The Biological and Biochemical Basis of Allogeneic Effect Factor (AEF) Activity: Relationship to T Cell Alloreactivity

Immunobiol., vol. 161, pp. 51-83 (1982)

Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada

The Biological and Biochemical Basis of Allogeneic Effect Factor (AEF) Activity: Relationship to T Cell Alloreactivity l T. L. DELOVITCH and M. L. PHILLlPS2

Introduction The immune system of a species is able to distinguish self from nons elf (1). Cell-surface associated antigens determined by the major histocompati­ bility gene complex (MHC) comprise a highly polymorphic family of glycoproteins which mediate this distinction of individuality within a species (2). It is known that during an immune response, T lymphocytes acquire the capacity to bind to both the given immunogen and to self-MHC antigens (3-6). In particular, it is reasoned that the immunological reper­ toire of T helper cells is dependent in part on the extent of diversity and specificity of their membrane receptors for epitopes of both the nominal antigen and Ia antigens (7). It is not yet known whether T cells bear two structurally distinct receptors, or otherwise a single receptor, for these two different groups of epitopes. The ability of T cells to distinguish between molecular complexes formed between antigen and either self- or allo-Ia molecules may regulate different phenotypic patterns of immune responsiveness (5-7, 9-12). Preliminary biochemical characterizations of the polymorphic Ia alloantigens suggest that an Ia molecule possesses both unique epitopes and epitopes common to other Ia polypeptides (13-15). This implies that T helper cell-derived self­ and allo-Ia antigen receptors may share some structural homology and/or cross-reactivity. If this is the case, it might provide an explanation for the high precursor frequency of alloreactive T cells (16) and may also identify a molecular basis for understanding the biological significance of T cell­ mediated alloreactivity (1, 12, 17-21). We have approached this problem of T cell alloreactivity by extensive biological and biochemical analyses of allogeneic effect factor (AEF). This is a multicomponent helper factor derived from a mixed lymphocyte reaction (MLR) of proliferating alloactivated responder splenic T cells and irradiated stimulator spleen cells that mediates I-region restricted in vitro immune 1 Supported in part by grant MT 5729 from the Medical Research Council of Canada, by a grant from the National Cancer Institute of Canada, and by grants from the University of Toronto. 2 Dr. M. L. PHILLIPS is the recipient of a C. H. Best Foundation Postdoctoral Fellowship.

52 .

T. L.

DELOVITCH

and M. L.

PHILLIPS

responses. The AEF analyzed can recognize and presumably interact with stimulator haplotype-derived allo-Ia molecules and as such provide an important signal (s) required for lymphocyte communication. The biochemical identification, serological characterization, cell( s) of origin, target cell(s) and possible mechanism(s) of action of the relevant AEF components will be presented in this review. Emphasis will be given to the alloreactive component of AEF, its possible relationship to a T cell Ia alloantigen receptor, and its capacity to activate an I-region restricted Immune response.

Production of AEF The production of AEF, a modification of the method originally described by ARMERDING et al. (22), involves an initial step of T cell activation in vivo during a graft versus host reaction (GVHR). About 108 donor strain thymocytes are injected i.v. into an irradiated (800 R) host mouse (Fig. 1). Truly allogeneic (reviewed in 23) and not semi-allogeneic (22) donor-host strain age and sex-matched combinations are routinely used. Moreover, the donor and host cells used are derived from H-2 congenic mouse strains unlike that of ARMERDING et al. (22). Five days after cell transfer, approximately 80-95 % of the 107 lymphocytes resident in the host spleen can be serologically demonstrated by micro cytotoxicity with anti-Thy-l.2 and anti-H -2K serum antibodies to be of donor T cell in origin (24, 25). More recently, the predominance of donor T cells in irradiated host spleens at this time post-transfer has also been confirmed using monoclonal anti-Thy-1.2 (26) and anti-H-2Kk (27) antibodies. The yield of viable donor T cells is optimum at day 5 and thus these cells are harvested at this time for use in the next step of AEF production. The GVHR phase of AEF production has proven to be a necessary and crucial requirement for the subsequent generation of MHC-restricted AEF prep­ arations (28). The second phase of AEF production involves an in vitro restimulation of GVHR alloactivated donor T cell (responder cells) proliferation by irradiated (3000 R) host spleen lymphocytes (stimulator cells) during an MLR (Fig. 1). 10 7 cells of each MLR cell population are mixed together in a volume of 1 ml of serum-free RPMI-1640 medium freshly supplemented with 100 V/ml penicillin, 100 !lg/ml streptomycin, 40 !lg/ml gentamycin, 2 mM glutamine, 50 !lM 2-mercaptoethanol and 10 % (v/v) Trasylol, a serine protease inhibitor. We initiated the use of a serum-free medium and protease inhibitor for helper factor production and this has provided both a greater yield and much increased biological activity of AEF (24, 28). Cultures contained in 35-mm plastic petri dishes are placed in a humidified 5 % CO 2 atmosphere and rocked at 4 cycles/min in a 37°C incubator. After 16-20 hr, the culture supernatants are harvested and pooled, centrifuged at

The Biological and Biochemical Basis . 53

100,000 g for 90 min, aliquotted and either used immediately or frozen at -70°C until use. These MLR supernatants represent the starting unfraction­ ated multicomponent form of AEF. They can be stored in this form under these conditions for as long as 3-4 months and still retain> 75 % biological activity when assayed. Immediately prior to assay, AEF preparations are diluted appropriately, centrifuged at 15,000 g to remove possible large aggregates and if necessary rendered sterile by millipore ultrafiltration. Note that the donor-host mouse strain combinations, doses of irradia­ tion, duration of GVHR and MLR and protease inhibitor described herein differ from those used by KATZ and co-workers to prepare AEF. The conditions employed by the latter investigators have been reviewed else­ where (29). Most importantly, it should be recognized that KATZ et al. use non-con genic mouse strains and transfer both donor parental thymocytes and irradiated (2000 R) F J spleen cells into irradiated (600 R) F J hosts, then harvest the GVHR-activated donor T cells from host spleens 7 days later (29). It is likely, therefore, that distinct starting donor alloactivated T cell subpopulations are used in these two methods of AEF preparation. This may in part account for the different biological and biochemical properties observed for these two types of AEF (reviewed in 23, 29). Graft versus host reaction alloactivated lymphocytes Since the GVHR represents the first phase of AEF production, it is likely that certain AEF components are products of GVHR-alloactivated T cells. To ultimately gain a better understanding of the origin and properties of various components of AEF, it is essential to determine the serological, biochemical and functional characteristics of the subpopulations of GVHR­ alloactivated T cells used in the preparation of AEF. A. Donor T cell origin

GVHR occur when immunocompetent donor lymphoid cells are trans­ ferred to an allogeneic host. When the host is rendered immunoincompe­ tent, e.g. by exposure to sublethal irradiation as shown in Fig. 1, the capacity of the host to mount a host versus graft response is reduced considerably and may even be virtually eliminated. Thus, proliferating T cells resident in the host spleen after a GVHR should be predominantly of donor origin. This was confirmed in our previous serological (see section IIA above) and immunofluorescence (25) studies in which we demonstrated that 80-95 % of lymphocytes present in the host spleen after a 5 day GVHR were donor T cells. About 50-60 % of these T cells were T cell blasts. Greater than 95 % of these T cell blasts were found to express donor haplotype controlled H-2K and H-2D antigens, i.e. these T blasts were of donor origin (25). In addition, when irradiated host spleen cells were recovered on day 5 of a GVHR and used as responders in an MLR response against irradiated (3000 R) donor spleen stimulator cells, no significant

54 . T. L.

DELOVITCH

and M. L.

PHILLIPS

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proliferation above the control syngeneic values was obtained (unpublished observations). This suggests that back-stimulation does not occur in this situation. Thus, if any residual radiation-resistant host T cells survive in the spleen of an irradiated host after a 5 day GVHR, they likely constitute less than 1-2 % of the total T cell population present at this time.

