Role of HLA-DR antigen on T-cell activation in visceral leishmaniasis

Ability of peripheral blood monocytes in association with HLA-DR molecules to support T-cell activation in response to soluble Leishmania donovani antigen was investigated. Adherent cells were stained with monoclonal antibodies. The increased number of cells with DR expression was more efficient in presenting L. donovani antigen to sensitized T-cells. The results suggest that quantitative variation in monocytes with expression of DR molecules, correlates with their ability to support T-cell response to L. donovani antigen, in vitro, as assessed by migration inhibition factor (MIF). However, it is not clear whether this is due to only HLA-DR antigen on the surface or whether other factors are involved.     

Role of primary and secondary protein structure in neurotransmitter receptor activation mechanisms

A proton transfer triggered by a ligand interacting with the receptor had been suggested as the initial step in the activation of a receptor for the neurotransmitter serotonin (5-hydroxy-tryptamine; 5-HT). To evaluate the role of the receptor macromolecule in modulating the primary molecular event in ligand-mediated activation, the process of proton transfer was analysed in the environment of a protein model for the 5-HT receptor. In the absence of a detailed receptor structure, the enzyme actinidin was chosen as the model for the receptor based on criteria obtained from structure-activity considerations on the ligands. The first simulation of a mechanism for receptor activation was performed on this model using methods of theoretical chemistry to study the effect of specific structural elements. The premise is that the role of the elements of secondary structure of soluble proteins (e.g. actinidin) in determining structure-function relations in these macromolecules is maintained when these elements are part of membrane-bound receptor proteins. Results from the calculations of the effects of the six alpha helices of actinidin on the proton transfer process from the imidazolium side chain of His 162 to the thiol side chain of Cys 25 in the protein show that the helices contribute in different ways to modulate the energy of proton transfer. The largest helix, A1, opposes the proton transfer through the effect of the helix dipole. The charged residues (primary structure) in helix A3 favor the proton transfer, and mask the effect of its helix dipole (secondary structure) which opposes the transfer. The direction of the proton transfer simulated for the activation mechanism is opposite to that assumed in the catalytic process of the thiol protease, and the entire protein environment opposes the transfer. This supports the specific role of the ligand in triggering the proton transfer as a response to its binding.     

Role of acid sphingomyelinase bioactivity in human CD4+ T-cell activation and immune responses

Acid sphingomyelinase (ASM), a lipid hydrolase enzyme, has the potential to modulate various cellular activation responses via the generation of ceramide and by interaction with cellular receptors. We have hypothesized that ASM modulates CD4⁺ T-cell receptor activation and impacts immune responses. We first observed interactions of ASM with the intracellular domains of both CD3 and CD28. ASM further mediates T-cell proliferation after anti-CD3/CD28 antibody stimulation and alters CD4⁺ T-cell activation signals by generating ceramide. We noted that various pharmacological inhibitors of ASM or knockdown of ASM using small hairpin RNA inhibit CD3/CD28-mediated CD4⁺ T-cell proliferation and activation. Furthermore, such blockade of ASM bioactivity by biochemical inhibitors and/or molecular-targeted knockdown of ASM broadly abrogate T-helper cell responses. In conclusion, we detail immune, pivotal roles of ASM in adaptive immune T-cell responses, and propose that these pathways might provide novel targets for the therapy of autoimmune and inflammatory diseases.Acid sphingomyelinase (ASM), a lipid hydrolase enzyme localized to lysosomes and cell membranes, converts sphingomyelin to ceramide,¹ an important lipid messenger mediating cell signaling.^{2, 3} Through the generation of ceramide, ASM appears to have an important role in regulating cell differentiation, proliferation, and apoptosis.^{1, 4} Abnormalities in ASM bioactivity result in multiple system disorders. As an example, patients with Niemann–Pick disease, who have mutations in the ASM gene, exhibit neurological symptoms at early age, and develop visceral organ abnormalities in later life.⁴ Patients with Niemann–Pick disease are at risk of infections,⁵ as can be modeled in ASM-deficient mice.^{6, 7} This phenotype has been attributed to phagocyte dysfunction.⁸ Recently, however, ASM function has also been described and noted in various other non-phagocytic immune cells, for example, regulating cytotoxic granule secretion by CD8⁺ T cells.⁹ASM has been reported to modulate T-cell receptor (TCR) signaling initiated by TNF,¹⁰ mediate CD28 signals,¹¹ and induce or rescue CD4⁺ T cells from apoptosis under certain circumstances.^{12, 13} By generating ceramide, ASM serves as a regulator of intracellular downstream signaling. However, the exact manner whereby ASM participates in TCR/CD3 or/and CD28 signaling remains controversial.^{10, 11, 14} Furthermore, the molecular mechanisms as to how ASM regulates CD4⁺ T-cell activation are still largely unexplored.Adaptive immune responses are important in the maintenance of human immune homeostasis. Imbalances in T-helper cell (Th) responses associated with aberrant CD4⁺ T-cell activation contribute to the development of inflammation as in human autoimmune diseases.^{15, 16} It remains unclear whether or how ASM might dictate Th responses during the progression of inflammatory diseases.In the present study, we confirm that ASM interacts with CD3 and CD28, and mediates intracellular signals that control CD4⁺ T-cell activation. ASM inhibition either by pharmacological inhibitors of ASM or knockdown of ASM results in decreased ceramide production. This leads to non-responsiveness of CD4⁺ T-cell to CD3/CD28 engagement, and causes globally diminished Th responses. These data suggest the pivotal role of ASM in CD3/CD28 intracellular signaling and adaptive immune responses, and also provide a potential target for the therapy of immune disease.     

