Impact of MHC Class I Gene on Resistance to Murine AIDS

Development of murine AIDS in mice following infection with LP-BM5 murine leukaemia virus (MuLV) is highly strain dependent, with strain differences determind by genes within and outside H-2. Among H-2 genes, the D^d gene is the most closely associated with resistance to LP-BM5 MuLV infection. However, the D^d-mediated resistance is highly influenced by outside H-2 genes, i. e. A lineage strains are more resistant than mice strains of B6/B10 lineage. In this study, the mice having BALB background were analysed and, similarly to A lineage mice, only D^d gene products were found to be required to provide resistance to LP-BM5 MuLV infection. Furthermore, BALB/c Kh mice bearing both D^d and L^d genes clearly showed obviously higher resistance than BALB/c-H-2^dm2 mice solely having the D^d gene. In addition, in the long-term observation of the effect of the D^d gene on B6/B10 background mice, D8 mice having the D^d gene as a transgene and expressing a high level D^d gene product showed higher resistance than naturally recombinant B10. A(18R) mice. These results suggest that the MAIDS resistance associated with the D end loci is dependent on the level of expression of an MHC class I gene.     

Comparison between the Specificity of Primary and Secondary Killer Cells against Alloantigens and Hapten-modified Syngeneic Lymphoid Cells

Cytotoxic responses of lymphoid cells from different mouse strains against syngeneic cells modified with the haptens fluorescein isothiocyanate and trinitrophenyl were investigated. Mice of the H-2^k strain demonstrated strong primary in vitro hapten-specific cytotoxicity reactions, which were H-2 restricted and involved the K^k specificity. However, cells from H-2^d and H-2^b mice developed hapten-specific cytotoxic reactions that showed H-2 cross-reactivity. This cross-reactivity, with regard to the restriction element, was particularly evident with cells from mice that had been immunized in vivo. No cross-reaction was observed between the two haptens, however. Genetic mapping experiments demonstrated that cross-reactions existed between D^b and D^d target cell antigens. Similar cross-reactions were demonstrated in in vitro experiments in which secondary in vitro responses were induced by stimulation with cross-reacting H-2 antigens. This finding was also investigated in allogeneic cytotoxicity. In vitro induced responses resulting in relatively weak specific cell-mediated lympholysis reactions were H-2 specific, whereas secondary in vitro responses demonstrated cross-reactivity between D^d and D^b antigens. In these test systems, cross-stimulation was also demonstrated in secondary in vitro responses. These results are discussed in terms of similarities of T cell recognition of hapten-modified self antigens and of alloantigens.     

Failure of a tumour to prime CTL specific for some of the minor H antigens it expresses but not others

The H-Y-expressing murine tumour, ET-5, specifically immunizes B6 female mice that have rejected it against H-Y-positive male skin grafts, yet fails to prime their spleen cells for the generation of H-Y-specific cytotoxic T lymphocytes (CTL). ET-5 also fails to prime relevant congenic hosts to generate CTL specific for H-3 or H-25 minor H antigens, or major histocompatibility complex (MHC) class I antigens, all of which are expressed in immunogenic form by ET-5. Nonetheless, C3H.SW mice, which are MHC-compatible with B6 mice in which ET-5 originated, but differ from B6 at many minor H loci, can be primed to generate CTL directed against one or more unidentified minor H antigens. These CTL are conventional MHC-restricted, CD8+ T cells, and require priming in vivo for their generation. Significantly, C3H.SW females can be primed by ET-5 to generate B6-specific CTL, but not H-Y-specific CTL. Thus CTL priming is selective in the sense that ET-5 primes CTL specific for some of the antigens it expresses but not others. The basis for this selectivity is not known but, in the case of H-Y antigen, it appears not to result from an inability of ET-5 to express either H-2Db-encoded antigens, the restriction element for H-Y-specific CTL in B6 females, or H-Y itself in vivo.     

Private specificity of H-2Ldx molecule detected serologically by a surface antigen redistribution method (capping)

The presence of H-2·1 positive-H-2·63 negative molecules, H-2L^dx, in the products of the ^dx region was originally detected in H-2^dx inbred strains GRS/A and LIS/A (Snoek et al. 1979).
This study confirms the existence of H-2L^dx molecules in 2 congeneic strains with a H-2^dx haplotype on C3H (C3H·LG strain) and B10 (B10·GR strain) background. Besides anti-H-2·1-like antibodies present in anti-D^dx allo-antiserum, monoclonal antibody d anti- k (H100-5/28, H-2·m3) was shown to react with H-2L^dx molecule. Furthermore, evidence is shown that H-2L^dx molecule has a unique specificity, which is detected serologically in a capping experiment.     

