Immunological Effects of ChlorinatedDibenzo-p-dioxinsNancy 1. KerkviietDepartment of Agricultural Chemistry, Oregon State University,Corvallis, Oregon

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally similar halogenated aromatic hydro-carbons cause a broad range of immunologic effects in experimental animals including decreasedhost resistance to infectious disease and suppressed humoral and cell-mediated immuneresponses. In the mouse, TCDD immunotoxicity has been shown to be an aryl hydrocarbon (Ah)receptor-dependent process. However, despite considerable research, the biochemical andmolecular alterations that occur subsequent to Ah receptor activation that lead to altered immunereactivity remain to be elucidated. In addition to immune suppression, TCDD promotes inflamma-tory responses. This effect may result from an upregulation of the production of inflammatorycytokines such as interleukin-1 and tumor necrosis factor. Nonhuman primates exposed to TCDDshow suppressed antibody responses and changes in lymphocyte subsets in the peripheral blood.The immunotoxic effects of TCDD in humans are poorly characterized, and few studies haveexamined the immune status of individuals with known, documented exposure to TCDD. It isimportant for laboratory research to focus on defining TCDD-sensitive immunologic biomarkers inanimal models that can also be used in human subjects. Understanding the mechanisms thatunderlie species differences in TCDD immunotoxicity is also of critical importance for extrapolationof effects seen in laboratory animals to man. Environ Health Perspect 1 03(Suppl 9):47-53 (1995)

Key words: dioxins, immunotoxicity of HAH, health effects of dioxins

Introduction2,3,7,8-Tetrachlorodibenzo-p -dioxin(TCDD) has posed much concern to boththe public and the scientific communitybecause of its toxic potency and widespreaddistribution in the environment. TCDD isthe most toxic member of a large class ofplanar halogenated aromatic hydrocarbons(HAH) that include other environmentalcontaminants such as polychlorinatedbiphenyls (PCBs) and dibenzofurans(PCDFs). These chemicals are unusual in

This paper was prepared for the Great Lakes HealthEffects Program which is part of a CanadianDepartment of Health Initiative established in 1989.Manuscript received 24 October 1994; manuscriptaccepted 19 April 1995.

Excerpts of this manuscript were used for thepreparation of the "Canada-U.S. Position Paper onGreat Lakes Health Effects" (Environmental Health,Directorate, Health Canada).

Address correspondence to Dr. Nancy I. Kerkvliet,Department of Agricultural Chemistry, ALS 1007,Oregon State University, Corvallis, OR 97331.Telephone: (503) 737-4387. Fax: (503) 737-0497. E-mail: kerkvlin@ccmail.orst.edu

Abbreviations used:TCDD, 2,3,7,8-tetrachloro-dibenzo-p-dioxin; HAH, halogenated aromatic hydro-carbons; PCBs, polychlorinated biphenyls; PCDFs,polychlorinated dibenzofurans; DREs, dioxin-responseelements; SRBC, sheep red blood cells; CTL, cyto-toxic T lymphocyte; NK, natural killer cell; LPS, endo-toxin; IL-1, interleukin-1; TNF, tumor necrosis factor;NOEL, no observable effect level; IFNy, interferon-y.

that most of their toxicity is elicited throughtheir initial binding to a specific intracellularprotein, the aryl hydrocarbon (Ah) receptor(AhR) (1,2). In a process similar to steroidhormone receptor-mediated responses, thereceptor-ligand complex is translocatedfrom the cytoplasm to the nucleus where itbinds to DNA at specific sequences calleddioxin-response elements (DREs) to modifytranscription of the DRE-containing genes(3,4). A widely held hypothesis is thataltered transcription leads to over produc-tion or underproduction of specific proteinproducts that mediate the ultimate bio-chemical and toxic effects of TCDD andrelated HAH. In support of the AhRhypothesis, differences in toxic potencybetween various chlorinated congeners ofdioxins, furans, and biphenyls generally cor-relate well with differences in their bindingaffinity for the Ah receptor (5).

Immunotoxicity StudiesLaboratory Animal Studies

The immune system appears to be one ofthe most sensitive targets for the toxicityof TCDD and related HAH. Studies inthe 1970s demonstrated that exposure oflaboratory rodents to low doses of TCDDby diet or by gavage resulted in involution

of the thymus, increased susceptibility tovarious infectious diseases, and suppressionof both cell-mediated and humoral immunefunctions (6,7). Subsequently, many differ-ent animal models in addition to rodentshave been used to demonstrate the immuno-toxicity of TCDD. Unfortunately, due todifferences in experimental design and out-comes, a defined TCDD-induced immunedeficiency syndrome has not emerged.Likewise, because of the difficulties associ-ated with the measurement of manyimmune functions in some species of ani-mals, there is no clear ranking of species sen-sitivity to TCDD immunotoxicity.