B. fa and Lyt surface antigen phenotype During the inductive phase of a GVHR, donor T lymphocytes recognize MHC encoded alloantigens on host cell membranes and are stimulated to proliferate. It is conceivable that this recognition is mediated by the binding of different donor T cell receptors to host H-2 or Ia antigens. If this were the case then one might expect to be able to identify donor T cell subpopulations that bind H-2 and/or Ia antigens of the host haplotype. Furthermore, they should be serologically distinguishable since subpopula-

The Biological and Biochemical Basis . 55

tions of alloactivated T cells that respond to either H-2K and H-2D or Ia antigens may be separated according to their Lyt surface antigen phenotype (30, 31). The Ia and Lyt antigen phenotype of donor T cells activated during a GVHR across either an H-2, I-region or I-subregion incompatibility was examined by immunofluorescence. Table 1 shows that 30-50 % of donor T cell blasts activated across an H-2 incompatibility have either H -2K and H2D or Ia host-derived molecules bound to their surface membrane. These two alloactivated donor T cell subpopulations are non-overlapping (25) and accounted for about 90 % of the T cells examined. Approximately 70-80 % of the donor T cells that bound host H-2 and Ia antigens were identified as T cell blasts. No positive staining above the control value was observed when activated donor T cells were reacted with anti-Ia sera directed against the donor haplotype. This suggests that GVHR activated donor T cell blasts do not express self-Ia antigens of the donor haplotype but do bind allo-Ia antigens of the host haplotype. Non-activated donor thymocytes and peripheral T cells do not specifically bind host-derived H-2 and Ia antigens (32). Treatment of GVHR donor T cells with anti-Lyt-1.2 plus rabbit comple­ ment (C') resulted in an increase in the percentage of cells that bind host H2K and H-2D antigens from 40 to 68 % and a decrease in cells that bind host Ia antigens from 50 to 15 % (Table 2). By contrast, treatment with anti­ Lyt-2.2 plus C' increased the percent of host Ia antigen binding donor T cells form 50 to 69 % but decreased the percent of H-2K,D binding cells from 40 to 11 %. These results parallel those previously obtained with binding studies of MLR-alloactivated responder T cells (33) and are consis­ tent with a similar Lyt phenotype and function for GVHR-activated donor T cells. Thus, Lyt-1 + 2- donor T cells bind host Ia antigens. Genetic mapping studies demonstrated that host Ia antigens encoded by the I-A subregion, but neither the I-J nor I-E subregions, are transferred from the surface of host cells to the surface of activated donor T cells (Table 1). Similar results were obtained when GVHR responses across various I-region or I-subregion differences were examined (Table 3). A protein A cellular radio immune binding assay (34) showed that activated donor T cells adsorb onto their surface approximately 1/5 to 1/2 the number of host-derived I-A encoded Ia molecules that may be found on a normal host spleen cell (Table 4). Our previous studies suggest that a resting spleen B cell expresses about 104 Ia molecules on its surface. Thus, if the number of surface Ia molecules is linearly related to the 125 I -protein A cpm bound, it is estimated that between 2 X 103 and 5 X 103 host Ia molecules may bind to a GVHR activated donor T cell. While the estimated absolute number of transferred host Ia molecules may ultimately be proven incorrect, it is important to note the relative proportion of host Ia molecules on host spleen cells and activated donor T cells. It is intriguing to speculate whether

56 . T. L. DELOVITCH and M. L. PHILLIPS Table 1. GVHR Across a Whole H-Z Complex Incompatibility Donor

Host

H-2Incom- H-2 Regions Percent Donor Lympatibility Detected by phocytes Stained" Antisera b Specific Control C

BI0 BI0 BI0 BI0.5(7R) BI0.5(7R) BI0.5(7R) BI0.5(7R) BI0.S(7R) BI0.5(7R) BI0.BR BI0.BR (BI0.BR X BI0.S)Fl (BI0.BRXBI0.S)F\

BI0.BR BI0.BR BI0.BR BI0.BR BI0.BR BI0.BR BI0.BR BI0.BR BI0.BR (BI0.BRx BI0.S)F\ (BI0.BRXBI0.S)F\ BI0.BR BI0.BR

H_2k H-2k H-2k H_2k H-2k H-2k H_Zk H_Zk H_Zk H-Z'

H-2' None None

Kk, Dk Ik,Sk

1',5' Kk, Dk Ik,Sk 1',5' I-Ak, I-Jk I-Ek, Sk I-Jk Ik,Sk 1',5' Ik,Sk 1',5'

40 50 46 33 38 49 36

8 9 8 9 3 5

Z

4 5 30 8 5

" GVHR ailoactivated T cells were generated (Fig. 1) using the indicated donor and host strains. Samples containing 2.5 X 106 viable cells were treated first with the IgG fractions of either anti-H-2 or anti-Ia sera and second with either fluorescein or rhodamine-conjugated F (ab')2 of rabbit anti-mouse Fab. Staining for Thy-1.2 positive cells was performed using rabbit anti-mouse Thy-l and rhodamine-conjugated F(ab')2 of goat anti-rabbit Fab. The percentage of stained cells was determined using a Leitz Orthoplan fluorescence microscope. k b Anti-K k, Dk was produced by extensive absorption of C3H.SW anti-C3H/DiSn (anti-H-2 ) with A.TL spleen cells as determined by a dye exclusion microcytotoxicity assay. Before use, anti-Kk, Dk; anti-I-Ak, I-f; anti-P, Sk; anti-I-E k; anti-I-Ek, Sk, and anti-I-f were absorbed with donor BI0, BI0.S(7R), and BI0.T(6R) spleen cells. Anti-I', 5' was absorbed with A.TL, BI0, and B10.T(6R) spleen cells. Absorptions were performed by incubating 150 III of serum twice with 5 X 107 cells of a given strain for 30 min at 4°C. Sera were spun at 100,000 g for 1 h immediately before use to remove possible aggregates. C Donor cells were injected into BI0.T(6R) recipients. From (Z5).

Table Z. Anti-Lyt Plus C' Treatment of Donor Cells Donor

Host

H-2Incompatibility

Anti-Lyt + C' Treatment

H-2 Regions Detected by Antisera

Percent Lymphocytes Stained"

BI0 B10 B10 BI0 BI0 BI0

B10.BR BI0.BR B10.BR BI0.BR BI0.BR BIO.BR

H_Zk H_Zk H_Zk H-2k H-2k H-2k

Anti-Lyt-1.Z Anti-Lyt-1.2 Anti-Lyt-Z.Z Anti-Lyt-2.2

Kk, Dk Kk, Sk Kk, Dk Ik,Sk Kk, Dk Ik,Sk

68 15 11 69 40 50

" The preparation and immunofluorescent staining of GVHR-activated donor T cells, and the mouse alloantibodies used were as described in Table 1. Cells were treated with either anti­ Lyt-1.Z or anti-Lyt-Z.Z plus complement, and were then stained. From (25).

The Biological and Biochemical Basis . 57 Table 3. GVHR Across an I-Region or I-Subregion Incompatibility Donor

Host

H-2Incom- H-2 Regions Percent Donor LymDetected by phocytes Stained patibility Antiseraa Controlb Specific

A.TH A.TH A.TH A.TH (A.TH>< B10.HTT)F J (A.TH X B10.HTI)F J (A.THX: B10.HTI)F J (A.THX B10.HTI)F J (A.THX: B10.HTT)F J B10.S(7R) B10.S(7R) B10.S(lR) B10.S(7R) B10.A(3R) B10.A(3R)

A.TL A.TL A.TL A.TL A.TL A.TL A.TL A.TL A.TL B10.HTI B10.HTT B10.HTI B10.HTT B10.A(5R) B10.A(5R)

Ik, Sk Ik, Sk Ik, Sk Ik, Sk I-Ak,I-Jk I-Ak,I-Jk I-Ak, I-Jk I-Ak,I-Jk I-Ak,I-Jk I_Ek, Sk I-Ek, Sk I-Ek, Sk I-Ek, Sk I-Jk I-Jk

a

b

Ik, Sk 1', S' I-Ak,I-J k I-Ek, Sk Ik, Sk 1', S' I-Ak,I-Jk I_Ek, Sk I-Jk Ik, Sk 1', S' I-Ek, Sk I-Ek, Sk Ik, Sk I-Jk

37 2 36 2 34 9

32 5 46 16 77 76 3 8 76

3 30 7 6 6 33 6 5 3 7 8

Absorption of anti-Ik, Sk anti-I-Ak,I-f anti-I-E k, Sk; and anti-I-f were performed with A.TH and B10.S(7R) spleen cells. Anti-I', 5' was absorbed with A.TL and B10 spleen cells. Absorptions were performed as described in the legend to Table 1. Controls consisted of reciprocal combinations, i.e., recipient and donor strains were interchanged. From (25).

the number of host IA molecules on a donor T cell reflects the relevant number of Ia alloantigen receptor molecules on an alloreactive T cell. More recently, using a monoclonal anti-E~(Ae):Ea antibody (34), it was found that I-E alloantigens may also be transferred from GVHR host cells to donor cells (32). It was estimated that the relative number of I-E molecules was about 5-fold less than the number of I-A molecules trans­ ferred. These results are compatible with those of previous analyses of GVHR responses which demonstrated that an I-A incompatibility induces a much stronger response than that obtained in an I-E incompatibility (35-38), and that an I-J disparity does not induce a detectable response (36). They are also consistent with the finding that the rate of shedding or secretion of I-A alloantigens from spleen cells is about 5-fold greater than that of I-E alloantigens (R. Cone, personal communication). More interest­ ingly, they agree with the notion that both I-A and I-E polypeptides mediate antigen presentation to T cells (11, 12).

