J. Ceuppens

Phenotype, cytokine production and cytolytic capacity of fresh (uncultured) tumour-infiltrating T lymphocytes in human renal cell carcinoma

We investigated the phenotype and functional capacities of tumour‐infiltrating lymphocytes (TIL), freshly isolated from primary renal cell carcinoma (RCC) specimens (n = 20). Three‐colour flow cytometry immunophenotyping revealed that RCC TIL consist mainly of CD3+ T cells, with a clear predominance of CD4−CD8+ over CD4+ CD8− T cells, and a marked population of CD4+ CD8+ T cells. Natural killer (NK) cells were also strongly represented (> 25% in 15 of 20 tumour samples), while B cells constituted a minor TIL subset (< 5% in 18 of 20 tumour samples). More importantly, the T and NK cells within the tumour displayed a significantly higher expression of the early activation marker CD69 than their counterparts in adjacent normal renal tissue and in peripheral blood. Expression of CD54 and of HLA‐DR was also elevated on CD3+ TIL, and HLA‐DR expression was further vigorously up‐regulated following ex vivo stimulation with anti‐CD3, all suggesting enhanced immune activity within the tumour microenvironment. CD3+ CD4+ TIL displayed a normal capacity to up‐regulate CD25 expression and to secrete both Th1‐type (IL‐2, tumour necrosis factor‐alpha (TNF‐α) and interferon‐gamma (IFN‐γ)) and Th2‐type (IL‐4, IL‐5 and IL‐10) cytokines upon triggering with anti‐CD3. Furthermore, cytokine production was susceptible to modulation by CD28 costimulation. CD3+ CD8+ TIL, on the other hand, consistently demonstrated a poor up‐regulation of CD25 upon triggering with anti‐CD3, and displayed poor ex vivo cytolytic activity in an anti‐CD3‐redirected 4‐h cytotoxicity assay against murine P815 cells. Collectively, our findings indicate that the CD3+ CD4+ TIL in RCC have normal functional capacities, whereas the proportionally major CD3+ CD8+ TIL are functionally impaired. The relevance of these findings to the in vivo local immune response in RCC is discussed.

Impact of lipoteichoic acid modification on the performance of the probiotic Lactobacillus rhamnosus GG in experimental colitis.

While some probiotic strains might have adjuvant effects in the therapy for inflammatory bowel diseases (IBD), these effects remain controversial and cannot be generalized. In this study, a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having a drastic modification in its lipoteichoic acid (LTA) molecules, was analysed for its effects in an experimental colitis model. Dextran sulphate sodium (DSS) was used to induce either moderate to severe or mild chronic colitis in mice. Mice received either phosphate‐buffered saline (PBS), LGG wild‐type or the dltD mutant via the drinking water. Macroscopic parameters, histological abnormalities, cytokine and Toll‐like receptor (TLR) expression were analysed to assess disease activity. LGG wild‐type did not show efficacy in the different experimental colitis set‐ups. This wild‐type strain even seemed to exacerbate the severity of colitic parameters in the moderate to severe colitis model compared to untreated mice. In contrast, mice treated with the dltD mutant showed an improvement of some colitic parameters compared to LGG wild‐type‐treated mice in both experimental models. In addition, treatment with the dltD mutant correlated with a significant down‐regulation of Toll‐like receptor‐2 expression and of downstream proinflammatory cytokine expression in the colitic mice. These results show that molecular cell surface characteristics of probiotics are crucial when probiotics are considered for use as supporting therapy in IBD.

