Granulysin: The attractive side of a natural born killer.

First discovered in the 1980's, granulysin has until recently been thought of solely as an effector molecule present within cytotoxic immune cell populations, and involved in the direct killing of pathogens and infected or transformed eukaryotic cells. However, recent research has suggested an additional role of granulysin, in particular the 15 kDa isoform. While 9 kDa granulysin is broadly cytotoxic and capable of the direct killing of bacteria and other pathogens, the 15 kDa isoform of this molecule has been shown to function as an immune 'alarmin', causing the maturation and migration of antigen-presenting cells and other cells of the immune system. This dual function of granulysin indirectly increases the immune response to an infection or tumour, and therefore escalates its importance in the immune system. Here we review the different roles of granulysin, both as a cytotoxic molecule, and as a modulator of the immune system, and discuss the impact this molecule may have on the response to tumour and infection.

Harnessing the power of Vδ2 cells in cancer immunotherapy.

γδ T cells are a subset of T lymphocytes that have been implicated in immunosurveillance against infections and tumours. In the peripheral blood of humans the γδ T cell pool is made up predominantly of Vδ2 cells, which can detect both foreign and self-metabolites of the isoprenoid biosynthesis pathway. This unique axis of antigen recognition enables Vδ2 cells to respond to a range of pathogenic infections as well as perturbations in endogenous isoprenoid biosynthesis that can occur during cell stress and malignant transformation. There has been growing interest in Vδ2 cells as a potential avenue for cancer immunotherapy, and a number of strategies have been utilized in an attempt to boost the anti-tumour response of Vδ2 cells in patients. In this review we discuss critically the evidence that Vδ2 cells contribute to the cytotoxic response against tumours and evaluate current immunotherapeutic approaches that target these cells in cancer patients, with specific focus on their shortcomings and how they may be improved.

Topical imiquimod and intralesional interleukin-2 increase activated lymphocytes and restore the Th1/Th2 balance in patients with metastatic melanoma.

BACKGROUND: Patients with metastatic skin disease in malignant melanoma are difficult to treat, with unresectable lesions proving the biggest challenge. We have recently published data showing a significant clinical response in patients with multiple in-transit melanoma metastases treated with a combination of topical imiquimod and intralesional interleukin (IL)-2. Here we report the results of immunological analysis with the aim of highlighting correlations with our clinical findings. OBJECTIVES: To investigate the systemic effects of our localized combination treatment in patients with accessible metastases of melanoma, and to correlate this with their clinical responses. METHODS: The peripheral blood mononuclear cells of patients were collected at various time points throughout the treatment. Using antibodies to T-cell subsets we measured the changes in cell populations, and along with polyclonal stimulation, changes in cytokine production from these cells over a treatment course. RESULTS: We report an increase in the mean CD4/CD8 ratio from 2.78 to 3.54 with treatment (P < 0.01), and a rise in the percentage of CD25+ cells in the CD4+ population from 14.52% to 38.56%. Furthermore, staining with activation and T-regulatory markers showed that the majority of this population is activated T cells. Cytokine analysis on polyclonally stimulated peripheral blood mononuclear cells showed an increase in the ability of cells to produce interferon (IFN)-gamma over the treatment course, with an initial rise in the IFN-gamma/IL-5 ratio in five of six patients. CONCLUSIONS: The results of this study provide evidence that, in the majority of patients with in-transit metastases of melanoma, therapy with a combination of topical imiquimod and intralesional IL-2 induces a systemic immunological effect by reversing some of changes noted in patients with malignant disease.

Phase I/II study of topical imiquimod and intralesional interleukin-2 in the treatment of accessible metastases in malignant melanoma.

