Out-of-Sequence Signal 3 Paralyzes Primary CD4(+) T-Cell-Dependent Immunity.

Primary T cell activation involves the integration of three distinct signals delivered in sequence: (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion. Strong immunostimulatory events such as immunotherapy or infection induce profound cytokine release causing "bystander" T cell activation, thereby increasing the potential for autoreactivity and need for control. We show that during strong stimulation, a profound suppression of primary CD4(+) T-cell-mediated immune responses ensued and was observed across preclinical models and patients undergoing high-dose interleukin-2 (IL-2) therapy. This suppression targeted naive CD4(+) but not CD8(+) T cells and was mediated through transient suppressor of cytokine signaling-3 (SOCS3) inhibition of the STAT5b transcription factor signaling pathway. These events resulted in complete paralysis of primary CD4(+) T cell activation, affecting memory generation and induction of autoimmunity as well as impaired viral clearance. These data highlight the critical regulation of naive CD4(+) T cells during inflammatory conditions.

Influenza infection results in local expansion of memory CD8(+) T cells with antigen non-specific phenotype and function.

Primary viral infections induce activation of CD8(+) T cells responsible for effective resistance. We sought to characterize the nature of the CD8(+) T cell expansion observed after primary viral infection with influenza. Infection of naive mice with different strains of influenza resulted in the rapid expansion of memory CD8(+) T cells exhibiting a unique bystander phenotype with significant up-regulation of natural killer group 2D (NKG2D), but not CD25, on the CD44(high) CD8(+) T cells, suggesting an antigen non-specific phenotype. We further confirmed the non-specificity of this phenotype on ovalbumin-specific (OT-I) CD8(+) T cells, which are not specific to influenza. These non-specific CD8(+) T cells also displayed increased lytic capabilities and were observed primarily in the lung. Thus, influenza infection was shown to induce a rapid, antigen non-specific memory T cell expansion which is restricted to the specific site of inflammation. In contrast, CD8(+) T cells of a similar phenotype could be observed in other organs following administration of systemic agonistic anti-CD40 and interleukin-2 immunotherapy, demonstrating that bystander expansion in multiple sites is possible depending on whether the nature of activation is either acute or systemic. Finally, intranasal blockade of NKG2D resulted in a significant increase in viral replication early during the course of infection, suggesting that NKG2D is a critical mediator of anti-influenza responses prior to the initiation of adaptive immunity. These results characterize further the local bystander expansion of tissue-resident, memory CD8(+) T cells which, due to their early induction, may play an important NKG2D-mediated, antigen non-specific role during the early stages of viral infection.

Delineation of antigen-specific and antigen-nonspecific CD8(+) memory T-cell responses after cytokine-based cancer immunotherapy.

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8(+) T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8(+) T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing. In contrast, exposure to cytokine alone in vitro or with immunotherapy in vivo did not up-regulate these markers but resulted in expanded memory CD8(+) T cells expressing NKG2D, granzyme B, and possessing broadly lytic capabilities. Blockade of NKG2D in mice also resulted in significantly diminished antitumor effects after immunotherapy. Treatment of TCR-transgenic mice bearing nonantigen expressing tumors with immunotherapy still resulted in significant antitumor effects. Human melanoma tissue biopsies obtained from patients after topically applied immunodulatory treatment resulted in increased numbers of these CD8(+) CD25(-) cells within the tumor site. These findings demonstrate that memory CD8(+) T cells can express differential phenotypes indicative of adaptive or innate effectors based on the nature of the stimuli in a process conserved across species.

Recommendations for the development and validation of flow cytometry-based receptor occupancy assays.

Receptor occupancy measurements demonstrate the binding of a biotherapeutic agent to its extra-cellular target and represent an integral component of the pharmacodynamic (PD) portfolio utilized to advance the development and commercialization of a therapeutic agent. Coupled with traditional pharmacokinetic (PK) assessments derived from serum drug concentration, receptor occupancy data can be used to model PK/PD relationships and validate dose selection decisions throughout the drug development lifecycle. Receptor occupancy assays can be even more challenging to develop than other flow cytometric methods (e.g. surface immunophenotyping). In addition to typical considerations regarding stability of the cell type of interest, stability of the target-bound therapeutic agent and stability of the target receptor must be taken into account. Reagent selection is also challenging as reagents need to be evaluated for the potential to compete with the therapeutic agent and bind with comparable affinity. This article provides technical guidance for the development and validation of cytometry-based receptor occupancy assays.

Role of receptor occupancy assays by flow cytometry in drug development.

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents.

Aging predisposes to acute inflammatory induced pathology after tumor immunotherapy.

