CD8+ T cells and NK cells from blister fluid of an EEM/SJS overlap highly express Fas ligand and Granulysin

INTRODUCTION Erythema exsudativum multiforme majus (EEMM) and Stevens-Johnson Syndrome (SJS) are severe cutaneous reaction patterns caused by infections or drug hypersensitivity. The mechanism by which widespread keratinocyte death is mediated by the immune system in EEMM/SJS are still to be elucidated. Here, we characterized the blister cells isolated from a patient with EEMM/SJS overlap and investigated its cause. METHODS Clinical classification of the cutaneous eruption was done according to the consensus definition of severe blistering skin reactions and histological analysis. Common infectious causes of EEMM were investigated using standard clinical techniques. T cell reactivity for potentially causative drugs was assessed by lymphocyte transformation tests (LTT). Lymphocytes isolated from blister fluid were analyzed for their expression of activation markers and cytotoxic molecules using flow cytometry. RESULTS The healthy 58 year-old woman suffered from mild respiratory tract infection and therefore started treatment with the secretolytic drug Ambroxol. One week later, she presented with large palmar and plantar blisters, painful mucosal erosions, and flat atypical target lesions and maculae on the trunc, thus showing the clinical picture of an EEMM/SJS overlap (Fig. 1). This diagnosis was supported by histology, where also eosinophils were found to infiltrate the upper dermis, thus pointing towards a cutaneous adverse drug reaction (cADR). Analysis of blister cells showed that they mainly consisted of CD8+ and CD4+ T cells and a smaller population of NK cells. Both the CD8+ T cells and the NK cells were highly activated and expressed Fas ligand and the cytotoxic molecule granulysin (Fig. 2). In addition, in comparison to NK cells from PBMC, NK cells in blister fluids strongly upregulated the expression of the skin-homing chemokine receptor CCR4 (Fig 4). Surprisingly, the LTT performed on PBMCs in the acute phase was positive for Ambroxol (SI=2.9) whereas a LTT from a healthy but exposed individual did not show unspecific proliferation. Laboratory tests for common infectious causes of EEMM were negative (HSV-1/-2, M. pneumoniae, Parvovirus B19). However, 6 weeks later, specific proliferation to Ambroxol could no longer be observed in the LTT (Fig 4.).

Vascular cell adhesion molecule 1 expression by biliary epithelium promotes persistence of inflammation by inhibiting effector T-cell apoptosis

Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding a4b1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic a4b1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by a4b1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 Tcell survival. Conclusions: VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to a4b1 expressing proinflammatory T lymphocytes in CLD.

Hypertrophy of infected Peyer’s patches arises from global, interferon-receptor, and CD69-independent shutdown of lymphocyte egress

Lymphoid organ hypertrophy is a hallmark of localized infection. During the inflammatory response, massive changes in lymphocyte recirculation and turnover boost lymphoid organ cellularity. Intriguingly, the exact nature of these changes remains undefined to date. Here, we report that hypertrophy of Salmonella-infected Peyer's patches (PPs) ensues from a global "shutdown" of lymphocyte egress, which traps recirculating lymphocytes in PPs. Surprisingly, infection-induced lymphocyte sequestration did not require previously proposed mediators of lymphoid organ shutdown including type I interferon receptor and CD69. In contrast, following T-cell receptor-mediated priming, CD69 was essential to selectively block CD4+ effector T-cell egress. Our findings segregate two distinct lymphocyte sequestration mechanisms, which differentially rely on intrinsic modulation of lymphocyte egress capacity and inflammation-induced changes in the lymphoid organ environment.

CX3CR1+ cells facilitate the activation of CD4 T cells in the colonic lamina propria during antigen-driven colitis

Dendritic cells (DCs) and macrophages populate the intestinal lamina propria to initiate immune responses required for the maintenance of intestinal homeostasis. To investigate whether CX3CR1(+) phagocytes communicate with CD4 T cells during the development of transfer colitis, we established an antigen-driven colitis model induced by the adoptive transfer of DsRed OT-II cells in CX3CR1(GFP/+) × RAG(-/-) recipients challenged with Escherichia coli expressing ovalbumin (OVA) fused to a cyan fluorescent protein (CFP). After colonization of CX3CR1(GFP/+) × RAG(-/-) animals with red fluorescent E. coli pCherry-OVA, colonic CX3CR1(+) cells but not CD103(+) DCs phagocytosed E. coli pCherry-OVA. Degraded bacterial-derived antigens are transported by CD103(+) DCs to mesenteric lymph nodes (MLNs), where CD103(+) DCs prime naive T cells. In RAG(-/-) recipients reconstituted with OT II cells and gavaged with OVA-expressing E. coli, colonic CX3CR1(+) phagocytes are in close contact with CD4 T cells and presented bacterial-derived antigens to CD4 T cells to activate and expand effector T cells.

AIDS defining opportunistic infections in patients with high CD4 counts in the combination antiretroviral therapy (cART) era: things ain't what they used to be.

