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Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/β receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4+ T-cell compartment. Here, we show low IFN-β-mediated inhibition of CD4+ T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4+ T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-β in contrast to adults. The distinct IFN-β-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses.

OBJECTIVE: Recent clinical trials of adoptive immunotherapy showed diminished reactivity of human T cells upon ex vivo manipulation. For a safe and effective clinical application of human T cells, it is necessary to improve ex vivo manipulation procedures and evaluate their impact on in vivo functionality. However, there is no preclinical model for quantitative assessment of in vivo functionality of human T cells. In this study, we investigated the feasibility of using the huPBMC- RAG2(-/-)gammac(-/-) xenogeneic mouse model. As a first example, we compared 3 different ex vivo culture conditions for human T cells. METHODS: RAG2(-/-)gammac(-/-) mice received cultured human T cells that were stimulated via CD3 alone or costimulated via CD28 (CD3/28) and/or human 4-1BB (CD3/28/4-1BB). Engraftment levels and survival of the cells were measured. The dynamics of the human T cell phenotypes were analyzed during culture and in vivo, as well as the mechanism of the xenoresponse. RESULTS: Engraftment potential was improved twofold for costimulation compared to CD3 alone (p < 0.001). Phenotypic analysis showed a strikingly similar pattern of development towards CD4(+) and CD8(+) effector and effector-memory cells, suggesting antigen-driven survival and expansion. All parameters used to analyze different effects on in vivo T-cell functionality, like culture condition, engraftment levels, survival of the cells over time, or xenogeneic graft-vs-host disease were absolutely independent of the distribution of the T cell population in vivo following contact with xeno-antigen. CONCLUSION: The huPBMC-RAG2(-/-)gammac(-/-) xenogeneic transplant model is the most sensitive to date for in vivo functional evaluation of human T cells

Purpose: Effective prevention of graft-versus-host disease (GvHD) is a major challenge to improve the safety of allogeneic stem cell transplantation for leukemia treatment. In murine transplantation models, administration of naturally occurring CD4+CD25+ regulatory T cells (Treg) can prevent GvHD. Toward understanding the role of human Treg in stem cell transplantation, we studied their capacity to modulate T-cell-dependent xenogeneic (x)-GvHD in a new model where X-GvHD is induced in RAG2-/- γc-/- mice by i.v. administration of human peripheral blood mononuclear cells (PBMC). Experimental Design: Human PBMC, depleted of or supplemented with autologous CD25+ Tregs, were administered in mice at different doses. The development of x-GvHD, in vivo expansion of human T cells, and secretion of human cytokines were monitored at weekly intervals. Results: Depletion of CD25+ cells from human PBMC significantly exacerbated x-GvHD and accelerated its lethality. In contrast, coadministration of Treg-enriched CD25+ cell fractions with autologous PBMC significantly reduced the lethality of x-GvHD. Treg administration significantly inhibited the explosive expansion of effector CD4+ and CD8 + T cells. Interestingly, protection from x-GvHD after Treg administration was associated with a significant increase in plasma levels of interleukin-10 and IFN-γ, suggesting the de novo development of TRI cells. Conclusions: These results show, for the first time, the potent in vivo capacity of naturally occurring human Tregs to control GvHD-inducing autologous T cells, and indicate that this xenogeneic in vivo model may provide a suitable platform to further explore the in vivo mechanisms of T-cell down-regulation by naturally occurring human Tregs.

The safe application of new strategies for the treatment of graft-versus-host disease (GVHD) is hampered by the lack of a clinically relevant model for preclinical testing. Current models are based on intraperitoneal transfer of human peripheral blood mononuclear cells (huPBMCs) into NOD-SCID (nonobese diabetic-severe combined immunodeficient)/SCID mice. Intravenous transfer would be preferred but this has always been ineffective. We developed a new model for xenogeneic GVHD (X-GVHD) by intravenous transfer of huPBMCs into RAG2-/- γc-/-mice. Our results show a high human T-cell chimerism of more than 20% (up to 98%) in more than 90% of mice, associated with a consistent development of XGVHD within 14 to 28 days and a total mortality rate of 85% shorter than 2 months. After murine macrophage depletion, engraftment was earlier and equally high with lower doses of huPBMCs. Human macrophages were also absent in these mice. Purified huCD3+ cells showed a similar X-GVH effect with contribution of both CD4 and CD8 phenotypes. Human immunoglobulins and cytokines were produced in diseased mice. One of 30 mice developed chronic X-GVHD with skin histology similar to human GVHD. In conclusion, we present a new model for X-GVHD by intravenous transfer of huPBMCs in RAG2-/- γc-/- mice. Murine and human macrophages do not seem to be necessary for acute X-GVHD in this model. (Blood. 2003;102:2522-2531)

