Type I IFN-dependent T cell activation is mediated by IFN-dependent dendritic cell OX40 ligand expression and is independent of T cell IFNR expression.

Type I IFNs are important for direct control of viral infection and generation of adaptive immune responses. Recently, direct stimulation of CD4(+) T cells via type I IFNR has been shown to be necessary for the formation of functional CD4(+) T cell responses. In contrast, we find that CD4(+) T cells do not require intrinsic type I IFN signals in response to combined TLR/anti-CD40 vaccination. Rather, the CD4 response is dependent on the expression of type I IFNR (IFNαR) on innate cells. Further, we find that dendritic cell (DC) expression of the TNF superfamily member OX40 ligand was dependent on type I IFN signaling in the DC, resulting in a reduced CD4(+) T cell response that could be substantially rescued by an agonistic Ab to the receptor OX40. Taken together, we show that the IFNαR dependence of the CD4(+) T cell response is accounted for exclusively by defects in DC activation.

Adamantinomatous craniopharyngioma associated with a compromised blood-brain barrier: patient series.

BACKGROUND: Adamantinomatous craniopharyngioma (ACP) is a highly morbid adult and pediatric brain tumor derived from epithelial remnants of the craniopharyngeal canal (Rathke's pouch), which gives rise to the anterior pituitary gland. Standard therapy includes maximal safe resection with or without radiation therapy. Systemic antitumor therapy remains elusive. Immune-related paracrine signaling involving the interleukin-6 receptor (IL-6R) may contribute to ACP pathogenesis. Tocilizumab, a recombinant humanized monoclonal antibody against IL-6R, is approved by the US Food and Drug Administration but does not cross an intact blood-brain barrier. OBSERVATIONS: In a phase 0 trial design, a single dose of tocilizumab was delivered intravenously before clinically indicated surgical intervention in 3 children with ACP. The presence of tocilizumab was assayed in plasma, tumor tissue, tumor cyst fluid, and cerebrospinal fluid (n = 1) using a novel enzyme-linked immunosorbent assay. Tocilizumab reached ACP tumor tissue and/or cyst fluid after one systemic dose in every patient. LESSONS: This finding helps explain extant data that indicate tocilizumab may contribute to ACP therapy. It further indicates that ACP does not reside behind an intact blood-brain barrier, dramatically broadening the range of potential antitumor therapies against this tumor. This has substantial implications for the design of future clinical trials for novel therapies against ACP in both children and adults.

High-Dimensional Analysis of Postsplenectomy Peripheral Immune Cell Changes.

Although the consequences of splenectomy are well understood in mice, much less is known about the immunologic changes that occur following splenectomy in humans. We sought to characterize the circulating immune cell populations of patients before and after elective splenectomy to determine if these changes are related to postsplenectomy survival outcomes. Retrospective clinical information was collected from 95 patients undergoing elective splenectomy compared with 91 patients undergoing pancreaticoduodenectomy (Whipple procedure). We further analyzed peripheral blood from five patients in the splenectomy group, collected before and after surgery, using single-cell cytometry by time-of-flight mass spectrometry. We compared pre- and postsplenectomy data to characterize both the major and minor immune cell populations in significantly greater detail. Compared with patients undergoing a Whipple procedure, splenectomized patients had significant and long-lasting elevated counts of lymphocytes, monocytes, and basophils. Cytometry by time-of-flight mass spectroscopy analysis demonstrated that the elevated lymphocytes primarily consisted of naive CD4+ T cells and a population of activated CD25+CD56+CD4+ T cells, whereas the elevated monocyte counts were mainly mature, activated monocytes. We also observed a significant increase in the expression of the chemokine receptors CCR6 and CCR4 on several cellular populations. Taken together, these data indicate that significant immunological changes take place following splenectomy. Whereas other groups have compared splenectomized patients to healthy controls, this study compared patients undergoing elective splenectomy to those undergoing a similar major abdominal surgery. Overall, we found that splenectomy results in significant long-lasting changes in circulating immune cell populations and function.

