Colin Thalhofer

Engagement of OX40 Enhances Antigen-Specific CD4+ T Cell Mobilization/Memory Development and Humoral Immunity: Comparison of αOX-40 with αCTLA-4

Increasing the long-term survival of memory T cells after immunization is key to a successful vaccine. In the past, the generation of large numbers of memory T cells in vivo has been difficult because Ag-stimulated T cells are susceptible to activation-induced cell death. Previously, we reported that OX40 engagement resulted in a 60-fold increase in the number of Ag-specific CD4+ memory T cells that persisted 60 days postimmunization. In this report, we used the D011.10 adoptive transfer model to examine the kinetics of Ag-specific T cell entry into the peripheral blood, the optimal route of administration of Ag and αOX40, and the Ag-specific Ab response after immunization with soluble OVA and αOX40. Finally, we compared the adjuvant properties of αOX40 to those of αCTLA-4. Engagement of OX-40 in vivo was most effective when the Ag was administered s.c. Time course studies revealed that it was crucial for αOX40 to be delivered within 24–48 h after Ag exposure. Examination of anti-OVA Ab titers revealed a 10-fold increase in mice that received αOX40 compared with mice that received OVA alone. Both αOX40 and αCTLA-4 increased the percentage of OVA-specific CD4+ T cells early after immunization (day 4), but αOX40-treated mice had much higher percentages of OVA-specific memory CD4+ T cells from days 11 to 29. These studies demonstrate that OX40 engagement early after immunization with soluble Ag enhances long-term T cell and humoral immunity in a manner distinct from that provided by blocking CTLA-4.

Anti-OX40 (CD134) administration to nonhuman primates: immunostimulatory effects and toxicokinetic study.

The immune-stimulatory properties of anti-CD134 (OX40) antibodies have been well documented in rodents, including their ability to enhance antitumor immunity. In this study, an anti-OX40 antibody (Ab) known to costimulate monkey T cells in vitro, was infused into rhesus macaque monkeys during immunization with the simian immunodeficiency virus protein, gp130. The draining lymph nodes from immunized monkeys treated with anti-OX40 were enlarged compared with immunized monkeys injected with mouse Ig. Anti-OX40-treated monkeys had increased gp130-specific Ab titers, and increased long-lived T-cell responses, compared with controls. There were no overt signs of toxicity in the anti-OX40-treated monkeys. The encouraging immune-stimulatory effects led to the good manufacturing practice production of an anti-OX40 Ab for clinical trials in cancer patients. A detailed toxicology study was performed with anti-OX40 in nonhuman primates. Three groups of 8 monkeys received anti-OX40 at 1 of 3 dose levels (0.4, 2.0, and 10 mg/kg) and a control group received saline. No clinical toxicity was observed, but acute splenomegaly and enlarged gut-associated lymph nodes were observed in the anti-OX40-treated animals; splenomegaly and lymphadenopathy resolved by day 28. These studies demonstrate the immune-stimulatory properties and safety of anti-OX40 in primates and provide a strong scientific rationale to pursue clinical trials in humans.

OX40-Mediated Memory T Cell Generation Is TNF Receptor-Associated Factor 2 Dependent

Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adapter protein that associates with the cytoplasmic tail of OX40, may play a critical role in OX40-mediated signal transduction. To investigate the in vivo role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we bred OVA-specific TCR transgenic mice to TRAF2 dominant-negative (TRAF2 DN) mice. Following Ag stimulation and OX40 engagement of TRAF2 DN T cells in vivo, the number of long-lived OVA-specific T cells and effector T cell function was dramatically reduced when compared with wild-type T cells. We also demonstrate that CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF2 DN defect was partially overcome by CTLA-4 blockade in vivo. The data provide evidence that TRAF2 is linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.

