Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.

Epithelial cells respond to physico-chemical damage with up-regulation of major histocompatbility complex-like ligands that can activate the cytolytic potential of neighboring intraepithelial T cells by binding the activating receptor, NKG2D. The systemic implications of this lymphoid stress-surveillance response, however, are unknown. We found that antigens encountered at the same time as cutaneous epithelial stress induced strong primary and secondary systemic, T helper 2 (T H 2)-associated atopic responses in mice. These responses required NKG2D-dependent communication between dysregulated epithelial cells and tissue-associated lymphoid cells. These data are germane to uncertainty over the afferent induction of T H 2 responses and provide a molecular framework for considering atopy as an important component of the response to tissue damage and carcinogenesis.A conserved feature of T lymphocytes is their subdivision into two main types. One, composed of CD4 + and CD8 + T cells specific for complexes of peptides and major histocompatbility complex (MHC) molecules, has been termed "conventional" and is largely responsible for clonal, pathogen-specific memory responses that are the hallmark of adaptive immunity. The second T cell type, composed of cells expressing the T cell receptor (TCR), is not generally specific for peptide-MHC complexes and may instead recognize cell surface microbial and/or self-encoded moieties, some of which are upregulated through cellular dysregulation. Although T cells exhibit some properties characteristic of adaptive immunity, a predominant function of these "unconventional" Tcells is thought to be lymphoid stress-surveillance because they do not require major clonal expansion, because their functional potentials are preprogrammed during development, and because some may be activated in vivo independently of the TCR, by cytokines or by ligands for activating natural killer (NK) receptors, such as NKG2D (1). Such cells are major components of large but enigmatic tissue-resident Tcell compartments, which include mouse dendritic epidermal T cells (DETCs) and human or mouse intraepithelial lymphocytes (IELs), among which CD4 − CD8 − T cells may also contribute to rapid lymphoid stress-surveillance.Because NK G2D ligands such as Rae-1 (mouse) and MIC class 1 chain-related protein A (MICA) (human) are activated by DNA damage (2) and are frequently expressed by tumor (3,4). However, whether lymphoid stress-surveillance describes a purely local response or whether it affects the systemic immune compartment remains a key question. To investigate this, transgenic Rae-1 expression was induced as described (4) specifically in keratinocytes by a doxycycline (dox)-dependent, bitransgenic (BTg) molecular switch ( fig. S1A). This mode of Rae-1 induction avoids pleiotropic effects of applying agents that induce a stressed state within the epidermis. Rae-1 induction on otherwise normal epithelium promoted rapid morphological rearrangements of DETCs and of epidermal Langerhans cells (LCs), whi...

Extracorporeal photochemotherapy (ECP) is employed for the management of cutaneous T cell lymphoma (CTCL). ECP involves the extracorporeal exposure of white blood cells (WBCs) to a photosensitizer, 8-methoxypsoralen (8-MOP), in the context of ultraviolet A (UVA) radiation, followed by WBC reinfusion. Historically, the therapeutic activity of ECP has been attributed to selective cytotoxicity on circulating CTCL cells. However, only a fraction of WBCs is exposed to ECP, and 8-MOP is inactive in the absence of UVA light, implying that other mechanisms underlie the anticancer effects of ECP. Recently, ECP has been shown to enable the physiological differentiation of monocytes into dendritic cells (DCs) that efficiently cross-present tumor-associated antigens (TAAs) to CD8 + T lymphocytes to initiate cognate immunity. However, the source of TAAs and immunostimulatory signals for such DCs remains to be elucidated. Here, we demonstrate that 8-MOP plus UVA light reduces melanoma cell viability along with the emission of ICD-associated danger signals including calreticulin (CALR) exposure on the cell surface and secretion of ATP, high mobility group box 1 (HMGB1) and type I interferon (IFN). Consistently, melanoma cells succumbing to 8-MOP plus UVA irradiation are efficiently engulfed by monocytes, ultimately leading to cross-priming of CD8 + T cells against cancer. Moreover, malignant cells killed by 8-MOP plus UVA irradiation in vitro vaccinate syngeneic immunocompetent mice against living cancer cells of the same type, and such a protection is lost when cancer cells are depleted of calreticulin or HMGB1, as well as in the presence of an ATP-degrading enzyme or antibodies blocking type I IFN receptors. ECP induces bona fide ICD, hence simultaneously providing monocytes with abundant amounts of TAAs and immunostimulatory signals that are sufficient to initiate cognate anticancer immunity.

