Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies

Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed.Key words: monoclonal antibodies, non-clinical testing, immunopharmacology, immunotoxicity, cytokine release, immunosuppression, autoimmunity, hypersensitivity, immunogenicity, anti-drug antibody, MABEL     

Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28

Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models.

We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release.

In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo.     

Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28

Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models. We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release. In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo.     

Screening of xenobiotics for direct immunotoxicity in an animal study

It has now been recognised that the immune system as a whole can be the target for xenobiotic induced toxicity. The discipline of immuntoxicology encompasses non specific direct immunotoxicity and immunostimulation, and specific responses like hypersensitivity and autoimmunity. Direct immunotoxicity can be determined in tiered studies, TIER 1 being a general toxicity study with emphasis on evaluation of organs belonging to the immune system, TIER 2 investigating the effects of xenobiotics on immune functionality in immunological challenge experiments. In the TIER 1 study, organ weights and histopathological evaluation of immune organs like spleen, thymus, lymph nodes, blood and bone marrow may detect the occurrence of direct immunotoxicity. The follow up studies in the TIER 2 phase can then determine the extent of the immunosuppression and identify which specific parts or cellular components of the immune system are involved. In view of the complexity of the immune system and the multitude of interactions within the immune system in vivo animal experiments are needed to investigate xenobiotics for their potential immunotoxicity. In vitro assays with in vivo exposed cells of the immune system may present additional information on the mechanisms involved in the observed direct immunotoxicity.     

Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies

Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease, and as such are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed.     

Design, synthesis, and biological evaluation of 8-biarylquinolines: a novel class of PDE4 inhibitors

The structure-activity relationship of a novel series of 8-biarylquinolines acting as type 4 phosphodiesterase (PDE4) inhibitors is described herein. Prototypical compounds from this series are potent and non-selective inhibitors of the four distinct PDE4 (IC(50)<10 nM) isozymes (A-D). In a human whole blood in vitro assay, they inhibit (IC(50)<0.5 microM) the LPS-induced release of the cytokine TNF-alpha. Optimized inhibitors were evaluated in vivo for efficacy in an ovalbumin-induced bronchoconstriction model in conscious guinea pigs. Their propensity to produce an emetic response was evaluated by performing pharmacokinetic studies in squirrel monkeys. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of efficacy over emesis.     

Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.     

Evidence for Clostridium perfringens Enterotoxin (CPE) Inducing a Mitogenic and Cytokine Response In Vitro and a Cytokine Response In Vivo

We investigated some immunogenic properties of Clostridium perfringens enterotoxin (CPE) in vitro using murine J774A macrophages (MΦ) and in vivo using Swiss Webster (SW) mice. CPE was a potent mitogen in vitro, where cell proliferation increased with CPE concentration. CPE was nonmitogenic when MΦ were concurrently incubated with CPE and interferon gamma (IFN-γ). MΦ incubated in the presence of CPE induced the synthesis of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ), but not interleukin-2 (IL-2). In vivo, CPE induced a pro-inflammatory cytokine response with striking production of IFN-γ, IL-1, and IL-6. Regardless of route of CPE entry, serum cytokine levels generally peaked within 1 h of administration and were maintained for 4–8 h. Although CPE engenders an intense immune response during toxicosis, the toxin does not appear to be a superantigen. Death from CPE-induced shock appears to result from various interrelating immunological mechanisms. Received: 25 April 1998 / Accepted: 2 September 1998     

"Cytokine storm" in the phase I trial of monoclonal antibody TGN1412: better understanding the causes to improve preclinical testing of immunotherapeutics

The CD28-specific mAb TGN1412 rapidly caused a life-threatening "cytokine storm" in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.     

