Andrew Bushell

A simple method to cure established tumors by inflammatory killing of normal cells

We describe a simple technology used to cure an established metastatic disease. Intradermal injection of plasmid DNA encoding a transcriptionally targeted cytotoxic gene, along with hsp70, not only promoted tissue-specific, inflammatory killing of normal melanocytes, but also induced a CD8+ T-cell–dependent, antigen-specific response in mice that eradicated systemically established B16 tumors. This CD8+ T cell response was subsequently suppressed in vivo within a few days. The data demonstrate that deliberate destruction of normal tissue can be exploited to generate immunity against a malignant disease originating from that tissue. This approach obviates the need to identify tumor antigens and does not require complex isolation of tumor cells or their derivatives. In addition, it provides a model system for studying the mechanisms underlying the etiology and control of autoimmune diseases. Finally, despite targeting normal tissue, therapy could be separated from development of overt autoimmune symptoms, suggesting that the strategy may be valuable against tumors derived from both non-essential and essential tissue types.*Note: In the version of this article originally published online, the name of one of the authors was spelled incorrectly. Mayoi Lai should be Maoyi Lai. This mistake has been corrected in the HTML version and will appear correctly in print.

Cytokines and peripheral tolerance to alloantigen

The induction of peripheral tolerance to alloantigen is accompanied in many cases by a decrease in the production of cytokines such as IL-2 and IFN gamma, yet a sustained production of cytokines such as IL-10 and IL-4. Whether or not this altered pattern of cytokine production in tolerant animals is causally related to the induction and/or maintenance of the tolerant state has yet to be fully determined, although experiments blocking selectively the action of IL-2 with CD25 antibodies suggest that manipulation of cytokine production may at least be a route to tolerance. Alternative methods for directly influencing the cytokine balance are sought and recent experiments on the CD28/CTLA-4-B7 interaction suggest a possible approach.

Regulatory T cells: first steps of clinical application in solid organ transplantation

Solid organ transplantation is the treatment of choice for patients with end‐stage organ failure. To prevent rejection of the transplanted organ continuous treatment with immunosuppressive medication is needed. Immunosuppression may be harmful to the transplant recipient, increasing the risk of cancer, infections and cardiovascular disease. To improve transplant and patient survival, there is a need for an immune‐modulatory regimen that is not only potent in preventing rejection of the transplanted organ, but has less side effects compared to current immunosuppressive regimens. Increasingly, transplantation research focusses on regulatory T cell (Treg) therapy to achieve this aim, in which Treg are used as a strategy to allow reduction of immunosuppression. Currently, the first clinical trials are underway investigating the safety and feasibility of Treg therapy in renal transplantation. This review gives an overview of the rationale of using Treg therapy in transplantation, previous experience with Treg therapy in humans, and the expected safety, potential efficacy and cost‐effectiveness of Treg therapy in solid organ transplantation.

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection.

CD4+CD25+Foxp3+ regulatory T (Treg) cells mediate immunological self‐tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF‐β. In this study, we show that the co‐culture of naive T cells from C57BL/6 mice with allogeneic antigen‐presenting cells (APCs) from BALB/c mice in the presence of TGF‐β, RA, and IL‐2 resulted in a striking enrichment of Foxp3+ T cells. These RA in vitro‐induced regulatory T (RA‐iTreg) cells did not secrete Th1‐, Th2‐, or Th17‐related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg‐cell suppressive potential. Accordingly, these RA‐iTreg cells suppressed T‐cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA‐iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA‐iTreg cells showed alloantigen‐specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic‐specific Treg cells may be generated using TGF‐β, RA, and IL‐2. Thus, RA‐iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.

Regulatory T Cell Enrichment by IFN-γ Conditioning

IFN-γ was originally characterized as a proinflammatory cytokine with T helper type 1 inducing activity, but it is now clear that it also has important immunoregulatory functions. Regulatory T cells play an important role in models of autoimmunity, GVHD, and transplantation, and offer potential as a cellular therapy. In rodent models, in vivo-generated CD25(+)CD4(+) T cells can prevent allograft rejection, but therapeutic exploitation of Treg will more likely depend on protocols that allow the generation or selection of Treg ex vivo. The experiments described in this chapter will show that alloantigen-reactive Treg can be generated/expanded ex vivo using IFN-γ, a cytokine more usually associated with allograft rejection. Although IFN-γ production has hitherto been generally regarded as nonpermissive for allograft survival, we believe this paradoxical good-bad role for IFN-γ may reflect an important physiological negative feedback loop.

Distinctive Expression of Bcl-2 Factors in Regulatory T Cells Determines a Pharmacological Target to Induce Immunological Tolerance.

Distinctive molecular characteristics of functionally diverse lymphocyte populations may represent novel pharmacological targets for immunotherapy. The intrinsic apoptosis pathway is differently regulated among conventional and regulatory T cells (Tregs). Targeted pharmacological modulation of this pathway with a small molecule Bcl-2/Bcl-xL inhibitor (ABT-737) caused a selective depletion of effector T cells and a relative enrichment of Tregs in vivo. Treatment with ABT-737 resulted in a tolerogenic milieu, which was exploited to alleviate graft-versus-host disease, to prevent allograft rejection in a stringent fully MHC-mismatched skin transplantation model and to induce immunological tolerance in combination with bone marrow transplantation. This concept has the potential to find various applications for immunotherapy, since it allows pharmacologic exploitation of the immunomodulatory properties of Tregs without the need for cell manipulation ex vivo.

