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AD-A267 729 /(1IIlI I I1 l Ii IIII Ii I AD_______________________
CONTRACT NO: DAMD17-86-C-6154
TITLE: EVALUATION OF IMMUNE RESPONSE MODIFYING COMPOUNDSUTILIZING VIRUS-SPECIFIC HUMAN T LYMPHOCYTE CLONES
PRINCIPAL INVESTIGATOR: Marjorie L. Cohn, Ph.D.
CONTRACTING ORGANIZATION: Georgetown UniversityWashington, D.C. 20007
DTIREPORT DATE: July 1, 1991 - i [wN UG 119 ,TYPE OF REPORT: Final Report •- U
PREPARED FOR: U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21702-5012
DISTRIBUTION STATEMENT: Approved for public release;distribution unlimited
The findings in this report are not to be construed as anofficial Department of the Army position unless so designated byother authorized documents.
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1. AGENCY USE ONLY (Leave biani) 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED
11 July 19901 Final Report (3/1/86 - 4/30/89)4. TITLE AND SUBTITLE S. FUNDING NUMBERS
Evaluation of Immune Response Modifying Compounds Contract No.Utilizing Virus-Specific Ehman T Lymphocyte Clones DAMDl7-86-C-6154
6. AUTHOR(S) 61102ACohn, Ph.D. 3M161102BS12.AD. 132
iJDA309562
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
REPORT NUMBER
Georgetown UniversityWashington, D.C. 20007
S. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESSUES) 10. SPONSORING, MONITORING
AGENCY REPORT NUMBERU.S. Army Medical Research and Development CommandFort DetrickFrederick, Maryland 21702-5012
11. SUPPLEMENTARY NOTES
12a. DISTRIBUTION AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE
Approved for public release; distribution unlimited
13. ABSTRACT (Maximum 200 words)
14. SUBJECT TERMS IS NUMBER OF PAGES
Immuno lodulator; RAI; BD; Surface antigen; T-lynm ocyte; 16 PRICE COoEVolunteers
17 SECURITY CLASSiFICATION 18 SECURITY CLASSIFICATION 19 SECURITY CLASSIFICATION 20 LIMITATION OF ABSTRACT
OF REPORT Of THIS PAGE OF ABSTRACT
Unclassified Unclassified I-n c 1:i,-- F_ _, fn_ -1;i _
NSN 7S40-1.180.5500 S',anoa'o ;ofm ;98 QeV 89."PI% 0"a at Ah4, sic 39
FOREWORD
For the protection of human subjects, the investigator(s)have adhered to policies of applicable Federal Law 45CFR56.
Citations of commercial organizations and trade names inthis report do not constitute an official Department of the Armyendorsement or approval of the products or services of theseorganizations.
Accession For
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Contents
Background 4
Materials and Methods 6
Results 8
Characterization of T Cell Clones 8
Evaluation of Immunomodulators 10
Conclusions 15
Literature Cited 17
Appendix I Tables 1 - 44
Appendix II Scientific Reviewer's Comments
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Background
A wide variety of viruses which pose little or no publichealth threat in the United States are significant problems formilitary personnel stationed in parts of the world where theseviruses are endemic. Many of these viruses are dependent on aninsect vector and are found primarily in tropical regions.Infections with these viruses result in symptoms which range fromtemporarily debilitating to extremely serious and many arepotentially fatal. The large number of these viruses and thedifficulties involved in working with many of them makevaccination an impractical goal at this time. Natural orsynthetic compounds which enhance the ability of the immunesystem to defend against these viruses would provide broaderspectrum management of such infections. The prophylactic and/ortherapeutic use of such agents could provide the criticaldifference in the outcome of situations involving large scalevirus exposure of military personnel.
Generation of an immune response involves complexinteractions among monocytes, antibody producing B lymphocytes,and effector and regulatory T lymphocytes, as well as a number offactors produced by these cells. Which aspects of the immuneresponse are most important in the defense against viralinfections differs for different viruses, depending on virusstructure, the cell types which the virus preferentially infects,and previous exposure and immunization of the host to relatedviruses. A choice of a compound or combination of compounds forin vivo use to enhance antiviral immunity will therefore dependon the types of viruses likely to be encountered, history ofprevious exposure of the individuals involved, and whether thecompounds are intended to be used prophylactically or as therapyin recently infected individuals.
It is clear that an understanding of the basic effects ofgiven immune response modulators on individual components of theimmune system is required before decisions can be made concerningwhich ones warrant further study. A compound may affect one ormore cellular subpopulations of the immune network and theeffects may be direct or indirect. Enhancement of immuneresponsiveness may occur by affecting cells in several possibleways, including: 1) increasing the number of antigen reactivecells, 2) decreasing the dose of antigen required for optimalresponsiveness, 3) altering the kinetics of the response, and4) increasing the level of function on the individual cellularlevel. In vitro assay systems are necessary for these basicstudies.
Over the past several years, technological advances havebeen made which now allow the long-term growth and maintenance offunctional antigen-specific T lymphocyte clones in culture. Theuse of these T cell clones offers numerous advantages over other
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in vitro techniques for the study of specific immune responses.One can obtain large quantities of cells responsive not only to asingle antigen, but to a single antigenic determinant. Thus, theresponse to several distinct antigenic determinants of a singleinfectious agent, or to a determinant common to a class ofinfectious agents (such as the flaviviruses), can be studied.
Each clone represents a single functional subpopulation.Such homogeneous cell populations allow the dissection of effectsof biochemical and cellular influences on individual cell types,and simplify the analyses of the cells themselves. Using T cellclones, the role of other cells of the immune system, such asantigen presenting cells and antibody producing cells can beanalyzed with great precision.
In both murine and human systems, T cell clones have beengenerated against a wide variety of antigens, including cellularalloantigens, tumor antigens, natural and synthetic solubleproteins, and bacterial and viral antigens (1-19). These cloneshave been used for analyses of fine antigen specificity,histocompatibility antigen (immune response genes) restriction ofcellular interactions, cell surface markers, T cell receptorstructure, factor production, and mechanisms of regulatory andeffector functions. Such analyses have resulted in extremelyrapid advancement in understanding the T cell at both cellularand molecular levels.
Studies of modulation of immune function have generallyemployed unfractionated lymphocyte, T cell, B cell, or macrophagepopulations. The use of virus-specific T cell clones, however,permits the dissection of effects of biochemical and cellularinfluences on individual cellular functions. Understanding therole the of each component of the immune response will greatlyfacilitate the analysis of effects of a single compound orcombination of compounds on complex systems of interacting celltypes.
The system used here allows comparisons of effects ofimmunomodulating compounds on responses to various viralantigenic determinants. One compound may improve the response tosome antigenic determinants and hinder the response to otherantigenic determinants. Clonal populations may be grown insufficient quantity to assay a variety of compounds, individuallyor in combination, on essentially identical populations of cells.This system allows analysis of responses of human cells, whichmay in some cases differ considerably from those of the animalmodel systems commonly used.
The studies described below employ human T cell clonesderived from a single donor and generated against influenza virusstrain A/Bangkok. The clones were all generated prior to theinitiation of this contract. To facilitate understanding of the
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system, however, details of the generation and characterizationof these clones are included in this report.
Materials and Methods
Viruses. Formalin fixed preparations of influenza virusstrains A/Bangkok RX 73 (H3N2), A/Brazil/X 71 (HINI), andB/Singapore were a generous gift of Dr. Lance Gordon, ConnaughtLaboratories, Swiftwater, PA. Before use, virus preparationswere dialyzed extensively against PBS to remove formalin.Following dialysis, each preparation was titered in aproliferation assay as described below.
Generation of T lymphocyte clones. Influenza virus-specificT cell clones were generated using slight modifications of ourpreviously described procedure (12). Peripheral blood leukocytes(PBL) (5x10 5 /ml) were incubated with an optimal dilution ofA/Bangkok influenza in 1.5 ml volumes in round bottom 5 mlculture tubes for 5 days. Blast cells were then isolated onFicoll-Hypaque gradients and cultured for 7 days at aconcentration of 1xl0 5 /ml with irradiated autologous PBL (feedercells - 5x10 5 /ml), virus, and conditioned medium (CM - 20%) toexpand the influenza-specific T cell population. Cells were thenwashed and cultured at ixl05 /ml with feeders and virus asdescribed above, in the absence of CM, in 5 ml tubes. Three dayslater, blast cells were again isolated and cloned at limitingdilution. These cultures were carried out in 60 well Terasakiplates in volumes of 0.02 ml/well; T cells were plated at aconcentration resulting in 1 cell/3 wells, with each well alsocontaining 104 feeders, virus, and 20% CM. Ten days later, thecontents of each well containing growing lymphocytes weretransferred individually to 0.2 ml flat-bottom wells containingfeeders (10 5 /well) and flu; CM (final concentration - 20%) wasadded the following day. After a week, cultures were expanded to2 ml wells containing 106 feeders and flu; again CM was added thenext day. One week later, each clone was assayed for influenza-specific proliferative responsiveness. Aliquots of each clonewere also frozen and maintained in liquid nitrogen.
Expansion of antigen-specific T cell clones. To expandclonal populations, cells were fed weekly as follows. Cells werecultured at ixl05 /ml in RPMI 1640 with 10% A+ plasma and 5x10 5
irradiated autologous PBL/ml and an optimal concentration ofantigen. The following day, CM was added (final concentration20%). On day 4, half the culture medium was removed and replacedwith fresh medium containing 20% CM.
Proliferation assays. T cell clones (5x10 3 cells/well) werecultured with irradiated autologous PBL (25x10 3 cells/well) in 96well U-bottom plates in a final volume of 0.2 ml. Each well alsocontained an appropriate concentration of antigen. Cultures were
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incubated for 48 hours, pulsed for 16 hours with 1.0 uCi of 3H-Thymidine and harvested onto glass-fiber filters. Filters weredried and counted using a liquid scintillation counter. Allassays were done in triplicate. Cells were cultured in theabsence of antigen to provide negative control values, with CM,which nonspecifically stimulates T cell proliferation, as apositive control, and an irrelevant antigen (generally aninfluenza B strain) as a specificity control.
Conditioned medium production. PBL were cultured at1x10 6 /ml in RPMI containing 1% A+ plasma and 0.1% PHA-P. After48 hours, supernatants were harvested and filtered. Lots of CMwere assayed for their ability to support proliferation of an IL-2 dependent cell line. Acceptable lots were pooled and thevolume adjusted for use at a final concentration of 20% inculture.
