes and in areas with epigenetic marks associated with active regulatory elements. In clones of equivalent abundance, integration sites in genes and active regions were significantly more frequent in ACs than patients with HAM/TSP, irrespective of HBZ binding and proviral load. Integration sites in genes were also more frequent in strong HBZ binders than weak HBZ binders. CONCLUSION: Clonal abundance is correlated with integration in a transcriptionally active genomic region, and these regions may promote cell proliferation. A clone that reaches a given abundance in vivo is more likely to be integrated in a transcriptionally active region in individuals with a more effective anti-HTLV-1 immune response, such those who can present HBZ peptides or those who remain asymptomatic.

Impact of isotype on the mechanism of action of agonist anti-OX40 antibodies in cancer: implications for therapeutic combinations.

BACKGROUND: OX40 has been widely studied as a target for immunotherapy with agonist antibodies taken forward into clinical trials for cancer where they are yet to show substantial efficacy. Here, we investigated potential mechanisms of action of anti-mouse (m) OX40 and anti-human (h) OX40 antibodies, including a clinically relevant monoclonal antibody (mAb) (GSK3174998) and evaluated how isotype can alter those mechanisms with the aim to develop improved antibodies for use in rational combination treatments for cancer. METHODS: Anti-mOX40 and anti-hOX40 mAbs were evaluated in a number of in vivo models, including an OT-I adoptive transfer immunization model in hOX40 knock-in (KI) mice and syngeneic tumor models. The impact of FcγR engagement was evaluated in hOX40 KI mice deficient for Fc gamma receptors (FcγR). Additionally, combination studies using anti-mouse programmed cell death protein-1 (mPD-1) were assessed. In vitro experiments using peripheral blood mononuclear cells (PBMCs) examining possible anti-hOX40 mAb mechanisms of action were also performed. RESULTS: Isotype variants of the clinically relevant mAb GSK3174998 showed immunomodulatory effects that differed in mechanism; mIgG1 mediated direct T-cell agonism while mIgG2a acted indirectly, likely through depletion of regulatory T cells (Tregs) via activating FcγRs. In both the OT-I and EG.7-OVA models, hIgG1 was the most effective human isotype, capable of acting both directly and through Treg depletion. The anti-hOX40 hIgG1 synergized with anti-mPD-1 to improve therapeutic outcomes in the EG.7-OVA model. Finally, in vitro assays with human peripheral blood mononuclear cells (hPBMCs), anti-hOX40 hIgG1 also showed the potential for T-cell stimulation and Treg depletion. CONCLUSIONS: These findings underline the importance of understanding the role of isotype in the mechanism of action of therapeutic mAbs. As an hIgG1, the anti-hOX40 mAb can elicit multiple mechanisms of action that could aid or hinder therapeutic outcomes, dependent on the microenvironment. This should be considered when designing potential combinatorial partners and their FcγR requirements to achieve maximal benefit and improvement of patient outcomes.

A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease more prevalent in people of African and Asian origin than Caucasian origin. FcgammaRIIb is an inhibitory Fc receptor with a critical role in immune regulation. Mouse data suggest that FcgammaRIIb deficiency increases susceptibility to autoimmune disease but protects against infection. We show that a SNP in human FCGR2B that abrogates receptor function is strongly associated with susceptibility to SLE in both Caucasians and Southeast Asians. The minor allele of this SNP is more common in Southeast Asians and Africans, populations from areas where malaria is endemic, than in Caucasians. We show that homozygosity for the minor allele is associated with substantial protection against severe malaria in an East African population (odds ratio = 0.56; P = 7.1 x 10(-5)). This protective effect against malaria may contribute to the higher frequency of this SNP and hence, SLE in Africans and Southeast Asians.

Copy number, linkage disequilibrium and disease association in the FCGR locus.

