Katharina Pfistershammer

Detection and Prediction of Active Tuberculosis Disease by a Whole-Blood Interferon-γ Release Assay in HIV-1–Infected Individuals

BACKGROUND The sensitivity of whole-blood interferon-gamma release assays to detect or predict active tuberculosis in individuals infected with human immunodeficiency virus type 1 (HIV-1) has as yet not been determined. Methods. In this prospective, longitudinal, single-center study, 830 HIV-1-infected patients underwent testing with the QuantiFERON-TB Gold In-Tube (QFT-GIT) assay. Clinical screening for active tuberculosis was performed at least every 3 months for a median follow-up time of 19 months. RESULTS At baseline, the QFT-GIT assay yielded positive or indeterminate results in 44 (5.3%) and 47 (5.7%) of the 830 patients, respectively. A positive QFT-GIT assay result occurred at significantly higher frequencies among black individuals than among white individuals (odds ratio, 4.84; 95% confidence interval, 2.25-9.97; P< .001), among patients from Africa than among patients from Austria (odds ratio, 6.57; 95% confidence interval, 2.99-14.25; P< .001), and among patients from high-prevalence countries than among patients from low-prevalence countries (odds ratio, 5.86; 95% confidence interval, 2.41-13.44; P< .001). In patients with indeterminate QFT-GIT assay results, both median actual and nadir CD4(+) T cell counts were significantly lower than in patients with interpretable QFT-GIT assay results (P< .001). At the time of baseline QFT-GIT screening, active tuberculosis was found in 7 (15.9%) of 44 individuals with a positive result and in 1 (0.1%) of 739 patients with a negative result. During the follow-up period, however, progression to active tuberculosis occurred exclusively in patients with a positive QFT-GIT assay result, at a rate of 8.1% (3 of 37 patients; P< .001). Collectively, the sensitivity of the QFT-GIT assay for active tuberculosis was 90.9% (95% confidence interval, 62.3%-98.4%). CONCLUSIONS Our results suggest that the QFT-GIT assay may be a sensitive tool for the detection and prediction of active tuberculosis in HIV-1-infected individuals.

Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains

In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-γ production.

B7-H3 is a potent inhibitor of human T-cell activation: No evidence for B7-H3 and TREML2 interaction

B7‐H3 belongs to the B7 superfamily, a group of molecules that costimulate or down‐modulate T‐cell responses. Although it was shown that B7‐H3 could inhibit T‐cell responses, several studies – most of them performed in murine systems – found B7‐H3 to act in a costimulatory manner. In this study, we have specifically addressed a potential functional dualism of human B7‐H3 by assessing the effect of this molecule under varying experimental conditions as well as on different T‐cell subsets. We show that B7‐H3 does not costimulate human T cells. In the presence of strong activating signals, B7‐H3 potently and consistently down‐modulated human T‐cell responses. This inhibitory effect was evident when analysing proliferation and cytokine production and affected naïve as well as pre‐activated T cells. Furthermore, we demonstrate that B7‐H3–T‐cell interaction is characterised by an early suppression of IL‐2 and that T‐cell inhibition can be reverted by exogenous IL‐2. Since the triggering receptor expressed on myeloid cells like transcript 2 (TREML2/TLT‐2) has been recently described as costimulatory receptor of murine B7‐H3 we have extensively analysed interaction of human B7‐H3 with TREML2/TLT‐2. In these experiments we found no evidence for such an interaction. Furthermore, our data do not point to a role for murine TREML2 as a receptor for murine B7‐H3.