Analysis of cell surface antigens. Cells were washed andresuspended to 101 cells/ml in RPMI with 10% horse serum and0.02% Na azide. Aliquots of cells (0.1 ml) were dispensed into 5ml tubes to which monoclonal antibody was added; the amount ofantibody used was determined by titration of each preparation.After 1 hour at 370 C, cells were washed and incubated with goatanti-mouse Ig for 30 min. at 370 C. Cells were again washed andthen incubated with fluorescein-conjugated rabbit anti-goat IgGfor 30 min. at 37 0 C. After two washes, cells were fixed in 2%paraformaldehyde and analyzed for fluorescence using afluorescence activated cell sorter.
Virus-Specific Cytotoxicity. Target cells. Target cellsare EBV transformed cells derived from the same donor as was usedto generate the T cell clones to be assayed. The use of EBVtransformed cells as targets allows the detection of CTL whichare HLA Class II restricted; cytotoxic effects of these Class IIrestricted clones often cannot be detected using PHA blasts astargets, due to insufficient levels of expression of Class IIantigens. At t=-24 hrs., target cells were diluted to 1 x 105cells/ml in RPMI 1640, 10% FCS, + influenza A (final dilution -1:250) and incubated at 370, 5% CO2 . For use as a specificitycontrol, target cells are incubated with medium rather thanvirus. At t= 0, target cells were harvested and viable cellspurified on a Ficoll-Hypaque gradient. Cells at the interfacewere collected and washed 3 times in RPMI 10% FCS. Cells werepelletted (up to 107 cells/tube) and 200 uCi 5 1 Cr (in 0.1 ml) wasadded. Cells were resuspended and incubated at 370, 5% C02 , for1 hr., mixing every 15 min. Cells were washed 3 times in RPMI10% FCS, counted, and resuspended at 1 x 105 cells/ml.
Effector cells. T cell clones were used as killers 7 days afterfeeding with antigen and antigen presenting cells. Cells werewashed once with RPMI 10% FCS, counted, and resuspended torequired concentration (determined for each clone to yield
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effective lysis of 104 targets; for most clones, 10 5 killercells/well was optimal)
Assay. Assays were carried out in 96 well U bottom plates. 0.1ml of killer cells and 0.1 ml of target cells were dispensed intoeach well. As controls, targets were incubated with 0.1 ml ofmedium (spontaneous 5 1 Cr release), or with 0.1 ml 0.1 N HCl(maximum 5 1 Cr release). Plates were centrifuged at 1000 rpm for30 sec. to lightly pellet the cells. Plates were then incubatedat 370, 5% CO2, for 4 hrs, centrifuged at 1000 rpm for 2 min.,harvested using Skatron supernatant harvesters, and counted.% specific lysis was calculated as:
Experimental cpm - Spontaneous cpm x 100Maximum cpm - Spontaneous cpm
For assays in which immunomodulators were added to killer ortarget cells on the day of the assay, cells were added at 2X thenormal concentration in half the normal volume to allow additionof the modulators without alteration of the total assay volume.
Results
Characterization of T cell clones
Screening of donors. To identify an appropriate donor forgeneration of T cell clones, PBL from several healthy volunteerswere assayed for in vitro proliferative responses to influenzavirus. PBL (5x10 5 /well) were cultured with medium or dilutionsof influenza A/Bangkok ranging from 0.1 to 50 HAU/ml for 6 days,after which incorporation of -H-thymidine was measured. Theresults of one such experiment are shown in Table 1; forsimplicity, only one virus concentration is presented. Donor 945clearly gave higher proliferative responses in this and otherexperiments and was therefore used for the generation of clonesfor the studies described here.
Determination of optimal virus dose for in vitro studies.Each preparation of virus must be tested for optimal dilution foruse in generating and maintaining clones and for proliferationassays. PBL from donor 945 were cultured with varying dilutionsof virus for 6 days and assayed for proliferation as describedabove. Data from the most recent titration of influenzaA/Bangkok are presented in Table 2; for this preparation, a1:1000 dilution results in optimal proliferation. Similarresults (not shown) were obtained with strains A/Brazil andB/Singapore.
Antigen specificity of T lymphocyte clones. A large numberof T lymphocyte clones were generated as described above, using
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PBL obtained from donor 945. Of these, 6 were chosen for theinitial phases of these studies. To determine their antigenspecificities, clor.es were incubated with irradiated autologousPBL, which serve as antigen presenting cells, and medium(background), 20% conditioned medium (a nonspecific stimuluswhich serves as a positive control), or optimal concentrations ofvirus in a 2 day proliferation assay. As can be seen in Table 3,all 6 clones proliferated well to conditioned medium andtherefore were healthy and able to divide after nonspecificstimulation. Clones TFE96 and TFE97 were able to respond to anH3N2 strain (A/Bangkok) of flu, but not to the HlNl strainA/Brazil, indicating that these clones recognize determinants oneither the hemaglutinin or neuraminadase components of the virus.Clones TFE40, TFE89, and TFE102 all responded to both strainsA/Bangkok and A/Brazil, indicating that these clones are specificfor antigenic determinants shared by A strain influenza viruses.Clone TFE88 responded to neither A/Bangkok nor A/Brazil; it wasincluded in some experiments as a control for nonantigen specificeffects of immunomodulators. In addition, but not shown here,all clones were assayed for responsiveness to strain B/Singaporeand in all cases incorporated less than 100 cpm.
Kinetics of responses of T cell clones. To determine theday of peak proliterative response of the T cell clones usedhere, clones were cultured with feeders and virus and pulsed with3H-thymidine after 2, 3, or 4 days of culture. The data in Table4 demonstrate that clones TFE40, TFE96, and TFE102 respondedoptimally 2 days after initiation of cultures. Clone TFE97responded optimally on day 3, but displayed only slightly lowerresponsiveness on day 2. All assays described below weretherefore pulsed on day 2 unless otherwise indicated.
Cell surface Phenotype of T cell clones. T cell clones werestained with monoclonal antibodies against the CD4 (OKT 4) andCD8 (OKT 8) determinants followed by fluorescein conjugated goatanti-mouse Ig antibodies and analyzed using a fluorescenceactivated cell sorter. The results in Table 5 indicate that allof these clones express the CD4 antigen and are negative for theCD8 marker.
Virus-specific cell mediat-d cytotoxicity. Of the largenumber of virus-specific T cell clones originally generated, fourcytotoxic clones were used in these studies (Table 6). In allassays, target cells which had been incubated with medium ratherthan Influenza virus were included as a specificity control. Anykilling due to recognition of EBV antigens or other cell surfaceantigens would be seen as killing of targets incubated withmedium as well as targets incubated with virus; using T cellclones as cytotoxic effectors, this nonspecific lysis has notbeen seen. In assays of immunomodulators, targets incubated withmedium also served to control for any nonspecific effects ofthese compounds on either the effector or target cells.
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Evaluation of immunomodulators
A total of 19 compounds (18 potential immunomodulators and 1placebo) were supplied for testing under this contract. Thesecompounds and their AVS numbers are listed in Table 7.
The first compound to be tested was CL 246,738. T cellclones were cultured with a series of ten-fold dilutions of themodulator. The modulator was included from the beginning of theculture and proliferation was assayed on day 2. The results ofthe first assay (Table 8) demonstrated that this compound wastoxic to the cells at a concentration of 10 ug/ml, withincorporation of 3 H-TdR into DNA eliminated, and lysis of cellsobserved on visual inspection of cultures. Partial inhibition ofcell division in response to CM and to flu compared to cultureEcontaining no modulator was observed when the compound wasincluded at a final concentration of 1 ug/ml. At lowerconcentrations of modulator, however, an increase in the responseto flu A was seen. Proliferation in response to the irrelevantantigen, flu B, was unaffected, and nonspecific responsiveness toCM was only marginally increased. The response of thenonspecific clone, TFE 88 was not appreciably altered under theseculture conditions. It thus appears that only the response tospecific antigen is enhanced. In further experiments similarresults were seen; results with antigen and modulatorconcentrations giving optimal enhancement of proliferation areshown in Table 9. It should be noted that the antigenconcentration which results in optimal stimulation appears to bedifferent for different clones.
The effect of CL 246,738 on virus-specific cytotoxicity wasthen tested over a wi4e range of concentrations. Initially,effectors were incubated with CL 246,738 for 1 hour before targetcells were added; partial inhibition of killing was seen at 10-1ug/ml and killing was completely eliminated at 1 ug/ml (Table10). Proliferation and cytotoxicity assays were carried outside-by-side, as described above. The data shown in Table 11demonstrate that at 10-1 ug/ml, the cytotoxic response was lessthan half of the control level for each clone, while theproliferative response was enhai'ced relative to the controlresponse, as was seen in initial experiments with this compound.The kinetics of inhibition of CTL responses by CL 246,738 wasalso examined. Killing by TFE 89 (Table 12) was slightlyinhibited when 10-1 ug/ml of CL 246,738 was added at the sametime as target cells, while a 1 hour or 24 hour preincubationresulted in a much greater inhibition. The effect of 1 ug/ml wassimilar with 0 or 1 hour incubation, while a 24 hour incubationresulted in total elimination of killing. In contrast, killingby clone TFE 23 (Table 13) was inhibited to a greater extent by10-1 ug/ml at 1 hour than at 24 hours, while 1 ug/ml totallyeliminated killing with either incubation time.
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The effect of OK-432 on virus-specific proliferation wasalso tested over a concentration range from 10 ug/ml to 10-6ug/ml with virus dilutions ranging from 1:1000 to 1:16,OCO.Optimal enhancement was seen in all cases with a virus dilutionof 1:2000; the concentration of OK-432 which induced optimalenhancement, however, varied for each clone (Table 14). Toexamine the effects of this compound on virus-specificcytotoxicity, OK 432 was incubated with cytotoxic clones for 1hour before target cells were added (Table 15). Killing by TFE18 was enhanced over the entire range of concentrations tested.The level of killing by TFE 23 was also enhanced at mostconcentrations of OK 432, but to a slightly lesser extent. Theresponses of TFE 79 ana TFE 89 were variable, with only modestenhancement and modest inhibition observed.
