Edward R. Kaminski

Abnormal T-cell Function in B-cell Chronic Lymphocytic Leukaemia

There is increasing evidence of T cell dysfunction in B cell chronic lymphocytic leukaemia (B-CLL) which may contribute to the aetiology and progress of the disease. An absolute CD8+ lymphocytosis correlates with disease progression and low expression of CD4 and CD8 (as found in autoimmune disease) is seen with abnormal expression of other surface molecules. Although the expression of T cell surface activation markers, CD25 and CD152, may be increased on culture in B-CLL serum, response to the common mitogens, PHA and PWM, is reduced. This and the excess of CD8 cells may explain partly the variable cooperation of T cells with B cell production of immunoglobulin in B-CLL. In the context of T cell cross-talk with antigen presenting cells, B-CLL B cells are poor antigen presenters. But the T cells themselves have significant abnormalities of expression of the many antigens and ligands necessary for this process. In particular, they exhibit variable expression of the low affinity and non-specific adhesion molecules LFA-1 and ICAM-1, variable, clonally restricted and skewed expression of the TCR repertoire (implying repeated antigenic stimulation possibly by CLL antigens), reduced CD28 and CD152 expression (implying impairment of ability to start or stop an immune response) and reduced IL2 and CD25 (IL2 R) expression (critical for positive feed-back in maintenance and expansion of the T cell response to antigen presentation). Although the production of IL2 and other cytokines by the T cell in B-CLL may be impaired, production of the anti-apoptotic cytokine IL4 is not and there may be a unique and expanded subset of CD8/CD30 cells capable of releasing IL4. The relationship of this T cell subset to the malignant B cell in vivo is unknown. However, T cells which are CD4+/CD152+/CCR4+ migrate selectively in vitro in response to the chemokine CCL22 (specific for the receptor CCR4) produced by the malignant B cells and are always seen amongst the malignant cells in bone marrow and lymph nodes from B-CLL patients. Other abnormalities of cytokine secretion are described. These findings suggest that the T cell in B-CLL may be unable to start, maintain and complete an immune response to the malignant B cell and other antigens and may be involved directly in sustaining the tumour. However, autologous tumour specific cytotoxicity has been shown in vitro and T cells which recognise tumour-derived heavy chain fragments circulate in vivo. If adoptive immunotherapy of any nature is to succeed in B-CLL, manipulation to optimise these CTL responses is needed to overcome the profound and variable T cell dysfunction in this disease.

Analysis of the expression of critical activation/interaction markers on peripheral blood T cells in B-cell chronic lymphocytic leukaemia: evidence of immune dysregulation.

B‐cell chronic lymphocytic leukaemia (B‐CLL) is characterized by an accumulation of clonal malignant B cells. The intrinsic characteristics that permit this accumulation have been extensively studied and described. However, it is possible that proliferation and survival of this malignant clone is facilitated by a disruption in the interaction between B and T cells that normally regulate the immune system. In this study, using flow cytometry and cell culture techniques, marked abnormalities of the expression of certain key activation and interaction molecules on the peripheral blood T cells of patients with B‐CLL were demonstrated. In particular, on comparison with normal controls, there was a marked reduction in the number of circulating T cells expressing CD25 (interleukin 2 receptor) (P = 0·007), CD28 (P = 0·01) and CD152 (CTLA‐4) (P = 0·001). There was also a reduction in the number of circulating T cells expressing CD4 (P = 0·03), CD5 (P = 0·05) and CD11a (P = 0·01). There was no difference in the number expressing T‐cell receptor αβ (P = 0·1), CD8 (P = 0·4), CD54 (P = 0·4) and CD154 (P = 0·5), and the only marker expressed on a greater number of circulating T cells in B‐CLL patients was HLA‐DR (P = 0·05). These results suggest that there is a profound T‐cell dysregulation that may contribute to the survival of the malignant B cells in patients with B‐CLL and to the related autoimmune phenomena of the disease.