The type of immune response that ismost sensitive to suppression followingTCDD exposure is also difficult to general-ize from animal studies. For example, theantibody response to sheep red blood cells(SRBC) in C57B1/6 mice is very sensitiveto suppression by TCDD, with a singledose of TCDD of 0.7 pg/kg sufficient tosuppress this response to 50% of the con-trol level (8-10). In contrast, a dose ofTCDD as high as 30 pg/kg does not sup-press this response in rats (11). However,even in mice, antibody responses to differ-ent antigens may differ more than 10-foldin sensitivity to suppression by TCDD ascompared to the response to SRBC (8,9),which indicates that the nature of the anti-genic stimulus is also an important factor.This is not unexpected given that differenttypes of antigens are known to evoke dif-ferent cellular interactions (e.g., antigen-presenting cells for soluble vs particulateantigens, requirement for different T-helpercell subtypes for humoral and cell-mediatedresponses, sensitivity to suppressor T-cellregulation, etc.). Understanding the mecha-nism(s) ofTCDD toxicity to these differentsubsets of cells of the immune system willbe necessary for understanding why certainimmune responses are more sensitive toTCDD than others.

Involution of the thymus gland follow-ing TCDD exposure is a hallmark ofTCDD toxicity in several animal species(2,12); however, no direct relationshipbetween the effects of TCDD on the thy-mus and effects on immune function havebeen demonstrated following exposure ofanimals to TCDD (13,14). In adult ani-mals, immune suppression occurs at dosesof TCDD below that required for thymicatrophy (15-17), suggesting that thymustoxicity and systemic immune toxicity areindependent. On the other hand, the effectsof prenatal exposure to TCDD on the fetal

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thymus gland could have serious and long-lasting consequences in terms of immunefunction since the thymus gland is criticalfor the selection of T cells that can appro-priately discriminate between self and non-self molecules (18). Several studies suggestthat prenatal exposure of mice or rats toTCDD is more immunosuppressive thancomparable exposure of adults (19-23).However, the specific role of the thymus inmediating such prenatal effects on immunefunction has not been determined, and theeffects of prenatal TCDD exposure on T-cell selection have not been examined. Onthe other hand, it was recently reported thatrecovery from a thymotoxic dose ofTCDDdid not affect intrathymic negative selectionof potentially autoreactive VP6+ T cells inadult Mls_1a mice (24).

Like other toxic responses to TCDD,the effects of TCDD on the immune sys-tem appear to be dependent on the AhR.Two lines of evidence support this conclu-sion: comparative studies using PCDD,PCDF, and PCB congeners show thatpotency to cause immune suppression cor-relates with the binding affinity for theAhR (25-29); and studies using mice witha defined Ah genotype show that mice withhigh affinity AhR (e.g., Ahbb C57BI/6 mice)are more sensitive to immune suppressionby these compounds than mice with loweraffinity AhR (e.g., Ahdd DBA/2 mice orAhdd congenic C57BI/6 mice)(8,25,26,29).AhR-dependent responses include suppres-sion of the T-cell-dependent antibodyresponse to SRBC (8,25-28,30,31), sup-pression of the T-cell-independent antibodyresponse to trinitrophenyl-lipopolysaccha-ride (8), suppression of the cytotoxic Tlymphocyte (CTL) response to allogeneictumor cells (29,32) bone marrow toxicity(33), and suppression of the cytotoxicresponses of activated neutrophils (34).However, while the data relating HAHimmunotoxicity to AhR-dependent eventsare convincing, it should be emphasizedthat all of the data have been obtained fromstudies in inbred mice using an acute orsubacute exposure regimen. Except forthymic atrophy, structure-immunotoxicityrelationships in other species including ratshave not been established, and otherspecies with defined Ah genotype are notcurrently available.