Dramatic differences among the clones were seen in theeffects of recombinant human gamma interferon (AVS 2-50 onantigen-specific proliferation (Table 16). Clone TF E89displayed enhanced proliferation at low interferon concentrationsand decreased proliferation at relatively high interferonconcentrations. Optimal enhancement of proliferation of clone TFE96 was seen at intermediate concentrations, while proliferationof clones TF E97 and TF E102 was optimally enhanced at highconcentrations of interferon. Decreased proliferation was notseen at any interferon concentration with any clone other than TFE89. These effects were sean at all antigen dilutions tested,although they were most driatic at suboptimal antigenconcentrations. Both the inhibition and enhancement were seenonly with antigen-specific responses; no effect was seen onnonspecific proliferation induced by conditioned medium or onnegative control responses. That inhibition of proliferation wasnot due to toxicity or interference with thymidine uptake by thecells was confirmed by the results with conditioned medium aswell as by trypan blue viability counts of the cells afterculture.
To study the effects of interferon on expression of surfacemarkers, clones were incubated with feeder cells and antigen fortwo days in the presence or absence of 1 ug/ml of interferonprior to cell sorter analysis (Tables 17 and 18). This timingand concentration of interLeron were chosen since theseconditions are those under which we found an effect of interferonon antigen-specific proliferation. After incubation with antigenand interferon, clone TF E89, which previously displayeddecreased antigen-specific proliferation in response to 1 ug/mlof interferon, was found to express slightly decreased levels ofCD4 antigens relative to cells incubated with antigen in theabsence of interferon. Clone TF E97, which displayed enhancedantigen-specific proliferation in the presence of 1 ug/mlinterferon, was found to have increased expression of CD4 and IL-2 receptor after incubation with antigen and interferon relativeto cells incubated with antigen alone. Clone TF E102 also showed
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enhanced CD4 expression after incubation with interferon;expression of IL-2 receptor, however, was not enhanced in thisclone. Each clone thus displays a unique pattern of alterationof surface marker expression in response to gamma interferon.
A series of experi.aents was carried out to examine theeffects of recombinant leukocyte A interferon (AVS 4625) onproliferation of influenza virus-specific T cell clones.Initially, AVS 4625 was tested over a range from 10-2 to 104U/ml. The results of these assays are shown in Tables 19a-19e.Some enhancement of antigen-specific proliferation was seen witheach clone under particular conditions (which differed for eachclone); only with TFE 96, however, could enhancement be detectedover a range of concentrations of AVS 4625 and antigen (Table1c). At concentrations of 103 and 104 U/ml, AVS 4625consistently suppressed proliferation of all 5 clones; thissupression was seen when proliferation was induced by specificantigen (Flu A) as well as when proliferation was nonspecificallyinduced by conditioned medium (CM). Similar levels ofsuppression were seen with optimal antigen concentrations andwith CM (15%-35% of control response remaining), with greatersuppression of responsiveness at lower antigen concentrations (upto 98% inhibition). To further examine enhancement ofproliferation by AVS 4625, this compound was included in anotherset of experiments at doses from 10-4 to 102 U/ml (Tables 20a-20e). In these experiments, enhancement of antigen-specific (butnot CM-induced) proliferation could be detected over the entirerange of antigen concentrations 4-ested. This enhancement wasseen primarily, but not exclusively, at lower concentrations ofAVS 4625, and again, maximal enhancement (up to 4.3 fold) wasseen at lower antigen doses. The effects of AVS 4625 on clonalcytotoxicity are shown in Table 21. At doses of 10-1 U/ml orless, cytotoxicity was either slightly inhibited or wasunchanged. Doses of 1 U/ml or more, however, caused slightenhancement of the response of clone TFE 23, which displayed ahigh control level of killing, and much greater enhancement ofthe levels of killing by clones TFE 79 and TFE 89, whichdisplayed lower control cytotoxicity.
The effects of recombinant hybrid human interferon alpha BD(AVS 5311) on proliferation of virus-specific T cell clones areshown in Tables 22a-22e. As was seen with AVS 4625 (recombinantleukocyte A interferon), both antigen-specific proliferation andnonspecific proliferation induced by conditioned medium weresuppressed by high concentrations (104 U/ml) of interferon. Atlower concentrations of interferon, modest enhancement was seenonly in isolated cases; our experience with AVS 4625 indicatesthat concentrations even lower than the lowest tested here (10-2U/ml) may be necessary to see consistent enhancement ofproliferation. When clone TFE 89 was incubated with AVS 5311 for1 hour prior to addition of Flu A coated targets, enhancement ofkilling is seen at concentrations of 10 U/ml or more (Table 23).
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Addition of AVS 5311 at the same time as target cells results ina slightly lower degree of enhancement, but optimal enhancementis seen lower concentrations of interferon (Table 24).
Poly ICLC (AVS 1761) was tested for effects on T cell clonalproliferation over concentrations ranging from 10 to 10- 5 ug/ml(Tables 25a-25e). As with AVS 4625, modest enhancement ofantigen-specific proliferation was seen under selectiveconditions with each clone; the response of TFE 96 to virus at1:16000, however, was enhanced more than 3 fold (Table 25c).High concentrations of Poly ICLC (1 and 10 ug/ml) suppressed theantigen-specific proliferative responses of all of the clonestested. Here again, the responses to lower antigenconcentrations were suppressed to a much greater extent than wasproliferation to optimal antigen concentration. Theproliferative responses to suboptimal antigen doses were alsoinhibited by lower concentrations of Poly ICLC. As contrastedwith AVS 4625, however, Poly ICLC only marginally inhibited thenonspecific proliferative response to conditioned medium. Theeffect of Poly ICLC on virus-specific cytotoxicity was similar tothat seen with AVS 4625; enhancement of specific killing by cloneTF E89 was seen even at concentrations of Poly ICLC whichmarkedly inhibited virus-specific proliferation by this clone(Table 26).
Poly I.Poly C 12U (AVS 2149) was tested for effects on Tcell clonal proliferative responses over the same range ofconcentrations and of antigen dilutions as were used for PolyICLC; results are presented in Tables 27a-27d. Enhancement ofproliferation of all clones was seen with AVS 2149 at 1 ug/ml atantigen dilutions ranging from 1:4000 to 1:16,000. Someenhancement was also seen at lower concentrations of AVS 2149and/or higher concentrations of virus, but this was notconsistent among the clones.
AVS 2149 was also tested for effects on virus-specificcytotoxicity. When cytotoxic effectors and drug were mixed atthe time of target cell addition, some decrease in killing wasseen (Table 28). When effectors and drug were incubated togetherfor 1 hour prior to addition of target cells (Table 29), killingwas inhibited to a much greater extent. In both situations,greater inhibition was seen at higher drug concentrations.Greater inhibition was also seen in clones which displayed highercontrol levels of killing.
Effects of liposomal muramyl tripeptide (AVS 2933) andcontrol placebo liposomes (AVS 4726) on T cell clonalproliferation are shown in Tables 30a,b-34a,b. In each case theeffects of MTP and the placebo were tested simultaneously witheach clone. Modest effects of both MTP and the placebo on thenonspecific response to conditioned medium were seen; in somecases proliferation was slightly enhanced, in others, slightly
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inhibited. At the highest concentrations tested, both MTP andplacebo liposomes caused marked inhibition of antigen-specificproliferative responses at all antigen concentrations. Thedegree of inhibition seen with MTP, however, was much greaterthan that seen with the placebo; this is particularly evident atlower antigen concentrations. No significant effect of MTP orplacebo liposomes on virus-s[ecific cytotoxicity was detected atany concentration tested (Table 35).
Two related compounds, l-isobutyl-4(iH)-imidazo[4,5-C]quinolinamine (AVS 1018) and its HCI salt (AVS 1300) were assayedin parallel. No significant effect of AVS 1018 on antigen-specific proliferation was seen with clones TF E40 or TF E102 atany antigen concentration or drug concentration, and only modestenhancement was detected with TF E89. Proliferation of clones TFE96 and TF E97 was enhanced up to 2 fold by drug concentrationsin the range of 10- 3 to 10-5 ug/ml at suboptimal antigenconcentrations (Table 36). A different pattern was seen withAVS-001300 (Table 37). This compound induced enhancedproliferation of all clones tested. Optimal enhancement was seenat low antigen concentrations and with drug concentrations in therange of 10-1 to 10-3 ug/ml, but some degree of enhancement wasseen over a relatively wide range of drug concentrations.
Inhibition of killing was seen when either AVS 1018 (Table38) or AVS 1300 (Table 39) were incubated with effectors for 1hour prior to addition of target cells. AVS 1018 inhibitedkilling by clones TFE 18 and TFE 23, which displayed high controllevels of killing, and by TFE 89, which in this experiment killedat an intermediate level; the drug dose which maximally inhibitedwas different for each clone. The low level of killing by TFE 79appeared to be enhanced by AVS 1018 at 1 ug/ml. A differentpattern was seen with AVS 1300. Killing by clones TFE 18 and TFE79 was inhibited by AVS 1300 at all concentrations tested.Virus-specific killing by clones TFE 23 and TFE 89, however, wasmoderately inhibited by some concentrations of AVS 1300 andmodestly enhanced by others.
In proliferation assays, AVS 2880 induced significantlyenhanced proliferation in only some of the clones; the mostdramatic enhancement was seen with clone TF E97 at suboptimalantigen concentrations (Table 40). This compound alone causedsignificant release of 51Cr from target cells, therefore it wasnot possible to assay its effects on virus-specific cytotoxicity.
Six related compounds, ABPP (AVS 2776), AIPP (AVS 2777),ABMP (AVS 2778), ACPP (AVS 3587), ABMFPP (AVS 3588), and ACDFPP(AVS 3589) all required high concentrations of DMSO to dissolve,but remained in solution when diluted in tissue culture mediumfor assay. All cultures therefore contained DMSO at the samefinal concentration (generally 1%) to control for effects of thesolvent on the clones. AVS 2776 induced enhanced proliferation
14
in four of the five clones tested (Table 41). This enhancementwas seen only at low antigen doses; the modulator dose requiredfor optimal enhancement, however, was variable. Enhancementgenerally was in the range of 1.7 to 3 fold, but in the case ofclone TFE102 at a 1:16,000 dilution of virus, up to 14.8 foldincrease in response was seen. AVS 2777 was able to increaseproliferation of all clones tested (Table 42). Optimalconditions varied, both in terms of antigen dilution andmodulator concentration, and in all cases enhancement was in therange of 1.5 to 4 fold. Similar results were obtained with AVS2778 (Table 43) and AVS 3587 (Table 44); neither of these,however, induced enhanced proliferation in all five clones. Noeffects on proliferation of any of the clones were seen with AVS3588 or AVS 3589 under any conditions tested. The relativelyhigh DMSO concentrations required to keep these compounds insolution resulted in considerable leakage of 5 1 Cr from targetcells, making cytotoxicity assays with these agents impossible.