Human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and B7-H1 expression

Dendritic cells (DC) are professional APCs with an unmatched ability to interact with and activate T cells. There is accumulating evidence that DC not only efficiently stimulate T cell activation but also regulate T cell responses. However, little is known about cell surface structures on DC involved in the regulation of T cell responses. We demonstrate that human rhinoviruses (HRV) can efficiently inhibit the accessory function of DC through induction of inhibitory cell surface receptors. We observed that treatment of DC with HRV14 (R-DC), a member of the major group HRV family, diminished their T cell stimulatory capacity and induced a promiscuous and deep anergic state in cocultured T cells despite high levels of MHC molecules as well as costimulatory molecules, e.g., B7-1 (CD80) and B7-2 (CD86), and independent of inhibitory soluble factors such as IL-10. In contrast, expression of inhibitory B7-H1 molecules was up-regulated and R-DC de novo expressed sialoadhesin (Sn). Most importantly, blocking of B7-H1 and Sn on R-DC with specific mAbs against both receptors reverted the inhibitory phenotype. Thus, inhibitory signals delivered from R-DC to T cells via B7-H1 and Sn were critical for the induction of anergy. These observations suggest that an altered accessory molecule repertoire on DC upon interaction with HRV down-modulates adaptive immune responses during the viral infection.

The capacity of the TNF family members 4-1BBL, OX40L, CD70, GITRL, CD30L and LIGHT to costimulate human T cells

Activating signals generated by members of the tumour necrosis factor receptor superfamily upon interaction with their cognate ligands play important roles in T‐cell responses. Members of the tumour necrosis factor family namely 4‐1BBL, OX40L, CD70, GITRL, LIGHT and CD30L have been described to function as costimulatory molecules by binding such receptors on T cells. Using our recently described system of T‐cell stimulator cells we have performed the first study where all these molecules have been assessed and compared regarding their capacity to costimulate proliferation and cytokine production of human T cells. 4‐1BBL, which we found to be the most potent molecule in this group, was able to mediate sustained activation and proliferation of human T cells. OX40L and CD70 were also strong inducers of T‐cell proliferation, whereas the costimulatory capacity of human GITRL was significantly lower. Importantly CD30L and LIGHT consistently failed to act costimulatory on human T cells, and we therefore suggest that these molecules might be functionally distinct from the costimulatory members of this family.

Recombinant factor VIII and factor VIII-von Willebrand factor complex do not present danger signals for human dendritic cells

Several lines of evidence have shown that antibody responses to coagulation factor VIII (FVIII) in patients with hemophilia A depend on the help of activated CD4(+) T cells. The primary activation of CD4(+) T cells requires interaction with mature dendritic cells (DCs) that present antigenic peptides in the context of MHC class II and express costimulatory molecules. Maturation of DCs requires danger signals provided by exogenous or endogenous stimuli such as pathogen-derived products or inflammatory cytokines. We asked the question whether FVIII itself, FVIII complexed with von Willebrand factor (VWF) or thrombin-activated FVIII contain danger signals for human DCs that induce the upregulation of costimulatory molecules or the expression of proinflammatory cytokines necessary for effective activation of CD4(+) T cells. Human peripheral monocytes were differentiated into DCs. FVIII, thrombin-activated FVIII, VWF, VWF-FVIII, lipopolysaccharide (LPS), LPS + FVIII, LPS + VWF or LPS + FVIII-VWF were added either on day 0 or on day 5 of differentiation cultures. Differentiation markers, cytokines in cell culture supernatants and the capacity of DCs to stimulate autologous and allogeneic T cells were analysed after seven days of differentiation cultures. Our results indicate that neither FVIII, thrombin-activated FVIII, VWF nor a complex of FVIII and VWF modulate the maturation of human DCs or their capacity to stimulate autologous or allogeneic T cells. We conclude that neither of these proteins present danger signals to human DCs.

CD63 as an Activation-Linked T Cell Costimulatory Element

Dendritic cells (DC) are unique in their capacity to either stimulate or regulate T cells, and receptor/ligand pairs on DC and T cells are critically involved in this process. In this study we present such a molecule, which was discovered by us when analyzing the functional effects of an anti-DC mAb. This mAb, 11C9, reacted strongly with DC, but only minimally with lymphocytes. In MLR it constantly reduced DC-induced T cell activation. Therefore, we assumed that mAb 11C9 primarily exerts its functions by binding to a DC-structure. This does not seem to be the case, however. Preincubation of DC with mAb 11C9 before adding T cells had no inhibitory effect on T cell responses. Retroviral expression cloning identified the 11C9 Ag as CD63. This lysosomal-associated membrane protein (LAMP-3), is only minimally expressed on resting T cells but can, as we show, quickly shift to the surface upon stimulation. Cross-linkage of that structure together with TCR-triggering induces strong T cell activation. CD63 on T cells thus represents an alternative target for mAb 11C9 with its binding to activated T cells rather than DC being responsible for the observed functional effects. This efficient CD63-mediated costimulation of T cells is characterized by pronounced induction of proliferation, strong IL-2 production and compared with CD28 enhanced T cell responsiveness to restimulation. Particularly in this latter quality CD63 clearly surpasses several other CD28-independent costimulatory pathways previously described. CD63 thus represents an activation-induced reinforcing element, whose triggering promotes sustained and efficient T cell activation and expansion.

