Hans J. Stauss

Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer

We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1–restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1–restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon expression of wild-type and partially humanized high-affinity T cell antigen receptor (TCR) genes derived from the transgenic mice. These results demonstrate that TCR gene transfer can be used to circumvent self-tolerance of autologous T lymphocytes to universal tumor antigens and thus provide the basis for a TCR gene transfer–based broad-spectrum immunotherapy of malignant disease.

Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.

Isolation of endothelial cells from murine tissue

The isolation and long-term culture of murine endothelial cells (ECs) has often proven a difficult task. In this paper we describe a quick, efficient protocol for the isolation of microvascular endothelial cells from murine tissues. Murine lung or heart are mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained is then incubated with an anti-CD31 antibody, anti-CD105 antibody and with biotinylated isolectin B-4. Pure EC populations are finally obtained by magnetic bead separation using rat anti-mouse Ig- and streptavidin-conjugated microbeads. EC cultures are subsequently expanded and characterised. The surface molecule expression by the primary cultures of murine EC obtained from lung and heart tissue is analysed and compared to that of a murine endothelioma and of primary cultures of murine renal tubular epithelial cells. The phenotype and morphology of these cultures remain stable over 10-15 passages in culture, and no overgrowth of contaminating cells of non-endothelial origin is observed at any stage.

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

Generation of human tumor-reactive cytotoxic T cells against peptides presented by non-self HLA class I molecules.

The cyclin‐D1 protein, which was found to be overexpressed in various human tumors, promotes cell cycle progression from the G1 into the S phase. It is normally expressed at low levels in several tissues and is likely to induce immunological tolerance. We have recently shown in a murine system that T cell tolerance to a widely expressed protein was circumvented by raising cytotoxic T lymphocytes (CTL) from major histocompatability complex mismatched donors. In this study, we tested whether it is possible to raise human allo‐restricted CTL against the cyclin‐D1 protein. The human cell line T2 is deficient in the genes encoding the transporter associated with antigen processing (TAP), resulting in inefficient loading of HLA‐A2 class I molecules with endogenous peptides. Thus, a large number of A2 molecules can bind exogenously supplied synthetic peptides. Peripheral blood mononu clear cells from HLA‐A2‐negative donors were stimulated with T2 cells presenting cyclin‐D1‐derived synthetic peptides. Cloning of bulk cultures revealed that a large proportion of CTL clones were peptide specific. One peptide induced CTL which lysed cyclin‐D1‐expressing breast cancer cells, but not control Epstein‐Barr virus‐transformed B lymphoid cells. The results show that HLA‐A2‐negative donors can be used to isolate tumor‐reactive CTL spe cific for cyclin‐D1 peptides presented by HLA‐A2 class I molecules.

Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines

BACKGROUND & AIMS Virus-specific T cells capable of controlling HBV and eliminating hepatocellular carcinoma (HCC) expressing HBV antigens are deleted or dysfunctional in patients with chronic HBV or HBV-related HCC. The goal of this study was to determine if T cell receptor (TCR) gene transfer can reconstitute HBV-specific T cell immunity in lymphocytes of chronic HBV patients and investigate whether HCC cells with natural HBV-DNA integration can be recognized by genetically modified T cells. METHODS We used vector-mediated gene transfer to introduce HLA-A2-restricted, HBV-specific TCRs into T cells of chronic HBV as well as HBV-related HCC patients. RESULTS The introduced TCRs were expressed on the cell surface, evidenced by Vβ and pentamer staining. TCR transduced T cells produced IFN-γ, TNF-α, IL-2, and lysed HBV infected hepatocyte-like cell lines. Furthermore, HCC cell lines with natural HBV-DNA integration could be recognized by HBV-specific TCR-re-directed T cells. CONCLUSIONS TCR re-directed HBV-specific T cells generated from PBMC of chronic HBV and HBV-related HCC patients were multifunctional and capable of recognizing HBV-infected cells and HCC tumor cells expressing viral antigens from naturally integrated HBV DNA. These genetically modified T cells could be used to reconstitute virus-specific T cell immunity in chronic HBV patients and target tumors in HBV-related HCC.

Adoptive therapy with redirected primary regulatory T cells results in antigen-specific suppression of arthritis.

Regulatory T cells (Tregs) can suppress a wide range of immune cells, making them an ideal candidate for the treatment of autoimmunity. The potential clinical translation of targeted therapy with antigen-specific Tregs is hampered by the difficulties of isolating rare specificities from the natural polyclonal T cell repertoire. Moreover, the initiating antigen is often unknown in autoimmune disease. Here we tested the ability of antigen-specific Tregs generated by retroviral gene transfer to ameliorate arthritis through linked suppression and therefore without cognate recognition of the disease-initiating antigen. We explored two distinct strategies: T cell receptor (TCR) gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs; and co-transfer of FoxP3 and TCR genes served to convert conventional CD4+ T cells into antigen-specific regulators. Following adoptive transfer into recipient mice, the gene-modified T cells engrafted efficiently and retained TCR and FoxP3 expression. Using an established arthritis model, we demonstrate antigen-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, we describe a robust strategy to rapidly generate antigen-specific regulatory T cells capable of highly targeted inhibition of tissue damage in the absence of systemic immune suppression. This opens the possibility to target Tregs to tissue-specific antigens for the treatment of autoimmune tissue damage without the knowledge of the disease-causing autoantigens recognized by pathogenic T cells.

Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette

The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRα- and β-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-γ secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRα- and β-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.

A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection.

Adoptive transfer of antigen-specific CD4+ and CD8+ T cells is one of the most efficient forms of cancer immunotherapy. However, the isolation of antigen-specific CD4+ T cells is limited because only few tumor-associated helper epitopes are identified. Here, we used T cell antigen receptor gene transfer to target CD4+ T cells against an MHC class I-presented epitope of a model tumor antigen. IFN-γ-producing CD4+ T cells were unable to expand in vivo and to provide help for tumor rejection. In contrast, CD4+ T cells producing high levels of IL-2 expanded in vivo, provided help for cytotoxic T lymphocyte-mediated tumor rejection, and developed T cell memory. The data demonstrate in vivo synergy between T cell antigen receptor-transduced CD4+ and CD8+ T cells specific for the same epitope resulting in long-term tumor protection.

Immunotherapy with CTLs restricted by nonself MHC

Abstract Allo-MHC-restricted cytotoxic T lymphocytes can be directed against peptides that are preferentially expressed in malignant cells. Here, Hans Stauss discusses how these cells can mediate a graft-versus-leukaemia reaction without causing graft-versus-host disease when used for immunotherapy in bone-marrow-transplanted leukaemia patients.