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Exposure to ionizing radiation often induces T helper (Th) cell differentiation, resulting in an imbalance of Th1 and Th2 cellular subtypes, which can affect the efficacy of cancer radiotherapy. The aim of this study was to analyze differential expression of Th1, Th2 and Th3/Type 1 regulatory T cell (Tr1) subtype-related genes and cytokines in mouse thymocytes after high- and low-dose systemic radiation, using functional classification gene arrays and Elisa assays, and to explore the molecular mechanisms underlying radiation's immune effects and their relationship with Th1/Th2 immunity. We found that expression of 8 genes was upregulated after LDR, while expression of 5 genes was downregulated. After HDR, 54 genes were upregulated and 3 genes were downregulated, including genes related to Th1, Th2 and Th3/Tr1 cellular subtypes, Th1/Th2-type immune response genes and transcription factor-related genes. In the foregoing results, LDR and HDR in the thymus induced opposite patterns of expression for Th1-, Th2- and Th3-type related cytokines TGF-β, C/EBP-β and TNF-α. We also found that expression of Interferon-γ (IFN-γ) and Interleukin-2 (IL-2), which have a moderating effect on immune function, was upregulated after LDR. Furthermore, the secretion of negative regulatory factors Interleukin-1β (IL-1β), Interleukin-4 (IL-4), transforming growth factor-β (TGF-β) and Interleukin-21 (IL-21) was reduced after LDR, but HDR produced the opposite effect and stimulated their expression. These findings suggest that LDR may induce a Th1-type immune response, while HDR may lead to a Th2-type immune response.

In the present study, we investigated the effect of tyndallized HY7712 (tHY7712) on the expression of Th cell specific transcription factors and cytokines in whole-body γ-irradiated mice. Oral administration of tHY7712 strongly recovered the γ-irradiation-suppressed expression of helper T (Th) cell- and regulatory T cell-related transcription factors and cytokines, such as T-bet, Foxp3, IFN-γ, TNF-α, and IL-10, and suppressed Th2 cell-associated transcription factor and cytokine GATA3 and IL-5, respectively. Furthermore, compared with the control, tHY7712 treatment also restored γ-irradiation-impaired natural killer and cytotoxic T cell activities against YAC-1 tumor cells to 97.8% and 98.6%, respectively.

Dendritic cell function is modulated by stromal cells, including fibroblasts. Although poorly understood, the signals delivered through this crosstalk substantially alter dendritic cell biology. This is well illustrated with release of TNF-α/IL-1β from activated dendritic cells, promoting PGE2 secretion from stromal fibroblasts. This instructs dendritic cells to up-regulate IL-23, a key Th17-polarizing cytokine. We previously showed that ionizing radiation inhibited IL-23 production by human dendritic cells in vitro. In the present study, we investigated the hypothesis that dendritic cell-fibroblast crosstalk overcomes the suppressive effect of ionizing radiation to support appropriately polarized Th17 responses. Radiation (1-6 Gy) markedly suppressed IL-23 secretion by activated dendritic cells (P < 0.0001) without adversely impacting their viability and consequently, inhibited the generation of Th17 responses. Cytokine suppression by ionizing radiation was selective, as there was no effect on IL-1β, -6, -10, and -27 or TNF-α and only a modest (11%) decrease in IL-12p70 secretion. Coculture with fibroblasts augmented IL-23 secretion by irradiated dendritic cells and increased Th17 responses. Importantly, in contrast to dendritic cells, irradiated fibroblasts maintained their capacity to respond to TNF-α/IL-1β and produce PGE2, thus providing the key intermediary signals for successful dendritic cell-fibroblasts crosstalk. In summary, stromal fibroblasts support Th17-polarizing cytokine production by dendritic cells that would otherwise be suppressed in an irradiated microenvironment. This has potential ramifications for understanding the immune response to local radiotherapy. These findings underscore the need to account for the impact of microenvironmental factors, including stromal cells, in understanding the control of immunity., (© The Author(s).)

Background: Vitiligo is a T cell mediated autoimmune depigmenting disease. Altered cytokine concentrations have been suggested in the pathogenesis of vitiligo., Methods: T helper and regulatory T cell cytokines (IL-2, IFN-γ, IL-10, IL-13, IL-17 and TGF-β) have been estimated by ELISA and their clinical correlation was determined. The study had 3 groups: group I with 80 vitiligo patients (60 active and 20 stable), group II with 25 narrow band ultraviolet B treated vitiligo and group III with 70 healthy controls., Results: Significant difference was found in the serum interleukin (IL)-10, IL-13, IL-17A and TGF-β1 concentrations among 3 groups (P<0.05). In group I, serum IL-2, IL-17A concentrations were significantly increased and TGF-β1 concentrations were decreased in active vitiligo compared to stable vitiligo (P<0.05). Concentrations of IL-2, IL-10 and IL-13 (rho=-0.307, rho=-0.407, rho=-0.351 and P<0.05; respectively) were negatively- and TGF-β1 concentrations were positively-correlated (rho=0.799, P=0.001) with disease duration. Interleukin-13 concentrations were negatively- and serum TGF-β1 concentrations were positively-correlated (rho=-0.326, rho=0.244 and P<0.05; respectively) with percentage of body surface area involvement., Conclusions: Increased concentrations of serum IL-10, IL-13, and IL-17A and decreased concentrations of TGF-β1 suggested altered cell-mediated immunity that may facilitate the melanocyte cytotoxicity in vitiligo., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Sonodynamic therapy (SDT) was developed as a localized ultrasound-activated cytotoxic therapy for cancer. The ability of SDT to destroy target tissues selectively is especially appealing for atherosclerotic plaque, in which selective accumulation of the sonosensitizer, protoporphyrin IX (PpIX), had been demonstrated. Here we investigate the effects of PpIX-mediated SDT on macrophages, which are the main culprit in progression of atherosclerosis., Methods and Results: Cultured THP-1 derived macrophages were incubated with PpIX. Fluorescence microscopy showed that the intracellular PpIX concentration increased with the concentration of PpIX in the incubation medium. MTT assay demonstrated that SDT with PpIX significantly decreased cell viability, and this effect increased with duration of ultrasound exposure and PpIX concentration. PpIX-mediated SDT induced both apoptosis and necrosis, and the maximum apoptosis to necrosis ratio was obtained after SDT with 20 μg/mL PpIX and five minutes of sonication. Production of intracellular singlet oxygen and secondary disruption of the cytoskeleton were also observed after SDT with PpIX., Conclusion: PpIX-mediated SDT had apoptotic effects on THP-1 macrophages via generation of intracellular singlet oxygen and disruption of the cytoskeleton. PpIX-mediated SDT may be a potential treatment to attenuate progression of atherosclerotic plaque.