CD57+/CD28- T cells in untreated hemato-oncological patients are expanded and display a Th1-type cytokine secretion profile, ex vivo cytolytic activity and enhanced tendency to apoptosis

Three-color flow cytometry immunophenotyping revealed significant increases of CD57+ and CD28− cells among both circulating CD4+ and CD8+ T lymphocytes of untreated hemato-oncological patients (n = 54) as compared to healthy donors (n = 55), with CD57 and CD28 expression on the patients’ T cells being largely reciprocal. Marked expansion of CD57+ cells among circulating CD4+ T lymphocytes was frequently detected in patients with chronic leukemia of B cell origin (B-CLL, hairy cell leukemia) but not in patients with chronic myeloid leukemia, suggesting a causal relation with the tumor’s major histocompatibility complex class II expression. Using immunomagnetic separation techniques, we further demonstrate that the patients’ CD57+/CD28− T cells display a typical Th1-type cytokine secretion profile upon anti-CD3 stimulation, with a markedly higher secretion of the Th1-type cytokines IL-2, IFN-γ, and TNF-α than their CD57−/CD28+ counterparts. Cytotoxic activity of circulating CD8+ T lymphocytes, measured ex vivo in an anti-CD3-redirected assay, was almost exclusively exerted by the CD57+/CD28− subset. Moreover, a marked cytotoxic activity was detected within CD4+CD57+ T cells from some B-CLL patients. Finally, the patients’ CD57+/CD28− T cells displayed an increased tendency to apoptosis in culture. Collectively, our results indicate that the expanded CD57+/CD28− T cells in hemato-oncological patients represent differentiated effector cells, similar to their (quantitatively minor) counterpart in healthy donors. The reason for their expansion and their pathophysiologic significance, however, remains unclear.

Interaction of CTLA‐4 (CD152) with CD80 or CD86 inhibits human T‐cell activation

Occupancy of CTLA‐4 (cytotoxic T‐lymphocyte antigen‐4 or CD152) negatively regulates the activation of mouse T lymphocytes, as indicated by the fate of CTLA‐4‐deficient mice, by the impact of anti‐CTLA‐4 monoclonal antibodies (mAbs) on mouse T‐cell activation in vitro and by the impact of CTLA‐4 blockade on the course of experimental tumoral, autoimmune, alloimmune or infectious disease in this animal. The function of human CTLA‐4, however, remains less clear. The expression and function of human CTLA‐4 were further explored. CTLA‐4 was expressed under mitogenic conditions only, its expression being, at least partially, dependent on the secretion of interleukin‐2. Memory T cells expressed CTLA‐4 with faster kinetics than naive T cells. The functional role of human CTLA‐4 was assessed utilizing a panel of four anti‐CTLA‐4 mAbs that blocked the interaction between CTLA‐4 and its ligands. These mAbs, in immobilized form, profoundly inhibited the activation of T cells by immobilized anti‐CD3 mAb in the absence of anti‐CD28 mAb, but co‐stimulated T‐cell activation in the presence of anti‐CD28 mAb. Finally, and importantly, blockade of the interaction of CTLA‐4 with its ligands using soluble anti‐CTLA‐4 mAbs, in intact form or as Fab fragments, enhanced T‐cell activation in several polyclonal or alloantigen‐specific CD80‐ or CD80/CD86‐dependent assays, as measured by cytokine production, cellular proliferation or cytotoxic responses. It is concluded that interaction of CTLA‐4 with its functional ligands, CD80 or CD86, can down‐regulate human T‐cell responses, probably by intracellular signalling events and independent of CD28 occupancy.

Regulation of the IL-10 Production by Human T Cells

Interleukin (IL)‐10, an immunomodulatory cytokine predominantly produced by monocytes/macrophages and T cells, inhibits several functions of dendritic cells (DC), monocytes and T cells including their cytokine production, but it stimulates B cell immunoglobulin (Ig) production and cytotoxic T lymphocyte (CTL) generation. A precise knowledge of the mechanisms that control the IL‐10 production is therefore highly important for understanding the immunoregulation. The IL‐10 production was studied in cultures of freshly isolated human T cells. A rise in intracellular calcium as well as the common γ‐chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL‐10 induction. CD80, CD58, rIL‐12 and rIFN‐α all had efficacious and independent costimulatory activities on the IL‐10 production, while PGE2 was inhibitory. Dependence on autocrine IL‐2 signalling was shown by the effects of anti‐IL‐2 and anti‐IL‐2R monoclonal antibodies (MoAb), but the IL‐10 production proceeded partly IL‐2‐independent when CD80 provided costimulation. Sensitivity to inhibition by CsA was not removed by CD80 or CD58 costimulation and/or by addition of rIL‐12 or rIFN‐α, pointing to the absolute requirement for calcineurin activity. These data reveal important differences in the regulatory pathways between IL‐10 (a cytokine‐inhibitory interleukin) and IL‐2 (a cytokine‐inducing interleukin), which can potentially be exploited therapeutically. The fact that CsA blocks the production of IL‐10, which itself has important immunosuppressive properties, should be taken into account in defining immunosuppressive treatment schedules which include the use of CsA.

