Adult T-cell leukaemia/lymphoma (ATL) arises from chronic non-malignant human T lymphotropic virus type-1 (HTLV-1) infection which is characterized by high plasma pro-inflammatory cytokines whereas ATL is characterized by high plasma anti-inflammatory (IL-10) concentrations. The poor prognosis of ATL is partly ascribed to disease-associated immune suppression. ATL cells have a CD4+CCR4+CD26-CD7- immunophenotype but infected cells with this immunophenotype (‘ATL-like’ cells) are also present in non-malignant HTLV-1 infection. We hypothesized that ‘ATL-like’ and ATL cells have distinct cytokine producing capacity and a switch in the cytokines produced occurs during leukemogenesis. Seventeen asymptomatic carriers (ACs), 28 patients with HTLV-1-associated myelopathy (HAM) and 28 with ATL were studied. Plasma IL-10 concentration and the absolute frequency of IL-10-producing CD4+ T cells were significantly higher in patients with ATL compared to AC. IL-10-producing ATL cells were significantly more frequent than ‘ATL-like’ cells. The cytokine-producing cells were only a small fraction of ATL cells. Clonality analysis revealed that even in patients with ATL the ATL cells were composed not only of a single dominant clone (putative ATL cells) but also tens of non-dominant infected clones (‘ATL-like’ cells). The frequency of cytokine-producing cells showed a strong inverse correlation with the relative abundance of the largest clone in ATL cells suggesting that the putative ATL cells were cytokine non-producing and that the ‘ATL-like’ cells were the primary cytokine producers. These findings were confirmed by RNAseq with cytokine mRNA expression in ATL cells in patients with ATL (confirmed to be composed of both putative ATL and ‘ATL-like’ cells by TCR analysis) significantly lower compared to ‘ATL-like’ cells in patients with non-malignant HTLV-1 infection (confirmed to be composed of hundreds of non-dominant clones by TCR analysis). A significant inverse correlation between the relative abundance of the largest clone and cytokine mRNA expression was also confirmed. Finally, ‘ATL-like’ cells produced less pro- and more anti-inflammatory cytokines than non ‘ATL-like’ CD4+ cells (which are predominantly HTLV uninfected). In summary, HTLV-1 infection of CD4+ T cells is associated with a change in cytokine producing capacity and dominant malignant clonal growth is associated with loss of cytokine producing capacity. Non-dominant clones with ‘ATL-like’ cells contribute to plasma cytokine profile in patients with non-malignant HTLV-1 infection and are also present in patient with ATL.
Human T- lymphotropic virus type-1 (HTLV-1) is a complex delta retrovirus infecting an estimated 10 million individuals worldwide [1]. In the majority, infection leads to a chronic asymptomatic carrier state (AC) but 2% to 6% develop adult T-cell leukaemia/lymphoma (ATL) and another 3% inflammatory disorders e. g. HTLV-1-associated myelopathy (HAM). The diagnosis of ATL is based on clinical features, morphology (lymphocytes with characteristic ‘flower cell’ morphology), immunophenotyping (CD3+, CD4+, CCR4+, CD25+, CD26- and CD7-) and demonstration of dominant HTLV-1 infected clones [2,3]. ATL is classified into four subtypes: smouldering, chronic, acute and lymphoma. Smouldering and chronic ATL are suggested to have an indolent course while acute and lymphoma an aggressive course [3]. Survival with chemotherapy is poor [4–7] due to primary chemo-refractory disease, or early relapse or opportunistic infections. [8–11]. Patients with ATL have high plasma concentrations of anti-inflammatory cytokines e. g. interleukin (IL) -10 [12,13]. IL-10 is secreted by regulatory CD4+ T cells[14]. CD4+ T cells in patients with ATL have been shown to have high IL-10 expression [13,15]. ATL cells express regulatory T cell-associated markers CD25 and FOXP3 [16–19]. This has led to the assumption that ATL cells are a regulatory T cell counterpart and mediate an immunosuppressive clinical state by secreting IL-10. In contrast HAM is characterized by organ damage due to immune activation, and high concentrations of plasma pro-inflammatory cytokines e. g. interferon γ (IFNγ) [20]. HTLV-1 infected cells secrete IFNγ [21,22] and directly contribute to the plasma cytokine profile in HAM. These findings suggest a distinct cytokine producing capacity of HTLV-1 infected cells in keeping with clinical state. ATL arises de novo in AC and patients with HTLV-1-associated inflammation such as HAM, hereafter referred to as non-malignant HTLV-1 infection. HTLV-1-infected cells have a