State Of T-cell Activation Research Articles

Complementary HLH susceptibility factors converge on CD8 T-cell hyperactivation

AbstractHemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes. Familial HLH is caused by genetic impairment of granule-mediated cytotoxicity (eg, perforin deficiency). MAS is linked to excess activity of the inflammasome-activated cytokine interleukin-18 (IL-18). Though individually tolerated, mice with dual susceptibility (Prf1⁻/⁻Il18tg; DS) succumb to spontaneous, lethal hyperinflammation. We hypothesized that understanding how these susceptibility factors synergize would uncover key pathomechanisms in the activation, function, and persistence of hyperactivated CD8 T cells. In IL-18 transgenic (Il18tg) mice, IL-18 effects on CD8 T cells drove MAS after a viral (lymphocytic choriomeningitis virus), but not innate (toll like receptor 9), trigger. In vitro, CD8 T cells also required T-cell receptor (TCR) stimulation to fully respond to IL-18. IL-18 induced but perforin deficiency impaired immunoregulatory restimulation-induced cell death (RICD). Paralleling hyperinflammation, DS mice displayed massive postthymic oligoclonal CD8 T-cell hyperactivation in their spleens, livers, and bone marrow as early as 3 weeks. These cells increased proliferation and interferon gamma production, which contrasted with increased expression of receptors and transcription factors associated with exhaustion. Broad-spectrum antibiotics and antiretrovirals failed to ameliorate the disease. Attempting to genetically “fix” TCR antigen-specificity instead demonstrated the persistence of spontaneous HLH and hyperactivation, chiefly on T cells that had evaded TCR fixation. Thus, drivers of HLH may preferentially act on CD8 T cells: IL-18 amplifies activation and demand for RICD, whereas perforin supplies critical immunoregulation. Together, these factors promote a terminal CD8 T-cell activation state, combining features of exhaustion and effector function. Therefore, susceptibility to hyperinflammation may converge on a unique, unrelenting, and antigen-dependent state of CD8 T-cell hyperactivation.

Linking antigen specific T-cell dynamics in a microfluidic chip to single cell transcription patterns

A new non-invasive screening profile has been realized that can aid in determining T-cell activation state at single-cell level. Production of activated T-cells with good specificity and stable proliferation is greatly beneficial for advancing adoptive immunotherapy as innate immunological cells are not effective in recognizing and eliminating cancer as expected. The screening method is realized by relating intracellular Ca2+ intensity and motility of T-cells interacting with APC (Antigen Presenting Cells) in a microfluidic chip. The system is tested using APC pulsed with OVA257-264 peptide and its modified affinities (N4, Q4, T4 and V4), and the T-cells from OT-1 mice. In addition, single cell RNA sequencing reveals the activation states of the cells and the clusters from the derived profiles can be indicative of the T-cell activation state. The presented system here can be versatile for a comprehensive application to proceed with T-cell-based immunotherapy and screen the antigen-specific T-cells with excellent efficiency and high proliferation.

Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies.

Hepato-pancreatico-biliary (HPB) malignancies are difficult-to-treat and continue to to have a high mortality and significant therapeutic resistance to standard therapies. Immune oncology (IO) therapies have demonstrated efficacy in several solid malignancies when combined with chemotherapy, whereas response rates in pancreatic ductal adenocarcinoma (PDA) are poor. While promising in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), there remains an unmet need to fully leverage IO therapies to treat HPB tumors. We therefore defined T cell subsets in the tumor microenvironment of HPB patients utilizing a novel, multiparameter flow cytometry and bioinformatics analysis. Our findings quantify the T cell phenotypic states in relation to checkpoint receptor expression. We demonstrate the presence of CD103+ tissue resident memory T cells (TRM), CCR7+ central memory T cells, and CD57+ terminally differentiated effector cells across all HPB cancers, while the anti-tumor function was dampened by expression of multiple co-inhibitory checkpoint receptors. Terminally exhausted T cells lacking co-stimulatory receptors were more prevalent in PDA, whereas partially exhausted T cells expressing both co-inhibitory and co-stimulatory receptors were most prevalent in HCC, especially in early stage. HCC patients had significantly higher TRM with a phenotype that could confer restored activation in response to immune checkpoint therapies. Further, we found a lack of robust alteration in T cell activation state or checkpoint expression in response to chemotherapy in PDA patients. These results support that HCC patients might benefit most from combined checkpoint therapies, whereas efforts other than cytotoxic chemotherapy will likely be necessary to increase overall T cell activation in CCA and PDA for future clinical development.

