Absence Of T Cell Receptor Stimulation Research Articles

Assessment of T-Cell Receptor (TCR) Mediated T-Cell Activation in Acute Myeloid Leukemia at Diagnosis Has Implications for Risk Stratification and Response to Immune Checkpoint Inhibitors

Background Relapse in patients with acute myeloid leukemia (AML) has been, in part, attributed to the ability of leukemic blasts to escape immune surveillance through mechanisms such as MHC class II antigen modulation, alteration of the cytokine milieu, and upregulation of inhibitory and immune checkpoint ligands that promote T-cell suppression. Yet, immune checkpoint inhibitors (ICI) used in AML have yielded variable and unpredictable results. Moreover, evaluation of T-cell function is not part of the diagnostic assessment of patients with AML. Methods To better characterize the T-cell dysfunction in AML, we conducted a prospective study on freshly harvested, bone marrow aspirates from patients with AML at presentation, collected under an Institutional IRB-approved banking protocol over the past two years. Using a multiparameter flow cytometry-based assay, we analyzed T-cell activation potential in response to CD3/CD28 (bead-based), T-cell receptor (TCR) stimulation, both alone (n= 40 AMLs and 10 healthy donors) or in combination with a library of ICI (n=15 AML and 5 healthy donors). We selected OX40 (CD134/TNFRSF4) and ICOS (Inducible T-cell costimulator) as surrogate markers for T-cell activation based on their expression pattern post bead-based stimulation in healthy donors' bone marrows. The ICI library comprised 31 therapeutic antibody inhibitors of known immune checkpoints, signaling pathways, and chemokines reported to be implicated in T-cell suppression in AML and other solid tumors. Results Healthy T-cells displayed the highest surface expression of OX40 and ICOS at 5 days post-bead-stimulation, which was selected as the endpoint for our in vitro analyses. We found that bone marrow-derived T-cells from healthy donors upregulated the expression of the markers ICOS and/or OX40 in at least 20% of T-cells post-stimulation (n= 10, ICOS: median 36.9%, range 20-72%; OX40: median 59.3%, range 21.4 -83%). Overall, AML samples displayed a lower degree of ICOS and OX40 T-cell expression in response to TCR stimulation (n=40, ICOS: median 20.5%, range 4-65%; OX-40: median 30.3%, range 0.3-80%). Based on this result, T-cell activation was defined as >20% CD3+ cells ICOS+/OX-40+ with stimulation, with greater than 2-fold changes from baseline ICOS and OX40 expression. Using this criterion, patients with AML were then classified as activators or non-activators. T-cell activation status correlated with ELN 2022 risk (1) (Figure 1A). For the intermediate risk group, we observed that the activators group had a significantly longer relapse-free survival when compared to the non-activators (Figure 1B, n=10, Mantel-Cox log-rank = 0.02, follow up 400 days at the time of writing). While a similar trend was observed in the favorable and the poor risk groups, it did not reach significance in these ELN categories. To assess if the T-cell activation could be rescued or augmented upon immune checkpoint inhibition, we tested the effect of each of the 31 compounds of the ICI library on the degree of T-cell activation in diagnosis bone marrow aspirates of 15 patients (7 activators and 8 non activators) and in 5 healthy donors. Matched peripheral blood (PB) samples were available for 5 of the 15 patients with AML. All tested normal donors reached the threshold for T-cell activation, which was not significantly affected by ICI treatment. The 7 AML activators behaved similarly to the healthy donors. In 2 of the 8 AML non-activators, selected ICI rescued T-cell activation in absence of TCR stimulation, while in the remaining 6 cases, T-cell activation was not achieved under any condition. Similar, but overall dampened activation trends, were observed in PB samples. Conclusions Consistent with the findings of our previous retrospective study (2), the ongoing prospective analysis of T-cell activation of diagnosis bone marrows of patients with AML suggests that testing TCR-mediated T-cell activation potential at diagnosis may help refine risk stratification of the heterogenous ELN intermediate risk group. AML immune suppression seems to be reversible in a minority of cases with the use of selected ICI inhibitors.

Identification of a distinct NK-like hepatic T-cell population activated by NKG2C in a TCR-independent manner.