CD4+CCR4+CD26- immunophenotype; the loss of CD7 expression by these cells differentiates ATL from non-malignant HTLV-1 infection [23–27]. We and others have shown that ‘ATL-like’ HTLV-1-infected (CD4+CCR4+CD26-CD7-) cells are present in patients with non-malignant HTLV infection. We hypothesize that the cytokine producing capacity of ATL and ‘ATL-like’ cells are distinct from each other and are directly responsible for the respective plasma cytokine profile in ATL and non-malignant HTLV-1 infection, and that the change in cytokine producing capacity reflects malignant transformation from non-malignant HTLV-1 infection to ATL. Pro- and anti-inflammatory cytokine were measured in plasma and cells (intracellular staining and gene expression by mRNA sequencing) in patients with four different HTLV diagnoses: AC; HAM; indolent ATL; aggressive ATL. Clonality analysis of ATL and ‘ATL-like’ cells was performed and the relationship between plasma, cellular cytokine, clonality and clinical state was studied.
The patient cohort is based at the National Centre for Human Retrovirology (NCHR) at St Mary’s Hospital, Paddington, London, UK and the University of Miami School of Medicine, Miami, USA. Diagnosis of HTLV-1 infection, HAM and ATL was made according to World Health Organization criteria. Patients’ samples are collected and stored in a Communicable Diseases Research Tissue Bank approved by the UK National Research Ethics Service (references 09/H0606/106 and 15/SC/0089). Samples at University of Miami School of Medicines are collected under IRB-approved study" Study of Blood, Tissue and Body Fluids of Viral Associated Malignancies" (EPROST No. 20030608). Samples are stored under license in accordance with the Human Tissues Act 2004. Samples were collected prior to any systemic therapy in patient with ATL or immune modulatory treatment in patients with HAM. Baseline demographic and clinical characteristics of the study population are shown in S1 Table. Peripheral blood mononuclear cells (PBMCs) and plasma were separated from fresh whole blood by density-gradient centrifugation on Histopaque-1077 (Sigma-Aldrich, St Louis, USA). Plasma was stored at -80°C. PBMCs were harvested from the interface, washed in phosphate buffer saline (PBS, Sigma-Aldrich), cryopreserved in 10% dimethyl sulphoxide (Sigma-Aldrich) and 90% heat inactivated foetal calf serum (FCS) (Gibco, Carlsbad, USA), and stored in liquid nitrogen until use. Plasma cytokine concentrations of the following nine cytokines/chemokines were measured using sensitive and specific V_PLEX immunoassays according to the manufacturer’s protocol (Meso Scale Discovery, Gaithersburg, USA): Human pro-inflammatory cytokines: interferon gamma (IFNγ), interleukin (IL) -2, IL-6, IL-7, IL-17α, tumour necrosis factor-alpha (TNFα); anti-inflammatory cytokine: IL-10 and chemokines: macrophage-derived chemokine (MDC) also known as CCL22 and C-X-C motif chemokine 10 (CXCL-10) also known as IFNγ-induced protein 10 (IP-10), . For samples with detectable concentrations below the limits of quantification missing values were replaced with 99% of the lowest detectable concentration. Thawed cryopreserved PBMCs were incubated in either complete media (CM) comprising 10% heat inactivated FCS in RPMI with L-glutamine plus 1% Penicillin only, CM with phytohemaggulutinin (PHA, final concentration of 5 μg/mL, Sigma) or CM with 2% cell activation cocktail (containing phorbol 12-myristate 13-acetate (PMA) and ionomycin, (BioLegend, San Diego, USA) ) at 37°C, in 5% CO2 at a concentration of 106 cells/ 50 μL for 6 hours. Brefeldin A (BioLegend) was added for the last five hours. All washes were done by suspending cells in PBS containing 1% FCS followed by centrifugation at 600g for 5 minutes twice. PBMCs were washed thrice at the end of incubation and stained sequentially with near infrared fixable viability stain followed by flurochrome conjugated monoclonal antibodies against cell surface markers (CD3, CD4, CD7, CD8 and CCR4) for 30 minutes at room temperature (RT). The cells were then fixed using fixation buffer from FoxP3 / Transcription Factor Staining Buffer Set (eBioscience, San Diego, USA). The fixed cells were washed with permeablization buffer followed by incubation with fluorochrome-conjugated monoclonal antibodies against IL-6, IL-10, TNFα and IFNγ for 15 minutes at RT. PBMCs were then washed twice and stored at 4°C in PBS 1% FCS overnight until analysis on Becton Dickinson Fortessa II. A minimum of 20,000 events were recorded for analysis. Compensation was computed using BD FACS diva software using single staining of Comp