Donor selection for adoptive cell therapy with CD45RA− memory T cells for patients with coronavirus disease 2019, and dexamethasone and interleukin-15 effects on the phenotype, proliferation and interferon gamma release

Background aimsWe have previously demonstrated the safety and feasibility of adoptive cell therapy with CD45RA− memory T cells containing severe acute respiratory syndrome coronavirus 2–specific T cells for patients with coronavirus disease 2019 from an unvaccinated donor who was chosen based on human leukocyte antigen compatibility and cellular response. In this study, we examined the durability of cellular and humoral immunity within CD45RA− memory T cells and the effect of dexamethasone, the current standard of care treatment, and interleukin-15, a cytokine critically involved in T-cell maintenance and survival. MethodsWe performed a longitudinal analysis from previously severe acute respiratory syndrome coronavirus 2–infected and infection-naïve individuals covering 21 months from infection and 10 months after full vaccination with the BNT162b2 Pfizer/BioNTech vaccine. ResultsWe observed that cellular responses are maintained over time. Humoral responses increased after vaccination but were gradually lost. In addition, dexamethasone did not alter cell functionality or proliferation of CD45RA- T cells, and interleukin-15 increased the memory T-cell activation state, regulatory T cell expression, and interferon gamma release. ConclusionsOur results suggest that the best donors for adoptive cell therapy would be recovered individuals and 2 months after vaccination, although further studies with larger cohorts would be needed to confirm this finding. Dexamethasone did not affect the characteristics of the memory T cells at a concentration used in the clinical practice and IL-15 showed a positive effect on SARS-CoV-2-specific CD45RA- T cells.

T-cell activation state differentially contributes to neuropsychiatric complications in women with HIV

Neuropsychiatric complications are common among women with HIV (WWH). The pathophysiological mechanisms underlying these complications are not fully known but likely driven in part by immune modulation. We examined associations between T-cell activation states which are required to mount an effective immune response (activation, co-stimulation/normal function, exhaustion, senescence) and neuropsychiatric complications in WWH. 369 WWH (78% HIV RNA undetectable/<20cp/mL) enrolled in the Women's Interagency HIV Study completed neuropsychological testing and measures of depression (Center for Epidemiological Studies Depression Scale-CES-D), self-reported stress levels (Perceived Stress Scale-10), and post-traumatic stress (PTSD Checklist-Civilian Scale). Multiparametric flow cytometry evaluated T-cell activation state. Partial least squares regressions were used to examine T-cell phenotypes and neuropsychiatric outcome associations after confounder adjustment. In the total sample and among virally suppressed (VS)-WWH, CD4+ T-cell exhaustion was associated with poorer learning and attention/working memory (P's < 0.05). In the total sample, CD4+ T-cell activation was associated with better attention/working memory and CD8+ T-cell co-stimulation and senescence was associated with poorer executive function (P's < 0.05). For mental health outcomes, in the total sample, CD4+ T-cell activation was associated with more perceived stress and CD4+ T-cell exhaustion was associated with less depressive symptoms (P's < 0.05). Among VS-WWH, CD4+ senescence was associated with less perceive stress and CD8+ T-cell co-stimulation and senescence was associated with higher depression (P's < 0.05). Together, results suggest the contribution of peripheral CD4+ and CD8+ T-cell activation status to neuropsychiatric complications in WWH.

Regulatory T-lymphocyte subsets in patients with HIV-infection receiving highly active antiretroviral therapy

Background. The reason why HIV-infected patients receiving highly active antiretroviral therapy (HAART) suffer from the increased immune activation remains elusive. Regulatory T-cells (Treg) are able to control immune activation, but their quantity may vary due to the infection. The aim of this work was to estimate the number and subsets of Tregs in HIV-positive patients receiving virologically effective HAART.Materials and methods. The CD4+ T-lymphocyte (CD3+CD4+) and Treg (CD3+CD4+FOXP3+) quantities were determined by flow cytometry. Treg subsets were assessed based on the FOXP3 expression level. The state of T-cell activation was established according to the simultaneous expression of CD38 and HLA-DR molecules.Results. It was shown that HIV-positive patients compared to healthy people have reduced CD4+ T-lymphocyte counts despite virologically effective HAART. At the same time in HIV-infected people, Treg absolute numbers were only slightly decreased. Moreover, the major part of Treg pool in their blood consisted of lymphocytes with a high level of FOXP3 expression that corresponded to the phenotype of cells with the highest suppressor activity. However, an increased relative amount of activated CD4+ T-lymphocytes was retained in the HIV-infected individuals’ blood.Conclusion. In HIV-infected patients who received HAART in time and whose treatment resulted in an effective HIV viral load suppression and a satisfactory CD4+ T-cell counts increase, a relatively large pool of peripheral Tregs is maintained. However, these lymphocytes are not enough to fully control immune activation that develops against the background of chronic lentivirus infection.

