Arie Voordouw

Functional CD47/signal regulatory protein alpha (SIRPα) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo

The homeostatic control mechanisms regulating human leukocyte numbers are poorly understood. Here, we assessed the role of phagocytes in this process using human immune system (HIS) BALB/c Rag2−/−IL-2Rγc−/− mice in which human leukocytes are generated from transplanted hematopoietic progenitor cells. Interactions between signal regulatory protein alpha (SIRPα; expressed on phagocytes) and CD47 (expressed on hematopoietic cells) negatively regulate phagocyte activity of macrophages and other phagocytic cells. We previously showed that B cells develop and survive robustly in HIS mice, whereas T and natural killer (NK) cells survive poorly. Because human CD47 does not interact with BALB/c mouse SIRPα, we introduced functional CD47/SIRPα interactions in HIS mice by transducing mouse CD47 into human progenitor cells. Here, we show that this procedure resulted in a dramatic and selective improvement of progenitor cell engraftment and human T- and NK-cell homeostasis in HIS mouse peripheral lymphoid organs. The amount of engrafted human B cells also increased but much less than that of T and NK cells, and total plasma IgM and IgG concentrations increased 68- and 35-fold, respectively. Whereas T cells exhibit an activated/memory phenotype in the absence of functional CD47/SIRPα interactions, human T cells accumulated as CD4+ or CD8+ single-positive, naive, resting T cells in the presence of functional CD47/SIRPα interactions. Thus, in addition to signals mediated by T cell receptor (TCR)/MHC and/or IL/IL receptor interactions, sensing of cell surface CD47 expression by phagocyte SIRPα is a critical determinant of T- and NK-cell homeostasis under steady-state conditions in vivo.

Treatment of low-grade non-Hodgkin's lymphoma with continuous infusion of low-dose recombinant interleukin-2 in combination with the B-cell-specific monoclonal antibody CLB-CD19

Seven patients with low-grade non-Hodgkins lymphoma were treated with a combination of a murine monoclonal antibody directed against the B-cell-specific antigen CD19 (CLB-CD10), given twice weekly, and continuous infusion of low-dose recombinant interleukin-2 (rIL-2). We demonstrated stable serum CLB-CD19 levels throughout the 12 weeks of treatment, and homing of the antibody into the tumour sites. A variable degree of antigenic modulation was noted. Prolonged treatment resulted in a sustained increase in the number of natural killer cells in the circulation with enhanced cytotoxic capacity, including antibody-dependent cellular cytotoxicity. During the first weeks of treatment, T cell activation occurred in the majority of patients. Toxicity was related to the rIL-2 treatment and consisted of transient constitutional symptoms and a flu-like syndrome without organ dysfunction. A partial remission occurred in one patient, and in another patient who was primarily leukaemic a greater than 50% reduction of circulating B cells was noted. An antitumour effect occurred early during treatment and could not be related to rIL-2-induced modulation of natural killer cell or T lymphocyte activation.

Human Telomerase Reverse Transcriptase-Transduced Human Cytotoxic T Cells Suppress the Growth of Human Melanoma in Immunodeficient Mice

