Fractionated Radiation Severely Reduces the Number of CD8+ T Cells and Mature Antigen Presenting Cells Within Lung Tumors.

PURPOSE: The combination of standard-of-care radiation therapy (RT) with immunotherapy is moving to the mainstream of non-small cell lung cancer treatment. Multiple preclinical studies reported on the CD8+ T cell stimulating properties of RT, resulting in abscopal therapeutic effects. A literature search demonstrates that most preclinical lung cancer studies applied subcutaneous lung tumor models. Hence, in-depth immunologic evaluation of clinically relevant RT in orthotopic lung cancer models is lacking. METHODS AND MATERIALS: We studied the therapeutic and immunologic effects of low-dose fractionated RT on lungs from C57BL/6 mice, challenged 2 weeks before with firefly luciferase expressing Lewis lung carcinoma cells via the tail vein. Low-dose fractionation was represented by 4 consecutive daily fractions of image guided RT at 3.2 Gy. RESULTS: We showed reduced lung tumor growth upon irradiation using in vivo bioluminescence imaging and immunohistochemistry. Moreover, significant immunologic RT-induced changes were observed in irradiated lungs and in the periphery (spleen and blood). First, a significant decrease in the number of CD8+ T cells and trends toward more CD4+ and regulatory T cells were seen after RT in all evaluated tissues. Notably, only in the periphery did the remaining CD8+ T cells show a more activated phenotype. In addition, a significant expansion of neutrophils and monocytes was observed upon RT locally and systemically. Locally, RT increased the influx of tumor-associated macrophages and conventional type 2 dendritic cells, whereas the alveolar macrophages and conventional type 1 DCs dramatically decreased. Functionally, these antigen-presenting cells severely reduced their CD86 expression, suggesting a reduced capacity to induce potent immunity. CONCLUSIONS: Our results imply that low-dose fractionated RT of tumor-bearing lung tissue shifts the immune cell balance toward an immature myeloid cell dominating profile. These data argue for myeloid cell repolarizing strategies to enhance the abscopal effects in patients with non-small cell lung cancer treated with fractionated RT.

IL-7 During Antigenic Stimulation Using Allogeneic Dendritic Cells Promotes Expansion of CD45RA-CD62L+CD4+ Invariant NKT Cells With Th-2 Biased Cytokine Production Profile.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes cells that recognize glycolipid antigens associated with CD1d, non-classical antigen presenting proteins. They can drive either pro-inflammatory (Th-1) or anti-inflammatory (Th-2) immune microenvironment through the production of both Th-1 and Th-2 type cytokines upon activation, thus play a vital role in cancer, infection, and autoimmune diseases. Adoptive cell therapy using ex vivo expanded iNKT cells is a promising approach to enhance anti-tumor immunity or immunosuppression. However, overcoming phenotypic and functional heterogeneity and promoting in vivo persistency of iNKT cells remains to be a challenge. Here, we compared various methods for ex vivo expansion of human iNKT cells and assessed the quality of expansion, phenotype, and cytokine production profile of expanded iNKT cells. While a direct stimulation of iNKT cells in peripheral blood mononuclear cells with agonist glycolipid led to the expansion of iNKT cells in varying degrees, stimulation of enriched iNKT cells by irradiated autologous peripheral blood mononuclear cells or allogeneic dendritic cells resulted in consistent expansion of highly pure iNKT cells. Interestingly, the mode of antigenic stimulation influenced the dominant subtype of expanded iNKT cells. Further, we evaluated whether additional IL-7 or IL-15 during antigenic stimulation with allogeneic dendritic cells can improve the phenotypic heterogeneity and modify cytokine production profile of iNKT cells expanded from 18 consecutive donors. The presence of IL-7 or IL-15 during antigenic stimulation did not affect the fold of expansion or purity of expanded iNKT cells. However, IL-7, but not IL-15, led to a better expansion of CD4+ iNKT cells, enhanced Th-2 type cytokine production of CD4+ iNKT cells, and maintained the expansion of central memory (CD45RA-CD62L+) CD4+ iNKT cells. Our results suggest the addition of IL-7 during antigenic stimulation with allogeneic dendritic cells can promote the expansion of CD62L+Th-2+CD4+ human iNKT cells that can be used as novel immunotherapeutic to control excessive inflammation to treat various autoimmune diseases.

