Goronzy, Jörg J.

Abstract We have studied the molecular diversity of the allogeneic TCR repertoire specific for the HLA-DR4 variant, DRB1*0404, by analyzing the V beta gene segment usage of CD4+ T cell clones from nine different donors and comparing the V beta repertoire in allospecific and unselected T cell populations. To investigate the forces shaping the repertoire of the alloreactive T cell response we compared the diversity of TCR specificities utilized in very similar and in disparate responder/stimulator combinations. Six of the responders shared the HLA-DRB1*0401 allele, which differs from the HLA-DRB1*0404 allele by only two amino acid substitutions at positions 71 and 86 of the HLA-DR beta 1 chain; three responders expressed HLA-DRB1 alleles with extensive polymorphisms compared with the stimulator HLA-DRB1 allele. In all nine responders, TCR specificities recruited to recognize HLA-DRB1*0404 were strongly biased toward the dominant usage of one to three V beta elements. Despite some degree of heterogeneity between different responders, the overall pattern was very similar with a hierarchy of TCR V beta elements expressed by HLA-DRB1*0404 specific T cells. A core group of V beta elements (V beta 6, 5, 2, 13.2, 18, and 7) was preferentially used, whereas other V beta elements including V beta 3, 4, and 8 were infrequently used or not used at all. Sequencing of the VDJ beta region from selected T cell clones showed no junctional bias associated with the preferential V beta gene segment usage. Surprisingly, the T cell diversity in focused and in complex alloreactive responses were equally biased. The finding that HLA-DR4+ and DR4- donors exhibited a similar bias supports the interpretation that the alpha-helical HLA-DR structure of the stimulator cell has a dominant contribution in shaping the alloreactive repertoire.

Abstract T lymphocytes are a major component of the inflammatory infiltrate in rheumatoid synovitis, but their exact role in the disease process is not understood. Functional activities of synovial T cells were examined by adoptive transfer experiments in human synovium-SCID mouse chimeras. Adoptive transfer of tissue-derived autologous CD8+ T cells induced a marked reduction in the activity of lesional T cells and macrophages. Injection of CD8+, but not CD4+, T cells decreased the production of tissue IFN-γ, IL-1β, and TNF-α by >90%. The down-regulatory effect of adoptively transferred CD8+ T cells was not associated with depletion of synovial CD3+ T cells or synovial CD68+ macrophages, and it could be blocked by Abs against IL-16, a CD8+ T cell-derived cytokine. In the synovial tissue, CD8+ T cells were the major source of IL-16, a natural ligand of the CD4 molecule that can anergize CD4-expressing cells. The anti-inflammatory activity of IL-16 in rheumatoid synovitis was confirmed by treating synovium-SCID mouse chimeras with IL-16. Therapy for 14 days with recombinant human IL-16 significantly inhibited the production of IFN-γ, IL-1β, and TNF-α in the synovium. We propose that tissue-infiltrating CD8+ T cells in rheumatoid synovitis have anti-inflammatory activity that is at least partially mediated by the release of IL-16. Spontaneous production of IL-16 in synovial lesions impairs the functional activity of CD4+ T cells but is insufficient to completely abrogate their stimulation. Supplemental therapy with IL-16 may be a novel and effective treatment for rheumatoid arthritis.

Abstract Patients with rheumatoid arthritis have a subset of CD4+ T lymphocytes that are characterized by a defect in CD28 expression. CD4+CD28− T cells frequently undergo clonal expansion in vivo. These clonotypes include autoreactive cells and persist over many years. The clonogenic potential and longevity of these T cells could be related to an altered response to apoptosis-inducing signals. To explore this possibility, CD4+CD28− T cell lines and clones were examined for their response pattern to stimuli inducing physiologic cell death. CD4+CD28− T cells were found to be resistant to apoptosis upon withdrawal of the growth factor, IL-2. To examine whether the altered sensitivity to this apoptotic signal was correlated with the expression of proteins of the bcl-2 family, the expression of bcl-2, bcl-x, and bax proteins was determined. CD28+ and CD28−CD4+ T cells could not be distinguished by the levels of bax or bcl-xL protein; however, CD4+CD28− T cells expressed higher amounts of bcl-2 protein than did CD4+CD28+ T cells. The increased bcl-2 expression in CD4+CD28− T cells was relatively independent of signals provided by exogenous IL-2. In CD28-deficient CD4+ T cells, bcl-2 was not significantly up-regulated by the addition of exogenous IL-2 and was maintained despite IL-2 withdrawal, as opposed to CD28-expressing CD4+ T cells. We propose that CD4+CD28− T cells are characterized by a dysregulation of the survival protein, bcl-2, which may favor the clonal outgrowth of autoreactive T cells and thus contribute to the pathogenesis of rheumatoid arthritis.