No evidence for dualism in function and receptors: PD-L2/B7-DC is an inhibitory regulator of human T cell activation.

The B7 family member programmed‐death‐1‐ligand 2 (PD‐L2/B7‐DC) is a ligand for programmed‐death‐receptor 1 (PD‐1), a receptor involved in negative regulation of T cell activation. Several independent studies have reported that PD‐L2, however, can also potently costimulate murine T cells via an additional yet unidentified receptor. In this study, we evaluated the contribution of PD‐L2 to the activation of human T cells using a novel system of engineered T cell stimulators that expresses membrane‐bound anti‐CD3 antibodies. Analyzing early activation markers, cytokine production and proliferation, we found PD‐L2 to consistently inhibit T cell activation. PD‐L2 inhibition affected CD4+ and CD8+ T cells and was not abrogated by costimulation via CD28. Blocking PD‐1 reverted the inhibitory effect of PD‐L2, demonstrating involvement of this pathway. In human T cells, we found no evidence for any of the costimulatory effects described for PD‐L2 in murine systems. In line with our functional data that do not point to stimulatory PD‐L2‐ligands, we show that binding of PD‐L2‐immunoglobulin to activated human T cells is abrogated by PD‐1 antibodies. Our results demonstrate that PD‐L2 negatively regulates human T cell activation and thus might be a candidate molecule for immunotherapeutic approaches aimed to attenuate pathological immune responses.

Host antimicrobial proteins as endogenous immunomodulators

Host defense mechanisms are multilayered and involve physical as well as chemical barriers, antimicrobial factors as well as a broad set of immunocompetent cells. The mode of action of antimicrobial factors is variable, ranging from opsonisation and agglutination to direct killing of pathogens. In the last years it has become increasingly clear that some of these factors act as endogenous ligands that bind to distinct host receptors, as for example pathogen recognition receptors (PRRs), thereby influencing distinct immunological processes like chemotaxis, modulation of phagocytosis, dendritic cell maturation or the production of cytokines. By that way, these factors are implicated to protect the host by preventing and clearing of microbial infections.

Allogeneic disparities in immunoglobulin-like transcript 5 induce potent antibody responses in hematopoietic stem cell transplant recipients

In hematopoietic stem cell transplant (HSCT) recipients, the recognition of polymorphic antigens by the donor-derived immune system is an important mechanism underlying both graft-versus-host disease and graft-versus-leukemia (GVL) effect. Here we show that a subset of HSCT recipients (13.9%, n = 108) have antibodies directed to surface molecules of dendritic cells. We have used one such serum in conjunction with retroviral expression cloning to identify the highly polymorphic surface molecule immunoglobulin-like transcript 5 (ILT5) as one of the targets of dendritic cell-reactive antibodies. ILT5 reactive antibodies were found in 5.4% of HSCT patients but not in solid organ transplantation recipients, patients with collagen diseases, multiparous women, or polytransfused or healthy persons. We show that ILT5-specific antibodies can mediate killing of ILT5-bearing cells and furthermore demonstrate ILT5 expression in some leukemic cells, indicating that it might be a target for GVL effects. Thus, our results represent the first description of potent allogeneic antibody responses to a non-major histocompatibility complex cell surface molecule in hematopoietic stem cell transplanted patients and warrant further studies to elucidate the role of antibodies to polymorphic cell surface molecules in GVL and graft-versus-host responses.