Complexity of minor histocompatibility loci

Allografts can be rejected as a result of major histocompatibility antigen disparity or as a result of differences at any of a number of minor histocompatibility antigens. In many cases, rejection due to multiple minor histoincompatibility is as difficult to control as that induced by major histoincompatibility. Although an understanding of the molecular, biochemical, and functional parameters of the major histocompatibility loci and their products is increasing at an exponential rate, little is known about these same facets of minor histocompatibility loci and their products. It is generally accepted that minor histocompatibility loci in the murine model have a degree of polymorphism similar to that of H-2K or H-2D. This conclusion was based on typing alleles by the classic F1-skin graft test. Based on these allelic assignments, numerous unexpected findings of CTL specificity were made. Therefore, a systematic analysis was made comparing CTL specificity, F1-complementation, and allograft rejection. Based on these three parameters, the data presented using strains of mice that were bred to, and therefore presumed to, differ only at H-3 indicate that the antigen disparity of these congenic strains and the parental B10 strain as defined by CTL specificity and skin graft rejection is much more complex than originally described. One especially interesting chromosomal region is H-3/beta 2-microglobulin in the fifth linkage group of chromosome 2. Using CTL, ten specificities are defined, three of which appear to be specific for beta 2-microglobulin-A, -B, and -C. These findings raise the question of whether any minor histocompatibility locus is polymorphic or is instead a composite of multiple minor H-loci which are masquerading as a single locus.     

A novel H-2s class I molecule expressed on a B-cell leukemia from SJL/J mice

Anti-H-2.33 [(B10.D2 X A)F1 anti-B10.A(5R)], which predominantly contains antibodies recognizing H-2Kb and IAb molecules, was found to be cytotoxic against DMLM 1678, a B-cell leukemia of SJL/J (H-2s) origin. The antiserum precipitated a typical class I (H-2-like) molecule from labeled tumor cell preparations as judged by molecular mass, papain susceptibility and association with beta 2-microglobulin. Sequential immunoprecipitation studies revealed that it was distinct from either H-2Ks or H-2Ds, the 2 molecules expressing the private antigens of the H-2s haplotype. Absorption analysis using congenic mice mapped the gene controlling the expression of the novel molecule telomeric to the S-region within the major histocompatibility complex.     

Herpes simplex virus-specific cytolytic T lymphocytes restricted to a normally low responder H-2 allele are protective in vivo

The herpes simplex virus (HSV)-specific cytolytic T lymphocyte (CTL) response in C57BL/6 (B6, H-2b) mice is restricted exclusively to the class I major histocompatibility complex (MHC) glycoprotein H-2Kb, with no detectable response restricted by H-2Db. However, analysis of the memory CTL population derived from B10.A(2R) (Kk Db) mice indicated that Db was a functional restriction element, and that failure to detect this subpopulation in B6 mice was not caused by the inability of HSV-derived antigens to associate with this self protein. Two long-term polyclonal CTL lines, one generated from B6 mice restricted by Kb and a second generated from 2R mice restricted by Db, were greater than 99% CD8+ and exhibited no natural killer (NK) cell activity. The adoptive transfer of either CTL line to naive recipients prior to infection in the hind footpads with HSV resulted in reduced levels of virus recovered from footpad tissue during the acute phase of infection and a reduction in latent HSV able to be reactivated from the sensory dorsal root ganglia (DRG) during the latent phase of infection. These results demonstrated not only that HSV antigen(s) may associate with Db, allowing restricted recognition controlled by this H-2 gene product, but also that functional Db-restricted CTL have the potential to exert biological activity in an environment in which they are not normally generated.     