BACKGROUND: Patients with metastatic skin disease in malignant melanoma can be difficult to treat effectively, often requiring repeated treatments with different modalities in an attempt to control their disease. Treatment of nonsurgically resectable melanoma deposits is unsatisfactory, as they are often multiple and recurring. Anecdotal evidence from individual use of imiquimod in superficial metastases and intralesional interleukin (IL)-2 in subcutaneous deposits suggests that the combination may be more effective in bulky subcutaneous disease. OBJECTIVES: To investigate the combination of topical imiquimod and, for selected lesions, intralesional IL-2, to treat a small cohort of patients with accessible melanoma metastases resistant to other treatments. METHODS: Thirteen patients were recruited: all had evidence of multiple cutaneous and/or subcutaneous metastases. Imiquimod was applied to the metastases on a daily basis for 4 weeks, before the introduction of intralesional IL-2. This was injected up to three times a week, into selected lesions, with 0.1 mL injected per lesion at a concentration of 3.6 MIU mL(-1), a total of 1 mL being given at each session. The treated lesions were assessed individually at intervals of 3 months. RESULTS: Thirteen patients were treated, with 10 being eligible for assessment. In total, 182 lesions were treated: 137 purely cutaneous lesions and 41 subcutaneous lesions. Overall, a clinical response was seen in 92 lesions (50.5%) with 74 (40.7%) of these being a complete response (CR) with 91% of the CRs being in the cutaneous lesions. New lesions did appear during the treatment course; however, patients with cutaneous disease experienced a marked slowing of the appearance of new cutaneous lesions. No cutaneous lesions that responded reappeared on cessation of treatment. CONCLUSIONS: The combination of imiquimod and IL-2 is effective in controlling this mixed cutaneous and subcutaneous disease, and is well tolerated. Imiquimod alone is often enough to elicit a response in purely cutaneous lesions. The addition of intralesional IL-2 increases the response rates in subcutaneous lesions, and in otherwise refractory cutaneous lesions.

Antibody response to the human stress protein BiP in rheumatoid arthritis.

OBJECTIVES: The human stress protein BiP (immunoglobulin binding protein) has been implicated in the pathogenesis of rheumatoid arthritis (RA) since BiP was found to stimulate synovial T-cell proliferation and anti-BiP antibodies are present in the serum of RA patients. The aim of this study was the development of a rapid and reproducible enzyme-linked immunosorbent assay (ELISA) to determine the specificity and sensitivity of anti-BiP antibodies in RA. METHODS: An ELISA was developed that detected antibodies to BiP. The prevalence of anti-BiP antibodies was determined in sera from patients with early and established RA, sera antedating the onset of RA and sera from patients with other inflammatory and autoimmune diseases and healthy controls. RESULTS: We have confirmed the increased prevalence of antibodies to BiP in the sera of a large cohort of patients with established RA (specificity 71% and sensitivity 73%) and early RA (specificity 65% and sensitivity 66%). In pre-disease sera, median 2.5 yr (interquartile range 1.1-4.7) before symptoms of joint disease, the sensitivity for anti-BiP antibodies was 45% and the specificity was 65% for the development of RA. CONCLUSION: Antibodies to BiP are found in the sera of patients with RA and in sera antedating the onset of RA.

BiP, a putative autoantigen in rheumatoid arthritis, stimulates IL-10-producing CD8-positive T cells from normal individuals.

OBJECTIVES: We have reported that synovial fluid T cells from patients with rheumatoid arthritis (RA) proliferate in response to the endoplasmic reticulum molecular chaperone immunoglobulin binding protein (BiP). The aim of the present work was to clone and define T cells responding to this protein. METHODS: T-cell clones were generated from the peripheral blood of an individual known to respond to BiP by limiting dilution of BiP-stimulated peripheral blood mononuclear cells. T-cell receptor usage of BiP-responsive clones was determined by monoclonal antibody staining followed by flow cytometric analysis. Cytokine production by the BiP-responsive clones was determined by analysis of post-stimulation supernatants by ELISA. Additional phenotyping was performed by flow cytometry. RESULTS: Of 49 clones isolated, six were shown to proliferate in response to BiP. Proliferation was low but consistent. One clone expressed CD4 and five were CD8-positive. Three clones, all CD8(+), grew strongly and were investigated further. T-cell receptor usage was determined in two clones (Vbeta 7.1 and Vbeta 12); the Vbeta element of the remaining clone was not recognized by the panel of antibodies used. All three clones produced interleukin 10 (IL-10) (80-380 pg/ml) and two of them produced IL-4 (10-80 pg/ml) and IL-5 (>5000 pg/ml). One clone produced both IL-10 and interferon gamma (>5000 pg/ml). Additional phenotyping of these clones showed them to express CD25, CD28, CD80 and 86 but not CD56 or 57. One clone constitutively expressed CTLA-4 cytoplasmically. CONCLUSIONS: This study demonstrates that a population of CD8(+) T cells with the cytokine profile of Tc2 cells can be stimulated by the chaperone BiP. These cells may perform a regulatory role in the normal response to inflammation. The increase in response to this antigen in the synovial joint in RA may indicate an attempt to regulate the ongoing inflammation.

