Requirements for Lysis of Activated T Cells by Class-II-Restricted Cytolytic T-Lymphocytes Assia Eljaafari, Isabelle Dorval, Dominique Zeliszewski, Sylvie Le Gac, and Ghislaine Sterkers

ABSTRACT: In the present study, we explored the specific requirements for lysis of human activated T cells by CD4 + CTLs. This was achieved by using human CD4 + T cell lines or clones specific for a peptidic fragment of influenza virus as both CTL effectors and target T cells (TTCs). Our results further establish that human activated T cells expressing HLA-DR molecules can present Ag to and be lysed by CD4 + HLA-DR restricted CTLs. This killing is Ag specific and HLA-DR restricted. It can be observed whether TTCs are heterologous or autologous, CD4 + or CD8 +. However, we find that in our model: (a) TTCs are able to present artificially processed peptidic fragments of Ag, but not the corresponding natural Ag in

the context of class II determinants, even if they can process whole virus in the context of class I determinants; (b) TTCs must express high density of HLA-DR molecules on their membrane; (c) preincubation of TTCs with high concentrations ofpeptide is required; and (d) interestingly enough, addition of free peptide at similar concentration during the cytolytic assay to replace TTC preincubation inhibits TTC lysis by at least two different mechanisms, i.e., cold-target inhibition in which CTLs serve as their own cold targets and inhibition at the effector cell level. From these results, one can conclude that stringent conditions are required for lysis of activated T cells by class-IIrestricted CTLs. Human Immunology 35, 50-59 (1992)

ABBREVIATIONS A-CTL aggressor CTL APC antigen-presenting cell CTL cytolytic T-lymphocyte EBV-BLCL Epstein-Barr-virus-transformed B-lymphoblastoid cell line Flu influenza virus HA hemagglutinin

HLA IL2 mAb MHC PHA TCR TTC

human leukocyte antigen interleukin 2 monoclonal antibody major histocompatibility complex phytohemagglutinin T-cell receptor target T cell

INTRODUCTION CD4 + T cells represent an heterogeneous population with respect to their functions. They induce antibody responses [ 1], mediate delayed-type hypersensitivity reactions [2], and in some cases suppress antibody secretion [3]. M o r e recently, their capacity to lyse targets expressing major histocompatibility complex (MHC) class II molecules has been described [ 4 - 7 ] . T h e physiologic relevance o f these class-II-restricted cytolytic Tlymphocytes (CTLs) has been a matter of controversy. From the Laboratory of Development and Maturation of the Immune System, CJF 90-15, H~pital Robert Debre, Paris, France. Address reprint requests to A. Eljaafari, Laboratory of Development and Maturation of the Immune System, CJF 90-15, H~pital Robert Debre, 48 Boulevard Serrurier, 75019 Paris, France. Received March 26, 1992; acceptedJune 17, 1992. 50 0198-8859/92/$5.00

Initially, it has been proposed that CD4 + CTL activity is an in vitro artifact, because this function is usually acquired in long-term culture [4]. However, this activity has also been found in freshly isolated T cells [8]. CD4 + CTLs recognize Ag almost exclusively in association with class II molecules [9]. It is therefore essential that antigen-presenting cells (APCs) express class II molecules on their surface m e m b r a n e to activate C D 4 + CTLs. Conventional APCs such as B cells or macrophages constitutively express modulable level o f Ia molecules in mice and humans [ 10-13]. H u m a n T cells also express M H C class II molecules after in vitro or in vivo activation [ 1 4 - 1 6 ] . As a consequence, they can potentially function as APCs for C D 4 + T cells [ 1 7 - 1 8 ] . Another requirement for antigen presentation is Ag processing, Human Immunology 35, 50-59 (1992) © American Society for Histocompatibility and Immunogenetics, 1992

Lysis of Activated T Cells by CD4 + T Cells

including unfolding and fragmentation leading to peptidic fragments that bind to MHC molecules. B cells or macrophages are efficient to process Ag and present the corresponding peptides to T cells [19-24]. Discordant results have been reported concerning these properties among T cells [17, 25-27]. A compilation of data suggests that complex Ag cannot be efficiently processed and subsequently presented by activated T cells to CD4 + helper or cytolytic T cells unless native Ag has affinity for some T-cell membrane structures [28, 29]. Such examples have been provided by the envelope glycoprotein SUgp120 of HIV, which is able to be processed through binding to CD4 molecules [28, 29]. The ability of CD4 + MHC class-II-positive T cells to present SUgp120 to C D 4 + CTLs has been proposed to contribute in the HIV-induced immune deficiency. However, immune responses to Ag exceptionally lead to systemic immune deficiency. Therefore, one should expect mechanisms protecting activated T cells from lysis. In the present report, we determined some of the requirements that would prevent lysis of activated T cells by class-II-restricted CTLs. This was achieved by using, as effector cells, human long-term oligoclonal T-cell lines or clones specific for a peptidic fragment of the hemagglutinin (HA) of the influenza virus. MATERIALS AND METHODS