Relating TCR-peptide-MHC affinity to immunogenicity for the design of tumor vaccines.

One approach to enhancing the T cell response to tumors is vaccination with mimotopes, mimics of tumor epitopes. While mimotopes can stimulate proliferation of T cells that recognize tumor-associated antigens (TAAs), this expansion does not always correlate with control of tumor growth. We hypothesized that vaccination with mimotopes of optimal affinity in this interaction will improve antitumor immunity. Using a combinatorial peptide library and a cytotoxic T lymphocyte clone that recognizes a TAA, we identified a panel of mimotopes that, when complexed with MHC, bound the TAA-specific TCR with a range of affinities. As expected, in vitro assays showed that the affinity of the TCR-peptide-MHC (TCR-pMHC) interaction correlated with activity of the T cell clone. However, only vaccination with mimotopes in the intermediate-affinity range elicited functional T cells and provided protection against tumor growth in vivo. Vaccination with mimotopes with the highest-affinity TCR-pMHC interactions elicited TAA-specific T cells to the tumor, but did not control tumor growth at any of the peptide concentrations tested. Further analysis of these T cells showed functional defects in response to the TAA. Thus, stimulation of an antitumor response by mimotopes may be optimal with peptides that increase but do not maximize the affinity of the TCR-pMHC interaction.

Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome.

People with Down syndrome (DS; trisomy 21) display a different disease spectrum relative to the general population, including lower rates of solid malignancies and higher incidence of neurological and autoimmune conditions. However, the mechanisms driving this unique clinical profile await elucidation. We completed a deep mapping of the immune system in adults with DS using mass cytometry to evaluate 100 immune cell types, which revealed global immune dysregulation consistent with chronic inflammation, including key changes in the myeloid and lymphoid cell compartments. Furthermore, measurement of interferon-inducible phosphorylation events revealed widespread hypersensitivity to interferon-α in DS, with cell-type-specific variations in downstream intracellular signaling. Mechanistically, this could be explained by overexpression of the interferon receptors encoded on chromosome 21, as demonstrated by increased IFNAR1 surface expression in all immune lineages tested. These results point to interferon-driven immune dysregulation as a likely contributor to the developmental and clinical hallmarks of DS.

Multiple innate signaling pathways cooperate with CD40 to induce potent, CD70-dependent cellular immunity.

We have previously shown that Toll-like receptor (TLR) agonists cooperate with CD40 to generate CD8 T cell responses exponentially larger than the responses generated with traditional vaccine formulations. We have also shown that combined TLR agonist/anti-CD40 immunization uniquely induces the upregulation of CD70 on antigen bearing dendritic cells (DCs). In contrast, immunization with either a TLR agonist or a CD40 stimulus alone does not significantly increase CD70 expression on DCs. Furthermore, the CD8(+) T cell response generated by combined TLR agonist/anti-CD40 immunization is dependent on the expression of CD70 by DCs, as CD70 blockade following immunization dramatically decreases the CD8 T cell response. Here we show that other innate pathways, independent of the TLRs, can also cooperate with CD40 to induce potent, CD70 dependent, CD8 T cell responses. These innate stimuli include Type I IFN (IFN) and alpha-galactosylceramide (alphaGalCer) or aC-GalCer, glycolipids that are presented by a nonclassical class I MHC molecule, CD1d, and are able to activate NKT cells. Furthermore, this combined IFN/anti-CD40 immunization generates protective memory against bacterial challenge with Listeria monocytogenes. Together these data indicate the importance of assessing CD70 expression on DCs as a marker for the capacity of a given vaccine formulation to potently activate cellular immunity. Our data indicate that optimal induction of CD70 expression requires a coordinated stimulation of both innate (TLR, IFN, alphaGalCer) and adaptive (CD40) signaling pathways.

Framework for evaluating anthrax risk in buildings.