It is generally accepted that there aremultiple cellular targets within theimmune system that can be altered byTCDD since both T-cell-mediated andantibody-mediated immune responses aresuppressed following TCDD exposure;

however, the specific cellular defectsinduced by TCDD have not been fullyelucidated despite considerable research.One of the problems has been a difficultyin demonstrating direct effects ofTCDDon in vitro responses of lymphoid cells(35,36). This is especially true with Tcells; direct effects ofTCDD on T cells invitro have not been observed even thoughT-cell functions in vivo are clearly altered byTCDD (14,32,33,36-38). Furthermore,when in vitro effects ofTCDD are observed,they may be inconsistent with the in vivoimmunotoxic effects of TCDD. For exam-ple, in vitro suppression of the antibodyresponse to SRBC has been reported bysome laboratories to be independent of theAhR (39,40), whereas suppression of thisresponse in vivo is clearly AhR-dependent.Although the basis for the discrepanciesbetween in vivo and in vitro effects is notknown, differences in culture conditionsmay be partially responsible since the invitro effects of TCDD on lymphoid cellsappear to be influenced by unknown fac-tors present in serum-supplemented tissueculture media (41,42). Interestingly,serum was also shown to modulate theinduction of P4501AI-dependent enzymeactivity by TCDD in primary cultures ofhepatocytes (42), demonstrating that theserum phenomenon is not restricted toeffects on lymphocytes.

While T-cell responses appear to beresistant to direct effects of TCDD, severallaboratories have reported that TCDDdirectly alters B-lymphocyte functions invitro (13,40,43,44). Studies using bothmurine and human B cells suggest thatTCDD alters the terminal differentiationof B cells into antibody-secreting plasmacells without altering B-cell proliferation(43-45). The induction of protein kinaseactivity (46-48) and altered calciumhomeostasis (49) have been implicated inthe immunotoxic effects of TCDD on Bcells. Snyder et al. (48) reported thatTCDD induced the phosphorylation of29, 45, 52, and 63 kDa proteins in B cells,which by density gradient were character-ized as activated B cells. Interferon-y(IFNy) was shown to antagonize theTCDD-induced phosphorylation and toreverse the TCDD-induced suppression ofthe antibody response to SRBC in vitro.Interestingly, suppression of IFNy produc-tion has recently been correlated with the invivo suppression of cytotoxic T-lymphocyteactivity in TCDD- and PCB-treated mice[NI Kerkvliet, unpublished data; (50)].Thus, changes in IFNy production may

represent an underlying mechanism forTCDD-induced immunotoxicity.

Activities associated with innate immunefunction have also been examined followingTCDD exposure and have generally beenfound to be resistant to suppression whenassessed ex vivo. Macrophage-mediatedphagocytosis, macrophage-mediated tumorcell cytolysis or cytostasis, oxidative reactionsof neutrophils and macrophages, and nat-ural killer (NK) cell activity were not sup-pressed following TCDD exposure, withdoses as high as 30 pg/kg failing to suppressNK and macrophage functions (51,52). Apotentially important exception is thereported inhibition of phorbol ester-acti-vated antitumor activity of neutrophils byTCDD (34). On the other hand, thepathology associated with TCDD toxicityoften includes neutrophilia and aninflammatory response in certain tissues(e.g., liver and skin) characterized by acti-vated macrophage and neutrophil accumu-lation (53-55). While these observationsmay simply reflect a normal inflammatoryresponse to tissue injury, there is increasingexperimental evidence to suggest thatinflammatory cells may be activated byTCDD exposure. For example, TCDDexposure has been shown to produce anenhanced inflammatory response in theperitoneal cavity of mice following SRBCinjection (56). This effect of TCDD wascharacterized by a 2- to 4-fold increase inthe number of neutrophils and macrophageslocally infiltrating the intraperitoneal site ofSRBC injection. However, the time courseof the cellular influx was not altered byTCDD exposure. Likewise, the expressionof activation markers (I-A and F4/80) andthe antigen-presenting function of the peri-toneal exudate cells was unaltered byTCDDexposure. Thus, the effect of TCDDappeared to reflect a quantitative rather thana qualitative change in the inflammatoryresponse. Interestingly, although enhancedantigen clearance/degradation caused by theincreased numbers of phagocytic cells couldresult in a decreased antibody response inTCDD-treated mice, increasing the amountof antigen used for sensitization did notalter the immunosuppressive effect ofTCDD (56). Thus, a relationship betweenthe inflammatory and immunosuppressiveeffects of TCDD in the SRBC model wasnot apparent.