Zinc inhibits the mixed lymphocyte culture

The mixed lymphocyte culture (MLC) is an established clinical method for bone marrow transplantation, as it serves as an in vitro model for allogenic reaction and transplantation. We previously showed that cytokine release into the supernatant is a more specific and sensitive parameter for cross-reactivity in the MLC than the common measurement of cell proliferation. Therefore we tried to find an inhibitor of the MLC in vitro with the least side effects in vivo, measuring interferon (IFN)-γ as one of the most important cytokines in posttransplant medicine. Earlier studies showed that zinc is an important trace element for immune function with both stimulatory and inhibitory effects on immune cells. We found that slightly elevated zinc concentrations (three to four times the physiological level), which do not decrease T-cell proliferation in vitro nor produce immunosuppressive effects in vivo, suppress alloreactivity in the mixed lymphocyte culture. In this report we analyzed the mechanism whereby zinc influences the MLC to possibly find a nontoxic way of immunosuppression.     

In vitro and in vivo activities of leukotriene B4-loaded biodegradable microspheres

Leukotriene B(4) (LTB(4)) is a potent inflammatory mediator and stimulates the immune response. In addition, LTB(4) promotes leukocyte functions such as phagocytosis, chemotaxis and chemokinesis of polymorphonuclear leukocytes, as well as modulates cytokine release. However, some physicochemical characteristics of leukotrienes, such as poor solubility in water and chemical instability, make them difficult to administer in vivo. The aim of this study was to develop LTB(4)-loaded microspheres (MS) that prolong and sustain the in vivo release of this mediator. An oil-in-water emulsion solvent extraction-evaporation method was chosen to prepare the lipid-loaded MS. We determined their diameters, evaluated the in vitro release of LTB(4), using enzyme immunoassay and evaluated in vitro MS uptake by peritoneal macrophages. To assess the preservation of neutrophil chemoattractant activity, LTB(4)-loaded MS were tested in vitro (in a modified Boyden microchamber) and in vivo, after intratracheal administration.     

Design and preparation of 2-benzamido-pyrimidines as inhibitors of IKK

The design, synthesis, and the biological evaluation of 2-benzamido-pyrimidines as novel IKK inhibitors are described. By optimization of the lead compound 3, compounds 16 and 24 are identified as good inhibitors of IKK2 with IC(50) values of 40 and 25 nM, respectively. Compound 16 also demonstrated significant in vivo activity in an acute model of cytokine release.     

The Registry of Cytotoxicity: toxicity testing in cell cultures to predict acute toxicity (LD50) and to reduce testing in animals

This is a translation of a report on the Registry of Cytotoxicity (RC), originally published in German in 1998. The report presented an advanced in vitro method, which can significantly reduce the number of animals needed for the toxicity testing of a broad range of compounds/xenobiotics. With the RC method, it was possible to predict the oral or intravenous acute toxicity (LD50)--which is a regulatory requirement for newly developed pharmaceuticals and industrial and household chemicals--from the cytotoxicity data (mean IC50 = IC50X) obtained with mammalian cells. The RC method can be used before the in vivo test, and it does not pose any additional harm or suffering to laboratory animals. The RC method is of broad practical use: it can be applied, for example, in the pharmaceutical industry or the chemical industry in regulatory testing or in research. It is ready for validation, and could then be incorporated into OECD guidelines, thus reducing the total number of animals needed for regulatory toxicity testing. The RC method is based on the comparison of the IC50X values and the LD50 values by using linear regression analysis. With the RC method, it was possible to predict, within a predefined dose range, the acute oral LD50 for 252 of 347 xenobiotics, and the intravenous LD50 for rats and/or mice for 117 of 150 xenobiotics. Comparative studies showed that these results are highly reproducible.     

Estrogen reduces pro-inflammatory cytokines in rodent adipose tissue: studies in vivo and in vitro.