Conclusions
A series of human T cell clones generated against InfluenzaA virus (strain A/Bangkok RX 73) was used as a model system toevaluate immunomodulatory potential of a variety of compounds.Included in the group were clones specific for various viralproteins as well as representatives of different functional Tcell subpopulations.
Many of the compounds tested affected antigen-specificproliferation of some or all of the T cell clones, and bothenhancement and suppression of proliferation were seen. Somecompounds affected proliferation under the same conditions forall clones, while with others, optimal effects were seen withdifferent concentrations of virus and/or immunomodulator for eachclone. The effects seen were generally most profound onsuboptimal control responses. Of particular interest was gammainterferon, which under identical conditions enhanced theproliferation of some clones while inhibiting proliferation ofothers. In most cases, only virus-specific proliferation wasaffected, but a few of the compounds altered nonspecificproliferation induced by IL-2 containing conditioned medium.
In assays of virus-specific cell mediated cytotoxicity, mostcompounds which enhanced proliferative responses suppressedcytotoxicity under similar conditions, while most of those whichsuppressed proliferation enhanced cytotoxicity. Poly ICLC (onlyat relatively low concentrations) and OK-432, however, were foundto enhance both virus-specific proliferation and cytotoxicityunder the same conditions. As was seen in proliferation assays,there was variation among the clones in their response todifferent compounds.
15
The unique patterns of responsiveness of different T cellclones to many of the immunomodulatory compounds tested indicatethat such a system can be useful in dissecting the basic effectsof such agents on individual components of the immune response.Since differences were seen both with different T cell functionalsubpopulations as well as among clones with specificity forvarious viral antigens, it will be critical in future studies touse clones generated against viruses of military interest. Itshould also be noted that all of the clones used here weregenerated from a single donor, thus no assessment of individualvariation in responsiveness to either the virus or theimmunomodulators was possible. The ultimate value of studies ofpotential immunomodulators will not be in the data generatedalone but in such data in conjunction with other studies such asthose which employ in vivo animal models.
16
Literature Cited
1. Fathman, C.G. and Hengartner, H. 1978. Clones ofalloreactive T cells. Nature 272:617.
2. Glasebrook, A.L and Fitch, F.W. 1980. Alloreactive clonedT cell lines I. Interactions between cloned amplifier andcytolytic T cell lines. J. Exp. Med. 151:876.
3. Von Boehmer, H., Hengartner, H., Nabholz, M., Lernhardt,W., Schreier, M.H., and Haas, W. 1979. Fine specificity ofa continuously growing killer cell clone specific for H-Yantigen. Eur. J. Immunol. 9:592.
4. Eckels, D.D. and Hartzman, R.J. 1982. Characterization ofhuman T lymphocyte clones (TLCs) specific for HLA-regiongene products. Immunogenetics 16:117.
5. Weiss, A., Brunner, K.T., MacDonald, H.R., and Cerottini,J. 1980. Antigenic specificity of the cytolytic Tlymphocyte response to murine sarcoma virus-induced tumorsIII. Characterization of cytolytic T lymphocyte clonesspecific for Moloney leukemia virus-associated cell surfaceantigens. J. Exp. Med. 152:1210.
6. Infante, A.J., Atassi, M.Z., and Fathman, C.G. 1981. T cellclones reactive with sperm whale myoglobin. Isolation ofclones with specificity for individual determinants onmyoglobin. J. Exp. Med. 154:1342.
7. Shigeta, M. and Fathman, C.G. 1981. I-region geneticrestrictions imposed upon the recognition of KLH by murineT-cell clones. Immunogenetics 14:415.
8. Meuer, S.C., Schlossman, S.F., and Reinherz, E.L. 1982.Clonal analysis of human cytotoxic T lymphocytes: T4+ andT8+ effector T cells recognize products of different majorhistocompatibility complex regions. Proc. Nat. Acad. Sci.USA 79:4395.
9. Barak, V., Fuks, Z., Galilli, N., and Treves, A.J. 1983.Selection and continuous growth of antigen-specific human Tcells by antigen-treated monocytes. Eur. J. Immunol.13:952.
10. Braciale, T.J., Andrew, M.E., and Braciale, V.L. 1981.Heterogeneity and specificity of cloned lines of influenzavirus-specific cytotoxic T lymphocytes. J. Exp. Med.153:910.
17
11. Braciale, T.J., Andrew, M.E., and Braciale, V.L. 1981.Simultaneous expression of H-2-restricted and alloreactiverecognition by a cloned line of influenza virus-specificcytotoxic T lymphocytes. J. Exp. Med. 153:1371.
12. Lamb, J.R., Eckels, D.D., Lake, P., Johnson, A.H.,Hartzman, R.J., and Woody, J.N. 1982. Antigen-specifichuman T lymphocyte clones: Induction, antigen specificity,and MHC restriction of influenza virus immune clones. J.Immunol. 128:233.
13. Lamb, J.R., Eckels, D.D., Phelan, M., Lake, P., and Woody,J.N. 1982. Antigen-specific human T lymphocyte clones:Viral specificity of influenza virus immune clones. J.Immunol. 128:1428.
14. Lamb, J.R., Eckels, D.D., Ketterer, E.A., Sell, T.W., andWoody, J.N. 1982. Antigen-specific human T lymphocyteclones: Mechanisms of inhibition of proliferative responsesby xenoantiserum to human nonpolymorphic HLA-DR antigens.J. Immunol. 129:1085.
15. Lamb, J.R., Woody, J.N., Hartzman, R.J., and Eckels, D.D.1982. In vitro influenza virus-specific antibody productionin man: Antigen-specific and HLA-restricted induction ofhelper activity mediated by cloned human T lymphocytes. J.Immunol. 129:1465.
16. Eckels, D.D., Lamb, J.R., Lake, P., Woody, J.N., Johnson,A.H., and Hartzman, R.J. 1982. Antigen-specific human T-lymphocyte clones. Genetic restriction of virus-specificresponses to HLA-D region genes. Human Immunol. 4:313.
17. Eckels, D.D., Lake, P., Lamb, J.R., Johnson, A.H., Shaw,S., Woody, J.N., and Hartzman, R.J. 1983. SB-restrictedpresentation of influenza and herpes simplex virus antigensto human T lymphocyte clones. Nature. 301:716.
18. Eckels. D.D., Sell, T.W., Bronson, S.R., Johnson, A.H.,Hartzman, R.J., and Lamb, J.R. 1984. Human helper T-cellclones that recognize different influenza hemagglutinindeterminants are restricted by different HLA-D regionepitopes. Immunogenetics 19:409.
19. Jacobson, S., Richert, J.R., Biddison, W.E., Satinsky, A.,Hartzman, R.J., and McFarland, H.F. 1984. Measles virus-specific T4+ human cytotoxic T cell clones are restrictedby class II HLA antigens. J. Immunol. 133: 754.
18
APPENDIX I
TABLES 1 - 44
19
Donor Medium Flu A/Bangkok
15 1,362 4,130(247) (814)
20 686 6,284(175) (2035)
44 570 16,625(113) (1132)
127 635 3,449(104) (1523)
769 305 15,123(39) (1017)
945 404 19,984(27) (135)
1719 401 8,175(43) (123)
2156 295 6,313(44) (925)
Table 1. Influenza Virus-Specific Proliferative Response ofPBL from 8 Normal Donors.
PBL from healthy volunteers were cultured for 6 days withinfluenza virus (5 HAU/ml). Cultures were pulsed with 3 H-thymidine for the final 18 hours of culture and incorporation ofradiolabel into DNA measured. Data are presented as mean cpm(SEM); all cultures were done in triplicate.
Flu dilution cpm (S.E.)
medium 2557 (559)
1:250 4884 (453)
1:500 7302 (1645)
1:1000 10,548 (1416)
1:2000 4457 (1436)
1:4000 4329 (620)
1:8000 5645 (952)
1:16,000 2140 (774)
Table 2. Dose-Response of PBL to Influenza A/Bangkok.
PBL (5xl0 5 /well) from donor 945 were cultured with the indicateddilutions of virus for 6 days and pulsed with 3 H-thymidine forthe final 18 hours of culture. Data are the mean of triplicatecultures (SEM).
Flu A/Bangkok Flu A/BrazilClone Medium CM (H3N2) (HlNl)
TFE40 161 18,578 21,337 26,025(50) (642) (240) (467)
TFE88 69 9,394 103 157(24) (755) (45) (36)
TFE89 52 7,573 10,906 12,517(19) (347) (52) (392)
TFE96 37 7,342 6,153 79(3) (578) (606) (29)
TFE97 31 6,415 6,919 502(5) (145) (551) (73)
TFE102 41 6,589 10,031 15,783(16) (411) (586) (571)
Table 3. Antigen Specificity of T Lymphocyte Clones.
T cell clones (1x10 4 /well) were cultured with autologousirradiated feeders (2.5x10 4 /well) and medium, 20% CM, or optimaldilutions of virus as indicated. After 2 days cultures werepulsed with 3 H-thymidine for 18 hours and incorporation of labelinto DNA was measured. Data are presented as mean of triplicatecultures (SEM).
Clone Day 2 Day 3 Day 4
TFE40 4295 3037 NT(725) (423)
TFE96 2121 1469 470(345) (215) (33)
TFE97 4196 5293 3631(324) (492) (585)
TFE102 1421 1273 659(154) (360) (131)
Table 4. Kinetics of Proliferative Response of T Cell Clones.
T cell clones (1xl0 4 /well) were cultured with irradiatedautologous feeders (2.5x10 4/well) and influenza A/Bangkok
1i:1000). After the indicated times cultures were pulsed withH-thymidine for 18 hours and incorporation of label into DNA was
measured. Data is presented as mean cpm for triplicate cultures(SEM). Background for clones cultured with feeders in theabsence of virus in all cases was less than 100 cpm.
