Mark P. Rubinstein

Selective Stimulation of T Cell Subsets with Antibody-Cytokine Immune Complexes

Interleukin-2 (IL-2), which is a growth factor for T lymphocytes, can also sometimes be inhibitory. Thus, the proliferation of CD8+ T cells in vivo is increased after the injection of a monoclonal antibody that is specific for IL-2 (IL-2 mAb), perhaps reflecting the removal of IL-2–dependent CD4+ T regulatory cells (T regs). Instead, we show here that IL-2 mAb augments the proliferation of CD8+ cells in mice simply by increasing the biological activity of preexisting IL-2 through the formation of immune complexes. When coupled with recombinant IL-2, some IL-2/IL-2 mAb complexes cause massive (>100-fold) expansion of CD8+ cells in vivo, whereas others selectively stimulate CD4+ T regs. Thus, different cytokine-antibody complexes can be used to selectively boost or inhibit the immune response.

Macrophage Expression of Hypoxia-Inducible Factor-1α Suppresses T-Cell Function and Promotes Tumor Progression

T cells can inhibit tumor growth, but their function in the tumor microenvironment is often suppressed. Many solid tumors exhibit abundant macrophage infiltration and low oxygen tension, yet how hypoxic conditions may affect innate immune cells and their role in tumor progression is poorly understood. Targeted deletion of the hypoxia-responsive transcription factor hypoxia-inducible factor-1α (HIF-1α) in macrophages in a progressive murine model of breast cancer resulted in reduced tumor growth, although vascular endothelial growth factor-A levels and vascularization were unchanged. Tumor-associated macrophages can suppress tumor-infiltrating T cells by several mechanisms, and we found that hypoxia powerfully augmented macrophage-mediated T-cell suppression in vitro in a manner dependent on macrophage expression of HIF-1α. Our findings link the innate immune hypoxic response to tumor progression through induction of T-cell suppression in the tumor microenvironment.

Antiviral CD4+ memory T cells are IL-15 dependent

Survival and intermittent proliferation of memory CD4+ and CD8+ T cells appear to be controlled by different homeostatic mechanisms. In particular, contact with interleukin (IL)-15 has a decisive influence on memory CD8+ cells, but not memory CD4+ cells. Past studies of memory CD4+ cells have relied heavily on the use of naturally occurring memory phenotype (MP) cells as a surrogate for antigen (Ag)-specific memory cells. However, we show here that MP CD4+ cells contain a prominent subset of rapidly proliferating major histocompatibility complex (MHC) II–dependent cells. In contrast, Ag-specific memory CD4 cells have a slow turnover rate and are MHC II independent. In irradiated hosts, these latter cells ignore IL-15 and expand in response to the elevated levels of IL-7 in the lymphopenic hosts. In contrast, in normal nonlymphopenic hosts where IL-7 levels are low, memory CD4 cells are heavily dependent on IL-15. Significantly, memory CD4+ responsiveness to endogenous IL-15 reflects marked competition from other cells, especially CD8+ and natural killer cells, and increases considerably after removal of these cells. Therefore, under normal physiological conditions, homeostasis of CD8+ and CD4+ memory cells is quite similar and involves IL-15 and IL-7.

Interleukin-15/Interleukin-15R{alpha} Complexes Promote Destruction of Established Tumors by Reviving Tumor-Resident CD8+ T Cells

Tumors often escape immune-mediated destruction by suppressing lymphocyte infiltration or effector function. New approaches are needed that overcome this suppression and thereby augment the tumoricidal capacity of tumor-reactive lymphocytes. The cytokine interleukin-15 (IL-15) promotes proliferation and effector capacity of CD8(+) T cells, natural killer (NK) cells, and NKT cells; however, it has a short half-life and high doses are needed to achieve functional responses in vivo. The biological activity of IL-15 can be dramatically increased by complexing this cytokine to its soluble receptor, IL-15R alpha. Here, we report that in vivo delivery of IL-15/IL-15R alpha complexes triggers rapid and significant regression of established solid tumors in two murine models. Despite a marked expansion of IL-2/IL-15R beta(+) cells in lymphoid organs and peripheral blood following treatment with IL-15/IL-15R alpha complexes, the destruction of solid tumors was orchestrated by tumor-resident rather than newly infiltrating CD8(+) T cells. Our data provide novel insights into the use of IL-15/IL-15R alpha complexes to relieve tumor-resident T cells from functional suppression by the tumor microenvironment and have significant implications for cancer immunotherapy and treatment of chronic infections.