Leukocytes Infiltrate the Skin and Draining Lymph Nodes in Response to the Protozoan Leishmania infantum chagasi

ABSTRACT The vector-borne protozoan Leishmania infantum chagasi causes minimal inflammation after inoculation into skin but disseminates to cause fatal visceral leishmaniasis. To define the inflammatory response at the parasite inoculation site, we introduced metacyclic L. infantum chagasi promastigotes intradermally into BALB/c mouse ears and studied inflammatory cells over 7 days. Ly6G+ neutrophils rapidly infiltrated the dermis, peaking after 6 to 24 h. Macrophages and NK cells next infiltrated the dermis, and NK followed by B cells expanded in draining lymph nodes. Parasite-containing phagocytes were tracked with fluorescent mCherry-labeled L. infantum chagasi. Ly6G+ neutrophils contained the greatest proportion of intracellular parasites 6 to 24 h after inoculation, whereas dermal macrophages harbored the majority of intracellular parasites after 2 to 7 days. These observations were validated microscopically. Low doses of antibody transiently depleted mice of neutrophils, leaving other cells intact. Combined results of in vivo imaging, flow cytometry, and quantitative PCR showed that neutrophil depletion slowed the clearance of extracellular (luciferase-positive) promastigotes during the first 24 h after inoculation yet decreased the numbers of leukocytes containing intracellular (mCherry-positive) parasites. From 3 days onward, total L. infantum chagasi-containing dermal leukocytes and total L. infantum chagasi parasites in draining lymph nodes were similar in both groups. Nonetheless, a second wave of L. infantum chagasi-containing neutrophils occurred 7 days after parasite inoculation into neutrophil-depleted mice, corresponding to the time of neutrophil recovery. Thus, neutrophils were recruited to the dermis even late after inoculation, and L. infantum chagasi trafficked through neutrophils in both neutrophil-depleted and control mice, albeit with different kinetics. Recruitment of neutrophils and transient parasite residence in neutrophils may play a role in nonulcerative forms of leishmaniasis.

The generation of T cell memory: a review describing the molecular and cellular events following OX40 (CD134) engagement

OX40 (CD134), a membrane‐bound member of the tumor necrosis factor‐receptor superfamily, is expressed primarily on activated CD4+ T cells. Following engagement on the cell surface, OX40 delivers a costimulatory signal that leads to potent, proinflammatory effects. Engagement of OX40 during antigen (Ag)‐specific stimulation of T cells leads to increased production of memory T cells, increased migration of Ag‐specific T cells, enhanced cytokine production by effector T cells, and the ability to break peripheral T cell tolerance in vivo. Therefore, OX40 engagement in vivo could have important ramifications for the enhancement of vaccine strategies and inhibition of unwanted inflammation. This review summarizes the molecular and cellular events that occur following OX40 engagement during Ag‐specific T cell activation.

In vivo imaging of transgenic Leishmania parasites in a live host.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

Oxidant Generation by Single Infected Monocytes after Short-Term Fluorescence Labeling of a Protozoan Parasite

ABSTRACT Leishmania spp. are intracellular protozoa residing in mononuclear phagocytes. Leishmania organisms are susceptible to microbicidal responses generated in response to phagocytosis. Assuming that both phagocyte and parasite populations are heterogeneous, it is advantageous to examine the response of individual cells phagocytosing living parasites. Because Leishmania spp. lose virulence during the raising of transfectants, we developed a method to label live Leishmania chagasi short-term with fluorescent dyes. Up to six parasite divisions were detected by flow cytometry after labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE), dioctadecyl-tetramethylindo carbocyanine perchlorate, or chloromethyl tetramethylrhodamine. Labeled parasites entered mononuclear phagocytes as determined by confocal and time-lapse microscopy. Dihydroethidium (DHE) was used to detect macrophage-derived oxidants generated during phagocytosis. Presumably Leishmania organisms are opsonized with host serum/tissue components such as complement prior to phagocytosis. Therefore, we investigated the effects of opsonization and found that this increased the efficiency of CFSE-labeled parasite entry into monocytes (84.6% ± 8.8% versus 20.2% ± 3.8% monocytes infected; P < 0.001). Opsonization also increased the percentage of phagocytes undergoing a respiratory burst (66.0% ± 6.3% versus 41.0% ± 8.3% of monocytes containing CFSE-labeled parasites; P < 0.001) and the magnitude of oxidant generation by each infected monocyte. Inhibitor data indicated that DHE was oxidized by products of the NADPH oxidase. These data suggest that opsonized serum components such as complement lead to more efficient entry of Leishmania into their target cells but at the same time activate the phagocyte oxidase to generate microbicidal products in infected cells. The parasite must balance these positive and negative survival effects in order to initiate a viable infection.

