Case ReportInduction of T-cell Tolerance in a Patient with Idiopathic Thrombocytopenic Purpura by Single Injection of Humanized Monoclonal Antibody to CD40 Ligand

Normal B cells can be induced to express immune costimulatory molecules by activated T cells, and activated CD4 T cells can express CD40 ligand, a molecule that can engage CD40 on the B-cell surface. CD40–CD40 ligand interaction plays an important role in the pathology of certain autoimmune diseases. We report a patient with chronic idiopathic thrombocyopenic purpura (ITP) who was effectively treated with a single injection of humanized monoclonal antibody to CD40 ligand (E6040). The patient was refractory to steroid therapy, and had baseline platelet counts below 30×10⁹/l during the 3-month period before antibody treatment. The patient's platelet counts have increased to more than 100×10⁹/l long-term after E6040 administration. Platelet-associated IgG was decreased with thrombocytosis. Compared with the initial period of E6040 administration, the number of anti-GPIIb/IIIa antibody-producing B cells decreased, and proliferative response of autoreactive T cells to GPIIb/IIIa was also improved. A single injection of humanized monoclonal antibody to CD40 ligand may induce T-cell tolerance in patients with ITP.     

Inter-mouse strain differences in the in vivo anti-CD3 induced cytokine release.

Triggering of the CD3 molecule by in vivo injection of the hamster anti-murine CD3 monoclonal antibody 145-2C11 in adult BALB/c mice leads to massive although transient T cell activation. High levels of tumour necrosis factor (TNF), interferon-gamma (IFN-gamma), IL-2, IL-3 and IL-6 are released into the circulation 1 to 8 h after a single 10 micrograms 145-2C11 i.v. injection. This release induces an impressive self-limited physical reaction associating hypothermia, hypomotility (as assessed by actimetry), diarrhoea, piloerection and even death when high doses (a single dose of greater than 100 micrograms/mouse injection) are administered. In vivo injection of 145-2C11 to other selected mouse strains, namely NZW, CBA/J and C3H/HeJ, induced both different cytokine release patterns and sickness. 145-2C11 induced significant release of TNF and IL-2 in all four strains. At variance, IFN-gamma was only detected in BALB/c mice sera which, in terms of physical reaction (hypothermia and hypomotility) were the most affected. Higher and long-lasting circulating IL-3/GM-CSF levels were present in CBA/J sera, correlating with a later recovery. These results underline heterogeneity in the in vivo cell activation pattern among different mouse strains, when triggering T lymphocytes via the CD3/Ti molecule as compared to exclusive targeting of monocyte/macrophages by means of lipopolysaccharide.     

γδ T‐cell conference 2012: Close encounters for the fifth time

The fifth international γδ T‐cell conference was held in Freiburg, Germany, from May 31 to June 2, 2012, bringing together approximately 170 investigators from all over the world. The scientific program covered topics such as thymic development and the mechanisms of ligand recognition and activation, the interaction of γδ T cells with other immune and non‐immune cells and its implications for homeostasis, infection, tissue repair and autoimmunity, and the role of γδ T cells in malignancy and their potential for novel immunotherapies. Here we discuss a selection of the oral communications at the conference, and summarise exciting new findings in the field regarding the development, mode of antigen recognition, and responses to microorganisms, viruses and tumours by human and mouse γδ T cells.     

Dual function of recombinant human CD58: inhibition of T cell adhesion and activation via the CD2 pathway.

To produce large quantities of recombinant CD58 (rCD58) glycoproteins for biochemical and functional studies, a cDNA clone containing the phosphatidylinositol-linked form of human CD58 was expressed in insect cells using the baculovirus system. Gel filtration showed rCD58 to form soluble oligomeric aggregates which were functionally, antigenically, and biochemically similar to their natural counterpart. Sequence analysis of the amino- and carboxy-terminal ends of released rCD58 protein revealed that the 28 amino acid signal peptide was accurately removed. In contrast, the hydrophobic C-terminal peptide was not removed. rCD58 binds to its natural ligand CD2 with a dissociation constant Kd = 5 x 10(-8) M, which is equivalent to the affinity of physiological T cell adhesion mediated by the membrane bound CD2-CD58 receptor-ligand pair. Rosette formation of human T lymphocytes with sheep and human erythrocytes was completely abrogated. In addition, the mixed lymphocyte reaction was significantly inhibited by rCD58. Moreover, cytotoxicity of human NK clones (CD2+CD3-) was inhibited by rCD58 similar to inhibition by CD58 mAbs. In contrast, rCD58 synergized with mitogenic CD2R mAbs in T cell triggering. These data demonstrate that rCD58 might serve as a biological immunomodulator which influences T cell adhesion and activation.     