ebeads (eBioscience) and checked manually. Fluorescence minus one control (antibody cocktail containing all antibodies except one) was used for gating. Data were analysed by Flowjo software. Magnetic cell sorting was performing by negative selection. Thawed PBMCs were incubated with primary biotinylated antibodies (CD7, CD26, CD8, CD14, CD15, CD16, CD19, CD36, CD 56, CD123, TCR γ/δ, CD235a [Miltenyi Biotec Ltd. , United Kingdom and Biolegend, USA]) at 4°C for 10 min and 107 cells/100 μl. All washes were done by suspending cells in 1% BSA (Sigma) followed by centrifugation at 600g for 5 minutes twice. Labelled PBMCs were washed and incubated with 30% dilution of streptavidin conjugated microbeads at a final concentration of 107 cells/100 μL. Microbead-conjugated PBMCs were washed and magnetically sorted twice on LS Columns (Miltenyi Biotec Ltd) according to the manufacturer’s directions. The flow-through containing CD3+CD4+CD7-CD26-cells, of which 99% were CCR4+, was washed twice. Of these cells, 104 cells were used to check for purity by flow cytometry and the remainder were used for HTLV-1 proviral load quantification and clonality analysis. Genomic DNA was extracted from PBMCs and sorted CD4+ T cell subsets using QIAamp DNA mini kit (Qiagen, Hilden, Germany). The proviral load was determined by real-time PCR as previously described [28]. Clonality analysis was performed using linker-mediated (LM) -PCR, high-throughput sequencing analysis of HTLV-1 integration sites according to the method previously described [29]. Random fragments of 1 μg genomic DNA (100 ng of sample DNA mixed with 900 ng of uninfected DNA from Jurkat cells) were generated by sonication and ligated to custom, partially double-stranded, DNA adaptors. Nested PCR using specific primers was performed to selectively amplify adaptor-ligated DNA fragments abutting the HTLV-1 3’ LTR. The PCR products from each sample had two unique 8bp multiplexing barcodes and were pooled for sequencing. Paired-end 150-base reads were generated on an Illumina MiSeq. The reads were de-multiplexed using the MiSeq reporter. The linker and primer sequences are listed in S2 Table. The in silico analysis was conducted in shell and R environment. Read 1 and Read 2 were aligned to reference human (hg38, UCSC) and HTLV-1 genomes using Bowtie2[30]. The number of unique integration sites (clones) and the relative and absolute abundance of each unique integration site (clonal size) were calculated as previously described [29]. RNA was extracted from MACS-sorted cells using Qiagen Allprep DNA/RNA column-based extraction kits (Qiagen) as per the manufacturer’s instructions. RNA-seq was performed on DNase-treated samples using TruSeq Stranded mRNA Library Prep Kit (Illumina, United States) as per the manufacturer’s instructions. All sorted cells had the highest RNA quality (> 100 ng RNA and RNA quality score ≥ 8). All sequencing was performed using 50 nucleotide paired-end reads on an Illumina HiSeq 4000 instrument at the Imperial biomedical research centre (BRC) genomics facility, London, United Kingdom. Statistical analysis was performed using Graphpad Prism software. The significance of difference in continuous variables between multiple patient groups was determined by a Kruskal-Wallis test with Dunn post-test analysis. The significance of difference in continuous variables between two cell subsets was determined using a Wilcoxon signed-rank test. The significance of difference in contingency variables was determined by a chi-squared test. Differences were considered statistically significant if p <0. 05. The correlation between two continuous variables was determined by a non-parametric Spearman test. The Spearman correlation was considered significant for p<0. 05 and showing a trend if the p value was between 0. 05 and 0. 1. A classification tree was constructed to identify the hierarchical organisation of plasma cytokine concentration in ACs, patients with HAM and patients with ATL. The classification tree was produced using a Recursive Partitioning and Regression Trees (RPART) analysis in R. The network analysis was performed using NodeXL. In the analysis, absolute CD3+, CD4+ and CD8+ cell counts and PBMC PVL were used as cellular variables, and plasma cytokine concentrations were used as cytokine variables. The cellular and cytokine variables were used as edges of the network while the Spearman correlates between nodes were used as vertices. Vertices with at least a significant trend on Spearman correlation (p <0. 1) were included. The layout was performed using Harel-Koren Fast multiscale.