DR3 signaling modulates the function of Foxp3+ regulatory T cells and the severity of acute graft-versus-host disease

AbstractCD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft-versus-host disease (GVHD) in murine models of allogeneic hematopoietic stem cell transplantation. We recently demonstrated that a single treatment of the agonistic antibody to DR3 (death receptor 3, αDR3) to donor mice resulted in the expansion of donor-derived Treg and prevented acute GVHD, although the precise role of DR3 signaling in GVHD has not been elucidated. In this study, we comprehensively analyzed the immunophenotype of Treg after DR3 signal activation, demonstrating that DR3-activated Treg (DR3-Treg) had an activated/mature phenotype. Furthermore, the CD25+Foxp3+ subpopulation in DR3-Treg showed stronger suppressive effects in vivo. Prophylactic treatment of αDR3 to recipient mice expanded recipient-derived Treg and reduced the severity of GVHD, whereas DR3 activation in mice with ongoing GVHD further promoted donor T-cell activation/proliferation. These data suggest that the function of DR3 signaling was highly dependent on the activation status of the T cells. In conclusion, our data demonstrated that DR3 signaling affects the function of Treg and T-cell activation after alloantigen exposure in a time-dependent manner. These observations provide important information for future clinical testing using human DR3 signal modulation and highlight the critical effect of the state of T-cell activation on clinical outcomes after activation of DR3.

Doses of Total Nucleated Cells and Activated T-Cells Transplanted Influence Survival and Graft Versus Host Disease Among AML and MDS Recipients of Reduced-Intensity Mobilized Peripheral Blood Allografts

Background: Patients undergoing allogeneic hematopoietic stem cell transplant with reduced-intensity conditioning (RIC-alloSCT) rely primarily on the graft-versus-tumor (GVT) effect to prevent relapse. Although relapse remains high in this setting, previous data suggest modifications of allograft composition could produce enhanced GVT effect and improve outcomes. Prior retrospective studies suggested higher total nucleated cell (TNC) and CD8 dose correlate with improved overall survival (OS) and a reduction in relapse among all patients undergoing RIC-alloSCT. The aim of our study was to further investigate this association by comparing transplant outcomes with detailed graft immunophenotyping data including T-cell and NK-cell activation and maturation status and CD34 cell doses in patients with AML and MDS undergoing RIC-alloSCT.Methods: We performed a retrospective analysis using data from consecutive patients with AML and MDS who underwent RIC-alloSCT at a single center from 2010-2015 who had graft immunophenotyping data available. We compared transplant outcomes with TNC per kilogram (kg) and dose of CD3, CD4, CD8, CD34, and NK cells along with selected activation and maturation subsets. A competing risk regression analysis was conducted to examine the association between cell dose/kg and risk of relapse and graft versus host disease (GVHD). Overall survival and relapse free survival (RFS) were assessed utilizing the Cox proportional hazard model. Estimates of survival probability were determined by the Kaplan-Meier survival function. The log-rank test was used to compare OS and RFS among patients. Univariable and multivariable regression analyses were performed. Disease Risk Index (DRI) was used to stratify risk among patients. To identify optimal cutoffs (OC) of continuous variables for a specific outcome, a Classification and Regression Tree (CART) algorithm was utilized.Results: Our study included 142 patients who underwent RIC-alloHSCT for AML (n=97, 68%) and MDS (n=45, 32%). The vast majority of patients (98%) received fludarabine and busulfan conditioning with 65% receiving anti-thymocyte globulin. All patients received tacrolimus-based GVHD prophylaxis. Overall relapse rate was 37% at 3 years. Overall survival was 49% with median follow-up of 3.2 years.Total Nucleated Cell Dose: In multivariable analysis controlling for disease, comorbidity index, and performance status, a high infused TNC dose (10.6 - 16.8 x 108 cells/kg) correlated with improved overall survival (HR 0.27, 95% CI [0.12 - 0.59], p=0.001) and relapse free survival (HR 0.31, 95% CI [0.15 - 0.64], p=0.002). However, a very high TNC dose (OC >16.8 x 108 cells/kg) correlated with significantly worse OS (HR 2.84, 95% CI [1.16 - 6.98], p=0.023) and trended toward worse RFS (HR 2.09, 95% CI [0.88 - 4.93], p=0.093). Higher TNC doses were also correlated with an increased risk of chronic GVHD (HR 2.16, 95% CI [1.28 - 3.67], p=0.004, OC 9.8 x 108 cells/kg).Cell Subsets: Very low CD3 cell doses (OC <1.06 x 108 cells/kg) correlated with worse OS (HR 3.30, 95% CI [1.23 - 8.85], p=0.017) and worse RFS (HR 2.64, 95% CI [1.01 - 6.88], p=0.047). High CD3 doses were correlated with an increase in acute GVHD (all grades) (HR 2.41, 95% CI [1.36 - 4.25], p=0.002, OC 4.1 x 108 cells/kg). NK, CD4, CD8, and CD34 cell dose was not associated with relapse, OS, or RFS in multivariable analysis. Utilizing a previously published (Reshef et al, JCO, 2015) cutoff of 7.2 x 108 cells/kg, there was a trend toward a reduction in relapse with higher CD8 cell dose, but this did not reach statistical significance (HR 0.67, 95% CI [0.39 - 1.16], p=0.15). A high dose of late activated T-cells (CD3+/HLA-DR+) correlated with increased risk of acute GVHD (HR 2.31, 95% CI [1.26 - 4.20], p=0.006, OC 2.01 x 107 cells/kg) and chronic GVHD (HR 1.85, 95% CI [1.12 - 3.07], p=0.016, OC 6.6 x 106 cells/kg).Conclusion: Among AML and MDS patients following RIC-alloSCT, higher infused TNC/kg was associated with improved RFS and overall survival, confirming findings from a recent study (Martin et al, Haematologica, 2016). However, very high TNC doses may be deleterious. T-cell activation state influences risk of GVHD. Further research needs to be pursued to corroborate these findings and potentially optimize TNC and T-cell doses. We were unable to identify a significant correlation between CD8 cell dose and relapse or overall survival. [Display omitted] [Display omitted] DisclosuresLozanski:Genentech: Research Funding; Boehringer Ingelheim: Research Funding; Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding.

