Mark A. Wright

Mechanisms of immune suppression in patients with head and neck cancer : Influence on the immune infiltrate of the cancer

Freshly excised human head and neck cancers (219 primary cancers; 64 metastatic lymph node cancers) were analyzed for the immune inhibitory mediators released from the cancer tissues and the immune infiltrate within the tumor. Significant levels of the immune inhibitory mediators transforming growth factor‐β (TGF‐β), prostaglandin E2 (PGE2) and interleukin‐10 (IL‐10) were released from these cancers. Also released was granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), whose secretion was associated with an intratumoral presence of CD34+ cells. We have previously shown that CD34+ cells within human head and neck cancers are immune inhibitory granulocyte‐macrophage progenitor cells. The presence of TGF‐β, PGE2 and IL‐10 was associated with a reduced content of CD8+ T‐cells within the cancers. The CD4+ cell content appeared to be less affected by these immune inhibitory mediators. Instead, parameters indicative of CD4+ cell function (p55 IL‐2 receptor expression, release of IL‐2 and IFN‐γ) were diminished in cancers that released higher levels of TGF‐β, IL‐10 and GM‐CSF and had a higher CD34+ cell content. Furthermore, metastatic cancers released higher levels of the soluble immune inhibitory mediators and lower levels of IFN‐γ and IL‐2 than did primary cancers, although CD34+ cells were similarly present in both primary and metastatic cancers. Our results show that human head and neck cancers have a multiplicity of non‐mutually exclusive mechanisms of immune suppression that are most prominently associated with reduced CD8+ cell influx and reduced influx and altered function of intratumoral CD4+ cells.

Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocyte-macrophage colony-stimulating factor and contained CD34+ natural suppressor cells

Human head and neck squamous cell carcinomas (HNSCC) that produce high levels of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) have been shown to contain CD34+ natural suppressor cells that inhibit the activity of intratumoral T‐cells. The present study evaluated whether GM‐CSF production and the presence of CD34+ cells within primary HNSCC would translate into increased recurrence, metastasis or cancer‐related death during the 2 years following surgical excision. Freshly excised primary HNSCC of 20 patients that subsequently developed disease, and of 17 patients that remained with no evidence of disease were analyzed for production of GM‐CSF and for CD34+ cell content. The cancers of patients that subsequently developed recurrences or metastatic disease produced almost 4‐fold the levels of GM‐CSF and had approximately 2.5‐fold the number of CD34+ cells as did cancers of patients that remained disease‐free. In a second method of analysis, the prognostic significance of high vs. low GM‐CSF and CD34+ cell values was evaluated. These analyses showed that patients whose cancers produced high GM‐CSF levels or had a high CD34+ cell content had a disproportionately high incidence of recurrence or metastatic disease (94% and 100%, respectively), while the majority of patients whose primary cancers produced low levels of GM‐CSF or had a low CD34+ cell content remained disease‐free (16% and 19%, respectively). Our results indicate that the presence of CD34+ cells in GM‐CSF‐producing HNSCC is associated with a poorer prognosis for the cancer patients and suggest the utility of these parameters as prognostic indicators of outcome. Mechanistically, our results suggest that the presence of immune suppressive CD34+ cells in GM‐CSF‐producing HNSCC leads to increased tumor recurrence or metastasis.Int. J. Cancer 74:69–74.

Increased presence of CD34+ cells in the peripheral blood of head and neck cancer patients and their differentiation into dendritic cells.

Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune deficiencies. In 65% of these patients, there is an increased intra‐tumoral presence of immune‐suppressive CD34+ progenitor cells. The goal of the present study was to determine whether CD34+ cell levels were also increased in the peripheral blood of HNSCC patients and if these immune‐suppressive cells could be differentiated into dendritic cells. Our results showed that CD34+ cell levels are increased in the peripheral blood of HNSCC patients. To assess if these CD34+ cells could differentiate into dendritic cells, they were isolated from the blood of HNSCC patients and cultured for 12 days with various cytokine combinations. Culturing CD34+ cells with stem cell factor (c‐kit ligand) plus granulocyte‐macrophage colony‐stimulating factor resulted in the appearance of a significant proportion of cells expressing phenotypic markers characteristic of dendritic cells. Also, including tumor necrosis factor‐α yielded a significant proportion of cells resembling the bi‐potential precursor cells for dendritic cells and monocytes (CD14+CD1a+), in addition to the dendritic‐like cells. When the differentiation inducer 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3] was added along with the cytokine combinations, the yield of cells having characteristics of dendritic cells was further increased. Cells that were derived from CD34+ cell cultures containing 1,25(OH)2D3 had a more potent capacity to present the recall antigen tetanus toxoid to autologous peripheral blood leukocytes and to stimulate a mixed leukocyte response compared to cultures to which 1,25(OH)2D3 had not been added. Our results show that CD34+ cells, whose frequency is increased in HNSCC patients, can be differentiated into cells that phenotypically and functionally resemble dendritic cells and that 1,25(OH)2D3 accentuates this differentiation. Int. J. Cancer 73:663–669, 1997.

Tumor-derived cytokines induce bone marrow suppressor cells that mediate immunosuppression through transforming growth factor β

SummaryNormal bone marrow cells become immunosuppressive when cultured with supernatants of metastatic Lewis lung carcinoma (LLC-LN7) cells. The suppressorinducing activities in the LLC-LN7 supernatants are interleukin-3 and granulocyte/macrophage-colony-stimulating factor. In the present study, the mechanisms by which these induced suppressor cells (LLCsup-BM) mediate their immunosuppression were investigated. The suppression by LLCsup-BM of splenic concanavalin CA blastogenesis was not dependent on cell contact since immunosuppression occurred regardless of whether the LLCsup-BM were separated from the responder spleen cells by a permeable membrane or if the LLCsup-BM were cocultured with the spleen cells. Culture supernatants of LLCsup-BM also inhibited T cell blastogenesis, being more suppressive than were supernatants of control bone marrow cells, which had been precultured with medium. The suppression by the soluble inhibitors elaborated from the LLCsup-BM was not restricted to the inhibition of T cell function as the supernatants also inhibited the natural killer activity of normal spleen cells. Studies to determine the identity of the suppressive activity produced by the LLCsup-BM showed increased levels of transforming growth factor β (TGFβ) in their supernatants. Immunosuppressive bone marrow and spleen cells obtained from mice bearing metastatic LLC-LN7 tumors also secreted more TGFβ than did the cells obtained from normal mice. When anti-TGFβ antibodies were added to the LLCsup-BM supernatants, the suppressive activity was diminished. These results suggest that the LLCsup-BM mediate at least part of their immunosuppression through production of TGFβ.

Treating tumor-bearing mice with vitamin D3 diminishes tumor-induced myelopoiesis and associated immunosuppression, and reduces tumor metastasis and recurrence

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colonystimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1α,25-dihydroxyvitamin D3 reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D3 reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D3 treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D3 after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D3 treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.

Antibodies to colony-stimulating factors block Lewis lung carcinoma cell stimulation of immune-suppressive bone marrow cells

SummaryProgressive growth of metastatic Lewis lung carcinoma (LLC) tumors results in a concurrent stimulation of myelopoiesis and the appearance of immune-suppressive bone marrow cells. The present study has shown that normal bone marrow cells could be induced to become immune-suppressive by 3 days of culture with supernatants of cloned metastatic LLC-LN7 variant cells. The capacity of the LLC-LN7 supernatants to stimulate the appearance of suppressor cells was directly proportional to the concentration of supernatant used in the bone marrow culture. When adoptively transferred with a LLC-LN7 tumor inoculum, the supernatant-induced suppressor bone marrow cells increased the rate of appearance of palpable tumors and the frequency of tumor establishment. The LLC-LN7 supernatants containing suppressor-cell-inducing activity also had colony-stimulating factor (CSF) activity. The CSF activity produced by the LLC-LN7 cells could be diminished with neutralizing antibodies to either granulocyte/monocyte(GM-) CSF or to interleukin-3 (IL-3). Likewise, the suppressor-inducing activity in the LLC-LN7 supernatants was diminished by pretreatment with anti-GM-CSF or anti-IL-3. The combination of anti-GM-CSF and anti-IL-3 completely neutralized all suppressor-inducing activity produced by the LLC-LN7 cells. These results suggest that the secretion of IL-3 and GM-CSF by LLC-LN7 tumor cells is a mechanism by which the tumors stimulate myelopoiesis and induce normal bone marrow cells to become immune-suppressive. Bone marrow cells that are induced to become immune-suppressive by culture with LLC-LN7 supernatants can, in turn, facilitate the establishment of tumor in vivo.