T-cell lines and clones. They were produced as already described [30, 31]. They were maintained in long-term culture by the addition of irradiated autologous peripheral blood mononuclear cells preincubated for 1½ hours with Ag every week. Recombinant interleukin 2 (IL-2) was added every 2 days. They are all specific for the influenza A/Texas virus and restricted by HLA-DR molecules (see Table 1) ([30, 32] and unpublished data). In all experiments described herein, except otherwise indicated, T cell lines were used 7 days after their last restimulation. B-cell lines. They were either issued from the Tenth Histocompatibility Workshop (New York, November 1987) or locally produced. Antigens. Influenza virus was kindly provided by Claude Hannoun (Institute Pasteur, Paris). Influenza A/Texas (AT) virus was grown in allantoic enbryonated chicken eggs. Synthetic peptides of AT virus hemagglutinin (H3), p57 and p66, were purchased from Neosystem (Strasbourg, France). HLA-specific monoclonal antibodies. Monoclonal antibodies (mAbs) L243 and B7.21.1 were locally produced from hybridomas that were purchased from the Ameri-

51

can Type Tissue Culture Collection (Rockville, MD). The specificity of these mAbs according to previous studies is indicated in Table 2. All mAbs were used as ascitic fluids at the 1% final dilution.

Culture medium. Effector cells were grown in RPMI 1640 supplemented with 1% glutamine, 1% pyruvate, 1% nonessential aminoacids, antibiotics, and 5% heatinactivated human AB serum. T-cellcytotoxicity assays. Cytotoxicity was assessed by the standard 51Cr-release assay described elsewhere [33]. Briefly, effector cells (5 × 104 cells per well), were seeded into round-bottomed microtiter plates in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). Target cells were preincubated with antigen for 1½ hours during labeling with 5~Cr. After washing, they were added to effector cells (5 × 10 ~ cells per well) in a total volume of 200/A. Then cells were incubated for 4 hours at 37°C in 5% CO2 atmosphere. Plates were centrifugated at 100 g, 100/~l of supernatant was harvested from each well, and the radioactivity was measured using a gamma counter. Results of cytotoxicity assay are expressed as percentage of specific cytotoxicity as calculated by the following formula: % cytotoxicity -- 100 × experimental 51Cr release - spontaneous release total 51Cr incorporation - spontaneous release

Flow-cytofluorometric analysis. A total of 5 x 105 cells were stained with CD3-FITC or DR-FITC mAbs for 30 minutes at 4°C. Control aliquots were stained with anti-mouse Ig-FITC (GAM-Ig). The labeled cells were then washed three times and resuspended in I ml phosphate-buffered saline (PBS) containing 1% paraformaldehyde; 5000 events from each sample were then analyzed on a flow cytometer (FacStar, Becton Dickinson, San Jose, CA) gated to exclude nonviable cells. Positive fluorescence was defined by comparison with the fluorescence from control samples. Mean fluorescence was calculated on the positive population.

RESULTS

Characteristics of CD4 + CTLs and target T cells used in the present study. Aggressor CTLs (A-CTLs) used in this study were CD4 + human T-cell lines or clones specific for the influenza A/Texas virus. They were issued from three different healthy donors. The fine restriction and precise specificity of these T cells were first extensively documented according to methods currently used in our laboratory and previously described [30-33]. As sum-

52

A. Eljaafari et al.

TABLE 1 T-cell characteristics: restriction and specificity Fine antigen specificity characterization Cells Flu

Viral HA

DRBI*1301 + DRBI*1302

+

DR3, DQw2, Dw3/

DRBI*130I +

+

DRwl3, DQwl, Dwl9

DRBI*1302 + DRB 1"0801

Name

Phenotype

M3ap20

DRwl3, DQwl, Dwl9/ DRwl3, DQwl, Dwl9

372ap20

HC12aA

HA peptide Genes involved in restriction

DR4, DQw3, Dw14/

+

Name

Sequence

+

p20 + p57

CPKYVKQNTLKLATGMRNVPEKQTR + CPKYVKQNTLKLATG

+

p20 +

CPKYVKQNTLKLATGMRNVPEKQTR +

p57

CPKYVKQNTLKLATG

-

HLA A2

Unidentified

DR8, DQw4, Dw8 CotC14

DRw13, DQw6, Dw19/ DRw13, DQw6, Dw19

154ap20

DR3, DQw2, Dw3/ DRB 1*0801 +

DRB1*1301 + DRBI*1302

+

DRBl*1101 +

+

Unidentified + p 57

+ DRB 1" 1601

p20 CPKYVKQNTLKLATGMRNVPEKQTR + + DRwl 1, DQw3, Dw5 CPKYVKQNTLKLATG + p66

CPKYVKSNTLKLATG

Characteristics were determined as currently performed in our laboratory [32, 33]. HLA phenotype was confirmed by oligotypingif not gene sequencing.