Purpose: To evaluate the utility and allogenicity of gamma-irradiated corneal allografts., Methods: Corneal buttons were harvested from C57BL/6 mice and decellularized with gamma irradiation. Cell viability was assessed using TUNEL and viability/cytotoxicity assays. Orthotopic penetrating keratoplasty was performed using irradiated or nonirradiated (freshly excised) C57BL/6 donor grafts and BALB/c or C57BL/6 recipients. Graft opacity was assessed over an 8-week period and graft survival was evaluated using Kaplan-Meier survival curves. Mixed-lymphocyte reactions and delayed-type hypersensitivity assays were performed to evaluate T-cell alloreactivity. Real-time PCR was used to investigate the corneal expression of potentially pathogenic T-helper 1, 2, and 17 cell-associated cytokines., Results: Corneal cells were devitalized by gamma irradiation as evidenced by widespread cellular apoptosis and plasma membrane disruption. Nonirradiated allograft and isograft rates of survival were superior to irradiated allograft and isograft rates of survival (P < 0.001). Mixed lymphocyte reactions demonstrated that T-cells from irradiated allograft recipients did not exhibit a secondary alloimmune response (P < 0.001). Delayed-type hypersensitivity assays demonstrated that irradiated allografts did not elicit an alloreactive delayed-type hypersensitivity response in graft recipients (P ≤ 0.01). The corneal expression of T-helper 1, 2, and 17 cell-associated cytokines was significantly lower in failed irradiated allografts than rejected nonirradiated allografts (P ≤ 0.001)., Conclusions: Gamma-irradiated corneas failed to remain optically clear following murine penetrating keratoplasty; however, gamma irradiation reduced the allogenicity of these corneas, potentially supporting their use in procedures such as anterior lamellar keratoplasty or keratoprosthesis implantation.

The objective of this research was quantitative assessment of serum and membrane regulatory proteins in blood from nuclear workers as markers of radiation-induced alterations in immune homeostasis in the late period after protracted exposure of nuclear workers with different doses. The effector and regulatory lymphocytes were measured using a flow cytofluorometer in workers from the main facilities of the Mayak PA (aged ∼60 y up to 80 y) in the late period after combined exposure to external gamma-rays and internal alpha-radiation from incorporated 239Pu. The control group included non-occupationally exposed members of the Ozyorsk population matched by gender and age to the group of Mayak workers. Thirty serum proteins involved in regulation of immune homeostasis, such as growth factors, multifunctional interleukins, pro- and anti-inflammatory cytokines, and their receptors, were measured using ELISA in blood serum specimens from the Radiobiology Human Tissue Repository. The dosimetry estimates were obtained using Doses-2005. The correlation analysis revealed a statistically significant direct relationship of T-killers and plutonium body burden and a decreasing level of T-helpers with accumulated external dose in exposed individuals. There were differences in expression of membrane markers in young regulatory cells (double null T-lymphocytes, NKT-lymphocytes, regulatory T-cells, and an increase of activated forms of T-lymphocytes), which indicated an active role of regulatory cells in maintaining immune homeostasis in terms of protracted exposure. The assessment of regulatory proteins in blood indicated that growth factors (EGF, TGF-β1, PDGF), multifunctional interleukins (IL-17A, IL-18), and pro-inflammatory cytokines (IL-1β and INF-γ) could be potential markers of radiation-induced alterations in protein status. An imbalance of pro- and antiinflammatory proteins in blood and variations of protein profiles at the lower exposure levels (gamma-ray dose <1 Gy, plutonium body burden <0.74 kBq) in the late period after protracted exposure were less pronounced than at the higher exposure levels, which was probably explained by compensatory-adaptive responses in the late period among senile individuals with polypathology.

Exposure to ultraviolet radiation (UVR) suppresses immunity by complex pathways, initiated by chromophores located in the skin and ending with the generation of specific subsets of T and B regulatory cells. The primary and memory (recall) immune response to a wide variety of antigens, including microorganisms, can be reduced by UVR, leading to the possibility that the efficacy of vaccination could be similarly reduced. A limited number of animal models of vaccination demonstrate that this may indeed be the case. The situation in human subjects has not been rigorously assessed but there are indications from a variety of sources that UVR adversely affects the immune responses to several vaccines. These studies are reviewed and the implications for vaccine administration discussed. As vaccination represents a major public health measure world-wide for the control of an increasing number of common infections, it is important to maximise its efficacy; therefore further evaluation of UVR in the context of vaccination is required and warranted., (This journal is © The Royal Society of Chemistry and Owner Societies 2011)

The goal was to investigate the T helper (Th) response in splenocytes of mice exposed to low-dose/low-dose-rate (LDR) γ-rays, simulated solar particle event protons (sSPE), or combination of both. C57BL/6 mice were exposed to LDR γ-radiation ((57)Co) to a total dose of 0.05 Gray (Gy) at 0.024 cGy/h, either with or without subsequent exposure to 2 Gy sSPE protons. Expression of genes related to Th cells was evaluated immediately after exposure (day 0). On day 21, intra- and extracellular cytokine production was assessed after activation with anti-CD3 monoclonal antibodies (mAb) or phorbol 12-myristate 13-acetate/ionophore (PMA/I). Five genes were significantly modulated on day 0 in one or more of the irradiated groups compared to controls (p < 0.05): Ccl11, Ccr5, Cd80, Inha, and Il9. On day 21, numbers of cells positive for interferon-γ were high in the LDR + sSPE group versus 0 Gy and LDR γ-rays (p < 0.05), but there was no difference in IL-2 and TNF-α. Levels of secreted cytokines after anti-CD3 mAb activation were high for 5 (MIP-1α, GM-CSF, IFN-γ, TNF-α, IL-13) and low for 2 (IL-7, IL-9) in all irradiated groups. Priming with LDR photons had a significant effect on IFN-γ and IL-17 compared to sSPE protons alone; IL-2 was low only in the LDR + sSPE group. The cytokine patterns after anti-PMA/I activation were different compared to anti-CD3 mAb and with fewer differences among groups. The data show that total-body exposure to space-relevant radiation has profound effects on Th cell status and that priming with LDR γ-rays can in some cases modulate the response to sSPE.