Histone Deacetylase 6 (HDAC6) Influences T-Cell Activation and Survival: Implications For Cancer Immunotherapy

The role of histone deacetylases (HDACs) as epigenetic regulators of immune function is becoming increasingly clear. Recently, the role of specific HDACs in orchestrating T-cell maturation, survival and function has begun to emerge, giving rationale to selective therapy to direct immune responses in different disease settings, including cancer. In particular, HDAC6 has recently been characterized as a negative regulator of regulatory T-cell suppressive activity (de Zoeten, Molecular and Cellular Biology, 2011). Here we report an expanded, novel role of HDAC6 in regulating T-cell survival and activation. First, the relative expression of the eleven classic HDACs was evaluated in resting and activated T-cells from mouse and human samples. It was found that the majority of HDACs decrease in expression following activation, including HDAC6. Next, in a HDAC6KO mouse model, it was found that T-cells lacking HDAC6 had skewed survival when compared to wild-type murine T-cells. This difference seems to be the result of an increased CD4+ T-cells population in the lymph nodes, with a concomitant decrease in viable CD8+ T-cells. To determine whether this population skewing was the consequence of defects in HDAC6KO mice T-cell development, wild-type murine T-cells were treated with an isotype-selective HDAC6 inhibitor. The results seen in HDAC6KO T-cells were recapitulated when wild-type T-cells were activated and treated with HDAC6 specific inhibitors, indicating a role of HDAC6 outside of thymic development in promoting CD4+ T-cell survival at the expense of CD8+ T-cells. Interestingly, it was found that activated CD4+ T-cells displayed decreased expression of the apoptosis signaling receptor FAS after HDAC6 inhibition while no differences were observed in CD8+ T-cells under the same conditions. In addition to these results implicating HDAC6 in regulating T-cell survival, expression of surface markers was altered in both CD8+ and CD4+ T-cells, including enhanced expression of the activation molecule CD69 in stimulated T-cells treated with an isotype-selective HDAC6 inhibitor. Finally, in vivo studies in tumor-bearing HDAC6KO mice revealed a significantly delayed in tumor progression. Similar results were observed in lymphoma-bearing mice treated with HDAC6 specific inhibitors. Taken together, this data shows that HDACs are dynamic in expression with regards to T-cell activation state. More specifically, we have unveiled hereto-unexplored roles of HDAC6 in regulating T-cell survival and function, pointing at this specific HDAC as an appealing target to harness T-cell immunity in hematologic malignancies. Disclosures:No relevant conflicts of interest to declare.

Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic

The concept of suicide gene therapy dates from the beginning of the field of gene therapy and was one of the first clinical applications. Initially described as an anticancer therapy, it quickly found a specific application in the practice of hematopoietic stem cell transplantation. A recent publication by Di Stasi et al. presents the first clinical data on a relatively new suicide gene system that targets caspase 9–mediated apoptosis in an inducible fashion.1 Specifically, five children undergoing haplo-identical stem cell transplantation for leukemia received donor T lymphocytes that had been genetically engineered with the inducible caspase 9 (iCasp9) gene. Four of the five patients developed graft-vs.-host disease (GVHD) caused by the donor lymphocytes that was quickly reversed by induction of the iCasp9 suicide gene. These results further validate the findings of others that suicide gene therapy for the control of GVHD is a valuable clinical procedure and, in the broader context, may have implications for a variety of other gene therapy applications in which a suicide safety switch may be useful.

Generation of Potent and Stable Human CD4+ T Regulatory Cells by Activation-independent Expression of FOXP3

Therapies based on enhancing the numbers and/or function of T regulatory cells (Tregs) represent one of the most promising approaches to restoring tolerance in many immune-mediated diseases. Several groups have investigated whether human Tregs suitable for cellular therapy can be obtained by in vitro expansion, in vitro conversion of conventional T cells into Tregs, or gene transfer of the FOXP3 transcription factor. To date, however, none of these approaches has resulted in a homogeneous and stable population of cells that is as potently suppressive as ex vivo Tregs. We developed a lentivirus-based strategy to ectopically express high levels of FOXP3 that do not fluctuate with the state of T-cell activation. This method consistently results in the development of suppressive cells that are as potent as Tregs and can be propagated as a homogeneous population. Moreover, using this system, both naïve and memory CD4(+) T cells can be efficiently converted into Tregs. To date, this is the most efficient and reliable protocol for generating large numbers of suppressive CD4(+) Tregs, which can be used for further biological study and developed for antigen-specific cellular therapy applications.

Coinhibitory T-Cell Signaling in Islet Allograft Rejection and Tolerance

Autoaggressive T cells directed against insulin secreting pancreatic beta-cells mediate the development of type 1 diabetes. Islet transplantation offers superior glycemic control over exogenous insulin, but chronic immunosuppression limits its broad application. Pathogenic T cells are also important in allograft rejection. Inducing and maintaining antigen-specific peripheral T-cell tolerance toward beta-cells is an attractive strategy to prevent autoimmune disease, and to facilitate treatment of diabetes with islet allografts without long-term immunosuppression. Recent efforts have focused on blocking costimulatory T-cell signals for tolerance induction. Although costimulatory blockade can prolong graft survival, true immunological tolerance remains elusive. Costimulatory signals may even be required for the maintenance of peripheral tolerance. The discovery of novel coinhibitory T-cell pathways, including CTLA-4, PD-1, and BTLA, offers an alternative approach. Stimulating negative T cell cosignals alone or in combination may help induce tolerance. The focus of this review is to summarize the strategies directed at turning off the immune response by exploiting these negative cosignaling pathways in tolerance induction in islet transplantation. Activating several coinhibitory pathways together may be synergistic in preventing pathogenic T-cell responses. Tolerance induction will likely rely on understanding the balance of positive and negative signals affecting the state of T-cell activation.

Human immunodeficiency virus type 1 Nef-mediated downregulation of CD4 correlates with Nef enhancement of viral pathogenesis.

The nef gene products encoded by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus type 1 (SIV-1) increase viral loads in infected hosts and accelerate clinical progression to AIDS. Nef exhibits a spectrum of biological activities, including the ability to downregulate surface expression of CD4 and major histocompatibility complex (MHC) class I antigens, to alter the state of T-cell activation, and to enhance the infectivity of viral particles. To determine which of these in vitro functions most closely correlates with the pathogenic effects of Nef in vivo, we constructed recombinant HIV-1 NL4-3 viruses carrying mutations within the nef gene that selectively impair these functions. These mutant viruses were evaluated for pathogenic potential in severe combined immunodeficiency (SCID) mice implanted with human fetal thymus and liver (SCID-hu Thy/Liv mice), in which virus-mediated depletion of thymocytes is known to be Nef dependent. Disruption of the polyproline type II helix (Pxx)4 within Nef (required for binding of Hck and p21-activated kinase-like kinases, downregulation of MHC class I, and enhancement of HIV-1 infectivity in vitro but dispensable for CD4 downregulation) did not impair thymocyte depletion in virus-infected Thy/Liv human thymus implants. Conversely, three separate point mutations in Nef that compromised its ability to downregulate CD4 attenuated thymocyte depletion while not diminishing viral replication. These findings indicate that the functional ability of Nef to downregulate CD4 and not MHC class I downregulation, Hck or PAK binding, or (Pxx)4-associated enhancement of infectivity most closely correlates with Nef-mediated enhancement of HIV-1 pathogenicity in vivo. Nef-mediated CD4 downregulation merits consideration as a new target for the development of small-molecule inhibitors.

