David Vermijlen

Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer

The increasing evidence that gammadelta T cells have potent antitumor activity suggests their value in immunotherapy, particularly in areas of unmet need such as metastatic carcinoma. To this end, we initiated a phase I clinical trial in metastatic hormone-refractory prostate cancer to examine the feasibility and consequences of using the gammadelta T-cell agonist zoledronate, either alone or in combination with low-dose interleukin 2 (IL-2), to activate peripheral blood gammadelta cells. Nine patients were enlisted to each arm. Neither treatment showed appreciable toxicity. Most patients were treated with zoledronate + IL-2, but conversely only two treated with zoledronate displayed a significant long-term shift of peripheral gammadelta cells toward an activated effector-memory-like state (T(EM)), producing IFN-gamma and perforin. These patients also maintained serum levels of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), consistent with a parallel microarray analysis showing that TRAIL is produced by gammadelta cells activated via the T-cell receptor and IL-2. Moreover, the numbers of T(EM) gammadelta cells showed a statistically significant correlation with declining prostate-specific antigen levels and objective clinical outcomes that comprised three instances of partial remission and five of stable disease. By contrast, most patients treated only with zoledronate failed to sustain either gammadelta cell numbers or serum TRAIL, and showed progressive clinical deterioration. Thus, zoledronate + IL-2 represents a novel, safe, and feasible approach to induce immunologic and clinical responses in patients with metastatic carcinomas, potentially providing a substantially increased window for specific approaches to be administered. Moreover, gammadelta cell phenotypes and possibly serum TRAIL may constitute novel biomarkers of prognosis upon therapy with zoledronate + IL-2 in metastatic carcinoma.

In vivo manipulation of Vgamma9Vdelta2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients.

The potent anti‐tumour activities of γδ T cells have prompted the development of protocols in which γδ‐agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vγ9Vδ2 T cell agonist, plus low‐dose interleukin (IL)‐2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vγ9Vδ2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vγ9Vδ2 T cell numbers emerged, as seven patients who failed to sustain Vγ9Vδ2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vγ9Vδ2 cell populations showed declining CA15‐3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vγ9Vδ2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low‐dose IL‐2 offers a novel, safe and feasible approach to enhance this in a subset of treatment‐refractory patients with advanced breast cancer.

Human cytomegalovirus elicits fetal γδ T cell responses in utero

The fetus and infant are highly susceptible to viral infections. Several viruses, including human cytomegalovirus (CMV), cause more severe disease in early life compared with later life. It is generally accepted that this is a result of the immaturity of the immune system. γδ T cells are unconventional T cells that can react rapidly upon activation and show major histocompatibility complex–unrestricted activity. We show that upon CMV infection in utero, fetal γδ T cells expand and become differentiated. The expansion was restricted to Vγ9-negative γδ T cells, irrespective of their Vδ chain expression. Differentiated γδ T cells expressed high levels of IFN-γ, transcription factors T-bet and eomes, natural killer receptors, and cytotoxic mediators. CMV infection induced a striking enrichment of a public Vγ8Vδ1-TCR, containing the germline-encoded complementary-determining-region-3 (CDR3) δ1–CALGELGDDKLIF/CDR3γ8–CATWDTTGWFKIF. Public Vγ8Vδ1-TCR–expressing cell clones produced IFN-γ upon coincubation with CMV-infected target cells in a TCR/CD3-dependent manner and showed antiviral activity. Differentiated γδ T cells and public Vγ8Vδ1-TCR were detected as early as after 21 wk of gestation. Our results indicate that functional fetal γδ T cell responses can be generated during development in utero and suggest that this T cell subset could participate in antiviral defense in early life.

Distinct cytokine-driven responses of activated blood gammadelta T cells: insights into unconventional T cell pleiotropy.