INTRODUCTION According to reports from observational databases, classic AIDS-defining opportunistic infections (ADOIs) occur in patients with CD4 counts above 500/µL on and off cART. Adjudication of these events is usually not performed. However, ADOIs are often used as endpoints, for example, in analyses on when to start cART. MATERIALS AND METHODS In the database, Swiss HIV Cohort Study (SHCS) database, we identified 91 cases of ADOIs that occurred from 1996 onwards in patients with the nearest CD4 count >500/µL. Cases of tuberculosis and recurrent bacterial pneumonia were excluded as they also occur in non-immunocompromised patients. Chart review was performed in 82 cases, and in 50 cases we identified CD4 counts within six months before until one month after ADOI and had chart review material to allow an in-depth review. In these 50 cases, we assessed whether (1) the ADOI fulfilled the SHCS diagnostic criteria (www.shcs.ch), and (2) HIV infection with CD4 >500/µL was the main immune-compromising condition to cause the ADOI. Adjudication of cases was done by two experienced clinicians who had to agree on the interpretation. RESULTS More than 13,000 participants were followed in SHCS in the period of interest. Twenty-four (48%) of the chart-reviewed 50 patients with ADOI and CD4 >500/µL had an HIV RNA <400 copies/mL at the time of ADOI. In the 50 cases, candida oesophagitis was the most frequent ADOI in 30 patients (60%) followed by pneumocystis pneumonia and chronic ulcerative HSV disease (Table 1). Overall chronic HIV infection with a CD4 count >500/µL was the likely explanation for the ADOI in only seven cases (14%). Other reasons (Table 1) were ADOIs occurring during primary HIV infection in 5 (10%) cases, unmasking IRIS in 1 (2%) case, chronic HIV infection with CD4 counts <500/µL near the ADOI in 13 (26%) cases, diagnosis not according to SHCS diagnostic criteria in 7 (14%) cases and most importantly other additional immune-compromising conditions such as immunosuppressive drugs in 14 (34%). CONCLUSIONS In patients with CD4 counts >500/ µL, chronic HIV infection is the cause of ADOIs in only a minority of cases. Other immuno-compromising conditions are more likely explanations in one-third of the patients, especially in cases of candida oesophagitis. ADOIs in HIV patients with high CD4 counts should be used as endpoints only with much caution in studies based on observational databases.

Kidney disease in antiretroviral-naïve HIV-positive adults with high CD4 counts: prevalence and predictors of kidney disease at enrolment in the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial.

OBJECTIVES HIV infection has been associated with an increased risk of chronic kidney disease (CKD). Little is known about the prevalence of CKD in individuals with high CD4 cell counts prior to initiation of antiretroviral therapy (ART). We sought to address this knowledge gap. METHODS We describe the prevalence of CKD among 4637 ART-naïve adults (mean age 36.8 years) with CD4 cell counts > 500 cells/μL at enrolment in the Strategic Timing of AntiRetroviral Treatment (START) study. CKD was defined by estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m(2) and/or dipstick urine protein ≥ 1+. Logistic regression was used to identify baseline characteristics associated with CKD. RESULTS Among 286 [6.2%; 95% confidence interval (CI) 5.5%, 6.9%] participants with CKD, the majority had isolated proteinuria. A total of 268 participants had urine protein ≥ 1+, including 41 with urine protein ≥ 2+. Only 22 participants (0.5%) had an estimated glomerular filtration rate < 60 mL/min/1.73 m(2) , including four who also had proteinuria. Baseline characteristics independently associated with CKD included diabetes [adjusted odds ratio (aOR) 1.73; 95% CI 1.05, 2.85], hypertension (aOR 1.82; 95% CI 1.38, 2.38), and race/ethnicity (aOR 0.59; 95% CI 0.37, 0.93 for Hispanic vs. white). CONCLUSIONS We observed a low prevalence of CKD associated with traditional CKD risk factors among ART-naïve clinical trial participants with CD4 cell counts > 500 cells/μL.

Viral load versus CD4⁺ monitoring and 5-year outcomes of antiretroviral therapy in HIV-positive children in Southern Africa: a cohort-based modelling study.

OBJECTIVES Many paediatric antiretroviral therapy (ART) programmes in Southern Africa rely on CD4⁺ to monitor ART. We assessed the benefit of replacing CD4⁺ by viral load monitoring. DESIGN A mathematical modelling study. METHODS A simulation model of HIV progression over 5 years in children on ART, parameterized by data from seven South African cohorts. We simulated treatment programmes with 6-monthly CD4⁺ or 6- or 12-monthly viral load monitoring. We compared mortality, second-line ART use, immunological failure and time spent on failing ART. In further analyses, we varied the rate of virological failure, and assumed that the rate is higher with CD4⁺ than with viral load monitoring. RESULTS About 7% of children were predicted to die within 5 years, independent of the monitoring strategy. Compared with CD4⁺ monitoring, 12-monthly viral load monitoring reduced the 5-year risk of immunological failure from 1.6 to 1.0% and the mean time spent on failing ART from 6.6 to 3.6 months; 1% of children with CD4⁺ compared with 12% with viral load monitoring switched to second-line ART. Differences became larger when assuming higher rates of virological failure. When assuming higher virological failure rates with CD4⁺ than with viral load monitoring, up to 4.2% of children with CD4⁺ compared with 1.5% with viral load monitoring experienced immunological failure; the mean time spent on failing ART was 27.3 months with CD4⁺ monitoring and 6.0 months with viral load monitoring. Conclusion: Viral load monitoring did not affect 5-year mortality, but reduced time on failing ART, improved immunological response and increased switching to second-line ART.