marized in Table 1, most of these cell lines were specific for peptidic fragments of the viral HA, the sequences of which are indicated. At least one of these peptides (p57) was recognized by most of cell lines in association with human leukocyte artigen (HLA) products encoded by the loci DRBl*1302 or 1101 (according to the new nomenclature). We also used two additional cell lines that did not react with HA; one of them was CD8 + and restricted by HLA class I antigens. The same CD4 + or CD8 + clonal or oligoclonal T cells were used as target T cells (TTCs). These lines or clones were maintained in culture under the same conditions, irrespective to their use as A-CTLs or TTCs (see Materials and Methods). Unless otherwise indicated, they were tested 7 days after their last stimulation by irradiated feeder cells plus Ag or phytohemagglutinin (PHA). At that time, they expressed a high density of HLA-DR molecules on their surface (as shown later). The polymorphism of their DRB1 encoded chains has been precisely identified by oligonucleotide typing or gene sequencing (not shown). Human HLA-DR-positive CD4 + as well as CD8 + T cells can present HA peptide to and be lysed by CD4 +, HLADR-restricted CTLs. This lysis is Ag specific and M H C class II restricted. In the experiment shown in Table 2,

M3ap20 CTLs specific for p57 and restricted by DRB1*1302 gene product (DRw13), were used as effectors. Various TTCs expressing different HLA-DR products were pulsed for 1½hours with the p57 peptide, which is specifically recognized by M3ap20 CTLs. As control, the same TTCs were treated in parallel either in the absence of peptide or with an irrelevant peptide, p66, differing from p57 by a single amino acid residue (see Table 1). After labeling with 5ICr, TTCs were tested for their susceptibility to lysis by A-CTLs in a short-term chromium-release assay. It must be added that pulsed TTCs were extensively washed before the chromiumrelease assay to avoid peptide carry over on autologous A-CTLs, which also express HLA-DR molecules. As shown in Table 2 (top panel), M3ap20 CTLs were able to lyse CD4+ TTCs expressing the proper peptide--HLA-DR complex, i.e., p57-DRw13. Interestingly, these A-CTLs were able to lyse TTCs from autologous or heterologous lines with close efficiency, indicating that T cells can be susceptible to self-lysis. Even though p57 and p66 have been shown to bind to DRwl 1 and DRw13 molecules (manuscript in preparation), TTCs expressing inappropriate HLA-DR products, i.e., DR3/DRw11, or pulsed with the irrelevant peptide, p66, were not killed. The involvement of HLA-DR in the presentation of

Lysis of Activated T Cells by CD4 + T Cells

TABLE 2

53

Lysis of TTCs by class-II-restricted CTLs is Ag specific and M H C restricted Target T cells

E - T cell ratio

Effector

Name

Specificity

Pulsed with

M3ap20 M3ap20 M3ap20 M3ap20 M3ap20 M3ap20

M3ap20 372ap20 CotC14 154ap20 M3ap20 M3ap20

DRw13/DRw13 DRw13/DRw13 DRw13/DRw13 DR3/DR11 DRw13/DRw13 DRw13/DRw13

p57 p57 p57 p57 p66

M3aP20 M3ap20 M3ap20 M3ap20

M3ap20 M3ap20 M3ap20 M3ap20

DRw13/DRw13 DRw13/DRw13 DRw13/DRw13 DRw13/DRw13

p57 p57 + anti-DP p57 + a n t i - D R --

--

10:1

3:1

1:1

0.3 : 1

33 19 13 0 0 0

21 10 9 0 0 0

5 2 4 0 0 0

3 0 0 0 0 0

37 31 3 5.5

24 28 1.5 1.3

11 13 2.2 2

7 7 2 0

Various TTCs were incubated with or without the indicated peptides (50/zg/ml) for 1½hours before the 51Cr-release assay (top panel). When indicated anti-DR (L243) or anti-DP (B7.21.1) mAbs were added at the beginning of the assay (bottom panel) at the 1:100 dilution of ascitis. Effector cells were seeded with 51Crlabeled targets at the indicated E-T cell ratio. Results of 51Cr release are expressed as percent of specific lysis calculated as described in Materials and Methods.