The most prevalent cancer diagnosed in the world is sunlight-induced skin cancer. In addition to being a complete carcinogen, UV radiation, the causative agent of skin cancer, induces immune suppression. Because UV-induced immune suppression is a well-recognized risk factor for skin cancer induction, it is crucial to understand the mechanisms underlying UV-induced immune suppression. Mast cells, which have recently emerged as immune regulatory cells, are particularly important in UV-induced immune suppression. UV exposure does not induce immune suppression in mast cell-deficient mice. We report that UV irradiation blocks germinal center (GC) formation, Ab secretion, and T follicular helper (Tfh) cell function, in part by altering the expression of transcription factors BCL-6 and BLIMP-1. No suppression of GC formation, Tfh cell IL-21 expression, or Ab secretion was observed in UV-irradiated mast cell-deficient (Kit(W-sh/W-sh)) mice. When mast cell-deficient mice were reconstituted with wild type mast cells, immune suppression was restored. Reconstituting the mast cell-deficient mice with bone marrow-derived mast cells from IL-10-deficient mice failed to restore the ability of UV radiation to suppress GC formation. Our findings demonstrate a function for mast cells, suppression of Tfh cell production, GC formation, and Ab production in vivo.

Were investigated the parameters of apoptosis of immune cells, the cell subpopulation composition and frequency of TCR-mutant lymphocytes in the peripheral blood of persons exposed to radiation. Increased number of peripheral mFasL-positive mononuclear cells and the accumulation of TCR-mutant T lymphocytes in a wide range of doses were found in the patients. It is doubtless that the effect of ionizing radiation is accompanied by a simulation of Fas-ligand expression on the surface of peripheral mononuclear cells and modification of the activating signal to apoptosis through the molecule CD4, CD8, CD3. The results can be caused by proapoptotic effect of radiation.

Background: Narrowband ultraviolet B (NB-UVB) has recently been used for the treatment of various skin disorders. Its effects on the production of cytokines and chemokines by keratinocytes are unknown., Objectives: To investigate the effect of NB-UVB on production of chemokines and proinflammatory cytokines by keratinocytes in comparison with broadband (BB)-UVB., Methods: Normal human epidermal keratinocytes (or the human keratinocyte cell line HaCaT in some experiments) at semiconfluency were irradiated with NB-UVB at 10, 100, 500 or 1000 mJ cm(-2) or BB-UVB at 10 or 100 mJ cm(-2). The cultures were maintained in the presence or absence of interferon (IFN)-gamma at 200 U mL(-1). The 72-h culture supernatants were analysed by enzyme-linked immunosorbent assay to quantify T helper (Th)1 chemokines (IFN-inducible protein 10 and monokine induced by IFN-gamma), Th2 chemokines [macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC)] and proinflammatory cytokines [interleukin (IL)-1alpha and tumour necrosis factor (TNF)-alpha]. The expression of mRNA for these molecules was simultaneously assessed by reverse transcriptase-polymerase chain reaction. The culture supernatants were also tested for their chemotactic activity for Th1 and Th2 cells. The two UVB sources were compared on the basis of their minimal erythemal doses and clinically used doses., Results: Although both NB-UVB and BB-UVB increased the production of IL-1alpha and TNF-alpha, the augmentative effect of NB-UVB was less than that of BB-UVB. Both wavelength ranges of UVB enhanced or had no effect on Th1 chemokine production, but suppressed the production of Th2 chemokines MDC and TARC. This was confirmed by chemotactic assay, which showed decreased chemotactic activity for Th2 cells by the culture supernatants from NB-UVB-irradiated keratinocytes., Conclusions: NB-UVB reduces the production of Th2 chemokines without excess production of proinflammatory cytokines, suggesting its therapeutic effectiveness on Th2-mediated skin disorders as well as its relative safety in clinical usage.

We studied effects of dry air radon baths (DARB) on bronchial hypersensitivity and serum levels of proinflammatory cytokines in patients with bronchial asthma (BA) with reference to the disease severity and radon therapy regimen. DARB with radon concentrations 40 nCi/l significantly raise the threshold of bronchial sensitivity to metacholine compared to control and DARB with radon concentration 20 nCi/l. The effect was the highest in patients with mild asthma. Comparison of serum levels of alpha- and gamma-interferons, tumor necrosis factor alpha, IL-2, IL-4 and IL-6 has shown that alpha radon radiation significantly increases serum gamma-interferon and reduces IL-4 concentration. These changes were more evident in the group with high radon content. Thus, radon therapy has immunomodulating activity.

Recent studies on "nonthermal" effects of mobile phone radiofrequency (RF) suggest that RF can interact with cellular functions and molecular pathways. To study the possible RF effects on human lymphocyte activation, we analyzed CD25, CD95, CD28 molecules in unstimulated and stimulated CD4+ e CD8+ T cells in vitro. Peripheral blood mononuclear cells (PBMCs) from young and elderly donors were exposed or sham-exposed to RF (1,800 MHz, Specific Absorption Rate 2 W/kg) with or without mitogenic stimulation. No significant changes in the percentage of these cell subsets were found between exposed and sham-exposed lymphocytes in both young and elderly donors. Nevertheless, after RF exposure we observed a slight, but significant, downregulation of CD95 expression in stimulated CD4+ T lymphocytes from elderly, but not from young donors. This age-related result is noteworthy given the importance of a such molecule in regulation of the immune response.

Purpose: The well-documented increases in malignant tumours in the A-bomb survivors have recently been supplemented by reports that non-cancer diseases, including cardiovascular disease, may also have increased in incidence with increasing radiation dose. Given that low-level inflammatory responses are widely accepted as a significant risk factor for such diseases, we undertook a detailed investigation of the long-term effects of ionizing radiation on the levels of the inflammatory markers C-reactive protein (CRP) and interleukin 6 (IL-6) in A-bomb survivors., Materials and Methods: Blood samples were taken from 453 participants in a long-term epidemiological cohort of A-bomb survivors. Plasma levels of CRP and IL-6 were measured using standard antibody-mediated procedures. Relationships between CRP or IL-6 levels and radiation dose were then investigated by multivariate regression analysis. Blood lymphocytes from each individual were used for immunophenotyping by flow cytometry with murine monoclonal antibodies to CD3, CD4 and CD8., Results: CRP levels were significantly increased by about 31% Gy(-1) of estimated A-bomb radiation (p=0.0001). Higher CRP levels also correlated with age, male gender, body mass index and a history of myocardial infarction. After adjustments for these factors, CRP levels still appeared to have increased significantly with increasing radiation dose (about 28% increase at 1Gy, p=0.0002). IL-6 levels also appeared to have increased with radiation dose by 9.3% at 1Gy (p=0.0003) and after multiple adjustments by 9.8% at 1Gy (p=0.0007). The elevated CRP and IL-6 levels were associated with decreases in the percentages of CD4(+) helper T-cells in peripheral blood lymphocyte populations., Conclusions: Our results appear to indicate that exposure to A-bomb radiation has caused significant increases in inflammatory activity that are still demonstrable in the blood of A-bomb survivors and which may lead to increased risks of cardiovascular disease and other non-cancer diseases.

