Immunology and Cell Biology

(2005)

83

, 175–181 doi:10.1111/j.1440-1711.2004.01306.x

© 2004 Australasian Society for Immunology Inc.

Research Article

Cooperation between CD4 T helper cells is required for the generation of alloantigen-specific, IFN-

γ

-producing human CD4 T cells

T A R A S T R U T T a n d P E T E R B R E T S C H E R

Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Summary

We have used MLR to assess the cell interactions that enable human PBL stimulated with irradiated,allogeneic PBL to give rise to allospecific, IFN-

γ

-producing CD4 T cells. We were able to generate alloantigen-specific, IFN-

γ

-producing T cells from peripheral blood. The responder and stimulator cell numbers required for theoptimal generation of such cells, enumerated using an enzyme-linked immunospot assay, were separatelydetermined for various combinations of responders and stimulators. The number of antigen-specific, IFN-

γ

-producing cells generated per 10

6

responder cells, seeded at the initiation of culture, is critically dependent on thedensity of the responder cells, with minimal generation of IFN-

γ

-producing T cells at low responder densities. Theseand further observations with fractionated responding PBL show that CD4 T-cell/CD4 T-cell interactions, which arecompletely independent of CD8 T cells, are necessary for the

in vitro

generation of alloantigen-specific, IFN-

γ

-producing CD4 T cells.

Key words:

alloantigen, helper T cells, human, interferon-gamma.

Introduction

Studying the mechanisms controlling the regulation of humanresponses presents unique difficulties in comparison tosimilar analyses of mechanisms controlling animal immuneresponses. The most frequently encountered difficulty is aninability to generate antigen-specific responses. This diffi-culty stems mainly from an inability to obtain large numbersof responding lymphocytes from volunteers. It is also oftenproblematical to carry out, and repeat,

in vitro

experimentswith lymphocytes from a genetically identical source. Thesecircumstances have led researchers to study the activation ofhuman lymphocytes using reagents that can act on a substan-tial fraction of the lymphocytes present, thus allowing thegeneration of measurable effects with relatively few lympho-cytes.

1

The most widely used reagents include lectins,calcium ionophores in combination with phorbol esters, andanti-CD3 antibodies immobilized on plastic or bound to APC,whose effects are believed to mimic the effects of receptorengagement.

2,3

Monoclonal antibodies against costimulatorymolecules, transfected cell lines expressing desired costimu-latory molecules and/or purified cytokines are frequentlyemployed in combination with these receptor-mimickingreagents.

1,4

Although such studies are interesting, it may behazardous to conclude that antigen-specific lymphocytesbehave in the same manner when exposed to their cognateantigen. For example, marked differences in lymphocyteresponses to nominal antigen and to different polyclonalstimulants are observed when T cells from normal and atopicindividuals are compared.

5–10

Another strategy frequently used to circumvent therequirement for large numbers of unprimed lymphocytes,which avoids the use of polyclonal stimulators for T-cellactivation, is to study immune responses against alloantigensbecause the frequency of lymphocytes specific for allo-antigens is approximately 100-fold greater than for conven-tional antigens. Numerous human systems rely on the use ofHLA antigens for

in vitro

stimulation; however, the antigen-specificity of responses is often difficult to ascertain whendetection and characterization of effector T cells dependson proliferation assays and/or the production of cytokinesfollowing polyclonal stimulation of the activated T cells.

In the present study, we used an

in vitro

MLR system toanalyse the cellular interactions required for the generation ofhuman immune responses to alloantigens. We used theenzyme-linked immunospot assay (ELISPOT) to detect thegeneration of antigen-specific effector T cells, primarilybecause of its sensitivity, that is, it is able to detect single,antigen-specific, cytokine-secreting cells.

11–13

This sensitivityallows one to quantify immune responses generated fromrelatively few lymphocytes. Using numerous PBL donors, wewere able to demonstrate repeatable, alloantigen-dependentactivation of CD4 T cells

in vitro

. Using this system weexamined the cellular interaction requirements for the activa-tion of human CD4 T cells.

