Inka Albrecht

Professional Memory CD4+ T Lymphocytes Preferentially Reside and Rest in the Bone Marrow

CD4(+) T lymphocytes are key to immunological memory. Here we show that in the memory phase of specific immune responses, most of the memory CD4(+) T lymphocytes had relocated into the bone marrow (BM) within 3-8 weeks after their generation-a process involving integrin alpha2. Antigen-specific memory CD4(+) T lymphocytes highly expressed Ly-6C, unlike most splenic CD44(hi)CD62L(-) CD4(+) T lymphocytes. In adult mice, more than 80% of Ly-6C(hi)CD44(hi)CD62L(-) memory CD4(+) T lymphocytes were in the BM. In the BM, they associated to IL-7-expressing VCAM-1(+) stroma cells. Gene expression and proliferation were downregulated, indicating a resting state. Upon challenge with antigen, they rapidly expressed cytokines and CD154 and efficiently induced the production of high-affinity antibodies by B lymphocytes. Thus, in the memory phase of immunity, memory helper T cells are maintained in BM as resting but highly reactive cells in survival niches defined by IL-7-expressing stroma cells.

Th memory for interleukin‐17 expression is stable in vivo

Based on the memory for the re‐expression of certain cytokine genes, different subsets of Th cells have been defined. In Th type 1 (Th1) and Th2 memory lymphocytes, the genes for the cytokines interferon‐γ and interleukin (IL)‐4 are imprinted for expression upon restimulation by the expression of the transcription factors T‐bet and GATA‐3, respectively, and epigenetic modification of the cytokine genes. In Th17 cells, IL‐17 expression is dependent on the transcription factors RORγt and RORα. Here, we analyze the stability and plasticity of IL‐17 memory in Th17 cells. We have developed a cytometric IL‐17 secretion assay for the isolation of viable Th cells secreting IL‐17. For Th17 cells generated in vitro, IL‐17 expression itself is dependent on continued TGF‐β/IL‐6 or IL‐23 signaling and is blocked by interferon‐γ and IL‐4 signaling. In response to IL‐12 and IL‐4, in vitro generated Th17 cells are converted into Th1 or Th2 cells, respectively. Th17 cells isolated ex vivo, however, maintain their IL‐17 memory upon subsequent in vitro culture, even in the absence of IL‐23. Their cytokine memory is not regulated by IL‐12 or IL‐4. Th17 cells generated in vivo are a stable and distinct lineage of Th cell differentiation.

IFN-γ and IL-12 synergize to convert in vivo generated Th17 into Th1/Th17 cells

Th1 and Th17 cells are distinct lineages of effector/memory cells, imprinted for re‐expression of IFN‐γ and IL‐17, by upregulated expression of T‐bet and retinoic acid‐related orphan receptor γt (RORγt), respectively. Apparently, Th1 and Th17 cells share tasks in the control of inflammatory immune responses. Th cells coexpressing IFN‐γ and IL‐17 have been observed in vivo, but it remained elusive, how these cells had been generated and whether they represent a distinct lineage of Th differentiation. It has been shown that ex vivo isolated Th1 and Th17 cells are not interconvertable by TGF‐β/IL‐6 and IL‐12, respectively. Here, we show that ex vivo isolated Th17 cells can be converted into Th1/Th17 cells by combined IFN‐γ and IL‐12 signaling. IFN‐γ is required to upregulate expression of the IL‐12Rβ2 chain, and IL‐12 for Th1 polarization. These Th1/Th17 cells stably coexpress RORγt and T‐bet at the single‐cell level. Our results suggest a molecular pathway for the generation of Th1/Th17 cells in vivo, which combine the pro‐inflammatory potential of Th1 and Th17 cells.

Autoregulation of Th1-mediated inflammation by twist1

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohns disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-γ, IL-2, and tumor necrosis factor-α, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

Persistence of effector memory Th1 cells is regulated by Hopx

Th1 cells are prominent in inflamed tissue, survive conventional immunosuppression, and are believed to play a pivotal role in driving chronic inflammation. Here, we identify homeobox only protein (Hopx) as a critical and selective regulator of the survival of Th1 effector/memory cells, both in vitro and in vivo. Expression of Hopx is induced by T‐bet and increases upon repeated antigenic restimulation of Th1 cells. Accordingly, the expression of Hopx is low in peripheral, naïve Th cells, but highly up‐regulated in terminally differentiated effector/memory Th1 cells of healthy human donors. In murine Th1 cells, Hopx regulates the expression of genes involved in regulation of apoptosis and survival and makes them refractory to Fas‐induced apoptosis. In vivo, adoptively transferred Hopx‐deficient murine Th1 cells do not persist. Consequently, they cannot induce chronic inflammation in murine models of transfer‐induced colitis and arthritis, demonstrating a key role of Hopx for Th1‐mediated immunopathology.