C. Immunochemical analysis One-dimensional (l-D) and two-dimensional (2-D) gel electrophoretic analyses illustrated that 125I-Iabelled a- and j3-polypeptide chains of both I-

58 . T. L. DELOVITCH and M. L. PHILLIPS Table 4. Estimation of Relative Number of Host I-A Coded la Antigens on Activated Donor T Cells a Group

Target Cell

Donor

1251-Protein A Cpm Boundc 10-2.16 b 10-3.6 b Host

BALB.B(H-2 b) BALB.K(H-2 k) a) unseparated activated spleen cells b) Ig- panned activated spleen cellsd BALB.K BALB.B a) unseparated activated spleen cells b) Ig- panned activated spleen cells 2

B10(H-2b) B10.BR(H-2k) a) unseparated activated spleen cells b) Ig- panned activated spleen cells B10 B10.BR a) unseparated activated spleen cells b) Ig- panned activated spleen cells

3

BALB.K normal spleen cells BALB.B normal spleen cells A.TL normal thymocytes

116± 28 160± 15

33± 35±

7 9

50±18 83± 8

±13 20± 7

2~

358±136 394± 36

150±21 335±64

54± 7 58± 13

38±13 36±10

872±130 39± 5 44± 11

600±58 28± 9 2~

± 6

The binding of mouse Ig to formalin-fixed lymphocytes was evaluated by an indirect cell binding assay. Formalin-fixed target cells (5)< 105) were incubated with a wide (6000-fold) concentration range of monoclonal anti-I-Ak antibody for 15 hr at 4°C in PVC plastic 96well microtitre plates. The binding buffer used was PBS containing 10 % FCS, 0.1 % gelatin, 10 mM Tris (pH 7.5) and 0.02 % Na azide. Plates were washed >< 3 and 1251-protein A (2.3>< 104 cpm = 50 ng) was added for 1 hr at 4°C. After a further 3 washes and drying of the plates, 125 1 cpm bound per well was determined. Background binding was defined by binding of 1251-protein A at each concentration of monoclonal antibody used to BALB.B normal spleen cells and A.TL thymocytes. Mean background binding (at saturating levels of binding to BALB.K spleen target cells) was 41 ±22 cpm for 10-2.16 and 32± 17 cpm for 10-3.6. Binding to target cells with one standard deviation of this basal binding were defined as background. b Monoclonal 10-2.16 and 10-3.6 anti-I-Ak antibodies were obtained from the Salk Institute Cell Distribution Center. They do not react with H-2b lymphocytes. C Mean binding of 1251-protein A in cpm ± standard error. Five observations were made for each value. d GVH activated donor T cells were harvested from irradiated host spleens and panned for Ig­ cells using plastic dishes coated with a polyvalent, affinity purified, goat-anti mouse Ig (40). From (32, 39). a

A and I-E encoded Ia molecules of the host haplotype, but not the donor haplotype, may be immunoprecipitated from GVHR activated donor T cells (32, 39). No structural alteration of the host Ia polypeptides is apparent after their transfer to the donor T cells, i.e., the same 2-D gel patterns were observed for host Ia molecules present on either host cells or

The Biological and Biochemical Basis . 59

211K-

Fig. 2. 2-D gel fluorograms of immunoprecipitated !25I-labelled host Ia antigens. 125I-labelled, lentil lectin bound and eluted glycoprotein fractions of NP-40 lysates were prepared from either normal A/WySn (H-2"; I_Ak, I-Ek) spleen cells (A) or donor A.SW (H-2'; I-A', I-E') T cells activated during a GVHR against host AlWySn H-2 antigens (B-D). After prec1earing the samples by treatment with normal mouse serum (NMS) and Staphylococcus aureus Cowan I strain (SaCI), they were reacted with either (A and B) A.TH anti-A.TL (anti-Ik ), (C)A.TL anti-A.TH (anti-I') or (D) NMS. Samples were separated by isoelectric focusing (lEF) in the first dimension (left to right) and by 10 % sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in the second dimension (top to bottom). The basic end (pH 7.5) is at the left and the acidic end (pH 4.5) at the right. Molecular weight (M.W.) marker positions of porcine lactate dehydrogenase (36,000) and Ig L-chain (25,000) are indicated by arrows. From (32, 39).

donor cells (Fig. 2). Immunoprecipitation of 35S-methionine labelled Nonidet-P40 (NP-40) solubilized proteins from activated donor T cells indicated that these cells do not synthesize Ia antigens of the donor haplotype (Fig. 3) (32, 39). These immunochemical data are in close agreement with the immunofluorescence data presented above. They lend further support to the concept that GVHR alloactivated donor T cells do not express self-Ia antigens but do synthesize receptors that bind to allo-Ia antigens.

D. I-region restricted interaction The antigen-binding studies of GVHR activated donor Lyt-l + r T cells pose several important questions with regards to the functional capacity of these cells. First, if the binding of host Ia molecules to GVHR activated donor Lyt-l + 2- T cells occurs via specific donor T cell allo-Ia receptors, does this postulated Ia:anti-Ia ligand:receptor type of interaction regulate the helper T cell activity of these cells in I-region restricted immune responses? Second, do subpopulations of GVHR donor T cells exist that recognize only self (donor)-Ia or only allo (host)-Ia, and if so, do they display an MHC preference in their ability to help B cells of either the donor or host haplotype, respectively? Third, does the repertoire of a self-

60 . T. L.

DELOVITCH

and M. L.

PHILLIPS

NORMAL A.SW SPLEEN CELLS

ACTIVATED DONOR A.SW T CELLS

anti-!'

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Fig. 3. I-D gel electrophoresis of immunoprecipitated 35S-methionine labelled donor A.SW (IS) cell Ia antigens. NP-40 Iysates containing glycoproteins were prepared as in Fig. 2 from 35S-methionine labelled normal A.SW spleen cells (A, B) or GVHR-activated A.SW donor T cells (c. D). They were precleared with NMS and SaCI, and then treated with either (A, C) A.TL anti-A.TH (anti-IS) or (B, D) A.TH anti-A.TL (anti-Ik). The 10 % SDS-polyacrylamide slab gel was run under reducing conditions for 16 hr at 10 rnA, and the radioactivity in 2 mm gel slices was determined. Migration positions of M.W. markers bovine serum albumin (68,000), Ig H-chain (55,000), ovalbumin (43,000), porcine lactate dehydrogenase (36,000) and Ig L-chain (25,000) are indicated. From (32, 39).

Ia reactive helper T cell overlap with that of an allo-Ia reactive helper T cell? Functional analyses of GVHR-activated donor T cells that either bind or do not bind host Ia molecules were carried out in an attempt to answer some of these questions. Donor A. SW (H-2S) thymus-derived T cells were activated as in Fig. 1 in a GVHR against host A/WySn (H-2a; carries I-Ak region) H-2 antigens. After 5 days, the viable lymphocytes were recovered from the spleens of irradiated hosts, panned (40) for the non-adherent Ig- fraction of T cells using plastic dishes coated with a polyvalent goat anti-mouse Ig (41), and then stained first with biotinylated monoclonal anti-I-Ak (11-5.2, Becton­ Dickinson, Mountain View, Calif.) and second with fluoresceinated-avidin (Becton-Dickinson). The fluorescence of scatter-gated viable c~lls was determined with Dr. R. O. Miller's OCI (Ontario Cancer Institute, Dept. of Medical Biophysics, University of Toronto) fluorescence-activated cell sorter (FACS) (42). About 30 % of the cells analyzed were specifically

The Biological and Biochemical Basis . 61

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Fig. 4. FACS separation of GVHR activated donor A.SW T cells that either bind or do not bind host AlWySn I-Ak Ia antigens. GVHR activated donor A.5W T cells were stained with either biotinylated monoclonal anti-I-Ak (11-5.2) and fluoresceinated-avidin (sorted sample, - ) , biotinylated anti-I-A' (IgG fraction of A.TL anti-A.TH antiserum) and fluoresceinated­ avidin (control, -- -) or left unstained (autofluorescence control, ------). The fluorescence profile obtained by staining with only fluoresceinated-avidin (second step control) was very similar to that shown for anti-I-A' plus fluoresceinated-avidin (unpublished observations). 30 X 106 panned Ig - activated donor A.SW T cell blasts were sorted. Approximately 20 % of the brightest viable cells were taken to be I_Ak<:±J. The dimmest viable cells showing no fluorescence below channel 10 (- 66 % of cells) were taken to be I-Ak 8. From (32, 39).