Materials and methods

Donors

All volunteers who participated in this study were healthy, unrelatedadults. The experimental protocols carried out were conducted withethical approval from the University of Saskatchewan AdvisoryCommittee on Ethics in Human Medical Experimentation.

Correspondence: Peter Bretscher, Department of Microbiologyand Immunology, University of Saskatchewan, Saskatoon, SK,Canada. Email: bretschr@duke.usask.ca

Received 23 August 2004; accepted 11 October 2004.

176

T Strutt and P Bretscher

Mononuclear cell separation

Adult peripheral blood from consenting volunteers was collected intoheparanized vacutainer tubes and was diluted 1:1 with completeRPMI culture medium (RPMI-1640 supplemented with L-glutamine[Gibco Laboratories, Grand Island, NY, USA], 10% pooled humansera [PHS], 100 U/mL penicillin, 100

µ

g/mL streptomycin and5

×

10

–5

mol/L

β

-mercaptoethanol). The PBMC were isolated onFicoll-Hypaque as previously described.

14

The number of viable cellsobtained was determined using trypan blue exclusion.

Prior to use the PHS sera, used as a media supplement, was heatinactivated at 56

°

C for 30 min. The quality of the PHS was thenassessed. A standard proliferation assay

14

in which the proliferationof unstimulated PBMC and PBMC stimulated with irradiated allo-geneic PBMC for 5 days was used to determine the suitability ofdifferent lots of PHS. The lots chosen supported at least as strong aproliferative response as that obtained with 10%, endotoxin free,heat-inactivated FCS (Gibco).

Primary culture

Responding PBMC were cultured at 37

°

C at a partial pressure of 5%CO

2

in 24-well costar trays in either the absence or presence ofallogeneic PBMC that had been exposed to 2000 Rads of

γ

-irradiationfrom a

60

Co source. To determine whether a specific response wasgenerated

in vitro

, 10

6

PBMC from a sole donor were independentlycultured with a similar number of allogeneic PBMC from three otherindividuals. After 5–6 days culture, lymphocytes were harvested andcellular debris removed by ficoll-density gradient centrifugation. Therecovery of viable cells from the hypaque procedure was typically70–90%. The isolated cells were assessed for cytokine production inthe ELISPOT in the presence of the PBMC from the originalstimulator, and separately in the presence of PBMC from two third-party donors.

In experiments to determine the optimal responder density andallogeneic PBMC dose, both the number of responding PBMC perwell and the dose of irradiated allogeneic stimulators were varied (seeResults). From these experiments it became evident that the optimalculture conditions varied with different responders and stimulators.The optimal responder density was used in subsequent experimentswith a dose of 10

6

stimulators, which was found to be capable ofstimulating significant responses from all pairs of responders andstimulators. The densities used for each donor pair for which dataare presented can be found in the legends of the relevant figures.

Enumeration of alloantigen-dependent IFN-

γ

-producing cells

A variation of the ELISPOT was used to enumerate the number ofalloantigen-dependent, IFN-

γ

-producing cells generated

in vitro

. Inbrief, 96-well nitrocellulose-bottomed Millipore plates (Millipore,Molsheim, France) were coated overnight with 5

µ

g/mL anti-IFN-

γ

antibody (Mabtech, Nacka, Sweden; Mab1-DIK) in 50 mmol/Lcarbonate buffer, pH 9.6. Prior to the addition of effector cells, theplates were washed with sterile PBS containing 0.05% Tween-20(PBST) and blocked with complete RPMI for a minimum of 2 h at37

°

C at a partial pressure of 5% CO

2

. Effector cells were seeded, intriplicate, at densities ranging from 0.1

×

10

4

to 1.0

×

10

4

PBMC/wellwith or without 1.5

×

10

5

PBMC/well from the responder, from thesame individual that provided the stimulators or from third-partyindividuals. All PBMC used as antigen in the ELISPOT werecollected at the same time as the responding populations and werestored at –70

°

C for the duration of the experiment, and then thawedas described previously.