Innate-Like Effector Differentiation of Human Invariant NKT Cells Driven by IL-7

Conventional MHC-restricted T lymphocytes leave thymus with a naive phenotype and require Ag-dependent stimulation coupled to proliferation to acquire effector functions. Invariant (i)NKT cells are a subset of T lymphocytes considered innate because they display an effector memory phenotype independent of TCR stimulation by foreign Ags. We investigated the effector differentiation program followed by human iNKT cells by studying cells from a relevant set of fetal thymi and umbilical cord blood samples. We find that human fetal iNKT cells have already started a differentiation program that activates the epigenetic and transcriptional control of ifng and il4 genes, leading at birth to cells that express these cytokines upon TCR signaling but independently of proliferation in vitro. Both ex vivo and in vitro analysis of fetal and neonatal iNKT cells delineate an effector differentiation program linked to cell division in vivo, and they identify IL-7 as one of the crucial signals driving this program in the apparent absence of Ag stimulation. Consistent with these data, human fetal and neonatal iNKT cells are hyperresponsive in vitro to IL-7 in comparison to conventional T cells, owing to an increased expression and signaling function of the IL-7 receptor α-chain. The innate nature of human iNKT cells could thus derive from lineage-specific developmental cues that selectively make these cells efficient IL-7 responders following thymic selection.

Twist1 and Hopx determine function and persistence of pathogenic memory/effector T cells

Introduction T helper (Th) lymphocytes support the initiation and maintenance of autoimmune inflammation through secretion of cytokines and provision of B cell help leading to the generation of longlived plasma cells secreting pathogenic autoantibodies. In particular Th type 1 (Th1) and Th17 cells, which promote inflammation through the secretion of IFN-γ, tumour necrosis factor-α and interleukin-17, have been implicated in the pathogenesis of autoimmunity. The critical role of CD4+ Th cells has been demonstrated by the clinical benefit achieved through the depletion of Th cells with an anti-CD4 antibody in refractory rheumatoid arthritis patients. However, unwanted side-effects of sustained CD4 lymphopenia and the associated induced immunodeficiency call for the development of more advanced approaches allowing the selective elimination of disease-causing Th cells. This requires a better understanding of the biology of pathogenic Th cells and the identification of selective biomarkers. Material and methods The authors wanted to investigate the mechanisms involved in the transition from acute to chronic inflammation and identify selective biomarkers allowing the identification of pathogenic Th cells. Following the rationale that Th cells involved in chronic autoimmune inflammation have a history of repeated (auto-)antigen contact, the authors have compared the transcriptomes of Th1 and Th2 cells which they have stimulated only once to Th cells which they have stimulated several times. Results The authors could identify the two genes, twist1 and hopx, to be specifically upregulated in repeatedly stimulated, pro-inflammatory Th1 cells. Twist1 is a helix-loop-helix transcription factor whose expression increments with each restimulation. Twist1 is highly expressed in CD4+ T cells isolated directly from the site of inflammation in patients with, for example, rheumatoid arthritis and inflammatory bowel disease, but not in T cells isolated from the peripheral blood. When twist1 is downregulated in Th1 cells by RNA interference in the murine ovalbumin-induced arthritis model, inflammation is augmented, indicating that twist1 is an intrinsic regulator of inflammation. Inhibition of hopx expression in Th1 cells leads to an impairment in their ability to persist in vivo. Accordingly, hopx-deficient mice have fewer effector/memory Th1 cells in steady-state. Th1 cells, which lack expression of hopx, are not able to induce inflammation in the murine transfer colitis model or the ovalbumin-induced arthritis model. Conclusions Twist1 and hopx qualify as selective biomarkers allowing the identification of pro-inflammatory, pathogenic Th1 cells. The importance of twist1 and hopx for the function and persistence of pathogenic Th1 cells may offer novel opportunities for the selective targeting of such cells.