Christoph Wülfing

Immune mobilizing monoclonal TCR against cancer (ImmTAC) are cancer therapeutics that activate T cells through recognition of a tumor-associated antigenic MHC/peptide complex. A first-in-class ImmTAC, Tebentafusp, is approved for the treatment of metastatic uveal melanoma. While clinical efficacy is thus established, the cellular mechanisms underpinning ImmTAC action are not fully resolved. Using a recently established experimental strategy to generate suppressed human primary cytotoxic T lymphocytes (CTL), we have investigated an ImmTAC that recognizes a peptide derived from the tumor associated antigen NY-ESO-1 in comparison to direct engagement of a TCR recognizing the same MHC/peptide complex. In response to endogenous antigen presentation, ImmTACs could elicit tumor cell cytolysis by suppressed CTL, but not IFNγ secretion, in a manner dependent on the engager affinity for CD3ε. ImmTACs enhanced the efficient execution of subcellular CTL polarization steps required for effective cytolysis and could trigger calcium signaling. These data establish that ImmTACs activate CTL similarly to direct engagement of a TCR by MHC/peptide and are likely to retain this capability under suppressive conditions such as in the tumor microenvironment.

Introduction: Synthetic T cell receptor (TCR) engagers are cancer therapeutics that activate T cells through recognition of antigens on the tumor cell surface. Immune-mobilizing monoclonal TCRs against cancer (ImmTAC) are composed of the extracellular domains of the alpha and beta chains of an affinity-enhanced TCR recognizing a tumor-associated antigenic peptide-major histocompatibility complex (MHC) complex linked to an scFv fragment of an antibody against CD3ε. A first-in-class ImmTAC, Tebentafusp, is approved for the treatment of metastatic uveal melanoma. Here we asked how ImmTACs activate human primary cytotoxic T lymphocytes (CTL) in response to antigen-presenting tumor target cells. Materials and methods: We used a recently established experimental strategy to generate active and matched suppressed CTL in vitro. Results: ImmTACs could elicit tumor cell cytolysis in response to endogenous antigen presentation by both active and suppressed CTL, but much less so for IFNγ secretion, in a manner dependent on the engager affinity for CD3ε. ImmTACs did so by enhancing the efficient execution of subcellular CTL polarization steps required for effective cytolysis and could trigger calcium signaling. Conclusion: These data establish that ImmTACs are powerful inducers of CTL cytotoxicity and do so with a comparable efficacy and mechanism as direct engagement of a TCR by peptide-MHC. ImmTACs retain this capability under suppressive conditions comparable to those in the tumor microenvironment.

TIM3 is a coregulatory receptor that is highly abundant on multiple immune cell types, including T cells in response to prolonged exposure to antigen, and it marks functionally suppressed cytotoxic T lymphocytes (CTLs) in the tumor microenvironment. TIM3 exhibits inhibitory function in vivo but paradoxically has costimulatory T cell signaling capability in vitro. Here, we found that TIM3 directly inhibited the function of murine and human CTLs in direct interaction with target tumor cell spheroids. TIM3 regulated the ability of suppressed CTLs to polarize their actin cytoskeleton as a required step in cytolysis. Whereas the expression of the proposed TIM3 ligands CEACAM1 and galectin 9 in trans on target tumor cells enhanced TIM3 function, expression of CEACAM1 in cis on CTLs had the opposite effect. TIM3 functioned as an inhibitory receptor on spheroid-suppressed CTLs but not on active CTLs in a two-dimensional tissue culture model. Together, these data suggest that TIM3 enhances T cell function, serving as either a coinhibitory or costimulatory receptor depending on the functional context of the T cell on which it is expressed.

Cytotoxic T lymphocytes (CTL) are key effectors in the anti-tumor immune response. However, their function is commonly suppressed in tumors in the form of exhausted CTL. Understanding molecular mechanisms of suppression and of therapeutics to overcome them is of substantial basic and translational importance yet hindered by limited access to large numbers of exhausted CTL in vitro. Here we use three-dimensional tissue culture to generate primary human CTL with suppressed function. Using a 21-antibody flow cytometry panel and determination of calcium signaling and cell couple maintenance, we show that these cells closely resemble exhausted CTL from tumors. For better understanding of in vitro human primary CTL as key tools in therapeutic development, before and after induction of suppression, we have determined the dependence of CTL function on technicalities of in vitro CTL generation, antigen dose and affinity across two T cell receptors and multiple tumor cell lines. We have investigated morphology and subcellular F-actin distributions of CTL as a key regulators of effector function. Primary human CTL formed cell couples with tumor target cells even in the absence of antigen. Yet, gradual stabilization of such cell couples was associated with increasing CTL effector function. Induction of suppression substantially destabilized CTL tumor cell couples. This comprehensive characterization of the phenotype of in vitro primary human CTL, including a suppressed state, should facilitate their wider use in basic and translational research.

Background: Cytotoxic T lymphocytes (CTL) are key effectors in the antitumor immune response. However, their function is commonly suppressed in tumors in the form of exhausted CTL. Understanding mechanisms of suppression and of therapeutics to overcome them is of substantial basic and translational importance yet hindered by limited access to large numbers of exhausted CTL in vitro. Methods: Here we use three-dimensional tissue culture to generate primary human CTL with suppressed function. Using functional assays, a 21-antibody flow cytometry panel and determination of calcium signaling and CTL tumor cell couple maintenance, we have characterized their phenotype. Results: We show that these cells closely resemble exhausted CTL from tumors. For a better understanding of in vitro human primary CTL as key tools in therapeutic development, before and after induction of suppression, we have determined the dependence of CTL function on methodology of generation, antigen dose, and affinity across two T–cell receptors and multiple tumor cell lines. As a further determination of their phenotype, we have investigated the morphology and subcellular F-actin distributions of CTL as key regulators of effector function. Primary human CTL formed cell couples with tumor target cells even in the absence of antigen. Yet, the gradual stabilization of such cell couples was associated with increasing CTL effector function. Induction of suppression substantially destabilized CTL tumor cell couples. Conclusion: This comprehensive characterization of the phenotype of in vitro primary human CTL, including a suppressed state, should facilitate their use in basic research, the development of CTL-targeting therapeutics and the determination of their mechanism of action.

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to a persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3, and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct, with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

TIM3 is a co-regulatory receptor that is highly expressed on multiple immune cell types, including on T cells after prolonged exposure to antigen. It marks functionally suppressed cytotoxic T lymphocytes (CTL) in the tumor microenvironment. However, it is unresolved whether TIM3 acts directly on suppressed CTL. Moreover, the nature of TIM3 ligands remains controversial. Paradoxically, TIM3 combines inhibitory function in vivo with costimulatory signaling capability in vitro. Here we have investigated TIM3 in the direct interaction of suppressed murine and human CTL with tumor target cell using spheroids. TIM3 directly inhibited the function of such CTL. TIM3 regulated the ability of suppressed CTL to polarize their cytoskeleton as a required step in cytolysis. Expression of CEACAM1 in cis, on the CTL, blocked TIM3 function, expression of CEACAM1 and galectin9 in trans, on the tumor target cells, enhanced TIM3 function. TIM3 only functioned as an inhibitory receptor on the spheroid-suppressed CTL, not on active CTL in a two-dimensional tissue culture model. These data suggest that TIM3 amplifies T cell function, serving as a co-inhibitory or co-stimulatory receptor depending on the functional context of the T cell it is expressed on.

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3 and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and a biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

Linker for activation of T cells (LAT) is a central adaptor protein in proximal T cell activation. A key element of its adaptor function is the efficiency with which LAT interacts with its binding p