the foreign peptide was further demonstrated in inhibition studies with HLA-specific mAbs. As shown in Table 2 (bottom panel), anti-DR, but not anti-DP, mAb inhibited the DR-restricted lysis of TTCs. This indicates that the H L A - D R molecules expressed on T cells are involved in the presentation of foreign peptide to the ACTL T-cell receptor, as previously published for B-cell targets [32]. In addition, we excluded bystander lysis in our model because peptide-pulsed cold targets did not

FIGURE 1 Susceptibility of TTCs to class-II-restricted lysis is phenotype independent. 154ap20 CD4+ CTLs ( I ) or HC12aA CD8 + CTLs ([2), expressing the proper HLA-DR product, were incubated with p57 for 1½hours before labeling w i t h 51Cr. Various numbers of effector cells were then mixed with targets at the indicated E-T cell ratio for 4 hours. Results of 51Cr release are expressed as percent of specific lysis, calculated as described in Materials and Methods. Dotted lines represent lysis of nonpulsed TTCs. 30

25

'~ 20"

~ 15:n

3

10-

1o';1

~;l

1;1 E/T ratio

o, ~/l

induce significant lysis of nonpulsed bystander targets (not shown). We then tested whether HLA class-II-compatible CD8 + CTLs could be lysed by CD4 + CTLs. This was achieved by using HC12aA CTLs as CD8 + TTCs and 154ap20 CTLs as CD4 + CTLs or TTCs. Figure 1 clearly shows that 154ap20 CTLs were able to lyse HC12aA CTLs to the same extent than themselves. This strongly supports that activated HLA-DR-positive T cells are susceptible to class-II-restricted lysis, regardless of their CD4 or CD8 phenotype.

Human activated T cells do not present complex Ag to classII-restricted T-cell lines. T o investigate the ability to activated T cells to process complex Ag, the whole virus rather than influenza virus (Flu) peptide was used as Ag. As shown in Table 3 (top panel), activated T cells were able to present p57, which does not require Ag processing, to class-II-restricted CTLs. In contrast, TTCs incubated with whole virus could not be lysed by classII-restricted CTLs. It must be added that even at concentrations 100 times higher than that required for B-cell killing, no presentation of virus occurred (not shown). As control of efficient infection, the same targets were able to present Flu to C D 8 + class-I-restricted CTLs (Table 3, bottom panel). As previously reported [34], conventional APCs, i.e., 20 Epstein-Barr virus (EBV) and HC12 EBV presented viral antigens to class-I- as well as to class-II-restricted CTLs. T T C susceptibility to class-II-restricted lysis is highly dependent on the density of H L A - D R molecules expressed at their surface. H L A - D R density required on TTCs for efficient presentation of Ag to A-CTLs was investigated as follows: M3ap20 TTCs were activated with P H A for either

54

A. Eljaafari et al.

TABLE 3

Activated T cells present complex Ag to class-I but not to class-II-restricted CTLs Target cells

Effector cells

Name

E-T cell ratio Pulsed with

10 : 1

3:1

1: 1

0.3 : 1

M3ap20 (CD4 + ) M3ap20 M3ap20 M3ap20 M3ap20 M3ap20

M3ap20 M3ap20 M3ap20 20 EBV 20 EBV 20 EBV

p57 Flu -p57 Flu --

33 0 0 33 25 2

21 0 0 12 13 1

5 0 0 2 1 1

3 0 0 2 0 2

HC12aA (CD8 + ) HC12aA HC 12aA HC12aA

M3ap20 M3ap20 HC 12EBV HC12EBV

Flu -Flu --

24 7 29 0

10 3 13 0

5 3 9 1

5 2 2 2

M3ap20 TTCs or 20 EBV and H C 1 2 EBV target B ceils were preincubated either with p57 (50/zg/ml) or active influenza A/Texas virus (10 IU/ml) for 1½ hours before the 5~Cr-release assay. CD4 + (M3ap20) or CD8 + (HC12aA) effector cells were mixed to target cells at the indicated E - T ratio. Results of 51Cr release are expressed as percent of specific lysis calculated as described in Materials and Methods.

3, 7, or 10 days. Each TTC was tested for D R expression by l a b e l i n g w i t h f l u o r e s c e i n a t e d a n t i - D R m A b s . T h e i r s u s c e p t i b i l i t y to M 3 a p 2 0 lysis was d e t e r m i n e d in parallel. A r e p r e s e n t a t i v e e x p e r i m e n t s h o w n in Fig. 2 d e m o n strates a d i r e c t c o r r e l a t i o n b e t w e e n H L A - D R d e n s i t y o n T T C m e m b r a n e and s u s c e p t i b i l i t y to c l a s s - I I - r e s t r i c t e d lysis. T h e r e f o r e , o p t i m a l T T C lysis c o u l d o c c u r o n l y