The presence of replication-competent retrovirus (RCR) in retroviral-based gene therapy products poses a potential safety risk for patients. Therefore, RCR testing of clinical gene therapy products and monitoring of patients enrolled in gene therapy trials is required to assure viral safety. The requirement to test ex vivo-transduced cells originates from the presumed amplification of adventitious RCR during the transduction procedure. However, data on the capacity of different cell types to do so are lacking. In this study, we sought to analyze the amplification potential of primary human T lymphocytes after infection with amphotropic MLV-based RCR. The total number of viral particles produced after 1 or 2 weeks was measured by a quantitative 4070A env-specific RT-PCR assay. The fraction of infectious replication-competent viral particles was analyzed in the PG-4 S+L- assay. From this study, we conclude that the total number of viral particles RCR produced by T lymphocytes is 2-4 logs lower than the number produced by NIH-3T3 cells. Surprisingly, less than 1% of the viral particles produced by primary T lymphocytes appeared to be infectious, while nearly all virions produced by NIH-3T3 were. We conclude that primary human T lymphocytes are low producers of MLV-based amphotropic RCR.

Top of pageAbstract Replication competent retrovirus (RCR) may arise through homologous recombination in virus producer cells. A seminal study by Donahue et al. showed that infection with RCR may lead to development of cancer in primates. Therefore, rigorous testing of clinical grade preparations of retroviral vectors is required to exclude the presence of RCR. Many regulatory agencies require RCR-testing of ex vivo transduced cells as well. The latter requirement originates from the presumed amplification of RCR during culture, when cells are infected by very low levels of RCR. In a previous study we challenged the requirement of RCR-testing of ex vivo transduced T cells (Ebeling et al., Gene Ther. 10: 1800, 2003). We found that T cells have a very low capacity to produce RCR. In this study we present the mechanism, which allow T cells to efficiently abort RCR-infection. Individual clones were established from the bulk cultures of RCR-infected T cells. None of the 19 clones tested produced RCR, but all clones had been infected initially, as evidenced by an LTR-specific PCR. Intriguingly, only two clones contained the env gene, suggesting that part of the proviral genome had been deleted. To establish the extent of the deletions, four PCR assays were designed, which together cover the entire proviral genome. Twelve clones were negative in all four PCR assays, suggesting that a very large part of the proviral genome had been deleted. One clone, #27, was positive in the PCR assay, which covers almost the entire env gene. A second clone, #36, was positive in two PCR assays, which together cover half of the pol and the entire env gene. These deletions were consistent with the activity of the recently discovered CEM15/APOBEC3G gene. The CEM15 gene product has been shown to be co-packaged in retro- and lentiviral particles produced by T cells. After infection of target cells with such CEM15+ virions, CEM15 converts C into U residues in the negative cDNA strand during viral replication. As a consequence, this negative strand is degraded. Alternatively, the mutated negative strand may remain (partly) intact and serve as a template for synthesis of the positive strand, resulting in G to A mutations in this strand (e.g. Harris et al., Cell 2003, and Mangeat et al., Nature 2003). Therefore, the provirus in clones #27 and #36 and the LTR of four other clones were sequenced. We found that the 3' end of the PCR product from clone #27 contained numerous G to A substitutions. We therefore conclude that RCR-infection in human T cells is aborted by the CEM15 protein. Additional analysis of the proviral integration sites through molecular cloning only revealed the 3' LTR. The 5' LTR was never found. These data add to our conclusion that the major part of the proviral genome has been deleted from these clones.Together, the results broaden our understanding of the precise mechanism of action of CEM15. In conclusion, we believe that these results will be valuable in evaluation of the safety profile of retro- and lentiviral vectors. In addition, these experiments provide sufficient reason to reconsider the necessity of RCR-testing of ex vivo transduced T cells.