Modulation of immune response to induced-arthritis by low-level laser therapy.

As low-level laser therapy immune cells responses are not always clarified, this study aimed to evaluate cytokines and immune cells profile after low-level laser therapy (LLLT) on arthritis-induced model. Arthritis was induced in C57BL/6 mice divided into five groups: euthanized 5 hours after inflammation induction; untreated; dexamethasone treated; LLLT at 3 Jcm-2 ; LLLT at 30 Jcm-2 . Cytokine measurements by enzyme-linked immunosorbent assay and mRNA cytokine relative levels by real-time quantitative polymerase chain reaction were performed with arthritic ankle (IL-1ß, IL-6, TNF-α, IL-10 and TGF-ß). Macrophages, dendritic cells, natural killer cells, lymphocytes CD4+ , CD8+ , Treg and costimulatory proteins were quantified in proximal lymph node by flow cytometry. Data showed decrease in all cytokine levels after LLLT and alteration in mRNA relative levels, depending on the energy density used. LLLT was able to increase of immune cell populations analyzed in the lymph node as well as costimulatory proteins expression on macrophages and dendritic cells. Treg TCD4+ and TCD8+ population enrichment were observed in LLLT at 3 and 30 Jcm-2 groups, respectively. Furthermore, Treg TCD8+ cells expressing higher levels of CD25 were observed at LLLT at 30 Jcm-2 group. Our results indicate that LLLT could change the inflammatory course of arthritis, tending to accelerate its resolution through immune cells photobiostimulation.

Effects of experimental desynchronosis on the organs of immune system in WAG and ISIAH rats.

We studied the influence of abnormal illumination regimen on cell composition of the central and peripheral organs of the immune system in ISIAH rats and control WAG rats. In ISIAH rats, 24-h illumination for 14 days led to more pronounced inhibition of cell proliferation and differentiation in the thymus and more pronounced decrease in splenocyte proliferation and T and B cell counts in the spleen in comparison with WAG rats; however, the level of antigen-presenting cells in the spleen of ISIAH increased. We concluded that ISIAH rats are more sensitive to abnormal illumination regimen than WAG rats. Twenty-four-hour illumination was associated with impairments of central differentiation of T cells and activation of systemic inflammation followed by impairment of differentiation regulation, which can aggravate metabolic dysfunctions in these animals.

Characterization of direct radiation-induced immune function and molecular signaling changes in an antigen presenting cell line.

Radiation therapy is a widely used cancer treatment and pre-transplantation conditioning regimen that has the potential to influence anti-tumor and post-transplantation immune responses. Although conventionally fractionated radiation doses can suppress immune responses by depleting lymphocytes, single high doses of local tumor radiation can enhance immune responses. Using phospho-flow cytometry analysis of a human monocytic cell line, we identified novel radiation-induced changes in the phosphorylation state of NFκB family members known in other cell types to maintain and regulate immune function. These phosphorylation changes were p53 independent, but were strongly dependent upon ATM activation due to DNA damage. We found that radiation promotes the activation and APC functional maturation through phosphorylation of NFκB Essential Modulator (NEMO). Our results and the analytic methods are especially well suited to the study of functional changes in APC when radiation is used for immune modulation in clinical protocols.

Mechanism of dichotomy between CD8+ responses elicited by apoptotic and necrotic cells.

Apoptotic cells are significantly more immunogenic than necrotic cells, even though both forms are identical in antigenic content. When a combination of apoptotic and necrotic cells are used to immunize, the phenotype conferred by apoptotic cells, i.e., high immunogenicity, is dominant. However, necrotic cells are not immunosuppressive or tolerogenic. Apoptotic and necrotic cells are taken up by antigen-presenting cells in an equivalent manner. The priming of naïve T cell response is also equivalent. However, the CD8+ T cells elicited by apoptotic cells expand, accumulate, and express effector function, while those primed by the necrotic cells do not. This dichotomy does not extend to CD4+ cells. Apoptotic and necrotic cells elicit equivalent CD4+ T cell priming, accumulation, and function. The deficit in CD8+ T cell function elicited by necrotic cells can be overcome to varying degrees by anti-CD40 antibody and ligands for TLR4 and TLR9; conversely, the immunogenicity of apoptotic cells can be abrogated by blocking anti-CD154 antibody. Our results indicate that immunization with apoptotic cells leads to engagement of CD40 on antigen-presenting cells; this is essential for their ability to elicit mature functional CD8+ cells. The necrotic cells fail to engage CD40, and this failure is the basis of their lack of immunogenicity. These differences have consequences for the understanding of mechanisms of cross-presentation and for efforts toward immunotherapy of cancers and autoimmune pathologies.

