Katharina Grabmeier-Pfistershammer

TIM-3 Does Not Act as a Receptor for Galectin-9

T cell immunoglobulin and mucin protein 3 (TIM-3) is a type I cell surface protein that was originally identified as a marker for murine T helper type 1 cells. TIM-3 was found to negatively regulate murine T cell responses and galectin-9 was described as a binding partner that mediates T cell inhibitory effects of TIM-3. Moreover, it was reported that like PD-1 the classical exhaustion marker, TIM-3 is up-regulated in exhausted murine and human T cells and TIM-3 blockade was described to restore the function of these T cells. Here we show that the activation of human T cells is not affected by the presence of galectin-9 or antibodies to TIM-3. Furthermore, extensive studies on the interaction of galectin-9 with human and murine TIM-3 did not yield evidence for specific binding between these molecules. Moreover, profound differences were observed when analysing the expression of TIM-3 and PD-1 on T cells of HIV-1-infected individuals: TIM-3 was expressed on fewer cells and also at much lower levels. Furthermore, whereas PD-1 was preferentially expressed on CD45RA−CD8 T cells, the majority of TIM-3-expressing CD8 T cells were CD45RA+. Importantly, we found that TIM-3 antibodies were ineffective in increasing anti-HIV-1 T cell responses in vitro, whereas PD-L antibodies potently reverted the dysfunctional state of exhausted CD8 T cells. Taken together, our results are not in support of an interaction between TIM-3 and galectin-9 and yield no evidence for a functional role of TIM-3 in human T cell activation. Moreover, our data indicate that PD-1, but not TIM-3, is a promising target to ameliorate T cell exhaustion.

Identification of PD-1 as a Unique Marker for Failing Immune Reconstitution in HIV-1-Infected Patients on Treatment

PD-1 expression on T cells correlates with T-cell exhaustion and disease progression in HIV-infected patients. Previous studies have shown that combinational antiretroviral therapy induced viral suppression results in immune restoration and reduced PD-1 expression. However, a significant number of patients fail to restore CD4 T cells despite suppression of HIV replication below limit of quantification. In this study, we have analyzed PD-1 expression on CD4 and CD8 T cells in patients with poor immune reconstitution despite successful highly active antiretroviral therapy. We found that T cells of such patients express significantly higher levels of PD-1 than patients who had normal recovery of CD4 cells after treatment. In contrast, failing immune reconstitution was not associated with the expression of activation markers, indicating that PD-1 is a unique marker for failing immune reconstitution despite viral suppression. Furthermore, we show that T cells from patients with poor immune recovery differ from T cells of elderly in respect of their marker profile. PD-1 expression negatively correlated with individual CD4 cell counts, and PD-1 expressing T cells were more prone to programmed death ligand-mediated inhibition of T-cell proliferation, indicating that PD-1-mediated T-cell suppression may have a role in impaired immune reconstitution in HIV patients.

Receptors and ligands implicated in human T cell costimulatory processes

It is well established that full activation of T cells that recognize antigens requires additional signals. These second signals are generated by the interaction of costimulatory ligands expressed on antigen presenting cells with their receptors on T cells. In addition, T cell activation processes are negatively regulated by inhibitory costimulatory pathways. Interaction of members of the B7 and the TNF superfamilies with members of the CD28 and TNF-R-superfamilies plays major roles in costimulatory processes. However, a large number of molecules that do not belong to these families have been reported to be involved in the generation of T cell costimulatory signals. In addition to well-defined costimulatory pathways, where both receptors and ligands are known, there are many T cell surface molecules that have been described to generate a second signal under certain experimental conditions, f.i. when ligated with antibodies. Furthermore there are several ligands that have been shown to positively or negatively modulate T cell activation by interacting with as of yet unknown T cell receptors. Here we give a comprehensive overview of molecules that have been implicated in human T cell activation processes and propose criteria that define genuine T cell costimulatory pathways.