% Positive
Clone CD 4 CD 8
TFE40 99.4 6.3
TFE88 95.1 3.5
TFE89 97.5 2.2
TFE96 95.9 3.7
TFE97 98.9 7.4
TFE102 98.3 1.9
Table 5. ExpressiLn of Cell Surface Markers on T Cell Clones.
Cells were incubated with OKT4 (CD4) or OKT8 (CD8) monoclonalantibodies followed by fluorescein-conjugated goat anti-mouse Igantibodies. Stained cells were analyzed using a fluorescenceactivated cell sorter.
Clone Medium Flu A 1:250
TFE 18 1.0 52.9
TFE 23 0.7 46.4
TFE 79 0 19.8
TFE 89 3.8 19.1
Table 6. Virus-Specific Cytotoxicity Mediated by T Cell Clones
Clones were assayed for the ability to kill syngeneic EBVtransformed target cells which had been incubated for 24 hourswith either medium or virus as indicated. Results are expressedas % specific lysis.
AVS Number Compound
-- OK-4321968 CL 246,7382269 Tumor Necrosis Factor2350 Human Recombinant Interferon Gamma4625 Recombinant Leukocyte A Interferon5311 Recombinant Hybrid Human Interferon Alpha BD1018 l-isobutyl-4(lH)-imidazo[4,5-C~quinolinamine1300 l-isobutyl-4(lH)-imidazo[4,5-C]quinolinamine,
HCl salt2776 2-amino-5-bromo-6-phenyl-4 (3H) -pyrimidinone
(Bropirimine, ABPP)2777 2-amino-5-iodo-6-phenyl-4 (3H) -pyrimidinone (AIPP)2778 2-amino-5-bromo-6-methyl-4 (3H) -pyrimidinone (ABMP)2880 DL-2,3,5,6,7,8-hexahydro-8,8-dimethoxy-2--
phenylimidazo[ 1, 2-A]pyridine hydrochoride3587 ACPP3588 ABMFPP3589 ACDFPP2149 PolyI.PolyC12U1761 PolyICLC2933 Liposomal Muramyl Tripeptide (Muramyl Tripeptide
Phosphatidylethanolamine)4726 Liposome (Placebo for AVS 2933)
Table 7. Compounds provided for testing of immunomodulatoryactivity.
Modulator Flu A/BangkokConcentration Medium CM 1:500 1:1000 1:2000
0 32 6810 3862 2419 1502(9) (463) (348) (241) (514)
10-3 ug/ml 23 7915 4480 2441 1765(10) (521) (202) (389) (101)
10-2 ug/ml 32 7427 4561 2693 1936(2) (314) (451) (121) (240)
10-1 ug/ml 49 6922 5054 3425 2678(26) (237) (291) (298) (596)
1 ug/ml 19 2474 1481 1008 539(8) (170) (163) (112) (133)
10 ug/ml 15 16 11 25 19(4) (5) (1) (6) (1)
Table 8. Toxicity of Modulator CL 246,738.
Clone TFE97 (1x10 4 cells/well) was cultured with irradiatedautologous feeders (2.5x104 /well) and the indicated antigen,along with the indicated concentration of CL 246,738. After 48hours, cultures were pulsed for 18 hours with 3 H-thymidine andincorporation of radiolabel into DNA was measured. Results areexpressed as mean cpm for triplicate cultures (SEM).
CL 246,738 Flu A Flu AClone Concentration Medium 1:2000 1:16.000
TFE40 0 43 3232 757(14) (186) (134)
10-1 ug/ml 25 3477 1645(8) (283) (228)
TFE89 0 55 4186 948(19) (133) (41)
10-1 ug/ml 54 4209 1642(22) (369) (158)
TFE97 0 28 3128 611(6) (313) (157)
10-1 ug/ml 37 4143 824(10) 440 (180)
Table 9. Effect of CL 246,738 on Proliferation of Virus-SpecificT Cell Clones.
T cell clones (ix10 4 /well) were cultured with irradiatedautologous feeders (2.5x10 4 /well) and the indicatedconcentrations of antigen and CL 246,738. After 48 hours,cultures were pulsed with 3 H-thymidine for 18 hours andincorporation of label into DNA was measured. Results areexpressed as mean cpm of triplicate cultures (SEM).
[CL 246,7381 TFE 18 TFE 23 TFE 79 TFE 89
0 36.0 49.1 3.4 10.1
10-7 ug/ml 43.1 49.6 3.8 10.2
10-6 41.4 48.4 2.2 9.0
10-5 40.9 53.5 1.5 9.1
10-4 38.2 55.5 2.7 8.1
10-3 47.3 53.8 6.2 10.7
10-2 37.6 49.4 4.3 9.5
10-1 23.7 31.1 2.4 4.1
1 2.3 2.5 0.2 1.8
Table 10. Effect of CL 246,738 on Virus-Specific Cytotoxicity
Effector cells were incubated with the indicated concentrations of
CL 246,738 for 1 hour before addition of target cells. Results are
expressed as % specific lysis.
Clone [CL 246,7381 Prolif.* % Specific Lysis
TFE 23 0 13,697 58.6
10-6 14,876 57.2
10-5 14,256 49.3
10-4 14,124 40.1
10-3 15,459 54.5
10-2 16,404 48.6
10-1 15,495 20.7
1 96 6.2
10 33 7.4
TFE 89 0 11,306 26.6
10-4 11,695 22.2
10- 3 12,865 22.1
10-2 13,181 16.0
10-1 14,308 12.2
1 101 8.5
10 59 11.1
Table 11. Effect of CL 246,738 on Proliferation and Cytotoxicity
* Flu A 1:4000
[CL 246,7381 0 hours 1 hour 24 hours
0 15.1 16.5 10.9
10-6 ug/ml 17.7 17.2 13.5
10-5 15.7 17.1 11.8
10-4 17.0 17.0 11.6
10-3 15.7 19.5 12.4
10-2 18.0 17.4 10.4
10-1 11.1 7.6 4.9
1 7.7 7.5 -0.3
Table 12. Kinetics of CL 246,738 Effect on Virus-SpecificCytotoxicity
Clone TFE 89 was preincubated with the indicated concentrations ofCL 246,738 for the indicated length of time prior to the addition ofinfluenza A coated target cells. Results are expressed as % specificlysis.
[CL 246,7381 1 hour 24 hours
0 41.0 38.1
10-6 ug/ml 46.3 47.3
10-5 43.4 46.3
10-4 42.1 44.0
10-3 47.3 44.1
10-2 40.1 47.8
10-1 10.7 24.5
1 1.2 -0.8
Table 13. Kinetics of CL 246,738 Effect on Virus-SpecificCytotoxicity
Clone TFE 23 was preincubated with the indicated concentrations ofCL 246,738 for the indicated lengths of time prior to the addition ofinfluenza A coated target cells. Results are expressed as % specificlysis.
Flu AClone Modulator Medium 1:2000
TFE89 - 72 3325(7) (101)
10-3 ug/ml 68 4805(10) (293)
TFE96 - 83 3277(17) (107)
10-1 ug/ml 67 4099(8) (98)
TFE97 - 95 2120(27) (194)
10-6 ug/ml 117 2799(2) (425)
TFE102 - 63 2531(5) (212)
10-3 ug/ml 47 3651(10) (365)
Table 14. Effect of Modulator 0K432 on Proliferation of Virus-Specific T Cell Clones.
T cell clones (1x10 4 cells/well) were cultured with irradiatedautologous feeders (2.5x10 4 /well) and the indicated concentrations ofantigen and OK432. After 48 hours, cultures were pulsed with 3 H-thymidine for 18 hours and incorporation of label into DNA wasmeasured. Results are expressed as mean cpm for triplicate cultures(SEM)
[OK 4321 TFE 18 TFE 23 TFE 79 TFE 89
0 58.1 51.1 9.5 22.0
10-6 ug/ml 73.6 57.2 6.7 17.7
10-5 70.3 50.7 7.5 18.2
10-4 69.2 54.7 12.0 22.2
10-3 68.6 49.4 5.9 24.0
10-2 62.9 6 2 8.7 22.3
10-1 65.8 57... 6.7 21.8
1 64.6 55.8 11.4 19.7
10 74.5 56.6 9.2 23.0
Table 15. Effect of OK 432 on Virus-Specific Cytotoxicity
T cell clones were incubated with the indicated concentrations of OK432 for 1 hour prior to the addition of target cells. Results areexpressed as % specific lysis.
IFN Conc. E40 E89 E96 E97 E102
0 286 1875 1361 310 110(108) (149) (360) (29) (14)
10-6 ug/ml 206 2833 1838 449 323(108) (432) (203) (67) (48)
10-5 ug/ml 177 2525 2507 667 175(79) (158) (261) (100) (47)
10- 4 ug/ml 447 2235 3575 714 317(245) (211) (860) (118) (57)
10-3 ug/mi 303 2275 1807 614 161(83) (143) (108) (70) (79)
10-2 ug/ml 208 1892 1785 767 239(79) (209) (274) (34) (61)
10-1 ug/ml 396 1253 1968 1152 180(61) (143) (377) (171) (48)
1 ug/ml 515 800 1877 879 347(169) (102) (108) (162) (77)
Table 16. Effect of Gamma Interferon on Proliferation of Virus-Specific T Cell Clones.
T cell clones (1xl0 4 cells/well) were cultured with irradiatedautologous feeders (2.5x104 /well), a suboptimal concentration ofinfluenza virus (1:8000), and the indicated concentration of gammainterferon. After 48 hours, cultures were pulsed with 3 H-thymidinefor 18 hours and incorporation of label into DNA was measured.Results are expressed as mean cpm for triplicate cultures (SEM).Clones incubated with medium rather than virus incorporated less than40 cpm.
Clone Interferon % Positive Mean Fl Channel
E40 50.2 244
+ 46.3 278
E89 62.5 367
+ 47.8 278
E96 53.6 254
+ 42.8 235
E97 61.5 295
+ 86.0 619
E102 62.8 365
+ 79.8 492
Table 17. Effect of Gamma Interferon on Cell Surface Expressionof CD4.
T cell clones (1x105 cellsýml) were cultured with irradiatedautologous feeders (2.5xl0 /ml), a suboptimal concentration ofinfluenza virus (1:8000), and 1 ug/ml gamma interferon. After 48hours, cells were analyzed for expression of surface CD4.