stained by comparison to control values (Fig. 4). The 20 % brightest viable cells were taken to be I-AkE8 cells, i.e. donor T cells which bound host I-A antigens. The dimmest 66 % of viable cells showing no specific fluorescence were taken to be I_AkG, i.e. donor T cells which did not bind host I-A antigens. The helper cell activity of I_AkG and I_AkE8 donor T cells for normal T cell-depleted spleen cells of either donor or host origin was examined in an in vitro primary anti-sheep erythrocyte (SRBC) plaque forming cell (PFC) response. T cell-depleted spleen cells were prepared by treatment with a monoclonal anti-Thy-1.2 antibody (26) plus rabbit C'. Fig. 5 shows that unsorted GVHR activated donor T cells helped host B cells but not donor B cells (groups (5-8). I_AkG donor T cells that did not bind host I-A antigens preferentially helped unprimed B cells of the donor haplotype (groups 9-12). I_AkE8 donor T cells that bound host I-A antigens helped unprimed B cells of only the host haplotype (groups 13-16). Both donor and host unprimed B cells were activated in cultures containing an equal number (10 5 ) of I_AkG and I-AkE8 donor T cells (groups 17-20). No suppressive effect was evident therefore upon mixing these two subpopulations of donor T cells; yet donor B cells were unresponsive when interacted with unsorted donor T cells (groups 5 and 6). It is possible that some donor haplotype specific suppressor T cells were deleted from the selected sorted

62 . T. L. DELOVITCH and M. L. PHILLIPS

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Fig. 5. H-2 restricted interaction of FACS sorted GVHR activated donor T cells with unprimed B cells. GVHR activated donor A.SW T cells were sorted (see Fig. 4) into subpopulations that either bind (I-Ak(!) or do not bind I_A k8 host Ia antigens. Cultures were set up containing 105 T cells from either one or both of these subpopulations and 4 X 105 anri­ Thy-1.2 plus C' treated spleen cells from either unprimed A.SW or A/WySn mice in the presence or absence of SRBC (106 ). Control culmres contained no antigen (Ag) and either no T cells or unsorted activated A.SW T cells. Direct PFC in triplicate 5 day cultures in 0.2 ml of RPMI-1640 medium containing 5 % FCS were enumerated and are presented as an arithmetic standard error of the mean. Data from 2 experiments are shown. Form (32, 39).

T cell subpopulations. In addition, it is likely that in the unsorted activated donor T cell population the precursor frequency of allo (host)-reactive helper T cells is much greater than that of self (donor)-reactive helper T cells. This may also account for the observed I-region restricted helper activity of unsorted donor T cells for host B cells. It should be noted that activated donor I_AkG and I-AkEEJ T cells cooperate equally well with antigen-primed B cells of the donor and host haplotypes (unpublished observations); no I-region restricted interactions occur, therefore, between GVHR-activated donor T cells and antigen-stimulated B cells (see section IVF for further discussion).

c..> W

a: 0

The Biological and Biochemical Basis . 63 Table 5. Properties of GVHR Alloactivated T cells 1. Comprised of 80-95 % donor T cells. 2. About 50-60 % of the donor cells are activated T cell blasts. 3. Greater than 95 % of donor T cell blasts express H-2K and H-2D antigens of the donor haplotype. They do not synthesize Ia antigens of the donor haplotype. 4. About 70 % of donor T cell blasts are Lyt-l +, 2- cells that bind Ia antigens determined mainly by the I-A subregion of the host haplotype.

5. Intercellular transfer of host-derived I-A, and about 5-fold fewer I-E, encoded Ia u- and [3polypeptides occurs from host antigen-presenting cells (presumably macrophages and B cells) to activated donor T cell blasts. These Ia polypeptides do not appear to be structurally altered after their release from host cells and binding to donor T cells. 6. Two major subpopulations of I-region restricted GVHR activated donor helper T cells exist. Donor T cells that do not bind host I-A molecules preferentially help donor B cells. Donor T cells that bind host I-A antigens preferentially help host B cells. These T cell subsets may express distinct self-Ia and allo-Ia receptors, respectively.

Thus, the Ia antigen-binding specificity of the two subpopulations of alloactivated donor T cells regulates their I-region restricted helper activity. This specificity is presumably mediated by allo-I-A and self-I-A receptors which, as suggested by the data presented in Fig. 5, could conceivably represent different molecules on distinct T cell subsets. It remains to be determined whether the self-reactive donor T cells can also help B cells of a haplotype(s) that differs from the host haplotype used in these studies. Several T cell clones that proliferate in response to both self-MHC plus antigen and allo-MHC in the absence of antigen have been identified (11, 12, 43, 44). The frequency observed thus far for such «heteroclitic» T cell clones is about 5 % of that observed for T cell clones that are only self­ reactive. Further genetic and biochemical experimentation with GVHR­ induced alloreactive T cell clones of either self-I-A and/or allo-I-A specific­ ity is required to resolve the contested issues concerning the nature and structural relationship of T cell antigen and alloantigen receptors (reviewed in 11, 12, 43). Such studies should also further clarify the biological significance of alloreactivity. Despite the several unknowns and points of controversy presently encircling alloreactive T cells, several interesting observations have emerged from our studies and are summarized in Table 5. These findings on GVHR alloactivated T cells bear directly on the biologi­ cal and biochemical properties of AEF presented in the next section.

Characterization of AEF To understand the intriguing observation that during an allogeneic effect, GVHR activated donor T cells bind H-2 and Ia antigens of host cell origin, it was considered important to study T cell alloactivation under conditions where the interacting cell types can be carefully selected and the factors

64 . T. L.

DELOVITCH

and M. L.

PHILLIPS

produced by both donor and host cells can be isolated and analysed. This may be accomplished by the characterization of products released from GVHR activated cells upon further proliferation in vitro.

A. Cell of origin During the second phase of AEF production, donor T cells alloactivated in a GVHR (section lIlA) are used as proliferating responder cells and irradiated spleen cells of the host haplotype are used as stimulator cells in MLR cultures. Soluble helper factors termed allogeneic effect factors (AEF) are present in the supernatants of MLR cultures of GVHR activated responder T cells and either H-2(24, 45), I-region (46), I-subregion (28, 47) or MIs-locus (48) incompatible irradiated stimulator cells. While AEF helper factors mimic the activity of helper T cells in vivo in their ability to recognize an immunogen in association with syngeneic Ia antigens (48), the molecular basis for this associative recognition is unknown. About 90-95 % of the alloactivated MLR responder cell population used to prepare AEF can be serologically demonstrated to be GVHR donor T cells (see section IlIA) (25). In addition, the Lyt antigen phenotype of both the GVHR and MLR activated T cell blasts is Lyt-l +r (25,33), which is identical to the Lyt antigen phenotype of antigen-specific helper T cells (49). Just as GVHR donor T cell blasts passively acquire host cell derived Ia antigens onto their surface (section IIIB), a similar intercellular exchange of Ia antigens occurs from stimulator cells to responder T cells during an MLR (33). An interesting parallel observation is that the activity of various AEF produced by such responder cells can be removed by immunoadsorption with either anti-I-A (23, 45, 46) or anti-I-J(23, 28) alloantisera that detect Ia determinants of the stimulator haplotype. It is, therefore, conceivable that unfractionated AEF contains a GVHR donor and MLR responder T cell derived membrane protein(s) that binds to host and stimulator I-A or I-J alloantigens. This protein, as well as others, may be present in AEF because they are either shed or secreted by the activated MLR responder T cell blasts. These observations identify the MLR activated Lyt-l +2- T cell blasts as the source of this and possibly other AEF components, and the irradiated stimulator cells as the source of AEF Ia antigens.

B. Biological and biochemical properties of two major components In view of the functional and serological complexity of AEF, fractiona­ tion and biochemical characterization of its biologically active components was necessary to understand the molecular basis of its activity. Since the broad difference in Ia antigenic specificities between the H-2k and H-2S haplotypes results in an AEF of strong helper activity, MLR cultures of A.SW (H-2S) GVHR activated responder T cells and A/WySn (H-2a) irradiated stimulator spleen cells were used to prepare an AEF for biochem­ ical analysis.