F I G U R E 2 Susceptibility of TTCs to class-II-restricted lysis is correlated with H L A - D R density. (A) M2ap20 TFCs activated in vitro with P H A for 3, 7, or 10 days were stained with DR-FITC mAbs (black histograms). Results are expressed as arbitrary normalized fluorescence histograms i.e., number of cells versus fluorescence intensity. Fluorescence of cells initially pulsed with control mAbs is indicated by white histograms. (B) Concomitantly, their susceptibility to M3ap20 lysis was tested by preincubating them with peptide ( ) or not ( - - - ) . Results of 51Cr release are expressed as percent of specific lysis calculated as described in Materials and Methods. A

B

w h e n high d e n s i t y o f H L A - D R pressed on TTC membrane.

m o l e c u l e s w e r e ex-

Prepulsing of TTCs with high concentrations of peptide p5 7 is required to observe efficient lysis. T h e effect o f p e p t i d e c o n c e n t r a t i o n o n t h e s u s c e p t i b i l i t y o f T T C s to class-IIr e s t r i c t e d lysis was also i n v e s t i g a t e d and c o m p a r e d to that o f E p s t e i n - B a r r - v i r u s - t r a n s f o r m e d B - l y m p h o i d cell lines ( E B V - B L C L s ) . F i g u r e 3 shows that the range o f p e p t i d e c o n c e n t r a t i o n r e q u i r e d to reach the p l a t e a u values o f T T C o r E B V - B L C L lysis was closely similar.

F I G U R E 3 TTC lysis by HLA class-II-restricted CTLs depends on Ag concentration. 372ap20 TTCs (I-1) or autologous EBV-BLCLs (ll) were incubated with various concentrations of the peptide p57 for 1½ hours before labeling with 51Cr. Effector cells were mixed with targets at a 10 : 1 ratio. Results of 51Cr release are expressed as percent of specific lysis calculated as described in Materials and Methods. 90"

% of 510r release 00"

J3

70"

20

,°l 1o 0

.Q

._~ 6o.

1011

.

.

.

3/1

111 ._o 5o"8 o

Z

J7

20

40-

10 ~,? 00-

.~

1011

n"

311

111 20.

30 20

,110

1

1011 Log Fluorescence

Intensity

311 E/T ratio

1/1

2

12 5

25

i 5

peptide

0 75

0 4

0 2

c'oneenLration

0 I

0 05 0 0 2 5 0 0 1 2 0 006

(~g/ml)

0

Lysis of Activated T Cells by CD4 + T Cells

55

60-

Activated T cells are no more susceptible to class-II-restricted lysis when peptide is added under a free form during the cytolytic assay. We next tried to replace the prepulsing

50"

of targets with peptide by adding free peptide to the assay at various concentrations. Results shown in Fig. 4 clearly indicate that in these conditions, no T-cell lysis was observed, irrespective o f the concentrations of peptide used. Interestingly, lysis o f EBV-BLCLs was maintained (Fig. 4), but at suboptimal levels, as compared with lysis of EBV-BLCLs that have been pretreated (not shown). In the presence o f free peptide, self-lysis of A-CTLs with each other could be responsible for the absence of T T C lysis. Indeed, because A-CTLs and TTCs are the same cells in our model, both are able to present peptide and to exert cytolytic activity when free peptide is added during the cytolytic assay. T o test this hypothesis, we labeled both A-CTLs and TTCs with 51Cr and tested whether addition of free peptide at various concentrations would result in specific self-lysis. As shown in Fig. 4, addition of free peptide did not result in specific 51Cr release by either labeled A-CTLs or labeled-TTCs in a 4-hour as well as in an 18-hour assay, whereas CTLs were still viable (not shown). This indicates that self-lysis cannot account for the absence o f T T C lysis observed in the presence o f free peptide.

40-

6 3o-

~

20"

10-

~0

~0

50

25

~0

~0

~0

~0

12.5

6.25

3.12

1.5

~ 0 /i ~ 0 0.75

peptide c o n c e n t r a t i o n

0.15

~.0 ~ 0 ~ " ~ . ~ 0 0.03

0.006

0

(pg/rn[)

FIGURE 4 Addition of peptide under a free form inhibits target cell lysis. 51Cr release was evaluated in two different sets of experiment: Cold 372ap20s were cultured in the presence of peptide (50 /zg/ml) and labeled 372ap20 ( I ) or labeled syngeneic EBV-BLCLs (rT) at a 10:1 E - T ratio in the first set, whereas, in the second set, labeled 372ap20s were cultured in the presence of peptide alone (50/zg/ml) for 4 hours (*) or 18 hours (o). Results of 51Cr release are expressed as percent of specific lysis calculated as described in Materials andMethods.