Clone Interferon % Positive Mean F1 Channel
E40 15.0 178
+ 14.7 186
E89 14.9 104
+ 11.5 173
E96 14.9 198
+ 22.0 223
E97 5.5 258
+ 40.5 254
E102 11.0 192
+ 10.2 204
Table 18. Effect of Gamma Interferon on Cell Surface Expressionof IL-2 Receptors.
T cell clones (1x10 5 cells4ml) were cultured with irradiatedautologous feeders (2.5xl0 /ml), a suboptimal concentration ofinfluenza virus (1:8000), and 1 ug/ml gamma interferon. After 48hours, cells were analyzed for expression of surface IL-2 receptors.
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10 66.2 16.7 25.4
102 67.6 19.0 25.4
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104 66.7 16.4 24.2
Table 21. Effect of Leukocyte A Interferon (AVS 4625) on Virus-Specific Cytotoxicity.
T cell clones were incubated with the indicated concentrations of AVS4625 for 1 hour prior to the addition of target cells. Results areexpressed as % specific lysis.
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104 32.8
Table 23. Effect of Hybrid Human Interferon Alpha BD (AVS 5311) onVirus-Specific Cytotoxicity.
Clone TFE 89 was preincubated with the indicated concentrations ofAVS 5311 for 1 hour prior to the addition of Flu A coated 5 1 Crlabelled target cells.
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102 37.8 38.4 27.6 39.5
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10 4 35.4 41.2 26.1 38.2
105 33.3 44.7 26.4 40.7
Table 24. Effect of Hybrid Human Interferon Alpha BD (AVS 5311) onVirus-Specific Cytotoxicity.
The indicated concentrations of AVS 5311 were added to cytotoxiceffectors at the time of target cell addition. Results are expressedas % specific lysis.
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ON ( (n N %D U) H4. U)
H H H 4
0 MP4. )
go M' 0 M' 0 00 LA (11 %D0 u3 .o 0 H LA 0 0'. v - H- to) to
!. a'. N N- N 0a% m LA N 4-4 p M 4
H** CN en en4 cn N~ H 0 4) 4
*H4 4-IH
0 WU4)
O N- H- (n %0 % 0 NV L A 00 0) 0 - 0
o m ~ M e L A % D 0 C H % D L A % .0 '4 4 W01 o ((A 0'. N co N- N. C Q) 00 -H 0 $ 4
C13 -e '.0 % '0 Iq ( c f -4 H r-H0 0 0
t~o 00ý
0 V~
o0' LA U LO N~ LA LA '.0 LO - v -) ao N, N v 0 LA v m4 ('. H (04.)
,- 4 0 H- , COD Co 0 W'%.- 440 H' 0%D 0 C O N0 CID N, '.0 m~ H- N, ru
r-4~. *0 H
V~ H00~~~ ~ ~ v*n. nUl Or4 U -4LJ
S 0 O'. -c LA %.0 H- m~ Iq 0 :9
u N- H (1) H- H- m LA %D u H-144'.0 W. '0 %.0 W. %0 LA Vj H 0O
1 04)
H- (04-4 00 a0 -eq
04 0 41.
:3o 0 00N ~ ~ ~ ~ ~ -N - - Q PP
0 0 1.0
z 4 U :30~4.4 C ~4-)
4 -I to 4-)
0~~~ ~ ~ 0*' 00 - qC-
(0 HO 4DPN4 H LA4 *j
rPolV ICLC1 % Specific Lysis
0 15.7
10-5 26.9
10-4 24.6
10-3 25.6
10-2 27.6
10-1 27.0
1 23.6
10 24.1
Table 26. Effect of Poly ICLC (AVS 1761) on Virus-Specific Cytotoxicity.
Clone TFE 89 was preincubated with the indicated concentrations of PolyICLC (ug/ml) for 2 hours prior to the addition of target cells.Targets were incubated with Flu A (1:250) for 24 hours prior to assay.
O N~ LO 0 N N, LO Ml N:o 0 l ( m~ Ch co ml m LO (11r-4H
(44
o 0- '-4nr- - -I rI r
o N- N N N~ (n LA 0 %D0(%D en 0 LA LA (Ai V N co 4)
E4 Ic -H (A
0~~~1 4.V) o N
'4.) to)a4 ON -4 4
o~~~~~ -H V oO 0 0
H (4 4 - 04 4
0 cn (o towo 4
00 LA Nl N LA %D fn N rn ý4 H-4. N- N, %0 a% Ný Co v N 4) 0,W
NT H~ H- '4-4 44 0 )
0 *1-4 v,
or- 0 a,o CN ON 0 4.r Co N- %0 4 0 ON-1o H- 4. LA W0 LA O~N r C 0 to :
N V4 CoD (A LA %0 v. v. H- H4 U
Hq H- H- H- H- H- N Cl ON 0 : t
H o MN 40
o LO 00 N- N H H 0 4. -
o mC N (A H4 %0 LO 0 o*r4.) Z
N~ ml N~ Ml N N Ml ml NHf H- w 0
W~ 4.)
H- 4.)HHI -0 4 r4Na)
ml Co 0'N N 0 O N N, r 3: .Q
Cl LA Co LA %D LA CO ml * ) (aO 0) 4. '0 LA ON 0 ON Hl 1-4 V>
Co N, N Co %.0 N , N -NL>1 4. 4-4H- (0(4-400 .40
U. - 04-4 r. -I-40 .414.
:j CO v. C) H N Ný H- NAU1*H ON N ON LA LA Cl IV %D .41 (L) tp w
44) 41~0
C -4 WOH
0 04-) 0
%D0 LA v.C N H4 N 4-) (0U
I I I 1 I P- r_ --4
0a 0 0 w
00oN a% m1 %i O D r- N r-
S0 N 4 LA) N N N N N ýrH 0 -i
4-4
0'
0o r- r- C-4 qtr co 4to%D N LO a% N co r, H- N 4)H- ýo co co ON r- ON 4q %D a% *.4 41 0
N co I
'4.) =
O) 9-4 W
om~a a ~ N OD ON H 0 0)O 4 r% LO m' LA LA LO m~ H- -d 4cc a% cc 19 cc LO 4w (1 r- 0
coH H- H- HA H- - N 4) mxH4-4 trir-4(1
o ~-4
4.4
0 4.1 -4)o r= m kO r- LA . m r- to to l W 4.)0 H- m~ N~ 0 LA H- in v 1-4 S-4q4 LA '.0 v LA Ci 0 H r, 0) 4) t
I r4 I H H N H- N~ N N m1 44 0) oWH "- 01-404
o 4.4 4Jý4 r-4
o r. 0 01)o c-, LA 0 LA CID M~ 00 4l 0 t~l .- $o ON H- N a% N a% H H 0 t)
N H N a% M' cc cc %D0 LA . I Hw t** m N -4 e1 cli N H (1) 0% 0 d0
H 04 4i000~~~ 0 0 m N c
'IT~~ ~ ~ ~ N lnr- %LOv4
> 0
or- 4J r-4 OrH-4rN cH N 0 A O0L -A C, '-0)
ýo 4n 4 l OlN N- a 3: 4~
Hq H 0 44 00 404~
U -4 0
o4 *-I -1
.1 ON 0 eC -H '0 LA r- 4J H) 0
i W . )
OO 0 4*T H Nl Ou p 0 H wd>
>Q 4.) 4.a4
C.) 0 0.4- H-.-I --
0 4j4
"a4 4~ CO Vl C C l 4.) '1 0
1 1 1 A '0 1 P-4 N. U-40) 0) 0 0 0 r- A
r--4. f3 r-4 r- $ 0>- W0-4
04- 0 w
(1 C 4-
o LA N ý,D LA (1 LA H4 (n 9-~O N t0 f N cl m~ m
H4.4 0144
41 -v.4
0 U -4
0~~ ~ ~ Hn Li Ln C) M m %0 V- "T o a% 0 r4 CO4 Iq(
H H '0 *'-E-4
o ON CO %D0 r- LA r-4 r- c'n 0 4)o co 0 IT Cl) 0 0% r- LA H- - 14.OD OD H- CO LA r- ml OD ON 04
H4 H- H- N H 0) JWxH4-4 r-4 (1
0 :34.41 044)
0 to '
o 0 %D ON C-- N C-- ai LA (t w3t4~0 H- N N LA N N ON %.0 5-i r-i
lw N N N OD %0 r) '0 a) M :5.. H H- Nq N H- H- H N 4-4 '(W)fM
0 4-44-)
or. 0 Wo LA a% IT (1 m ( 0 ml 0 15'-$-p
o o ON CN 0% H- 'I m' m 0 to::N 0 %D LA CN lq~ C- N* N - 4 P$1M
Ml N H4 N CN N1 CN m0 :3 0 toH- :3 4.10R
CN (0
O H '0 N N~ 0 (7% C- Ho 0 LA r H LA co H- 0 V 44Z
H OD C-- cl) Cl) LA co Ir D ~44Oen cn ml m -d' en M N (a 4
U _4 4 .)
ON -v.4
o -14-IN (0 - H H- N1 ON '.0 LA a4 :3
Z LO O'N N CN f N %0 '.0 LO(U N CN co C-- 0 OD OD '.0 >- r-4
LA %.0 LA LA) '0 LA LA LA>1 4) 4-4
0 .0 0
U -0
0 .- 4 r:4-)
-.14 CO a% H- (1) C- 0 r-- H 4-) 0) p (L)
C,4 N n %D N N LA r- m pl j S 02p
14.4 3C -4-4 U) 0H
c uu z
0 4-i 0U H'4J
Cl L N H- (N 43I~H I 1 1 -
] 0 0 0 0 0 H r-4 ) u 0
0 0 U ý-40-0E4 r
o Ho
~O ( O N Cl) M ON %0 LOHH I l r.4 () m 0 LO N -W m l
4-4
4-) -4
0V
o (11 LA %DO (1) co (7% %D0 le%. 0 %D LA w0 0 CA~ LA
H N~ C ~ H N - -- 14.1
.am IVH~P Vx W
Co -W N L A N HD HO OD 0- 0
a) to
4-)
o to 4 o'041) - 4)W
o N ml N N, H- N- VJ Ml CO V):30 co 0 N- 1w m LA 0 N w- M. H-r- 0 co 0 %0 H- N- (A m N 0)4 0W
r -4 VW :
o 44 4.)$4 W -I
0 UVo w - ,r, N c 0 tr-
oA H -1 N N0 N- M %D 0 to -
o -N -O r N N N. rH Cl C-4 (7 0W: (N~q 41 0 N l 0 C ) S
H 4 R
M a, qwto i~4) 3:
o (V) OD N- ml H %0 r- LA W40io OD %D0 0 %.0 0 0 lid 0 V 4-ZH- H- 0 (IN H- (A 0 co N , V440
M*ClC Cl) 4r V V M N N1 tv 4
5-4 V.