Immunotherapy of melanoma by adoptive transfer of tumor-reactive T lymphocytes aims at increasing the number of activated effectors at the tumor site that can mediate tumor regression. The limited life span of human T lymphocytes, however, hampers obtaining sufficient cells for adoptive transfer therapy. We have shown previously that the life span of human T cells can be greatly extended by transduction with the human telomerase reverse transcriptase (hTERT) gene, without altering antigen specificity or effector function. We developed a murine model to evaluate the efficacy of hTERT-transduced human CTLs with antitumor reactivity to eradicate autologous tumor cells in vivo. We transplanted the human melanoma cell line melAKR or melAKR-Flu, transduced with a retrovirus encoding the influenza virus/HLA-A2 epitope, in RAG-2−/− IL-2Rγ −/− double knockout mice. Adoptive transfer of the hTERT-transduced influenza virus-specific CTL clone INFA24 or clone INFA13 inhibited the growth of melAKR-Flu tumors in vivo and not of the parental melAKR melanoma cells. Furthermore, the hTERT-transduced CTL clone INFA13 inhibited tumor growth to the same extent in vivo as the untransduced CTL clone, as determined by in vivo imaging of luciferase gene-transduced melAKR-Flu tumors, indicating that hTERT did not affect the in vivo function of CTL. These results demonstrate that hTERT-transduced human CTLs are capable of mediating antitumor activity in vivo in an antigen-specific manner. hTERT-transduced MART-1-specific CTL clones AKR4D8 and AKR103 inhibited the growth of syngeneic melAKR tumors in vivo. Strikingly, melAKR-Flu cells were equally killed by the MART-1-specific CTL clones and influenza virus-specific CTL clones in vitro, but only influenza-specific CTLs were able to mediate tumor regression in vivo. The influenza-specific CTL clones were found to produce higher levels of IFNγ on tumor cell recognition than the MART-1-specific CTL clones, which may result from the higher functional avidity of the influenza virus-specific CTL clones. Also, melAKR-Flu tumors were growing faster than melAKR tumors, which may have surpassed the relatively modest antitumor effect of the MART-1-specific CTL, as compared with the influenza virus-specific CTL. Taken together, the adoptive transfer model described here shows that hTERT-transduced T cells are functional in vivo, and allows us to evaluate the balance between functional activity of the CTL and tumor growth rate in vivo, which determines the efficacy of CTLs to eradicate tumors in adoptive transfer therapy.

Genetic Control of T-Cell and NK-Cell Protection Against Lethal Sendai Virus Infection

The in vivo importance of class I MHC regulation of the cytotoxic T cell (Tc) response to a natural pathogenic agent of high virulence was studied on the basis of our demonstration of a major difference in the capacity to generate a Sendai virus-specific Tc response between C57BL/6 (B6, H-2b) mice and H-2Kb mutant B6.C-H-2bm1 (bm1) mice. These two mouse strains differ from each other only in three amino acids in the crucial H-2Kb restriction element for this response. Bm1 mice, in contrast to B6 mice, are Tc nonresponders against this virus, but show Sendai-specific T cell proliferation, antibody production, and delayed type hypersensitivity (DTH) reactions, as well as natural killer (NK) cell activity, equal to those of B6 mice. Another H-2b mouse strain, the 129/J, also shows equal Sendai virus-specific Tc, T helper cell (Th), B cell and DTH responses compared to B6, but is virtually deficient in generating an NK cell response. B6, Sendai Tc-deficient bm1, T cell-deficient B6 nu/nu and NK low-responder 129/J mice differ from each other in susceptibility to lethal pneumonia induced by intranasal (i.n.) inoculation of virulent Sendai virus. The lethal dose (LD50) in B6 mice averages 152 TCID50, in bm1 mice 14 TCID50, in B6 nu/nu mice 0.5 TCID50 and in 129/J mice 0.2 TCID50. The importance of Tc is shown by the difference in susceptibility between B6 and bm1 mice and also by the complete protection of B6 nu/nu mice against infection with a lethal virus dose by i.v. injection of a Sendai virus-specific, IL-2 dependent and H-2Kb restricted B6 Tc clone. In vivo protection by this Tc clone is H-2Kb restricted. Apart from Tc, an important role for virus-specific Th cells is evident from the difference in susceptibility between bm1 and B6 nu/nu mice. This conclusion is supported by the demonstration that the mean survival time of B6 nu/nu and bm1 nu/nu mice can be significantly prolonged, in an I-Ab restricted manner, by the injection of in vitro-propagated, Sendai-specific B6 or bm1 Th clones after a lethal dose of Sendai virus, and by the demonstration that inoculation of these Th clones provides help to virus-specific Tc by means of IL-2 production. Strikingly, Th and Tc cooperate in anti-Sendai virus immunity, since permanent survival of lethally infected nu/nu mice is only achieved by inoculation of a mixture of Tc and Th clones or a mixture of a Tc clone and rIL-2. Furthermore, the difference in susceptibility to Sendai virus infection between B6 and bm1 mice provides a unique model for the study of MHC-disease associations. The importance of NK cells is revealed by the high susceptibility of 129/J mice to Sendai virus infection, although all other immune parameters measured appear to be normal.