Reflections on the field of photoimmunology.

Photoimmunology evolved from experiments carried out in the 1970s on the immunology of cancer. In studying the antigenic properties of skin cancers induced in mice by UV radiation, I found that most of these tumors failed to grow when transplanted into normal, syngeneic mice but grew progressively in immunosuppressed mice. Thus, these UV-induced skin cancers were highly antigenic. The critical question was, how can these antigenic skin cancers escape immune rejection in their primary host? The answer was that exposing their skin to UV radiation prevented mice from triggering an immune response against their tumors. The failure to reject these tumors was owing to the development of UV tumor-specific regulatory T cells during the course of irradiation. In unraveling the mechanisms of this effect of UV, much has been learned about the immunology of the skin, including the function of Langerhans cells, the migration of immune cells in skin, the role of antigen-presenting cells in directing the immune response, and the role of keratinocytes as producers of immunological mediators. Thus, photoimmunology helped demonstrate that skin is an important immunological organ, and that the immune system can be influenced by the external environment via the skin.

Lipoteichoic acid from Lactobacillus rhamnosus GG as an oral photoprotective agent against UV-induced carcinogenesis.

Probiotics are live micro-organisms that when administered in adequate amounts confer a health benefit on the host. Cell surface molecules of these micro-organisms are being studied in relation to their ability to interact with the host. The cell wall of lactobacilli possesses lipoteichoic acids (LTA) which are molecules with immunomodulatory properties. UV radiation (UVR) has been proposed as the main cause of skin cancer because of its mutagenic and immunosuppressive effects. Photoprotection with some nutrition interventions including probiotics has recently been shown. The aim of the present study was to investigate whether the oral administration of purified LTA from Lactobacillus rhamnosus GG can modulate the immune-suppressive effect of UVR and skin tumour development in female Crl:SKH-1-hrBR mice. For this purpose, two irradiation models were studied: (1) a chronic irradiation scheme consisting of daily irradiations during twenty consecutive days and (2) a long-term irradiation schedule, irradiating the animals three times per week, during 34 weeks for tumour development. The results showed that T-cells in the inguinal lymph node of LTA-treated mice produced higher levels of (1) interferon-γ and (2) a number of total, helper and cytotoxic T-cells compared with non-treated mice. Moreover, a significant delay in tumour appearance was found in LTA-treated mice. An increased IgA⁺ cell number was found in the small intestine together with a higher number of activated dendritic cells in the mesenteric lymph nodes. The latter results might be indicative of a direct effect of LTA in the gut, affecting the cutaneous immune system and restoring homeostasis through the gut-skin axis.

Induction of acute GVHD by sex-mismatched H-Y antigens in the absence of functional radiosensitive host hematopoietic-derived antigen-presenting cells.

It is currently thought that acute GVHD cannot be elicited in the absence of Ag presentation by radiosensitive host hematopoietic-derived APCs after allogeneic BM transplantation. Because clinical data suggest that sex-mismatched H-Y Ags may be important minor histocompatibility Ags for GVH responses, we directly tested their relevance and ability to initiate GVHD when presented by either the hematopoietic- (host or donor) or the nonhematopoietic-derived APCs. H-Y minor Ag incompatibility elicited both CD4(+) and CD8(+) T-cell driven GVHD lethality. Studies with various well-established BM chimera recipients, in contrast to the current views, have reported that in the absence of functional radiosensitive host hematopoietic-derived APCs, H-Y Ag presentation by either the donor hematopoietic-derived or the host nonhematopoietic-derived APCs is sufficient for inducing GVHD. Our data further suggest that infusion of sufficient numbers of alloreactive donor T cells will induce GVHD in the absence of radiosensitive host hematopoietic-derived APCs.