15

The seeded plates were incubated for48 h at 37

°

C at a partial pressure of 5% CO

2

, rinsed twice with

ddH20 and thoroughly washed with PBST. A concentration of1.0

µ

g/mL of biotinylated anti-IFN-

γ

antibody (Mabtech, Mab7-B6-1-biotin) diluted in PBS was then added. Following 3 h of incubation,at 37

°

C with 5% CO

2

, the plates were thoroughly washed with PBSTand 0.2

µ

g/mL of streptavidine-alkaline phosphatase (Mabtech) wasadded and incubated for a period of 1.5 h. The plates were thenwashed with PBST and rinsed thoroughly with PBS to remove anyresidual Tween-20. The IFN-

γ

spots were developed using theaddition of a 5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium chloride (BCIP/NBT) substrate solution (Sigmafast;Sigma, St Louis, MA, USA) prepared using the manufacturer’sinstructions. After 20 min the plates were washed with dH

2

0 to stopthe development reaction. When the plates were dry the spots werecounted under a dissecting microscope with the assistance of aneyepiece grid.

Cell subset depletion

T-cell subsets were depleted at the initiation or effector phase ofculture using the appropriate antibody-coated magnetic beads. CD4,CD8, both CD4 and CD8, or all T cells (CD2

+

) were depleted,according to the manufacturer’s instructions, from responding cellpopulations by negative selection with M-450 Dynabeads (Dynal,Oslo, Norway). The APC were similarly depleted from effector orthawed allogeneic PBMC populations via the use of a combination ofanti-CD14 and anti-CD19 antibody-coated M-450 Dynabeads(Dynal). CD14 is a membrane antigen that is predominately expressedon human monocytes and macrophages and CD19 is a B-cellrestricted membrane antigen.

16

For all depletions, the number ofcells/mL was determined prior to manipulation and was not adjustedto compensate for cell loss. Depletions were confirmed using flowcytometry. A volume ratio was used in experiments in whichfractionated populations were mixed. For example, 0.5 mL of afractioned population would be combined with 0.5 mL of another toreconstitute the original 1 mL of the untreated control population.

Statistics

Statistical analysis of the relevant data was carried out using a two-sample

t

-test for related values.

17

A 95% confidence interval waschosen.

Results

Alloantigen can induce the generation of IFN-

γ

-producing T cells

in vitro

We set out to establish an

in vitro

system in which antigenstimulates the generation of antigen-specific human cytokine-producing CD4 T cells. Preliminary experiments had revealedthat 10

6

PBL/well in a 24-well tray, stimulated by 10

6

irradiated, allogeneic stimulators, produce, on day 5 of cul-ture, many more spot-forming cells in the presence of thestimulating antigen in the ELISPOT than unstimulatedcontrol cultures. Our first concern was to assess whether thisincreased number reflected the generation of a specificimmune response. Therefore, we stimulated one responderpopulation with either no antigen or with one of threedifferent stimulators. The number of specific cytokine-producing cells generated on incubating the cells harvested onday 5 with either no antigen or with each of the threeallogeneic targets in the ELISPOT was assessed. The numberof IFN-

γ

spots per 10

6

input cells generated in the presence

Th cooperation for human Th effectors

177

of the stimulating alloantigen was found to be consistentlygreater than, or at least equal to, the number generated in thepresence of third-party alloantigens (Table 1). The observationsreported in this table are from a single experiment, which isrepresentative of seven similar experiments carried out withdifferent volunteers. The number of spots detected afterstimulation with third-party alloantigens was also found to beconsistent in that the pattern of cross-reactive IFN-

γ

spotsdetected was repeatable. We have found that the detection ofantigen-specific cells is optimal on day 6 of culture (Fig. 1A).After serial dilution, the number of IFN-

γ

-producing cellsdetected in the ELISPOT was found to decrease in a linearfashion (Fig. 1B) independently of the day the cells wereharvested. Thus, an alloantigen-specific, IFN-

γ

response isgenerated from PBMC

in vitro

under the culture conditionsdescribed.

