David J. Morgan

Prevention of Both Direct and Cross-Priming of Antitumor CD8+ T-Cell Responses following Overproduction of Prostaglandin E2 by Tumor Cells In vivo

Defects in antitumor immune responses have been associated with increased release of prostaglandin E(2) (PGE(2)) as a result of overexpression of cyclooxygenase (COX)-2 by tumors. In this report, we examine the effects of PGE(2) on antitumor CD8(+) T-cell responses generated both by cross-presenting dendritic cells and by direct priming by tumor cells. Our data show that PGE(2) inhibits dendritic cell maturation, resulting in the abortive activation of naive CD8(+) T cells, and is dependent on interleukin-10 production by dendritic cells. Interaction of tumor cells with naïve CD8(+) T cells in the presence of PGE(2) in vitro results in the induction of CD8(+) CD28(-) T cells, which fail to proliferate or exhibit effector function. In vivo, overexpression of COX-2 by tumor cells results in a decrease in number of tumor-infiltrating dendritic cells and confers the ability of tumor cells to metastasize to the tumor draining lymph nodes.

CD4+ T cells pass through an effector phase during the process of in vivo tolerance induction.

An important process in the generation of tolerance to peripheral self-Ags is the induction of unresponsiveness in mature specific T cells. Although the end stage of this process, termed anergy, is well defined, the pathway by which naive T cells become anergic remains to be elucidated. Using an in vivo self-tolerance model, we demonstrate that CD4+ T cells pass through a significant effector stage on their way to an anergic state. This stage is characterized by production of effector cytokines, provision of help for CD8+ T cells, and induction of in vivo pathology within organs that express cognate Ag. These results suggest that the initial activation stage in T cell tolerance is similar to that seen in memory induction. They also suggest that autoimmune pathology can result during the natural process of tolerance induction rather than requiring that tolerance be broken.

Characterization of CD8+ T Lymphocytes That Persist After Peripheral Tolerance to a Self Antigen Expressed in the Pancreas

As a result of expression of the influenza hemagglutinin (HA) in the pancreatic islets, the repertoire of HA-specific CD8+ T lymphocytes in InsHA transgenic mice (D2 mice expressing the HA transgene under control of the rat insulin promoter) is comprised of cells that are less responsive to cognate Ag than are HA-specific CD8+ T lymphocytes from conventional mice. Previous studies of tolerance induction involving TCR transgenic T lymphocytes suggested that a variety of different mechanisms can reduce avidity for Ag, including altered cell surface expression of molecules involved in Ag recognition and a deficiency in signaling through the TCR complex. To determine which, if any, of these mechanisms pertain to CD8+ T lymphocytes within a conventional repertoire, HA-specific CD8+ T lymphocytes from B10.D2 mice and B10.D2 InsHA transgenic mice were compared with respect to expression of cell surface molecules, TCR gene utilization, binding of tetrameric KdHA complexes, lytic mechanisms, and diabetogenic potential. No evidence was found for reduced expression of TCR or CD8 by InsHA-derived CTL, nor was there evidence for a defect in triggering lytic activity. However, avidity differences between CD8+ clones correlated with their ability to bind KdHA tetramers. These results argue that most of the KdHA-specific T lymphocytes in InsHA mice are not intrinsically different from KdHA-specific T lymphocytes isolated from conventional animals. They simply express TCRs that are less avid in their binding to KdHA.