High Concentration of Soluble HLA-DR in the Synovial Fluid: Generation and Significance in “Rheumatoid-like” Inflammatory Joint Diseases

In the search for its role in inflammatory joint diseases, soluble HLA-DR (sHLA-DR) was quantitated in 72 synovial fluids (SF) by a newly established immunoenzyme assay. Unlike other soluble receptors which accumulated only moderately (sCD25, sCD4) or negligibly (sHLA class I, sCD8) in the SF, SF sHLA-DR levels exceeded serum levels by up to 3 orders of magnitude and varied disease dependently from “control” values (traumatic synovitis and osteoarthritis: 9.9 ± 6.1 ng/ml). Clear-cut different SF sHLA-DR values in HLA-DR-associated “rheumatoid-like” (136.5 ± 130.0 ng/ml) vs HLA-B27-associated “spondylarthropathy-like” arthritic forms (28.4 ± 29.1 ng/ml) were most significant comparing oligoarticular juvenile chronic arthritis type I (147.6 ± 112.6 ng/ml) and type II (3.3 ± 1.1 ng/ml), thus offering a new classification marker. Also ex vivo, large amounts of sHLA-DR were released spontaneously by SF mononuclear cells and found to be related to the T-cell activation state. SF sHLA-DR may be shed in large complexes or micelles, as it eluted mainly at >450 kDa on gel filtration. Western blotting revealed that the majority of SF sHLA-DR consisted of full-length α- and β-chains. Minor fractions of smaller sized antigens seemed to be generated by proteolytic cleavage rather than by alternative splicing, since only minute amounts of HLA-DRB mRNA lacking the transmembrane exon could be amplified by RT–PCR. Distinct forms of high-dose sHLA-DR, able to provoke rather than to suppress T-cell responses, are discussed as contributing to some HLA-DR disease association.

Immunobiology of left ventricular assist devices

The increasing use of implanted biomaterial devices has made it evident that no material is biologically inert. As a result of direct contact with elements of the blood circulation, such as during hemodialysis or after left ventricular assist device (LVAD) implantation, significant changes in systemic immunologic and thrombostatic functions occur. The clinical success of LVAD implantation has, nevertheless, been accompanied by complications arising from an aberrant state of monocyte and T-cell activation, leading to heightened susceptibility of circulating CD4 T cells to undergo activation-induced cell death; this results in progressive defects in cellular immunity and an increased risk of serious infection. Because of the increased state of T-cell activation and the selective loss of Th1 cytokine producing CD4 T cells, LVAD recipients also develop B-cell hyperreactivity and dysregulated immunoglobulin syntheses by unopposed production of Th2 cytokines and increased CD40 Ligand-CD40 interactions. LVADs are currently being evaluated as a permanent therapy for end-stage heart failure. Because these immune dysfunctions appear to be related to the effects of excessive biomaterial associated T-cell activation, future efforts will need to be directed at either altering the physical properties of the materials interacting with the host circulation or pharmacological intervention aimed at inhibiting T-cell activation. Copyright © 2000 by W.B. Saunders CompanyProgress in Cardiovascular Diseases, Vol. 43, No. 1 (July/August), 2000: pp 67-80

