We have investigated the importance of interleukin-6 (IL-6) in promoting tumor growth and metastasis. In human primary breast cancers, increased levels of IL-6 were found at the tumor leading edge and positively correlated with advanced stage, suggesting a mechanistic link between tumor cell production of IL-6 and invasion. In support of this hypothesis, we showed that the IL-6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3) pathway drives tumor progression through the stroma and metastatic niche. Overexpression of IL-6 in tumor cell lines promoted myeloid cell recruitment, angiogenesis, and induced metastases. We demonstrated the therapeutic potential of interrupting this pathway with IL-6 receptor blockade or by inhibiting its downstream effectors JAK1/2 or Stat3. These clinically relevant interventions did not inhibit tumor cell proliferation in vitro but had profound effects in vivo on tumor progression, interfering broadly with tumor-supportive stromal functions, including angiogenesis, fibroblast infiltration, and myeloid suppressor cell recruitment in both the tumor and pre-metastatic niche. This study provides the first evidence for IL-6 expression at the leading edge of invasive human breast tumors and demonstrates mechanistically that IL-6/JAK/Stat3 signaling plays a critical and pharmacologically targetable role in orchestrating the composition of the tumor microenvironment that promotes growth, invasion, and metastasis.

Natural variability in abundance of signaling regulators can lead to divergence in cell fate, even within genetically identical cells sharing a common differentiation state. To leverage this observation, we introduce cell-to-cell variability analysis (CCVA), an experimental and computational methodology to quantify the correlation between variability in signaling regulator abundance and variation in sensitivity to stimuli. Here, we apply CCVA to investigate the unexpected effects of the interleukin 2 (IL-2) receptor α chain (IL-2Rα) on the sensitivity to common-gamma chain (γc) cytokines in primary T lymphocytes. Our work demonstrates that increased IL-2Rα abundance decreases the concentration of IL-2 but increases the concentrations of IL-7 and IL-15 required for a half-maximal activation (EC50) of downstream signal transducer and activator of transcription 5 (STAT5), without affecting the EC50 of other γc cytokines. To probe the mechanism of IL-2Rα's effect on γc family cytokine EC50s, we introduce a Bayesian-inference computational framework that models the formation of receptor signaling complexes using prior biophysical measurements. Using this framework, we demonstrate that a model in which IL-2Rα drives γc depletion through pre-assembly of complete IL-2 receptors is consistent with both CCVA data and prior measurements. The combination of CCVA and computational modeling yields quantitative understanding of the crosstalk of γc cytokine signaling in T lymphocytes.

Variability within isogenic T cell populations yields heterogeneous ‘local’ signaling responses to shared antigenic stimuli, but responding clones may communicate ‘global’ antigen load through paracrine messengers, such as cytokines. Such coordination of individual cell responses within multicellular populations is critical for accurate collective reactions to shared environmental cues. However, cytokine production may saturate as a function of antigen input, or be dominated by the precursor frequency of antigen-specific T cells. Surprisingly, we found that T cells scale their collective output of IL-2 to total antigen input over a large dynamic range, independently of population size. Through experimental quantitation and computational modeling, we demonstrate that this scaling is enforced by an inhibitory cross-talk between antigen and IL-2 signaling, and a nonlinear acceleration of IL-2 secretion per cell. Our study reveals how time-integration of these regulatory loops within individual cell signaling generates scaled collective responses and can be leveraged for immune monitoring.DOI: http://dx.doi.org/10.7554/eLife.01944.001

IntroductionThe dendritic cell (DC) hematopoietic lineage comprises heterogeneous subsets that share the capacity for potent initiation and control of innate and adaptive immunity, 1-5 but with sufficient plasticity to orchestrate the quantity and quality of lymphocyte responses. Indeed, defined subsets of human DCs generated with cytokines in vitro also yield sufficient numbers with counterparts in vivo to discern distinct consequences to T cells interacting with one or another subtype. [6][7][8][9][10][11][12] Human Langerhans-type DCs derived from CD34 ϩ hematopoietic progenitor cells (HPCs) have consistently proven superior to other conventional DC subsets as stimulators of CD8 ϩ cytolytic T lymphocytes (CTLs) in vitro. 9,11 This has led some investigators to advocate inclusion of CD34 ϩ HPC-derived Langerhans cells (LCs) in DC vaccines 13,14 rather than relying solely on the more commonly used monocyte-derived DCs (moDCs). The mechanisms underlying LC potency have remained elusive, but IL-15 has emerged as an important cytokine mediator. 9,14 First, LCs' potent stimulation of CD8 ϩ CTLs occurs in the complete absence of IL-12p70, 9,15 which has proven more critical for the activation of NK cells 15 than for CD8 ϩ CTLs. 9 Despite several shared functions, IL-15 also has important contrasting roles with IL-2. [16][17][18] IL-2 controls autoreactive T cells through their activation-induced cell death, or apoptosis, and the maintenance and expansion of Tregs. In contrast, IL-15 is critical to the generation of durable, high-avidity, memory CD8 ϩ T cells, which could include autoimmune populations with specificity for self-differentiation tumor antigens. [16][17][18] LCs secrete more IL-15 than any other conventional DC subtype, 9,15 but investigators have not previously identified IL-15R-␣ on the surface of LCs. This ␣-subunit is required for transport of IL-15 to the cell membrane to bind ␤ (CD122) and ␥ (CD132) chains on responding lymphocytes. Only then is the biologically active, heterotrimeric receptor-cytokine complex complete, supporting subsequent lymphocyte signaling and activation. [16][17][18] We therefore undertook alternative approaches to define the expression of IL-15R-␣ by resident populations of primary human LCs emigrating from cultured human epidermal sheets, as well as LCs, dermal-interstitial DCs (DDC-IDCs), and moDCs generated with recombinant human cytokines in vitro. 9 Eliminating exposure to IL-15 during DC development allowed comparison of tumorspecific CTLs stimulated by LCs with those elicited by moDCs, as Methods Media and noncytokine supplementsComplete RPMI 1640 included 10mM HEPES, 1% penicillin/streptomycin (Media Lab, Memorial Sloan-Kettering Cancer Center [MSKCC]), 50M 2-mercaptoethanol (Invitrogen), 1% L-glutamine (Invitrogen), and 1% or 10% volume/volume heat-inactivated, pooled normal human serum (NHS; Atlanta Biologicals). X-VIVO 15 (BioWhittaker; Lonza Walkersville) was used as manufactured without additives. All media and reagents were endotoxin-free. Human cells, media...