We analyzed the indices of antioxidant and immune systems in 69 patients with cervical carcinoma T3NxMo at different stages of radiotherapy. Radiation doses of 10-20 Gy were followed by certain normalization of antioxidant enzyme levels, increased concentrations of vitamins E and A in immunocompetent blood cells and improved the penetration and elastic properties of formed cells. The dosage stimulated the activity of helper-inducing and cytotoxic T-cells. Such effects should be taken into account when working out regimens of complex treatment of locally-advanced cervical carcinoma which include cytostatic drugs and radiation.

The article presents results of treatment of 78 patients with the history of coronary bypass operations for ischemic heart disease. There were 48 patients with high risk factors of the development of pyo-septic complications. Among them there were 27 patients treated by complex postoperative therapy including intravascular photomodification of blood. The intravascular irradiation of blood was shown to have a positive influence upon the development of the early postoperative period in this category of patients. The immunomodulating effect was noted due to a greater amount of activated lymphocytes and T-helpers in blood. The positive dynamics of immunological indices were in correspondence with the data of the clinical state, pyo-inflammatory complications were not registered in this group of patients. Early (on the 3rd-5th days) inclusion of the intravascular irradiation of blood in the complex postoperative therapy is thought to be expedient for the patients with high risk of pyo-inflammatory complications.

Helper T lymphocyte precursor (HTLp) frequencies determined by limiting dilution analysis (LDA) have a predictive value for alloreactivity in allogeneic bone marrow transplantation. Methodological problems in LDA include autoreactivity in the responder or stimulator cell populations and interleukin 2 (IL-2) production by the stimulator cells as a response to the responder cells (backstimulation). The extent and impact of these aspects for IL-2 production and HTLp frequency determination were studied by autologous and allogeneic mixed lymphocyte reactions with healthy volunteers and HTLp determinations from bone marrow transplantation donor/recipient pairs. We found that autoreactivity occurred in the unirradiated cells with a reproducible inter-individual variation. The immunogenicity of the stimulator cells was preserved after gamma irradiation with 50 Gy and the risks of autoreactivity and backstimulation were limited. Higher doses of irradiation decreased the immunogenicity. Immune reactions to antigens present in the serum supplement of the culture medium were seen with foetal calf serum and to a lesser extent with pooled human sera. This could be avoided by the use of autologous serum. We were unable to ensure satisfactory culture conditions in serum-free medium. The reproducibility of the HTLp frequency determinations was tested for intra- and inter-assay variation. The coefficients of variation were estimated as 24% and 35%, respectively. This was acceptable considering the range of the HTLp frequencies (1:10(2) to 1:10(7)). The influence of the extent of autoreactivity of the bone marrow donors was investigated in 28 HLA-identical sibling transplantations. We found no correlation between the autoreactivity of the donors and the HTLp frequencies. The extent of autoreactivity of the donor did not correlate with the clinical outcome in terms of acute graft-versus-host disease, treatment-related mortality, risk of relapse and overall survival. In spite of methodological difficulties and interference from autoreactivity and backstimulation, reproducible quantification of clinically significant alloreactivity can be attained.

Background: It has been suggested that prolonged exposure to sunlight may induce systemic or local immune alterations, which may facilitate the development of skin cancer and, perhaps, non-Hodgkin's lymphona. The effects of prolonged sunlight exposure on peripheral blood cells were studied., Methods: Leukocytes and lymphocyte subpopulations of 12 volunteers aged 10-45 were investigated before and after a 3-week summer holiday in seaside resorts in Greece. Lymphocyte phenotypes were estimated using monoclonal antibodies and flow cytometry., Results: There were no significant differences with respect to total numbers of T cells, T-helper/inducer, T-suppressor/cytotoxic, B cells or HLA-Dr+ cells. However, we have found evidence of lymphocyte stimulation, reflected in an increase in cells expressing the interleukin-2 receptor (IL-2R) and, more specifically, an increase in the T cells expressing IL-2R and HLA-Dr antigens. An increase in natural killer cells has also been noticed., Conclusions: These findings suggest that prolonged intense exposure to sunlight may be associated with immunostimulation, rather than immunosuppression.

Significant decreases in the fraction of lymphocytes that are CD4(+) and increases in serum levels of some classes of immunoglobulin have been reported to occur in atomic bomb (A-bomb) survivors and in victims of the Chernobyl nuclear plant accident. To investigate the long-term effects of nuclear radiation on cellular immunity in more detail, we used limiting dilution assays with peripheral blood mononuclear cell preparations to analyze the T-cell responses of 251 A-bomb survivors exposed to less than 0.005 Gy and 159 survivors exposed to more than 1.5 Gy. The percentages of CD2-positive cells that were capable of proliferating in response to phytohemagglutinin (PHA) in the presence of exogenous interleukin 2 (IL2) did not differ substantially between distally exposed and more heavily exposed survivors. The heavily exposed survivors appeared to possess fewer T cells that were capable of proliferating in response to concanavalin A (Con A) or of producing interleukin 2. Assuming that CD4 T cells were the ones primarily responsible for producing IL2 in response to Con A, we were able to estimate how many cells in any given CD4 T-cell population were actually producing IL2. The results indicated that peripheral blood samples from heavily exposed survivors contained significantly fewer IL2-producing CD4 T cells than did similar samples from distally exposed survivors, indicating that significant exposure to A-bomb radiation may have a long-lasting negative effect on the capacity of CD4 T-cell populations to produce IL2.

The immunosuppressive effects of UVB irradiation have been well documented. The production of cytokines by keratinocytes is considered to play a major role in the induction of local as well as systemic immunosuppression. It is thought that partly due to the interaction of locally produced cytokines with antigen-presenting cells (APC) systemic effects, like antigen-specific tolerance, can be induced. In this study we examined the effect of UVB irradiation on cytokine profiles of peripheral APC as well as the functional consequences. Our results indicate that UVB irradiation impairs T(h)1-mediated immune responses in vivo by suppression of the systemic IL-12p70 production. Splenic APC from UVB-exposed mice showed an enhanced production of prostaglandin E(2), IL-1, IL-6 and tumor necrosis factor-alpha after in vitro stimulation. Also, spleen cells from UVB irradiated IL-4(-/-) mice showed increased IL-6 levels. These APC were less efficient in inducing IFN-gamma production by CD4(+) T cells and suppressed IgM production by B cells. We conclude that the altered cytokine profile of peripheral APC can be responsible for the systemic effects of UVB irradiation on the T(h)1/T(h)2 balance as well as on B cell responses.