Regulation of T-cell activation by the cytoskeleton

To become activated, T cells must efficiently recognize antigen-presenting cells or target cells through several complex cytoskeleton-dependent processes, including integrin-mediated adhesion, immunological-synapse formation, cellular polarization, receptor sequestration and signalling. The actin and microtubule systems provide the dynamic cellular framework that is required to orchestrate these processes and ultimately contol T-cell activation. Here, we discuss recent advances that have furthered our understanding of the crucial importance of the T-cell cytoskeleton in controlling these aspects of T-cell immune recognition.     

T-cell activation: the power of one

Adaptive immunity depends on antigen-specific activation of resting lymphocytes. Using high-resolution live-cell imaging, a single ligand has been found to trigger a biochemical response in T cells. On the basis of this and other recent findings, a 'pseudodimer' with one foreign- and one self-antigen-engaged receptor linked via a CD4 molecule has been proposed as the fundamental unit of effective T-cell signaling.     

On the dynamics of T-cell activation in lymph nodes

The immune system is a complex network comprising many different organs and cell types, all of which have to work together in a highly accurate manner to exert their function. How is it, then, that the key players of adaptive immunity, T cells, B cells and dendritic cells (DC) move through this network? How is compartmentalization maintained and how do they interact? Over the past decade much attention has been paid to how and where T-cell/DC interactions take place, but only recently--with the advent of new techniques--has research been directed to investigate 'live' T-cell/DC interactions ex vivo and in situ. Whereas the overall sequence of events leading to T-cell activation is largely undisputed, many of the cellular and molecular details of early T-cell priming remain undefined or controversial. This review will focus on recent findings and discuss their implications for T-cell activation.     

Studies of T-cell activation in chronic inflammation

The strong association between specific alleles encoded within the MHC class II region and the development of rheumatoid arthritis (RA) has provided the best evidence to date that CD4+ T cells play a role in the pathogenesis of this chronic inflammatory disease. However, the unusual phenotype of synovial T cells, including their profound proliferative hyporesponsiveness to TCR ligation, has challenged the notion that T-cell effector responses are driven by cognate cartilage antigens in inflamed synovial joints. The hierarchy of T-cell dysfunction from peripheral blood to inflamed joint suggests that these defects are acquired through prolonged exposure to proinflammatory cytokines such as tumour necrosis factor (TNF)-alpha. Indeed, there are now compelling data to suggest that chronic cytokine activation may contribute substantially to the phenotype and effector function of synovial T cells. Studies reveal that chronic exposure of T cells to TNF uncouples TCR signal transduction pathways by impairing the assembly and stability of the TCR/CD3 complex at the cell surface. Despite this membrane-proximal effect, TNF selectively uncouples downstream signalling pathways, as is shown by the dramatic suppression of calcium signalling responses, while Ras/ERK activation is spared. On the basis of these data, it is proposed that T-cell survival and effector responses are driven by antigen-independent, cytokine-dependent mechanisms, and that therapeutic strategies that seek to restore T-cell homeostasis rather than further depress T-cell function should be explored in the future.     