Hybridization analysis reveals novel DQw3 α-β associations suggesting a recombination point between DQα and DQβ on the HLA-DR4 haplotype

Southern blotting and hybridization with locus-specific probes in the analysis of HLA class II specificities provides a useful complementary technique to conventional serological typing. Several laboratories have demonstrated that under appropriate conditions the restriction fragment patterns obtained from well-characterized homozygous cell lines and randomly selected individuals correlate with their serologically defined DR and DQ specificities (Anderson et al. 1984, Bell et al. 1987, MacMurray et al. 1987). Similar observations have recently been reported for the HLA-DP subregion (HeyldigNielsen et al. 1987, Bodmer et al. 1987). Restriction fragment analysis has also been of considerable value in the identification of subtypes within existing serological specificities. In an analysis of 24 cell lines homozygous for DQw3 Kim and co-workers (1985) showed by DQ~ hybridization that two subtypes exist within the serological specificity DQw3. Functional support for their restriction fragment (r. f.) data was provided by the observation that the split correlated exactly with the reactivity of a monoclonal antibody (A-10-83). Their data showed that although the predominant association of these two subtypes was ofDQw3.1 with DR5 and DQw3.2 with DR4, DQw3.1 was found on the DR4 haplotype in 3/16 cases. We have examined a number of DQw3 homozygous cell lines (HCL) by restriction enzyme digestion and hybridization with DRy, DQ~, and DQ~ probes. Our data show that the split of DQw3 first clearly demonstrated by hybridization with DQ¢ can also be seen with the DQ~ probe and that some DR4 haplotypes carry novel DQ o~-/3 associations. Digested DNA was electrophoresed in 0.7% agarose gels in Tris-acetate-ethylenediaminetetraacetate (EDTA) buffer (Maniatis et al. 1982), transferred to nylon membranes (Amersham International, Amersham, United Kingdom), and hybridized at 42 °C for 20 h with DRy,

An interactive computer program for the analysis of HLA class II restriction fragment patterns

There is considerable current interest in the application of Southern blotting and hybridization, with locus-specific HLA class II probes, to HLA typing. The work of several laboratories has shown that, under suitable conditions, restriction fragment patterns that correlate with DR and DQ serology and with DP cellular reactivities can be obtained from homozygous cell lines. Although the identification of class II specificities from restriction fragment patterns is relatively straightforward in homozygotes, the increased complexity of the patterns obtained from heterozygotes makes the interpretation considerably more difficult; this difficulty is compounded by even slight variation between and within gels. We have developed an interactive computer program that allows HLA-DR and DQ typing by the visual matching of restriction fragment pattern data with standard data derived from a panel of well-characterized homozygous cell lines. The program also uses a simple algorithm (after Southern) to estimate the fragment sizes in the unknown track. The screen display can be printed out to give a convenient record of the match; a numerical measure is also given of the goodness of fit of the new data.

A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

The concept of regulatory T cell (Treg)-based immunotherapy has enormous potential for facilitating tolerance in autoimmunity and transplantation. Clinical translation of Treg cell therapy requires production processes that satisfy the rigors of Good Manufacturing Practice (GMP) standards. In this regard, we report our findings on the implementation of a robust GMP compliant process for the ex vivo expansion of clinical grade Tregs, demonstrating the feasibility of this developed process for the manufacture of a final product for clinical application. This Treg isolation procedure ensured the selection of a pure Treg population that underwent a 300-fold expansion after 36 days of culture, while maintaining a purity of more than 75% CD4+CD25+FOXP3+ cells and a suppressive function of above 80%. Furthermore, we report the successful cryopreservation of the final product, demonstrating the maintenance of phenotype and function. The process outlined in this manuscript has been implemented in the ONE study, a multicenter phase I/IIa clinical trial in which cellular therapy is investigated in renal transplantation.

Intentional killing of normal nelanocytes by gene transfer generates curative anti melanoma immunity without obligate autoimmunity

Most tumor vaccination strategies depend upon the molecular identification of specific tumor rejection antigens and/or the use of patient derived tumor materials. In addition, there is a strong correlation between the use of cancer vaccines and the induction of autoimmune reactivity. We hypothesised that in situ, inflammatory killing of a normal tissue from which a tumor derives may generate autoimmune reactivity to self antigens expressed in that tissue as well as in the tumor cells. Here we report that simple intradermal injection of 2 plasmids promoted tissue specific, inflammatory killing of melanocytes and induced an immune response that eradicated systemically established B16 tumors. The therapeutic response was CD8+T cell dependent but, significantly, was rapidly suppressed in vivo and did not induce autoimmune disease in animals in which tumors had been cured. The T cell response was characterized by selection of B16 tumor variants which grow extremely aggressively in vivo, are amelanotic and which have lost expression of the tyrosinase and TRP-2 antigens. The mechanism of antigen loss was, at least in part, through epigenetic mechanisms. These could be reversed by long term growth in culture or, more rapidly, by treatment with the de-methylating agent 5-azacytidine. Depletion of putative regulatory T cells within the CD25+ T cell population improved therapeutic efficacy. Finally, vaccination could be further improved using adoptive transfer of activated T cells with specificity for an antigen that was not a target of the initial T cell response raised by melanocyte killing. These data demonstrate that inflammatory killing of a normal tissue can lead to the immunoselection of tumor cell growth variants. They also raise the prospect that such vaccinations could be effectively used in combination with other conventional anti tumor treatment modalities.