Reversible phosphorylation of cyclin T1 promotes assembly and stability of P-TEFb

The positive transcription elongation factor b (P-TEFb) is a critical co-activator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.

Conversion of Anergic T Cells Into Foxp3- IL-10+ Regulatory T Cells by a Second Antigen Stimulus In Vivo

T cell anergy is a common mechanism of T cell tolerance. However, although anergic T cells are retained for longer time periods in their hosts, they remain functionally passive. Here, we describe the induction of anergic CD4+ T cells in vivo by intravenous application of high doses of antigen and their subsequent conversion into suppressive Foxp3- IL-10+ Tr1 cells but not Foxp3+ Tregs. We describe the kinetics of up-regulation of several memory-, anergy- and suppression-related markers such as CD44, CD73, FR4, CD25, CD28, PD-1, Egr-2, Foxp3 and CTLA-4 in this process. The conversion into suppressive Tr1 cells correlates with the transient intracellular CTLA-4 expression and required the restimulation of anergic cells in a short-term time window. Restimulation after longer time periods, when CTLA-4 is down-regulated again retains the anergic state but does not lead to the induction of suppressor function. Our data require further functional investigations but at this stage may suggest a role for anergic T cells as a circulating pool of passive cells that may be re-activated into Tr1 cells upon short-term restimulation with high and systemic doses of antigen. It is tentative to speculate that such a scenario may represent cases of allergen responses in non-allergic individuals.

Assembly and levels of P-TEFb depend on reversible phosphorylation of cyclinT1

The positive transcription elongation factor b (P-TEFb) is a critical co-activator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, it is absent in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions require phosphorylation of two threonine residues (Thr143 and Thr149) in CycT1 by PKC. Conversely, PP1 dephosphorylates these sites. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminal differentiation of cells.

Cbl-b deficiency prevents functional but not phenotypic T cell anergy

T cell anergy is an important peripheral tolerance mechanism. We studied how T cell anergy is established using an anergy model in which the Zap70 hypermorphic mutant W131A is coexpressed with the OTII TCR transgene (W131AOTII). Anergy was established in the periphery, not in the thymus. Contrary to enriched tolerance gene signatures and impaired TCR signaling in mature peripheral CD4 T cells, CD4SP thymocytes exhibited normal TCR signaling in W131AOTII mice. Importantly, the maintenance of T cell anergy in W131AOTII mice required antigen presentation via MHC-II. We investigated the functional importance of the inhibitory receptor PD-1 and the E3 ubiquitin ligases Cbl-b and Grail in this model. Deletion of each did not affect expression of phenotypic markers of anergic T cells or T reg numbers. However, deletion of Cbl-b, but not Grail or PD-1, in W131AOTII mice restored T cell responsiveness and signaling. Thus, Cbl-b plays an essential role in the establishment and/or maintenance of unresponsiveness in T cell anergy.

Immunotherapy for Multiple Myeloma

Despite therapeutic advances over the past decades, multiple myeloma (MM) remains a largely incurable disease with poor prognosis in high-risk patients, and thus new treatment strategies are needed to achieve treatment breakthroughs. MM represents various forms of impaired immune surveillance characterized by not only disrupted antibody production but also immune dysfunction of T, natural killer cells, and dendritic cells, although immunotherapeutic interventions such as allogeneic stem-cell transplantation and dendritic cell-based tumor vaccines were reported to prolong survival in limited populations of MM patients. Recently, epoch-making immunotherapies, i.e., immunomodulatory drug-intensified monoclonal antibodies, such as daratumumab combined with lenalidomide and chimeric antigen receptor T-cell therapy targeting B-cell maturation antigen, have been developed, and was shown to improve prognosis even in advanced-stage MM patients. Clinical trials using other antibody-based treatments, such as antibody drug-conjugate and bispecific antigen-directed CD3 T-cell engager targeting, are ongoing. The manipulation of anergic T-cells by checkpoint inhibitors, including an anti-T-cell immunoglobulin and ITIM domains (TIGIT) antibody, also has the potential to prolong survival times. Those new treatments or their combination will improve prognosis and possibly point toward a cure for MM.

