Angus G. Dalgleish

The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain

The isolation of clones encoding the human surface protein T4, and the expression of the T4 gene in new cellular environments, have enabled us to examine the role of this protein in the pathogenesis of AIDS. Our studies support a mechanism of AIDS virus infection that initially involves the specific interaction of the AIDS virus with T4 molecules on the cell surface. This association can be demonstrated on T4+ transformed T and B lymphocytes as well as epithelial cells. Furthermore, the presence of T4 on the surface of all human cells examined is sufficient to render these cells susceptible to AIDS virus infection. Our data suggest that the T4-AIDS virus complex is then internalized by receptor-mediated endocytosis. Finally, we find that the T4 gene is expressed in the brain as well as in lymphoid cells, providing an explanation for the dual neurotropic and lymphotropic character of the AIDS virus. In this manner, a T lymphocyte surface protein important in mediating effector cell-target cell interactions has been exploited by a human retrovirus to specifically target the AIDS virus to populations of T4+ cells.

SLIM DISEASE: A NEW DISEASE IN UGANDA AND ITS ASSOCIATION WITH HTLV-III INFECTION

A new disease has recently been recognised in rural Uganda. Because the major symptoms are weight loss and diarrhoea, it is known locally as slim disease. It is strongly associated with HTLV-III infection (63 out of 71 patients) and affects females nearly as frequently as males. The clinical features are similar to those of enteropathic acquired immunodeficiency syndrome as seen in neighbouring Zaire. However, the syndrome is rarely associated with Kaposis sarcoma (KS), although KS is endemic in this area of Uganda. Slim disease occurs predominantly in the heterosexually promiscuous population and there is no clear evidence to implicate other possible means of transmission, such as by insect vectors or re-used injection needles. The site and timing of the first reported cases suggest that the disease arose in Tanzania.

Chronic immune activation and inflammation as the cause of malignancy.

Several chronic infections known to be associated with malignancy have established oncogenic properties. However the existence of chronic inflammatory conditions that do not have an established infective cause and are associated with the development of tumours strongly suggests that the inflammatory process itself provides the prerequisite environment for the development of malignancy. This environment includes upregulation of mediators of the inflammatory response such as cyclo-oxygenase (COX)-2 leading to the production of inflammatory cytokines and prostaglandins which themselves may suppress cell mediated immune responses and promote angiogenesis. These factors may also impact on cell growth and survival signalling pathways resulting in induction of cell proliferation and inhibition of apoptosis. Furthermore, chronic inflammation may lead to the production of reactive oxygen species and metabolites such as malondialdehyde within the affected cells that may in turn induce DNA damage and mutations and, as a result, be carcinogenic. Here it is proposed that the conditions provided by a chronic inflammatory environment are so essential for the progression of the neoplastic process that therapeutic intervention aimed at inhibiting inflammation, reducing angiogenesis and stimulating cell mediated immune responses may have a major role in reducing the incidence of common cancers.

Novel thalidomide analogues display anti-angiogenic activity independently of immunomodulatory effects

The anti-tumour effects of thalidomide have been associated with its anti-angiogenic properties. Second generation thalidomide analogues are distinct compounds with enhanced therapeutic potential. Although these compounds are beginning to enter trials for the treatment of cancer there is very little information regarding the anti-angiogenic activity of these clinically relevant compounds. Furthermore, it is not known how the various immunomodulatory activities of these compounds relate to anti-angiogenic activity. In this study we assessed the anti-angiogenic activity of compounds from both IMiD™ and SelCID™ classes of analogues using a novel in vitro multicellular human assay system and the established rat aorta assay. Our results show that both the IMiDs and SelCIDs tested are significantly more potent than thalidomide. The anti-angiogenic potency of the analogues was not related to inhibition of endothelial cell proliferation, nor their TNF-α/PDE type 4 inhibitory properties. However, anti-migratory effects in vitro and inhibition of tumour growth in vivo was observed with the analogue IMiD-1 (clinically known as REVIMID™). Our results show that anti-angiogenic activity spans both currently defined classes of thalidomide analogue and is not related to their previously described immunomodulatory properties. Identification of the differential effects of these compounds will enable targeting of such compounds into the appropriate clinical setting.

The anti-cancer agents lenalidomide and pomalidomide inhibit the proliferation and function of T regulatory cells

Lenalidomide (Revlimid®; CC-5013) and pomalidomide (CC-4047) are IMiDs® proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-α is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependant adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-β or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

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.