IL-7 and IL-15 differentially regulate CD8+ T-cell subsets during contraction of the immune response.

Although it is known that interleukin-7 (IL-7) and IL-15 influence the survival and turnover of CD8+ T cells, less is known about how these cytokines affect different subsets during the course of the immune response. We find that IL-7 and IL-15 differentially regulate CD8+ T-cell subsets defined by KLRG1 and CD127 expression during the contraction phase of the immune response. The provision of IL-15, or the related cytokine IL-2, during contraction led to the preferential accumulation of KLRG1(hi)CD127(lo) CD8+ T cells, whereas provision of IL-7 instead favored the accumulation of KLRG1(lo)CD127(hi) cells. While IL-7 and IL-15 both induced proliferation of KLRG1(lo) cells, KLRG1(hi) cells exhibited an extraordinarily high level of resistance to cytokine-driven proliferation in vivo despite their dramatic accumulation upon IL-15 administration. These results suggest that IL-15 and IL-2 greatly improve the survival of KLRG1(hi) CD8+ T cells, which are usually destined to perish during contraction, without inducing proliferation. As the availability of IL-15 and IL-2 is enhanced during periods of extended inflammation, our results suggest a mechanism in which a population of cytokine-dependent KLRG1(hi) CD8+ T cells is temporarily retained for improved immunity. Consideration of these findings may aid in the development of immunotherapeutic strategies against infectious disease and cancer.

An intense form of homeostatic proliferation of naive CD8+ cells driven by IL-2

In conditions of T lymphopenia, interleukin (IL) 7 levels rise and, via T cell receptor for antigen–self–major histocompatibility complex (MHC) interaction, induce residual naive T cells to proliferate. This pattern of lymphopenia-induced “homeostatic” proliferation is typically quite slow and causes a gradual increase in total T cell numbers and differentiation into cells with features of memory cells. In contrast, we describe a novel form of homeostatic proliferation that occurs when naive T cells encounter raised levels of IL-2 and IL-15 in vivo. In this situation, CD8+ T cells undergo massive expansion and rapid differentiation into effector cells, thus closely resembling the T cell response to foreign antigens. However, the responses induced by IL-2/IL-15 are not seen in MHC-deficient hosts, implying that the responses are driven by self-ligands. Hence, homeostatic proliferation of naive T cells can be either slow or fast, with the quality of the response to self being dictated by the particular cytokine (IL-7 vs. IL-2/IL-15) concerned. The relevance of the data to the gradual transition of naive T cells into memory-phenotype (MP) cells with age is discussed.

Defining the ability of cyclophosphamide preconditioning to enhance the antigen-specific CD8+ T-cell response to peptide vaccination: creation of a beneficial host microenvironment involving type I IFNs and myeloid cells.

Although cyclophosphamide (CTX) has been clearly shown to enhance active specific and adoptive immunotherapies, the mechanism(s) underlying these beneficial effects have not been clearly defined. To define the impact of CTX preconditioning on the antigen-specific CD8 T-cell response to peptide vaccination, we used an adoptive transfer model based on the OT-1 T-cell receptor transgenic mouse. CTX preconditioning dramatically enhanced the antigen-specific CD8 T-cell response to peptide vaccination. Specifically, CTX significantly enhanced the expansion and function of responding CD8 T cells as demonstrated by flow cytometry and cytokine production. In parallel experiments, we attempted to define the mechanism(s) underlying these beneficial effects of CTX therapy. CTX therapy increased the relative number and activation status of myeloid dendritic cells, and was associated with the induction of significant levels of the inflammatory cytokines interferon-α, monocyte chemoattractant protein-1, and IL-6. Adoptive transfer experiments into type I IFNR−/− and CR3−/− mice confirmed that the beneficial effects of CTX were at least partially dependent on type I interferons and myeloid cells. Adoptive transfer of up to 150×106 naive spleen cells at the time of antigen-specific CD8 T-cell transfer did not abrogate the effects of CTX therapy, suggesting that the creation of a niche in the immune system may not be required. CTX decreased the absolute, but not relative number of CD4+CD25+ Treg cells, consistent with the possibility that regulatory T cells may be targeted by CTX therapy. Of note, combination therapy with CTX and a synthetic TLR3 agonist further enhanced the antigen-specific CD8+ T-cell response. Taken together, our data suggest that CTX modulates specific components of the innate immune system resulting in a beneficial host microenvironment. Specific targeting of these components may enhance the effectiveness of CTX preconditioning for adoptive immunotherapy.