The survival of memory T cells is critical to vaccination strategies for infectious diseases and cancer, whereas their elimination may be crucial for treatment of autoimmune states. We examined the consequences of gamma-irradiation, which induces apoptosis of memory T cells in vitro, on the memory response to MHC class I alloantigen in vivo. Sublethal gamma-irradiation of primed mice eliminated accelerated rejection of skin allografts but failed to induce tolerance. Accelerated rejection was restored in irradiated mice by infusion of bone marrow cells expressing the priming alloantigen on immunostimulatory APCs (dendritic cells), whereas the memory response was not restored by infusion of bone marrow cells expressing the priming alloantigen on nonstimulatory APCs (B cells). Strikingly, irradiated mice infused with nonstimulatory bone marrow APCs exhibited long-term survival or tolerance to skin grafts expressing the priming MHC class I alloantigen. The mechanism of tolerance in this setting is explored.

Ultraviolet A (UVA) irradiation is effectively used to treat patients with atopic dermatitis and other T cell mediated, inflammatory skin diseases. In the present study, successful phototherapy of atopic dermatitis was found to result from UVA radiation-induced apoptosis in skin-infiltrating T helper cells, leading to T cell depletion from eczematous skin. In vitro, UVA radiation-induced human T helper cell apoptosis was mediated through the FAS/FAS-ligand system, which was activated in irradiated T cells as a consequence of singlet oxygen generation. These studies demonstrate that singlet oxygen is a potent trigger for the induction of human T cell apoptosis. They also identify singlet oxygen generation as a fundamental mechanism of action operative in phototherapy.

In the determination of alloreactive helper T lymphocyte precursor frequencies (HTLpf) by limiting dilution analysis (LDA), peripheral blood mononuclear cells (PBMC) are used as stimulating cells. Interleukin-2 (IL-2) production by stimulating T cells constitute a source of error in these assays. We found that 100-150 Gy of gamma irradiation was required to abrogate IL-2 production by stimulating PBMC. This dose of irradiation, however, greatly reduced their allostimulatory capability. Here we describe how non-irradiated PBMC, immunomagnetically depleted of T cells and thus of IL-2 producing cells, can be used as stimulators in assays to determine alloresponsive HTLpf.

We describe a limiting dilution assay for the enumeration of alloreactive helper T lymphocyte precursor frequencies in human peripheral blood. The proliferation rate of the murine indicator cell line, cytotoxic T lymphoblastic line 2 (CTLL-2) induced by interleukin-2 (IL-2) culture supernatants was determined by staining with the fluorescent DNA dye propidium-iodide. Lymphocytes from healthy individuals as well as from patients with end stage kidney disease and no previous allosensitization exhibited a relative radioresistance of their IL-2 production up to gamma irradiation doses of 40-60 Gy. This differs from previous findings in the literature, showing a total inhibition of the IL-2 production in unsensitized individuals using a gamma irradiation dose of 20 Gy. The consequences of this relative radioresistance are that for a reliable stimulator cell inactivation in assays for the enumeration of helper T lymphocyte precursors gamma irradiation doses of at least 50 (-60) Gy are needed. Increasing the gamma irradiation dose for the inactivation of the stimulator cells can result in a decrease of the antigen presenting capacity of these cells.

It is confirmed that large amounts of IgM, IgG, and IgA are produced when human B cells are cultured with T cells activated by immobilized CD3 antibody (CD3 system). IL-2 was essential; lower levels of Ig production with different isotype ratios were obtained if IL-4 or IL-6 replaced IL-2. Depletion of sIgG+ or sIgA+ cells from the B population to be cultured markedly reduced production of IgG or IgA. Cultures of B cells selected with the pan-B markers CD19, CD72, or CD21 contained similar levels of Ig of all three isotypes, whereas B cells selected for sIgM or sIgD expression produced IgM but very little IgG or IgA indicating that little isotype switching was occurring. Production of IgG or IgA from cells expressing these isotypes was more efficient than production of IgM from IgM+IgD+ cells. These results are considered in the light of the demonstration by others of the production of multiple isotypes from single sIgM+-selected B cells. Cloned human T cells from a single donor induced production of all three isotypes, but the proportions varied indicating that the potent T-B cell interactions inducing B cell activation may override and conceal the operation of isotype specific cell interactions. Some T clones used at an optimal dose were as effective untreated as X-irradiated, whereas with other clones maximum Ig production was not achieved without irradiation.

The lobes of thymus of newborn mice irradiated on 17 day of embryonic life or on 1 day after the birth were transplanted under the renal capsule of adult mice. The recipients were thymectomized, lethally irradiated and reconstituted by syngeneic bone marrow cells treated by monoclonal anti-Thy-1-antibody and complement 1 month before the transplantation of thymus. Colonization of the thymus transplants, number and functional activity of T cells in peripheral lymphoid organs of recipients were measured 30 day after the transplantation of thymus. The colonization of the thymus transplants was decreased after irradiation of the thymus donors in doses of 6 and 8 Gy. The number of L3T4+ cells (T helpers) in the lymph nodes of the recipients and the level of the humoral immune response on thymus-dependent antigen were decreased after the exposure of the thymus donors to dose of 1 Gy or higher and the level of graft-versus-host reactivity of lymph node cells--after the dose of 2 Gy and higher. The degree of the suppression of the humoral immune response was higher when the donors of thymus were irradiated in embryonal period of development than after the birth. Thus the damage of the components of thymic microenvironment responsible for the T cell development can be arisen by relatively low doses of ionizing irradiation.

The micronucleus expression in T-helper, T-suppressor and B lymphocytes of the peripheral blood was studied after in vitro exposure to high (2.5 Gy and 5 Gy) and low (0.5 Gy and 1 Gy) doses of ionizing radiation. Investigations were carried out by combining the micronucleus assay with immunofluorescence staining using subpopulation specific antibodies. While in the higher dose range B cell proliferation was inhibited nearly completely-so that micronuclei could not be expressed-we found after exposure to lower doses that B cells were the lymphocyte subpopulation which was most sensitive to micronucleus induction. Among the T cell population, the T-suppressor subset revealed a higher yield of micronuclei than T-helper cells, whereas with regard to the effect of radiation on proliferative ability, T-helper cells reacted more sensitivity than the T-suppressor lymphocytes. Our studies provide insight into the effect of radiation exposure on the micronucleus expression of lymphocyte subpopulations and new information which may be useful for the further development of biological dosimetry.

Peripheral blood lymphocytes were obtained from eight untreated patients suffering from long-standing plaque-type psoriasis 1 h following UVB treatment and at the end of 15 UVB treatments. Before treatment and following in vivo treatment with 0.01-0.3 J/cm2, the patients' lymphocytes were tested for interleukin-2 (IL-2) production, and helper activity with and without addition of the immunomodulator AS101. AS101 is a synthetic organotellurium compound, ammonium trichloro-(O,O'-dioxyethylene) tellurate. UVB at a flow of 0.01 J/cm2 did not affect any of these functions. However, after 15 UVB treatments, when the dose given had reached 0.3 J/cm2, a complete inhibition of IL-2 production and helper activity was observed. In vitro addition of AS101 at 0.1 micrograms/ml to the in vivo UVB-treated lymphocytes raised IL-2 production to approximately double the initial level, and improved the helper activity of CD4 cells.