The liver provides a unique niche of lymphocytes enriched with a large proportion of innate-like T cells. However, the heterogeneity and functional characteristics of the hepatic T-cell population remain to be fully elucidated. We obtained liver sinusoidal mononuclear cells from the liver perfusate of healthy donors and recipients with HBV-associated chronic liver disease (CLD) during liver transplantation. We performed a CITE-seq analysis of liver sinusoidal CD45+ cells in combination with T cell receptor (TCR)-seq and flow cytometry to examine the phenotypes and functions of liver sinusoidal CD8+ T cells. We identified a distinct CD56hiCD161-CD8+ T-cell population characterized by natural killer (NK)-related gene expression and a uniquely restricted TCR repertoire. The frequency of these cells among the liver sinusoidal CD8+ T-cell population was significantly increased in patients with HBV-associated CLD. Although CD56hiCD161-CD8+ T cells exhibit weak responsiveness to TCR stimulation, CD56hiCD161-CD8+ T cells highly expressed various NK receptors, including CD94, killer immunoglobulin-like receptors, and NKG2C, and exerted NKG2C-mediated NK-like effector functions even in the absence of TCR stimulation. In addition, CD56hiCD161-CD8+ T cells highly respond to innate cytokines, such as IL-12/18 and IL-15, in the absence of TCR stimulation. We validated the results from liver sinusoidal CD8+ T cells using intrahepatic CD8+ T cells obtained from liver tissues. In summary, the current study found a distinct CD56hiCD161-CD8+ T-cell population characterized by NK-like activation via TCR-independent NKG2C ligation. Further studies are required to elucidate the roles of liver sinusoidal CD56hiCD161-CD8+ T cells in immune responses to microbial pathogens or liver immunopathology. The role of different immune cell populations in the liver is becoming an area of increasing interest. Herein, we identified a distinct T-cell population that had features similar to those of natural killer (NK) cells - a type of innate immune cell. This distinct population was expanded in the livers of patients with chronic liver disease and could thus have pathogenic relevance.

IL-15 enhances CCR5-mediated migration of memory CD8+ Tcells by upregulating CCR5 expression in the absence of TCR stimulation.

During microbial infection, bystander CD8+ Tcells that are not specific to infecting pathogens can be activated by interleukin (IL)-15. However, the tissue-homing properties of bystander-activated CD8+ Tcells have not been elucidated. Here, we examine the effects of IL-15 on the expression of chemokine receptors on CD8+ Tcells and their migration. IL-15 upregulates CCR5 in memory CD8+ Tcells in the absence of Tcell receptor (TCR) stimulation and enhances CCR5-dependent migration. IL-15-induced CCR5 upregulation is abrogated by TCR stimulation, indicating that CCR5 is upregulated in bystander-activated CD8+ Tcells. Moreover, CCR5 signals increase proliferation and cytotoxic protein expression in IL-15-treated memory CD8+ Tcells, although the increase has a small extent. CCR5 upregulation in bystander-activated CD8+ Tcells is associated with severe liver injury in patients with acute hepatitis A. Altogether, the results indicate that CCR5 upregulation by IL-15 mediates the migration of bystander-activated CD8+ Tcells.

Selective inhibition of gelatinases mitigates T-cell receptor signaling in CD4+ T-cells resulting in reduced clinical severity in a murine model of Multiple Sclerosis.

Abstract Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix components. The gelatinases, MMP-2 and MMP-9, have functions essential for normal physiological processes such as leukocyte trafficking, but their dysregulation is associated with various pathologies including Multiple Sclerosis (MS). During MS, CD4+ T-cells activated in the periphery penetrate the blood-brain barrier (BBB), recruit immune cells, initiate destruction of the myelin sheath, and cause axonal loss. Experimental Autoimmune Encephalomyelitis (EAE) is a well-established murine model of MS. In EAE, gelatinases are required for penetration of the BBB and generation of auto-antigens. Recent studies have demonstrated that they contribute to normal intracellular function of various cell types including antigen activated T-cells; however, their intracellular role in immune cell activation during EAE pathogenesis is not known. In our studies, we demonstrate that gelatinases are important for effective T-cell receptor (TCR) signaling. Upon activation, CD4+ T-cells lacking gelatinase activity demonstrated a reduced ability to express early activation markers, proliferate, produce cytokines, and undergo metabolic reprogramming. However, in the absence of TCR stimulation, CD4+ T-cells have no proliferative defects. In addition, RNA-seq analysis highlight key proteins impacted by gelatinase inhibition that are associated with the TCR signaling. Furthermore, EAE mice demonstrated reduced clinical severity following gelatinase inhibition. These results emphasize the importance of the gelatinases as therapeutic targets in CD4+ T-cell mediated autoimmune diseases.