Generation of an IFN-

γ

response requires a substantial responder cell density and substantial dose of allogeneic stimulators

We wished to assess whether interactions between respondercells are required to generate allospecific responses. One wayto indirectly assess this is to determine whether the respondercell density influences the size of the response generatedwhen the responses are expressed per 10

6

input respondercells. A number of different responder and stimulator pairswere tested. The results from one representative pair areshown in Figure 2. The dose of stimulators was held constantat a number known to be able to stimulate the generation ofsignificant IFN-

γ

-producing cells, and the responding PBMCdensity was varied by twofold increments. The number ofIFN-

γ

-producing cells detected per 10

6

input responding cells

Table 1

In vitro

generated human IFN-

γ

-producing cells are allospecific

Alloantigen in culture

†

Restimulating alloantigen in the ELISPOT

None 1 2 3

None 2

±

1 16

±

3 12

±

3 8

±

11 3

±

1

73

±±±±

6

40

±

3 24

±

32 14

±

2 83

±

3

170

±±±±

19

84

±

93 18

±

2 79

±

9 134

±

6

219

±±±±

19

†

The PBMC from a sole donor were cultured at a density of 0.5

×

10

6

cells/well in 24-well trays with or without 10

6

irradiated, allogeneic PBMCfrom donors 1, 2 or 3. Cells were harvested and a known number plated alone or with PBMC from donors 1, 2 or 3. The number of IFN-

γ

-producingcells recorded is presented as the number of spots generated per 10

6

unprimed input cells. The errors represent the SD of three triplicate wells in theELISPOT. These results are representative of experiments carried out with seven donor groups.

Figure 1

Linearity in the IFN-

γ

response at different times of

in vitro

culture. The PBMC were cultured in 24-well trays with(

�

) or without (

�

) an equal number of irradiated allogeneicPBMC. (A)

The number of spot-forming cells detected on days 5,6 and 7 in the absence (

�

,

�

) and presence (

�

,

�

) of antigenrestimulation in the ELISPOT. (B)

Day 5 (

�

), 6 (

�

) and 7 (

�

)effector populations were also serially diluted threefold andthe number of IFN-

γ

-producing cells detected in the absence(

�

,

�

,

�

) and presence (

�

,

�

,

�

) of allogeneic stimulationdetermined. Error bars represent the SD of triplicate wells in theELISPOT.

Figure 2

Induction of an

in vitro IFN-γ response is dependent onresponder culture density and stimulator dose. The PBMC wereserially diluted twofold and cultured with or without 106 irradiatedallogeneic PBMC (A) or were cultured at a density found to beoptimal for the particular donor, 0.125 × 106 cells/well in thiscase, with (�) or without (�) a serially diluted number of irradi-ated allogeneic PBMC (B). The number of antigen-dependent,IFN-γ-producing cells present on day 6 of culture was enumeratedusing ELISPOT and is presented as the number of spots detectedper 106 unprimed input cells. Similar results to those shown inpanels A and B were obtained in independent experiments carriedout with four other responder and stimulator pairs. The error barsshown represent the SD of the number of antigen-dependent spots.

178 T Strutt and P Bretscher

was found to dramatically increase when the number ofresponders per well was increased from 0.6 to 1.2 × 105

(Fig. 2A). It was also evident that the size of the responsegenerated is dependent on the presence of an optimal numberof stimulator cells (Fig. 2B). Observations from a number ofdifferent responders and stimulators reveal that the criticalresponder cell density required to support the generation ofsignificant responses after stimulation with an optimalnumber of stimulator cells is unique to each responder andstimulator pair (data not shown).

At very low responder densities, the number of alloantigen-specific, IFN-γ-producing cells detected was consistentlynegligible for all responders. The inability to detect signifi-cant alloantigen-specific, IFN-γ responses at these densitiescould conceivably result from the generation of suppressorcells that act at the effector stage. To examine this possibility,effector populations were harvested from cultures at densitiessuboptimal and optimal for the generation of IFN-γ-producingcells and assessed for alloantigen-dependent spot formationwhen plated separately or in combination. The number ofantigen-dependent spots produced by primed cells harvestedfrom cultures optimal for the generation of alloantigen-specific, IFN-γ-producing cells was unaffected by the pres-ence of effector cells harvested from low responder densitycultures (data not shown). The inability to detect significantnumbers of IFN-γ spot-forming cells from low responderdensity cultures is, therefore, unlikely to result from thesuppression of cytokine secretion. Effector populations werealso harvested at later time points to determine if a lack ofsignificant numbers of IFN-γ-producing cells at too low aresponder density was the result of a kinetic delay. Anincrease in the number of IFN-γ-producing cells was notobserved after 8 days of culture. Thus, something other than asuppressive element is most probably preventing the detec-tion of significant numbers of alloantigen-specific, IFN-γresponses at too low a responder density. The observation thata critical responder density is required to support the genera-tion of significant responses might indicate that responder cellcooperation is required to generate responses.