Induction of CD8+ CTL Recognizing Mycobacterial Peptides

Mycobacterium tuberculosis is the single, most important cause of morbidity attributable to a single infectious organism. CD8⁺ T cells play an important role in anti-tuberculous immune responses in both mice and humans. Data concerning the identity of mycobacterial antigens recognized by CD8⁺ T cells is limited; consequently, few CTL epitopes have been characterized. The authors identified allele-specific (H-2^{b and d}) MHC class I binding motifs in six prominent M. tuberculosis protein antigens (the 19 and 38 kDa lipoglycoproteins and the 10, 16, 65 and 70 kDa stress proteins). These predicted epitopes were tested for MHC binding as well as their ability to elicit peptide-specific CTL following in vivo priming. The authors were able to identify eight previously undescribed mycobacterial CTL epitopes by using spleen cells from peptide-immunized mice. In addition, CTL specific for at least one of these epitopes also recognized the naturally processed epitope presented on transfected EL4 target cells. These mycobacteria-derived CTL epitopes could be important for future analysis of the involvement of CD8⁺ T cells in M. tuberculosis infection, pathogenesis and vaccine development.     

Localization of two cytotoxic T lymphocyte epitopes and three anchoring residues on a single nonameric peptide that binds to H-2Ld and is recognized by cytotoxic T lymphocytes against mouse tumor P815

Mouse mastocytoma P815 expresses tumor antigens P815A and P815B encoded by a single gene called P1A and carried by a single peptide named P1A 35–43 (NH₂-Leu-Pro-Tyr-Leu-Gly-Trp-Leu-Val-Phe-COOH). P1A 35–43 is presented to anti-P815A and anti-P815B cytotoxic T lymphocytes (CTL) by major histo-compatibility complex (MHC) H-2L^d molecules. In order to determine the individual role played by each amino acid residue of P1A 35-43 in binding to H-2L^d and in recognition by anti-A and anti-B T cell receptors (TcR), a series of P1A 35-43 peptides substituted by alanine at single positions was synthesized and tested for binding to H-2L^d and for CTL recognition. Binding to H-2L^d was estimated by measuring the ability of the peptide to up-regulate cell surface expression of H-2L^d. We found that three residues were important for interaction of P1A 35-43 with H-2L^d. Two of them, Pro at position 2 and Phe at position 9 were consistent with the described H-2L^d binding motif. A third residue, Trp at position 6, was also required for effective MHC binding of the tumor antigen. CTL sensitization assays showed that alanine substitution at position 7 (Leu) or at position 8 (Val) dramatically affected peptide recognition by anti-A CTL while positions 3 (Tyr) and 4 (Leu) were critical for recognition by anti-B CTL. We conclude that Pro², Trp⁶ and Phe⁹ constitute the anchor residues of P1A 35-43 to H-2L^d, whereas the dipeptidyl sequences Tyr³-Leu⁴ and Leu⁷-Val⁸ form the core epitopes recognized by the TcR of anti-P815B and anti-P815A CTL, respectively.     

Rabbit Antisera with Specificity for Isolated Mouse T-Lymphocyte Receptors

Rabbit antibodies obtained after Immunization of mouse Immunoglobulin (Mig)-tolerant rabbits with B6 anti-CBA IgG and having specificity for B6 anti-CBA IgG and T-cell receptors (antiserum 5936) were used to isolate 5936-reactive molecules from B6 anti-CBA mixed lymphocyte culture supernatants. Such 5936-reactive molecules were produced by the B6 T cells, and they did not react with rabbit anti-MIg antisera. They had a mol. wt of 50,000–75,000, and were single-chain polypeptides that did not react with concanavalin A (Con A) Sepharose. These molecules were in turn injected into rabbits, and the antisera thus obtained had the following characteristics: (1) they reacted against B6 anti-CBA T-cell receptor material but not against B6 anti-CBA IgG; (2) they reacted with about 35% of B6 (H-2^b. Ig-1^b) anti-CBA T cells, 25% of B6 Con A blasts and 0 10%; of normal B6 T cells but not with B6 lipopolysaccharide (LPS) blasts, C3H.B10 (H-2^b, Ig-I) anti CBA or CBA anti-B6 T cells, CBA Con A blasts or normal CBA T cells: and (3) they reacted with the same 50,000–75.000 mol. wt, T-cell-derived molecules as did antiserum 5936. The implications of these findings are discussed in relation to the nature of T-cell receptors.     