T helper cells plasticity in inflammation

CD4+ T cells can be subdivided from a functional point of view into two main subsets: effector cells, which provide protection against exogenous offending agents, and regulatory T (Treg) cells whose function is to avoid autoimmune reactions and to stop the effector response against exogenous antigens, when the response itself becomes dangerous for the host. Human effector CD4+ T lymphocytes can be additionally classified into lineages based mainly on their immunological functions that are supported by distinct profile of cytokine, transcription factor, and homing receptors expression. In the last years, beyond the well known populations of human T helper (Th) lymphocytes, Th1 and Th2 cells, other populations have been discovered and phenotypically characterized. These include the Th17 subset, which is certainly the most intensively studied, but also Th22, Th9, and T follicular helper (Tfh) lymphocytes. In addition to their protective functions, these T helper populations are also involved in the pathogenesis of several inflammatory immune-mediated disorders. Th1 and Th17 cells are involved in the pathogenesis of organ-specific autoimmune diseases and other chronic inflammatory disorders, whereas allergen-specific Th2 lymphocytes play a crucial role in allergy. Although classically viewed as distinct lineages, recent evidence indicate that CD4+ T cells, particularly the Th17 subset, are more plastic than previously thought. It is not fully understood how often such plasticity occurs in the course of physiologic responses to pathogens and what its importance is in protective immunity, but in inflammatory conditions Th17 lymphocytes that have shifted towards a Th1 or Th2 phenotype, acquiring the ability to produce IFN-γ or IL-4, and seem to be particularly aggressive and more pathogenic than the unshifted cells. In this context, the possibility to interfere with this modulation of phenotype can be considered a possible target for developing novel therapeutic strategies in the above mentioned diseases.

Establishment of HIV Latency in Primary CD4+Cells Is due to Epigenetic Transcriptional Silencing and P-TEFb Restriction

The development of suitable experimental systems for studying HIV latency in primary cells that permit detailed biochemical analyses and the screening of drugs is a critical step in the effort to develop viral eradication strategies. Primary CD4(+) T cells were isolated from peripheral blood and amplified by antibodies to the T-cell receptor (TCR). The cells were then infected by lentiviral vectors carrying fluorescent reporters and either the wild-type Tat gene or the attenuated H13L Tat gene. After sorting for the positive cells and reamplification, the infected cells were allowed to spontaneously enter latency by long-term cultivation on the H80 feeder cell line in the absence of TCR stimulation. By 6 weeks almost all of the cells lost fluorescent protein marker expression; however, more than 95% of these latently infected cells could be reactivated after stimulation of the TCR by alpha-CD3/CD28 antibodies. Chromatin immunoprecipitation assays showed that, analogously to Jurkat T cells, latent proviruses in primary CD4(+) T cells are enriched in heterochromatic markers, including high levels of CBF-1, histone deacetylases, and methylated histones. Upon TCR activation, there was recruitment of NF-kappaB to the promoter and conversion of heterochromatin structures present on the latent provirus to active euchromatin structures containing acetylated histones. Surprisingly, latently infected primary cells cannot be induced by tumor necrosis factor alpha because of a restriction in P-TEFb levels, which can be overcome by activation of the TCR. Thus, a combination of restrictive chromatin structures at the HIV long terminal repeat and limiting P-TEFb levels contribute to transcriptional silencing leading to latency in primary CD4(+) T cells.