Antigen-dependent, IFN-γ-producing cells are CD4 T cells

We wished to ascertain the phenotype of the antigen-specific,IFN-γ-producing cells generated in culture. Using the optimalculture conditions for each responder, effector cells weregenerated and depleted of CD4, CD8 or both CD4 and CD8 Tcells using negative selection with magnetic beads. In contrastto the complete loss of IFN-γ-producing cells observed whenCD4 or both CD4 and CD8 T cells are depleted, the depletionof CD8 T cells alone had no effect on the number of antigen-dependent, IFN-γ-producing cells detected (Fig. 3A). Theobservation that the primary IFN-γ-producing cell generatedis a CD4 T cell conforms with the findings of researchers thatthe majority of cells proliferating in human MLR culture areCD4 T cells.18

Recognition of alloantigen occurs primarily through a direct rather than an indirect pathway

We wished to determine if the stimulator-specific, IFN-γ-producing CD4 T cells primarily recognize allogeneic MHC

class II molecules directly, or if they recognize processedalloantigen in the context of self MHC class II.19 To achievethis we depleted effector populations and the ELISPOT allo-antigen of B cells and monocyte-derived APC.16 The numberof IFN-γ spots detected in the ELISPOT was significantlydecreased after depletion of these APC from the allogeneic,stimulating PBMC population (P < 0.001). In contrast, thenumber of IFN-γ spots decreased only slightly when effectorphase responders were similarly depleted of CD19+ B cellsand CD14+ monocytes (Fig. 3B). We conclude that the major-ity of IFN-γ-producing CD4 T cells recognize alloantigen bya direct pathway.

Figure 3 The IFN-γ-producing cell is a CD4 T cell that recog-nizes alloantigen directly. The PBMC were cultured at a densityof 0.25 × 106 cells/well with or without a dose of 106 cells/well ofirradiated allogeneic PBMC. Responder cells were harvested atday 6 and were untreated or depleted of CD4, CD8 or both CD4and CD8 T cells to determine the phenotype of the cytokine-producing cell (A). In separate experiments, responding effectorcells (R) and the restimulating ELISPOT alloantigen (Ag) wereuntreated or depleted of both CD14+ monocytes and CD19+ Bcells to determine the nature of alloantigen recognition in theELISPOT (B). The results of replicate cultures are also shown inpanel B. The error bars shown represent the SD of the number ofantigen-dependent spots. Observations from one of three similarexperiments are shown.

Th cooperation for human Th effectors 179

Generation of IFN-γ-producing CD4 T cells is independent of CD8 T cells

To assess whether IFN-γ-producing CD4 T cells can begenerated independently of the presence of CD8 T cells,PBMC populations were either untreated or depleted of CD4or CD8 T cells before culture. The results presented inFigure 4 show that when CD8 T cells are removed the numberof antigen-specific, IFN-γ-producing cells generated is com-parable to that of the control population. In contrast, minimalnumbers are generated when CD4 T cells are removed. Thegeneration of antigen-dependent, IFN-γ-producing CD4 Tcells can be restored by mixing the fractionated CD4 and CD8T-cell depleted populations at a volume ratio of 1:1 prior toculture, showing that the process of depletion does notdamage the cells in a manner that precludes their activation.The slightly greater response of the combined populationcompared to the unfractionated population may result fromthe presence of double the number of non-T cells. Alloantigen-specific, IFN-γ CD4 T cells are, therefore, generated inde-pendently of the presence of CD8 T cells.