However, the levels o f T T C lysis were reproducibly much lower than those o f E B V - B L C L ones. As a consequence, significant T T C lysis was only observed when high concentrations of peptide were used, as compared with E B V - B L C L lysis.

TABLE 4

Inhibition of lysis by excess of peptide results from peptide presentation by A-CTLs to each other. We next tested the effect of effector cell prepulsing with peptide. A representative experiment shown in Table 4 (top panel) illus-

Inhibition of lysis by excess o f peptide results from peptide presentation by A-CTLs to each other Effectors

Targets

E - T cell ratio

Name

Pulsed with

Name

Pulsed with

10:1

3:1

M3ap20 M3ap20 M3ap20 M3ap20 M3ap20 M3ap20 M3ap20 M3ap20

---

M3ap20 M3ap20 M3ap20 M3ap20 20 EBV 20EBV 20EBV 20 EBV

p57 -p57 -p57 -p57 --

27 0 7 0 67 0 41 0

14 0 8 0 43 0 30 0

p57 p57 --p57 --

1:1 4 0 5 0 14 0 11 0

0.3 : 1 0 0 4 0 13 0 4 0

M3ap20 M3ap20 M3ap20

-p57 p66

M3ap20 M3ap20 M3ap20

p57 p57 p57

19 0 21

8 0 19

1.5 0 4

0 0 7

M3ap20 M3ap20 M3ap20

-p57 p57 + anti-DR

M3ap20 M3ap20 M3ap20

p57 p57 p57

33 5 26

21 0 20

5 0 9

3 5 5

Target cells were incubated either with p57 (50 tzg/ml) or not for 1½hours. Effector cells were pulsed either with p57 (50/zg/ml), p66 (50 v.g/ml), or not for 1½ hours. When indicated, a DR-specific mAB, L243, was added during the pulse time at the 1 : 100 final dilution of ascites. After washing, effector and target cells were mixed at the indicated E-T ratio. Results of 51Cr release are expressed as percent of specific lysis calculated as described in Materials and Methods.

A. Eljaafari et al.

56

Hot TTC

40

?

Hot EBV-BLCL

100~

A

~_ s01 ,o o01

/

30

.~ 20

-It

~

:

4Ol

"E

10

2ol

0|

0 ~Q"

0

I/I

3/I

10/I 30/I 100/I



25 C Hot TTC

0

[D

I/I

3/I

10/I 30/I 100/I

Hot EBV-BLCL

! I 0

1)1

3/1

10/1 30/1 100/1

Cold-target competition experiments. 51Cr-labeled target cells were pulsed with p57 for 1½ hours and mixed at the cold-hot cell ratios indicated with unlabeled cold targets, which were either pulsed with p57 ( ) or nonpulsed (---). M3ap20 CTLs were used as effector cells and added into wells at the 10:1 effector-hot-target cell ratio. (A) Hot target, M3ap20s; and cold target, M3ap20s. (B) Hot target, EBV-BLCLs; and cold target, M3ap20s. (C) Hot target, M3ap20s; and cold target, EBV-BLCI_s. (D) Hot target, EBV-BLCLs; and cold target, EBV-BLCLs. FIGURE 5

"o

0

1)1

3'/1

10/1 30/1 100/1

C/H ratio trates that lysis of autologous TTCs was abolished when A-CTLs were pulsed with the specific peptide p57 for 1½hours and that killing of autologous EBV-BLCLs was decreased in the same conditions. We then demonstrated that this inhibition was Ag specific and resulted from self-presentation of Ag by DR molecules because (a) it was not observed when A-CTLs were pretreated with p66, an irrelevant peptide (Table 4, middle panel); and (b) it was completely abrogated if A-CTLs preincubation with peptide was carried out in the presence of DR-specific mAbs (Table 4, bottom panel).

Mechanisms involved in lysis inhibition. All the experiments described previously strongly argue against several explanations like (a) self-lysis and/or suicide of ACTLs in the presence of p57; (b) lysis of peptide-pulsed A-CTLs by targets; and (c) direct binding of peptide to the T-cell receptors (TCRs), which could interfere with target cell killing or provide offsignals. Therefore, other mechanisms were investigated. At once, we tested whether peptide-pulsed A-CTLs could act as cold target inhibitors of recognition and lysis of labeled peptidepulsed targets. Indeed, the effector themselves need not be lysed in order to act as cold targets. Figure 5A shows that lysis of peptide-pulsed TTCs could be competed away in a dose-dependent fashion by the pretreated cold A-CTLs. Inhibition ofTTC lysis was also observed when peptide-pulsed cold EBV-BLCLs were added (Fig. 5C). When peptide-pulsed EBV-BLCLs were used as hot targets, however, peptide-pulsed cold A-CTLs were not efficient at all in competing away their lysis (Fig. 5B), probably because A-CTLs are just not as good targets as EBV-BLCLs. As control, EBV-BLCL lysis was competed away in a dose-dependent fashion by cold