Cytotoxic T lymphocytes generated across and I-Ab mutant difference are directed against a molecule bearing Ia antigens

The recently discovered B6.C-H-2bm12 (bm12) histocompatibility mutant bears a mutation of the I-A subregion of the H-2b haplotype. We have studied cytotoxic T lymphocytes (CTL) generated in secondary mixed lymphocyte culture (MLC) between the bm12 mutant and the strain of origin C57BL/6 (B6). In both directions, strong CTL responses were generated against lipopolysaccharide-stimulated, but not against concanavalin A-stimulated target cells. Studies of the specificity of the bm12 anti-B6 CTL response, using target cells from a panel of H-2 recombinants, showed that the CTL were directed against specificities of the I-Ab subregion. This conclusion was confirmed by the observation that the bm12 anti-B6 CTL could be specifically blocked by anti-Iab, but not by anti-Kb antisera. EL4 tumor cells, which express Kb and Db but not Iab antigens, were not lysed by the CTL. The combined data confirm that bm12 is an I-A subregion mutant and indicate that the I-A subregion antigens, recognized by antibody and those recognized by CTL, are probably present on the same molecules. The CTL effector cells generated in secondary MLC across the I-Ab mutant difference had the surface phenotype Lyt 1- 2+.     

Dendritic Cells Engineered to Express Defined Allo-HLA Peptide Complexes Induce Antigen-specific Cytotoxic T Cells Efficiently Killing Tumour Cells

Most tumour-associated antigens (TAA) are non-mutated self-antigens. The peripheral T cell repertoire is devoid of high-avidity TAA-specific cytotoxic T lymphocytes (CTL) due to self-tolerance. As tolerance is major histocompatibility complex-restricted, T cells may be immunized against TAA presented by a non-self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo-restricted CTL of high-avidity and low cross-reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA-A*0201, efficiently present externally loaded peptides from the antigen, Melan-A/MART-1 to T cells from HLA-A*0201-negative donors. CD8⁺ T cells binding HLA-A*0201/MART-1 pentamers were detected already after 12 days of co-culture in 11/11 donors. The majority of cells from pentamer⁺ cell lines were CTL and efficiently killed HLA-A*0201⁺ melanoma cells, whilst sparing HLA-A*0201⁺ B-cells. Allo-restricted CTL specific for peptides from the leukaemia-associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo-HLA peptide complexes are highly efficient in generating CTL specifically reacting with tumour-associated antigens.     

Adoptive transfer of unresponsiveness to allogeneic skin grafts with hepatic gamma delta + T cells.

C3H/HEJ mice injected with irradiated multiple minor incompatible B10.BR lymphoid cells via the portal vein showed delayed rejection of subsequent B10.BR skin grafts. Similar delayed rejection was produced by lateral tail vein injection of B10.BR hepatic mononuclear cells or H-2k cells pulsed in vivo with B10 minor histocompatibility antigens. Inhibition of C3H anti-B10.BR immunity in vivo (assessed by delayed graft rejection) and in vitro (assessed by B10.BR-induced lymphokine production) can be transferred by radioresistant, plastic-adherent F4/80+33D1-CD4-CD8-alpha beta TcR-gamma delta TcR- mononuclear hepatic cells from (C3H/HEJ x C3H.SW)F1 mice injected 36 hr earlier with 100 x 10(6) irradiated spleen cells. By 10 days post-injection, cells transferring delayed rejection are radiosensitive, plastic non-adherent, F4/80-33D1-CD4-CD8- alpha beta Tc+- gamma delta TcR+ cells. Injection of interleukin-2 (IL-2) in vivo into mice receiving pretreatment with B10.BR cells via the portal vein, or adoptive transfer into such mice of immune anti-B10.BR lymphoid cells, abolished delayed rejection on subsequent skin grafting. Delayed rejection or modulation of lymphokine production was associated in all cases with suppression of IL-2 production and preferential retention of IL-4 production from cells stimulated in vitro.     

Expression of the H-2 Antigenic Complex on Murine Haematopoietic Stem Cells

We used hybridoma-derived monoclonal antibody to H-2K^k antigens and xenoantibodies to β₂-microglobulin (β₂) to study the expression of the H-2 antigenic molecular complex on murine hematopoietic stem cells and the effect of long-term culture in vitro on the expression of these antigens. Monoclonal anti-H-2K^k antibody produced potent complement-dependent inhibition of pluripotent (CFU-S), myeloid (CFU-C), and erythroid (CFU-E) stem cells from the bone marrow of C3H and AKR (H-2^k) mice and was without effect on stem cells from Balb/C (H-2^d) mice. Anti-β₂m xenoantibodies inhibited stem cells from all three strains. Stem cells could not be 'rescued' from the inhibitory effects of the antibodies by the addition of thymocytes to marrow cells after antibody treatment. Both the anti-H-2K^k monoclonal antibody and the anti-β₂m xenoantibodies produced potent inhibition of AKR (H-2^k) CFU-C that had been maintained in culture for up to 6 weeks. These results indicate that murine CFU-S, CFU-C, and CFU-E express H-2 antigens and that the expression of these antigens by CFU-C is not altered during long-term culture.     