The relative and absolute frequencies of CD3+, CD4+ T cells and HTLV-1 PVL in PBMCs were significantly higher in patients with ATL compared to ACs or patients with HAM (Table 1). Patients with HAM had significantly higher plasma concentrations of IFNγ, CXCL10, IL-2 and IL-17 (pro-inflammatory cytokines) compared to ACs and patients with ATL (Fig 1A–1D). Patients with ATL and patients with HAM had significantly higher plasma concentrations of the anti-inflammatory cytokine IL-10 compared to ACs (Fig 1E). There was no significant difference in plasma concentrations of IL-6, IL-7, CCL22 and TNFα between the three patient groups (Fig 1F–1I). The median plasma concentrations of IL-6 and TNFα in all three patient groups were near the upper limit of the manufacturer’s normal human range while those of CCL22, IL-2 and IL-7 were near the lower limit. Patients with aggressive ATL had significantly higher TNFα, IL-6 and IL-10 plasma concentrations than those with indolent ATL (Fig 2A–2C). There was no significant difference in plasma concentrations of IFNγ, CXCL10, CCL22, IL-2 and IL-7 between ATL subtypes as shown in S1 Fig. The plasma chemokine and cytokine concentrations, of four patients with HAM who developed de novo aggressive ATL and two patients with indolent ATL who progressed to aggressive ATL, were measured at aggressive ATL diagnosis and at 3–12 months earlier. Plasma TNFα, IL-6 and IL-10 concentrations were significantly higher at diagnosis, with median increases of 1. 9-fold; 3. 7-fold and 7. 1-fold respectively (Fig 2D). There was a large variance in changes of plasma IFNγ concentrations. In summary, patients with HAM have the highest concentrations of pro-inflammatory cytokine and ACs had the lowest IL-10 plasma concentrations. Patients with aggressive ATL had higher plasma concentrations of TNFα, IL-6 and IL-10 compared to patients with indolent ATL. These higher plasma concentrations did not precede malignant progression. Network analysis was performed to better understand the interaction between cellular and plasma immune markers. The absolute frequency of CD3+, CD4+ T cells and PVL were significantly positively correlated with each other in patients with HAM or ATL (Fig 3A and 3B). There were also significant positive correlations between the plasma concentrations of TNFα, IL-6 and IL-10 in all three diagnostic groups (Fig 3A–3C). The plasma concentration of IL-10 correlated significantly with IFNγ in patients with ATL and to a lesser extent with both IFNγ and IL-17 in patients with HAM. A classification tree analysis was performed on plasma cytokine concentrations to identify the cytokine profile which best differentiated AC, HAM and ATL states. A plasma IL-10 concentration < 0. 16 pg/mL identified AC with a 64. 7% sensitivity and 73% specificity as shown in Fig 3D. A plasma IL-17 concentration <1 pg/mL or if the IL-17 concentration was >1 pg/mL an IL-10 concentration of >0. 8 pg/mL identified ATL with 78. 5% sensitivity and 85% specificity whilst an IL-17 concentration of ≥ 1 pg/mL with an IL-10 concentration between 0. 16 and 0. 8 pg/mL identified HAM with 82. 1% sensitivity and a specificity of 71. 8%. In summary, a positive correlation between the plasma concentrations of specified pro- and anti-inflammatory cytokines was present in all three HTLV-1 patient groups. Together, the plasma concentrations of IL-10 and IL-17 discriminated between the clinical states associated with HTLV-1 infection. To identify the cellular source of the cytokines identified in plasma, the cytokine producing capacity of monocytes, CD4+ and CD8+ T cells was studied. Intracellular cytokine staining for TNFα, IFNγ, IL-6 and IL-10 was performed in 10 ACs, 11 patients with HAM and 10 with ATL (four with indolent and six with aggressive ATL). The gating strategy to detect cytokine producing cells is shown in S2 Fig. The relative and absolute frequencies of cells secreting each cytokine are shown as percentages and cell count per litre. Although there was no difference in the relative