Biphasic control of nuclear factor-χB activation by the T cell receptor complex: role of tumor necrosis factor α

The regulation of nuclear factor (NF)-χB activation by the T cell receptor (TcR)/CD3 complex in primary human T cells has been studied at various times after activation. Only p50 NF-χB protein bound the χB element of interleukin-2 receptor (IL-2R) α chain promoter on resting T cells. However, immediately after TcR/CD3 cross-linking (after approximately 1 h; immediate) binding of p50.p65 heterodimers was observed. p50.c-rel heterodimers were also detected bound to this sequence at early time points (7–16 h; early), and both remained active at later time points (40 h; late) after activation. This regulation takes place mainly at the level of nuclear translocation of p65 and c-rel, at immediate and early time points. Activation also induced c-rel and p105/p50 mRNA synthesis, but not p65 mRNA whose expression was constitutive. Interestingly, all those early and late events, but not the immediate ones, were inhibited by a neutralizing anti-tumor necrosis factor α (TNF-α) monoclonal antibody. Similarly, cycloheximide prevented the p65 and c-rel translocation and consequent formation of active binding heterodimers, at early and late times. Cyclosporin A impaired not only early and late, but also immediate events; however, addition of TNF-α prevented all inhibition. These results indicate that the regulation of NF-χB activation during T cell activation by TcR/CD3 signals is biphasic: TcR/CD3 triggers its immediate translocation, which is transient if no TNF-α is present. TNF-α, therefore, emerges as the main factor responsible for a second phase of NF-χB regulation, controlling both translocation of p65 and c-rel, and new mRNA synthesis for c-rel and p105/p50.     

Engineering higher affinity T cell receptors using a T cell display system

The T cell receptor (TCR) determines the cellular response to antigens, which are presented on the surface of target cells in the form of a peptide bound to a product of the major histocompatibility complex (pepMHC). The response of the T cell depends on the affinity of the TCR for the pepMHC, yet many TCRs have been shown to be of low affinity, and some naturally occurring T cell responses are poor due to low affinities. Accordingly, engineering the TCR for increased affinity for pepMHC, particularly tumor-associated antigens, has become an increasingly desirable goal, especially with the advent of adoptive T cell therapies. For largely technical reasons, to date there have been only a handful of TCRs engineered in vitro for higher affinity using well established methods of protein engineering. Here we report the use of a T cell display system, using a retroviral vector, for generating a high-affinity TCR from the mouse T cell clone 2C. The method relies on the display of the TCR, in its normal, signaling competent state, as a CD3 complex on the T cell surface. A library in the CDR3alpha of the 2C TCR was generated in the MSCV retroviral vector and transduced into a TCR-negative hybridoma. Selection of a high-affinity, CD8-independent TCR was accomplished after only two rounds of flow cytometric sorting using the pepMHC SIYRYYGL/Kb (SIY/Kb). The selected TCR contained a sequence motif in the CDR3alpha with characteristics of several other TCRs previously selected by yeast display. In addition, it was possible to directly use the selected T cell hybridoma in functional assays without the need for sub-cloning, revealing that the selected TCR was capable of mediating CD8-independent activity. The method may be useful in the direct isolation and characterization of TCRs that could be used in therapies with adoptive transferred T cells.     

A rat anti-mouse kappa chain specific monoclonal antibody, 187.1.10: purification, immunochemical properties and its utility as a general second-antibody reagent

A rat IgG1 monoclonal antibody, produced by hybridoma 187.1.10, exhibits specificity for mouse immunoglobulins containing kappa light chains (Yelton et al., 1981). The 187.1.10 hybridoma cell line secreted upwards of 200 micrograms/ml of monoclonal antibody in tissue culture and the secreted product was purified in a single step by antigen-immunoadsorbent affinity chromatography. The homogeneity of the purified 187.1.10 protein was determined by isoelectrofocusing and SDS gel electrophoresis. Equilibrium binding analyses of the radioiodinated 187.1.10 antibody indicated a strong interaction with its antigen of KA = 2 X 10(9) l/mole. The 187.1.10 antibody did not readily bind to Staph. aureus protein A unless it was complexed with antigen. The binding of immune complexes of 187.1.10 to protein A was shown to be dependent on the Fc region of the antigen. The utility of the 187.1.10 monoclonal antibody as a general second antibody reagent for studying mouse immunoglobulins was demonstrated in a rapid solid phase immunoprecipitation assay to detect and analyze radioiodinated membrane proteins of a human cytotoxic T cell line.     

NK cell expression of Tim-3: First impressions matter

Given the heightened interest in manipulation of co-signaling cascades for cancer immunotherapy, we sought to determine how/whether tumors decorated with therapeutic monoclonal antibodies (mAbs) impact the expression of co-signaling molecules on human NK cells. Stimulation of NK cells with aggregated IgG1 resulted in the upregulation of HAVCR2 – the gene encoding T-cell immunoglobulin and mucin-containing domain (Tim)-3 – known to be involved in the induction of peripheral T cell tolerance. This upregulation of HAVCR2 was recapitulated at the protein level, following NK cell stimulation by either mAb opsonized tumors, recombinant human IgG1 Fc multimer, and/or non-Fc stimuli e.g. IL-12/IL-18. The patterns of Tim-3 expression were temporally distinct from the FcR mediated induction of the co-signaling molecule, 4-1BB (CD137), with Tim-3 increases observed twenty minutes following exposure to Fc multimers and remaining at high levels for at least six hours, while increases in CD137 expression were first observed at the four-hour time point. Importantly, these Tim-3+ NK cells were functionally diverse, as evidenced by the fact that their ability to produce IFN-γ in response to an NK cell responsive tumor was strictly dependent upon the stimuli employed for Tim-3 induction. These data suggest that Tim-3 upregulation is the common end-result of NK cell activation by a variety of unique and overlapping stimuli and is not an independent marker of NK cell exhaustion. Furthermore, our observations potentially explain the diverse functionality attributed to Tim-3+ NK cells and should be considered prior to use of anti-Tim-3 inhibitory mAbs for cancer immunotherapy.     

