Stephen Todryk

Suppression of Antitumor Immunity by IL-10 and TGF-β-Producing T Cells Infiltrating the Growing Tumor: Influence of Tumor Environment on the Induction of CD4+ and CD8+ Regulatory T Cells

We examined the hypothesis that a failure of the immune system to eradicate tumors is due to the immunosuppressive environment created by the growing tumor, which is influenced by the site of tumor growth. We demonstrated that T cell responses to a bystander Ag in mice were suppressed by a growing CT26 tumor. T cells purified from the growing tumor expressed mRNA for IL-10, TGF-β, and Foxp3. Intracellular cytokine staining revealed a high frequency of IL-10-secreting macrophages, dendritic cells, and CD4+ and CD8+ T cells infiltrating the tumor. In contrast, T cell IFN-γ production was weak and CD8+ CTL responses were undetectable in mice with CT26 lung metastases and weak and transient following s.c. injection of CT26 cells, but were enhanced in the presence of anti-IL-10 and anti-TGF-β. Consistent with this, removal of CD8+ T cells abrogated CTL responses and promoted progression of the s.c. tumor. However, in the lung model, depletion of CD8+ T cells significantly reduced the tumor burden. Furthermore, depletion of CD4+ or CD25+ T cells in vivo reduced tumor burden in s.c. and lung models, and this was associated with significantly enhanced IFN-γ production by CD8+ T cells. These findings suggest that tumor growth facilitates the induction or recruitment of CD4+ regulatory T cells that secrete IL-10 and TGF-β and suppress effector CD8+ T cell responses. However, CD8+ T regulatory cells expressing IL-10 and TGF-β are also recruited or activated by the immunosuppressive environment of the lung, where they may suppress the induction of antitumor immunity.

Protective antitumor immunity induced by a costimulatory thalidomide analog in conjunction with whole tumor cell vaccination is mediated by increased Th1-type immunity.

Thalidomide and its novel T cell costimulatory analogs (immunomodulatory drugs) are currently being assessed in the treatment of patients with advanced cancer. However, neither tumor-specific T cell costimulation nor effective antitumor activity has been demonstrated in vivo. In this study, we assessed the ability of an immunomodulatory drug (CC-4047/ACTIMID) to prime a tumor-specific immune response following tumor cell vaccination. We found that the presence of CC-4047 during the priming phase strongly enhanced antitumor immunity in the vaccinated group, and this correlated with protection from subsequent live tumor challenge. Protection was associated with tumor-specific production of IFN-γ and was still observed following a second challenge with live tumor cells 60 days later. Furthermore, CD8+ and CD4+ splenocyte fractions from treated groups secreted increased IFN-γ and IL-2 in response to tumor cells in vitro. Coculture of naive splenocytes with anti-CD3 mAb in the presence of CC-4047 directly costimulated T cells and increased Th1-type cytokines. Our results are the first to demonstrate that a costimulatory thalidomide analog can prime protective, long-lasting, tumor-specific, Th1-type responses in vivo and further support their ongoing clinical development as novel anti-cancer agents.

Selective Expansion and Partial Activation of Human NK Cells and NK Receptor-Positive T Cells by IL-2 and IL-15

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56+ T cells, but not CD56− lymphocytes. All NK and CD56+ T cell subpopulations tested (CD4+, CD8+, CD4−CD8−, αβTCR+, γδTCR+, CD16+, CD161+, CD158a+, CD158b+, KIR3DL1+, and CD94+) expanded in response to both cytokines, whereas all CD56− cell subpopulations did not. Therefore, previously reported IL-15-induced γδ and CD8+ T cell expansions reflect proliferations of NK and CD56+ T cells that most frequently express these phenotypes. IL-15 also expanded CD8α+β− and Vα24Vβ11 TCR+ T cells. Both cytokines stimulated cytotoxicity by NK and CD56+ T cells against K562 targets, but not the production of IFN-γ, TNF-α, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56+ T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.