Vitamin D3 treatment of tumor bearers can stimulate immune competence and reduce tumor growth when treatment coincides with a heightened presence of natural suppressor cells

By secreting granulocyte-macrophage colony-stimulating factor (GM-CSF), Lewis lung carcinoma tumors induce immune suppressive granulocyte-macrophage progenitor cells. Treating mice having established tumors and high levels of suppressor activity with vitamin D3 eliminated suppressor activity, increased anti-tumor immunity, induced an immune stimulatory cell population, and reduced tumor growth. When instead, the vitamin D3 treatment was initiated earlier, when implanted tumors first became detectable and when natural suppressor activity was less prominent, the treatment had no effect. Thus, vitamin D3 treatment can stimulate the immune competence of tumor bearers when treatment is targeted to coincide with a heightened presence of GM-CSF-induced suppressor cells.

Skewed differentiation of bone marrow CD34+ cells of tumor bearers from dendritic toward monocytic cells, and the redirection of differentiation toward dendritic cells by 1α, 25-dihydroxyvitamin D3

Tumor presence is detrimental to the development of antigen-presenting dendritic cells. Since dendritic cells can arise from CD34+ precursor cells, the present study assessed the capacity of bone marrow CD34+ cells from tumor bearers to develop into dendritic cells when cultured in the absence of either tumor cells or their products. Culturing bone marrow CD34+ cells from mice bearing Lewis lung carcinomas yielded a lower number of dendritic cells than arose from CD34+ cells of normal mice. This reduced yield of dendritic cells was associated with a shift to development of monocytic cells and a reduced antigen presenting capability by the cultures. When the CD34+ cell cultures from tumor bearers were supplemented with the differentiation-inducing hormone 1alpha,25-dihydroxyvitamin D3, there was the restoration of dendritic cell development and antigen presenting ability. These results show that CD34+ cells from tumor bearers remain defective in their development into dendritic cells even when cultured outside the tumor environment, but development of dendritic cells can be restored with 1alpha,25-dihydroxyvitamin D3.

Stimulation of immune suppressive CD34+ cells from normal bone marrow by Lewis lung carcinoma tumors

Abstract Progressive growth of metastatic Lewis lung carcinoma (LLC-LN7) tumors is associated with increased levels of bone-marrow-derived CD34+ cells having natural suppressor (NS) activity toward T cells. The present studies determined whether tumor-derived products are responsible for this induction of NS activity. Culturing normal bone marrow cells with LLC-LN7-conditioned medium (LLC-CM) or with recombinant granulocyte/macrophage-colony-stimulating factor (GM-CSF) resulted in the appearance of NS activity. The development of NS activity coincided with a prominent increase in the levels of CD34+ cells. That the CD34+ cells were responsible for the NS activity of the bone marrow cultures containing LLC-CM was shown by the loss of NS activity when CD34+ cells were depleted. The stimulation of CD34+ NS cells by LLC-CM was attributed to tumor production of GM-CSF, since neutralization of GM-CSF within the LLC-CM reduced its capacity to increase CD34+ cell levels. Studies also showed that the induction of CD34+ NS cells by LLC-CM and GM-CSF could be overcome by including in the cultures an inducer of myeloid differentiation, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. These results demonstrate that the mechanism by which the LLC-LN7 tumors stimulate increased levels of CD34+ NS cells from normal bone marrow is by their production of GM-CSF and that this can be blocked with the myeloid differentiation inducer 1,25(OH)2D3.

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells.

Abstract Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D3 eliminated natural suppressor activity. In mice that were first treated with vitamin D3 and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D3/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon γ (IFNγ), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D3/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D3 to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFNγ production and cytolysis by regional lymph node cells of autologous tumor.