One mechanism by which TCDD couldaugment inflammatory responses is throughenhanced production of inflammatorymediators. For example, recent evidencesuggests that the hypersusceptibility of

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TCDD- and PCB-treated animals toendotoxin (57,58) and the increasedinflammatory response to SRBC (59) maybe related to an increased production oftumor necrosis factor (TNF). The ability ofmethylprednisolone to reverse the mortalityassociated with TCDD/endotoxin treatmentis also consistent with an proinflammatorymechanism (60). Similarly, increasedinflammatory mediator production mayunderlie the enhanced rat paw edemaresponse to carrageenan and dextran inTCDD-treated rats (61,62). A primaryeffect ofTCDD on inflammatory mediatorproduction is supported by the recentfindings that keratinocytes exposed toTCDD in vitro have increased mRNA forinterleukin-1p, plasminogen activatorinhibitor-2, and transforming growth factora and decreased mRNA for transforminggrowth factor 02 (63,64). Interestingly,mRNA for TNF was not altered by TCDDtreatment (64). The effects ofTCDD onkeratinocytes are similar to the effects ofTCDD on the macrophage cell line IC-21in that TCDD treatment increased endo-toxin induction ofmRNA for interleukin-1but not TNF (65).

The influence of TCDD exposure oninflammatory mediator production andaction is an important area for furtherstudy. In this regard, it is relevant to notethat treatment of mice with a soluble TNF-binding protein, under conditions thatresolved the hyperinflammatory response toSRBC induced by TCDD exposure, didnot affect TCDD-induced suppression ofthe anti-SRBC antibody response (66).Similarly, daily treatment of mice withaminoguanidine, an inhibitor of induciblenitric oxide synthase, did not influence thesuppression of the anti-SRBC antibodyresponse by TCDD (66). Thus, the rela-tionships, if any, between the proinflam-matory and immunosuppressive effects ofTCDD remain to be elucidated.

The ability of TCDD to augmentthe production of certain inflammatorychemoattractive mediators suggests thatTCDD exposure could result in enhancedhost resistance to pathogenic infection sincethe rapid influx of phagocytes to the site ofpathogen invasion is an important factor inhost resistance. However, since TCDDexposure is at the same time immunosup-pressive, which results in decreased specificimmune responses generated by T and Blymphocytes, the overall impact ofTCDDexposure on disease susceptibility willprobably vary depending on the nature ofthe pathogen and on the major mode of host

response to the specific infectious agent.These divergent effects ofTCDD on inflam-mation and immunity may, in fact, help toexplain the disparate effects ofTCDD in dif-ferent host resistance models that have beenpreviously reported (20,52,67,68).

Studies in Nonhuman PrimatesA limited number of studies using nonhu-man primates have been conducted toassess TCDD immunotoxicity. Fewimmunologic effects were found in rhesusmonkeys and their offspring that werechronically exposed to TCDD in food atlevels of 5 or 25 parts per trillion (ppt) for4 years (69). Although T-cell numbersdecreased in the TCDD-fed mothers (witha selective decrease in CD4+ cells), T-cellfunction as measured by proliferation tomitogens, alloantigens, or xenoantigenswas not affected. NK cell activity and theantibody response to tetanus toxoid werealso normal. Interestingly in the offspring,T-cell numbers were increased as was theantibody response to tetanus toxoid. [It isrelevant to note that the antibody responseto SRBC was not measured in these stud-ies because the antibody response to SRBCbut not to tetanus toxoid was decreased inmonkeys exposed to much higher levels ofPCB (70)].

In other studies, a single injection ofTCDD in marmosets (Callithrixjacchus)resulted in a decrease in the percentages ofCD20+ B cells and CD4+ T cells and anincrease in the percentage of CD8+ T cellsin the blood without affecting the totalnumbers of these cells (71). The CD4+ sub-set that was most affected was the CDo29+helper-inducer or memory subset, with sig-nificant effects observed after a TCDD doseof 10 ng/kg but not after a dose of 3 ng/kg.The changes in the T-cell subsets wereintensified following culture of the cellswith mitogens (72). Paradoxically, how-ever, chronic exposure of young marmosetsto lower levels ofTCDD (0.3 ng/kg/weekfor 24 weeks) produced the opposite effectof acute exposure on the CD4+CDw29+subset, with TCDD treatment resulting ina significant increase in this population(73). Upon transfer of the animals to ahigher dose ofTCDD (1.5 ng/kg/week) for3 weeks, the enhancing effect was reversedand suppression of the CD4+CDw029+ sub-set was observed. After discontinuation ofdosing, the reduction in the percentage andabsolute number of CD4+CDw029+ cellspersisted for 5 weeks, reaching normal range7 weeks later. Based on these results theauthors concluded that "extrapolations of