>4Hq 4j HH -o -4-C4N4
LA Ml LA) M 0 0 U) LA 04 .0S 0 (1) 0 al CO ml IT N1 U) (a
co r, 0 W. OD "T %0 LA H V -4LA LA %.0 LA LA LA U) LA
> 4 4J 44IH- M04-4 0o .0o
04 :3 rU - 0
4-4 c -f -r40 --4R 4
Wl N, N- LO N , LAoL-H 0 N co co m -T l 4.) (1) $O)
V H z S0 -iO) 4) w- Q,
4-4 3 4 54-)4-4 WOH r-
Cz A r. u4)0 Cr. -H -40 4.) a)
0) r-4 (0 04-)
CN US-4 a
.0 ur 3-(a 0 05wE- E- ~4 V
fAVS 21491 TFE 23 TFE 79 TFE 89
0 66.5 5.3 17.0
10- 5 ug/ml 62.1 1.8 11.2
10-4 60.0 2.6 10.0
10-3 60.1 4.5 15.6
10-2 63.6 1.5 10.6
i0-1 62.6 3.4 11.2
1 60.3 0.9 8.9
10 51.4 1.3 12.3
Table 28. Effect of Poly I.Poly C 12U (AVS 2149) on Virus-SpecificCytotoxicity.
The indicated concentrations of A;S 2149 were added to cytotoxic effectorat the time of target cell addition. Results are expressedas % specific lysis.
[AVS 21491 TFE 18 TFE 23 TFE 79 TFE 89
0 47.8 78.2 15.3 48.6
10-5 ug/ml 62.3 20.5 43.4
10-4 65.2 18.3 41.5
10- 3 67.9 14.2 44.2
10-2 62.7 8.8 41.8
10-1 32.9 64.1 18.0 43.2
1 21.7 52.8 15.3 37.3
10 27.2 48.7 14.3 35.9
Table 29. Effect of Poly I.Poly C 12U (AVS 2149) on Virus-SpecificCytotoxicity.
Effector cells were incubated with the indicated concentrations ofAVS 2149 for 1 hour before addition of target cells. Results areexoressed as % specific lysis.
".D (n ýD (I) r- C: r-4 (1) ':? r-oLC r-4 11 L' co 'I 1 r~ I"
0- r Li) ui) (0 0) U') --I I
I-4
0) CT) (1i (-V CO a,. ") (A 0 Y
LD C0 11 0) r- co CO 01, wD (1- Clj r-4 r-
4 1-1 Cl\J cl r-
4
0) -.1 m'~ o Li r-r G) c12- (C:) C r co C o CO CO ) U i cl .--C) m 0' 0) C 0 1-- . - co
-: CO r. -4 cl l C '4 C
.--- . ) .. ~ 4 ~ ~ ~
"r4 1-4
(yC)
-4..4
~C 0
-4
ý4-kC)
r-4 0.4.
ý4-
r-44
00
0 (') 0 C, m 7 r- C") ON 0ý
(Q C r 4
('1 c'J '-: 0 co 'V' cJ i0~ ~~~ C' rJC) C -4 LA ) -
r-i
o) cj-) r' L -q () 00 LA -')
o (YC) kC. -4 fl U-.o 0 'ý: '- ' LA .- 4 a)'
- (\: 4 4 ~ ,4 r
o r~ -4 r-4 c-4 G) 0'
~~~0U L) c U
~co Cr -4 " -A cli
[j4 r-4
OD 0 C0 CO LA ) '
S0 r-4 co C '] '
.0 1•) () '.D 10 0N (Y) ~0'' (D ti~ C") C) U) 0" -- 5
--- 4
~J.4 Ll.-- 4-
0 LA CJ C-4 LA C Jn'T
~~~C-- r-4 M' 1A " r ~ ~ ) -LI))
~L4 D~ '-
r--4
r-4 0' D J - ") .
~r4 U') ~ i) -4 LA) I- . ' ' -- -
-- 4..
K7)
1--4
0) LI) r-4 m CA r.c-q 0C) C' -q -4 kD (.O ) Uli lz
"r-4
o H - " C) '.0yi C'i -)o t~o in 00 co CA~ ID LOiC) r- r- (TI r-~ .1i tD f o
- C-4 CN m- N- -A-
r-4.
o 1- i V) C;%. CA] ifl <v1 C4: m) (n '.0 Co r'o --- 0a-, CO
C) OD 0) uLr 0 (NJ ii to Cri
CD-4.. r 0 c
4: C), L.- 0'~ r-) o co 4) oC) c"i (Y') a) C '3 Ch co C
"r4 1--I
0]
0 (n r- ,. -' o C' 4'~ -4:- 0 i'r (1C) (11 rmC) CO C) C'-
C) co C" Ch CD C' 1- o
-4
C) ) '' - C) Co (7. r- .r) C'
4C-) CO) C'10~ i ~ ] C0 Hl ::) C) - , C C '
[-4 -4 -4- 4.iO
-44
'44
co C) Li IDo C 3 r
3'i 0O CI CO CO C) 0- (1
c I ai C) -4 ,4 C- '-' -4 r -
'-4
o 4l i ~ J ) c (fl cm ID E)o r C r-4 r-4 c~i m n )
[14 -- A ~ ¶
C:)
.C0c)r- * ~ ' 4~ LO 0o co ci) i CO r ' c co
~Co rq J (Ii co N) r
03
r- c5 (TC" i "
(44
0
o ~ r' cmj o-) 31 C;l 4- )
oo CO) U-) C' C CID I--t*
4-4H
U -4
0.
CI Co (n- CO ui 0 Co
-~ ~~ ~ (Y) 0 c O O Lr-4 (DO I'
4 44
'-' G ~ U' ) -4j n C
-. 0 Icm cmD cm c:DoCor- -4,I r- -
r- z C11 kN Li) mi U1')
0 (n H n --4 S -
0Q (1) CA4 (NJ H H4 CC) N 'S Co oD 0 m~ '.0 (n CS U') S
- (e) ý (n mS ( C, (N H H
(n U') H4 r-4S ' H CAC)4 0 14 0 0 H- co COZ cr, H (n
ZO '0 O i r- ri U)i OD o'S 00 lz
I1-4 c
10 0 tn {J) C". r- N- (n co IQ (Y) (13 OD co G". 'D . - -
O H- m4 1) LT. H4 H4 r-H05 C '0 c %D N L Li I 59
-44C
IH
ý-41
CO 0 Lco 0- O -- ,.(I. I'D .~ N '-J' ro C) -
0 l N CO Cl) L-) ~ CO , cCO 0 - C U)
H 4-ý
4-4
C .0 C) N - 0 CD CS C
- 4 S i Cl H I T r-4 1 -4 -
o- r - - l r- co 0 r-
0 ~ ~ - 1JC ]C lC -4 C0 Cl
1-44
(-Y) C r- CD ~l C C~0 co 03 r-4 1-1 00 - '~ A C
0 0 r- (1) Gc Lfl Lf) 0) 0 coZO Cl Cl ~(e1 C'] C C -
4 C
44
C) 0 L r ' )L 0 L-) C 00 cmo ml U-1 () co (n 1-4 0 Ir- Coo f LI- LA - LA el) M
'-.4E1
LA. >l 0 ~ C 'i 0.
-4 A-4-
0
-4 CA Cl C r S -4 C .I -H-4 LI Co ' - '' ~ C- 4
0 H- If U-1 -V -I CJ :13 CA
;: c co -) C ' C co 0 Ln
-4
(Y) Cl ) :D 0 r-- 0D Co
~0 9 C LA -4 CA L:) m0 W C
ZZ 0 -- CO ,4 C- Cf) m) co) co
'--40U)J0
c1 0 9 0' IT c0 r- 1-4 -4 Cl -
'o C." co 10 O o Co ra CO Co 0
J)
C) C)-
- cw)~ f l 0. C' > L
[-. (Nj UL r4
i I'D U-) () Li -IC)0 r- C l') (Y) mn CO\ kl ()coo -4 L u") U' IC> MO (1 CO) (N
z0
44
0 0) CNJ co (Y) CY Ui .] In IoI C0i -4 m~ .V (13 ýv (N-j f -oD .,o co 0 CC) G% (N' Li r- ID
- ('13 mN 14 r-4 1-4 4
1-4w
r-4 'A ' O O (N L
~~~~J~Y 0) 0N N - C) cJ (J C0 (1O 00 :r m
4 ~ i 0 1 0
ca fn CA m ~I LI) CD q4
t0r- (- co U') m. MO 0. -i r-0 t-l 0l (AJ (N 'j - 0 Q) co
44
-44
~' -4 C~l i 0 L C'. ~ . r-4
0 4 0 cT~ ' 0 ) C) COCI
0-4 1- -
ý4-
-4-4 -4
co C'O co 0O cf' U' I C
'0j zli i (N CA r- 0ý r- rrIn 4Dn ýID 9-4 0o (J 0', ~J o CO- ~ C') CO CA COCAU N-
U4-4
-41 O -4 r-4 rN 4 r- 4 r-
11E-
C)la Lf l CO C - C0 CO CV) LI 1-4 r -
4 CD Ur) Cl (1) .C)~ - c * -4 Lr rv 1-4
-4-4q
roC- co c Li co m~ co oT-1 wN U') -1 03 col
-4 k
r-4
r-40)
44 4 -l ~ - ) C o - -
-4-
r~(C) F C. c ' C Cl .0 c) com)C. a '' '0~ r- C- C Ci
::I ) t.0 L..) z- -~ LI ;Z IV
1L44
0
co- LI a) r. ~ - 1-4 .- 10C 1- . 0 r- r- co 'ý. CI i) 4-C) a) t-0 %i .D 0) 1V m L,') (3) '
1-4 "-4
0;-4
14 Co co m' 0) m0 a - - ~0 ) C <V U-) Clj L:) c ci
C) m) 1-1 C' LI ) 0 .o C r-SC -4 00 Co -I CA C) C) C) r-
-44
0oo c.! c i r, r- C- C i -I
0)~ '0 C '0 C) '0 mi -4C--4 Cl c C-4 111 Cl --
,D m0 a ' Co C) m Cn- a'I Q0 0
a4
cl a'T G)~' a r-. - '- C'! (n '4-4:5 ý3 co C- !:4" 1- 1 Co C-- -4.1 (1 '-1 1-4 C". l Clf Cl (j
4-j
CD1 0 '-I
-4- 1- 4 - ,-4 1 "
C) (Y) -4 r, 0N L'5 0Y C) 3C Q o Le) N- 0 (1) (n L) *.,C CO N
r-4
0 ) ~ ~ N C r-4 N- %DfrC -4 tN co N- Oi 00 -4 a)i LA
o CO Nl 111 Cl lzr r-4 i
-~= 4 ,H r-4 rH
r-4 wl (J~ ~ C -
44-4
-4) Cl) ci 03 I'D (n LA CT)o~ co r- f..- ml r- o4 CL Cl
C) Le) a) C'! L-) r-4 (NJ C C'! -
P4 -!
r-44
:o co wi I'D (n fl ~ CC) CO 1! C 10 N~~' CoC L-1 C C') I~ en
44 0-- 4
-4-4
r- UA r-4 (1' (Y) (n 00
<0 r~ C-4 00- -A C."LA C!
i CY) 1-1 U'0 ) k O N .