frequency of IL-10+ CD4+ cells (Fig 4E) their absolute frequency was higher in patients with ATL compared to AC and HAM (Fig 4A). The relative and absolute frequency of IL-6+ CD4+ cells did not differ by disease state (Fig 4B–4F). The absolute frequencies of TNFα+ CD4+ T-cells were the same in each group (Fig 4C) but TNF+ cells made up a significantly lower percentage of all CD4+ T cells in patients with ATL compared to non-malignant HTLV-1 infection (Fig 4G). Finally, although the absolute frequency of CD4+ T-cells secreting IFNγ was increased in ATL (Fig 4D), the relative frequency of these cells was significantly lower in patients with ATL than in ACs and patients with HAM (Fig 4H). In patients with ATL the median relative frequencies of TNFα, IFNγ, IL-6 and IL-10 secreting CD4+ cells were 5%, 1. 7%, 0. 6% and 0. 3% respectively. The frequencies of TNFα, IFNγ, IL-6 and IL-10-secreting CD8+ cells and monocytes (except IFNγ, which is not secreted by monocytes) did not differ between diagnostic groups as shown in S3 Fig. In summary, although the absolute frequency of the cytokine-producing CD4+ T-cells was greater in patients with ATL these make up only a minority of their CD4+ T cells suggesting that ATL cells are in general not secreting these cytokines. CD4+ T cells are the dominant reservoir of infected cells in both non-malignant HTLV-1 infection and ATL [23,35]. The infected cells are derived from thousands of non-dominant clones in non-malignant HTLV-1 infection and from a dominant clone on a polyclonal background of non-dominant clones in ATL [29,36,37]. To further characterise the ATL and ‘ATL-like’ cells, their cytokine producing capability was determined. ATL cells have a CD4+CCR4+CD26-CD7- immunophenotype and ‘ATL-like’ cells are present in non-malignant HTLV-1 infection. The CD4+CCR4+CD7- immunophenotype was used to study the cytokine producing capacity of ATL cells as >99% of CD4+CCR4+CD7- cells were also CD26-. The relative (Fig 5A) and absolute frequencies (Fig 5B) of CD4+CCR4+CD7- T cells were significantly higher in patients with ATL compared to non-malignant HTLV-1 infection whilst there was no difference in the absolute frequency of non-CCR4+CD7- CD4+ T cells (Fig 5B). HTLV-1 PVL significantly and positively correlated with the relative (rho = 0. 87, p<0. 0001) and absolute (rho = 0. 88, p<0. 0002) frequencies of CD4+CCR4+CD7- cells but not the absolute frequency of non-CCR4+CD7- CD4+ T cells (rho = 0. 20, p = 0. 27). This confirms CD4+CCR4+CD7- cells as marker of ATL and ‘ATL-like’ HTLV-1 infected cells in patients with ATL and non-malignant HTLV-1 infection respectively. HTLV-1 infection leads to an absolute increase in ‘ATL-like’ cells in patients with non-malignant HTLV-1 infection. The relative and absolute frequency of ATL cells was actually higher than non-ATL CD4+T cells in patients with ATL but there was no difference between ‘ATL-like’ and non- ‘ATL-like’ CD4+ T cells in non-malignant HTLV-1 infection. The absolute frequency of IL-10-producing CD4+CCR4+CD7- cells in patients with ATL was significantly higher compared to ACs and HAM (Fig 6A). The relative frequency of IL-10-producing CD4+CCR4+CD7- cells in patients with ATL was lower compared to AC and HAM (Fig 6E). The absolute frequency of IL-6-producing CD4+CCR4+CD7- cells in patients with ATL was higher compared to ACs and patients with HAM (Fig 6B). The relative frequency of IL-6-producing CD4+CCR4+CD7- cells in patients with ATL was low compared to AC and patients with HAM (Fig 6F). The absolute frequency of CD4+CCR4+CD7- T cells producing TNFα (Fig 6C) or IFNγ (Fig 6D) was significantly higher in patients with ATL compared to ACs whilst there was a trend when compared to patients with HAM (p = 0. 11 and p = 0. 10 respectively). However, the relative frequencies of TNFα and IFNγ producing CD4+CCR4+CD7- cells were significantly lower in patients with ATL compared to ACs and HAM as shown in Fig 6G and 6H. The median relative frequencies of TNFα, IFNγ, IL-6 and IL-10 secreting CD4+CCR4+CD7- T cells in patients with ATL were 3. 