Naive CD8+ T cells differentiate into protective memory-like cells after IL-2–anti–IL-2 complex treatment in vivo

An optimal CD8+ T cell response requires signals from the T cell receptor (TCR), co-stimulatory molecules, and cytokines. In most cases, the relative contribution of these signals to CD8+ T cell proliferation, accumulation, effector function, and differentiation to memory is unknown. Recent work (Boyman, O., M. Kovar, M.P. Rubinstein, C.D. Surh, and J. Sprent. 2006. Science. 311:1924–1927; Kamimura, D., Y. Sawa, M. Sato, E. Agung, T. Hirano, and M. Murakami. 2006. J. Immunol. 177:306–314) has shown that anti–interleukin (IL) 2 monoclonal antibodies that are neutralizing in vitro enhance the potency of IL-2 in vivo. We investigated the role of IL-2 signals in driving CD8+ T cell proliferation in the absence of TCR stimulation by foreign antigen. IL-2 signals induced rapid activation of signal transducer and activator of transcription 5 in all CD8+ T cells, both naive and memory phenotype, and promoted the differentiation of naive CD8+ T cells into effector cells. IL-2–anti–IL-2 complexes induced proliferation of naive CD8+ T cells in an environment with limited access to self–major histocompatibility complex (MHC) and when competition for self-MHC ligands was severe. After transfer into wild-type animals, IL-2–activated CD8+ T cells attained and maintained a central memory phenotype and protected against lethal bacterial infection. IL-2–anti–IL-2 complex–driven memory-like CD8+ T cells had incomplete cellular fitness compared with antigen-driven memory cells regarding homeostatic turnover and cytokine production. These results suggest that intense IL-2 signals, with limited contribution from the TCR, program the differentiation of protective memory-like CD8+ cells but are insufficient to guarantee overall cellular fitness.

Induction of surface CCR4 and its functionality in mouse Th2 cells is regulated differently during Th2 development

T helper cell type 1 (Th1) and Th2 cells express distinct sets of chemokine receptors. In contrast to Th1 chemokine receptors, it is largely unknown how Th2 chemokine receptors such as CC chemokine receptor 4 (CCR4) are induced during Th2 differentiation. Here, we investigated the induction of CCR4 surface expression and ligand responsiveness evaluated by functional assays such as chemokine binding and chemotaxis. This was done in comparison with those of a Th1 chemokine receptor, CXC chemokine receptor 3 (CXCR3). Resting T cells expressed neither CXCR3 nor CCR4. CXCR3 expression and ligand responsiveness were observed when resting T cells were stimulated with anti-CD3 plus anti-CD28 in the presence of [interleukin (IL)-12+anti-IL-4] and then recultured without T cell receptor (TCR) stimulation. Unlike CXCR3, CCR4 was induced immediately after anti-CD3/anti-CD28 stimulation in the presence of (IL-4+anti-interferon-gamma+anti-IL-12). However, these CCR4-positive cells failed to exhibit chemokine binding and chemotaxis. Although the levels of surface CCR4 expression were not increased after the subsequent reculture in the absence of TCR stimulation, CCR4 responsiveness was induced in this stage of Th2 cells. The induction of CCR4 expression and the acquisition of CCR4 responsiveness did not occur in IL-4-deficient (IL-4(-/-)) and signal transducer and activator of transcription (STAT)6(-/-) T cells. CCR4 expression and functionality were regained in IL-4(-/-) but not in STAT6(-/-) T cells by the addition of recombinant IL-4. Although surface expression and functionality of CCR4 are induced depending on the IL-4/STAT6 signaling pathway, the present results indicate that the functionality of CCR4 does not correlate with CCR4 expression but emerges at later stages of Th2 differentiation.

Interleukin-12 and interleukin-2-induced invariant natural killer T-cell cytokine secretion and perforin expression independent of T-cell receptor activation.

Human invariant natural killer (iNK) T cells expressing an invariant Valpha24-Jalpha15 T-cell receptor (TCR) are thought to be important regulators of autoimmunity and tumour surveillance. Two major subsets of iNK T cells, CD4+ or CD4- CD8- are known to exist, but the in vivo importance of CD4 expression is unclear. Since interleukin-12 (IL-12) is a key iNK T-cell-activating cytokine, the effect of IL-12 plus or minus the T-cell growth factor IL-2 on a large panel of CD4+ versus CD4- CD8- iNK T-cell clones was examined. Strikingly, IL-12 and IL-2 significantly activated iNK T cells to secrete IL-4, interferon-gamma and granulocyte-macrophage colony-stimulating factor, and up-regulated perforin expression in the absence of TCR stimulation. Furthermore, IL-2 and IL-12 treatment resulted in a preferential increase in apoptosis of CD4- CD8- clones. Thus, independent of TCR activation, IL-2 and IL-12 can directly activate iNK T cells and provide a selective advantage to the CD4+ iNK T-cell population.