-44
-44
Cm ~ i-- 0 L '-4 Cl C(75
"N O C 4 O C '
r-4 r-4 "0 C') r-. -4 0 L rC) ý44 * r-- (Y) c'i U-) r c
-J4
LA (n ýoCl~ 4 :V r-4 LI) f~0 r1 - -. Nl LA 0 CA C110 - Ln w 0 r- oo - t '
-4
0 4 -4 14r4 -
'--4
Ft C) 0I J.1 Al co L) -4CC: 0~ kcO CI C') LA L 'Ycm 0
m0 (~i f-4 ,-4 ("J4 -4
(
-400 0
(-') G- -- L C) a
0) ,D u) a ('1 N '.)' l C0 m L- l j C) (") r-4 Ci 4 C
-4 -4
-I, a)) LA) Cm C' 4 LrCI 0 . A r
4 0 a ) -4 co 0 I-
-4. I'D (I W ) C
U)
'3. - (4 r- 0 CO 0 A 'LA 0) co co 0- CO 0l
0 -C. -4 LAo 0 0- r-4 c-i (I0 I'D-0 0 r- .o 0 m. 0-) LA LA. r
,J))
-4444
(1)
LA L '-4 C') -- C Co4
C- 0 0O 0 - (0 N
1--4
% Specific Lysis
MTP Placebo
0 23.5 23.5
10-7 ug/ml 20.5 18.8
10-6 22.8 20.4
10-5 19.8 19.3
10-4 23.3 20.5
10-3 21.3 20.7
10-2 21.5 20.4
10-1 23.2 21.0
1 21.1 17.9
Table 35. Effect of MTP on Virus-Specific Cytotoxicity.
Clone TFE 89 was preincubated with the indicated concentrations of MTP(AVS 2933) or placebo liposomes (AVS 4726) for 1 hour prior to theaddition of Flu A coated 5 1 Cr labelled target cells.
Clone [AVS 10181 Medium Flu 1:8000 Flu 1:16,000
TFE89 0 104 2077
10-5 64 2783
TFE96 0 65 531 396
10-5 31 1339 622
10-4 36 967 720
TFE97 0 69 1852 1256
10-5 72 3112
10-3 122 2583
Table 36. Effect of AVS 1018 on T Cell Clonal Proliferation.
No significant effect on antigen-specific proliferation of clones TFE40or TFE102 was seen at any concentration of AVS 1018.
Clone [AVS 13001 Medium Flu 1:8000 Flu 1:16.000
TFE40 0 99 5613
10-3 60 10,793
TFE89 0 88 7883 6685
10-5 65 12,384 8394
10- 4 49 9257 8681
10-1 117 9644 10,746
TFE96 0 97 5627 3560
10-3 50 7743 6356
TFE97 0 221 5076 3248
10-5 67 8761 5583
10-1 77 9306 4645
TFE102 0 981 10,083 5901
10- 6 263 14,713 9437
10- 2 224 12,617 9899
Table 37. Effect of AVS 1300 on T Cell Clonal Proliferation.
[AVS 10181 TFE 18 TFE 23 TFE 79 TFE 89
0 63.8 46.4 3.6 14.6
10-7 ug/ml 60.3 33.0 4.9 10.3
10-6 59.0 23.0 2.3 7.8
10-5 58.1 27.4 2.1 10.8
10- 4 51.8 33.4 1.2 8.8
10-3 69.4 34.3 3.9 10.6
10-2 57.3 37.6 3.5 11.3
10-1 61.3 36.2 1.2 16.1
1 48.9 33.4 8.8 11.1
Table 38. Effect o' AVS 1018 on Virus-Specific Cytotoxicity.
Effector cells were incubated with the indicated concentrations ofAVS 1018 for 1 hour before addition of target cells. Results areexpressed as % specific lysis.
[AVS 13001 TFE 18 TFE 23 TFE 79 TFE 89
0 63.8 46.4 3.6 14.6
10-7 ug/ml 35.8 30.8 0.2 8.1
10-6 47.6 27.3 -4.3 9.0
10-5 41.2 39.7 0.8 20.6
10-4 44.4 41.0 -4.9 19.9
10- 3 51.6 52.5 -1.0 10.9
10- 2 49.0 52.7 -1.2 13.0
10-1 37.5 51.8 -1.4 9.9
1 46.4 45.1 -0.8 14.3
Table 39. Effect of AVS 1300 on Virus-Specific Cytotoxicity.
Effector cells were incubated with the indicated concentrations ofAVS 1300 for 1 hour before addition of target cells. Results areexpressed as % specific lysis.
Clone [AVS 28801 Medium Flu 1:8000 Flu 1;16,000
TFE96 0 40 3209 1687
10-4 85 4387 2300
10-3 18 4352 2017
TFE97 0 240 3213 1153
10-5 167 4747 2118
10-4 151 3504 2611
TFE102 0 518 3447 1817
10-6 156 6341 2434
10-5 74 4288 3119
Table 40. Effect of AVS 2880 on T Cell Clonal Proliferation.
No significant effect was seen on proliferation of TFE40 or TFE89.
Clone [AVS 27761 Medium Flu 1:8000 Flu 1:16,000
TFE89 0 56 811
10-4 31 2,435
10-3 18 1,589
TFE96 0 27 1,149
10-2 29 1,961
TFE97 0 15 378
10-6 34 792
10-4 18 639
TFE102 0 36 606 67
10-6 59 1,759 277
10-5 90 1,272 581
10-1 30 993
Table 41. Effect of AVS 2776 on T Cell Clonal Proliferation.
Clone [AVS 27771 Medium Flu 1:2000 Flu 1:4000 Flu 1:16000
TFE40 0 37 2,743
10-3 33 4,065
TFE89 0 23 3,281 2,096
10-3 23 3,168
1 41 5,266
TFE96 0 35 831 155
10-6 33 599
10-5 19 1,583 411
TFE97 0 23 1,167 157
10-6 25 636
10-3 17 1,703
TFE102 0 19 381
10-5 19 1,165
Table 42. Effect of AVS 2777 on T Cell Clonal Proliferation.
Clone fAVS 27781 Medium Flu 1:1000 Flu 1:2000 Flu 1:16000
TFE96 0 38 2,679
10-6 216 4,157
TFE97 0 383 8,580
10- 4 269 11,490
TFE102 0 483 11,875 567
10-3 159 15,602 1,893
Table 43. Effect of AVS 2778 on T Cell Clonal Proliferation.
Clone [AVS 35871 Medium Flu 1:4000 Flu 1:8000 Flu 1:16000
TFE40 0 131 1,861 563
10-4 144 1,145
10-1 39 2,943
TFE89 0 64 779
10-4 21 1,491
TFE96 0 53 259
10-3 131 593
TFE102 0 73 1,922 487
10-3 151 3,523
10-2 312 1,084
Table 44. Effect of AVS 3587 on T Cell Clonal Proliferation.
APPENDIX II
Scientific Reviewer's Comments
SCIENTIFIC REVIEWER'S COMMENTSFINAL REPORT, JULY 1, 1991
CONTRACT NO. DAMD17-86-C-6154P.I. - MAJORIE L. COHN, Ph.D.
Subject: Review of Final Report Contract DAMDl7-86-C-6154.
1. Interesting data was generated on the in vitro biologicalactivity of immunomodulators, most of them have marked antiviralefficacy. In this study, influenza virus specific human T cellclones were used to assess some of the biological activity of thecompounds. The lymphoproliferative response of T cell clones tovarious influenza virus strains was not uniform, which indicated,that these clones recognized determinants on either thehemagglutinin or neuraminidase components of the virus. Likewise,the response of the clones to the same immunomodulator was notuniform, which could mean, that different signals are required byvarious determinant-expressing T cells. Although, the P.I.documented the lymphoproliferative response evoked by theimmunomodulators with each of the various clones, it is verydifficult to draw conclusion from more than 40 tables. A summarychart would help to sort out if indeed various signals are requiredby the clones expressing different antigenic determinants of theinfluenza virus.
2. The P.I.'s claim, that certain immunomodulators inhibit thecytolysis of EB virus-shedding T cell clone, requires a set ofcontrols which, has not been done according to the methodologyprovided in the report. Incubation of the target cells with testconcentrations of the substances in the absence of the attackercells would show, if the decrease in cytotoxicity is due toinhibition, or, to toxicity. The fact, that the decrease incytotoxicity was always obtained only by high dose(s), makes thetoxicity the most likely reason. The P.I. should provide the data,if she has them, to exclude toxicity.
3. A summary chart for the stimulation or depression of T cell-mediated cytotoxicity by the immunomodulators, would help to findthe kind of signaling required for evoking the effector mechanism.Cytokines stimulated by most of the tested substances are known,and documented in the literature, or I can provide most of them.
4. The contract generated useful information regarding thebiological responses stimulated by selected immunomodulators, butmore can be learn with a little more input. The final draft shouldcontain the requested revisions.
Meir Kende Ph.D.COR.Virology Division.
Note: These changes/suggestions were notmade by the Principal Investigator.