Loss of CTL Function among High-Avidity Tumor-Specific CD8+ T Cells following Tumor Infiltration

A major problem in generating effective antitumor CTL responses is that most tumors express self-antigens to which the immune system is rendered unresponsive due to mechanisms of self-tolerance induction. CTL precursors (CTLp) expressing high-affinity T-cell receptors (TCR) are often functionally deleted from the repertoire, leaving a residual repertoire of CTLp having only low-affinity TCR. Furthermore, even when unique antigens are expressed, their presentation by dendritic cells (DC) may predispose to peripheral tolerance induction rather than the establishment of CTL responses that kill tumor cells. In this study, we examined both high-avidity (CL4) and low-avidity (CL1) CD8(+) T-cell responses to a murine renal carcinoma expressing, as a neoantigen, high and low levels of the hemagglutinin (HA) protein from influenza virus A/PR/8 H1N1 (PR8; RencaHA(high) and RencaHA(low)). Our data show that, following encounter with K(d)HA epitopes cross-presented by bone marrow-derived DC, low-avidity CL1 cells become tolerized within tumor-draining lymph nodes (TDLN), and in mice bearing either RencaHA(high) or RencaHA(low) tumors, very few form tumor-infiltrating lymphocytes (TIL). In marked contrast, high-avidity CL4 cells differentiate into effector CTL within the TDLN of mice bearing either RencaHA(high) or RencaHA(low) tumors, and although they form TIL in both tumors, they lose CTL effector function. Critically, these results show that anticancer therapies involving either adoptive transfer of high-avidity tumor-specific CTL populations or targeting of preexisting tumor antigen-specific memory CD8(+) T cells could fail due to the fact that CTL effector function is lost following tumor infiltration.

The Role of Intercellular Adhesion Molecule-1/LFA-1 Interactions in the Generation of Tumor-Specific CD8+ T Cell Responses

The activation of naive CD4+ T cells requires both TCR engagement and a second costimulatory signal mediated by the interaction of CD28 with CD80/CD86 expressed on professional APC. However, the situation for naive CD8+ T cells is less clear. Although evidence indicates that induction of CD8+ T cell responses is also dependent on professional APC, the ability of some tumors, which do not express CD80/CD86, to induce CTL suggests that other pathways of costimulation exist for the activation of CD8+ T cells. We examined the ability of tumor cells expressing different levels of a tumor-specific Ag to directly prime CD8+ T cells. We demonstrate that CD8+ T cells are directly activated by tumor cells in a CD80/CD86-CD28 independent manner. In this system, costimulation requires ICAM-1/LFA-1 interaction. This results in the generation of CTL capable of inhibiting tumor growth in vivo, and maintaining long-term survival.

Ly6C supports preferential homing of central memory CD8(+) T cells into lymph nodes

Ly6C is a murine cell‐surface antigen expressed by plasma cells, subsets of myeloid cells and many T cells, including memory T cells. We previously documented that Ly6C crosslinking induces LFA‐1 clustering on naïve CD8+ T cells. Here, we show that in vitro and in vivo differentiation of naïve CD8+ T cells into central (Tcm) but not effector (Tem) memory T cells enhances Ly6C expression, and its crosslinking induces strong LFA‐1 clustering on Tcm. Blocking Ly6C function inhibits in vivo Tcm homing to LNs as efficiently as blocking L‐selectin but it does not potentiate the inhibition provided by blocking either L‐selectin or LFA‐1 function. Thus, Ly6C, L‐selectin and LFA‐1 all appear to be part of a common homing pathway. In vitro, Ly6C crosslinking enhances Tcm adherence to ICAM‐1 in the presence of CCL21. In summary, Tcm homing involves Ly6C, in addition to L‐selectin and LFA‐1, and appears to potentiate firm adhesion of Tcm to ICAM‐1 in synergy with a chemokine. We propose that Ly6C augments Tcm compartmentalization into LNs during their homing.

Self-tolerance and the composition of T cell repertoire.

T cell recognition of self-major histocompatibility complexpeptide complexes dictates the composition of the T cell receptor repertoire. Research projects in our laboratory deal with the mechanisms that regulate the composition of the repertoire specific for self-antigens and the defects that can result in autoimmunity. Two different types of disease models are under investigation: juvenile (type 1) diabetes and cancer. Both of these diseases are impacted by the presence of anti-self CD8 cells, yet in opposite ways. By understanding the mechanisms of peripheral tolerance and the reasons they fail in autoimmunity, we may learn how to prevent undersirable autoimmunity and how to encourage an autoimmune response when it is needed to eliminate tumor cells.