the results obtained at higher doses to verylow exposures is not justified with respectto the effects induced by TCDD on theimmune system of marmosets" (73). Therelevance of these changes in subset distrib-utions to immune function in the marmosethave not been determined. Interestingly, asimilar reduction in the "memory" CD4+ Tcell subset was observed in C57B1/6 micetreated once a week for 60 weeks with 0.2pg/kg TCDD (74), suggesting that thememory CD4+ T cell may represent a verysensitive biomarker of exposure to TCDD.A reduction in the memory T-cell popula-tion is consistent with the immunosuppres-sive effects ofTCDD.

Human StudiesThe immunotoxicity ofTCDD in humanshas been the subject of a limited number ofstudies in which cohorts were exposed toTCDD either occupationally or as a resultof residence in a TCDD-contaminatedarea. Mocarelli et al. (75) reported on theimmune status of 44 children, 20 ofwhomhad chloracne, that were exposed to TCDDfollowing an explosion at a herbicide fac-tory in Seveso, Italy. No abnormalitieswere found in serum immunoglobulin con-centrations, levels of circulating comple-ment, or lymphoproliferative responses toT- and B-cell mitogens. Interestingly, in astudy conducted 6 years after the explo-sion, a different cohort of TCDD-exposedchildren exhibited a significant increase incomplement protein levels, which corre-lated with the incidence of chloracne, aswell as increased numbers of peripheralblood lymphocytes and increased lympho-proliferative responses (76). No specifichealth problems were correlated withdioxin exposure in these children.

Webb et al. (77) reported the findingsfrom immunologic assessment of 41 per-sons from Missouri with documented adi-pose tissue levels ofTCDD resulting fromoccupational, recreational, or residentialexposure. Of the participants, 16 had tissueTCDD levels less than 20 ppt, 13 had lev-els between 20 and 60 ppt, and 12 had lev-els greater than 60 ppt. The highest levelwas 750 ppt. Data were analyzed by multi-ple regression based on adipose tissue leveland the clinical-dependent variable.Increased TCDD levels were correlatedwith an increased percentage and totalnumber ofT lymphocytes. CD8+ and Ti 1 +

T cells accounted for the increase, whileCD4+ T cells were not altered in percent ornumber. Lymphoproliferative responses toT-cell mitogens or tetanus toxoid were not

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altered nor was the cytotoxic T-cell response.Serum immunoglobulin A (IgA) wasincreased but IgG was not. No adverse clin-ical disease was associated with theseTCDD levels in these subjects. Only 2 ofthe 41 subjects reported a history of chlo-racne. These findings differ from thosereported for the Quail Run Mobile HomePark residents (tissue levels unknown) inwhich decreased T-cell numbers (T3+,CD4+, and T 1+) and suppressed cell-mediated immunity was reported (78).However, subsequent retesting of theseanergic subjects failed to confirm the sup-pressed immunity (79). On the other hand,when sera from some of these individualswere tested for levels of the thymic peptidethymosin a-1, the entire frequency distrib-ution for the TCDD-exposed group wasshifted toward lower thymosin a-i levels(80). A statistically significant differencebetween the TCDD-exposed persons andcontrols remained after controlling for age,sex, and socioeconomic status, with a trendof decreasing thymosin a-I levels as thenumber of years of residence in theTCDD-contaminated residential areaincreased. The thymosin a-I levels werenot correlated with changes in otherimmune system parameters or with anyincreased incidence of clinically diagnosedimmune suppression. The decrease in thy-mosin a-I levels in this cohort contrastswith the increase in thymosin a-I seen inPCB-treated monkeys (81).

Two studies have evaluated theimmunologic function of Vietnam veteransexposed to TCDD via use of the pesticideAgent Orange. When U.S. Army groundtroops were matched with a comparisonpopulation, no differences in lymphocytesubsets or serum immunoglobulins werefound (82). In the U.S. Air Force RanchHand Study, comprehensive immunologicprofiles were developed for each participantand correlated with serum TCDD concen-trations (83). The only significant positiveassociation with TCDD exposure wasincreased serum IgA level. Roegner et al.(83) suggested that the increase in serumIgA was consistent with a subclinical inflam-matory response, but no other evidence foran inflammatory response was obtained.