Involvement of 4-1BB (CD137)–4-1BBligand interaction in the modulation of CD4+ T cell-mediated inflammatory colitis

4‐1BB ligand (4‐1BBL) expressed on antigen‐presenting cells interacts with 4‐1BB on activated T cells (especially CD8+ cells) and co‐stimulates the latter to secrete cytokines and to proliferate. The role of 4‐1BB−4‐1BBL interaction was studied here in a model of colitis based on naive CD4+ T cell transfer to SCID mice, a disease model in which CD8 cells do not take part. We found that CD4+ T cells from 4‐1BB‐deficient mice, after transfer in SCID mice, proliferated more rapidly compared to wild‐type CD4+ T cells. Mice reconstituted with naive CD4+ T cells from 4‐1BB‐deficient mice developed colitis, however, with a mixed Th1/Th2 response, in contrast to the Th1‐type response in mice reconstituted with wild‐type naive CD4+ T cells. Importantly, this altered cytokine response did not temper colitis severity. Although it has been reported previously that 4‐1BB co‐stimulation may contribute to regulatory T cell functioning, we found that CD4+CD25+ regulatory T cells from 4‐1BB‐deficient mice were perfectly able to prevent naive CD4+ T cell‐induced colitis. In conclusion, our data provide evidence that 4‐1BB−4‐1BBL interaction modulates the effector CD4+ T cell‐driven immune response and cytokine production in experimental colitis without affecting regulatory T cell function.

Inhibition of costimulation allows for repeated systemic administration of adenoviral vector in rhesus monkeys

Immunogenicity of recombinant adenoviral (Ad) vectors severely hampers the clinical development of gene therapy protocols using repeated vector administrations. Inhibition of costimulation by APCs was explored as a strategy to circumvent the immune response against Ad particles. This strategy was tested in rhesus monkeys, treated transiently with chimeric anti-human CD40 and anti-human CD86 antagonist monoclonal antibodies (MAbs) at the time of systemic administration of a recombinant Ad vector. After Ad vector administration in the absence of immunosuppressive treatment, transgene expression in the serum lasted about 3–4 weeks. All control animals developed a strong neutralizing antibody (NAb) response to the Ad particles, which totally prevented efficient administration of a second vector, as shown by the lack of transgene expression. Treatment with anti-CD40 and anti-CD86 chimeric MAbs delayed or blocked the development of a humoral response against Ad and the infiltration of CD8+ lymphocytes into the liver. This resulted in (i) increased persistence of Ad-transduced cells after injection of a first vector encoding a nonimmunogenic transgene, and (ii) the possibility of readministering a second Ad vector with significant efficacy. In both respects, the combined blockade of CD40 and CD86 was more efficient than treatment with anti-CD40 alone. This study shows for the first time in non-human primates that blocking CD40 and CD86 costimulatory molecules represents a promising strategy to inhibit immune responses against an Ad vector injected systemically.