Human cytolytic T lymphocytes and NK cells can limit tumor growth and are being increasingly harnessed for tumor immunotherapy. One way cytolytic lymphocytes recognize tumor cells is by engagement of their activating receptor, NKG2D, by stress-antigens of the MICA/B and ULBP # Correspondence: adrian.hayday@kcl.ac.uk; adrian.hayday@cancer.org.uk. Authors' contributions: P.V designed and performed experiments, analysed data and co-wrote and revised the manuscript; C.W and A. Turner performed experiments, analysed data and revised the manuscript. C.S provided key reagents, designed the experiments relating to mRNA stability, analysed data and revised the manuscript; Y.H and O.S. performed experiments and analysed data; A.G and A. Tutt provided access to and analysed the array data generated from primary breast cancer samples and revised the manuscript. A.H designed the study, analysed and interpreted data and wrote and edited the manuscript. Figure S1. Induction of MICA by UVB and EGF is not due to heat-shock, DNA damage or cell proliferation. Figure S2. NKG2D ligands are upregulated at the cell surface following EGF treatment. Figure S3. Cell surface increase in NKG2D ligand expression by EGF is detected by cytotoxic lymphocytes. Figure S4. NKG2D ligand induction by various stress components of the exposome is EGFR-dependent. Figure S5. NKG2D ligand expression is regulated post-transcriptionally. Figure S6. NKG2D ligand mRNAs contain ARE sequences in their 3′UTRs. Figure S7. The ARE sequence and the MEK pathway regulate NKG2D ligand expression. Figure S8. AUF1 regulates NKG2D ligand expression. Figure S9. The EGFR/MEK pathway regulates AUF1 localization. Figure S10. NKG2D ligand expression correlates with EGFR expression levels and is abrogated by Erlotinib. Figure S11. EGF-induced upregulation of cell surface NKG2D ligand expression by confluent differentiated Caco-2 cells is abrogated by EGFR and MEK inhibitors. Figure S12. Cetuximab inhibits NKG2D ligand expression. Table S1. Primers used in this study. List of Supplementary Materials Competing interests:The authors declare that they have no competing interests. families. This study shows that surface upregulation of NKG2D ligands by human epithelial cells in response to ultraviolet irradiation, osmotic shock, oxidative stress, and growth factor provision, is attributable to activation of the EGF-receptor (EGFR). EGFR activation causes intracellular relocalisation of AUF1 proteins that ordinarily destabilise NKG2D ligand mRNAs by targeting an AU-rich element conserved within the 3′ ends of most human but not murine NKG2D ligand genes. Consistent with these findings, NKG2D ligand expression by primary human carcinomas positively correlated with EGFR expression that is commonly hyper-activated in such tumours, and was reduced by clinical EGFR inhibitors. Thus, stress-induced activation of EGFR not only regulates cell growth but concomitantly regulates the cells' immunological visibility. Thus, therapeutics designed to limit cancer cell growth should also ...