Irradiation enhances human T-cell function by upregulating CD70 expression on antigen-presenting cells in vitro.

In addition to the direct killing of tumor cells, radiation therapy can alter the balance of immune cells in vivo due to the differential radiosensitivity of different cell types. The addition of adjuvant radiation therapy before adoptive cell transfer therapy has been shown to enhance antitumor responses in both mouse models and clinical trials. This study examines the effects of in vitro irradiation on the phenotype and function of human antigen-presenting cells. The results indicated that irradiation upregulated CD70 expression on both B cells and mature dendritic cells (DCs). Expression of CD70 on mature DCs was enhanced in a dose-dependent manner, whereas under the same conditions, no significant upregulation of CD80, CD86, or CD40 was observed. The levels of expression of CD70 induced on mature DC by irradiation correlated highly with the ability of those cells to stimulate T-cell proliferation and interferon-γ production. Furthermore, significant reductions in T-cell proliferation and interferon-γ production were seen when CD70 expression on DCs was partially reduced using shRNA, as well as when DCs were incubated with a blocking anti-CD70 antibody. Radiation therapy may therefore enhance T-cell activation in vivo through the CD27 pathway by virtue of its ability to upregulate the expression of CD70 on antigen-presenting cells.

Effects on antigen-presenting cells of short-term interaction with the human host defence peptide ß-defensin 2.

ß-Defensins are antimicrobial peptides that exert their host-defence functions at the interface between the host and microbial biota. They display a direct, salt- and medium-sensitive cidal activity, in vitro, against a broad spectrum of bacteria and fungi, and there is increasing evidence that they also play a role in alerting and enhancing cellular components of innate and adaptive immunity. Their interaction with biological membranes plays a central role in both of these types of activities. In the present study, we have investigated the interaction of fluorescently labelled hBD2 (human ß-defensin 2) with monocytes, macrophages and iDCs (immature dendritic cells), observing a differential capacity to be rapidly internalized into these cells. Complementary microscopy techniques [TEM (transmission electron microscopy), optical microscopy and IR microspectroscopy] were used to explore the functional and biological implications of these interactions on iDCs. Short-term exposure to the peptide resulted in significant alterations in membrane composition and re-organization of the endomembrane system, with the induction of degranulation. These events may be associated with the antigen-presenting activities or the chemotaxis of iDCs, which appears to occur via both CCR6 (CC chemokine receptor 6)-dependent and -independent mechanisms.

UVR-induced regulatory T cells switch antigen-presenting cells from a stimulatory to a regulatory phenotype.

UVR-induced regulatory T cells (UVR-Treg) inhibit sensitization in an antigen-specific manner. The migratory behavior of UVR-Treg can be reprogrammed by antigen-presenting cells (APCs), indicating a cross-talk between these cells. Hence, we sought to investigate whether in turn UVR-Treg can influence APCs. Bone marrow-derived dendritic cells (DCs) were co-incubated with DNFB-specific UVR-Treg. DCs were isolated, coupled with DNBS, and injected into naive mice. Contrary to untreated dinitrobenzenesulfonic acid (DNBS)-coupled DC, DCs from the cocultures failed to induce sensitization in the recipients. Antibody blocking and transwell experiments indicated that both IL-10 and cellular contact are required during the co-incubation to induce inhibition. UVR-Treg downregulated B7-2 and major histocompatibility complex class II but induced the negative regulatory molecules B7-H3 and B7-H4 on DC. To suppress, UVR-Treg had to be activated in an antigen-specific manner. However, the suppression was not antigen-specific as activated DNFB-specific UVR-Treg inhibited DCs to sensitize also against trinitrochlorobenzene. Adoptive transfer experiments revealed that injection of hapten-coupled DCs, which were co-incubated with UVR-Treg, further induced Treg in the recipients. Together, this indicates that activated UVR-Treg can alter APCs in such a way that they lose their sensitizing capacity but in turn induce Treg. Thus, UVR-Tregs switch APCs from a stimulatory to a regulatory phenotype.