Detection of self-reactive CD8+T cells with an anergic phenotype in healthy individuals

Immunological tolerance to self requires naturally occurring regulatory T (Treg) cells. Yet how they stably control autoimmune T cells remains obscure. Here, we show that Tregcells can render self-reactive human CD8+T cells anergic (i.e., hypoproliferative and cytokine hypoproducing upon antigen restimulation) in vitro, likely by controlling the costimulatory function of antigen-presenting cells. Anergic T cells were naïve in phenotype, lower than activated T cells in T cell receptor affinity for cognate antigen, and expressed several coinhibitory molecules, including cytotoxic T lymphocyte–associated antigen-4 (CTLA-4). Using these criteria, we detected in healthy individuals anergic T cells reactive with a skin antigen targeted in the autoimmune disease vitiligo. Collectively, our results suggest that Tregcell–mediated induction of anergy in autoimmune T cells is important for maintaining self-tolerance.

Protective Cytotoxic Clonal T-Cells in Myeloma Have the Characteristics of Telomere-Independent Senescence Rather Than an Exhausted or Anergic Phenotype: Implications for Immunotherapy

Dysfunctional T-cells associated with human tumors can be identified utilizing multi-parameter flow cytometry and studied for intracellular signalling checkpoints using phospho-flow or time-of-flight mass spectrometry. Whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent has not yet been determined. Anergic T-cells are hypo-responsive with low cytokine production and induce tolerance to protect the host from autoimmune disease. T-cell exhaustion is usually associated with chronic viral infection such as CMV but also with some cancers. Both exhausted and anergic T-cells express PD-1, LAG-3, Tim-3, CD160 and CD28. Hypo-responsive T-cells associated with melanoma have the phenotype of exhausted T-cells which can be reactivated using targeted immune check point blockade, resulting in clinical benefit. Senescent T-cells accumulate in an oligoclonal manner with ageing and chronic antigen exposure. Senescence can be characterised by telomere shortening, the expression of CD57, KLRG-1, CD160 and the absence of CD28. However, not all senescent cells have shortened telomeres as telomere independent senescence involving the p21-p53 and p16-pRb pathways and senescence associated secretory phenotype (SASP) T-cells have been described. The aim of this study was to determine whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent and to identify targetable checkpoints to restore T-cell function. Cytotoxic T-cell clones (CD57+ CD28- TCRVβ restricted) determined by BetaMark TCRVβ analysis were present in 51% of patients with myeloma (n=264), are protective (OS χ2=6.2; p<0.01) and maintain high cytokine secretion. CFSE proliferation assays of flow sorted T-cell clones demonstrated a significant lack of proliferation after stimulation with MACS iBeads (n=12; p<0.0001). They failed to respond in vitro to cytokines such as IL- 2, IL-12, IL-15 and OX40. In contrast, the T-cell clones were proliferative in a group of long term myeloma survivors in whom such T-cells are universally present suggesting that the impaired proliferation may be reversible. Geneset enrichment analysis of mRNA expression from Affymetrix U133 plus 2.0 arrays demonstrated pleiotropic dysfunction in multiple intracellular signalling pathways involved with proliferation and T cell inactivation. Utilizing phospho-flow techniques we demonstrated reduced phosphorylated ERK levels in clonal vs non-clonal T-cells (n=8; p<0.002) as well as elevated levels of phosphorylated Smad2/3 (n=10; p<0.02) and Bcl-xL (n=7; p<0.04) .These findings suggest that the ERK pathway, associated with proliferation, was suppressed, the TGFβ pathway was activated and that the dysfunctional T-cell clones have a survival advantage due to apoptotic resistance. In contrast, normal SHP-2 and phosphorylated ZAP-70 levels suggested there was no defect in upstream TCR signaling. Clonal T-cells lacked CD28, PD-1, CTLA-4, LAG-3, CD160 and Tim-3 expression and therefore are neither exhausted nor anergic. However, telomere assessment by qPCR demonstrated telomere T/S ratios were normal length for age and not significantly less than autologous CD57+TCR-Vβ- cells. T-cell clones also had normal p38 expression suggesting that the senescence was not due to TNF-α inhibiting the p38/MAPK pathway. The cytotoxic T-cell clones in patients with myeloma have the characteristics of telomere-independent senescence. The normal telomere length suggests that the hypo-responsiveness is induced independent of telomeres, and is therefore potentially reversible, though the pleiotropic nature of this dysfunction might require the targeting of multiple intracellular signalling pathways. The lack of PD-1 and CTLA-4 expression suggests that immune checkpoint blockade in myeloma would be unable to reverse the T cell dysfunction. Disclosures No relevant conflicts of interest to declare.

Functional characterization of the human dendritic cell immunodeficiency associated with the IRF8K108E mutation

Key Points IRF8K108E mutation causes dendritic cell depletion, defective antigen presentation, and anergic T cells. IRF8K108E mutant protein is functionally null and shows defective nuclear targeting and increased proteasomal degradation.