Molecular specificity of a monoclonal anti-Ik alloantibody identifying a highly conserved determinant on mouse I-A, I-E and human DR antigens

We have investigated, using serological and biochemical assays, the specificity of an A.TH anti-A.TL-derived monoclonal antibody (mAb), designated 40.B, directed at a highly conserved antigenic determinant expressed on the majority of murine and human MHC class II antigens. In the mouse, mAb 40.B defines a new specificity expressed on the Ia products of the H-2 haplotypes k, d, b, v, r, p, u, j and w3. Analysis of its reactivity with H-2 recombinant strains and the results of the competitive binding inhibition of ¹²⁵I-labeled mAb 40.B to B10.BR cells with I-A^k and I-E^k specific mAb suggested recognition of a shared Ia determinant expressed on both I-A^k and I-E^k molecules. This has been confirmed by sequential immunoprecipitation studies which demonstrated the specificity of mAb 40.B for the A_β^k A_α^k, A_β^b A_α^b, A_β^d A_α^d, E_β^k E_α^k, E_β^s E_β^k, and E_β^d E_α^d dimers. In humans, this mAb bound to and immunoprecipitated HLA-D/DR molecules expressed on lymphoblastoid cell lines carrying the MTl and MT2 supertypic specificities. The possible implications of these findings with regard to an evolutionary model of MHC class II antigens are discussed.     

Role of major and minor histocompatibility antigens in the suppression of alloreactive cytotoxic responses induced by alloantigen pretreatment.

We have recently shown that priming mice with allogeneic strain A spleen cells before immunization with (A x B)F1 spleen cells strongly suppresses the cytotoxic T-lymphocyte (CTL) response directed against linked strain B alloantigens. This specific decrease in the CTL responses against the second immunizing alloantigen is associated with a high CTL response against the first priming alloantigen. The suppression of CTL responses against the strain B alloantigens is, however, not due to killing of F1 spleen cells by anti-A CTL, since it was observed after immunization of primed mice with a mixture of (A x B)F1 and B cells. In the present study, attempts were made to determine the relative contribution of H-2 and minor histocompatibility background antigens towards induction of suppression. Our results demonstrate that priming and immunizing spleen cells have only to share H-2 antigens in order to induce a downregulation of CTL responses directed against the linked alloantigens. This indicates that immunity against H-2 antigens is sufficient to induce suppression. However, priming against minor histocompatibility antigens also induces suppression, but only if spleen cells used for priming and immunization share H-2 antigens with the recipient strain. Therefore, the suppression can be induced by priming with non-H-2 antigens but is H-2-restricted. This study has also demonstrated that suppression can be induced by intraperitoneal or subcutaneous administration of allogeneic cells.     

The Localized Primary Cytotoxic T-cell Response to Cells Expressing Minor Histocompatibility Differences

Cytotoxic T lymphocytes (CTL) are able to eliminate P815 (DBA/2) mastocytoma cells growing in cerebrospinal fluid of BALB/c H-2-compatible but minor histocompatibility (H) antigen-different mice and in H-2-incompatible C3H/He mice. We examined the magnitude of the primary CTL response to multiple, minor H antigens and to determinants of the major histocompatibility complex (MHC) by using a direct cytolytic assay and limiting-dilution analysis to estimate CTL frequency. By these criteria, no obvious differences emerged, and the responses appeared comparable at the site of inflammatory process, despite differences in the number of clonal progenitors. Experiments with radiation chimeras showed evidence of a strong cytotoxic T-cell response against P815 cells in [(ddd X bbb)F1----ddd] and (F1----bbd), but not in (F1----bbb) radiation chimeras. Therefore, this cytotoxic T-cell response against minor H antigens obeys the postulated rules for thymic restriction of precursors. Compatibility at the H-2 D-end of the MHC is apparently sufficient to ensure a strong response.     