A Comprehensive and Quantitative Analysis of the Major Specificities in Rabbit Antithymocyte Globulin Preparations

Antithymocyte globulin (ATG) preparations are used for treatment and prevention of graft rejection episodes, graft versus host disease and aplastic anemia. The immunomodulatory and immuosuppressive properties of ATGs are mediated by their interaction with a large variety of antigens expressed on immune and nonimmune cell populations. We have conducted a comprehensive analysis on antibody specificities contained in rabbit ATGs in clinical use, ATG‐Fresenius (ATG‐F) and Thymoglobulin (THG). We have used retroviral expression cloning to identify novel ATG antigens and demonstrate that together with ATG antigens described earlier, these molecules account for the majority of ATG antibodies directed to human cells. Moreover, we have employed cell lines engineered to express antigens at high levels to quantify the antibodies directed to each ATG antigen. We have used cell lines expressing the T cell receptor complex, CD2 and CD28 to remove antibodies to these antigens from ATG preparations and demonstrate that this treatment abrogated the ability of ATGs to induce activation and forkhead box P3 expression in T cells. Comprehensive information and differences on the antigens targeted by ATG‐F and THG as well as novel approaches to assess their functional properties are the basis for a better understanding of their immunomodulatory capacities and might eventually translate into improved ATG‐based regimen.

Five-year on-treatment efficacy of lamivudine-, tenofovir- and tenofovir + emtricitabine-based HAART in HBV-HIV-coinfected patients.

Summary.  Data on the efficacy of lamivudine (LAM)‐, tenofovir (TDF)‐ and emtricitabine (FTC)‐based antiretroviral therapy (HAART) in HBV–HIV coinfection are limited. We completed a retrospective analysis of HBV–HIV‐coinfected patients treated at the Medical University of Vienna. One‐hundred and ten coinfected patients were included, with 57% being initially HBV e‐Antigen (HBeAg) positive. Baseline HBV load was significantly higher in HBeAg+ than in HBeAg− patients (5962 ± 3663 vs 20 ± 19 × 106 IU/mL; P < 0.0001). Over a median observation period of 83 month (range: 26–183), 87% received HAART and 91% showed a suppression of HBV replication. After 5 years of continuous treatment, HBeAg seroconversion was achieved in 21% of LAM‐, 50% of TDF‐ (P = 0.042 vs LAM) and in 57% of TDF + FTC (P = 0.008 vs LAM)‐treated patients, respectively. HBsAg loss after 5 years was found in 8% (LAM), 25% (TDF; P = 0.085 vs LAM) and 29% (TDF + FTC; P = 0.037 vs LAM) of HBeAg+ patients. In HBeAg− patients, HBsAg loss was achieved in 11% (LAM), 27% (TDF; P = 0.263 vs LAM) and 36% (TDF + FTC; P = 0.05 vs LAM), respectively. Pretreatment CD4+ counts did not influence rates of HBeAg seroconversion and of HBsAg loss. Patients with HBsAg loss had lower baseline HBV‐DNA levels and higher AST/ALT levels than patients without HBsAg loss. Transient HAART‐related hepatotoxicity was found in 32% (Grade I: 21%; II:7%; III:2%; IV:0%). Most HBV–HIV‐coinfected patients achieve complete suppression of HBV replication despite high baseline viremia. TDF‐based HAART leads to high rates of HBeAg seroconversion and HBsAg loss after 5 years of continuous exposure. One‐third of HBV–HIV‐coinfected patients may experience transient HAART‐related hepatotoxicity.

Targeting CD20 in Melanoma Patients at High Risk of Disease Recurrence

Melanomas contain distinct cell subpopulations. Several of these subpopulations, including one expressing CD20, may harbor stem cell-like or tumor-initiating characteristics. We hypothesized that patients at high risk of disease recurrence could benefit from an adjuvant anti-CD20 therapy. Therefore, we initiated a small pilot trial to study the effect of the anti-CD20 antibody rituximab in a group of melanoma patients with stage IV metastatic disease who had been rendered without evident disease by way of surgery, chemotherapy and/or radiation therapy. The major objective was safety, while secondary objectives were description of recurrence-free intervals (RFI) and overall survival (OS). Nine patients received rituximab at 375 mg/m(2) qw for 4 weeks followed by a maintenance therapy every 8 weeks. Treatment was discontinued after 2 years or with disease recurrence. Treatment was well tolerated. After a median observation of 42 months, the median neither of RFI nor of OS has been reached. Despite therapy that ended after 2 years, six out of nine patients are still alive and five of them are recurrence-free. Though the patient number is too small for definitive conclusions, our data may represent a first example of the potential therapeutic value of targeting CD20(+) cell populations-at least for a subset of patients.