A study of cytokine gene polymorphisms and protein secretion in renal transplantation

Although there is evidence that cytokine gene polymorphisms are associated with varying quantities of cytokine protein production, the exact role of these polymorphisms in allograft rejection remains unclear. In a previous study, we demonstrated a significant association between high IL-10 secretion in mixed lymphocyte culture (MLC), together with HLA mismatching for at least 4-6 antigens, with the occurrence of acute rejection following renal transplantation. We, therefore, wished to ascertain whether cytokine gene polymorphisms are associated with varying levels of protein secretion and/or allograft rejection in the same group of patients. Cytokine protein secretion in MLC for IL-4, IL-6, IL-10 and IFN-gamma was measured by ELISA in 49 patient-donor pairs. Protein secretion for the above cytokines was also measured in phytohaemagglutinin (PHA) stimulated cultures in 30 normal controls. In both patient and control groups, single nucleotide polymorphism analysis for IL-4 G(-590)T, IL-6 G(-174)C, IL-10 G(-1082)A, IL-10 C(-819)T, IL-10 C(-592)A, TNF-alpha G(-308)A and microsatellite analysis for IFNG (CA repeat) was performed. No correlation was found between cytokine gene polymorphisms and cytokine protein secretion in either mitogen stimulated cultures (control group) or MLC (patient group). In addition, no correlation was demonstrated between cytokine gene polymorphisms and renal allograft rejection.

Generation in vitro of B-cell chronic lymphocytic leukaemia-proliferative and specific HLA class-II-restricted cytotoxic T-cell responses using autologous dendritic cells pulsed with tumour cell lysate

Immunotherapy using dendritic cells has shown encouraging results in both haematological and non‐haematological malignancies. In this study, monocyte‐derived dendritic cells from patients with B‐CLL were cultured for 6 days in the presence of IL‐4 and GM‐CSF. Autologous B‐CLL T‐cells were cultured alone or with B‐CLL lysate‐pulsed and unpulsed autologous dendritic cells. IFN‐γ secretion was assessed using ELISA. Cytotoxicity was assessed, after 21 days in culture and re‐stimulation, using flow cytometry with and without blockade by anti‐HLA class I, anti‐HLA class II, anti‐CD4, anti‐CD8 and anti‐TCRαβ monoclonal antibodies. B‐CLL T cells stimulated with B‐CLL lysate‐pulsed autologous dendritic cells showed a significant (P = 0·0004) increase in IFN‐γ secretion and a significant (P = 0·0008) increase in specific cytotoxicity to autologous B‐cell targets, but none to autologous T cell or B cell targets from healthy individuals. B‐CLL T cells cultured with (non‐B‐CLL) B‐cell lysate‐pulsed B‐CLL dendritic cells showed no significant response. Pulsing dendritic cells from healthy volunteers with an autologous (non‐B‐CLL) B‐cell lysate did not stimulate proliferation, cytokine production or cytotoxicity by autologous T cells. Pulsing B‐CLL dendritic cells with allogeneic B‐CLL lysates and culturing with autologous T‐cells elicited cytotoxicity against autologous B‐CLL targets in some cases, but not in others. Cytotoxicity was significantly reduced by blocking with anti‐HLA class II (P = 0·001), anti‐TCRαβ (P = 0·03) and anti‐CD4 (P = 0·046) antibodies. Phenotyping of the responding T‐cell population demonstrated the majority to be CD4 positive. Our data demonstrate that HLA class II‐restricted proliferative and cytotoxic T‐cell responses to B‐CLL can be generated using autologous dendritic cells pulsed with tumour cell lysate.

Cannabinoid influence on cytokine profile in multiple sclerosis

Cannabinoids have been suggested as possessing immunomodulatory properties, and cannabinoid receptors are present on leucocytes. Clinically, there is some evidence that cannabinoids may be therapeutically useful in treating multiple sclerosis, which is generally believed to be an autoimmune condition. This paper reports data derived from the Cannabinoids in MS (CAMS) study, which was the largest randomized controlled trial yet conducted to evaluate the therapeutic efficacy of cannabinoids. We found no evidence for cannabinoid influence on serum levels of interferon (IFN)‐γ, interleukin (IL)‐10, IL‐12 or C‐reactive protein as measured using enzyme‐linked immunosorbent assay (ELISA), in comparison to control values. Mitogenic stimulation experiments also failed to demonstrate any significant reduction in percentage of CD3+, IFN‐γ producing cells after exposure to cannabinoids in vivo, although numbers were small. Further work is needed to establish the functional significance of cannabinoid receptors on immune cells.