Retinal laser burn disrupts immune privilege in the eye.

Immune privilege allows for the immune protection of the eye in the absence of inflammation. Very few events are capable of overcoming the immune-privileged mechanisms in the eye. In this study, we report that retinal laser burn (RLB) abrogates immune privilege in both the burned and nonburned eye. As early as 6 hours after RLB, and as late as 56 days after RLB, antigen inoculation into the anterior chamber of the burned eye failed to induce peripheral tolerance. After RLB, aqueous humor samples harvested from nontreated eyes but not from either the burned or the contralateral eye, down-regulated the expression of CD40 and up-regulated interleukin-10 mRNA in peritoneal exudate cells, and converted peritoneal exudate cells into tolerogenic antigen-presenting cells (APCs). Unlike F4/80(+) APCs from nontreated mice, F4/80(+) APCs from RLB mice were unable to transfer tolerance after anterior chamber inoculation of antigen into naïve mice. The increased use of lasers in both the industrial and medical fields raises the risk of RLB-associated loss of immune regulation and an increased risk of immune inflammation in the eye.

A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402-based photodepletion technique.

Selective allodepletion is a strategy to eliminate host-reactive donor T cells from hematopoietic stem cell allografts to prevent graft-versus-host disease while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were cocultured with responder cells from HLA-matched or -mismatched donors. Optimal results were achieved when cocultured cells were incubated with 7.5 muM TH9402, followed by dye extrusion and exposure to 5 Joule/cm(2) light energy at 5 x 10(6) cells/mL. In mismatched stimulator-responder pairs, the median reduction of alloreactivity was 474-fold (range, 43-fold to 864-fold) compared with the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9-fold to 3.3-fold) reduction. In matched pairs, alloreactive helper T-lymphocyte precursors were reduced to lower than 1:100 000, while third-party responses remained higher than 1:10 000. This establishes a clinical-scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current Good Manufacturing Practice. This procedure is currently being investigated in a clinical trial of allotransplantation.

Decreased human epidermal antigen-presenting cell activity after ultraviolet A exposure: dose-response effects and protection by sunscreens.

BACKGROUND: Ultraviolet (UV) exposure of human skin causes immunosuppression that contributes to the growth of skin cancer. The contribution of UVA in these processes is still a matter of debate. OBJECTIVES: The purpose of our study was first to find a dose-response effect of UVA exposure on human epidermal antigen-presenting cell (APC) activity and to evaluate the protective capacity of two sunscreen formulations against a high level of acute UVA exposure. We also tried to evaluate the protective capacity afforded by the same sunscreens against UVA-induced clinical changes such as redness and pigmentation. METHODS: The functional assessment of the alloantigen-presenting capacity of epidermal cells prepared from skin keratotome samples 3 days after UVA exposure was measured with a mixed epidermal cell-lymphocyte reaction (MECLR) in each healthy volunteer (n = 16). Redness and pigmentation were assessed by chromametry 24 h after exposure to a single UVA dose. RESULTS: In vivo UVA exposure to 15, 30 and 60 J cm(-2) resulted in a dose-dependent decrease in purified allogeneic T cell (CD4+ T cells) proliferation induced by UVA-irradiated epidermal cells. The epidermal APC function was significantly decreased with a suberythemal exposure corresponding to 15 J cm(-2). The decrease, partial and not statistically different between 30 and 60 J cm(-2), exhibits a plateau-response effect. There was no correlation between the decrease of the epidermal APC function and the intensity of erythema and persistent pigment darkening. Both sunscreen formulations strongly inhibited the UVA-induced reduction of MECLR at 90 J cm(-2). CONCLUSION: Our results clearly demonstrate that UVA impairs the APC activity of the epidermal cells and thus may contribute to UV-induced immunosuppression in humans. They also indicate that erythema and immunosuppression have different dose-response curves in the UVA range. The two sunscreen formulations afforded a significant protection against the decrease in epidermal APC activity induced by exposure to a high UVA dose (90 J cm(-2)).

Effect of PUVA, narrow-band UVB and cyclosporin on inflammatory cells of the psoriatic plaque.