3%, 1. 7%, 0. 2% and 0. 3% respectively. The CD4+ T cells which are not CCR4+CD7- are a mixture of predominantly uninfected (82%) and infected cells (18%) [23]. The uninfected non-CCR4+CD7- CD4+T cells are a mix of naïve and memory CD4+T cells. CD4+naïve T cells have been shown to be depleted in HTLV-1 infection. In addition, naïve CD4+T cells produce less cytokine than memory T cells suggesting that the cytokine producing capacity of non-CCR4+CD7- CD4+T cells is mainly derived from uninfected memory T cells. The absolute and relative frequencies of pro- inflammatory cytokine (TNFα, IFNγ and IL-6) producing CCR4+CD7- cells (i. e. ‘ATL-like’ infected cells) was lower than in non-CCR4+CD7- CD4+ T cells (i. e. predominantly HTLV-1 uninfected memory cells, Fig 6F–6H) in AC and patients with HAM. The absolute and relative frequencies of CD4+CCR4+CD7- cells producing the anti-inflammatory cytokine IL-10 was higher compared to non-CCR4+CD7- CD4+T cells in ACs and patients with HAM as shown in Fig 6A–6H. The relative frequency of pro-inflammatory cytokine producing CD4+CCR4+CD7- cells was lower than non-CCR4+CD7- CD4+T cells whilst the absolute frequency of IL-10 secretion was higher in patients with ATL. The absolute and relative frequencies of TNFα, IFNγ, IL-6 and IL-10 secreting non-CCR4+CD7- CD4+cells did not differ significantly between the three clinical groups. In summary, patients with non-malignant HTLV-1 infection have an increased frequency of CD4+CCR4+CD7- (‘ATL-like’ infected cells) which correlates strongly with PVL. There is a further increase in these cells in patients with ATL. These cells are capable of producing pro- and anti-inflammatory cytokines. However, CD4+CCR4+CD7- cells had lower pro- and higher anti-inflammatory cytokine producing capacity compared to non-CCR4+CD7- CD4+T cells (predominantly uninfected cells) in non-malignant HTLV-1 infection. The absolute frequencies of not only IL-10 cytokine secreting CD4+CCR4+CD7- cells but also TNFα, IFNγ and IL-6 secretors were higher in ATL compared to non-malignant HTLV-1 infection. However, the cytokine producing cells made up only a tiny fraction of CD4+CCR4+CD7- T cells in patients with ATL. This suggests that at some point in the transformation of ‘‘ATL-like’ to ‘ATL’ cells CD4+CCR4+CD7- lose their cytokine producing capacity and that they are not the source of the plasma cytokines observed in ATL. The question remains whether the cytokine-secreting ‘ATL’ cells are a sub-population of the malignant clone or ‘ATL-like’ cells. Patients with ATL have a putative dominant clone on a polyclonal background. The dominant cell population in ATL is CD4+CCR4+CD7-. In order to determine the likely clonal origin of cytokine secreting CD4+CCR4+CD7- T cells clonality analysis was performed within sorted CD4+CCR4+CD7- T cells in four patients with aggressive ATL. In these patients, the CD4+CCR4+CD7- cells population had a median of 41 clones with the largest clone contributing a median 88% of the HTLV-1 infection burden as shown in Table 2. Thus, CD4+CCR4+CD7- cells in patients with ATL are derived not exclusively from a single dominant clone (putative ATL cells) but also from tens of non-dominant infected clones i. e. ‘ATL-like’ cells. There was a perfect negative correlation (rho = -0. 99, p<0. 0001) between the relative frequencies of cytokine secreting cells and the relative abundance of the largest clone further supporting the suggestion that ‘ATL-like’ infected cells from non-dominant clones are cytokine producing whilst the cells from the dominant clone secrete little or no cytokines in patients with ATL. In order to determine the global and confirm the deferential cytokine profile of CD4+CCR4+CD7- cells in patients with non-malignant HTLV-1 infection and ATL, we studied the expression of cytokines, chemokines and their receptors by mRNA sequencing of sorted CD4+CCR4+CD7- T cells in eight patients with non-malignant (four AC and four patients with HAM) and eight with ATL.