Type I interferon is the primary regulator of inducible Ly-6C expression on T cells.

Ly-6C has been proposed as a marker of memory CD8+ T cells. Reports have indicated that Ly-6C is upregulated after T cell receptor (TCR) stimulation or exposure to proinflammatory cytokines. This study examined the relative roles of proinflammatory cytokines and TCR engagement in Ly-6C induction. In vitro experiments tested the effects of cytokines on Ly-6C expression and confirmed interferon-alpha (IFN-alpha) as a primary cytokine that induces Ly-6C expression on CD4+ and CD8+ T cells. The amount and duration of Ly-6C expression were examined on T cells after in vivo induction of proinflammatory cytokines (CpG oligodeoxynucleotides [ODN]) or TCR activation (staphylococcal enterotoxin B [SEB]). In vivo, proinflammatory cytokines transiently upregulated Ly-6C on T cells in the absence of TCR stimulation. TCR stimulation by SEB transiently upregulated Ly-6C expression on antigen-specific and antigen-nonspecific T cells but did not cause long-term upregulation of Ly-6C expression in either population. IFN-alpha was confirmed as a primary inducer of Ly-6C in vivo, as CpG ODN were unable to induce Ly-6C expression in IFN-alphaRI(-/-) mice. Thus, inducible Ly-6C expression on CD4+ and CD8+ T cells is largely due to IFN-alpha in the environment and appears not to be directly correlated with the development of T cell memory.

Regulation of CD31 expression and interleukin-4 production by human cord blood CD4+ T cells with interleukin-4 and interleukin-7.

T helper 2 cell type cytokines, such as interleukin (IL)-4 and IL-5, play pivotal roles in the development of allergic diseases. However, the mechanism by which naive CD4+ T cells acquire the ability to produce these cytokines remains unclear. Recently, it was reported that IL-7 induces the ability to produce IL-4 as well as interferon (IFN)-gamma and IL-5 in naive CD4+ T cells without TCR stimulation. To further analyze the mechanism of acquiring IL-4-producing ability by naive CD4+ T cells, the effects of IL-7 on human cord blood CD4+ T cells were compared with those of IL-4, which induced the ability to produce IFN-gamma but not IL-4. Interleukin-7 preserved the population of CD4+CD31- T cells in cord blood and induced their IL-4-producing ability without T cell receptor (TCR) stimulation, while IL-4 induced CD31 on CD31- T cells and could not induce their IL-4-producing ability. Both the CD31-inducing effect and the inhibitory priming effect for IL-4-production by IL-4 were also observed after cord blood CD4+ T cells had been primed with IL-7 and acquired the IL-4-producing ability. Interleukin-7 induced the IL-4-producing ability in naive CD4+CD31- T cells without TCR stimulation, suggesting that the signal transduction via CD31 may have an inhibitory effect on the acquisition of the IL-4-producing ability by cord blood CD4+ T cells in the absence of TCR stimulation.

TCR-independent CD28-mediated gene expression in peripheral blood lymphocytes from donors chronically infected with HIV-1.

Complete activation of peripheral blood T cells requires both T-cell receptor (TCR) stimulation and CD28 costimulation. Signalling pathways associated specifically with CD28 are not well understood, however, because ligation of CD28 in the absence of TCR stimulation does not give rise to cellular responses in normal cells. In peripheral blood lymphocytes (PBL) from donors chronically infected with human immunodeficiency virus-1 (HIV-1), CD28 can induce viral replication through an alternative pathway that does not require TCR ligation. We have exploited this observation to study CD28-mediated signal transduction using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) to amplify viral RNA. Independent ligation of CD28 on donor PBL induced expression of the HIV-1 tat gene but not the interleukin-2 (IL-2) gene. Viral induction did not occur following pretreatment of cells with actinomycin D, suggesting it was mediated through transcriptional activation of the viral long terminal repeat (LTR). tat was induced in the presence of the protein kinase C inhibitor H-7, but was inhibited by cyclosporin A. Our results demonstrate that CD28 is linked directly to specific signalling pathways leading to de novo induction of genes in PBL.