In vitro dendritic cell‐induced T cell responses to B cell chronic lymphocytic leukaemia enhanced by IL‐15 and dendritic cell–B‐CLL electrofusion hybrids

HLA class II‐restricted proliferative and cytotoxic T cell (CTL) responses to B cell chronic lymphocytic leukaemia (B‐CLL) can be generated using autologous dendritic cells (DCs) pulsed with tumour cell lysate. In this study a number of different approaches were used to optimize further the in vitro system. First, the effects of a variety of maturation agents were studied. The addition of TNF‐α, polyriboinosinic polyribocytidylic acid (Poly(I:C)) and LPS to autologous DCs resulted in the emergence of only a small percentage of CD83+ DCs, IFN‐α having no demonstrable effect. Only the addition of Poly(I:C) to DCs resulted in modestly increased specific cytotoxicity to B‐CLL targets, IFN‐α and LPS having no effect. Secondly, T cells were pretreated with IL‐15, prior to culturing with lysate‐pulsed autologous DCs. A significant increase in T cell activation (P = 0·038), IFN‐γ secretion (P = 0·030) and specific cytotoxicity to B‐CLL targets (P = 0·006) was demonstrated compared to untreated T cells. Thirdly, monocyte derived DCs electrofused with B‐CLL B cells were compared with lysate‐pulsed DCs. T cells stimulated by fused DCs generated higher levels of specific cytotoxicity to autologous B‐CLL B cell targets than those stimulated by lysate pulsed DCs (P = 0·013). Blocking studies demonstrated inhibition of this cytotoxicity by both anti‐CD4 (P = 0·062) and anti‐CD8 monoclonal antibodies (P = 0·018), suggesting the generation of both HLA class I‐ and HLA class II‐restricted CTL responses. In summary, in vitro B‐CLL‐specific T cell responses can be enhanced further by preincubating T cells with IL‐15 and using autologous fused DC–B‐CLL hybrids instead of autologous lysate‐pulsed DCs. These preliminary data require confirmation with larger numbers of patients. Such an approach, however, may eventually provide effective immunotherapy for treatment of B‐CLL.

Humoral immunity and bronchiectasis

Bronchiectasis is a common complication of primary antibody deficiency but the incidence of antibody deficiency as an underlying cause of bronchiectasis is largely undefined. In this study the humoral immune status of a cohort of bronchiectatic patients was investigated to detect the frequency of significant antibody deficiency and to determine the extent of immunological investigation which is appropriate for routine assessment of bronchiectasis patients. Fifty‐six out‐patients (with a mean age of 59·6 years) had serum immunoglobulins, IgG subclasses and specific antibodies to capsular polysaccharides of Haemophilus influenzae and Streptococcus pneumoniae measured. Where specific antibody levels were low, where possible, appropriate immunization with pneumococcal or conjugated Haemophilus polysaccharide vaccines was offered and the responses quantified. Three of 56 patients had low total serum IgG levels. Thirteen of 56 had deficiencies of either a single IgG subclass or combinations of two or more subclasses, with IgG4 being most frequently implicated (9/56). Twenty‐nine of 56 had low basal specific polysaccharide antibody levels. Test immunization, where performed, produced satisfactory responses in all cases except one, where a specific defect of responsiveness to pneumococcal polysaccharide was identified. This study indicates that antibody deficiency is an uncommon aetiological/underlying factor in the causation of bronchiectasis beyond the fourth decade and that detailed investigation of humoral immune status as a routine in bronchiectasis patients, at least at this age, is not generally justified.