T cell receptor usage in patients with non-progressing HIV infection.

It is still unclear why some patients with HIV progress more slowly than others to developing full blown AIDS. In this study using flow cytometry we have investigated the TCRBV repertoire of peripheral blood T lymphocytes in 17 long-term non-progressing HIV patients (LTNP) to determine if there is a biased usage of T cell receptor V gene products. Patients were identified from hospital records and entered into the study. Three colour flow cytometry was used to determine the expression of the TCRBV3S5, BV5S1, BV5S2, BV5S3, BV6S1, BV7S1, BV9, BV11, BV12, BV13, BV14, BV16, BV17, BV18, BV20, BV21S3, BV22, and BV23 by CD8 and CD4 positive cells isolated from the peripheral blood of patients and controls. Increases in the absolute numbers of CD8+ T cells expressing TCRBV2 and 8 were observed in the HIV-LTNP population (P < 0.05 in both cases). No differences were seen in numbers of CD8+ T cells expressing other TCRBV or in any TCRBV within the CD4+ T cell popu-lation. At follow up (1-2 years later), those patients in which CD4 levels were below 500 x 106/l were those initially found to have lower levels of TCRBV8 +ve CD8 cells. A significant increase in the absolute numbers of T cells coexpressing the gamma delta (gammadelta) T cell receptor and CD8 were also seen in the HIV-LTNP patients compared with controls (P = 0.002). The increase in CD8+ T cells in the HIV-LTNP patients may be interpreted as either an antigen specific, or group of antigen specific responses to viral antigen, or less likely a viral superantigen. A low level of TCRBV8, CD8+ T cells might be predictive of a more rapid disease progression and might indicate a protective role for this population in HIV infected patients. The increase in gammadeltaT cells bearing the CD8 coreceptor suggests a role for this cell type in the response to HIV infection.

The human endoplasmic reticulum molecular chaperone BiP is an autoantigen for rheumatoid arthritis and prevents the induction of experimental arthritis.

Rheumatoid arthritis (RA) is the most common, crippling human autoimmune disease. Using Western blotting and tandem mass spectroscopy, we have identified the endoplasmic reticulum chaperone BiP, a 78-kDa glucose-regulated protein, as a possible autoantigen. It preferentially stimulated increased proliferation of synovial T cells from patients with RA but not from patients with other arthritides. Mice with established collagen- or pristane-induced arthritis developed IgG Abs to BiP. Although BiP injected in CFA failed to induce arthritis in several strains of rats and mice, including HLA-DR4(+/-)- and HLA-DR1(+/+)-transgenic animals, it completely inhibited the development of arthritis when given i.v. 1 wk before the injection of type II collagen arthritis. Preimmunization with BiP suppressed the development of adjuvant arthritis in Lewis rats in a similar manner. This is the first report of a mammalian chaperone that is an autoantigen in human RA and in experimental arthritis and that can also prevent the induction of experimental arthritis. These findings may stimulate the development of new immunotherapies for the treatment of RA.

Decreased expression of FcgammaRIII (CD16) by gammadelta T cells in patients with rheumatoid arthritis.

Some gammadelta T cells express a receptor for the Fc portion of immunoglobulin G (FcgammaRIII - CD16). The relevance of this Fc receptor to gammadelta T-cell function is at present unclear. Our previous studies have shown that gammadelta T cells express activation markers in patients with rheumatoid arthritis (RA). In this study we have examined the relative proportions of CD16+ gammadelta T cells in the blood and synovial fluid of these patients compared with control blood. CD16+ gammadelta T cells from RA patients were significantly reduced in synovial fluid compared with the circulation. That this was due to blocking of antibody binding to CD16 was unlikely as treatment of blood gammadelta T cells with RA synovial fluid (known to contain immune complexes) failed to alter expression of CD16. Treatment of blood gammadelta T cells with phytohaemagglutinin in vitro, resulted in a time-dependent decrease in expression of CD16, with a concomitant increase in expression of human leucocyte antigen-DR, at the single cell level. We conclude that expression of CD16 by gammadelta T cells is lost in the synovial compartment as the result of activation.