Facets of heat shock protein 70 show immunotherapeutic potential

Amongst the families of intracellular molecules that chaperone and assist with the trafficking of other proteins, notably during conditions of cellular stress, heat shock protein (hsp) 70 is one of the most studied. Although its name suggests that expression is exclusively induced during cellular hyperthermia, members of the hsp70 family of proteins can be constitutively expressed and/or induced by a range of other cellular insults. The ubiquitous presence of hsp70 in eukaryotic and prokaryotic cells, combined with its high degree of sequence homology and intrinsic immunogenicity, have prompted the suggestion that inappropriate immune reactivity to hsp70 might lead to pro‐inflammatory responses and the development of autoimmune disease. Indeed, hsp70 has been shown to be a potent activator of innate immunity and aberrant expression of hsp70 in certain organs promotes immunopathology. However, studies also suggest that hsp70 might have immunotherapeutic potential, as hsp70 purified from malignant and virally infected cells can transfer and deliver antigenic peptides to antigen‐presenting cells to elicit peptide‐specific immunity and, in contrast to its reported pro‐inflammatory effects, the administration of recombinant hsp70 can attenuate experimental autoimmune disease. This review focuses on the immunoregulatory capacity of hsp70 and its potential therapeutic value.

Durable Human Memory T Cells Quantifiable by Cultured Enzyme-Linked Immunospot Assays Are Induced by Heterologous Prime Boost Immunization and Correlate with Protection against Malaria

Immunological memory is a required component of protective antimalarial responses raised by T cell-inducing vaccines. The magnitude of ex vivo IFN-γ T cell responses is widely used to identify immunogenic vaccines although this response usually wanes and may disappear within weeks. However, protection in the field is likely to depend on durable central memory T cells that are not detected by this assay. To identify longer-lived memory T cells, PBMC from malaria-naive vaccinated volunteers who had received prime boost vaccinations with a combination of DNA and/or viral vectors encoding the multiepitope string-thrombospondin-related adhesion protein Ag were cultured in vitro with Ag for 10 days before the ELISPOT assay. Ex vivo T cell responses peaked at 7 days after the final immunization and declined substantially over 6 mo, but responses identified after T cell culture increased over the 6-mo period after the final immunization. Moreover, individual cultured ELISPOT responses at the day of challenge time point correlated significantly with degree of protection against malaria sporozoite challenge, whereas ex vivo responses did not, despite a correlation between the peak ex vivo response and magnitude of memory responses 6 mo later. This cultured assay identifies long-lasting protective T cell responses and therefore offers an attractive option for assessments of vaccine immunogenicity.

Depletion of NK cells results in disseminating lethal infection with Bordetella pertussis associated with a reduction of antigen-specific Th1 and enhancement of Th2, but not Tr1 cells.

IFN‐γ plays a critical role in protection against Bordetella pertussis, but Th1 cells are only detectable after the infection has started to resolve, suggesting a protective role for innate IFN‐γ early in infection. Here, we demonstrate significant recruitment of NK cells and NKT cells into the lungs following respiratory challenge with B. pertussis. Furthermore, NK cells are the primary source of IFN‐γ in the lungs during the acute stage of infection. Stimulation of IFN‐γ production by NK cells was indirect through B. pertussis‐activated IL‐12 or IL‐23 production by dendritic cells. Depletion of NK cells with anti‐asialo ganglio‐N‐tetraosylceramide antibody resulted in a lethal infection, with enhancement of bacterial load in the lungs and dissemination of the bacteria to the liver via the blood. NK cell‐depleted mice had significantly reduced B. pertussis‐specific IFN‐γ and enhanced IgG1 and IL‐5, but not IL‐10 production, suggesting that regulatory T cells are induced simultaneously with Th1 cells, but the absence of NK cells resulted in enhancement of Th2‐type responses. These findings suggest that NK cells confer resistance to B. pertussis by activating IL‐12‐mediated production of IFN‐γ, which enhances the anti‐bacterial activity of macrophages, but also promotes the differentiation of Th1 cells.