T cell stimulator cells, an efficient and versatile cellular system to assess the role of costimulatory ligands in the activation of human T cells

It is well established that full activation of T cells requires the interaction of the TCR complex with the peptide–MHC complex (Signal 1) and additional signals (Signal 2). These second signals are generated by the interaction of costimulatory ligands expressed on antigen presenting cells with activating receptors on T cells. In addition, T cell responses are negatively regulated by inhibitory costimulatory pathways. Since professional antigen presenting cells (APC) harbour a plethora of stimulating and inhibitory surface molecules, the contribution of individual costimulatory molecules is difficult to assess on these cells. We have developed a system of stimulator cells that can give signal 1 to human T cells via a membrane bound anti-CD3 antibody fragment. By expressing human costimulatory ligands on these cells, their role in T cell activation processes can readily be analyzed. We demonstrate that T cell stimulator cells are excellent tools to study various aspects of human T cell costimulation, including the effects of immunomodulatory drugs or how costimulatory signals contribute to the in vitro expansion of T cells. T cell stimulator cells are especially suited for the functional evaluation of ligands that are implicated in costimulatory processes. In this study we have evaluated the role of the CD2 family member CD150 (SLAM) and the TNF family member TL1A (TNFSF15) in the activation of human T cells. Whereas our results do not point to a significant role of CD150 in T cell activation we found TL1A to potently costimulate human T cells. Taken together our results demonstrate that T cell stimulator cells are excellent tools to study various aspects of costimulatory processes.

HIV coreceptor tropism in antiretroviral treatment-naive patients newly diagnosed at a late stage of HIV infection

Objective:A substantial number of HIV infections worldwide are diagnosed at a late stage of disease. Mortality in late presenters is high, and their treatment is a specific challenge. We have determined the relative proportions of HIV-1 strains of different coreceptor tropism (CRT) in this group of patients and investigated the impact of CRT on progression markers such as CD4 cell counts and viral load, and on the clinical presentation of the patients. Design and methods:Plasma samples from 50 treatment-naive patients with a late HIV diagnosis (CD4 cell counts of <200 cells/μl at the time of diagnosis) were analyzed. HIV strains were sequenced, and for CRT determination, the internet tool geno2pheno[coreceptor] was used, with a 20% false-positive rate as the cutoff. Differences in progression markers, patient characteristics and HIV subtype distribution between the R5-infected and X4/DM-infected patient groups were evaluated statistically. Results:CRT predictions indicated that 62% of the patients had only R5-tropic strains. CRT was not associated with CD4 cell counts or viral load at the time of diagnosis. Only in very late presenters (CD4 cell counts <50 cells/μl) was there a significant difference in disease stage at the time of presentation, showing that patients with R5 more often were at Centers for Disease Control and Prevention stage C3 compared with those with X4/DM strains (P = 0.04). Conclusion:A substantial number of patients diagnosed at a late stage of HIV-1 infection may be infected exclusively with R5-tropic virus strains, making this specific patient group a possible candidate for coreceptor antagonist treatment.

Revisiting liver disease progression in HIV/HCV-coinfected patients: the influence of vitamin D, insulin resistance, immune status, IL28B and PNPLA3

To perform a comprehensive study on independent modulators of liver fibrosis progression and determinants of portal pressure considering immune status, insulin resistance (IR), serum 25‐hydroxyvitamin D (25(OH)D) levels, genetic variants of patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) and interleukin 28B (IL28B) in a thoroughly documented cohort of HIV/hepatitis C‐coinfected (HIV/HCV) patients.

Interaction of Antithymocyte Globulins with Dendritic Cell Antigens

The polyclonal rabbit antithymocyte globulins (ATGs), Thymoglobulin and ATG‐Fresenius S, are widely used for prevention and therapy of allograft rejection and graft versus host disease. Dendritic cells (DC) govern immune responses and thus the interaction of ATGs with these cells could potentially contribute to the clinical effects of ATG therapy. Currently there is little information on the DC‐antigens targeted by ATGs. In this study we have used a new methodology to identify DC surface antigens recognized by ATGs. By screening an eukaryotic expression library generated from DC with ATGs we could identify several novel ATG antigens including CD81, CD82, CD98, CD99 and CD147. Furthermore, we engineered cells to express previously described ATG antigens and probed them with Thymoglobulin and ATG‐Fresenius S. Our results demonstrated strong binding to some but not all of these molecules. We show that previously described antigens and antigens identified in this study account for around 80% of the DC reactivity of ATGs. Analysis of molecules induced by ATG–DC interaction are more in support for an activation of these cells by ATGs than for a specific induction of a tolerogenic DC phenotype.