IFNγ-Dependent Interactions between ICAM-1 and LFA-1 Counteract Prostaglandin E2–Mediated Inhibition of Antitumor CTL Responses

Robust antitumor CTL responses require adhesion of the killer cell to tumor cells. PGE2 suppresses CTL function, but this could be overcome by the IFNγ-induced upregulation of ICAM-1, which drove CTL generation and limited tumor growth in vivo. Tumor-expressed ICAM-1 interaction with LFA-1 on naïve tumor-specific CD8+ T cells not only stabilizes adhesion, but, in the absence of classical B7-mediated costimulation, is also able to provide potent alternative costimulatory signaling resulting in the production of antitumor cytotoxic T lymphocyte (CTL) responses. This study shows that overproduction of prostaglandin (PG) E2 by metastatic murine renal carcinoma (Renca) cells inhibited direct priming of tumor-specific CTL responses in vivo by preventing the IFNγ-dependent upregulation of ICAM-1 that is vital during the initial priming of naïve CD8+ T cells. The addition of exogenous IFNγ during naïve CD8+ T-cell priming abrogated PGE2-mediated suppression, and overexpression of ICAM-1 by tumor cells restored IFNγ production and proliferation among PGE2-treated tumor-specific CD8+ T cells; preventing tumor growth in vivo. These findings suggest that novel anticancer immunotherapies, which increase expression of ICAM-1 on tumor cells, could help alleviate PGE2-mediated immunosuppression of antitumor CTL responses. Cancer Immunol Res; 4(5); 400–11. ©2016 AACR.

In a Transgenic Model of Spontaneous Autoimmune Diabetes, Expression of a Protective Class II MHC Molecule Results in Thymic Deletion of Diabetogenic CD8+ T Cells

H-2d mice expressing both the influenza virus hemagglutinin (HA) as a transgene-encoded protein on pancreatic islet β cells (InsHA), as well as the Clone 4 TCR specific for the dominant H-2Kd-restricted HA epitope, can be protected from the development of spontaneous autoimmune diabetes by expression of the H-2b haplotype. Protection occurs due to the deletion of KdHA-specific CD8+ T cells. This was unexpected as neither the presence of the InsHA transgene nor H-2b, individually, resulted in thymic deletion. Further analyses revealed that thymic deletion required both a hybrid MHC class II molecule, Eβb Eαd, and the Kd molecule presenting the HA epitope, which together synergize to effect deletion of CD4+CD8+ thymocytes. This surprising example of protection from autoimmunity that maps to a class II MHC molecule, yet effects an alteration in the CD8+ T cell repertoire, suggests that selective events in the thymus represent the integrated strength of signal delivered to each cell through recognition of a variety of different MHC-peptide ligands.

Dynamic Control of Self-Specific CD8+ T Cell Responses via a Combination of Signals Mediated by Dendritic Cells

It is acknowledged that T cell interactions with mature dendritic cells (DC) lead to immunity, whereas interactions with immature DC lead to tolerance induction. Using a transgenic murine system, we have examined how DC expressing self-peptides control naive, self-reactive CD8+ T cell responses in vitro and in vivo. We have shown, for the first time, that immature DC can also stimulate productive activation of naive self-specific CD8+ T cells, which results in extensive proliferation, the expression of a highly activated cell surface phenotype, and differentiation into autoimmune CTL. Conversely, mature DC can induce abortive activation of naive CD8+ T cells, which is characterized by low-level proliferation, the expression of a partially activated cell surface phenotype which does not result in autoimmune CTL. Critically, both CD8+ T cell responses are determined by a combination of signals mediated by the DC, and that altering any one of these signals dramatically shifts the balance between autoimmunity and self-tolerance induction. We hypothesize that DC maintain the steady state of self-tolerance among self-specific CD8+ T cells in an active and dynamic manner, licensing productive immune responses against self-tissues only when required.