Mary A. Markiewicz

The Balance between T Cell Receptor Signaling and Degradation at the Center of the Immunological Synapse Is Determined by Antigen Quality

The role of the center of the immunological synapse (the central supramolecular activation cluster or cSMAC) is controversial. One model suggests that the role of the cSMAC depends on antigen quality and can both enhance signaling and receptor downregulation, whereas a second model proposes that the sole function of the cSMAC is to downregulate signaling. An important distinction between the models is whether signaling occurs in the cSMAC. Here, we demonstrate that at early time points, signaling occurs outside the cSMAC, but occurs in the cSMAC at later time points. Additionally, we show that cSMAC formation enhances the stimulatory potency of weak agonists for the TCR. Combined with previous studies showing that cSMAC formation decreases the signaling by strong agonists, our data support a model proposing that signaling and receptor degradation both occur in the cSMAC and that the balance between signaling and degradation in the synapse is determined by antigen quality.

Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression

Integrins mediate many biological processes, including tumor-induced angiogenesis and metastasis. The arginine–glycine–aspartic acid (RGD) peptide sequence is a common recognition motif by integrins in many proteins and small peptides. While evaluating a small library of RGD peptides for imaging αVβ3 integrin (ABI)-positive tumor cell line (A549) by optical methods, we discovered that conjugating a presumably inactive linear hexapeptide GRDSPK with a near-infrared carbocyanine molecular probe (Cypate) yielded a previously undescribed bioactive ligand (Cyp-GRD) that targets ABI-positive tumors. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay with A549 cells showed that Cyp-GRD was not cytotoxic up to 100 μM in cell culture. The compound was internalized by cells, and this internalization was blocked by coincubation with a cyclic RGD peptide (cyclo[RGDfV], f is d-phenylalanine) that binds ABI with high affinity. In vivo, Cyp-GRD selectively accumulated in tumors relative to surrounding normal tissues. Blocking studies with cyclo[RGDfV] inhibited the in vivo uptake of Cyp-GRD, suggesting that both compounds target the same active site of the protein. A strong correlation between the Cyp-GRD peptide and mitochondrial NADH concentration suggests that the new molecule could also report on the metabolic status of cells ex vivo. Interestingly, neither a Cypate-labeled linear RGD peptide nor an 111In-labeled DOTA-GRD conjugate was selectively retained in the tumor. These results clearly demonstrate the synergistic effects of Cypate and GRD peptide for molecular recognition of integrin expression and suggest the potential of using carbocyanines as optical scaffolds for designing biologically active molecules.

Costimulation through NKG2D Enhances Murine CD8+ CTL Function: Similarities and Differences between NKG2D and CD28 Costimulation

Multiple studies have demonstrated that the NK cell activating receptor NKG2D can function as a costimulatory receptor for both mouse and human CD8+ T cells. However, it has recently been suggested that stimulation through NKG2D is insufficient for costimulation of CD8+ T cells. To aid in the delineation of NKG2D function in CTL responses, we investigated whether stimulation of NKG2D by the natural ligand RAE1ε was able to costimulate effector functions of a murine CTL line generated from DUC18 TCR transgenic mice. We found that NKG2D was able to costimulate DUC CTL responses and did so in a manner similar to CD28 costimulation. The T cells exhibited increased proliferation, IFN-γ release, and cytotoxicity when presented antigenic peptide by P815 cells expressing RAE1ε or B7-1 compared with untransfected P815. In addition, both RAE1ε and B7-1 enhanced Ag-independent IFN-γ secretion in response to IL-12 and IL-18 by DUC CTL. However, only costimulation through CD28 allowed for DUC CTL survival upon secondary stimulation, whereas ligation of NKG2D, but not CD28, induced DUC CTL to form an immune synapse with target cells in the absence of TCR stimulation. Understanding the outcomes of these differences may allow for a better understanding of T cell costimulation in general.

Spontaneous Rejection of Poorly Immunogenic P1.HTR Tumors by Stat6-Deficient Mice

Experimental evidence suggests that a type 1 T cell response may result in optimal tumor rejection in vivo. This phenotype is determined in part by cytokines that influence T cell differentiation. In transplantable tumor models such as P1.HTR, tumors grow progressively despite expression of defined tumor Ags. We hypothesized that this failure to reject may be due to poor generation of a type 1 phenotype, through a dominant influence of the type 2-promoting cytokines IL-4 and/or IL-13. This hypothesis was tested by implanting P1.HTR tumors into mice deficient in Stat6. In contrast to progressive growth of P1.HTR tumors in wild-type mice, and aggressive growth even of IL-12-transfected P1.HTR in Stat1−/− mice, P1.HTR was spontaneously rejected by Stat6−/− mice. Rejection was accompanied by augmented tumor-specific IFN-γ production and CTL activity. These results suggest that pharmacologic inhibition of Stat6 signaling could potentiate anti-tumor immunity in vivo.

Progress in the Development of Immunotherapy of Cancer Using Ex Vivo-Generated Dendritic Cells Expressing Multiple Tumor Antigen Epitopes

Immunotherapy with tumor-associated antigen-pulsed, ex vivo-generated dendritic cells (DCs) is a promising approach for the treatment of cancer that has shown efficacy in animal models and is now being tested in the clinic. The majority of studies performed to date make use of a single tumor-associated epitope. However, because of the high rate of mutation in tumor cells allowing for loss of expression of a single antigen, it is likely that use of multiple antigenic epitopes will induce a broader, longer-lasting, and effective tumor-specific immune response. Multiple vehicles for loading DCs with multiple antigenic epitopes are under investigation to determine the most effective method for vaccination, with many of these methods showing promise. These loading methods, as well as other critical considerations for making DC vaccination as efficacious as possible, are discussed in this article.