Effect of aging on T-cell tolerance induction

In the MHC compatible rat strain combination AS → HS, the same ⁵¹Cr-labeled lymph node suspension behaves totally differently depending on whether it is injected into syngeneic or allogeneic recipients. Using ⁵¹Cr-labeled lymph node suspensions from AS or (AS × HS)F1 donors, and AS, HS, and F₁ hosts, the distribution of label over a 72-hr period was studied. Evidence has been obtained that recognition of self-components results in the homing of cells to the lymph nodes in syngeneic hosts, while recognition of foreignness impairs homing of the same cells to the lymph nodes in allogeneic hosts. The spleen is the major organ in which these recognition processes occur. Substantially more cells are destroyed by nonimmune allogeneic hosts than by syngeneic hosts, a clear difference being apparent as early as 6 hr after injection. Some lymphoid cells are obligatory spleen seekers and do not enter the lymph nodes.     

Role of feeding in the reproductive 'group effect' in females of the German cockroach Blattella germanica (L.)

We have found that whether a female German cockroach, Blattella germanica (L.), is kept alone or in the presence of another female has a major impact on how fast it reproduces and how much it eats. By the sixth day of adulthood, females paired since adult eclosion had substantially larger o?cytes than did females isolated during the same time, and females paired with intact females, or with ones rendered incapable of feeding, consumed more rat chow in the first six days of adulthood than did isolated females. The stimulatory effect of pairing on reproduction was, however, partially independent of feeding because the o?cytes of solitary and paired females differed in size on day 6 even when they were given, and had consumed, the same amount of food. This result was confirmed with analysis of covariance using the total food intake of a female as the covariate in the analysis. A female's social condition probably influenced the development of its o?cytes by affecting the quantity of juvenile hormone synthesized by its corpora allata. The corpora allata of paired females produced more hormone than did those of isolated ones, even when all females had consumed an equivalent amount of food. Moreover, females treated with a juvenile hormone analog, fenoxycarb, reproduced more quickly than identically reared and fed control females, showing that juvenile hormone could influence reproduction independently of feeding. We conclude that both group rearing and food intake accelerate o?cyte development by diminishing the brain's inhibition on the synthesis of juvenile hormone.     

The role of peripheral T-cell deletion in transplantation tolerance

The apoptotic deletion of thymocytes that express self-reactive antigen receptors is the basis of central (thymic) self-tolerance. However, it is clear that some autoreactive T cells escape deletion in the thymus and exist as mature lymphocytes in the periphery. Therefore, peripheral mechanisms of tolerance are also crucial, and failure of these peripheral mechanisms leads to autoimmunity. Clonal deletion, clonal anergy and immunoregulation and/or suppression have been suggested as mechanisms by which 'inappropriate' T-lymphocyte responses may be controlled in the periphery. Peripheral clonal deletion, which involves the apoptotic elimination of lymphocytes, is critical for T-cell homeostasis during normal immune responses, and is recognized as an important process by which self-tolerance is maintained. Transplantation of foreign tissue into an adult host represents a special case of 'inappropriate' T-cell reactivity that is subject to the same central and peripheral tolerance mechanisms that control reactivity against self. In this case, the unusually high frequency of naive T cells able to recognize and respond against non-self-allogeneic major histocompatibility complex (MHC) antigens leads to an exceptionally large pool of pathogenic effector lymphocytes that must be controlled if graft rejection is to be avoided. A great deal of effort has been directed toward understanding the role of clonal anergy and/or active immunoregulation in the induction of peripheral transplantation tolerance but, until recently, relatively little progress had been made towards defining the potential contribution of clonal deletion. Here, we outline recent data that define a clear requirement for deletion in the induction of peripheral transplantation tolerance across MHC barriers, and discuss the potential implications of these results in the context of current treatment modalities used in the clinical transplantation setting.     

CD3 pathway of T-cell activation. II. Role of HLA-class I molecules in early events

The role of distinct regions of HLA class I molecules in regulating T-cell activation via the CD3-antigen receptor complex was investigated. Monoclonal antibodies (MoAbs) which recognize monomorphic and polymorphic epitopes on HLA Class I molecules were shown to inhibit T-cell proliferation to OKT3. These MoAbs have differential effects on the synthesis of interleukin-2 (IL-2) and IL-2 receptor expression. Cell cycle analysis demonstrated that these MoAbs function both in inhibiting cell cycle entry (G0-G1 shift) and in blocking cell cycle progression (G1-S shift) of activated T cells. Furthermore, these MoAbs have regulatory effects on the alternate pathway of T-cell activation via the CD2 molecule, T-cell activation induced by PHA, and activation induced by the phorbol ester PMA in conjunction with the calcium ionophore Ionomycin. Thus these MoAbs have different effects depending upon the pathway of T-cell activation. The results indicate that HLA class I molecules are selectively involved in the sequence of intracellular events leading to T-cell activation and proliferation.     