Immunotherapeutic potential of whole tumour cells

Abstract. Despite the identification of tumour antigens and their subsequent generation in subunit form for use as cancer vaccines, whole tumour cells remain a potent vehicle for generating anti-tumour immunity. This is because tumour cells express an array of target antigens for the immune system to react against, avoiding problems associated with major histocompatibility complex (MHC)-restricted epitope identification for individual patients. Furthermore, whole cells are relatively simple to propagate and are potentially efficient at contributing to the process of T cell priming. However, whole cells can also possess properties that allow for immune evasion, and so the question remains of how to enhance the immune response against tumour cells so that they are rejected. Scenarios where whole tumour cells may be utilised in immunotherapy include autologous tumour cell vaccines generated from resected primary tumour, allogeneic (MHC-disparate) cross-reactive tumour cell line vaccines, and immunotherapy of tumours in situ. Since tumour cells are considered poorly immunogenic, mainly because they express self-antigens in a non-stimulatory context, the environment of the tumour cells may have to be modified to become stimulatory by using immunological adjuvants. Recent studies have re-evaluated the relative roles of direct and cross-priming in generating anti-tumour immunity and have highlighted the need to circumvent immune evasion.

Heat shock protein derived from a non-autologous tumour can be used as an anti-tumour vaccine.

Antigenic cross‐reactivity between certain tumours has allowed the development of more widely applicable, major histocompatibility complex‐disparate (allogeneic) whole‐cell vaccines. This principle should also allow heat shock proteins (hsp) derived from certain tumours (and carrying cross‐reactive antigens) to be used as vaccines to generate anti‐tumour immunity in a range of cancer patients. Here, hsp70 derived from gp70‐antigen+ B16 melanoma generated cytotoxic‐T‐lymphocyte‐mediated immune protection in BALB/c mice against challenge with gp70‐antigen+ CT26 colorectal tumour cells. Using ovalbumin as a model tumour antigen, it is shown that hsp70 enhances peptide re‐presentation by dendritic cells via class I over equimolar whole ovalbumin antigen. However, while transfection of tumour cells with inducible hsp70 increases hsp yield from tumours, it does not enhance antigen recognition via purified hsp70 nor via whole cells or their lysate.

Comparing human T cell and NK cell responses in viral-based malaria vaccine trials.

Vaccination with viral-based vaccines continues to hold promise for the prevention of malaria. Whilst antigen-specific T cell responses are considered a major aim of such an approach, a role for induced NK cells as anti-malarial effector cells, or in shaping T cell responses, has received less attention. In this study naïve human volunteers were vaccinated in a prime-boost vaccination regimen comprising recombinant viral vectors fowlpox (FP9) and modified vaccinia Ankara (MVA) encoding liver-stage antigens, or a virosome vaccine. Significant T cell responses specific for the vectored vaccine antigens were demonstrated by IFNgamma ELISPOT and intracellular cytokine staining (ICS) for IFNgamma and IL-2, the ICS being associated with increased time to parasitaemia following subsequent challenge. Numbers of CD56(bright) lymphocytes increased significantly following vaccination, as did CD3(+) CD56(+) lymphocytes, whilst CD56(dim) cells did not. No such increases were seen with the virosome vaccine. There was no significant correlation of these CD56(+) populations with the antigen-specific T cell responses nor time to parasitaemia. To investigate pathways of immune activation that could contribute to these lymphocyte responses, viral vectors were shown in vitro to efficiently infect APCs but not lymphocytes, and stimulated inflammatory cytokines such as type I interferons. In conclusion, measuring antigen-specific T cells is more meaningful than NK cells in these vaccination regimens.

Cell death associated with genetic prodrug activation therapy of colorectal cancer

Genetic prodrug activation therapy (GPAT) is a form of cancer gene therapy that has potential use against tumours such as colorectal malignancy. The characterization of such therapies using laboratory models provides a basis for clinical trials. In this study the gene encoding Herpes Simplex Virus thymidine kinase (HSVtk) was delivered to colorectal tumour cells using an Adenoviral (Ad) vector in vitro. In this way the cells were made susceptible to killing with the prodrug ganciclovir to various degrees depending on cell infectability with Ad. Bystander killing effect appeared minimal both in vitro and when transduced cells were injected in vivo. Mechanisms of cell death, measured in vitro using anti-BrDU (DNA-break labelling) and propidium iodide staining variously showed a combination of apoptosis in the G1 cell cycle phase and late apoptotic or necrotic sub-G1 DNA fragmentation, depending on the tumour cell line. These findings suggest that gene therapy of colorectal cancer by GPAT gives rise to therapeutic forms of direct cell death, but requires improvements in transduction, and possibly immune augmentation.