The Biological and Biochemical Basis . 65 Fractionation of AEF on ACA 54 BALB/c

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Fraction Number

Fig. 6. Assay of AEF activity after gel filtration on ACA 54. Approximately 15 ml of AEF was applied to a 2- X 90- cm column of Ultrogel ACA 54 previously equilibrated with 0.9 % NaCI. (A) Column fractions (8 ml) were assayed for their capacity to promote at a 1 :10 dilution a primary in vitro anti-SRBC direct PFC response of BALB/c nude spleen cells (A) or to stimulate at a 1:5 dilution CTLL cell growth (e). PFC results are expressed in terms of the fraction of responding microcultures in the 120 cultures tested for each column fraction (41). Results of cell growth are expressed as [3Hlthymidine counts per minute incorporated into trichloroacetic acid-precipitable DNA. The molecular weight markers used to calibrate the column were human serum albumin (67,000), ovalbumin (43,000), TCGF (- 30,000), and cytochrome c (12,500). (B) Column fractions were assayed at a 1 :10 dilution for their ability to stimulate an anti-SRBC response of either BI0.S (e) or BI0.A (A) T cell-depleted spleen cells. In (A) and (B) above, two distinct components, pools I and II, were identified. From (23).

66 . T. L. DELOVITCH and M. L. PHILLIPS

Gel filtration under non-dissociating conditions on ACA 54 (Fig. 6), resolves AEF into two distinct components which differ both biochemi­ cally and biologically (23). Component I chromatographs in the 50,000 to 70,000 molecular weight (M.W.) range and component II elutes in the 30,000 to 35,000 M.W. range. In a direct PFC assay, component I displays helper activity for H-2d nude spleen cells (Fig. 6A), for T cell-depleted spleen cells of the stimulator (H-2a) but not the responder (H-2S) haplotype (Fig. 6B), and for T cell depleted spleen cells of those haplotypes which share only an I-A subregion identity with the stimulator haplotype (23). Thus, the helper activity of component I is H-2 restricted. AEF component II also stimulates a primary anti-SRBC response of nude mouse spleen cells and of T cell-depleted spleen cells. However, this component provides help to both the responder and stimulator strains and is therefore not H-2 restricted in its activity. In addition, component II stimulates the growth of a long-term cytotoxic T cell line (CTLL) (50); the growth of this CTLL line is dependent on T Cell Growth Factor (TCGF) [also known as Costim­ ulator (51, 52) or Interleukin-2 (53, 54) and hereafter referred to as IL-2]. Component II also promotes thymocyte mitogenesis in the presence of non-mitogenic doses of Concanavalin A (Con A) and generates alloreactive cytotoxic T cells in either nude spleen cell or thymocyte cultures. The latter three properties are not manifested by component 1. The observation that the helper activity of AEF component I is about two-fold greater than that of component II might explain why most AEF preparations when tested at an appropriate dilution manifest H-2 restricted help. We suggest that the

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Fig. 7. Assay of activity of AEF component I after isoelectric focusing. After elution from DEAE Sephacel, component I was further purified by IEF on a 110 ml LKB preparative column that was stabilized with a gradient of 0-60 % glycerol. Ampholytes in the pH range of 3-10 were added to the gradient at a 1 :40 dilution. Electrophoresis was carried out for 25 hr at 4°C. 40 2.5 ml fractions were collected, their pH determined, and then neutralized to pH 7.2 using 1 M Tris base. Fractions were diluted 1:3 and assayed for their helper activity with A.SW (0) or A/WySn (e) T cell-depleted spleen cells. From (23).

The Biological and Biochemical Basis . 67

H-2 restricted activity of a particular AEF is dependent in part upon its relative amounts of components I and II. In addition, it may be seen in Fig. 7 that AEF component I, after fractionation by IEF, elicits a strong PFC response by stimulator B cells and a much weaker PFC response by responder B cells. Thus, the I-A restricted helper activity of component I results in quantitative rather than qualitative differences in the PFC

AB

68K-55K-

43K36K--

25KFig. 8. 1-D gel fluorogram of 1251-labelled AEF com­ ponent 1 obtained after 1EF. Fractions 17-19 of Fig. 7 were dialyzed against phosphate-buffered saline to remove Ampholytes, concentrated 100 X, adjusted to pH 8.5, and labelled with 1251 using the Bolton-Hunter reagent (> 1375 Ci/mmole). Samples were elec­ trophoresed on a 1-D SDS-polyacrylamide slab gel under reducing (A) or nonreducing (B) conditions. M.W. markers are as in Fig. 3. From (23).

68 . T. L. DELOVITCH and M. L. PHILLIPS

responses observed. This might depend on its capacity to bind with high affinity to stimulator (nons elf) B cells and/or macrophages and perhaps with lower affinity to responder (self) B cells and/or macrophages. Further characterization of component I (23) demonstrated that it elutes from DEAE Sephacel between 0.05 M and 0.1 M NaCl, does not bind to lentil lectin and therefore may not be a glycoprotein, and has a pI of approximately 5.5-6.0 (Fig. 7). It seems to consist of a single major protein of about 68,000 M.W. as examined after radioiodination by 1-D sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis under reducing and non-reducing conditions (Fig. 8). No subunit structure component I was detectable by acid-urea gel electrophoresis in the absence of SDS (23). 2-D gel electrophoresis of 12sI-labelled component I confirmed that it has a M.W. of - 68,000 and a pI of - 5.8; it also indicated that component I was purified to apparent electrophoretic homogeneity (23). Component II elutes from DEAE Sephacel between 0.15 M and 0.2 M NaCl, does not bind to lentil lectin, has a pI of 4.3 and 4.9 and a M.W. of about 30,000 (23, 50). It appears to consist of two 15,000 M.W. subunits when electrophoresed under reducing conditions (53). Thus, it is clear that AEF is comprised of at least two major components that differ markedly in their biological and biochemical properties, as summarized in Table 6. Table 6. Biochemical, Serological, and Biological Properties of AEF Property

Component I

Component II

Biochemical Molecular weight (ACA 54) Molecular weight (SDS-PAGE) Salt elution from DEAE-Sephacel pI Lentil lectin affinity

50,000-70,000 68,000··b 0.05-0.1 M 5.8

30,000-35,000 30,000'; 15,000b 0.15-0.2 M

4.3,4.9

Serological Ia Ig (isotypic and idiotypic) Biological Helper activity H-2 restricted H-2 nonrestricted Stimulation of CTLL growth Stimulation of thymocyte mitogenesis Generation of cytotoxic T cells Role

+

T cell Ia receptor?

• Electrophoresis under nonreducing conditions (53). Electrophoresis under reducing conditions (53). From (23).

b

+ + + +

IL-2

The Biological and Biochemical Basis . 69

C. Serological analysis

A number of antigen-specific T helper and T suppressor cell derived molecules have been shown to express Ig heavy-chain variable-region determinants (VH)' cross-reactive idiotypic determinants and Ia determin­ ants (reviewed in 7, 55, 56). Some of these T cell products are similar in size to AEF component I (57-63). To investigate the relationship of component I to these T cell products, it was subjected to an extensive serological analysis (23). Immunoprecipitation of 125I-labelled component I showed that it does not carry Ig isotypic H-chain and L-chain determinants or Ig idiotypic determinants (Table 6). Although similar in size to mouse serum albumin and gp 70, the major envelope glycoprotein of murine leukemia RNA viruses (64), component I does not react with antibodies directed against these two proteins. Furthermore, all three proteins have different pI values. Previous affinity chromatography studies of several unfractionated AEF have shown them to consist of la-positive helper components (23, 24, 28, 45, 65). The AEF Ia antigens were found to be stimulator B cell and/or macrophage derived. However, a more sensitive radioimmunoprecipitation assay demonstrated component I to be an la-negative molecule (Table 6) (23). Furthermore, spots corresponding to Ia antigens are not detectable on a 2-D gel fluorogram of component I (23). It is possible that stimulator cell derived Ia antigens are active components of AEF when AEF is in its unfractionated form. Extensive manipulation of AEF during its fractiona­ tion may result in the dissociation of Ia antigens from component I. Component II shares many biological and biochemical properties with IL-2 (sections IVB and IVE). IL-2 has recently been shown to be an Ia­ negative and Ig-negative molecule (51-53).