OX40, PD-1 and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head and neck cancer

The tumor microenvironment of squamous cell carcinoma of the head and neck (SCCHN) has been shown to be immune suppressive. Therefore, strategies aimed at overcoming this issue could have a positive therapeutic impact. Hence, we investigated the expression of the known immune‐modulatory proteins OX40, programmed cell death protein 1 (PD‐1) and cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) in SCCHN on different T‐cell subsets of tumor‐infiltrating lymphocytes (TIL) to ascertain whether these proteins could potentially be targeted alone or in combination for future clinical trials. T cells from peripheral blood (PBL) and tumor were analyzed for the expression of OX40, PD‐1 and CTLA‐4 in 29 patients undergoing surgery. These proteins were all expressed significantly higher in T‐cell subsets isolated from tumors compared with PBL of the same patient. OX40 expression was significantly greater in the TIL regulatory T‐cell (Treg) population relative to conventional CD4 and CD8 TIL or the Treg isolated from PBL. PD‐1 expression was increased in all T‐cell subsets relative to PBL. CTLA‐4 was also increased in all TIL subsets relative to blood, and similar to OX40, its highest level of expression was observed in the Treg TIL. The highest frequency of PD‐1, CTLA‐4 and OX40 triple‐positive cells were found in the Treg population isolated from the tumor. We analyzed both human papilloma virus‐positive and ‐negative patients and found similar levels and expression patterns of these two patient populations for all three proteins. These data suggest that there may be therapeutic advantages of targeting these pathways independently or in combination for patients with this disease.

OX40 and other immunoregulatory molecules are highly expressed on tumor infiltrating lymphocytes in oral, head and neck squamous cell carcinoma

OX40 is a potent co-stimulatory receptor on the surface of T lymphocytes. An OX40 agonist was recently tested in a Phase I clinical trial and was found to be well tolerated and enhanced both humoral and cellular immunity in patients with metastatic cancer. Both PD-1 and CTLA-4 blockade are showing therapeutic clinical activity in late stage cancer patients. Hence, we hypothesize that the immune suppressed tumor microenvironment that characterizes oral, head and neck squamous cell carcinoma (OHNSCC) can be overcome by strategies that favor antitumor inflammatory T cell responses and may be an ideal tumor site for immune modulation. This investigation aims to provide a better understanding of the expression of OX40, PD-1, CTLA-4 and CD103 in OHNSCC tumor infiltrating lymphocytes (TIL). Additionally we evaluated whether phenotypic differences existed in patients with HPV positive vs. negative status. Methods TIL and autologous blood were isolated from 29 patients undergoing surgery for OHNSCC. A sample of tissue was obtained from the resection specimen of the primary tumor and sent to the laboratory with autologous blood. Tumor was minced and subjected to digestive enzymes. Blood and tumor samples were separated over ficoll gradient to enrich for lymphocytes. Samples were frozen at -140°c and later thawed in batches for analysis. Proteins were then labeled with fluorescently conjugated antibodies and analyzed by flow cytometry. Results The percentage of FoxP3+CD25+CD4+ (Tregs) of CD4+ T cells is significantly higher in OHNSCC Tumor Infiltrating Lymphocytes (TIL) compared to blood (p < .0005), regardless of HPV status. OX40 expression is significantly higher in the TIL Treg population relative to blood Tregs (p < .0005), also independent of HPV status. PD1 expression is increased in the Tregs (p < .0005), conventional CD4 (p=.0066), and CD8 T cells (p < .0005), in TIL relative to blood. CTLA-4 expression is increased in TIL relative to blood in all subsets with the highest expression observed in the Treg population (.0056). CD103 expression is increased on CD8 T cells in TIL relative to blood (p < .0005). CD8s from HPV positive patients have higher percentage CD103 positive cells than CD8s from HPV negative patients (p=.0056). The highest percentages of PD-1, CTLA-4 and OX40 triple positive cells were found in the Treg population of TIL compared to blood (p < .0005). Due to increased expression of OX40, CTLA-4 and PD-1 in TIL of OHNSCC patients we hypothesize that future combination clinical trials that target these pathways will be therapeutically beneficial.