The cloning rate of PHA-stimulated T lymphocytes after treatment with 8-methoxypsoralen plus UVA irradiation described by Wunder and Reischmann (1983) gives a linear dose-effect relationship at low dosages. However, with increasing doses a flattening of the negative gradient occurs. This relationship deviates from the classical exponential curve which can be observed when fibroblasts are treated with mutagens and which is explainable by a 'recovery plateau' at lower dosages. In this study we show that some subpopulations of T lymphocytes, in particular the T-helper and T-suppressor cells, influence the overall dose-effect relationship. These isolated subpopulations exhibit varying sensitivities in comparison with their depleted cell populations. It may be assumed that heterogeneous cell populations exist within each isolated subpopulation which may be separated into further subclasses according to their specific sensitivity.

Lethally irradiated rhesus monkeys were treated with autologous bone marrow that had either been (1) nontreated, i.e., normal; (2) depleted of T lymphocytes with a monoclonal antibody directed against rhesus T lymphocytes; (3) fractionated with the soybean agglutinin (SBA- fraction); or (4) fractionated with SBA and further depleted of T cells by E-rosetting. There was no difference in hematologic reconstitution among the animals, but all showed a marked lowering of the T helper/T suppressor ratio during the first 10 months posttransplant and reduced capability of their peripheral blood leukocytes (PBL) to produce Ig upon stimulation with pokeweed mitogen. This subnormal ability of PBL to produce Ig, as measured by plaque-forming cells in a reverse hemolytic plaque assay, was not explained entirely by the altered T-H/T-S ratio but was correlated with the functional status of the T-H, T-S, and B lymphocytes. Isolated populations of the different lymphocyte subsets from PBL of the experimental animals were cocultured with normal cells of the appropriate subset to obtain Ig synthesis when stimulated with PWM. Animals treated with normal bone marrow showed recovery of T-H cell function after 5 months, but their T-S cells showed excessive suppressor activity that persisted for 20 months posttransplant. In contrast, those animals receiving treated marrow (mAb plus complement, or SBA) showed a much-delayed (12 months or more) return to normal T-H cell function and an earlier return of T-S cells expressing a normal level of suppressor activity. Since the SBA- fraction of marrow contains very few or no T-H cells and T cell depletion of marrow with mAb also removes these cells, it is suggested that the kinetics of immune recovery of the different lymphocyte subsets of PBL is influenced by the presence or absence of T-H cells in the marrow inocula.

Four to six weeks after total lymphoid irradiation (TLI), there is a selective deficit in the CD4+ T cells which secrete IL-2, proliferate in the MLR, and induce GVHD (Th1-like functions). A similar deficit in CD4+ T cells which secrete IL-4 and help antibody responses (Th2-like functions) is not observed. In the present study, shielding of the thymus with lead during TLI increased the Th1-like functions of CD4+ cells. Mice without thymus shields showed a marked selective reduction in the medullary stromal cells identified with the monoclonal antibody, MD1, and the severe reduction was prevented with thymus shields. Thus, shielding the thymus prevents the depletion of thymic medullary stromal cells and allows for a rapid recovery of Th1-like functions in the mouse spleen after TLI. Th2-like functions recover rapidly after TLI whether or not the thymus is irradiated.

Merocyanine 540 (MC 540) is a photoactive dye used to purge bone marrow of tumor cells in autologous bone marrow transplantation. The effects of MC 540 on the lymphoid components in the marrow are unknown. This study evaluates the treatment of lymphocytes by MC 540 (15 micrograms/mL) and light (70 W/m2) on: (1) phytohemagglutinin and Con A-induced proliferation; (2) allogeneic mixed lymphocyte cultures (MLC); (3) the regulation of Ig synthesis by T cells; and (4) the ability of B cells to produce polyclonal Igs as measured by an enzyme-linked immunosorbent assay-plaque assay. The results show that MC 540 and light treatment reduced Con A-stimulated T-cell proliferation greater than 50% after 30 minutes and greater than 80% after 60 minutes of MC 540-sensitized photoirradiation. Ninety minutes of MC 540 and light exposure (designated treatment) inhibited MLC greater than 90%. In polyclonal Ig synthesis, T-cell helper activity could be abrogated by 90 minutes of treatment in cocultures containing untreated B cells. Purified B cells treated for 90 minutes cocultured with normal T cells did not produce Ig. Treatment of B cells completely inhibited Epstein-Barr virus-stimulated Ig synthesis. These data show that T- and B-cell immunity is suppressed by the MC 540-sensitized photoirradiation. Treatment of bone marrow with MC 540 and light may have profound effects on immune reconstitution in autologous marrow graft recipients. More provocative is the fact that the same immunomodulatory effects may be applicable to partially mismatched marrow transplant situations as a means of reducing graft-versus-host reactions.

We have detected and characterized a subpopulation of immunoregulatory cells, i.e., B-helpers capable to enhance the activity of Td-lymphocytes and controlling differentiation of syngeneic hemopoietic stem cells in mouse spleen and bone marrow. B-helpers found in the spleen and lymphatic nodes are resistant to radiation (at a dose of 6 Gr) but are impaired when irradiated at 9 Gr. Manifestation of the helper activity does not require either DNA or RNA synthesis but depends on protein synthesis and is mediated by soluble transmitter substances. Initial activation of B-helpers by lipopolysaccharide or alloantigens does not affect their helper functions. In the absence of T-lymphocytes B-cells do not affect differentiation of hemopoietic stem cells; interaction of B-helpers with differentiating Td-lymphocytes is not genetically restricted. Using preparative electrophoresis, we could isolate fractions of Td-lymphocytes which require or do not require B-helper cells in order to induce change in differentiation of hemopoietic stem cells from mainly erythroid to preferentially granulocyte pathway.

We have recently demonstrated that a single dose (200 J/m2) of UVB radiation abrogates the capacity of mouse epidermal Langerhans cells (LC) or splenic adherent cells (SAC) to present keyhole limpet hemocyanin (KLH) to Ag-specific, MHC-restricted CD4+ Th1 cells. In the present study we determined whether such Th1 unresponsiveness represented long-lasting immunologic tolerance. To address this question, Th1 were preincubated with KLH-pulsed UVB-LC or UVB-SAC, then isolated and restimulated with unirradiated APC (LC or SAC) plus KLH or with exogenous rIL-2 in the absence of APC. Preincubation with KLH and UVB-LC or UVB-SAC rendered Th1 unresponsive to subsequent restimulation with APC and KLH. In addition, such Th1 were defective in their autocrine IL-2 production, but could respond normally to exogenous rIL-2, indicating that unresponsiveness was due to functional inactivation and not to cell death. Th1 unresponsiveness was Ag-specific, MHC-restricted, and long lasting (greater than 16 days). In addition, it appears that Th1 unresponsiveness is not due to the release of soluble suppressor factors from UVB-LC or UVB-SAC because supernatants from such cells had no effect on Th1 proliferation. Addition of unirradiated allogeneic SAC during preincubation prevented the induction of unresponsiveness by UVB-LC or UVB-SAC, suggesting that UVB interferes with the capacity of LC or SAC to deliver a costimulatory signal(s) that can be provided by allogeneic SAC. We conclude that UVB can convert LC or SAC from immunogenic to tolerogenic APC.