Human Vγ9/Vδ2 T cells comprise a small population of peripheral blood T cells that in many infectious diseases respond to the microbial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), expanding to up to 50% of CD3+ cells. This “transitional response,” occurring temporally between the rapid innate and slower adaptive response, is widely viewed as proinflammatory and/or cytolytic. However, increasing evidence that different cytokines drive widely different effector functions in αβ T cells provoked us to apply cDNA microarrays to explore the potential pleiotropy of HMB-PP-activated Vγ9/Vδ2 T cells. The data and accompanying validations show that the related cytokines, IL-2, IL-4, or IL-21, each drive proliferation and comparable CD69 up-regulation but induce distinct effector responses that differ from prototypic αβ T cell responses. For example, the Th1-like response to IL-2 also includes expression of IL-5 and IL-13 that conversely are not induced by IL-4. The data identify specific molecules that may mediate γδ T cell effects. Thus, IL-21 induces a lymphoid-homing phenotype and high, unexpected expression of the follicular B cell-attracting chemokine CXCL13/BCA-1, suggesting a novel follicular B-helper-like T cell that may play a hitherto underappreciated role in humoral immunity early in infection. Such broad plasticity emphasizes the capacity of γδ T cells to influence the nature of the immune response to different challenges and has implications for the ongoing clinical application of cytokines together with Vγ9/Vδ2 TCR agonists.

Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway

Hepatic natural killer (NK) cells are located in the liver sinusoids adherent to the endothelium. Human and rat hepatic NK cells induce cytolysis in tumor cells that are resistant to splenic or blood NK cells. To investigate the mechanism of cell death, we examined the capacity of isolated, pure (90%) rat hepatic NK cells to kill the splenic/blood NK‐resistant mastocytoma cell line P815. Cell death was observed and quantified by fluorescence and transmission electron microscopy, DNA fragmentation, and 51Cr release. RNA and protein expression were determined by real time reverse transcription‐polymerase chain reaction and Western blotting. Compared with splenic NK cells, hepatic NK cells expressed higher levels of perforin and granzyme B and readily induced apoptosis in P815 cells. Although P815 cells succumbed to recombinant Fas ligand (FasL) or isolated perforin/granzyme B, hepatic NK cells used only the granule pathway to kill this target. In addition, hepatic NK cells and sinusoidal endothelial cells strongly expressed the granzyme B inhibitor, protease inhibitor 9 (PI‐9)/serine PI‐6 (SPI‐6), and P815 cells and hepatocytes were negative. Transfection of target cells with this inhibitor resulted in complete resistance to hepatic NK cell‐induced apoptosis. In conclusion, hepatic NK cells kill splenic/blood NK‐resistant/FasL‐sensitive tumor cells exclusively by the perforin/granzyme pathway. Serine protease inhibitor PI‐9/SPI‐6 expression in liver sinusoidal endothelial cells may protect the liver microenvironment from this highly active perforin/granzyme pathway used to kill metastasizing cancer cells.

The integration of conventional and unconventional T cells that characterizes cell-mediated responses.

This review builds on evidence that cell-mediated immune responses to bacteria, viruses, parasites, and tumors are an integration of conventional and unconventional T-cell activities. Whereas conventional T cells provide clonal antigen-specific responses, unconventional T cells profoundly regulate conventional T cells, often suppressing their activities such that immunopathology is limited. By extrapolation, immunopathologies and inflammatory diseases may reflect defects in regulation by unconventional T cells. To explore the function of unconventional T cells, several extensive gene expression analyses have been undertaken. These studies are reviewed in some detail, with emphasis on the mechanisms by which unconventional T cells may exert their regulatory functions. Highlighting the fundamental nature of T-cell integration, we also review emerging data that the development of conventional and unconventional T cells is also highly integrated.

Effect of resuscitative mild hypothermia on glutamate and dopamine release, apoptosis and ischaemic brain damage in the endothelin-1 rat model for focal cerebral ischaemia

The relationship between glutamate and dopamine release, apoptosis and ischaemic damage was studied following induction of transient focal cerebral ischaemia under normothermic (37°C) and postischaemic (resuscitative) mild hypothermic (34°C for 2 h) conditions in sevoflurane anaesthetized male Wistar rats. Focal ischaemia was induced by infusing endothelin‐1 adjacent to the middle cerebral artery. In vivo microdialysis was used to sample glutamate and dopamine from striatum and parietal cortex of the ipsilateral hemisphere. The volume of ischaemic damage and the degree of apoptosis were determined 24 h after the insult. In both striatum and cortex of the normothermic group an initial increase in extracellular glutamate and dopamine levels following endothelin‐1 infusion was observed. Striatal glutamate levels remained enhanced (250% of baseline) throughout the experiment, while the other neurotransmitter levels returned to baseline values. Hypothermia significantly attenuated the endothelin‐1 induced glutamate release in the striatum. It also reduced apoptosis and infarct volume in the cortex. These results indicate that: (i) postischaemic mild hypothermia exerts its neuroprotective effect by inhibiting apoptosis in the ischaemic penumbral region; and (ii) this effect is not associated with an attenuation of glutamate or dopamine release in the cortex.