A cytotoxic humanized anti-ganglioside antibody produced in a murine cell line defective of N-glycolylated-glycoconjugates

Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors.     

Activation requirements and responses to TLR ligands in human CD4 T cells: Comparison of two T cell isolation techniques

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4⁺ T cells isolated either by IMACS (IMACS-CD4⁺) or by IMACS followed by FACS (IMACS/FACS-CD4⁺). As expected, IMACS-CD4⁺ were less pure than IMACS/FACS-CD4⁺ (92.5% ± 1.4% versus 99.7% ± 0.2%, respectively). Consequently, IMACS-CD4⁺ proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4⁺. In addition IMACS-CD4⁺ but not IMACS/FACS-CD4⁺ responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4⁺ and highly purified IMACS-/FACS-CD4⁺. Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.     

T cell receptor-associated protein tyrosine kinases: The dynamics of tolerance regulation by phosphorylation and its role in systemic lupus erythematosus

There are different abnormalities that lead to the autoreactive phenotype in T cells from systemic lupus erythematosus (SLE) patients. Proximal signaling, involving the T-cell receptor (TCR) and its associated protein tyrosine kinases (PTKs), is significantly affected in SLE. This ultimately leads to aberrant responses, which include enhanced tyrosine phosphorylation and calcium release, as well as decreased IL-2 secretion. Lck, ZAP70 and Syk, which are PTKs with a major role in proximal signaling, all present abnormal functioning that contributes to an altered T cell response in these patients. A number of other molecules, especially regulatory proteins, are also involved. This review will focus on the PTKs that participate in proximal signaling, with specific emphasis on their relevance in maintaining peripheral tolerance, their abnormalities in SLE and how these contribute to an altered T cell response.     

Making sense of regulatory T cell suppressive function

Several types of regulatory T cells maintain self-tolerance and control excessive immune responses to foreign antigens. The major regulatory T subsets described over the past decade and novel function in transplantation will be covered in this review with a focus on CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells. Multiple mechanisms have been proposed to explain how Treg cells inhibit effector cells but none can completely explain the observed effects in toto. Proposed mechanisms to explain suppressive activity of Treg cells include the generation of inhibitory cytokines, induced death of effector cells by cytokine deprivation or cytolysis, local metabolic perturbation of target cells mediated by changes in extracellular nucleotide/nucleoside fluxes with alterations in intracellular signaling molecules such as cyclic AMP, and finally inhibition of dendritic cell functions. A better understanding of how Treg cells operate at the molecular level could result in novel and safer therapeutic approaches in transplantation and immune-mediated diseases.     

Identification of germ cells at risk for neoplastic transformation in gonadoblastoma: an immunohistochemical study for OCT3/4 and TSPY

Carcinoma in situ (CIS) is the precursor of malignant testicular germ cell tumors (GCTs) of adolescents and young adults, being the neoplastic counterpart of primordial germ cells/gonocytes. Carcinoma in situ cells will develop into invasive seminoma/nonseminoma. Gonadoblastoma (GB) is the precursor of invasive GCTs in dysgenetic gonads, predominantly dysgerminoma (DG). In this process, part of the Y chromosome (GBY region) is involved, for which TSPY is a candidate gene. A detailed immunohistochemical survey was performed for the known diagnostic markers, germ cell/placental alkaline phosphatase (PLAP), c-KIT, and OCT3/4, as well as testis-specific protein on the Y chromosome (TSPY) on a series of GBs, and adjacent invasive DGs. All 5 patients were XY individuals (4 females and 1 male). In contrast to c-KIT, PLAP was positive in all cases. The immature germ cells of GBs were positive for OCT3/4, whereas the mature germ cells were negative for this marker, but positive for TSPY. In every GB, a minor population of germ cells positive for both markers could be identified, similar to most CIS cells and early invasive DG. On progression to an invasive tumor, TSPY can be lost, a process that is also detectable in invasive testicular GCTs compared to CIS. These results indicate that GB is a heterogeneous mix of germ cells, in which the OCT3/4-positive cells have the potential to undergo progression to an invasive tumor. These early invasive stages are initially also positive for TSPY (like CIS), supporting a positive selection mechanism. Therefore, OCT3/4 in combination with TSPY is valuable to identify malignant germ cells in dysgenetic gonads. This could allow better prediction of the risk of progression to a GCT. In addition, the data support the model that GB represents the earliest accessible developmental stage of malignant GCTs.