Definition of alien H-2 determinants on a Friend leukaemia by analysis of alloreactivity of CTL from primary MLC

A Friend virus-induced tumour of BALB/c (H-2d) origin, HFL/d, was examined for the expression of alien H-2 antigens. The alloantigens on HFL/d were typed by generating CTL in primary MLC with HFL/d as stimulator and measuring reactivity to targets with known H-2 antigens, and confirmed by assessing recognition of HFL/d targets by CTL generated in primary MLC with stimulators expressing known H-2 antigens. Potential cross-reactivities between alloantigens were analysed by cold-target inhibition experiments. BALB/c cells stimulated with HFL/d lysed H-2b targets, and BALB/c anti-H-2b CTL lysed HFL/d; analysis with recombinant haplotypes demonstrated both H-2Kb and H-2Db alien antigens antigens on HFL/d. C57BL/6 (H-2b) cells stimulated with HFL/D recognized H-2Kd, H-2Dd, and an additional determinant unique to HFL/d. (BALB/c x B6)F1 cells also recognised a unique HFL/d determinant not of H-2b or H-2d origin. These unique determinants, which induced a strong cytotoxic response in primary MLC, were not shared by BALB/c or B6 tumours induced by cross-reactive FMR viruses. Thus, HFL/d expressed the K and D antigens of its strain of origin, two typed alien H-2 antigens, and at lest one other untyped antigen which may represent an additional H-2 determinant. These studies further demonstrate the utility of examining the reactivity of CTL generated in primary MLC to probe for the presence of alien H-2 antigens.     

Immunodominant deters the response to other tumor antigens thereby favoring escape: prevention by vaccination with tumor variants selected with cloned cytolytic T cells in vitro

Variant cancer cells which arise from the parent tumor during tumor progression can escape immunity but retain antigens. We have mixed highly immunogenic (A⁺B⁺) murine parental cancer cells with less immunogenic (A^-B⁺) variant cancer cells to construct a model of a cancer containing escape variants. When such mixtures of cancer cells were injected into normal mice, the variant cells grew out because immune responsiveness to the B antigen on the variant was hindered by dominance of the A antigen on the surrounding parental tumor cells. However, A^-B⁺ variant cells inoculated alone at a separate site induced B specific cytolytic T cells and were rejected. Moreover, mice immunized with A^-B⁺ cells rejected a challenge which contained a mixture of variant and parental cancer cells, while immunization with A⁺B⁺ cells was ineffective. Thus, variant tumor cells selected from parental tumor cells by cytolytic T cells in vitro can be used to induce protective immunity against variants expected to escape tumor immunity in vivo. The immunodominance of the A antigen may be related to its ability to induce a much more rapid CTL response than the B antigen, since we show in another model that the preexistence of a CTL response to one antigen prevented the subsequent induction of CTL to another antigen injected at the same site, even if both antigens were equally efficient at inducing CTL. These results indicate that immunodominance can affect strong as well as weak antigens. Vaccination with individual antigens at separate sites rather than with multiple antigens at one site may, therefore, be needed to prevent tumor escape and tumor recurrence or to counteract infectious diseases.     

Induction of Cell-Mediated Cytotoxic Self Responses by Epidermal Cells Modified with a Haptenic Sulphydryl Reagent

Murine epidermal cells (EC) act as stimulator cells in the generation of ullogeneic cyto-toxic T lymphocytes (CTL) in cell-mediated lympholysis (CML) and are suitable targets for allogeneic and hapten-self CTL. To analyse the role of EC in the generation of and recognition by ami-self CTL, syngeneic hapten-modified murine EC were used as in vivo and in vitro stimulating populations and as target cells in a hapten-self CML system. Epidermal cells were modified with the sulphydryl-reactive haptenic reagent N-iodoacetyl-N'-(5-sulphonic-l-naphthyl)ethylenediamine (I-AED). C3H.SW (H-2^b) AED-self CTL responses were generated by stimulation with syngeneic AED-modified EC and were readily demonstrated when tested on syngeneic hapten-modified EC. These CML responses were hapten-specific and H-2-restricted. No substantial difference was detected in the ability of AED-modified EC and spleen cells (SC) to stimulate the generation of secondary AED-self CTL. Cold target inhibition experiments with hapten-modified EC and SC blockers did not reveal tissue-specific recognition of hapten-modified EC or SC targets by AED-self CTL. These findings demonstrate that hapten-modified EC, when used for priming in vivo and subsequently for in vitro sensitization, can induce hapten-specific self CTL that are reactive against syngeneic hapten-modified EC.