CD4 T cell–CD4 T cell interactions are required to generate antigen-dependent, IFN-γ-producing CD4 T cells

Two PBMC cell populations were prepared from respondercells, one was depleted of T cells to generate a populationconsisting primarily of APC and the other was unaltered, tocritically examine the cell interactions required to generatein vitro alloresponses,. These two populations were mixedtogether in various proportions so that the total cells seeded

in culture were derived from the same optimal number ofresponder cells. The resulting cultures contained a constantnumber of APC and a systematically varied number ofresponding T cells. The number of IFN-γ-producing CD4 Tcells present, on day 6, per 106 T cells plated in culture isshown in Figure 5A. It is evident that substantial numbers ofantigen-dependent IFN-γ-producing cells are generated oncea sufficient density of T cells is present. This dramaticdependence of the response on T-cell density is similar to thedependence observed previously when different densities ofunfractionated responder cells are stimulated with alloantigen(Fig. 2A). One can infer from this observation that a coopera-tive process between T cells is involved in the generation ofsignificant numbers of alloantigen-specific, IFN-γ-producingCD4 T cells.

We repeated the mixture experiment with fractionatedresponder T-cell populations to determine whether the T-cellinteractions critical for the generation of alloresponses arethose between CD4 T cells. A responder population depletedof either CD4 or CD8 T cells was mixed with an APCpopulation to generate a series of mixed populations thatsystematically varied the number of CD4 or CD8 T cells fromresponder PBMC. In the absence of CD4 T cells, CD8 T cellsfail to initiate the generation of antigen-dependent humanIFN-γ responses in vitro (Fig. 5C). The dramatic increase inthe generation of IFN-γ-producing T cells/106 CD4 T cells inculture with the increasing numbers of CD4 T cells present/well (Fig. 5B) allows one to infer that the generation of CD4T cells producing IFN-γ in response to allogeneic stimulationrequires CD4 T cell–CD4 T cell cooperation.

Discussion

Observations in animal systems, murine in vitro and in vivosystems in particular, have led to different models for theactivation requirements of CD4 T cells. Some envisage thatan interaction of a resting CD4 T cell with a threshold numberof appropriately mature or appropriately activated APC,bearing the pertinent processed antigen, is sufficient.20–22

Other studies suggest that this requirement by itself isinsufficient23,24 and that other specific cells, in particular CD4T cells, may be involved. For example, conditions can befound in which the antigen-dependent in vitro generation ofT cells mediating delayed type hypersensitivity (DTH) occursonly when antigen-specific ‘helper’ T cells are provided.23

The CD4/CD8 phenotype of the interacting T cells in theseexperiments was not determined. However, more recent invivo experiments employing DNA immunization24 show thatthe activation of CD4 T cells specific for a tumour antigencan be ‘helped’ by CD4 T cells specific for another antigen,and that this help is optimal when the DNA encoding the twoantigens are on the same construct. Recent observations fromour own laboratory have shown that the generation ofpeptide-specific CD4 T cells, obtained after immunizingnaïve mice with heat-aggregated, intact protein antigen inalum, also requires CD4 T-cell cooperation (NC Peters andPA Bretscher, unpubl. data). These observations suggest thatcooperation between T cells is optimal when antigens arelinked.23,24

The question of whether CD4 T-cell cooperation is requiredto activate unprimed CD4 T helper cells is important and may

Figure 4 Induction of IFN-γ production by CD4 T cells isindependent of CD8 T cells. Identical populations of PBMC at aconcentration of 0.125 × 106 cells/mL were untreated or depletedof CD4 or of CD8 T cells before culture. Cultures were set upwith the untreated, with the CD4 T cell depleted, with the CD8T cell depleted populations, or with a combination of the twodepleted populations (Mix). On day 6 an ELISPOT was used toenumerate the generation of IFN-γ-producing cells. The numberof spots presented is the number detected per 106 unprimed inputcells and the corresponding error bars represent the SD of thenumber of antigen-dependent spots. Observations from one ofthree similar experiments are shown.