The response of a leukocyte to immune complexes (ICs) is modulated by receptors for the Fc region of IgG (FcgammaRs), and alterations in their affinity or function have been associated with risk of autoimmune diseases, including systemic lupus erythematosus (SLE). The low-affinity FcgammaR genomic locus is complex, containing regions of copy number variation (CNV) which can alter receptor expression and leukocyte responses to IgG. Combined paralogue ratio tests (PRTs) were used to distinguish three intervals within the FCGR locus which undergo CNV, and to determine FCGR gene copy number (CN). There were significant differences in FCGR3B and FCGR3A CNV profiles between Caucasian, East Asian and Kenyan populations. A previously noted association of low FCGR3B CN with SLE in Caucasians was supported [OR = 1.57 (1.08-2.27), P = 0.018], and replicated in Chinese [OR = 1.65 (1.25-2.18), P = 4 x 10(-4)]. There was no association of FCGR3B CNV with vasculitis, nor with malarial or bacterial infection. Linkage disequilibrium (LD) between multi-allelic FCGR3B CNV and SLE-associated SNPs in the FCGR locus was defined for the first time. Despite LD between FCGR3B CNV and a variant in FcgammaRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcgammaRIIB-232T allele were individually strongly associated with SLE risk. Thus CN of FCGR3B, which controls IC responses and uptake by neutrophils, and variations in FCGR2B, which controls factors such as antibody production and macrophage activation, are important in SLE pathogenesis. Further interpretations of contributions to pathogenesis by FcgammaRs must be made in the context of LD involving CNV regions.

Confirmation of the genetic association of CTLA4 and PTPN22 with ANCA-associated vasculitis.

BACKGROUND: The genetic contribution to the aetiology of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is not well defined. Across different autoimmune diseases some genes with immunomodulatory roles, such as PTPN22, are frequently associated with multiple diseases, whereas specific HLA associations, such as HLA-B27, tend to be disease restricted. We studied ten candidate loci on the basis of their immunoregulatory role and prior associations with type 1 diabetes (T1D). These included PTPN22, CTLA4 and CD226, which have previously been associated with AAV. METHODS: We genotyped the following 11 SNPs, from 10 loci, in 641 AAV patients using TaqMan genotyping: rs2476601 in PTPN22, rs1990760 in IFIH1, rs3087243 in CTLA4, rs2069763 in IL2, rs10877012 in CYP27B1, rs2292239 in ERBB3, rs3184504 in SH2B3, rs12708716 in CLEC16A, rs1893217 and rs478582 in PTPN2 and rs763361 in CD226. Where possible, we performed a meta-analysis with previous analyses. RESULTS: Both CTLA4 rs3087243 and PTPN22 rs2476601 showed association with AAV, P = 6.4 x 10-3 and P = 1.4 x 10-4 respectively. The minor allele (A) of CTLA4 rs3087243 is protective (odds ratio = 0.84), whereas the minor allele (A) of PTPN22 rs2476601 confers susceptibility (odds ratio = 1.40). These results confirmed previously described associations with AAV. After meta-analysis, the PTPN22 rs2476601 association was further strengthened (combined P = 4.2 x 10-7, odds ratio of 1.48 for the A allele). The other 9 SNPs, including rs763361 in CD226, showed no association with AAV. CONCLUSION: Our study of T1D associated SNPs in AAV has confirmed CTLA4 and PTPN22 as susceptibility loci in AAV. These genes encode two key regulators of the immune response and are associated with many autoimmune diseases, including T1D, autoimmune thyroid disease, celiac disease, rheumatoid arthritis, and now AAV.

Integration site and clonal expansion in human chronic retroviral infection and gene therapy.

Retroviral vectors have been successfully used therapeutically to restore expression of genes in a range of single-gene diseases, including several primary immunodeficiency disorders. Although clinical trials have shown remarkable results, there have also been a number of severe adverse events involving malignant outgrowth of a transformed clonal population. This clonal expansion is influenced by the integration site profile of the viral integrase, the transgene expressed, and the effect of the viral promoters on the neighbouring host genome. Infection with the pathogenic human retrovirus HTLV-1 also causes clonal expansion of cells containing an integrated HTLV-1 provirus. Although the majority of HTLV-1-infected people remain asymptomatic, up to 5% develop an aggressive T cell malignancy. In this review we discuss recent findings on the role of the genomic integration site in determining the clonality and the potential for malignant transformation of cells carrying integrated HTLV-1 or gene therapy vectors, and how these results have contributed to the understanding of HTLV-1 pathogenesis and to improvements in gene therapy vector safety.