The utility of the ISAC allergen array in the investigation of idiopathic anaphylaxis

A diagnosis of idiopathic anaphylaxis following a detailed clinical assessment remains very challenging for patients and clinicians. Risk reduction strategies such as allergen avoidance are not possible. This study investigated whether the (ISAC) allergen array with 103 allergens would add diagnostic value in patients with idiopathic anaphylaxis. We extended the specific immunoglobulin (Ig)E testing in 110 patients with a diagnosis of idiopathic anaphylaxis from five UK specialist centres using ISAC arrays. These were divided into three groups: score I identified no new allergen sensitization beyond those known by previous assessment, score II identified new sensitizations which were not thought likely to explain the anaphylaxis and score III identified new sensitizations felt to have a high likelihood of being responsible for the anaphylaxis. A proportion (50%) of score III patients underwent clinical reassessment to substantiate the link to anaphylaxis in this group. The results show that 20% of the arrays were classified as score III with a high likelihood of identifying the cause of the anaphylaxis. A wide range of major allergens were identified, the most frequent being omega‐5‐gliadin and shrimp, together accounting for 45% of the previously unrecognized sensitizations. The ISAC array contributed to the diagnosis in 20% of patients with idiopathic anaphylaxis. It may offer additional information where a careful allergy history and follow‐on testing have not revealed the cause of the anaphylaxis.

A study of cytokine protein secretion, frequencies of cytokine expressing cells and IFN-G gene polymorphisms in normal individuals.

BACKGROUND Cytokines are major regulators of immune responses, and there is evidence that they play a role in allograft rejection. Before embarking on a detailed study of pretransplant cytokine profiles in renal allograft recipients, we wished to investigate variations in cytokine protein secretion, numbers of cytokine expressing T cells, and cytokine gene polymorphisms in normal volunteers. METHODS Twenty normal healthy volunteers were studied. Cytokine protein secretion [interleukin- (IL) 2, IL-4, IL-10, and interferon- (IFN) y] and numbers of cytokine expressing CD3+ T cells (IL-2, IL-4, IL-10, and IFN-gamma) were quantified by means of enzyme-linked immunosorbent assay and two-color flow cytometry respectively. IFN-gamma gene polymorphisms were determined by polymerase chain reaction and autoradio graphy. RESULTS Large interindividual variations in both the quantity of IL-2, IL-4, IL-10, and IFN-gamma cytokine protein secreted and numbers of IL-2 and IFN-gamma expressing T cells were demonstrated. However, numbers of IL-4 and IL-10 expressing cells were found to be below detectable limits by flow cytometry. In the case of IFN-gamma, a bi-modal distribution was seen for the quantity of protein secreted. In addition, correlations were observed between IL-2 protein and frequency of IL-2 expressing T cells. However, no relationship was found between IFN-gamma protein levels, numbers of IFN-gamma expressing cells and IFN-gamma gene polymorphisms. CONCLUSIONS We have demonstrated large differences in both numbers of T helper 1 cytokine expressing cells and the quantity of T helper 1 and T helper 2 cytokine protein secreted between normal individuals. Although the amount of IL-2 protein secreted appeared to be determined by the frequency of IL-2 expressing cells, this was not the case for IFN-gamma.

Cytokine secretion in mixed lymphocyte culture: a prognostic indicator of renal allograft rejection in addition to HLA mismatching

We have previously demonstrated significant inter-individual variations in cytokine protein secretion between normal individuals and patients prior to renal transplantation. In this study, pre-transplant patient vs. donor mixed lymphocyte cultures (MLC) were set up between 57 renal allograft patient/donor pairs, and secretion of cytokine protein (IL-2, IL-4, IL-6, IL-10 and IFN-gamma) into the culture supernatant measured by ELISA. Significant inter-individual variations in protein secretion in MLC were observed for all cytokines studied. Univariate analysis demonstrated that high levels of IFN-gamma and IL-10 in MLC and spontaneous IL-4, together with female donor sex and a high degree of HLA mismatching (especially HLA-DR) were significantly associated with rejection. However, multivariate analysis revealed the greatest risk of rejection (RR = 25.5, P = 0.003) was associated with a combination of high IL-10 secretion in MLC and mismatching for at least four HLA antigens (HLA-A, -B and -DR). It remains to be determined whether cytokine secretion in MLC is linked to cytokine gene polymorphisms. In future, assays for measuring either cytokine secretion or genetic polymorphisms may prove to be useful in aiding donor selection and tailoring immunosuppressive therapy.