Graft-versus-host disease (GVHD) is the most important determinant for long-term morbidity and mortality in allogeneic stem cell transplantation, affecting approximately 50% of patients. Acute (a)GVHD is a distinctive syndrome of dermatitis, hepatitis and enteritis, while the term chronic (c)GVHD describes a more pleiotropic syndrome. Many clinical manifestations of cGVHD are very similar to those of autoimmune diseases such as lupus erythematosus and scleroderma. In addition, in cGVHD autoantibody formation is a common feature. To investigate the pathogenesis of cGVHD and to study the effect of new treatment modalities, clinically relevant models are of great importance. Recently, we were able to develop a model for GVHD by intravenous transfer of huPBMC in RAG2−/− γc−/− mice (van Rijn et al., Blood102: 2522, 2003). In this study we report on the whole spectrum of histological and immunohistochemical findings in the skin, bowel, tongue, liver, kidney, spleen, bone marrow and lung of 3 mice with acute and 7 with chronic X-GVHD and compare them with those seen in patients with GVHD. All mice showed a ruffled fur and 3 acute and 2 chronic mice had an erythematous skin with loss of hair. Lymphocytic infiltrates and tissue damage were observed in all organs. These lymphocytic infiltrates consisted of T lymphocytes with a surprising predominance of CD4+ cells (75–85%) that was not reflected in the peripheral blood. The kidneys contained variable numbers of lymphocytes, mainly localized in the interstitium. The lungs contained a mild to severe lymphoplasmacellular infiltrate. In three mice there was damage of the bronchial epithelium. 5/7 of the chronic mice showed peribronchial fibrosis. All mice showed a mild to moderate fibrosis in the bone marrow. The bowels contained a very mild lymphocytic infiltrate, but there were no signs of epithelial damage or diarrhoea. Chronic mice demonstrated less lymphocytic infiltrate, more plasma cells and more fibrosis than acute mice. Fibrosis was found in the skin, liver, lungs, spleen and bone marrow. One mouse had severe fibrosis of the liver, spleen, skin and lungs. In later experiments, five more mice sacrificed with chronic GVHD showed a similar histology. Importantly, the livers of the chronic mice showed an auto-immune hepatitis with large amounts of plasma cells. In 5/7 chronic mice even a deposition of human IgA and/or IgG was observed in liver and/or skin. A honeycomb-like pattern was present in the IgA staining, with an IgA-positive surface of the periportal hepatocytes in the liver of 5/7 chronic mice. Ig deposits were also observed in the skin of 4/7 mice. The presence of Ig- and complement-deposits were confirmed in the skin of 3/30 patients cGVHD patients without and 3/3 with blistering diseases. These data provide evidence for a participating role of B cells in the pathogenesis of cGVHD. The beneficial effect of anti-CD20 Ab in cGVHD reported in small scale clinical studies is also consistent with a critical role of B cells in cGVHD-pathology. In summary, the histopathological findings in chronic mice are very similar to the findings in cGVHD. As a consequence, the huPBMC/RAG2−/− γc−/− mouse model provides the unique opportunity to study the contribution of B cells to the pathogenesis of cGVHD.