Systemic Administration of IL-15 Augments the Antigen-Specific Primary CD8+ T Cell Response Following Vaccination with Peptide-Pulsed Dendritic Cells

The systemic administration of IL-2 can act as a potent adjuvant for T cell-directed vaccine strategies. However, not only is the administration of IL-2 potentially toxic, but recent evidence suggests that it may also paradoxically limit the duration and magnitude of the cytotoxic T cell response. A recently identified cytokine, IL-15, shares many properties with IL-2 and may provide a preferential means of augmenting T cell-directed vaccine responses. Although well characterized in vitro, there are few data on the ability of IL-15 to augment T cell-mediated responses in vivo. We therefore evaluated the ability of systemic IL-15 to function as a T cell adjuvant in a murine vaccine model. To establish a population of easily identifiable Ag-responsive T cells, naive CD8+ (OT-1) T cells were first adoptively transferred into mice. Vaccination with peptide-pulsed dendritic cells induced a modest expansion of OT-1 T cells. The addition of systemic IL-15 for 7 days following vaccination resulted in a significant increase in the expansion of responding T cells in the PBL, spleen, and lymph nodes. Importantly, the responding T cells were cytotoxic and maintained a Tc1-biased phenotype. We did not observe either enhanced resistance to activation-induced cell death or preferential generation of memory T cells as a result of treatment with IL-15 compared with IL-2. These studies show for the first time that IL-15 is capable of augmenting the primary CD8+ T cell response to vaccination and contribute to the basis for future experiments exploring the clinical role of IL-15.

Efficacy and Mechanism-of-Action of a Novel Superagonist Interleukin-15: Interleukin-15 Receptor αSu/Fc Fusion Complex in Syngeneic Murine Models of Multiple Myeloma

ALT-803, a complex of an interleukin (IL)-15 superagonist mutant and a dimeric IL-15 receptor αSu/Fc fusion protein, was found to exhibit significantly stronger in vivo biologic activity on NK and T cells than IL-15. In this study, we show that a single dose of ALT-803, but not IL-15 alone, eliminated well-established 5T33P and MOPC-315P myeloma cells in the bone marrow of tumor-bearing mice. ALT-803 treatment also significantly prolonged survival of myeloma-bearing mice and provided resistance to rechallenge with the same tumor cells through a CD8(+) T-cell-dependent mechanism. ALT-803 treatment stimulated CD8(+) T cells to secrete large amounts of IFN-γ and promoted rapid expansion of CD8(+)CD44(high) memory T cells in vivo. These memory CD8(+) T cells exhibited ALT-803-mediated upregulation of NKG2D (KLRK1) but not PD-1 (PDCD1) or CD25 (IL2RA) on their cell surfaces. ALT-803-activated CD8(+) memory T cells also exhibited nonspecific cytotoxicity against myeloma and other tumor cells in vitro, whereas IFN-γ had no direct effect on myeloma cell growth. ALT-803 lost its antimyeloma activity in tumor-bearing IFN-γ knockout mice but retained the ability to promote CD8(+)CD44(high) memory T-cell proliferation, indicating that ALT-803-mediated stimulation of CD8(+)CD44(high) memory T cells is IFN-γ-independent. Thus, besides well-known IL-15 biologic functions in host immunity, this study shows that IL-15-based ALT-803 could activate CD8(+)CD44(high) memory T cells to acquire a unique innate-like phenotype and secrete IFN-γ for nonspecific tumor cell killing. This unique immunomodulatory property of ALT-803 strongly supports its clinical development as a novel immunotherapeutic agent against cancer and viral infections.

Surviving the crash: Transitioning from effector to memory CD8+ T cell

One outcome of infection is the formation of long-lived immunological memory, which provides durable protection from symptomatic re-infection. In response to infection or vaccination, T cells undergo dramatic proliferation and differentiate into effector T cells that mediate removal of the pathogen. Following pathogen clearance, the majority of effector cells die, restoring lymphocyte homeostasis. However, a small number of antigen-specific cells survive and seed the memory T cell population. Here, we focus on recent advances in identifying the key proteins and transcription factors that allow a portion of effector CD8(+) T cells to persist after contraction of the immune response, forming a memory cell population programmed for long-term self-renewal and survival. We also examine new findings addressing the role of environmental cues such as cytokines and co-stimulatory molecules in CD8(+) memory T cell formation and how the cell-extrinsic cues influence the molecular players of intracellular pathways important for memory formation.