D. Relationship of component I to a T cell alloantigen receptor Our studies have shown that AEF component I is derived from Lyt1 +2- T cells, possesses an H-2 restricted helper activity, functions as an anti-I-A- like molecule that binds to I-A determinants on macrophages and/or B cells, and is la-negative. These properties are shared by helper T cells. It is therefore compelling to conceive of AEF component I as an Lyt1 +2- helper T cell derived I-A alloantigen receptor. If AEF component I is indeed a T cell-derived anti-l-A-Iike molecule, it is interesting to note that it differs in its properties from that of a B cell­ derived anti-I-A alloantibody. Whereas component I elicits helper activity at the level of the macrophage (section IVF), anti-I-A antibody inhibits helper activity upon binding to macrophages (66, 67). Furthermore, while an anti-I-A antibody bears Ig idiotypic and isotypic determinants, compo­ nent I of AEF may be idiotype- and isotype-negative (Table 6). Alterna­ tively, it is possible that these two types of molecules express different idiotypic and isotypic determinants. Irregardless of which of these two alternatives is correct, the important implications that arise are that T cell

70 . T. L.

DELOVITCH

and M. L.

PHILLIPS

and B cell derived «anti-I-A» molecules differ with respect to their structure and the manner in which they regulate immune responses. Finally, note also the close resemblance of AEF component I to a previously identified 70,000 M.W., la-negative, apparently idiotype-posi­ tive, product of alloactivated T cells. This product has also been suggested to represent a T cell alloantigen receptor (60).

E. Relationship of AEF component II to other AEF, IL-2 and TRF The relationship between the AEF described above and that described initially by ARMERDING and coworkers (68), and more recently by ALT­ MAN et al. (69), has been reviewed elsewhere (23, 29). Briefly, chromatogra­ phy of the latter AEF under dissociating conditions shows that it consists of 40,000 and 12,000 M.W. subunits, neither of which display the H-2 restricted helper activity of the 68,000 M.W. AEF component I (charac­ terized in section IVB). The 40,000 M.W. subunit, however, is of similar molecular weight to our AEF component II (30,000-35,000 M.W.) and both have H-2 non-restricted T cell stimulatory functions. The functional properties of component II are identical to those previously reported for IL-2 (52, 53) and both are produced by an I-A negative T lymphocyte (49). Thus, IL-2 appears to be a major component of our AEF. The AEF produced by ALTMAN et al. also possesses considerable IL-2 activity (29). The 40,000 M.W. AEF component(s) originally identified by ARMER­ DING et al. has many of the helper activities attributable to IL-2, but might have at least one additional function not attributable to that lymphokine. The AEF of ALTMAN et al. is able to autonomously induce a primary self­ reactive cytotoxic T cell response in vitro in the absence of stimulating target cells during the sensitization phase (29). The presence of Ia determi­ nants in this AEF may regulate its ability to induce self-reactivity (29). Further biochemical and biological analyses of the putative 40,000 M.W. AEF subunit may reveal it to be derived from a larger M.W. molecule and to mediate the self-reactivity observed. Thus, it may ultimately be shown to be a 68,000 M.W. (or even larger) self-Ia receptor. A second lymphokine, T cell replacing factor (TRF) (70) is, like IL-2, found in Con-A activated spleen cell supernatants. It has a M.W. of 30,000, a pI of 3-4, and activates a non-genetically restricted anti-SRBC response of T cell-depleted spleen cells. Although it does not have the T cell stimulatory activities of IL-2, it still might be a constituent of component II. Since TRF can be distinguished from IL-2 by isoelectric focusing (50, 70), further experimentation should confirm the suspected presence of TRF in AEF component II.

F. Target cells and mechanisms of action Previous studies demonstrated that 9 out of 10 AEF generated across either an H-2 (24,45), I-region (46), I-subregion (28, 47), or Mis-locus (48) incompatibility elicited an I-region restricted helper activity for B cells of

The Biological and Biochemical Basis . 71

the stimulator haplotype. These results are summarized in Table 7. The only exception noted was for AEF-1 and this may be attributable to differences in its method of production (23). The I-region restricted helper activities of unfractionated AEF 2-10 were generally detected in an optimum concentration range of 0.001 %-0.0001 % (v/v); no I-region restricted activity was evident beyond this range. To identify the possible restricted helper activity of a given AEF, it is therefore essential that a dose response analysis of this AEF be performed. All AEF preparations examined in this manner restrict their help to stimulator B cells. It was therefore apparent that the AEF target cell(s) of action is present in the stimulator cell population. Genetic mapping studies indicated that AEF 1-10 also help B cells of haplotypes that share the relevant I-region or I-subregion identity with the stimulator haplotype. Moreover, the activity of AEF 2-8 could, prior to their fractionation, be absorbed by alloantibodies reactive with the respec­ tive I-A (23, 45, 46) or I-J(28) products of the stimulator haplotype but not of the responder haplotype. Taken together, these findings suggested that the active helper component of AEF is comprised of Ia antigens determined by the stimulator haplotype and/or a receptor-like molecule that binds to Ia antigens on stimulator haplotype derived antigen-presenting target cells. It was not possible to decide between these two alternatives when unfraction­ ated AEF preparations were used. Neither was it possible to identify precisely the type of stimulator target cell involved. Since the helper activity of AEF is routinely assayed in T cell-depleted spleen cultures, the AEF Table 7. Helper Activities of Various AEF Strain" Responder 1. BlO.BR 2. BlO.BR(Ia-) 3. BlO.S 4.A.SW 5.A.TH 6. (A.THXBlO.HTI)F j 7. BlO.A(3R) 8. BI0.A(5R) 9. BlO.S(7R)

10. C3H/HeJ

Incompati­ bilityb Stimulator BI0.S H-2' BlO.S(Thy-1.2-) H-2' H-2k BlO.BR H_2k AlWySn Ik, TL?, Qa? A.TL A.TL I-Ak, I-Jk I-Jk BI0.A(5R) BI0.A(3R) I-Jb BI0.HTT I-Ek, TL?, Qa? Mlsb BI0.BR

ActivityC

H-2 RestricResponder Stimulator tion ++++

± ±

+ +

±

±

+++ ++++ ++++ ++++ ++++ +++ ++ ++ +++ ++

No Yes Yes Yes Yes Yes Yes Yes Yes Yes

" Strains used for the production of AEF. b Genetic incompatibility in the GVHR and MLR phases of AEF production. C Helper activity with T-cell depleted spleen cells of either responder or stimulator haplotype. Relative strengths of AEF helper activity are shown as: + + + +(2,000-3,000 PFC/l0 7 cells), + + + (1,000-2,000 PFC/107 cells), + + (300-1,000 PFC/10 7 cells), + (100-300 PFC/107 cells) ± (50-100 PFC/10 7 cells), and - «50 PFC/I0 7 cells). From (23).

72 . T. L. DELOVITCH and M. L. PHILLIPS Table 8. I-Region Restricted Interaction of AEF Component I with Macrophages and not B Cells of the Stimulator Haplotype Group

2 3 4 5 6 7 8 a

b

C

AEF' Ag Component I (106 SRBC)

++ ++ ++ ++

+ +

Macrophages b

B Cells b

Direct PFC perc 107 Cultured Cells

BI0.S BI0.A BI0.S BI0.A BI0.S BIO.A BI0.S BI0.A

BI0.S BI0.A BI0.S BI0.A B10.S BI0.A BI0.A BI0.S

0 0 2±11 9±18 110±37 803±36 73±19 638±18

AEF component I was partially by ACA 54 chromatography (see Fig. 6). Active fractions were pooled and used at a 1 : 10 final dilution in RPMI 1640 medium containing 5 % FCS. Macrophages and B cells were prepared from either BI0.S (H-2', syngeneic to responder A.SW) or BI0.A(H-2a, syngeneic to stimulator A/WySn) spleen cells that were treated with monoclonal anti-Thy-1.2 plus C. The T-depleted spleen cells were then separated into macrophages and B cells by 1 g velocity sedimentation on35 % FCS according to (71). 104 macrophages and 5 X 105 B cells were added to cultures (0.21 ml) containing 106 SRBC as antigen. Direct (IgM) PFC in triplicate 5 day cultures are presented as the arithmetic mean ± standard error.

target cell is most likely a macrophage and/or a B cell. For AEF 1-6, 9 and 10, it was proposed that both types of cells could be involved because both Ia and Mis antigens are expressed on macrophages and B cells (24, 28, 45-48). Further experimentation with purified AEF components and iso­ lated macrophages and B cells was required to resolve the nature of the AEF target cell. The ideal AEF component to be used in this type of experiment is component I, since it likely represents an alloactivated responder T cell­ derived receptor for I-A alloantigens on a stimulator target cell (see section IVD). Preliminary experiments have been carried out in collaboration with Dr. R. GORCZYNSKI, Dept. of Medical Biophysics, University of Toronto, using AEF component I obtained after ACA 54 chromatography (see Fig. 6) and either BI0.S (H-2S; histocompatible with A.SW responder haplotype) or BIO.A (H-2a; histocompatible with AlWySn stimulator haplotype) T cell-depleted spleen cells that were further separated by 1 g velocity sedimentation into macrophages and B cells (71). It is evident from Table 8 that maximal anti-SRBC PFC responses were obtained in the presence of antigen, component I and macrophages of the stimulator haplotype (groups 6 and 8). Either stimulator or responder B cells could be present, however, an increased response was achieved when stimulator macrophages and syngeneic stimulator B cells (group 6) rather than allogeneic responder B cells (group 8) were used. It follows from these data that an I-region restricted helper activity of AEF component I is operative at the level of recognition of allo-Ia

