Transient immunological and clinical effectiveness of treating mice bearing premalignant oral lesions with PD-1 antibodies.

A carcinogen-induced premalignant oral lesion model that progresses to oral cancer was used to examine the impact of blocking PD-1 on cytokine expression and on progression of lesions to cancer. The results of this study show increased production of IL-2 and the inflammatory cytokines IL-6, IL-17 and TNF-α by spleen cells of lesion-bearing mice that were treated with PD-1 antibody for 1 week compared to cytokine production by spleen cells of lesion-bearing mice treated with control antibody. Production of IFN-γ increased at 3 weeks of PD-1 antibody treatment, although production of the other Th1 and inflammatory mediators declined. By 5 weeks, levels of these cytokines declined for both control and PD-1 antibody-treated mice. Flow cytometric analysis for IFN-γ-expressing cells showed shifts in CD4+ cells expressing IFN-γ consistent with the changes in cytokine secretion. Whether or not treatment generated reactivity to lesions or HNSCC was determined. Spleen cells from PD-1 antibody-treated mice were stimulated by lysates of premalignant lesion and HNSCC tongue tissues to produce increased levels of Th1 and select inflammatory cytokines early in the course of PD-1 antibody treatment. However, with continued treatment, reactivity to lesion and HNSCC lysates declined. Analysis of clinical response to treatment suggested an early delay in lesion progression but, with continued treatment, lesions in PD-1 antibody-treated mice progressed to the same degree as in control antibody-treated mice. Overall, these results show an early beneficial response to PD-1 antibody treatment, which then fails with continued treatment and lesion progression.

Treatment to sustain a Th17-type phenotype to prevent skewing toward Treg and to limit premalignant lesion progression to cancer.

While immune suppression is a hallmark of head and neck squamous cell carcinoma (HSNCC), the immunological impact of premalignant oral lesions, which often precedes development of HNSCC, is unknown. The present study assessed the changes in splenic and draining lymph node CD4(+) cell populations and their production of select cytokines that occur in mice with carcinogen-induced premalignant oral lesions and the changes that occur as lesions progress to oral cancer. These studies found skewing toward Th1 and Th17-type phenotypes in the spleen and lymph nodes of mice with premalignant oral lesions and a shift to Treg as lesions progress to cancer. Since the role of Th17 cells in the progression from premalignant lesions to cancer is not clear, studies determined the immunological and clinical effect of treating mice bearing premalignant oral lesions with a TGF-ß type 1 receptor inhibitor plus IL-23 as an approach to sustain the Th17 phenotype. These studies showed that the treatment approach not only sustained the Th17 phenotype, but also increased distal spleen cell and regional lymph node cell production of other stimulatory/inflammatory mediators and slowed premalignant lesion progression to cancer.

Local Immune Responsiveness of Mice Bearing Premalignant Oral Lesions to PD-1 Antibody Treatment.

A carcinogen-induced premalignant oral lesion model that progresses to oral cancer was used to examine the immunological impact of a 5-week treatment regimen to block programmed cell death protein 1 (PD-1). PD-1 antibody treatment resulted in concurrent, but transient, increases in interleukin (IL)-2, IFN-γ and IL-17, and delayed increases in IL-6 and IL-10 within the lesion-bearing tongue epithelium. In contrast, cytokine secretion by lymph node cells of PD-1 antibody-treated mice was lower than for mice treated with control antibodies, with the exception of interferon (IFN)-γ, whose secretion increased late in the treatment period. This delayed secretion of IFN-γ coincided with an increase in CD4⁺ lymph node cells expressing IFN-γ. Lymph node cells of PD-1 antibody-treated mice reacted to a challenge with lysates of lesions or cancer by early production of IFN-γ, but this rapidly subsided. There also was increased production IL-17 and tumor necrosis factor (TNF)-α in response to the challenge, but the response was greatest by cells of control lesion-bearing mice. Clinical assessment showed an early but transient, stabilization of disease in mice treated with PD-1 antibody. These results show an early beneficial, but time-limited, response to PD-1 antibody treatment, which then fails with continued lesion progression.

An exploratory approach demonstrating immune skewing and a loss of coordination among cytokines in plasma and saliva of Veterans with combat-related PTSD.

Studies have suggested PTSD to be associated with an inflammatory state, although few studies have examined the balances between stimulatory and inhibitory immune mediators in PTSD. An exploratory approach was taken to assess the immune imbalances between Th1 stimulatory, inflammatory and inhibitory mediators associated with PTSD. This approach focused on a tightly-controlled and relatively homogeneous population of Veterans, all with similar levels of combat exposure in the Afghanistan and Iraq wars, but some testing negative and others testing positive for PTSD. Although the sample size was small (6 controls and 7 with PTSD) and a limitation of this study, the results showed significant imbalances in immune cytokines favoring a Th1 and inflammatory state, with reduced levels of inhibitory cytokines in Veterans with PTSD. This was particularly prominent in the saliva of PTSD subjects compared to in their plasma.