Abstract The self-restriction of Ag-specific T cell responses is interpreted as the result of a positive selection of the individual’s T cell specificities for their compatibility with self-MHC molecules. If the T cell receptor (TCR) specificities in any given individual have an affinity for syngeneic MHC molecules, it is unclear how they interact with allogeneic MHC structures. To approach this question, we analyzed 123 alloreactive HLA-DR4 Dw4 or Dw14 specific T cell clones that were generated from responder/stimulator combinations with defined disparities in the HLA-DR beta 1-chain. Sets of T cell clones were established from three different HLA-Dw4+ responders and compared for their fine specificities. The majority of HLA-DR4 Dw14 specific T cell clones co-recognized HLA-DR1 Dw1+ (33 to 36% of all T cell clones) or HLA-DRw14 Dw16+ (26 to 33%) stimulators, both of which share very similar sequences in the third hypervariable region of the HLA-DR beta 1-chain with the HLA-DR4 alleles Dw4 and Dw14. These data suggest that sequence and structural similarities in the alpha-helical portions of the HLA-DR molecule impose a strong bias on the recognition of allotargets. The second haplotype of the responder did not appear to affect the typical fingerprint of T cell recognition except for the deletion of self-reactive TCR specificities. Nonrandom usage of TCR specificities in anti-HLA-DR responses was also found for HLA-DRw11/DRw13+ and HLA-DRw11/DR7+ T cell donors who did not share any obvious polymorphic sequence stretches with the allostimulators HLA-DR4 Dw4 or Dw14. T cell clones from an HLA-DRw11/DRw13+ responder functionally resembled the TCR specificities derived from the HLA-DR4 Dw4+ donors. T cell clones derived from an HLA-DRw11/DR7+ individual were characterized by a distinct cross-reactivity pattern preferring HLA-DRw13 Dw19+ (50 to 60%) and HLA-DR3+ (43 to 57%) stimulator cells. These findings suggest that the responder HLA-DR alleles influence the structural constraints in the recognition of allo-HLA-DR molecules in closely related and in completely disparate responder/stimulator combinations.

SummaryMitochondria are the controllers of cell metabolism and are recognized as decision makers in cell death pathways, organizers of cytoplasmic signaling networks, managers of cellular stress responses, and regulators of nuclear gene expression. Cells of the immune system are particularly dependent on mitochondrial resources, as they must swiftly respond to danger signals with activation, trafficking, migration, and generation of daughter cells. Analogously, faulty immune responses that lead to autoimmunity and tissue inflammation rely on mitochondria to supply energy, cell building blocks and metabolic intermediates. Emerging data endorse the concept that mitochondrial fitness, and the lack of it, is of particular relevance in the autoimmune disease rheumatoid arthritis (RA) where deviations of bioenergetic and biosynthetic flux affect T cells during early and late stages of disease. During early stages of RA, mitochondrial deficiency allows naïve RA T cells to lose self-tolerance, biasing fundamental choices of the immune system toward immune-mediated tissue damage and away from host protection. During late stages of RA, mitochondrial abnormalities shape the response patterns of RA effector T cells engaged in the inflammatory lesions, enabling chronicity of tissue damage and tissue remodeling. In the inflamed joint, autoreactive T cells partner with metabolically reprogrammed tissue macrophages that specialize in antigen-presentation and survive by adapting to the glucose-deplete tissue microenvironment. Here, we summarize recent data on dysfunctional mitochondria and mitochondria-derived signals relevant in the RA disease process that offer novel opportunities to deter autoimmune tissue inflammation by metabolic interference.

The ectonucleotidase CD39 functions as a checkpoint in purinergic signaling on effector T cells. By depleting eATP and initiating the generation of adenosine, it impairs memory cell development and contributes to T cell exhaustion, thereby causing defective tumor immunity and deficient T cell responses in older adults who have increased CD39 expression. Tuning enzymatic activity of CD39 and targeting the transcriptional regulation of ENTPD1 can be used to modulate purinergic signaling. Here, we describe that STAT6 phosphorylation downstream of IL-4 signaling represses CD39 expression on activated T cells by inducing a transcription factor network including GATA3, GFI1, and YY1. GATA3 suppresses ENTPD1 transcription through prevention of RUNX3 recruitment to the ENTPD1 promoter. Conversely, pharmacological STAT6 inhibition decreases T cell effector functions via increased CD39 expression, resulting in the defective signaling of P2X receptors by ATP and stimulation of A2A receptors by adenosine. Our studies suggest that inhibiting the STAT6 pathway to increase CD39 expression has the potential to treat autoimmune disease while stimulation of the pathway could improve T cell immunity.