If Bacillus anthracis (BA), the organism that causes anthrax, is known or suspected to have contaminated a building, a critical decision is what level of contamination is unacceptable. This decision has two components: (1) what is the relationship between the degree of contamination and the risk to occupants, (2) and what is an acceptable risk to occupants? These lead to a further decision: (3) how many samples must be taken to determine whether a building is unacceptably contaminated? We discuss existing data that bear on these questions, and introduce a nomogram that can be used to investigate the relationship between risk of contracting anthrax, the surface concentration of BA, the probability of detection, and the number of samples needed to ensure detection with a given degree of certainty. The same approach could be used for other agents that are dangerous due to resuspension of deposited particles.

Age-dependent tolerance to an endogenous tumor-associated antigen.

Immunologic tolerance to endogenous antigens reduces antitumor responses. Gp70 is an endogenous tumor-associated antigen (TAA) of the BALB/c-derived colon carcinoma CT26. We found that expression of gp70 mRNA is detectable in tissues of mice 8 months of age and older. We showed that expression of gp70 establishes immunologic tolerance and affects antitumor immunity in a similarly age-dependent manner using gp70-deficient mice. We found that tumors grew in all gp70-sufficient mice, while approximately half of gp70-deficient mice controlled tumor growth with endogenous T-cell responses. Protection in gp70-deficient mice correlated with more robust gp70-specific CTL responses, and increased numbers and avidity of responding antigen-specific T cells after vaccination. We conclude that immunosurveillance may decline with age due to increased or de novo peripheral expression of endogenous TAAs.

Combined TLR/CD40 stimulation mediates potent cellular immunity by regulating dendritic cell expression of CD70 in vivo.

We previously showed that immunization with a combination of TLR and CD40 agonists (combined TLR/CD40 agonist immunization) resulted in an expansion of Ag-specific CD8 T cells exponentially greater than the expansion observed to immunization with either agonist alone. We now show that the mechanism behind this expansion of T cells is the regulated expression of CD70 on dendritic cells. In contrast to previous results in vitro, the expression of CD70 on dendritic cells in vivo requires combined TLR/CD40 stimulation and is not significantly induced by stimulation of either pathway alone. Moreover, the exponential expansion of CD8(+) T cells following combined TLR/CD40 agonist immunization is CD70 dependent. Thus, the transition from innate stimuli (TLRs) to adaptive immunity is controlled by the regulated expression of CD70.

A modified tyrosinase-related protein 2 epitope generates high-affinity tumor-specific T cells but does not mediate therapeutic efficacy in an intradermal tumor model.

The generation of tumor-specific T cells is hampered by the presentation of poorly immunogenic tumor-specific epitopes by the tumor. Here, we demonstrate that, although CD8+ T cells specific for the self/tumor Ag tyrosinase-related protein 2 (TRP2) are readily detected in tumor-bearing hosts, vaccination of either tumor-bearing or naive mice with an epitope derived from TRP2 fails to generate significant numbers of tetramer-staining TRP2-specific T cells or antitumor immunity. We identified an altered peptide epitope, called deltaV, which elicits T cell responses that are cross-reactive to the wild-type TRP2 epitope. Immunization with deltaV generates T cells with increased affinity for TRP2 compared with immunization with the wild-type TRP2 epitope, although TRP2 immunization often generates a greater number of TRP2-specific T cells based on intracellular IFN-gamma analysis. Despite generating higher affinity responses, deltaV immunization alone fails to provide any greater therapeutic efficacy against tumor growth than TRP2 immunization. This lack of tumor protection is most likely a result of both the deletion of high affinity and functional tolerance induction of lower affinity TRP2-specific T cells. Our data contribute to a growing literature demonstrating the ability of variant peptide epitopes to generate higher affinity T cell responses against tumor-specific Ags. However, consistent with most clinical data, simple generation of higher affinity T cells is insufficient to mediate tumor immunity.