NOVEL IMMUNOTHERAPEUTIC APPROACHES FOR THE TREATMENT OF ALLERGIC DISEASES

Advances in the knowledge of the cellular and molecular basis of immunity have led to an enhanced thorough understanding of the immunologic features that characterize an allergic response. A hallmark of an allergic response is a persistently elevated level of specific IgE antibodies to a variety of antigens, also referred to as allergens, to which the affected individual is regularly exposed by inhalation, ingestion, or contact with the skin. 48 Allergic sensitization involves processing of the antigen by an antigen-presenting cell (APC) and presentation, in association with a class II major histocompatibility complex (MHC) protein, to a T-cell receptor. Whereas dendritic cells (and macrophages) predominantly act as APCs in primary immunization, B cells may also participate in secondary immune responses to allergens. Activation of T cells requires the first signal from this trimolecular interaction and an additional costimulatory signal involving the ligation of B-7 on an APC (CD80/86) with CD28 or CTLA4 on a T cell. 27 When stimulated by antigens, helper T cells produce an array of specific cytokines, all of which have been designated as either T H 1 cytokines (interleukin [IL]-2, interferon [IFN]-γ, and tumor necrosis factor [TNF]-β) or T H 2 cytokines (IL-4, IL-5, IL-9, IL-10, and IL-13). The state of T-cell activation depends on several factors. 27,85 The first of these factors is the affinity of the interaction between the antigen and the T cell. The site of the antigen recognized by the T cell is termed the epitope. The affinity of epitope–T cell interaction is affected by the concentration of the antigen and the type of APC. 80 It also depends on the cytokine milieu of the T cell during antigen interaction. Thus, IFN-γ and IL-12 promote a T H 1-like response, whereas IL-4 promotes a T H 2-like response. 12 Additionally, host immune response genes may bias the overall immune responsiveness of an individual to favor a T H 1- or T H 2-like response. A T H 2-like cytokine profile is associated with the induction of IgE antibody (Ab) production in vitro and in vivo. 73 Specifically, IL-4 favors the development of T H 2-like cells from uncommitted T cells, and both IL-4 and IL-13 play a role in IgE antibody production. Manifestation of an allergic reaction depends on the specific IgE levels and the amount of exposure at the time of the reaction. Although an allergic condition is a risk factor for asthma, about 20% to 30% of asthmatics do not show positive skin tests to allergens. In general terms, asthma is defined as an inflammatory disease where not only lymphocytes, but also other cells, such as mast cells, basophils, eosinophils, and epithelial cells, play a role. Studies to date suggest that T H 2-like cytokines, such as IL-4 and IL-5, also play an important role in nonatopic asthma. 3,85 Developing immunotherapeutic approaches against allergic diseases, including asthma, may be pragmatically divided into two categories based on how soon they may make an inroad to the clinic, as shown in Figure 1. The first category includes therapies that are already in phase II/phase III clinical trials and are expected to be available in clinics within the next 1 to 3 years; such therapies include antihuman IgE antibodies and the soluble IL-4 receptor. The second category includes experimental approaches that are mainly at the stage of preclinical research in model systems and may proceed to clinical trials and the clinic within the next 5 to 10 years; these therapies include various allergen-specific and allergen-nonspecific approaches.

Selective resistance of mucosal T-cell activation to immunosuppression in Crohn's disease.

The inappropriately high state of T-cell activation found in Crohn's disease could be due to failure to respond to inhibitory signals. We tested the hypothesis that Crohn's disease mucosal T-cells are resistant to the immunosuppressive action of interleukin4. Patients with Crohn's disease, ulcerative colitis, and other malignant and non-malignant conditions undergoing bowel resection. The effect of interleukin-4 on lamina propria mononuclear cells from Crohn's disease, ulcerative colitis and control mucosa was assessed on various T-cell functions: interleukin-2-induced cytotoxicity, soluble interleukin-2 receptor and interleukin-2 production, and expression of mRNA for interleukin-2R and interferon-gamma. Cytotoxicity of control and ulcerative colitis cells was markedly decreased by interleukin-4, whereas Crohn's disease cells failed to be inhibited. Addition of interleukin-4 to interleukin-2-stimulated cultures decreased soluble interleukin-2R production significantly less in Crohn's disease and ulcerative colitis than control cells. In the same cultures, residual levels of interleukin-2 were significantly increased in control and ulcerative colitis, but not Crohn's disease cultures. Finally, Crohn's disease cells were significantly more resistant to interleukin-4-mediated inhibition of spontaneous and interleukin-2-induced expression of interleukin-2Ralpha and interferon-gamma mRNA compared to control cells. The effector function, receptor expression and cytokine production of Crohn's disease mucosal T-cells are resistant to interleukin4-mediated inhibition. Failure to respond to down-regulatory signals may contribute to persistent T-cell activation and chronicity of inflammation in Crohn's disease.

Paradoxical inhibition of T-cell function in response to CTLA-4 blockade; heterogeneity within the human T-cell population.