Interactions between rat colon carcinoma cells and Kupffer cells during the onset of hepatic metastasis

Liver sinusoids harbor populations of 2 important types of immunocompetent cells, Kupffer cells (KCs) and natural killer (NK) cells, which are thought to play an important role in controlling hepatic metastasis in the first 24 hr upon arrival of the tumor cells in the liver. We studied the early interaction of KCs, NK and CC531s colon carcinoma cells in a syngeneic rat model by confocal laser scanning microscopy. Results showed a minority of KCs (19% periportal and 7% pericentral) involved in the interaction with 94% of tumor cells and effecting the phagocytosis of 92% of them. NK cell depletion decreased the phagocytosis of tumor cells by KCs by 33% over a period of 24 hr, leaving 35% of the cancer cells free, as compared to 6% in NK‐positive rats. Surviving cancer cells were primarily located close to the Glisson capsule, suggesting that metastasis would initiate from this region.

Effector Vγ9Vδ2 T cells dominate the human fetal γδ T-cell repertoire

Significance Despite their enormous potential for diversity (in excess of 1015 theoretical receptor specificities), the human γδ T-cell repertoire is dominated by a specific subset expressing the T-cell receptor containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2) known to react to a set of pathogen-derived small molecules (phosphoantigens). Overrepresentation of this restricted set of γδ T cells in adults has been thought to reflect an antigen-specific selection process resulting from postnatal exposure to pathogens. However, we demonstrate here that restricted Vγ9Vδ2 cells with preprogrammed effector function represent the predominant γδ T-cell subset circulating in human fetal blood. This observation suggests that, despite developing in a sterile environment, the human fetal γδ T cell repertoire is enriched for pathogen-reactive T cells well before pathogen exposure. γδ T cells are unconventional T cells recognizing antigens via their γδ T-cell receptor (TCR) in a way that is fundamentally different from conventional αβ T cells. γδ T cells usually are divided into subsets according the type of Vγ and/or Vδ chain they express in their TCR. T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are the predominant γδ T-cell subset in human adult peripheral blood. The current thought is that this predominance is the result of the postnatal expansion of cells expressing particular complementary-determining region 3 (CDR3) in response to encounters with microbes, especially those generating phosphoantigens derived from the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid synthesis. However, here we show that, rather than requiring postnatal microbial exposure, Vγ9Vδ2 T cells are the predominant blood subset in the second-trimester fetus, whereas Vδ1+ and Vδ3+ γδ T cells are present only at low frequencies at this gestational time. Fetal blood Vγ9Vδ2 T cells are phosphoantigen responsive and display very limited diversity in the CDR3 of the Vγ9 chain gene, where a germline-encoded sequence accounts for >50% of all sequences, in association with a prototypic CDR3δ2. Furthermore, these fetal blood Vγ9Vδ2 T cells are functionally preprogrammed (e.g., IFN-γ and granzymes-A/K), with properties of rapidly activatable innatelike T cells. Thus, enrichment for phosphoantigen-responsive effector T cells has occurred within the fetus before postnatal microbial exposure. These various characteristics have been linked in the mouse to the action of selecting elements and would establish a much stronger parallel between human and murine γδ T cells than is usually articulated.

Functionally Mature CD4 and CD8 TCRαβ Cells Are Generated in OP9-DL1 Cultures from Human CD34+ Hematopoietic Cells

Human CD34+ hematopoietic precursor cells cultured on delta-like ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4+ and cytotoxic CD8+ TCRαβ cells is not expected. Phenotypically mature CD27+CD1− TCRγδ as well as TCRαβ cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCRαβ cells were observed. Mature CD8 single-positive cells consisted of two subpopulations: one expressing mainly CD8αβ and one expressing CD8αα dimers. TCRαβ CD8αα and TCRγδ cells both expressed the IL2Rβ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8αβ+ and CD4+ TCRαβ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8αβ+ and CD4+ T cells. These T cells had the characteristics of conventional T cells: CD4+ cells expressed ThPOK, CD40L, and high levels of IL-2 and IL-4; CD8+ cells expressed Eomes, Runx3, and high levels of granzyme, perforin, and IFN-γ. Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8+ cells. Similarly, the presence of dendritic cells was not required for the generation of mature CD4+ or CD8+ T cells. These data suggest that positive selection of these cells is induced by interaction between T precursor cells.