Spleen cells from BALB/c mice treated with total lymphoid irradiation (TLI) and from normal, unirradiated mice were compared in the mixed leukocyte reaction (MLR). Although the percentage of CD4+ cells in the spleen was close to normal, 4 to 6 weeks after TLI, the MLR of unfractionated spleen cells from irradiated mice was more than 10-fold lower than controls. A similar reduction was observed when purified CD4+ cells were used as responders in the MLR. Secretion of IL-2 by cells from irradiated mice was also about 10-fold lower than controls. However, the percentage of CD4+ and CD8+ cells which expressed IL-2 surface receptors during the MLR was similar using spleen cells from irradiated and control mice. Addition of an exogenous source of IL-2 restored the proliferative capacity of the irradiated cells and suggests that the lack of IL-2 secretion is the likely explanation of the marked deficit in the MLR of CD4+ spleen cells after TLI.

Mitogen driven differentiation of normal human mononuclear cells is a well-established model for the study of antibody synthesis in man. In certain rare individuals who are clinically normal, unfractionated mononuclear cells or a mixture of purified B plus T lymphocytes differentiate into immunoglobulin producing cells in response to purified protein derivative of tuberculin (PPD) but not in response to pokeweed mitogen (PWM). To evaluate this observation we have irradiated T cells from such individuals to eliminate naturally occurring suppressor T cell activity and then added the irradiated T cells back to autologous B cells before culture. The B cells then responded to PWM. The original PPD responses of cells from these individuals were now significantly reduced. Although, there was no difference between PWM nonresponders and responders in the number of OKT-8 positive cells, elimination of OKT-8 positive cells in the PWM nonresponders with OKT-8 monoclonal antibody and complement resulted in a significantly increased response to PWM. This study indicates that there are suppressor T cells which specifically inhibit B cell response to PWM without affecting the PPD response. These results also show that the helper T cells involved in the PWM response are radioresistant and those involved in the PPD response are radiosensitive.

The functional effects of anti-Ig antibodies have been investigated, using an experimental system where B cell activation is brought about by direct and specific interactions with T helper (Th) cells without participation of surface Ig receptors on the responding B cell. We have used Th cell lines and clones directed to class II major histocompatibility complex antigens of the responding B cells, and titrated into cooperative cultures either purified rabbit anti-mouse mu, or monoclonal mouse anti-delta antibodies. Both types of antibodies greatly enhanced B lymphocyte responses to suboptimal concentrations of functionally efficient Th cells, while they had no effect in cultures containing optimal Th:B cell ratios. In contrast, helper activity by low efficiency Th was, at all Th:B cell ratios, enhanced by appropriate concentrations of anti-Ig antibodies. Anti-Ig effects were exclusively observed when B cells were the targets for "cognate" recognition by Th cells. We conclude that ligand binding to surface Ig receptors on resting B cells fails, in our experimental conditions, to overcome "linked" collaboration, but it greatly facilitates productive Th-B cell interactions. Whatever the mechanisms underlying this facilitation, the observations imply roles of surface Ig in Th-dependent B lymphocyte activation other than either passive "focusing" of antigen or activation into reactivity to soluble, unspecific factors.

Cyclosporin A (CsA) is an undecapeptide fungal metabolite and is generally regarded as a new generation of immunosuppressive drugs. We uncovered a novel immunomodulatory property of CsA as a potent immunologic stimulator in the murine IgE antibody system. The enhancement of IgE responses was observed in mice receiving as few as three daily i.m. injections before Ag priming. Our studies demonstrate the three points listed below. First, CsA potentiates murine IgE responses regardless of Ag specificities in inbred mice. A hierarchy of immunopotentiation by CsA follows the order of low, intermediate, and high IgE responder mice. Second, CsA, when administered along with Ag, exerts a thorough and long lasting impact on the Ag-specific IgE antibody response, and leads to an Ag-specific breakthrough of IgE antibody synthesis in mice rendered tolerant in the IgE antibody system by soluble Ag pretreatment or neonatal IgE treatment. Third, IgE enhancer cells become sensitive to a low dose of irradiation. Two enhancer cellular components are identified, those of the Th cells and B cells, which appear to favor the induction of IgE responses. Understanding the cellular basis of the immunopotentiating effect of CsA will provide further insight into the murine IgE antibody system.

We studied the influence of radiation therapy on lymphocyte subpopulations in 17 patients undergoing adjuvant radiation therapy for primary breast cancer, and eight patients receiving brachytherapy and external beam irradiation for primary cancer of the uterus. Radiation therapy reduced B- and T-lymphocytes in proportion to the total lymphocyte population so that their percentages remained unchanged. Determination of helper and suppressor T-lymphocytes before, during and 6 months after completion of radiotherapy revealed that in both groups of patients suppressor T-lymphocytes were more resistant to and recovered faster after radiotherapy. This resulted in a decline of the "immunoregulatory balance" (helper/suppressor ratio). Although this ratio had been higher in both groups of patients than in healthy age- and sex-matched controls before therapy, it became normal and subnormal during and after radiotherapy. The clinical significance of the differential influence of radiotherapy on T-lymphocyte subpopulations remains to be determined.