Skewing of immune cell cytokine production by mediators from adipocytes and endothelial cells.

Since adipose tissue is composed of adipocytes as well as other cell types including endothelial cells, this study sought to determine how mediators from adipocytes and from endothelial cells impact on immune cell production of cytokines. A minimalistic design was used in which media conditioned by adipocytes or by endothelial cells were added individually or as a mixture to normal spleen cells. Media from adipocytes or endothelial cells each stimulated spleen cell production of Th1 cytokines, Th2 cytokines, most of the measured inflammatory cytokines, and some chemokines. However, a mixture of media conditioned by adipocytes and by endothelial cells inhibited production of Th1 cytokines and skewed reactivity toward a Th2 and inflammatory phenotype. Adiponectin, but not leptin, was shown to contribute to the skewing of immune responsiveness to endothelial cell-derived mediators.

Premalignant lesions skew spleen cell responses to immune modulation by adipocytes.

Obesity can promote a chronic inflammatory state and is associated with an increased risk for cancer. Since adipocytes can produce mediators that can regulate conventional immune cells, this study sought to determine if the presence of premalignant oral lesions would skew how immune cells respond to adipocyte-derived mediators to create an environment that may be more favorable for their progression toward cancer. While media conditioned by adipocytes stimulated normal spleen cell production of the T helper (Th) type-1 cytokines interleukin (IL)-2, interferon-γ (IFN-γ), IL-12 and granulocyte-monocyte colony-stimulating factor (GM CSF), media from premalignant lesion cells either blocked or had no added affect on the adipocyte-stimulated Th1 cytokine production. In contrast, media conditioned by premalignant lesion cells exacerbated adipocyte-stimulated spleen cell production of the Th2 cytokines IL-10 and IL-13, although it did not further enhance the adipocyte-stimulated spleen cell production of IL-4 and TGF-ß. The premalignant lesion environment also heightened the adipocyte-stimulated spleen cell production of the inflammatory mediators IL 1α, IL-1ß, IL-6 and IL-9, although it did not further increase the adipocyte-stimulated production of tumor necrosis factor-α (TNF-α). IL 17 production was unaffected by the adipocyte-derived mediators, but was synergistically triggered by adding media from premalignant lesion cells. These stimulatory effects on spleen cell production of Th2 and inflammatory mediators were not induced in the absence of media conditioned by adipocytes. In contrast, media conditioned by adipocytes did not stimulate production of predominantly monocyte-derived chemokine C-X-C motif ligand (CXCL)9, chemokine C-C motif ligand (CCL)3 or CCL4, although it stimulated production of CCL2 and the predominantly T cell-derived chemokine CCL5, which was the only chemokine whose production was further increased by media from premalignant lesions. These results suggest that the responsiveness of spleen cells to adipocyte-derived mediators is influenced by mediators from premalignant lesion cells to favor conventional immune cell production of a Th2 and inflammatory cytokines.

Adipocytes as immune regulatory cells.

Obesity is a chronic inflammatory state and adipocytes are capable of contributing to this inflammation by their production of inflammatory mediators. The present study used fibroblast-derived adipocytes and normal spleen cells as a model to determine if adipocytes can also serve as immune regulatory cells by modulating the functions of conventional immune cells. Media conditioned by the adipocytes stimulated release of the Th1-type cytokines IL-2, IFN-γ and GM-CSF from cultures of normal spleen cells. The adipocytes also stimulated spleen cell release of inhibitory cytokines, although to varying degrees. This included IL-10, IL-13 and, to a lesser extent, IL-4. Spleen cell production of the inflammatory cytokines IL-6, TNF-α and IL-9 was stimulated by adipocytes, although production of the Th17-derived cytokine, IL-17, was not stimulated. The adipocyte-conditioned medium did not stimulate production of predominantly monocytes-derived chemokines CXCL9, CCL2, CCL3, CCL4, but stimulated production of the predominantly T-cell-derived chemokine CCL5. In all cases where cytokine/chemokine production from spleen cells was stimulated by adipocytes, it was to a far greater level than was produced by the adipocytes themselves. Studies initiated to determine the identity of the adipocyte-derived mediators showed that the spleen cell modulation could not be attributed to solely adiponectin or leptin. Studies to determine the source of some of the cytokines whose production was stimulated by adipocytes showed that expression of the inflammatory cytokine IL-6 was not increased in either CD4(+) or CD8(+) T-cell. When the splenic T-cells were examined for IFN-γ, the adipocyte stimulation of IFN-γ was within CD8(+) T-cells, not CD4(+) T-cells. These studies show that adipocytes may be able to serve as immune regulatory cells to stimulate conventional immune cells to release a spectrum of immune mediators.