CD4 + T CELL MATTERS IN TUMOR IMMUNITY

CD4 + T cells have been shown to be able to affect tumor growth through both direct and indirect means. In addition, a requirement has been demonstrated for CD4 + T cells in the regulation and induction of T cell memory, and CD4 + suppressor T cells have been identified, stressing a role for CD4 + T cells in the induction and maintenance of antitumor immune responses. A review of the involvement of CD4 + T cells at different stages of tumor immunity is provided, and based on these data we discuss how CD4 + T cell response induction could be incorporated into tumor immunotherapy strategies. dendritic cells suppressor T cells T cell memory CD4 + T cells tumor immunology

RAE1ε Ligand Expressed on Pancreatic Islets Recruits NKG2D Receptor-Expressing Cytotoxic T Cells Independent of T Cell Receptor Recognition

The mechanisms by which cytotoxic T lymphocytes (CTLs) enter and are retained in nonlymphoid tissue are not well characterized. With a transgenic mouse expressing the NKG2D ligand retinoic acid early transcript 1ε (RAE1ε) in β-islet cells of the pancreas, we found that RAE1 expression was sufficient to induce the recruitment of adoptively transferred CTLs to islets. This was dependent on NKG2D expression by the CTLs and independent of antigen recognition. Surprisingly, the recruitment of CTLs resulted in the subsequent recruitment of a large number of endogenous lymphocytes. Whereas transgenic mice did not develop diabetes, RAE1 expression was sufficient to induce insulitis in older, unmanipulated transgenic mice that was enhanced by viral infection and pancreatic inflammation. These results demonstrate that the expression of an NKG2D ligand in islets is sufficient to recruit CTLs regardless of their antigen specificity and to induce insulitis.

IL-12 Enhances CTL Synapse Formation and Induces Self-reactivity

Immunological synapse formation between T cells and target cells can affect the functional outcome of TCR ligation by a given MHC-peptide complex. Although synapse formation is usually induced by TCR signaling, it is not clear whether other factors can affect the efficiency of synapse formation. Here, we tested whether cytokines could influence synapse formation between murine CTLs and target cells. We found that IL-12 enhanced synapse formation, whereas TGFβ decreased synapse formation. The enhanced synapse formation induced by IL-12 appeared to be functional, given that IL-12-treated cells could respond to weak peptides, including self-peptides, to which the T cells were normally unresponsive. These responses correlated with expression of functionally higher avidity LFA-1 on IL-12-treated CTLs. These findings have implications for the function of IL-12 in T cell-mediated autoimmunity.

ICAM-1 Contributes to but Is Not Essential for Tumor Antigen Cross-Priming and CD8+ T Cell-Mediated Tumor Rejection In Vivo

ICAM-1 has been described to provide both adhesion and costimulatory functions during T cell activation. In the setting of antitumor immunity, ICAM-1/LFA-1 interactions could be important at the level of T cell priming by APCs in draining lymph nodes as well as for transendothelial migration and tumor cell recognition at the tumor site. To determine the contribution of ICAM-1 to tumor rejection in vivo, we performed adoptive transfer of 2C TCR-transgenic/RAG2−/− T cells into TCRα−/− vs ICAM−/−/TCRα−/− recipient animals. ICAM-1-deficient mice successfully rejected HTR.C tumors expressing Ld recognized by the 2C TCR, albeit with a kinetic delay. Inasmuch as HTR.C tumor cells themselves express ICAM-1, a second model was pursued using B16-F10 melanoma cells that lack ICAM-1 expression. These cells were transduced to express the SIYRYYGL peptide recognized by the 2C TCR in the context of Kb, which is cross-presented by APCs in H-2b mice in vivo. These tumors also grew more slowly but were eventually rejected by the majority of ICAM-1−/−/TCRα−/− recipients. Delayed rejection in ICAM-1−/− mice was associated with diminished T cell priming as assessed by ELISPOT. In contrast, T cell penetration into the tumor was comparable in wild-type and ICAM-1−/− hosts, and adoptively transferred primed effector 2C cells rejected normally in ICAM-1−/− recipients. Our results suggest that ICAM-1 contributes to but is not absolutely required for CD8+ T cell-mediated tumor rejection in vivo and dominantly acts at the level of priming rather than the effector phase of the antitumor immune response.

Death of peripheral CD8+ T cells in the absence of MHC class I is Fas-dependent and not blocked by Bcl-xL

Productive immune responses require an appropriate environment to support peripheral CD8+ T cell survival. Although host MHC class I molecules appear to be required for this process, the cellular and molecular requirements have not been comprehensively studied. Using adoptive transfer of 2C/recombinase‐activating gene‐2 (RAG‐2)–/– TCR‐transgenic T cells, we found that the survival of both naive and effector CD8+ T cells was dependent upon host expression of the same MHC class I alleles that supported thymic selection. Expression of appropriate MHC class Iby either bone marrow‐ or non‐bone‐marrow‐derived cells was sufficient, suggesting that professional antigen‐presenting cells were not mandatory. In contrast to MHC class I, neither T cell expression of CD28 nor host expression of ICAM‐1 was required for peripheral T cell survival. Finally, T cell death in the absence of appropriate host MHC class I was overcome by elimination of Fas signaling but not by overexpression of Bcl‐xL by CD8+ T cells. These results suggest that, in the absence of a survival signal provided by engagement of host MHC/self peptide complexes, CD8+ T cells die via a Fas‐dependent, mitochondria‐independent pathway.