T-cell co-stimulation delivered by the molecules B7-1 or B7-2 through CD28 has a positive effect on T-cell activation, whereas engagement of cytotoxic T-lymphocyte antigen 4 (CTLA-4) by these molecules inhibits activation. In vivo administration to mice of blocking monoclonal antibodies or Fab fragments against CTLA-4 can augment antigen-specific T-cell responses and, thus, therapy with monoclonal antibody against CTLA-4 has potential applications for tumor therapy and enhancement of vaccine immunization. The effects of B7-1 and B7-2 co-stimulation through CD28 depend on the strength of the signal delivered through the T-cell receptor (TCR) and the activation state of T cells during activation. Thus, we sought to determine whether these factors similarly influence the effect of B7-mediated signals delivered through CTLA-4 during T-cell activation. Using freshly isolated human T cells and Fab fragments of a monoclonal antibody against CTLA-4, we demonstrate here that CTLA-4 blockade can enhance or inhibit the clonal expansion of different T cells that respond to the same antigen, depending on both the T-cell activation state and the strength of the T-cell receptor signal delivered during T-cell stimulation. Thus, for whole T-cell populations, blocking a negative signal may paradoxically inhibit immune responses. These results provide a theoretical framework for clinical trials in which co-stimulatory signals are manipulated in an attempt to modulate the immune response in human disease.

Activation-induced T-cell death and immune dysfunction after implantation of left-ventricular assist de vice

Cardiac transplantation is a limited option for end-stage heart failure because of the shortage of donor organs. Left-ventricular assist devices (LVADs) are currently under investigation as permanent therapy for end-stage heart failure, but long-term successful device implantation is limited because of a high rate of serious infections. To examine the relation between LVAD-related infection and host immunity, we investigated immune responses in LVAD recipients. We compared the rate of candidal infection in 78 patients with New York Heart Association class IV heart failure who received either an LVAD (n=40) or medical management (controls, n=38). Fluorochrome-labelled monoclonal antibodies were used in analyses of T-cell phenotype. Analysis of T-cell function included intradermal responses to recall antigens and proliferative responses after stimulation by phytohaemagglutinin, monoclonal antibodies to CD3, and mixed lymphocyte culture. We measured T-cell apoptosis in vivo by annexin V binding, and confirmed the result by assessment of DNA fragmentation. Activation-induced T-cell death was measured after T-cell stimulation with antibodies to CD3. All immunological tests were done at least 1 month after LVAD implantation. Between-group comparisons were by Kaplan-Meier actuarial analysis and Student's t test. By 3 months after implantation of LVAD, the risk of developing candidal infection was 28% in LVAD recipients, compared with 3% in controls (p=0.003). LVAD recipients had cutaneous anergy to recall antigens and lower (<70%) T-cell proliferative responses than controls after activation via the T-cell receptor complex (p<0.001). T cells from LVAD recipients had higher surface expression of CD95 (Fas) (p<0.001) and a higher rate of spontaneous apoptosis (p<0.001) than controls. Moreover, after stimulation with antibodies to CD3, CD4 T-cell death increased by 3.2-fold in LVAD recipients compared with only 1.2-fold in controls (p<0.05). LVAD implantation results in an aberrant state of T-cell activation, heightened susceptibility of CD4 T cells to activation-induced cell death, progressive defects in cellular immunity, and increased risk of opportunistic infection.

Low versus High Density of Immobilized Anti-CD3 Influences IL-4 Regulation of T-Cell Immune Responses

Varying the concentration of anti-CD3 immobilized on Sepharose beads allowed us to study both the inhibitory, and the stimulatory effects of IL-4 on purified T cells and contrast IL-4- versus IL-2-driven T-cell proliferative responses. In the presence of low density immobilized anti-CD3. IL-4 was unable to stimulate purified T cells and was inhibitory to IL-2-driven T-cell responses. The inhibitory, effects of IL-4 were enhanced by preincubation of T cells with IL-4 prior to stimulation. In contrast, the inhibitory effects of IL-4 could be avoided by delaying the addition of IL-4 until Days 3-5 of culture or they could be reversed by the addition of IL-1. In the presence of high-density anti-CD3, IL-4 elicited an IL-2-independent proliferative response by purified T cells or sorted CD8 + cells. Comparison of IL-4-driven versus IL-2-driven T-cell responses demonstrated that IL-2 was able to upregulate mRNA for IL-4 receptors and interferon-γ while IL-4 had minimal effects on upregulating mRNA for either the p55 or the p75 IL-2 receptor subunits or interferon-γ. The timing of the presence of IL-4, the state of T-cell activation, and the nature and strength of the stimulatory signal influenced the regulatory effect of IL-4 on the immune response.