Mingli Li

Transduction with the Antioxidant Enzyme Catalase Protects Human T Cells against Oxidative Stress

Patients with diseases characterized by chronic inflammation, caused by infection or cancer, have T cells and NK cells with impaired function. The underlying molecular mechanisms are diverse, but one of the major mediators in this immune suppression is oxidative stress caused by activated monocytes, granulocytes, or myeloid-derived suppressor cells. Reactive oxygen species can seriously hamper the efficacy of active immunotherapy and adoptive transfer of T and NK cells into patients. In this study, we have evaluated whether enhanced expression of the antioxidant enzyme catalase in human T cells can protect them against reactive oxygen species. Human CD4+ and CD8+ T cells retrovirally transduced with the catalase gene had increased intracellular expression and activity of catalase. Catalase transduction made CD4+ T cells less sensitive to H2O2-induced loss-of-function, measured by their cytokine production and ability to expand in vitro following anti-CD3 stimulation. It also enhanced the resistance to oxidative stress-induced cell death after coculture with activated granulocytes, exposure to the oxidized lipid 4-hydroxynonenal, or H2O2. Expression of catalase by CMV-specific CD8+ T cells saved cells from cell death and improved their capacity to recognize CMV peptide-loaded target cells when exposed to H2O2. These findings indicate that catalase-transduced T cells potentially are more efficacious for the immunotherapy of patients with advanced cancer or chronic viral infections.

Identification of a hepatitis C virus–reactive T cell receptor that does not require CD8 for target cell recognition

Hepatitis C virus (HCV) has been reported to elicit B and T cell immunity in infected patients. Despite the presence of antiviral immunity, many patients develop chronic infections leading to cirrhosis, hepatocellular carcinoma, and liver failure that can require transplantation. We have previously described the presence of HLA‐A2–restricted, HCV NS3–reactive cytotoxic T lymphocytes (CTL) in the blood of HLA‐A2− liver transplantation patients that received an HLA‐A2+ liver allograft. These T cells are analogous to the “allospecific” T cells that have been described in hematopoietic stem cell transplantation patients. It has been speculated that allospecific T cells express high‐affinity T cell receptors (TCRs). To determine if our HCV‐reactive T cells expressed TCRs with relatively high affinity for antigen, we identified and cloned a TCR from an allospecific HLA‐A2–restricted, HCV:NS3:1406‐1415–reactive CD8+ T cell clone and expressed this HCV TCR in Jurkat cells. Tetramer binding to HCV TCR–transduced Jurkat cells required CD8 expression, whereas antigen recognition did not. In conclusion, based on the reactivity of the TCR‐transduced Jurkat cells, we have identified a TCR that transfers anti‐HCV reactivity to alternate effectors. These data suggest this high affinity HCV‐specific TCR might have potential new immunotherapic implications. (HEPATOLOGY 2006;43:973–981.)

A Coreceptor-Independent Transgenic Human TCR Mediates Anti-Tumor and Anti-Self Immunity in Mice

Recent advancements in T cell immunotherapy suggest that T cells engineered with high-affinity TCR can offer better tumor regression. However, whether a high-affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope-reactive, CD8-independent, high-affinity TCR isolated from MHC class I-restricted CD4+ T cells obtained from tumor-infiltrating lymphocytes (TIL) of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2–restricted TCR was positively selected on both CD4+ and CD8+ single-positive cells. However, when the TCR transgenic mouse was developed with a HLA-A2 background, the transgenic TCR was primarily expressed by CD3+CD4−CD8− double-negative T cells. TIL 1383I TCR transgenic CD4+, CD8+, and CD4−CD8− T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2+/human tyrosinase TCR double-transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high-affinity TIL 1383I TCR alone in CD3+ T cells is sufficient to control the growth of murine and human melanoma, and the presence or absence of CD4 and CD8 coreceptors had little effect on its functional capacity.

Influence of Human CD8 on Antigen Recognition by T-Cell Receptor-Transduced Cells

The CD8 coreceptor on T cells has two functions. Namely, CD8 acts to stabilize the binding of the T-cell receptor (TCR) to the peptide-MHC complex while localizing p56(lck) (lck) to the TCR/CD3 complex to facilitate early signaling events. Although both functions may be critical for efficient activation of a CTL, little is known about how the structural versus signaling roles of CD8, together with the relative strength of the TCR, influences T-cell function. We have addressed these issues by introducing full-length and truncated versions of the CD8alpha and CD8beta chains into CD8(-) Jurkat cell clones expressing cloned TCRs with known antigen specificity and relative affinities. Using a combination of antigen recognition and tetramer-binding assays, we find that the intracellular lck-binding domain of CD8 is critical for enhanced T-cell activation regardless of the relative strength of the TCR. In contrast, the extracellular domain of CD8 seems to be critical for TCRs with lower affinity but not those with higher affinity. Based on our results, we conclude that there are different requirements for CD8 to enhance T-cell function depending on the strength of its TCR.