Background: The risk for cardiovascular disease is multifold higher in patients with the autoimmune disease rheumatoid arthritis, but underlying mechanisms remain poorly defined. We have explored whether patients with rheumatoid arthritis (RA) and patients with coronary artery disease (CAD) share pro-inflammatory effector cells that sustain tissue inflammation, thus maintaining synovial inflammation and atherosclerotic plaque formation. Methods: Tissue cytokines expressed in RA synovial lesions were analyzed by cytokine array technology. Cytokine production in macrophages was verified with RT-PCR, immunohistochemistry and flow cytometry. Mitochondrial function was evaluated by Seahorse analyzer. Results: In highly inflamed synovial tissues, 21 of 60 cytokines tested were significantly elevated, with the small cytokine CCL18 being strongly associated with tissue inflammation. Immunohistochemical examination assigned CCL18 to CD68 + tissue macrophages in both the atherosclerotic plaque and in rheumatoid synovitis. In monocyte-derived macrophages (MΦ), CCL18 enhanced production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and stimulated production of the tissue-injurious enzyme cathepsin K. Pro-inflammatory effector functions were linked to CCL18 induced activation of mitochondrial metabolism, quantified by oxygen consumption rates and generation of ATP. Macrophage stimulation with recombinant CCL18 resulted in the inactivating phosphorylation of the metabolic sensor GSK-3β. Immunoprecipitation experiments localized GSK-3β to the mitochondrial surface and imaging studies revealed excessive formation of mitochondria-endoplasmic reticulum connections in patient-derived CCL18 hi macrophages. Conclusions: Pro-inflammatory MΦ in CAD and RA patients share a metabolic profile of increased mitochondrial oxygen consumption and ATP production. Mitochondrial activation depends on inactivation of the metabolic sensor GSK-3β and promote pro-inflammatory effector functions, such as production of cytokines and proteases. Inhibiting GSK-3β activation provides a novel therapeutic strategy to suppress pro-inflammatory macrophages in CAD and RA.

Abstract Cellular senescence is an essential tumor suppressive mechanism that prevents the propagation of oncogenically activated, genetically unstable, and/or damaged cells. Induction of tumor cell senescence is also one of the underlying mechanisms by which cancer therapies exert antitumor activity. However, an increasing body of evidence from preclinical studies demonstrates that radiation and chemotherapy cause accumulation of senescent cells (SnCs) both in tumor and normal tissue. SnCs in tumors can, paradoxically, promote tumor relapse, metastasis, and resistance to therapy, in part, through expression of the senescence-associated secretory phenotype. In addition, SnCs in normal tissue can contribute to certain radiation- and chemotherapy-induced side effects. Because of its multiple roles, cellular senescence could serve as an important target in the fight against cancer. This commentary provides a summary of the discussion at the National Cancer Institute Workshop on Radiation, Senescence, and Cancer (August 10-11, 2020, National Cancer Institute, Bethesda, MD) regarding the current status of senescence research, heterogeneity of therapy-induced senescence, current status of senotherapeutics and molecular biomarkers, a concept of “one-two punch” cancer therapy (consisting of therapeutics to induce tumor cell senescence followed by selective clearance of SnCs), and its integration with personalized adaptive tumor therapy. It also identifies key knowledge gaps and outlines future directions in this emerging field to improve treatment outcomes for cancer patients.

Significance Antigen recognition by the immune system triggers rapid, specific, and protective responses, which are counterbalanced by inhibitory checkpoints to minimize potentially harmful immunity. The programmed death-1/ programmed death ligand-1 (PD-1/PD-L1) checkpoint is overreactive in cancer patients, curbing antitumor immunity. Whether a failing PD-1/PD-L1 checkpoint contributes to spontaneous autoimmune disease in humans is unknown. Here, we found that in patients with the autoimmune vasculitis giant cell arteritis, antigen-presenting cells provide insufficient negative signaling; unleashing highly activated T cells to infiltrate and damage the walls of large arteries. Thus, immunoinhibitory signals protect large arteries against inflammatory attack and checkpoint activation may be a suitable strategy to treat autoimmune vasculitis.