Prolonged kidney and liver allograft survival was produced in baboons by low cumulative doses (500 to 1200 rad) of total lymphoid irradiation (TLI). Continuing normal graft function for more than 1 yr after transplantation was seen in 10 animals in this series. The longest survivor (greater than 4 years) rejected a third party kidney allograft in typically acute fashion 1 yr after the original transplantation. The dose-response effect was parabolic, with a cumulative dose of 800 rad given as twice weekly fractions of 100 rad each being the most effective. With smaller cumulative doses (500 or 600 rad) results were improved if TLI was administered as a larger number of smaller fractions. These regimens are free of the radiation related mortality observed in earlier studies with larger cumulative doses. As in previous studies, the injection of donor bone marrow was not essential for prolonged and continuing graft survival. The field irradiated was extensive, and included a substantial part of the of the hemopoietic bone. Restricting the field irradiated to that approximating what is used clinically in patients with Hodgkin's disease resulted in markedly poorer graft survival. Alterations in T cell subpopulations were studied in these baboons with the use of the anti-human monoclonal antibodies OKT11 (anti-total T cells), OKT4 (anti-T helper cells; Th), and OKT8 (anti-T suppressor/cytotoxic cells, Tsc), which cross-react with baboon lymphocytes. After completion of TLI but before transplantation, the relative percentage of Tsc cells fell and the percentage of Th and the Th/Tsc ratio rose. Transplantation was followed by an inversion of this ratio due to reciprocal changes in the percentages of Th and Tsc cells. This pattern persisted and increased until 6 mo after transplantation, when it reverted to that seen in untreated control animals. This was maintained in long surviving animals up to 17 mo after transplantation. Transplantation was also followed by a marked early increase in the percentage of Ia+ (OKIa1 anti-human monoclonal antibody) total T, T4+, and T8+ cells. If findings in the human, in which activated suppressor but no cytotoxic T cells are Ia+, can be extrapolated to the baboon, a possible explanation for the serial changes observed in the present studies may be that transplantation after TLI results in a polyclonal expansion of nonspecific suppressor T cells. These disappear with time leaving specific suppressor T cells that may be responsible for continuing graft acceptance. Formal proof of this hypothesis will require appropriate functional studies.

The different requirements of human T lymphocytes of different densities for accessory cells and helper factors (Interleukin 1 (Il-1) and Interleukin 2 (Il-2) ) in the response to phytohaemagglutinin (PHA) were investigated. Human T lymphocytes were subfractionated by discontinuous density gradient centrifugation. The various T-cell subsets were stimulated by PHA to form colonies in an agar micro-culture in the presence or absence of additional adherent cells or crude preparations of Il-1 or Il-2. The results show that the higher the density of the fractionated T lymphocytes and the lower the number of cells cultured, the greater is the number of adherent cells or the amount of helper factors required for the stimulation of colony-forming T lymphocytes. The results are consistent with the assumption that monocytes provide positive modulating activity during mitogenic stimulation of colony-forming T lymphocytes. The number of monocytes necessary for exerting an optimal modulating activity depends on the number of T cells cultured and the density of the T-cell fraction. This may reflect a distinct susceptibility of T cells of different densities to monocyte-mediated helper effects.

We have used cloned alloreactive helper T cells to determine if direct T cell-B cell interaction can induce differentiation of human peripheral blood B cells which do not respond to pokeweed mitogen (PWM). T-cell clone 2F8 was derived from a one-way mixed lymphocyte reaction. 2F8 cells are T3+T4+T8-IL-2R+ and proliferate in response to irradiated stimulator cells, but not autologous cells, in the absence of exogenous interleukin-2. 2F8 cells provide allospecific help for polyclonal proliferation and differentiation of B cells in the absence of any other stimulus. The magnitude of this response is comparable to that of the response of the same B cells to PWM and fresh autologous T cells. 2F8 cells could also provide nonspecific help for unrelated donor B cells in the presence of PWM, with no requirement for costimulation by irradiated stimulator cells. Allospecific stimulation of B cells was completely inhibited by antibodies to class II major histocompatibility complex (MHC) framework determinants and was abrogated by 1000-rad irradiation. Cloned 2F8 T cells stimulated differentiation of both small, high-density B cells and larger B cells, generating up to 30% plasma cells with either fraction. B cells forming rosettes with mouse erythrocytes were also induced to differentiate by the helper T cell clone. As found previously, neither small, high-density B cells nor mouse rosette+ B cells responded well to PWM. Direct interaction with allospecific T cells induces differentiation of a broader spectrum of B cells than soluble growth and differentiation factors in conjunction with polyclonal activators such as PWM and protein A containing staphylococci.

Popliteal lymph node T cells from mice footpad-primed with azobenzenearsonate (ABA)-protein conjugates were able to help the anti-trinitrophenyl (TNP) and anti-ABA plaque-forming cell (PFC) responses of normal syngeneic spleen cells cultured in vitro with TNP-ABA-keyhole limpet haemocyanin. Enrichment in ABA-specific helper cells was obtained by positive selection of ABA-primed T cells on ABA-pulsed syngeneic macrophages. The ABA-specific T cells induced by ABA-protein priming are able to help the anti-TNP PFC response of normal B cells through recognition of the ABA determinant either unlinked to TNP (ABA and TNP separately presented to T and B cells) or linked to TNP (ABA and TNP presented as moieties of the same molecule). These two mechanisms of T-B cooperation are mediated by two different ABA-specific helper T-cell subpopulations, which can be distinguished by their different radiosensitivities: the former mechanism is mediated by radioresistant T cells, whereas the latter is mediated by radiosensitive T cells. Both helper T-cell subpopulations bind the ABA-pulsed syngeneic macrophages, demonstrating the presence of ABA-specific receptors on both cell types.

Clones of inducer cells activated by antigen and class II major histocompatibility complex (MHC) gene products synthesize large amounts of several distinct mRNA species not detected in other activated cell types, including antigen-activated suppressor or killer cell clones. Although inducer cells continued to synthesize and secrete peptides at about the same rate for 5 days after activation by adherent cells and antigen, they expressed a new set of mRNAs approximately 3 days after activation. We therefore tested the functional activity of purified Ly1 cells at different days after activation by adherent cells and antigen. We wished to find out (a) whether there was a qualitative or quantitative change in the level of inducer activity, and if so, (b) whether this functional change was an intrinsic property of Ly1 cells or, rather, was dependent on signals from the cellular environment, in particular from adherent antigen-presenting cells. We incubated purified Ly1 cells and splenic adherent cells for 1-7 days in vitro, and tested inducer activity in cultures containing highly purified B cells and sheep erythrocytes (SRBC). Anti-SRBC plaqueforming cells were counted 5 days later. We found that inducer cells that have been incubated with adherent cells in culture for more than 72 h (a) did not induce B cells to produce antibody, and (b) prevented virgin but not immune B cells from receiving T-helper signals. We term this latter phenomenon "paralysis." Acquisition of the ability to paralyze virgin B cells required an I-E gene-regulated interaction between inducer cells and in vitro-activated adherent cells. This interaction did not require antigen and was associated with transition from Ly1:Qa1- to Ly1:Qa1+ cell-surface phenotype. Taken together these findings indicate that (a) interactions between inducer cells and adherent antigen-presenting cells result first in classical inducer ("helper") activity and later in expression of paralytic activity and (b) sequential expression of these inducer activities depends on two distinct signals, supplied by resting and activated adherent cells, respectively. The signal supplied by autologous activated adherent cells is regulated by I-E gene products and is independent of corecognition of foreign proteins. The physiologic significance of this paralytic inducer activity in the prevention of potentially harmful immune reactions to foreign microbial agents is discussed.