TCR gene-modified T cells can efficiently treat established hepatitis C-associated hepatocellular carcinoma tumors

Abstract The success in recent clinical trials using T cell receptor (TCR)-genetically engineered T cells to treat melanoma has encouraged the use of this approach toward other malignancies and viral infections. Although hepatitis C virus (HCV) infection is being treated with a new set of successful direct anti-viral agents, potential for virologic breakthrough or relapse by immune escape variants remains. Additionally, many HCV+ patients have HCV-associated disease, including hepatocellular carcinoma (HCC), which does not respond to these novel drugs. Further exploration of other approaches to address HCV infection and its associated disease are highly warranted. Here, we demonstrate the therapeutic potential of PBL-derived T cells genetically engineered with a high-affinity, HLA-A2-restricted, HCV NS3:1406-1415-reactive TCR. HCV1406 TCR-transduced T cells can recognize naturally processed antigen and elicit CD8-independent recognition of both peptide-loaded targets and HCV+ human HCC cell lines. Furthermore, these cells can mediate regression of established HCV+ HCC in vivo. Our results suggest that HCV TCR-engineered antigen-reactive T cells may be a plausible immunotherapy option to treat HCV-associated malignancies, such as HCC.

Functional cloning of a gp100-reactive T-cell receptor from vitiligo patient skin.

We isolated gp100‐reactive T cells from perilesional skin of a patient with progressive vitiligo with superior reactivity toward melanoma cells compared with tumor‐infiltrating lymphocytes 1520, a melanoma‐derived T‐cell line reactive with the same cognate peptide. After dimer enrichment and limited dilution cloning, amplified cells were subjected to reverse transcription and 5′ RACE to identify the variable TCRα and TCRβ subunit sequences. The full‐length sequence was cloned into a retroviral vector separating both subunits by a P2A slippage sequence and introduced into Jurkat cells and primary T cells. Cytokine secreted by transduced cells in response to cognate peptide and gp100‐expressing targets signifies that we have successfully cloned a gp100‐reactive T‐cell receptor from actively depigmenting skin.

The transfer of genetically engineered lymphocytes in melanoma patients: a Phase I dose escalation study

Meeting abstracts Genetically engineered T cells have broadened the opportunity for use of T cell immunotherapy in cancer patients. The possibility of improving T cell efficacy via introduction of additional genes potentially gives them an advantage over TIL. Studies in adoptive T cell transfer

IL-12 conditioning improves retrovirally mediated transduction efficiency of CD8 + T cells

The ability to genetically modify T cells is a critical component to many immunotherapeutic strategies and research studies. However, the success of these approaches is often limited by transduction efficiency. As retroviral vectors require cell division for integration, transduction efficiency is dependent on the appropriate activation and culture conditions for T cells. Naive CD8+ T cells, which are quiescent, must be first activated to induce cell division to allow genetic modification. To optimize this process, we activated mouse T cells with a panel of different cytokines, including interleukin-2 (IL-2), IL-4, IL-6, IL-7, IL-12, IL-15 and IL-23, known to act on T cells. After activation, cytokines were removed, and activated T cells were retrovirally transduced. We found that IL-12 preconditioning of mouse T cells greatly enhanced transduction efficiency, while preserving function and expansion potential. We also observed a similar transduction-enhancing effect of IL-12 preconditioning on human T cells. These findings provide a simple method to improve the transduction efficiencies of CD8+ T cells.

Therapeutic implications of autoimmune vitiligo T cells

Vitiligo is an autoimmune disease presenting with progressive loss of skin pigmentation. The disease strikes 1% of the world population, generally during teenage years. The progressive loss of melanocytes from depigmenting vitiligo skin is accompanied by cellular infiltrates containing both CD4+ and CD8+ T lymphocytes. Infiltrating cytotoxic T cells with high affinity T cell receptors have likely escaped clonal deletion in the thymus, allowing such T cells to enter the circulation. Through the expression of CLA, these T cells home to the skin where they express type 1-cytokine profiles and mediate melanocyte apoptosis via the granzyme/perforin pathway. T cells found juxtapositionally apposed to remaining melanocytes can be isolated from the skin. Vitiligo T cells have demonstrated reactivity to antigens previously recognized as target antigens for T cells infiltrating melanoma tumors. In a comparison to existing melanoma-derived T cells, vitiligo T cells displayed superior reactivity towards melanoma cells. It is thought that genes encoding the TCRs expressed by vitiligo skin infiltrating T cells can be cloned and expressed in melanoma T cells, thereby generating a pool of circulating T cells with high affinity for their targets that can re-direct the immune response towards the tumor.