SummaryComplement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While “tonic” intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.

Generation and maintenance of an effective repertoire of T cell antigen receptors are essential to the immune system, yet the number of distinct T cell receptors (TCRs) expressed by the estimated 10(12) T cells in the human body is not known. In this study, TCR gene amplification and sequencing showed that there are about 10(6) different beta chains in the blood, each pairing, on the average, with at least 25 different alpha chains. In the memory subset, the diversity decreased to 1 x 10(5) to 2 x 10(5) different beta chains, each pairing with only a single alpha chain. Thus, the naïve repertoire is highly diverse, whereas the memory compartment, here one-third of the T cell population, contributes less than 1 percent of the total diversity.

Dasatinib, a broad-spectrum tyrosine kinase inhibitor (TKI), predominantly targets BCR-ABL and SRC oncoproteins and also inhibits off-target kinases, which may result in unexpected drug responses. We identified 22 patients with marked lymphoproliferation in blood while on dasatinib therapy. Clonality and immunophenotype were analyzed and related clinical information was collected. An abrupt lymphocytosis (peak count range 4-20 Â 10 9 /l) with large granular lymphocyte (LGL) morphology was observed after a median of 3 months from the start of therapy and it persisted throughout the therapy. Fifteen patients had a cytotoxic T-cell and seven patients had an NK-cell phenotype. All T-cell expansions were clonal. Adverse effects, such as colitis and pleuritis, were common (18 of 22 patients) and were preceded by LGL lymphocytosis. Accumulation of identical cytotoxic T cells was also detected in pleural effusion and colon biopsy samples. Responses to dasatinib were good and included complete, unexpectedly long-lasting remissions in patients with advanced leukemia. In a phase II clinical study on 46 Philadelphia chromosome-positive acute lymphoblastic leukemia, patients with lymphocytosis had superior survival compared with patients without lymphocytosis. By inhibiting immunoregulatory kinases, dasatinib may induce a reversible state of aberrant immune reactivity associated with good clinical responses and a distinct adverse effect profile.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4+CD25hi T (Treg) cells. Based on transcriptional profiling of ex vivo activated Treg and helper CD4+CD25− T (Th) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human Treg cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific Th cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other Treg-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in Treg cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-β induced Treg cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in Treg cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.