The Biological and Biochemical Basis . 73

molecules on antigen-presenting macrophages. This finding is compatible with the notion that T cells recognize a nominal antigen in the context of self-Ia antigens on accessory cells (antigen-presenting macrophages) and not B cells, as previously reported (72). While it does not eliminate the possibility that macrophage-B cell interactions are I-region restricted, it suggests that AEF component I does not mediate such an interaction. Nevertheless, the reason for the ability of AEF component I to elicit a response by allogeneic responder B cells (Table 8, group 8) is presently unclear. In the latter response, an la-mediated recognition between compo­ nent I and stimulator macrophages was necessary but the ensuing mac­ rophage-B cell interaction was not I-region restricted. This may either reflect the true physiological conditions under which macrophages com­ municate with B cells, or may simply be due to the fact that the preparation of AEF component I used after ACA 54 chromatography was somewhat cross-contaminated with IL-2 and TRF; these two lymphokines co­ chromatograph on ACA 54 (53). If this were the case, then TRF in particular may have activated responder B cells in a nonrestricted fashion. Ongoing studies with more homogeneous preparations of AEF component I obtained following isoelectric focusing (see section IVB) should clarify whether B cells serve as target cells of AEF component I, and if so, whether this level of interaction is I-region restricted. Thus far, the question of I-region restricted interaction at the level of the B cell has been addressed by examining the helper activity of AEF compo­ nent I for stimulator and responder haplotype-derived antigen-primed and mitogen-primed B cells. Data presented in Table 9 and 10 show that AEF Table 9. H-2 Unrestricted Interaction of Antigen-Primed B Cell Blasts with AEF Com­ ponentI B Cell Blasts'

AEFb

AntigenC

A.SW

+ +

+

234± 47 2368± 86

AlWySn

+ +

+

323± 23 2163±114

Indirect (IgG) PFC perd 107 Cultured Cells

• Spleen cells were derived from mice immunized 5 days previously with 0.1 ml of a 20 % (v/v) solution of SRBC. They were depleted of T cells by treatment with monoclonal anti-Thy-1.2 plus C'. Residual B cell blasts were isolated by ficoll-hypaque (cp = 1.077) density centrifugation. b AEF component I fractions 17-19 obtained by AEF (see Fig. 7) were pooled, dialysed and lyophilized to remove Ampholytes, and used at a 1 : 3 final dilution in RPMI -1640 medium containing 5 % FCS. 6 C 10 SRBC were added as antigen to appropriate culture wells. d Indirect (IgG) PFC in triplicate 5 day cultures are presented as the arithmetic mean ± standard error. Direct (IgM) PFC were approximately 100-200 PFC/10 7 cultured cells at this time.

74 . T. L. DELOVITCH and M. L. PHILLIPS Table 10. H-2 Unrestricted Interaction of LPS-Activated B Cell Blasts with AEF Component I LPS Blasts'

AEFb

Antigen

A.SW

+ +

+

84± 37 1891 ±201

A/WySn

+ +

+

133± 49 1508±186

C

Direct (lgM) PFC perd 107 Cultured Cells

• Spleen cells derived from non-immunized mice were incubated at 2.5 x 106 cells/ml with LPS (50 Ilg/ml) for 48 hr at 37° in the presence of 5 % CO 2 • Blasts were recovered from cultures by ficoll-hypaque (cp = 1.077) density centrifugation. 4x 105 blasts were added to cultures (0.21 ml) containing 105 syngeneic irradiated (3000 R) adherent spleen cells, as a source of macrophages. b AEF component I was obtained after IEF and used as described in Table 9. 106 SRBC were added as antigen to appropriate culture wells. d Direct (lgM) PFC in triplicate 5 day cultures are presented as the arithmetic mean ± standard error. Less than 100 indirect (lgG) PFC/10 7 cultured cells were detected at this time. C

component I is not restricted in its interaction with either antigen-primed (Table 9) or antigen-unprimed but E. coli bacterial lipopolysaccharide (LPS)-primed (Table 10) B cells. Syngeneic irradiated stimulator spleen cells were used as a source of macrophages in these experiments. In both instances, AEF component I provided equal help to A.SW responder­ derived and AlWySn stimulator-derived B cells. Thus, once B cells have been stimulated to divide by either an antigenic or mitogenic stimulus, recognition of their surface Ia antigens by cooperating T cells and macro­ phages is no longer required for effective interaction. IL-2, TRF and possibly other hormone-like lymphocyte derived molecules (53, 54) might non-specifically enhance IgM and IgG antibody secretion by already dividing B cells. These observations directly confirm those of MELCHERS et al. (73) who demonstrated that I-region restricted immune responses occur with small, resting, antigen-unprimed B cells but not with large, dividing, antigen-primed and/or LPS-primed B cell blasts. Interestingly, this group of investigators has also shown that a 70,000 M.W. component semi­ purified from the supernatant of an SRBC-specific cloned T helper cell line is antigen-nonspecific but is I-region restricted in its helper activity for non­ dividing unprimed B cells and not dividing primed B cell blasts (63). The similarity between this cloned T cell helper molecule and AEF component I is striking. The composite findings presented above for AEF component I, together with those previously reported for IL-1, a 15,000 M.W. macrophage­ derived lymphokine (74), IL-2 and TRF (52-54, 75), may be incorporated into a model which attempts to explain their role(s) in lymphocyte com­ munication (Fig. 9). Understandably, this is a minimal model which serves

The Biological and Biochemical Basis . 75 ~

c:z •

~

c:: C

Antigen

CamerPortlon Hapten Portion

rn0· ProcessedAg

- - Ial!=6orHl ---(

T Cell Receptor for mq;- Processed Camer Portion T Cell Receptor for Ia (~ AEF compor.ent I?) B Cellig Receptor for Hapten

Antibody Secretion

I' I' I' I'

i

~

~ and/or

'~~~

cbcbcbcb

TAF. AO"bOdY!cOdUCtiOO Synth~ I ~

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Fig. 9. A schematic model of the role of AEF and other lymphokines in certain pathways of lymphocyte communication.

to identify only those pathways of interaction that are likely to be mediated by T helper cells, macrophages, B cells and some of their known products. After immunization, macrophages bind and enzymatically fragment the nominal antigen. Antigen-presenting macrophages then interact in an I-A restricted manner with specifically recruited T helper cells. The recognition by T helper cells of I-A alloantigens on macrophages is mediated by AEF component I, i.e. a T helper cell I-A alloantigen receptor. This is followed by the synthesis of IL-l by activated macrophages. Subsequently, IL-l induces the responsive T helper cells to produce IL-2 (52-54); the produc­ tion of IL-2 therefore requires an I-A restricted interaction. In support of this notion are the recent observations that the recognition of self-Ia on macrophages by either cloned T helper cells (76) or T cells that proliferate in a syngeneic MLR (77) stimulates these T cells to produce IL-2. IL-2 acts in a nonrestricted fashion to expand clones of antigen-activated and/or allo­ antigen-activated T cells and T cell precursors (52-54). In this manner, the frequency of T helper cells that bear the appropriate antigen and alloantigen receptors is increased considerably. Due to uncertainty and simplicity, no distinction is made here between an antigen and alloantigen receptor. Clonally expanded T helper cells then cooperate with B cells and this step is both antigen-specific and I-region restricted. The latter requirement may be achieved by the binding of AEF component I to la on B cells; component I is likely present now in a greater amount and on a larger number of interacting T cells. I-region restricted macrophage-B cell interac­ tions may also occur at this time. This would suggest that macrophage and/ or B cell derived molecules are possibly involved in early stages of B cell activation. TRF, a T cell product, is known to act in an antigen-nonspecific and genetically nonrestricted manner during the late stages of B cell

76 . T. L.

DELOVITCH

and M. L.