Genetic variation, Fcγ receptors, KIRs and infection: the evolution of autoimmunity.

Recent work has emphasised the marked genetic variability that exists in the Fc receptor locus. This variation can contribute to the risk of autoimmune disease in both mice and humans, but can also have a profound impact on defence against infection. Using FcγRIIB and FcγRIIIB as examples, we demonstrate that variations associated with increased susceptibility to autoimmunity may be maintained in populations for their beneficial effect against infection. We examine the KIR locus from the same perspective and highlight similarities between the two loci. Intense selection pressure by pathogens presumably accounts for the marked variability within both regions and leads to susceptibility to autoimmunity for some alleles.

FcgammaRIIB, FcgammaRIIIB, and systemic lupus erythematosus.

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by the deposition of immune complexes in organs such as the kidney. This occurs as a result of multiple immunological abnormalities, including the production of high levels of autoantibody and dysregulated handling of immune complexes. Receptors for the Fc portion of IgG are critically involved in immune complex handling and clearance and in the regulation of B-cell activation. Polymorphisms in the low-affinity Fcgamma receptors have been associated with susceptibility to a number of autoimmune diseases, including SLE. We review the role of two such receptors in the pathogenesis of lupus-the inhibitory receptor FcgammaRIIB and the glycosylphosphatidylinositol-linked activatory receptor FcgammaRIIIB. Recent work has enhanced our understanding of the mechanism of action of the FcgammaRIIB I232T polymorphism and the overall role of this receptor in SLE. The human neutrophil antigen-1 allotypes of FcgammaRIIIB and the role of the receptor in SLE are discussed with regard to the recent determination of copy number variation in FCGR3B and the association of low copy number with SLE.

Distinct cell-specific control of autoimmunity and infection by FcgammaRIIb.

FcgammaRIIb is an inhibitory Fc receptor expressed on B cells and myeloid cells. It is important in controlling responses to infection, and reduced expression or function predisposes to autoimmunity. To determine if increased expression of FcgammaRIIb can modulate these processes, we created transgenic mice overexpressing FcgammaRIIb on B cells or macrophages. Overexpression of FcgammaRIIb on B cells reduced the immunoglobulin G component of T-dependent immune responses, led to early resolution of collagen-induced arthritis (CIA), and reduced spontaneous systemic lupus erythematosus (SLE). In contrast, overexpression on macrophages had no effect on immune responses, CIA, or SLE but increased mortality after Streptococcus pneumoniae infection. These results help define the role of FcgammaRIIb in immune responses, demonstrate the contrasting roles played by FcgammaRIIb on B cells and macrophages in the control of infection and autoimmunity, and emphasize the therapeutic potential for modulation of FcgammaRIIb expression on B cells in inflammatory and autoimmune disease.

A homozygous diploid subset of commercial wine yeast strains.

Genetic analysis was performed on 45 commercial yeasts which are used in winemaking because of their superior fermentation properties. Genome sizes were estimated by propidium iodide fluorescence and flow cytometry. Forty strains had genome sizes consistent with their being diploid, while five had a range of aneuploid genome sizes that ranged from 1.2 to 1.8 times larger. The diploid strains are all Saccharomyces cerevisiae, based on genetic analysis of microsatellite and minisatellite markers and on DNA sequence analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA of four strains. Four of the five aneuploid strains appeared to be interspecific hybrids between Saccharomyces kudriavzevii and Saccharomyces cerevisiae, with the fifth a hybrid between two S. cerevisiae strains. An identification fingerprint was constructed for the commercial yeast strains using 17 molecular markers. These included six published trinucleotide microsatellites, seven new dinucleotide microsatellites, and four published minisatellite markers. The markers provided unambiguous identification of the majority of strains; however, several had identical or similar patterns, and likely represent the same strain or mutants derived from it. The combined use of all 17 polymorphic loci allowed us to identify a set of eleven commercial wine yeast strains that appear to be genetically homozygous. These strains are presumed to have undergone inbreeding to maintain their homozygosity, a process referred to previously as 'genome renewal'.