Extracorporeal photochemotherapy (ECP) is a cancer immunotherapy for cutaneous T-cell lymphoma (CTCL) operative in more than 350 centers worldwide. Although its efficacy and favorable safety profile have driven its widespread use, elucidation of its underlying mechanism has been difficult. In this study, we identify the principal contributors to the anticancer immunotherapeutic effects of ECP, with the goal of enhancing potency and broadening applicability to additional malignancies. First, we scaled down the clinical ECP leukocyte-processing device to mouse size. Second, we used that miniaturized device to produce a cellular vaccine that regularly initiated therapeutic antimelanoma immunity. Third, we individually subtracted key factors from either the immunizing inoculum or the treated animal to ascertain their contribution to the antimelanoma response. Platelet-signaled monocyte-to-dendritic cell (DC) differentiation followed by sorting/processing/presentation of tumor antigens derived from internalized apoptotic tumor cells were absolute requirements. As in clinical ECP, immunogenic cell death of tumor cells was finely titrated by DNA cross-linkage mediated by photoactivated 8-methoxypsoralen (8-MOPA). ECP-induced tumor-loaded DC were effective immunotherapeutic agents only if they were spared exposure to 8-MOPA, indicating that healthy DC are required for ECP. Infusion of responder T cells into naïve tumor-challenged mice established the protective role of stimulated T-cell antitumor immunity. Collectively, these results reveal that selective antitumor effects of ECP are initiated by tumor antigen-loaded, ECP-induced DC, which promote potent collaboration between CD4 and CD8 tumor-specific T cells. These mechanistic insights suggest potential therapeutic applicability of ECP to solid tumors in addition to CTCL. These findings identify principal cellular contributors to the anticancer immunotherapeutic impact of ECP and suggest this treatment may be applicable to a broad spectrum of immunogenic malignancies. .

Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an “adhesion synapse,” a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor κB. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies.

The selectins expressed on activated endothelial cells (E-and P-selectin), leukocytes (L-selectin), and platelets (P-selectin) play crucial roles in the rolling and tethering of leukocytes. We explored the importance of donor and recipient selectins in acute and chronic cardiac allograft rejection using mice deficient in all three selectins (ELP Ϫ/Ϫ ). In BALB/c recipients, survival of fully allomismatched hearts from ELP Ϫ/Ϫ C57BL/6 donors was almost double that of wild-type grafts. In ELP Ϫ/Ϫ cardiac allografts, mononuclear cell infiltration and vasculitis of intramyocardial coronary arteries were significantly reduced. Interestingly, ELP Ϫ/Ϫ grafts were rejected similarly in both the presence and the absence of recipient selectins, and both wild-type and ELP Ϫ/Ϫ recipients promptly rejected wild-type hearts.Alternative adhesive molecules such as ␣4␤7 integrin may compensate for the lack of selectins and may mediate rejection in ELP Ϫ/Ϫ recipients. Chronic rejection was evaluated in a major histocompatibility complex (MHC) class II mismatch model using C57BL/6.C-H2 bm12 mice. While lack of selectins in recipients did not offer protection against chronic rejection, luminal stenosis of coronary arteries in ELP Ϫ/Ϫ grafts was markedly diminished. In conclusion, donor-derived selectins contribute to the development of both acute and chronic cardiac allograft rejection, and targeting donor selectins may open novel therapeutic approaches in clinical transplantation. 18: 292918: -293618: , 200718: . doi: 10.1681 Selectins play critical roles in inflammatory responses by mediating transient adhesion of leukocytes to the endothelium during the process of rolling. The binding affinity between selectins and selectin-ligands is relatively weak on the resting endothelium; however, once endothelial cells and leukocytes are activated in response to inflammatory stimuli, chemokines and integrins assist to form potent adhesive bridges between leukocytes and the endothelium. 1 Selectins are three structurally related proteins, which were named after the tissue in which they were first identified. 2-4 L-selectin is expressed by leukocytes and mediates the attachment of lymphocytes to high endothelial venules of peripheral lymph nodes, thereby promoting naïve T cell homing. 5 L-selectin also mediates leukocyte recruitment to the site of inflammation. 6 E-selectin is found on endothelial cells, and P-selectin is stored in ␣-granules of platelets and Weibel-Palade bodies J Am Soc Nephrol

No abstract