BACKGROUND: Because antigen presenting is necessary for T-cell activation, antigen-presenting cells should be involved in the pathogenesis of psoriasis. In this study, our purpose was to evaluate and compare effects of PUVA, cyclosporine A and narrow-band UVB on dendritic cells and activated lymphocytes in the psoriatic lesions. METHODS: Forty-five volunteered patients (15 patients in each treatment group as PUVA, cyclosporin A and narrow-band UVB) were enrolled in this study. Lesional skin biopsies were taken from each patient before and after treatments. Fresh frozen biopsies were studied for the expressions of CD1a, CD68, CD86, CD4, CD8 and HLA-DR proteins by immunohistochemistry. RESULTS: There was no correlation between severity of the lesions and expressions of the antigens. Only PUVA significantly decreased CD1a+ epidermal Langerhans cells' (LCs) counts. Treatment modalities decreased expression of costimulator CD86, and most of them decrease antigen-presenting capacity of skin by decreasing HLA class-II expression. CONCLUSIONS: All treatment modalities equally reduce lymphocytes, macrophages and dendritic cells. PUVA is the only treatment that decreases epidermal LCs. All treatments effectively diminish expression of CD86 and inhibit this step of inflammation.

Primary defect in UVB-induced systemic immunomodulation does not relate to immature or functionally impaired APCs in regional lymph nodes.

UVB irradiation of the shaved dorsal skin of mice can cause both local and systemic suppression of contact hypersensitivity responses; the former demonstrated by administration of the sensitizing Ag/hapten to the irradiated site and the latter by its administration at least 72 h later to distal unirradiated sites. The immunological basis of systemic immunomodulation is not clear. When haptens (trinitrochlorobenzene, FITC) were administered to the shaved ventral skin 4 days after irradiation (8 kJ/m(2)) to the shaved dorsum of BALB/c mice, CD11c(+)/FITC(+) cells in the skin-draining lymph nodes from control and irradiated mice produced on a per cell basis similar levels of IL-12 and PGE(2) were phenotypically mature and efficient at presenting FITC to lymphocytes from FITC-sensitized mice. Ag presentation by FACS-sorted CD11c(+) lymph node cells isolated 4 days after UVB irradiation was as efficient as were cells from unirradiated mice at presentation in vitro of an OVA peptide (OVA(323-339)) to CD4(+) cells from OVA-TCR-transgenic DO11.10 mice. Further, IFN-gamma levels were increased in the cultures containing CD11c(+) cells from UVB-irradiated mice, suggesting that inflammation may precede downstream immunosuppression. These results suggest that the primary cause of reduced contact hypersensitivity responses in mice in which UV irradiation and the sensitizing Ag are applied to different sites several days apart must originate from cells other than CD11c(+) APCs that directly or by production of soluble mediators (IL-12, PGE(2)) affect cellular responses in the nodes of UVB-irradiated mice.

B cells activated in lymph nodes in response to ultraviolet irradiation or by interleukin-10 inhibit dendritic cell induction of immunity.

Ultraviolet (UV) radiation suppresses systemic immunity. We explored these cellular mechanisms by exposing mice to systemically immunosuppressive doses of UV radiation and then analyzing cell phenotype and function in the lymphoid organs. Although UV radiation increased total cell number in the draining lymph nodes (DLN), it did not alter the activation state of dendritic cells (DC). Rather, UV radiation selectively activated lymph node B cells, with these cells being larger and expressing higher levels of both anti-major histocompatibility complex II and B220 but not co-stimulatory molecules. This phenotype resembled that of a B cell geared toward immune tolerance. To test whether UV radiation-activated B cells were responsible for immunosuppression, DC and B cells were conjugated to antigen ex vivo and transferred into naive hosts. Although DC by themselves activated T cells, when the B cells from UV radiation-irradiated mice were co-injected with DC, they suppressed DC activation of immunity. Interleukin (IL)-10-activated B cells also suppressed DC induction of immunity, suggesting that IL-10 may be involved in this suppressive effect of UV radiation. These results demonstrate a new mechanism of UV radiation immunosuppression whereby UV radiation activates B cells in the skin-DLN that can suppress DC activation of T cell-mediated immunity.