Cancer commonly occurs in the elderly and immunotherapy (IT) is being increasingly applied to this population. However, the majority of preclinical mouse tumor models assessing potential efficacy and toxicities of therapeutics use young mice. We assessed the impact of age on responses to systemic immune stimulation. In contrast to young mice, systemic cancer IT regimens or LPS given to aged mice resulted in rapid and lethal toxicities affecting multiple organs correlating with heightened proinflammatory cytokines systemically and within the parenchymal tissues. This inflammatory response and increased morbidity with age was independent of T cells or NK cells. However, prior in vivo depletion of macrophages in aged mice resulted in lesser cytokine levels, increased survival, and decreased liver histopathology. Furthermore, macrophages from aged mice and normal human elderly volunteers displayed heightened TNF and IL-6 production upon in vitro stimulation. Treatment of both TNF knockout mice and in vivo TNF blockade in aged mice resulted in significant increases in survival and lessened pathology. Importantly, TNF blockade in tumor-bearing, aged mice receiving IT displayed significant anti-tumor effects. These data demonstrate the critical role of macrophages in the age-associated hyper-inflammatory cytokine responses to systemic immunostimulation and underscore the importance of performing preclinical assessments in aged mice.

Regulatory and conventional CD4+ T cells show differential effects correlating with PD-1 and B7-H1 expression after immunotherapy.

Recently, our laboratory reported that secondary CD8+ T cell-mediated antitumor responses were impaired following successful initial antitumor responses using various immunotherapeutic approaches. Although immunotherapy stimulated significant increases in CD8+ T cell numbers, the number of CD4+ T cells remained unchanged. The current investigation revealed a marked differential expansion of CD4+ T cell subsets. Successful immunotherapy surprisingly resulted in an expansion of CD4+Foxp3+ regulatory T (Treg) cells concurrent with a reduction of conventional CD4+ T (Tconv) cells, despite the marked antitumor responses. Following immunotherapy, we observed differential up-regulation of PD-1 on the surface of CD4+Foxp3+ Treg cells and CD4+Foxp3- Tconv cells. Interestingly, it was the ligand for PD-1, B7-H1 (PDL-1), that correlated with Tconv cell loss after treatment. Furthermore, IFN-gamma knockout (IFN-gamma-/-) and IFN-gamma receptor knockout (IFN-gammaR-/-) animals lost up-regulation of surface B7-H1 even though PD-1 expression of Tconv cells was not changed, and this correlated with CD4+ Tconv cell increases. These results suggest that subset-specific expansion may contribute to marked shifts in the composition of the T cell compartment, potentially influencing the effectiveness of some immunotherapeutic approaches that rely on IFN-gamma.

The synthetic triterpenoid, CDDO, suppresses alloreactive T cell responses and reduces murine early acute graft-versus-host disease mortality.

Acute graft-versus-host disease (aGVHD) still remains one of the life-threatening complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Immunomodulation of alloreactive donor T cell responses, as well as cytokine secretion is a potential therapeutic approach for the prevention of aGVHD. The synthetic triterpenoid, CDDO (2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid), exhibits potent antitumor activity and has also been shown to mediate anti-inflammatory and immunomodulatory effects. We therefore wanted to assess the effects of CDDO on early lethal aGVHD. In this study, we found that CDDO significantly inhibited in vitro mixed lymphocyte responses and preferentially promoted the apoptosis of proliferating but not resting alloreactive T cells. Using a full major histocompatibility complex (MHC)-disparate murine aGVHD model, we found that the administration of CDDO immediately after transplantation significantly decreased liver pathology as determined by histologic assessment and prolonged survival in mice. Importantly, administration of CDDO did not adversely impair donor myeloid reconstitution as determined by peripheral blood cell count and the extent of donor chimerism. These findings indicate that CDDO has a significant immunomodulatory effects in vitro and on early lethal aGVHD development, particularly affecting the liver, in a murine allo-HSCT model.

Differential effects of proteasome inhibition by bortezomib on murine acute graft-versus-host disease (GVHD): delayed administration of bortezomib results in increased GVHD-dependent gastrointestinal toxicity.

We have recently demonstrated that the proteasome inhibitor, bortezomib, administered immediately following murine allogeneic bone marrow transplantation (BMT) resulted in marked inhibition of acute graft-versus-host disease (GVHD) with retention of graft-versus-tumor effects. We now assessed the effects of delayed bortezomib administration (5 or more days after BMT) on GVHD. Recipient C57BL/6 (H2b) mice were lethally irradiated and given transplants of bone marrow cells and splenocytes from major histocompatibility complex (MHC)-disparate BALB/c (H2d) donors. In marked contrast to the effects of bortezomib on GVHD prevention when administered immediately after BMT, delayed bortezomib administration resulted in significant acceleration of GVHD-dependent morbidity. No toxicity was observed following delayed bortezomib administration in models where donor T cells were not coadministered, indicating that these deleterious effects were critically dependent on GVHD induction. The increase in GVHD susceptibility even occurred when late administration of bortezomib was preceded by early administration. Pathologic assessment revealed that significant increases in gastrointestinal lesions occurred following delayed bortezomib administration during GVHD. This pathology correlated with significant increases of type 1 tumor necrosis factor alpha (TNF-alpha) receptor transcription in gastrointestinal cells and with significant increases of TNF-alpha, interleukin 1beta (IL-1beta), and IL-6 levels in the serum. These results indicate that the differential effects of proteasome inhibition with bortezomib on GVHD are critically dependent on the timing of bortezomib administration.