There is an absolute increase in ‘ATL-like’ cells made up of non-dominant infected clones in patients with non-malignant HTLV-1 infection (AC and patients with HAM). There is a further expansion of these ‘ATL-like’ cells in ATL along with the presence of ATL cells. The ‘ATL-like’ cells from non-dominant clones have a distinct cytokine producing pattern and contribute directly to the plasma cytokine profile in both non-malignant HTLV-1 infection and possibly in ATL. The ‘ATL-like’ cells of the dominant clone (the putative ATL cells) possess little or no cytokines producing capability. There was progressive loss of pro-inflammatory cytokine producing capacity from non-ATL (uninfected) through ‘ATL-like’ (infected) to ATL (malignant) cells and we hypothesise that this represents stages in the transformation process.
Human T-cell lymphotropic virus type-1 (HTLV-1) infection of CD4+ T cells is associated with a change in their cytokine producing capacity and is responsible for the different plasma cytokine profiles in patients with adult T-cell leukaemia/Lymphoma (ATL) and non-malignant HTLV-1 infection. Dominant malignant clonal growth of the infected CD4+ T cells is associated with loss of cytokine producing capacity. ACs, patients with HAM and patients with ATL have a common cytokine cluster with positive correlations between pro- (TNFα and IL-6) and anti- (IL-10) inflammatory cytokines. Plasma IL-10 was higher in the HAM and ATL states compared to AC whilst there was no difference in pro-inflammatory cytokines. Patients with HAM have raised plasma concentrations of IFNγ, IL-10 and IL-17 suggesting a complex interaction between these cytokine in HAM which was not seen in ATL. Aggressive ATL is associated with raised plasma concentrations of pro- and anti-inflammatory cytokines compared to indolent ATL. This cytokine profile did not precede or predict aggressive ATL. The ‘ATL-like’ infected cells in ACs and in patients with HAM have lower pro- and higher anti-inflammatory cytokine secretion than non- ‘ATL-like’ cells which are predominantly HTLV-1 uninfected. Putative ATL cells have little or no cytokine producing capacity. ‘ATL-like’ infected cells from non-dominant infected clones were present not only in patients with non-malignant HTLV-1 infection but also ATL. ‘ATL-like’ cells have cytokine producing capacity and contribute to plasma cytokine profile in patients with non-malignant HTLV-1 infection and possibly also in ATL.
blood cells
innate immune system
medicine and health sciences
immune physiology
cytokines
pathology and laboratory medicine
immune cells
body fluids
pathogens
immunology
microbiology
opportunistic infections
cloning
retroviruses
viruses
developmental biology
rna viruses
signs and symptoms
molecular development
molecular biology techniques
research and analysis methods
infectious diseases
white blood cells
inflammation
animal cells
medical microbiology
htlv-1
gene expression
microbial pathogens
t cells
molecular biology
blood plasma
immune response
immune system
diagnostic medicine
blood
cell biology
anatomy
physiology
viral pathogens
genetics
biology and life sciences
cellular types
organisms
Switching and loss of cellular cytokine producing capacity characterize in vivo viral infection and malignant transformation in human T- lymphotropic virus type 1 infection