Obesity is associated with an increased risk of developing insulin resistance, Type 2 diabetes, and cardiovascular disease. Reports have suggested that adipose tissue-derived cytokines such as tumor necrosis factor-alpha, interleukin-6 and interleukin-8 could be involved in the development of these health complications. Since estrogen has been suggested to attenuate the development of atherosclerosis and cardiovascular disease, we investigated whether ovariectomy affected the production and release of these three adipose tissue-derived cytokines with and without estrogen replacement in vivo and in vitro. Female Wistar rats were submitted to either a) ovariectomy, b) ovariectomy and estrogen replacement, or c) sham operation. After five months, animals were sacrificed and parametrial adipose tissue was removed and incubated for up to 24 hours with either interleukin-1beta (IL-1beta) (5 micro g/l), dexamethasone (50 nM) or estrogen (50 nM). Ovariectomy significantly increased interleukin-6 gene expression (p < 0.05) as well as interleukin-8 protein levels (p < 0.05) and gene expression (p < 0.05) in the adipose tissue, and estrogen replacement significantly reversed this increase (p < 0.05). However, no direct effects of estrogen were found in in vitro adipose tissue incubations. Neither ovariectomy nor estrogen replacement had any effects on tumor necrosis factor-alpha protein levels or gene expression. In conclusion, estrogen-deficient rats were found to have increased production of interleukin-6 and interleukin-8, which could be attenuated by estrogen-replacement. Since estrogen is suggested to be anti-atherosclerotic, this effect might be caused by a reduction in cytokine production from the adipose tissue.     

Organotypic brain slice cultures: an efficient and reliable method for neurotoxicological screening and mechanistic studies

This paper reviews the current state of the use of organotypic brain slice cultures for neurotoxicological and neuropharmacological screening and mechanistic studies, as exemplified by excitotoxin application. At present, no in vitro systems have been approved by the regulatory authorities for neurotoxicity testing. For the evaluation of the slice culture method, organotypic hippocampal slice cultures were exposed to toxic doses of the excitotoxins, glutamate, N-methyl-D-aspartate (NMDA), kainic acid and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and the glial toxin, DL-alpha-aminoadipic acid (DLAAA). Neuronal cell death was quantified by propidium iodide (PI) uptake, and visualised by Fluoro-Jade (FJ) staining. General cell death was monitored by lactate dehydrogenase (LDH) release into the culture medium. EC50 values for the different compounds, based on PI uptake after exposure for 48 hours in entire cultures, were: glutamate, 3.5 mM; DL-AAA, 2.3 mM; kainic acid, 13 microM; NMDA, 11 microM; and AMPA, 3.7 microM. In the slice cultures, the hippocampal subfields displayed the same differences in vulnerability as those observed in vivo. When subfield analysis was performed on the cultures, the CA1 subfield was most susceptible to glutamate, NMDA and AMPA, while CA3 was most susceptible to kainic acid. The amount of LDH release for DL-AAA was about four times that of L-glutamate, in accordance with the additional toxic effect on glial cells, which was also found by confocal microscopy to stain for FJ. In conclusion, it was found that organotypic brain slice culture, combined with standardised protocols and quantifiable markers, such as PI and FJ staining, is a relevant and feasible in vitro system for neurotoxicity testing. Considering the amount and quality of the available published data, it is recommended that the brain slice culture method could be subjected to pre-validation and formal validation for inclusion in a tiered in vitro neurotoxicity testing scheme to supplement and replace conventional animal tests.     

Janus kinase inhibition suppresses PKC-induced cytokine release without affecting HIV-1 latency reversal ex vivo

Background

Despite the durable viral suppression afforded by antiretroviral therapy, HIV-1 eradication will require strategies to target latently infected cells that persist in infected individuals. Protein kinase C (PKC) activation is a promising strategy to reactivate latent proviruses and allow for subsequent recognition and clearance of infected cells by the immune system. Ingenol derivatives are PKC agonists that induce latency reversal but also lead to T cell activation and the release of pro-inflammatory cytokines, which would be undesirable in vivo. In this work, we sought to identify compounds that would suppress pro-inflammatory cytokine production in the context of PKC activation.