180 T Strutt and P Bretscher

be critical to understanding whether antigen activates orinactivates CD4 T cells. Furthermore, it may also be impor-tant in determining the Th1/Th2 phenotype of CD4 T cells,25

which is an essential consideration for the development ofefficient vaccination strategies for tumour or pathogen-specific antigens. Therefore, we decided to attempt to developan experimental system in which we could address whetheror not the antigen-dependent generation of human antigen-specific CD4 T cells is CD4 T-cell cooperation dependent.

Although it has been reported that anti-CD3 stimulatedT-cell proliferation is dependent on T–T cell interactions,26

we described in the introduction why we consider it valuableto study the activation of human CD4 T cells in a manner thatis dependent on antigen rather than exposure to polyclonalstimulators. We believe that we have rigorously demonstratedthat alloantigen-specific CD4 T cells can be reliably generatedin vitro because the number of spots developed on exposingthe cultured cells to the stimulating alloantigen in theELISPOT for detecting antigen-specific IFN-γ-producingcells is always greater than the number developed on expos-ing the cultured cells to third-party alloantigens. We exam-ined the size of the response as a function of the cell densityof the responder cells. The absolute response per well is ofcourse expected to decrease as responder density decreases.However, we calculated the response per 106 responder cellsplated in culture. This parameter is not expected to change asresponder cell density is varied if a single responder CD4 Tcell is activated by alloantigen-presenting APC without anyinfluence from other responder cells. We find, in fact, that this‘normalized response’ vanishes, within experimental error, incultures with lower densities of responder cells. We inferunder these circumstances that the generation of all thealloantigen-specific CD4 T cells generated at higher celldensities is dependent on cell cooperation between respondercells. Our additional in vitro observations reveal that thiscooperation is mediated between CD4 T cells. These findings

provide a reasonable explanation for the in vivo dependencyof graft outcome on the size of the graft and precursorfrequency of graft specific CD4 T helper cells.27–29

We have not attempted in this study to analyse themechanism of this cooperation. There is precedence for twonon-exclusive mechanisms. One is the upregulation ofcostimulatory molecules on APC on recognition of antigen byactivated CD4 T cells.20 This upregulation can allow the moreefficient delivery of the second signal required for CD4 T-cellactivation to the responding T cell. The other is the natureand/or amount of CD4 T cell growth-supporting cytokinesproduced in an autocrine fashion that act on the respondingCD4 T cells. We consider both possibilities plausible. Ananalysis of the contribution of these two different mecha-nisms in a human in vitro system, and confirmation of suchfindings in vivo, employing protocols that do not rely on theuse of polyclonal stimulators presents a considerable chal-lenge; however, the insight gained may aid the design ofefficacious therapeutic and vaccination strategies.

Acknowledgements

We thank Canadian Blood Services for generously providingPHS. This work was supported equally by grants MOP-14121and MOP-37868 from the Canadian Institutes of Health Researchto P Bretscher. Tara Strutt was supported by a PostgraduateAward from the Natural Sciences and Engineering ResearchCouncil of Canada. We are indebted to Calliopi Havele forhelpful advice. We thank both Karl Kai McKinstry andCalliopi Havele for their thorough reviews of the manuscript.

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Figure 5 The induction of IFN-γ-producing cells occurs through CD4 T-cell cooperation. Responding PBMC were prepared andseparated into four identical populations that were subject to fractionation. One population was depleted of all T cells to generate apopulation consisting primarily of APC and the other populations were untreated, or depleted of CD8 or CD4 T cells. The APC populationwas mixed together with one of the other populations before culture at the percentages indicated in the figure. The resulting cultures containa constant number of APC and a varying number of responding T cells (A), CD4 T cells (B) or CD8 T cells (C). Responding lymphocyteswere either unstimulated or stimulated with 106 cells/well of irradiated allogeneic PBMC. The number of IFN-γ-producing cells generatedby day 6 of culture was determined in the absence and presence of antigen restimulation in the ELISPOT. The number of antigen-dependentspots per 106 unprimed PBL after correcting for loss of CD4 T cells is presented. The error bars shown represent the SD of the number ofantigen-dependent spots. Observations from one of two similar experiments are shown and results identical to those shown were obtainedin independent experiments in which alternate blood donors were used.

Th cooperation for human Th effectors 181

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