Thalidomide and its analogues have distinct and opposing effects on TNF-α and TNFR2 during co-stimulation of both CD4+ and CD8+ T cells

Thalidomide (Thd) is clinically useful in a number of conditions where its efficacy is probably related to its anti‐TNF‐α activity. More recently, Thd has also been shown to co‐stimulate T cells and second generation co‐stimulatory (IMiD™) analogues are currently being assessed in the treatment of cancer patients. However, in contrast to their known suppressive effects during inflammatory stimuli, the effects of Thd/IMiDs on TNF‐α and TNF receptors (TNFRs) during T cell co‐stimulation are not known. We sought to determine the effect of Thd, two clinically relevant IMiDs (CC‐4047, ACTIMID™ and CC‐5013, REVIMID™) and a non‐stimulatory SelCID analogue (CC‐3052) on TNF‐α production and on the expression and shedding of TNFRs during co‐stimulation. We found that co‐stimulation of PBMC with Thd/IMiDs, but not CC‐3052, prevented αCD3‐induced T cell surface expression of TNFR2 and thereby reduced soluble TNFR2 (sTNFR2) levels. However, there was no effect on total (surface/intracellular) TNFR2 protein expression, suggesting inhibition of trafficking to the cell membrane. The extent of co‐stimulation by Thd/IMiDs (assessed by CD69/CD25 expression and IL‐2/sIL‐2Rα production) was similar for CD4+ and CD8+ T lymphocytes and correlated with TNFR2 inhibition. Co‐stimulation, but not the early inhibitory effect on TNFR2, was IL‐2‐dependent and led to increased TNF‐α production by both CD4+ and CD8+ T lymphocytes. The clinical relevance of this observation was confirmed by the elevation of serum TNF‐α during REVIMID™ treatment of patients with advanced cancer. Together, these results suggest a possible role for TNF‐mediated events during co‐stimulation and contrast with the TNF inhibitory effects of Thd and its analogues during inflammatory stimuli.

PREVALENCE OF ANTIBODY TO HUMAN T-LYMPHOTROPIC VIRUS TYPE III IN AIDS AND AIDS-RISK PATIENTS IN BRITAIN

2000 persons in the UK were examined serologically for antibodies to human T-lymphotropic virus type III (HTLV-III). Sera reacting in a membrane immunofluorescence assay (IFA) to HTLV-III were also positive when tested against cells infected with lymphadenopathy virus (LAV-1), and cross-adsorption tests indicated that these retroviruses are probably identical. A competitive radioimmunoassay (RIA), which was wholly concordant with IFA, was used to screen the sera. 30/31 patients with the acquired immunodeficiency syndrome (AIDS) were seropositive, as were 89% patients with persistent generalised lymphadenopathy (PGL), 17% symptomless homosexual men, 34% haemophiliacs receiving pooled clotting factors, and 1.5% intravenous drug abusers. None of more than 1000 unselected blood donors was seropositive. These data confirm the close association between HTLV-III and AIDS and PGL and show that infection with HTLV-III is also prevalent in the populations in whom these syndromes are most likely to develop. However, it would be unwise to presume that AIDS will necessarily develop in seropositive subjects.

Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: Receptor modulation and differentiation induced by phorbol ester

The monocytic leukemic cell line U937 can be infected with human immunodeficiency virus type 1 (HIV-1) to become permanently infected virus producers. Uninfected U937 cells express T4 (CD4) antigen and form syncytia when mixed with HIV-1 producing cells. Anti-T4 monoclonal antibodies block syncytium formation indicating that the HIV-1 receptors on U937 cells include T4 antigen. The promyelocytic leukemic cell line HL60, while expressing only low amounts of surface T4 and not forming syncytia on exposure to HIV-1, can be infected by HIV-1 at lower efficiency than U937 and T-cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment induces the differentiation of U937 cells into macrophages. HIV-infected U937 cells retain the ability to differentiate, though less efficiently, as shown by the appearance of monocyte/macrophage surface markers. T4 antigen on both U937 and T-cell lines is down regulated by TPA treatment. Functional receptors for HIV-1, assayed by syncytium induction and pseudotype plating, are lost concomitantly with T4 antigen following TPA treatment of U937 cells and T cells.

Heat shock proteins refine the danger theory

Deficiencies of the self–non-self paradigm in explaining immune phenomena, such as the existence and breaking of immune tolerance, have lead the likes of Janeway1 and Matzinger2 to suggest additional levels of control of antigen-specific immune responses. In simple terms, the immune system is thought to be turned on by ‘danger’ associated with certain molecules of infectious organisms, or cell products released during tissue damage or stress. Antigen-presenting cells (APC) possess pattern-recognition receptors that can recognize such molecules. This recognition is thought to cause upregulation of costimulatory molecules on local APC, which is the second signal [in addition to antigenic peptide with major histocompatibility complex (MHC)] that is required to initiate an antigen-specific immune response. Candidate danger signals from infectious organisms include lipopolysaccharide, mannans, glycans and CpG DNA motifs, whilst those from mammalian cells include heat shock proteins (hsp; inducible and constitutive), mitochondria, mannose, RNA and DNA. With respect to hsp, recent evidence now suggests that their role in signalling danger may be wide ranging and, not surprisingly for immunology, more complicated than first thought.