Effects of haptoglobin polymorphisms and deficiency on susceptibility to inflammatory bowel disease and on severity of murine colitis

Background Haptoglobin (Hp) is a haemoglobin-binding protein with immunomodulatory properties. Its gene (16q22) harbours a common polymorphism with two different alleles: Hp1 and Hp2. Genotype Hp22 has been shown to be over-represented in different immune diseases. Results in Crohns disease (CD) are contradictory. Aims To determine whether Hp plays a role in inflammatory bowel disease, both genetically and functionally. Methods 1061 patients with CD, 755 with ulcerative colitis (UC) and 152 with primary sclerosing cholangitis, as well as 452 healthy controls, were genotyped using touch-down PCR. To confirm association results, 464 CD trios and 151 UC trios were genotyped. Serum Hp concentrations were determined in 62 individuals of different genotype. Colitis was induced in mice with dextran sulphate sodium (DSS) and oxazolone (Oxa). Cytokine production was evaluated by mRNA quantification in colonic tissue and ELISA on supernatants of mesenteric lymph node cells. Results Prevalence of Hp2 was higher in CD and UC than in controls. In the confirmatory cohorts, Hp2 was over-transmitted to the affected offspring. Serum Hp concentrations were higher in individuals with genotypes Hp11 and Hp21 than in those with Hp22 (1.38 vs 0.89 g/l). DSS- and Oxa-induced colitis were more severe in Hp-deficient mice than in control mice and accompanied by higher concentrations (although not statistically significantly different) of tissue mRNA for cytokines. Interleukin-17 production was significantly higher in the presence of Hp-deficient serum compared with wild-type serum. Conclusions The Hp gene may play a role in susceptibility to inflammatory bowel disease. Its implication in other immune diseases underscores the common pathways between these diseases. Experimental models of colitis showed that Hp has a protective role in inflammatory colitis, most likely by inhibiting the production of Th1 and Th17 cytokines.

Differences in regulatory pathways identify subgroups of T cell-derived Th2 cytokines

We analysed regulatory mechanisms involved in the production of Th2 cytokines by freshly isolated human T cells. We used an in vitro culture system in which the primary signal was provided by a cross‐linking anti‐CD3 MoAb presented on the Fc receptors of P815 cells. Both CD80 and CD86, expressed on transfected P815 cells, were able to provide efficient costimulation for the production of IL‐4, IL‐5 and IL‐13. IL‐2 was also highly important for induction of all three Th2 cytokines. However, differences between IL‐4 on the one hand and IL‐5 and IL‐13 on the other hand were observed when sensitivity to cyclosporin A (CsA) was studied. CsA (an inhibitor of calcineurin phosphatase activity) strongly inhibited IL‐4 production, but it did either not affect or even increased IL‐5 and IL‐13 production. In accordance with this, CD80 and phorbol myristate acetate (PMA) (without anti‐CD3 or calcium ionophore) were sufficient to induce production of IL‐5 and IL‐13, but not of IL‐4. The subgrouping of Th2 cytokines was further confirmed at another level on the basis of differences in cell sources: IL‐4 was predominantly produced by CD4+ T cells, while IL‐5 and IL‐13 were produced by both CD4+ and CD8+ T cells. Thus, differences in cell sources and in the requirement of the calcium/calcineurin‐signalling pathway allowed us to identify two subgroups (IL‐4 and IL‐5/IL‐13) among human Th2‐type T cell cytokines.

T Helper‐Independent Activation of Human CD8+ Cells: the Role of CD28 Costimulation

The concept that activation of MHC class I‐restricted CD8+ cells entirely depends on help from MHC class II‐restricted CD4+ T cells has recently been supplemented with an alternative model in which CD8+ cells can directly be activated by MHC class I‐expressing professional antigen‐presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28‐mediated costimulation for T helper cell‐independent activation of purified human CD8+ T cells in two different in vitro models. Freshly isolated CD8+ cells could be activated (proliferation, IL‐2 production and cytotoxic activity) by anti‐CD3‐presenting FcγR+ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8+ cells by allogeneic MHC class I on EBV‐transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti‐CD80 and anti‐CD86 MoAb or CTLA‐4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T‐cell activation. CsA and CD80/CD86‐blocking agents were synergistic in completely inhibiting activation of CD8+ cells in the MLR with allogeneic B‐cell lines. This combination also induced non‐responsiveness of CD8+ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8+ T cells, the signal derived from CD80/CD86 is unique in providing CsA‐resistance.