Rituximab-induced direct inhibition of T-cell activation

Background

Rituximab, an anti-CD20 monoclonal antibody, is reported to increase the T-cell-dependent infection risk. The current study was designed to investigate whether rituximab interferes with T-cell activation.

Patients and methods

Patients with non-Hodgkin lymphoma receiving 4–6 courses of 375?mg/m² rituximab underwent detailed assessment of T-cell activation pre- and post-rituximab. A similar analysis assessed the in vitro effect of rituximab on T-cell activation in response to allogeneic dendritic cells (allo-DCs) and other stimuli.

Results

Patients receiving rituximab exhibited a significant decline in IL-2 and IFN-γ levels in peripheral blood, most prominent after repeated rituximab courses. Evaluation at 3?months after rituximab therapy showed restoration of inflammatory cytokine production. Similarly, in vitro stimulation of peripheral blood mononuclear cells in the presence of rituximab resulted in a significant decrease in T-cell activation markers, inflammatory cytokine production and proliferative capacity. These effects were also observed using B-cell-depleted T cells (CD3⁺CD25^?CD19^?) and were accompanied with disappearance of CD3⁺CD20^dim T-cell population.

Conclusion

Rituximab administration results in transient, dose-dependent T-cell inactivation. This effect is obtained even in B-cell absence and may increase the infection risk.     

Mathematical modeling of T-cell activation kinetic

T-cell activation is a crucial step in mounting of the immune response. The dynamics of T-cell receptor (TCR) specific recognition of peptide presented by major histocompatibility complex (MHC) molecule decides the fate of the T cell. Several biochemical interactions interfere resulting in a highly complex mechanism that would be difficult to understand without computer help. The aim of the present study was to define a mathematical model in order to approach the kinetics of monoclonal T-cell-specific activation. The reaction scheme was first described and the model was tested using experimental parameters from the published data. Simulations were concordant with experimental data showing proportional decrease of membrane TCR and production of interleukin-2 (IL-2). Agonist and antagonist peptides induce different levels of intracellular signal that could make the yes or no decision for entry to cell cycle. Different conditions (peptide concentrations, initial TCR density and exogenous IL-2 levels) can be tested. Several parameters are missing for parameters estimation and adjustment before it could be adapted for a polyclonal T-cell reaction model. However, the model should be of interest in setting experiments, simulation of clinical responses and optimization of preventive or therapeutic immunotherapy.     

Feedback control of T-cell receptor activation

The specificity and sensitivity of T-cell recognition is vital to the immune response. Ligand engagement with the T-cell receptor (TCR) results in the activation of a complex sequence of signalling events, both on the cell membrane and intracellularly. Feedback is an integral part of these signalling pathways, yet is often ignored in standard accounts of T-cell signalling. Here we show, using a mathematical model, that these feedback loops can explain the ability of the TCR to discriminate between ligands with high specificity and sensitivity, as well as provide a mechanism for sustained signalling. The model also explains the recent counter-intuitive observation that endogenous 'null' ligands can significantly enhance T-cell signalling. Finally, the model may provide an archetype for receptor switching based on kinase-phosphatase switches, and thus be of interest to the wider signalling community.     

Polyclonal T-cell activation protocol stimulates preferential expansion of EBV-specific T-cell clones in vitro

Purpose Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukemia. The most important anti-leukemic effect may be mediated by the T-cells contained within the graft; however, the allogeneic T-cells may also give rise to graft-vs-host disease (GVHD). One way to control GVHD might be to transduce the donor T-cells with a drug-inducible suicide gene. If a retrovirus vector is to be used for this transduction, activation of the T-cells is required for integration of the transgene to occur. The activation protocol should ensure expansion of a broad repertoire of donor T-cells. Notably, T-cells specific for herpes virus family antigens are important for adoptive immunoprotection.Methods To define optimal activation conditions for retrovirus-mediated suicide gene transduction of donor T-cells, we examined the repertoire of CD8+ T-cells in general, and Epstein-Barr virus (EBV) specific T-cells in particular, following two different activation and expansion procedures.Results We found that repeated CD3/CD28 stimulation resulted in a high level of activation-induced T-cell death, affecting in vivo expanded clones, some of which were specific for EBV, in particular. In contrast, initial CD3/CD28 activation followed by proliferation in interleukin-2 lead to expansion of EBV-specific clones over and above the expansion observed for CD8+ T-cells in general.Conclusion These results should impact on protocols for ex vivo activation of T-cells prior to suicide gene transduction.     

Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance.

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.