Abdo Konur

Rapid identification and sorting of viable virus-reactive CD4+ and CD8+ T cells based on antigen-triggered CD137 expression

Current methods for the detection and isolation of antigen-specific CD4(+) and CD8(+) T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. Here we show that de novo cell surface expression of the TNF-receptor family member CD137 (4-1BB) identifies recently activated, but not resting, human CD4(+) and CD8(+) memory T cells. Maximum CD137 expression level is uniformly observed in both T-cell subsets at 24h after stimulation with antigen. In experiments with CMV and EBV-reactive T cells, we confirmed the specificity of CD137 expression by co-staining with peptide/HLA tetramers. Substantial proportions of CD137(+) T cells did not produce IFN-gamma, suggesting that CD137 detects a broader repertoire of antigen-specific T cells. Activated CD137(+) T cells could be easily purified by MACS and expanded in vitro thereafter. This CD137-based enrichment method was capable of isolating 2-fold higher numbers of anti-viral CD4(+) and CD8(+) T cells compared to the IFN-gamma secretion assay. In conclusion, antigen-triggered CD137 expression allows the rapid detection and sorting of virus-reactive CD4(+) and CD8(+) T cells. The CD137 assay is most attractive for the simultaneous targeting of anti-viral T helper and effector cells in monitoring studies and adoptive immunotherapy trials.

Excessive CpG 1668 stimulation triggers IL-10 production by cDC that inhibits IFN-α responses by pDC

Upon stimulation with a wide range of concentrations of CpG oligodeoxynucleotide 2216 (CpG 2216), plasmacytoid DC are induced to produce type I IFN (IFN‐α/β). In contrast, CpG 1668 shows a bell‐shaped dose–response correlation, i.e. only intermediate but not high doses of CpG 1668 induce IFN‐α/β. Interestingly, high‐dose CpG 1668 completely inhibited IFN‐α responses induced by CpG 2216. Experiments using supernatant of high‐dose CpG‐1668‐treated cells indicated that secreted inhibitor(s) mediated the IFN‐α shut‐off. Among modulating cytokines, IL‐10 turned out to be one important negative regulator. In line with this, supernatants of IL‐10‐deficient DC cultures stimulated with high‐dose CpG 1668 did not inhibit IFN‐α production. Interestingly, high‐dose CpG 1668 also inhibited IFN‐α responses induced by the DNA‐encoded mouse cytomegalovirus, whereas IFN‐α responses induced by negative‐strand RNA‐encoded vesicular stomatitis virus were only marginally affected. Experiments with DC cultures devoid of TLR9 indicated that TLR9 was critically required to mediate stimulatory and modulatory signals by low and high concentrations of CpG 1668, respectively. Analysis of purified DC subsets showed that conventional DC were the main IL‐10 producers, whereas plasmacytoid DC hardly produced any IL‐10.

Functional TCR Retrieval from Single Antigen-Specific Human T Cells Reveals Multiple Novel Epitopes

Simon, Omokoko, and colleagues developed an integrated approach to retrieve and functionally characterize TCRs from single viral or tumor Ag-reactive T cells and isolated 56 unique Ag-specific TCRs against 39 different epitopes, supporting rational design of T cell-based immunotherapies using this approach. The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-specific CD8+ or CD4+ T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro–transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8+ and CD4+ T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes. Cancer Immunol Res; 2(12); 1230–44. ©2014 AACR.

The Programmed Death (PD)-1/PD-Ligand 1 Pathway Regulates Graft-Versus-Host-Reactive CD8 T Cells After Liver Transplantation

Acute graft‐versus‐host disease (aGVHD) is a life‐threatening complication after solid‐organ transplantation, which is mediated by host‐reactive donor T cells emigrating from the allograft. We report on two liver transplant recipients who developed an almost complete donor chimerism in peripheral blood and bone marrow‐infiltrating T cells during aGVHD. By analyzing these T cells directly ex vivo, we found that they died by apoptosis over time without evidence of rejection by host T cells. The host‐versus‐donor reactivity was selectively impaired, as anti‐third‐party and antiviral T cells were still detectable in the host repertoire. These findings support the acquired donor‐specific allotolerance concept previously established in animal transplantation studies. We also observed that the resolution of aGVHD was not accompanied by an expansion of circulating immunosuppressive CD4/CD25/FoxP3‐positive T cells. In fact, graft‐versus‐host‐reactive T cells were controlled by an alternative negative regulatory pathway, executed by the programmed death (PD)‐1 receptor and its ligand PD‐L1. We found high PD‐1 expression on donor CD4 and CD8 T cells. In addition, blocking PD‐L1 on host‐derived cells significantly enhanced alloreactivity by CD8 T cells in vitro. We suggest the interference with the PD‐1/PD‐L1 pathway as a therapeutic strategy to control graft‐versus‐host‐reactive T cells in allograft recipients.

Liposome-Encapsulated Adjuvants are Potent Inducers of Antigen- Specific T-Cells in Vivo

As shown previously, encapsulation of a peptide derived from tyrosinase-related protein2 (TRP2) into liposomes (artificial virus envelope (AVE) 3) resulted in combination with CpG-oligodeoxynucleotides in the induction of higher numbers of antigen-specific T cells compared to vaccination with free TRP2. Here, we present further data with regard to optimal antigen dose, the relevance of vaccine injection site and on the T cell stimulatory synergism of liposomal adjuvant combinations. Compared to an aqueous solution liposomal TRP2 was more potent in the induction of TRP2-specific T cells at an optimal dose but showed a narrow dose optimum with profoundly impaired T cell responses at higher vaccine doses. Higher T cell numbers were induced when mice were vaccinated into their hint foodpads compared to intradermal vaccination, the site used routinely in murine tumor vaccination models. A synergistic adjuvant effect was observed when CpG-oligodeoxynucleotides were admixed with liposomal monophosphoryl lipid A (MPLA) and the lipopeptide Pam3Cys, respectively. In summary our data demonstrate that liposomes as carriers for peptide-antigen and adjuvant induce a strong antigen-specific T cell response and are superior over vaccine formulations composed of free peptide and adjuvant.