Design and methods

We performed an in vitro screen to identify compounds that could dampen pro-inflammatory cytokine release associated with T cell activation, using IL-6 as a model cytokine. We then tested the ability of the most promising screening hit, the FDA-approved Janus Kinase (JAK) inhibitor ruxolitinib, to diminish release of multiple cytokines and its effect on latency reversal using cells from HIV-1-positive, aviremic participants.

Results

We demonstrate that co-administration of ruxolitinib with ingenol-3,20-dibenzoate significantly reduces pro-inflammatory cytokine release without impairing latency reversal ex vivo.

Conclusion

The combination of ingenol compounds and JAK inhibition represents a novel strategy for HIV-1 eradication.

     

A novel therapeutic approach targeting articular inflammation using the filarial nematode-derived phosphorylcholine-containing glycoprotein ES-62

Understanding modulation of the host immune system by pathogens offers rich therapeutic potential. Parasitic filarial nematodes are often tolerated in human hosts for decades with little evidence of pathology and this appears to reflect parasite-induced suppression of host proinflammatory immune responses. Consistent with this, we have previously described a filarial nematode-derived, secreted phosphorylcholine-containing glycoprotein, ES-62, with immunomodulatory activities that are broadly anti-inflammatory in nature. We sought to evaluate the therapeutic potential of ES-62 in vitro and in vivo in an autoimmune disease model, namely, collagen-induced arthritis in DBA/1 mice. ES-62 given during collagen priming significantly reduced initiation of inflammatory arthritis. Crucially, ES-62 was also found to suppress collagen-induced arthritis severity and progression when administration was delayed until after clinically evident disease onset. Ex vivo analyses revealed that in both cases, the effects were associated with inhibition of collagen-specific pro-inflammatory/Th1 cytokine (TNF-alpha, IL-6, and IFN-gamma) release. In parallel in vitro human tissue studies, ES-62 was found to significantly suppress macrophage activation via cognate interaction with activated T cells. Finally, ES-62 suppressed LPS-induced rheumatoid arthritis synovial TNF-alpha and IL-6 production. Evolutionary pressure has promoted the generation by pathogens of diverse mechanisms enabling host immune system evasion and induction of "tolerance." ES-62 represents one such mechanism. We now provide proof of concept that parasite-derived immunomodulatory strategies offer a novel therapeutic opportunity in inflammatory arthritis.     

The ECVAM international validation study on in vitro tests for acute skin irritation: report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test

ECVAM sponsored a formal validation study on three in vitro tests for skin irritation, of which two employ reconstituted human epidermis models (EPISKIN, EpiDerm), and one, the skin integrity function test (SIFT), employs ex vivo mouse skin. The goal of the study was to assess whether the in vitro tests would correctly predict in vivo classifications according to the EU classification scheme, "R38" and "no label" (i.e. non-irritant). 58 chemicals (25 irritants and 33 non-irritants) were tested, having been selected to give broad coverage of physico-chemical properties, and an adequate distribution of irritancy scores derived from in vivo rabbit skin irritation tests. In Phase 1, 20 of these chemicals (9 irritants and 11 non-irritants) were tested with coded identities by a single lead laboratory for each of the methods, to confirm the suitability of the protocol improvements introduced after a prevalidation phase. When cell viability (evaluated by the MTT reduction test) was used as the endpoint, the predictive ability of both EpiDerm and EPISKIN was considered sufficient to justify their progression to Phase 2, while the predictive ability of the SIFT was judged to be inadequate. Since both the reconstituted skin models provided false predictions around the in vivo classification border (a rabbit Draize test score of 2), the release of a cytokine, interleukin-1alpha (IL-1alpha), was also determined. In Phase 2, each human skin model was tested in three laboratories, with 58 chemicals. The main endpoint measured for both EpiDerm and EPISKIN was cell viability. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1alpha release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. For EPISKIN, the sensitivity was 75% and the specificity was 81% (MTT assay only); with the combination of the MTT and IL-1alpha assays, the sensitivity increased to 91%, with a specificity of 79%. For EpiDerm, the sensitivity was 57% and the specificity was 85% (MTT assay only), while the predictive capacity of EpiDerm was not improved by the measurement of IL-1alpha release. Following independent peer review, in April 2007 the ECVAM Scientific Advisory Committee endorsed the scientific validity of the EPISKIN test as a replacement for the rabbit skin irritation method, and of the EpiDerm method for identifying skin irritants as part of a tiered testing strategy. This new alternative approach will probably be the first use of in vitro toxicity testing to replace the Draize rabbit skin irritation test in Europe and internationally, since, in the very near future, new EU and OECD Test Guidelines will be proposed for regulatory acceptance.     