The basis for the lack of consistent orsignificant exposure-related effects toTCDD in these human populations isunknown and may be dependent on severalfactors. Most notable in this regard is theinherent difficulties in assessing subclinicalimmunomodulation in an outbred humanpopulation. Most immunologic assays have

a very broad range of normal responses thatreduce the sensitivity to detect smallchanges. Similarly, the assays used to exam-ine immune function in humans exposedto TCDD have unfortunately been basedto a greater extent on what was clinicallyfeasible (e.g., lymphocyte phenotype, mito-gen responsiveness) rather than on assaysthat have been shown to be sensitive toTCDD in animal studies (e.g., antibodyresponse to SRBC). Thus, the lack of con-sistent or significant immunotoxic effects inhumans resulting from TCDD exposuremay be as much a function of the assaysused as the immune status of the cohort. Inaddition, few studies have examined theimmune status of individuals with known,documented exposure to TCDD. Rather,cohorts based on presumption of exposurehave been studied. There is some evidenceto suggest that the lack of consistent, signi-ficant effects may sometimes be due to theinclusion of subjects that had little or noactual exposure to TCDD (77). Likewise,the important role that Ah phenotype playsin TCDD immunotoxicity has not beenconsidered when addressing human sensi-tivity. Finally, in most studies, the assess-ment of immune function in exposedpopulations was carried out long after expo-sure to TCDD ceased. Thus, recovery fromany immunotoxic effects of TCDD mayhave occurred by the time of testing.

As an alternate approach to evaluatingthe sensitivity of the human immune sys-tem to TCDD, several laboratories haverecently reported on the direct in vitroeffects of TCDD on human lymphocytes.Neubert et al. (72) reported that TCDDreduced the percentage of CD20+ B cellsand CD4+CDo29+ T cells in pokeweedmitogen-stimulated cultures of peripheralblood lymphocytes at concentrations aslow as 10-12 to 10-14 M TCDD. Theseresults, however, were not corroborated insimilar studies reported by Lang et al. (35)in which concentrations ofTCDD rangingfrom 10-7 to 10-11 M were tested. Inanother model, Wood and Holsapple (84)reported that proliferation and antibodysecretion by pokeweed mitogen-stimulatedhuman tonsillar lymphocytes were notaltered by exposure to TCDD at concen-trations ranging from 3 x 10-8 to 10-io M.Yet these same concentrations of TCDDsignificantly suppressed the ability ofhuman tonsillar B cells of some donors toproduce antibodies in response to toxicshock syndrome toxin (85). Because of thelimited amount of data available and thelack of corroboration between laboratories,

no conclusions can yet be drawn regardingthe relative sensitivity of human lymphoidcells to TCDD.

Research NeedsFor the field of immunotoxicology ingeneral, there is a strong need to establish abroad database of normal values for the clin-ical immunology end points that may be ofuse as biomarkers of immune function inimmunotoxicity assessments. To validatethese biomarkers, there is a parallel need foranimal research to identify TCDD-sensitiveimmune end points in animals that can alsobe measured in humans in order to establishcorrelative changes in the biomarker andimmune function. In particular, it will beimportant to determine in animal modelshow well changes in immune function inthe lymphoid organs (e.g., spleen, lymphnodes) correlate with changes in the expres-sion of lymphocyte subset/activation mark-ers in peripheral blood. Also limited at thepresent time are good correlative databetween changes in immune function mea-surements and changes in host resistance tospecific disease challenges induced by xeno-biotic exposure. Until such correlations areestablished, the interpretation of changesobserved in subsets/activation markers inhuman peripheral blood lymphocytes interms of health risk will be limited to specu-lation. Research must also continue todevelop and characterize immune modelsusing multiple animal species that will leadto an understanding of the underlyingmechanisms of HAH immunotoxicity. Forexample, there is a clear need to documentAh receptor involvement in the immuno-toxicity of TCDD and related HAH inspecies other than mice. These studies needto go beyond descriptive immunotoxicityassessment to determine the mechanisticbasis for differences in species sensitivity toTCDD immunotoxicity following bothacute and chronic exposure. Until then, riskassessment must be based on the best avail-able data derived from well-controlled ani-mal studies on TCDD immunotoxicity.Because the antibody response to SRBC hasbeen widely studied and has been shown tobe dose-dependently suppressed by TCDDand related HAH in several animal species,including nonhuman primates, this data-base would appear to be best suited for cur-rent application to risk assessment. Theapproaches used to establish acceptableexposure levels for humans for immuntoxic-ity should be based on the same proceduresthat are used for other noncarcinogenictoxic end points.

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