Immanuel F. Luescher

Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen–specific CD8+ T cell dysfunction in melanoma patients

The paradoxical coexistence of spontaneous tumor antigen–specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1–specific CD8+ T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1–specific CD8+ T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1+ NY-ESO-1–specific CD8+ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3+PD-1+ NY-ESO-1–specific CD8+ T cells are more dysfunctional than Tim-3−PD-1+ and Tim-3−PD-1− NY-ESO-1–specific CD8+ T cells, producing less IFN-γ, TNF, and IL-2. Tim-3–Tim-3L blockade enhanced cytokine production by NY-ESO-1–specific CD8+ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3–Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1–specific CD8+ T cells upon prolonged antigen stimulation and acted in synergy with PD-1–PD-L1 blockade. Collectively, our findings support the use of Tim-3–Tim-3L blockade together with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.

Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide–major histo-compatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen. These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

CD8+ T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

Essential Role of CD8 Palmitoylation in CD8 Coreceptor Function

To investigate the molecular basis that makes heterodimeric CD8αβ a more efficient coreceptor than homodimeric CD8αα, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252–260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8β and that this allows partitioning of CD8αβ, but not of CD8αα, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8β abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3ζ in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8β is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

Cis association of Ly49A with MHC class I restricts natural killer cell inhibition

Natural killer (NK) cell function is negatively regulated by inhibitory receptors interacting with major histocompatibility complex class I molecules expressed on target cells. Here we show that the inhibitory Ly49A NK cell receptor not only binds to its H-2Dd ligand expressed on potential target cells (in trans) but also is constitutively associated with H-2Dd in cis (on the same cell). Cis association and trans interaction occur through the same binding site. Consequently, cis association restricts the number of Ly49A receptors available for binding of H-2Dd on target cells and reduces NK cell inhibition through Ly49A. By lowering the threshold at which NK cell activation exceeds NK cell inhibition, cis interaction allows optimal discrimination of normal and abnormal host cells.

Antigenicity and immunogenicity of Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma.

Summary: Some cancer patients mount spontaneous T‐ and B‐cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T‐cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure‐function properties of the melanocyte/melanoma tumor antigen Melan‐A/MART‐1, the assessment of the T‐cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine‐induced CTL responses to this antigen in patients with metastatic melanoma.

PD-1 Is a Regulator of NY-ESO-1-Specific CD8+ T Cell Expansion in Melanoma Patients

The programmed death 1 (PD-1) receptor is a negative regulator of activated T cells and is up-regulated on exhausted virus-specific CD8+ T cells in chronically infected mice and humans. Programmed death ligand 1 (PD-L1) is expressed by multiple tumors, and its interaction with PD-1 resulted in tumor escape in experimental models. To investigate the role of PD-1 in impairing spontaneous tumor Ag-specific CD8+ T cells in melanoma patients, we have examined the effect of PD-1 expression on ex vivo detectable CD8+ T cells specific to the tumor Ag NY-ESO-1. In contrast to EBV, influenza, or Melan-A/MART-1-specific CD8+ T cells, NY-ESO-1-specific CD8+ T cells up-regulated PD-1 expression. PD-1 up-regulation on spontaneous NY-ESO-1-specific CD8+ T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines. Importantly, blockade of the PD-1/PD-L1 pathway in combination with prolonged Ag stimulation with PD-L1+ APCs or melanoma cells augmented the number of cytokine-producing, proliferating, and total NY-ESO-1-specific CD8+ T cells. Collectively, our findings support the role of PD-1 as a regulator of NY-ESO-1-specific CD8+ T cell expansion in the context of chronic Ag stimulation. They further support the use of PD-1/PD-L1 pathway blockade in cancer patients to partially restore NY-ESO-1-specific CD8+ T cell numbers and functions, increasing the likelihood of tumor regression.

Restoring the Association of the T Cell Receptor with CD8 Reverses Anergy in Human Tumor-Infiltrating Lymphocytes

For several days after antigenic stimulation, human cytolytic T lymphocyte (CTL) clones exhibit a decrease in their effector activity and in their binding to human leukocyte antigen (HLA)-peptide tetramers. We observed that, when in this state, CTLs lose the colocalization of the T cell receptor (TCR) and CD8. Effector function and TCR-CD8 colocalization were restored with galectin disaccharide ligands, suggesting that the binding of TCR to galectin plays a role in the distancing of TCR from CD8. These findings appear to be applicable in vivo, as TCR was observed to be distant from CD8 on human tumor-infiltrating lymphocytes, which were anergic. These lymphocytes recovered effector functions and TCR-CD8 colocalization after ex vivo treatment with galectin disaccharide ligands. The separation of TCR and CD8 molecules could be one major mechanism of anergy in tumors and other chronic stimulation conditions.

T cells maintain an exhausted phenotype after antigen withdrawal and population reexpansion

During chronic infection, pathogen-specific CD8+ T cells upregulate expression of molecules such as the inhibitory surface receptor PD-1, have diminished cytokine production and are thought to undergo terminal differentiation into exhausted cells. Here we found that T cells with memory-like properties were generated during chronic infection. After transfer into naive mice, these cells robustly proliferated and controlled a viral infection. The reexpanded T cell populations continued to have the exhausted phenotype they acquired during the chronic infection. Thus, the cells underwent a form of differentiation that was stably transmitted to daughter cells. We therefore propose that during persistent infection, effector T cells stably differentiate into a state that is optimized to limit viral replication without causing overwhelming immunological pathology.

A peptide encoded by the human MAGE3 gene and presented by HLA-B44 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE3.

The humanMAGE3 gene is expressed in a significant proportion of tumors of various histological types, but is silent in normal adult tissues other than testis and placenta. Antigens encoded byMAGE3 may therefore be useful targets for specific antitumor immunization. Two antigenic peptides encoded by theMAGE3 gene have been reported previously. One is presented to cytolytic T lymphocytes (CTL) by HLA-A1, the other by HLA-A2 molecules. Here we show that MAGE3 also codes for a peptide that is presented to CTL by HLA-1344.MAGE3 peptides containing the HLA-1344 peptide binding motif were synthesized. Peptide MEVDPIGHLY, which showed the strongest binding to HLA-1344, was used to stimulate blood T lymphocytes from normal HLA-1344 donors. CTL clones were obtained that recognized not only HLA-B44 cells sensitized with the peptide, but also HLA-B44 tumor cell lines expressingMAGE3. The proportion of metastatic melanomas expressing theMAGE3/HLA-1344 antigen should amount to approximately 17% in the Caucasian population, since 24% of individuals carry theHLA-B44 allele and 76% of these tumors express MAGE3.