Heterogeneous cytokine production by acutely stimulated bronchoalveolar T lymphocytes in reovirus 1/Lang-infected mice

While the in vitro properties of CD4(+) and CD8(+) cytokine-producing lymphocytes have been well studied, the in vivo cytokine production patterns and relative roles of CD4(+) and CD8(+) T lymphocytes during a primary in vivo immune response remain unclear. In this study, mice were inoculated intranasally with reovirus 1/L, and respiratory T lymphocyte populations were analyzed using multicolor flow cytometric analysis for the production of cytokine within and between classical type 1/type 2 patterns. Cytokine production observed in vivo following infection did not correlate with classical T cell cytokine expression patterns; instead, multiple types of lymphocyte populations that produced one of several possible cytokine combinations were present. Cytokine production by CD4(+) lymphocytes appears in the early and middle stages of the immune response, while CD8(+) lymphocytes produce more cytokine in the later stages. Early cytokine responses occurred predominantly in the whole lung and lung-associated lymph node populations. The complex patterns of cytokine expression seen in this study likely influence local cell-mediated immunity as well as the complex interaction of T cell subsets and the interaction of T cells with B cells which are necessary for the generation of cell-mediated and humoral immune responses required for effective broad-spectrum immunity.     

Lymphocyte subset analyses in blood, spleen and lymph nodes of female Sprague-Dawley rats after short or prolonged exposure to a 50 Hz 100-microT magnetic field.

Based primarily on the results of in vitro studies, it has been suggested that power-line (50 or 60 Hz) magnetic fields (MFs) may reduce immune function, which could lower resistance to infection or cancer. This study was conducted to evaluate the influence of acute and chronic in vivo exposure to a linearly polarized 50 Hz MF on immune function in female Sprague-Dawley rats. Groups of rats were exposed continuously to the MF at a flux density of 100 microT for periods of 3 days, 14 days or 13 weeks. For each exposure period, one control group of rats was sham-exposed together with each MF-exposed group. Experimental end points included analyses of T-lymphocyte subsets as well as other immune cells involved in cell-mediated immune responses, i.e. natural killer (NK) cells, B lymphocytes, macrophages, and granulocytes in blood, spleen and mesenteric lymph nodes. In addition, immunohistochemical methods were used to detect proliferating and apoptotic cells in the various compartments of spleen tissue. The results obtained failed to demonstrate a significant effect of short or prolonged MF exposure on different types of leukocytes, including lymphocyte subsets. Furthermore, the experiments on the in vivo proliferation activity of lymphocytes and the extent of apoptosis in spleen samples did not indicate a difference between the MF-exposed and sham-exposed groups, indicating that MF exposure does not affect the mechanisms involved in the control of lymphocyte homeostasis. The lack of MF effects in the immune tests used in the present in vivo study makes it highly unlikely that MF exposure induces immunotoxicity, at least under the experimental conditions used. However, the data do not exclude the possibility that functional alterations in T-cell responses to mitogens and in NK cell activity as recently described for MF-exposed rodents may be one mechanism involved in the carcinogenic effects of MF exposure observed in some models of co-carcinogenesis.