EBV-BLCLs that had been pulsed with the same peptide (Fig. 5D). Of note, unpulsed cold A-CTLs increased specific lysis of hot TTC and EBV-BLCL targets because of their lytic capacity (Figs. 5A and B). These results strongly suggest that the abolition ofTTC lysis observed when A-CTLs were pulsed with peptide could at least partly result from cold-target inhibition. However, at least one additive mechanism might be involved in this lysis inhibition because the reproducible decrease in EBV-BLCL killing observed when A-CTLs were pulsed with peptide could not be explained by coldtarget inhibition. TCR modulation has been reported to inhibit helper function [35-37]; therefore, to carry on with the investigation of the inhibitory mechanism, we measured both the cytolytic activity and the CD3-TCR expression of A-CTLs pretreated with medium or with peptide for 1½ hours or 18 hours. As shown in Fig. 6, pulsing A-CTLs with peptide resulted in a time-dependent TCR modulation (Figs. 6A) that was correlated with a decrease of B-cell lysis (Fig. 6B). Note that 18-hour treatment with peptide did not affect T-cell viability because A-CTLs were still fully responsive to IL-2 treatment (Fig. 6C). It must be added that abolition of TTC lysis was observed at both times of pulsing, i.e., 1½hours and 18 hours (not shown), even though TCR modulation was moderate during 1½-hour pulse. DISCUSSION Among several functions, CD4 + T cells have been reported to kill class-II-positive cells expressing the proper MHC-peptide complex [38-40]. In human, activated T cells that express MHC class II molecules as a consequence of their activation have been shown under certain

Lysis of Activated T Cells by CD4 + T Cells

A ~,,,,,

57

1 B "~ 40

.

.

.

.

.

=

Z

~

o

1/1

I

.>o_ "~

T

~_~

...

i

~"

',

30 20

I

. ._-I.

iiiiii 1

701

o so =40

= ,

Log F l u o r e s c e n c e Intensity

5

25

2.5

IL-2 Concentration

6.25

3.125

(IU/ml)

FIGURE 6 Effect of excess of peptide on CTL responsiveness and CD3-TCR expression. (A) CD3-TCR expression of 372ap20 A-CTLs nonpulsed or pulsed with p57 for either 1½ hours or 18 hours was determined by their labeling with CD3-FITC mAbs. (B) A-CTLs nonpulsed ( i ) or pulsed with peptide for either 1½ hours ( i ) or 18 hours (+) were concomitantly tested for their ability to kill EBV-BLCLs preincubated with peptide or without (0). (C) A-CTLs were unpulsed (white histograms) or pulsed with peptide for 1½ hours (black histograms) or 18 hours (hatched histograms). After washing, they were tested for their sensitivity to recombinant interleukin 2 in a ~H incorporation assay.

circumstances to present Ag to and be lysed in vitro by CD4 + CTLs [28, 29, 41]. Ifoccuring in vivo, this killing is expected to generate systemic immune deficiency as recently proposed in the H I V model [29]. Experiments described herein were designed to explore the specific requirements of activated T-cell lysis by CD4 + CTLs. In the influenza model, we show that CD4 + cytolytic T cells, specific for a short synthetic peptide of H A and restricted by class II molecules, lyse human activated T cells bearing the proper HLA class I I - p e p t i d e complex, as reported by O t t e n h o f f and Mutis [41] in another infectious model. This lysis is the consequence of antigen presentation by T cells to each other, because it is antigen specific, HLA class II restricted, inhibited by anti-DR mAbs, and does not result in bystander lysis. Discrepant results concerning the capacity of CD4 + CTLs to lyse bystander targets have been reported so far [41, 42]; this might reflect differences in the models used or experimental conditions. The ability of activated T cells to present Ag in the context of class II determinants is still open to debate. Some studies indicate that they can only present peptidic fragments of Ag [17, 41]; others report that they are able to present native Ag [26, 27]. More recently, it has been proposed that the efficient capture of Ag through