Thromboxane A2 acts as tonic immunoregulator by preferential disruption of low-avidity CD4+ T cell-dendritic cell interactions

Interactions between dendritic cells (DCs) and T cells control the decision between activation and tolerance induction. Thromboxane A2 (TXA2) and its receptor TP have been suggested to regulate adaptive immune responses through control of T cell-DC interactions. Here, we show that this control is achieved by selectively reducing expansion of low-avidity CD4(+) T cells. During inflammation, weak tetramer-binding TP-deficient CD4(+) T cells were preferentially expanded compared with TP-proficient CD4(+) T cells. Using intravital imaging of cellular interactions in reactive peripheral lymph nodes (PLNs), we found that TXA2 led to disruption of low- but not high-avidity interactions between DCs and CD4(+) T cells. Lack of TP correlated with higher expression of activation markers on stimulated CD4(+) T cells and with augmented accumulation of follicular helper T cells (TFH), which correlated with increased low-avidity IgG responses. In sum, our data suggest that tonic suppression of weak CD4(+) T cell-DC interactions by TXA2-TP signaling improves the overall quality of adaptive immune responses.

Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.

Thyroid signaling in immune organs and cells of the teleost fish rainbow trout (Oncorhynchus mykiss).

Thyroid hormones are involved in modulating the immune system in mammals. In contrast, there is no information on the role played by these hormones in the immune system of teleost fish. Here we provide initial evidence for the presence of active thyroid signaling in immune organs and cells of teleosts. We demonstrate that immune organs (head kidney and spleen) and isolated leukocytes (from head kidney and peripheral blood) of the rainbow trout (Oncorhynchus mykiss) express both thyroid receptor α (THRA) and β (THRB). Absolute mRNA levels of THRA were significantly higher than those of THRB. THRA showed higher expression in immune organs and isolated immune cells compared to the reference organ, liver, while THRB showed the opposite. In vivo exposure of trout to triiodothryronine (T3) or the anti-thyroid agent propylthiouracil (PTU) altered THR expression in immune organs and cells. Effect of T3 and PTU over the relative expression of selected marker genes of immune cell subpopulations was also studied. Treatments changed the relative expression of markers of cytotoxic, helper and total T cells (cd4, cd8a, trb), B lymphocytes (mIgM) and macrophages (csf1r). These findings suggest that the immune system of rainbow trout is responsive to thyroid hormones.

Pre-therapy inflammation and coagulation activation and long-term CD4 count responses to the initiation of antiretroviral therapy

OBJECTIVES Pre-antiretroviral therapy (ART) inflammation and coagulation activation predict clinical outcomes in HIV-positive individuals. We assessed whether pre-ART inflammatory marker levels predicted the CD4 count response to ART. METHODS Analyses were based on data from the Strategic Management of Antiretroviral Therapy (SMART) trial, an international trial evaluating continuous vs. interrupted ART, and the Flexible Initial Retrovirus Suppressive Therapies (FIRST) trial, evaluating three first-line ART regimens with at least two drug classes. For this analysis, participants had to be ART-naïve or off ART at randomization and (re)starting ART and have C-reactive protein (CRP), interleukin-6 (IL-6) and D-dimer measured pre-ART. Using random effects linear models, we assessed the association between each of the biomarker levels, categorized as quartiles, and change in CD4 count from ART initiation to 24 months post-ART. Analyses adjusted for CD4 count at ART initiation (baseline), study arm, follow-up time and other known confounders. RESULTS Overall, 1084 individuals [659 from SMART (26% ART naïve) and 425 from FIRST] met the eligibility criteria, providing 8264 CD4 count measurements. Seventy-five per cent of individuals were male with the mean age of 42 years. The median (interquartile range) baseline CD4 counts were 416 (350-530) and 100 (22-300) cells/μL in SMART and FIRST, respectively. All of the biomarkers were inversely associated with baseline CD4 count in FIRST but not in SMART. In adjusted models, there was no clear relationship between changing biomarker levels and mean change in CD4 count post-ART (P for trend: CRP, P = 0.97; IL-6, P = 0.25; and D-dimer, P = 0.29). CONCLUSIONS Pre-ART inflammation and coagulation activation do not predict CD4 count response to ART and appear to influence the risk of clinical outcomes through other mechanisms than blunting long-term CD4 count gain.