Replication competent retrovirus (RCR) may arise through homologous recombination in virus producer cells. A seminal study by Donahue (J.Exp.Med. 1992) showed that infection with RCR may lead to development of cancer in primates. Therefore, rigorous testing of clinical grade preparations of retroviral vectors is required to exclude the presence of RCR. Many regulatory agencies require RCR-testing of ex vivo transduced cells as well. The latter requirement originates from the presumed amplification of RCR during culture, if cells are infected by very low levels of RCR. However, in a previous study we found that T lymphocytes have a very low capacity to produce RCR (Ebeling, Gene Ther. 2003). In this study we present the mechanism, which permit T cells to efficiently abort RCR-infection. Individual clones were established from the bulk cultures of RCR-infected T cells. None of the 19 clones tested produced RCR, although all clones had been infected initially, as evidenced by an LTR-specific PCR on genomic DNA. Intriguingly, only two clones contained the env gene, suggesting that part of the proviral genome had been deleted from the remainder of the clones. To establish the extent of the deletions, four PCR assays were designed, which together cover the entire proviral genome. Four clones were negative in all four PCR assays, suggesting that a very large part of the proviral genome had been deleted. One clone, #27, was positive in the PCR assay, which covers almost the entire env gene. A second clone, #36, was positive in two PCR assays, which together cover half of the pol and the entire env gene. Sequence analysis revealed that the 3′ end of the PCR product from clone #27 contained numerous G to A substitutions, which is consistent with the activity of the recently discovered antiviral APOBEC3G gene (e.g. Harris, Cell 2003, and Mangeat, Nature 2003). We therefore conclude that APOBEC3G is involved in abortion of the RCR-infection in human T cells. The extent of the proviral deletions was established through analyis of the retroviral integration sites in six clones via LM-PCR. In clones #26, #37, and #27 3′ end retroviral sequences were preceded by host DNA, definitively confirming deletion of the proviral genome. Intriguingly, in clones #26 and #31, aberrant proviral sequences were found in which part of the provirus was present in an ‘upside down’ orientation. In the case of clone #31, this ‘flipped’ sequence had also changed its position in the provirus. Such flipped sequences were never found during our analysis of > 156 independent integration sites of recombinant retroviruses in hematopoietic stem cells (Laufs, Blood 2003). To our knowledge, deletions or aberrant proviral configurations were never found to be associated with APOBEC3G in more than 30,000 bp of retro- or lentiviral DNA, synthesized in the presence of APOBEC3G. In summary, our work suggests the existence of a novel intracellular defense mechanism, beside APOBEC3G, in primary T lymphocytes, which leads to an efficient deletion of murine retroviral sequences. We believe that these experiments provide sufficient reason to reconsider the necessity of RCR-testing of ex vivo transduced T lymphocytes for gene therapy purposes. If this mechanism is also operational on lentiviral sequences, this may open up novel antiviral strategies for treatment of AIDS.

T cells retrovirally modified to express therapeutic genes encoding cytokines, exogenous TCRs or suicide molecules represent a novel class of immune therapeutics of great potency. However, recent clinical trials using retrovirally-modified T cells have indicated that T cells exhibit a diminished reactivity upon ex vivo manipulation. In addition, virus-specific memory T cells seem to be lost during gene transfer. In a BNML rat model we have shown that the culture procedure is one of the critical parameters. To preserve T cell reactivity, reliable models are required which permit readout of human T cell activity. We recently developed a huPBMC-RAG2−/−γc−/− mouse model for xenogeneic graft-versus-host disease (xGVHD), in which iv injection of 15 x 106 human T cells into RAG2−/−γc−/− mice consistently leads to high level engraftment and lethal xGVHD within 3 weeks in 80% of mice (van Rijn et al, Blood 2003). We have now used this model to analyze in vivo functionality of human T cells following different ex vivo culture procedures. For this, we cultured human T cells for 7 days with either of the two currently available clinically applicable stimulation conditions: 1) via CD3 and 2) via CD3/CD28. In addition, we included CD3/CD28/4-1BB stimulation to explore the effect of extensive costimulation. Mice were injected with escalating doses T cells. HuCD45+ cells in peripheral blood were measured by FACS. Lethal xGVHD occurred at only 6 times (90.106) the dose of fresh cells for CD3-stimulated T cells and 3 times for CD3/28- or CD3/28/4-1BB-stimulated cells. About 20% of surviving mice developed chronic xGVHD, independent of culture method. While lethal xGVHD was always associated with very high levels of engraftment (up to 95%) engraftment levels in chronic mice ranged from 1–75%. To compare the impact of the different culture conditions on in vivo T cell function, we analyzed engraftment potential. The fraction of huCD45+ cells was plotted against the time and the areas under the curves were compared. Based on a total of 68 mice, statistical analysis showed a 2-fold improvement of engraftment potential for C28-costimulated human T cells compared to CD3-stimulated cells (P