T-cell surface receptors determines whether Ag is processed [28, 29]. The data shown in the present report clearly indicate that activated T cells are able to present p57, a Flu peptidic fragment corresponding to the antigenic site, which does not require any processing (unpublished results). In contrast, they are unable to process the corresponding complex Ag, i.e., the living Flu virus, whatever the concentrations used. This defect in antigen presentation is not due to resistance to lysis, because the same cells, if pulsed with peptide, are efficiently lysed. Additionnally, efficient presentation of Flu Ag in the context of class I determinants, clearly eliminates a possible resistance to infection by whole virus. Therefore, the unability of our activated T cells to present complex Ag in association with class II molecules can be attributed to qualitative or quantitative defects in processing. Moreover, because Flu has been demonstrated to infect T cells through a binding to sialic acid containing membrane receptors and through an endocytosis in the endosomal compartment [43], these results lead to the conclusion that Ag uptake to the endosomes by activated T cells is not always sufficient for efficient presentation of Ag in the context of class II determinants. This conclusion appears to differ from reports that, by using SUgp120 as Ag, suggest that T cells can process and present antigens only if these antigens bind to cell surface receptors [28, 29]. This discrepancy cannot be explained by the use of whole virus in our model and protein in theirs, because the same unability of processing has been observed by using the native H A protein (not shown). Therefore, the different natures of Ag and receptor may account for these different abilities of processing. It is noteworthy that even clones requiring 1000-fold less peptide have been unable to present Flu in association with class II molecules (not shown) and that same unability o f C D 4 + CTLs to process native Ag has been observed by others [41]. Whatever the precise mechanism responsible, the unability of our T cells to present complex Ag strongly suggests that lysis of activated T cells by CD4 + CTLs is likely to occur under certain circumstances only. The unability o f T cells to always process complex Ag in vitro cannot, however, exclude that in some situations Ag could be degraded to peptides and then be available for direct binding to class II determinants. H e r e we show that, in addition to the requirement for artificial processing there appear to be other requirements for the killing of activated T cells by CD4 + CTLs: (a) The density of H L A - D R molecules expressed on activated T cells seems to be a limiting factor because their optimal lysis is dependent on their D R expression (Fig. 2). Therefore, it seems likely that such a requirement would avoid the class-II-restricted lysis of activated T cells expressing low amounts of D R molecules at their mere-

58

brane. (b) Activated T cells have to be pulsed with high concentrations of peptide. Moreover, peptide added under a free form during the cytolytic assay that approximates to more physiologic situations inhibits target cell lysis. This inhibition cannot be related to CTL death, as assessed by the chromiumrelease assay o f 51Cr-labeled activated CTLs (Fig. 4), by blue trypan exclusion test, and by sensibility to exogenous IL2 (Fig. 6C). It results from peptide presentation by T cells to each other because (a) the same specific peptide is required for both induction and inhibition of cytolysis, and (b) anti-DR mAbs both inhibit peptide presentation by target cells and abrogate peptide-induced inhibition of A-CTL cytolytic activity. One interpretation could be that peptide-pulsed CTLs are being inhibited as the result of cold-target inhibition. However, this hypothesis cannot explain by its own all the observed results because cold pulsed A-CTLs, able to compete for T T C killing, are inefficient to compete for B-cell killing (Fig. 5). An inhibition at the effector cell level may also be involved because pulsing A-CTLs with peptide results in a time-dependent C D 3 - T C R modulation (Fig. 6). This T C R modulation could explain the decrease o f E B V - B - c e l l lysis and the abolition of the poorly efficient T-cell lysis. Alternatively, the small degree of T C R modulation that occurs when T cells are pulsed for 1½ hours could be not sufficient to explain the marked inhibition that has been observed. Therefore, one cannot exclude that pulsing A-CTLs with peptide could deliver an off signal that would induce T-cell unresponsiveness irrespective o f T C R - C D 3 expression, as previously suggested by others [44]. Finally presentation o f peptide by T cells usually induces lower cytotoxic responses than presentation by EBV-BLCLs. These different patterns of response to an individual peptide presented by T cells, as compared with EBV-BLCLs, would reflect differences in susceptibility to lysis, Ag presentation, and/or T - T versus T - B interactions. Our cold competition experiments shown in Fig. 5 confirm that A-CTLs have a preference for Bcell targets because E B V - B L C L s are able to compete for T-cell killing (Fig. 5C) but not reciprocally (Fig. 5B). This preference would therefore limit the lytic T - T interactions in any situation where classic APCs are present. The physiologic relevance o f CD4 + CTLs is still unknown; by killing activated T cells, they could act as suppressor T cells. The stringent requirements described herein suggest that CD4 ÷ CTLs would essentially regulate overenthusiastic immune responses.

ACKNOWLEDGMENTS

This work was partly supported by grants from La Ligue nationale contre le Cancer, L'Association pour la recherche contre le

A. Eljaafari et aL

Cancer, and La Fondation de France. The authors thank Claude

Hannoun for kindly providing influenza virus, Marc Sitbon and Michel Seman for helpful comments on the manuscript, and Isabelle Rivenez for typing the manuscript.

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Requirements for lysis of activated T cells by class-II-restricted cytolytic T-lymphocytes.

In the present study, we explored the specific requirements for lysis of human activated T cells by CD4+ CTLs. This was achieved by using human CD4+ T...
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