PHILLIPS

activation (75). Thus, after I-region restricted T cell-B cell and macrophage­ B cell interactions, TRF may be synthesized by T cells and then induce B cell clonal expansion. Enhanced levels of antibody formation and secretion would ensue and hence a formidable immune response may result. The requirements for antigen and Ia recognition influence the activation of small, resting, antigen-unprimed B cells. They need not be fulfilled for the triggering of large, dividing, antigen-primed B cells, as discussed above. We have previously shown that an AEF generated across an I-J-subre­ gion disparity (AEF 7 and 8 of Table 7) elicits helper activity for an I-J­ restricted response of stimulator-haplotype derived T cell-depleted spleen cells (28). Since T cells are absent from these cultures and B cells do not express I-J determinants (78), this AEF presumably acts directly on macro­ phage target cells. This notion of an I-region restricted response between a T cell and a macrophage is compatible with the data presented for AEF component I in Table 8 and the model of lymphocyte interaction shown in Fig. 9. Recently, we have obtained further evidence in support of this model by demonstrating that an alloactivated responder T cell derived 50,000-70,000 M.W. component(s) of an AEF produced in an I-J incom­ patibility preferentially helps a primary anti-SRBC PFC response of B cells of only the stimulator haplotype (unpublished observations). It is suspected that this I-J AEF helper component possessed an anti-I-J-like activity, and it will prove interesting to compare its biological and biochemical proper­ ties with those of the I-A reactive AEF component I analyzed above. In collaboration with Drs. J. BROMBERG and M. GREENE, Dept. of Pathology, Harvard University, this semi-purified 50,000-70,000 M.W. T cell-derived anti-l-J-Iike component of an I-J AEF has been shown to profoundly influence the generation of T cells that suppress contact sen­ sitivity to either 2, 4, 6-trinitrobenzene sulfonic acid or azobenzenearsonate derivatized syngeneic lymphoid cells (79, 80, unpublished observations). Activation of the suppressor T cells involved the associative recognition of the modified-self antigenic determinants and allogeneic I-J determinants. Thus, binding of the anti-l-J-Iike component of AEF to I-J molecules on precursors of suppressor T cells may promote the maturation of these precursors to suppressor T cells (79). If this is the case, it would suggest that AEF mediates an important anti-I-J:I-J receptor: ligand type of interaction that regulates the role of suppressor T cells in different pathways of suppressor T cell circuits (81). Studies to test these possibilities are currently in progress. While B cell-derived anti-I-J antibodies ablate the effect of suppressor T cells (82), T cell-derived anti-I-J like molecules enhance the activity of suppressor T cells. This functional difference between T cell- and B cell-derived I-J alloantigen binding molecules is similar to that previously discussed for T cell- and B cell-derived I-A alloantigen binding molecules (section IVD). Finally, it should be mentioned that the above two types of experiments performed with a T cell-derived anti-I-I-like molecule(s) suggest that the

The Biological and Biochemical Basis . 77

same molecule may bind to both I-J-bearing macrophages and suppressor T cell precursors. If this is indeed the case, then such an anti-l-J-Iike molecule, depending on which of its target cells it chooses to bind to, could result in the subsequent activation of either B cells or suppressor T cells. Thus, an allogeneic effect elicited across an I-J disparity could prove crucial to the regulation of self versus nonself reactivity, as previously proposed (80). It remains to determine the biological significance and relationship between the recognition of allo-I-J and self-I-J determinants.

Conclusions The genetic and molecular basis of alloreactivity within the immune system still remains enigmatic. To unravel this enigma, we attempted to identify an alloreactive T lymphocyte-derived product(s) that mediates this type of immune response. It was also considered important to use immature thymocytes as the T lymphocyte source in order that their acquisition of alloreactivity be examined during T cell differentiation in vivo. Thus, products of thymocytes that were alloactivated in vivo during a GVHR and then restimulated in vitro during an MLR were characterized. One such MLR supernatant product, component I of AEF, was iden­ tified as a molecule that confers the alloreactivity of alloactivated, responder haplotype-derived, T helper cells. Although, it displays an I-A restricted helper activity for B cells of the stimulator haplotype, it also provides some, albeit much reduced, helper activity for B cells of the responder haplotype (Fig. 7). This suggests that it binds to allo-Ia antigens with high affinity and to self-Ia antigens with considerably lower affinity. Accordingly, it may also induce the appearance of cytolytic self-reactive T cells despite the previous claim by ALTMAN and KATZ (29) that an AEF prepared by our method described in Fig. 1 does not possess this property. Should further biochemical analysis of AEF component I confirm that it indeed consists of a single, homogeneous, plasmamembrane associated molecule, it will be evident that a T helper cell surface protein which is alloreactive is also self­ reactive. If it can also be shown to bind directly to la antigens (allo and self), e.g. when la antigens are present in liposomes or membrane-vesicles, this will lend added strong support to the claim that AEF component I is a T cell la antigen receptor. Such experiments will undoubtedly unravel the com­ plexities surrounding the relationship of alloreactivity to self-reactivity. This potential interrelationship should also be further clarified by our current analyses of cloned alloreactive T cells and their membrane-associ­ ated la-binding molecules. Despite the present uncertainty over the la receptor status of AEF component I, our studies have clearly shown that it exhibits la alloantigen specificity. Thus, whereas AEF has formerly been regarded as an antigen-

78 . T. L.

DELOVITCH

and M. L.

PHILLIPS

nonspecific factor, it should now be classified as an alloantigen-specific factor. It is not known whether allospecific T helper cells recognize allo-Ia antigens independently or allo-Ia antigens in association with other cell surface antigens. In the latter case, these T cells could be regarded as allo-Ia restricted T helper cells. From our studies on GVHR activated T helper cells (section III) and those of others on T helper cells obtained from chimaeric mice (83-86), it is clear that the phenotype of H-2 restriction is not determined by the H-2 genotype of T helper cells. It follows then from our analyses of several H-2 restricted AEF (Table 7), that each AEF probably consists of a structurally distinct 68,000 M.W. component that has binding specificity for different allo-Ia determinants. This infers the exist­ ence of a large repertoire of polymorphic T helper cell receptors for Ia antigens, and is quite compatible with the observation that the available repertoire of Lyt-l +2- T helper cells is heavily biased towards the recogni­ tion of Ia antigens (87). A wide spectrum of different molecular sizes has been reported for the various antigen-binding T cell derived molecules currently under study (reviewed in 7,55). They range in size from an apparent M.W. of 200,000 (88) down to as low as a M.W. of 25,000 (89). Surely, T cell receptors are more judicious in their selection of their molecular size. In this regard, it should be mentioned that while AEF component I has an apparent M.W. of 68,000, it may be released from a T helper cell by the enzymatic cleavage of a larger membrane-associated molecule. The latter molecule might consist of a hydrophobic portion which remains firmly embedded in the mem­ brane, and only its 68,000 M.W. hydrophilic portion may be shed or secreted. Some T cell products of higher M.W. (70,000-200,000 M.W.) (63, 88, 90) may be more hydrophilic than others and therefore more readily released from the membrane in an intact form. By contrast, smaller size molecules of 20,000-50,000 M.W. (56,68, 89) could be cleavage products of larger M.W. molecules that, due to their tertiary structure and/or method of preparation, are more susceptible to proteolysis. It is likely that further structural analyses of this great variety of T cell-derived molecules will reveal them to be of a similar molecular size. We have shown that AEF is, as previously anticipated (28, 48), a multi­ component factor. Several AEF components such as IL-2 and possibly TRF, and perhaps even others, seem to require I-region restricted interac­ tions for their production. However, they each act as a hormone-like molecule in a nonrestricted and antigen-nonspecific manner, as previously reported (52-54). Thus, once antigen and Ia specificity is achieved, it is left to these growth-promoting molecules to propagate an immune response. In summary, continued comparative biological and biochemical analyses of the various components of AEF, in particular component I, and the other T cell-derived antigen-binding molecules discussed above, should eventu­ ally determine the structural relationship between an alloantigen receptor and an antigen receptor. It remains to equate the in vitro activity of such

The Biological and Biochemical Basis . 79

putative lymphocyte receptors with their physiological role in vivo. Only then will it be possible to surmount the formidable task of delineating the molecular mechanisms of lymphocyte communication. Acknowledgments We especially thank all of our colleagues, in particular, Drs. J. F. HARRIS, R. G. MILLER and R. GORCZYNSKI, who have provided valuable advice and have made significant contributions to our studies presented in this review. We also thank Ms. R. BA'ITISTELLA, J. ROBERTS and K. KAUFMAN for their excellent technical assistance with various aspects of this work, and Ms. K. LOUSTE for her expert devotion in the maintenance and care of our mouse colony. Weare also grateful to Ms. D. CRAWFORD for her cheerful assistance with the preparation of this manuscript.

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