Differential responsiveness to MIs locus antigens in graft-versus-host and host-versus-graft reactions

After (semi)allogeneic transplantation of lymphoid cells into lethally irradiated mice, the development of anti-host directed T effector cells can be demonstrated by means of a simple delayed-type hypersensitivity (DTH) assay. Using this assay we have shown that in H-2 compatible combinations Mls locus antigens can induce the generation of such T effector cells during a graft-versus-host (GvH) reaction. Other non-H-2 alloantigens are probably of minor importance. The capacity of Mls locus antigens to induce distinct anti-host DTH reactivity correlated with the capacity to induce a one-way mixed lymphocyte culture (MLC) response. Mls^a and Mls^c locus antigens initiated a positive MLC response as well as distinct GvH-related DTH reactivity. On the other hand, in the combination DBA/2 versus (BALB/c × DBA/2) F₁, the Mls^b locus antigen was not able to initiate in vitro proliferation, a lack of response which coincided with a marginal and short-lasting GvH-related DTH reactivity. In contrast, the host-versus-graft (HvG) DTH reaction of BALB/c and DBA/2 mice to subcutaneously injected (BALB/c × DBA/2) F₁ spleen cells was equally strong. Here antigens other than those coded for by the Mls locus were mainly responsible for the antigraft DTH response. These results suggest that T effector cells generated in GvH and HvG reactions are specific for largely different sets of minor histocompatibility antigens, with a selective stimulation by Mls locus antigens under GvH conditions.     

Suppressor T cell recognition of major and minor histocompatibility alloantigens: selected suppression of MHC-directed responses by minor alloantigen Ts

The induction of suppression by i.v. administered alloantigens in the murine host was analyzed as a model of the possible effects of blood transfusion on transplant survival. The results indicated that suppressor T cells (Ts) specific for minor histocompatibility alloantigens could be readily induced by the i.v. presentation of minor alloantigen-disparate spleen cells. In contrast, similar priming with cells differing solely at the H-2 major histocompatibility complex stimulated only positive T cell immunity, with no evidence of suppression. The induction of H-2 directed Ts activity could be accomplished only by i.v. priming with major plus minor incompatible donor cells, suggesting that suppressor cell recognition of minor alloantigens may have facilitated the generation of Ts against H-2-encoded major transplantation antigens. A role for minor histocompatibility antigens in the regulation of H-2-specific immunity at the effector level was also indicated. Ts induced by i.v. pretreatment with minor antigen-disparate donor cells not only suppressed the delayed-type hypersensitivity (DTH) response to the relevant minor alloantigens, but also inhibited DTH against unrelated H-2 alloantigens introduced during subsequent intradermal immunization. Suppression of H-2-directed T cell reactivity was specific in that the presence of the Ts-inducing minor alloantigens was also required and occurred only when the minor and unrelated major alloantigens were presented within the same inoculum, if not on the same cell surface. The capacity of Lyt-2+Ts or Ts-derived suppressive factors specific for one set of cell surface molecules to modulate responses to an unrelated group of surface antigens does not appear to represent a general phenomenon, because similar suppression of immunity to unrelated tumor-specific transplantation antigens by minor-specific Ts was not observed. These results are discussed with respect to the possible mechanism of H-2-directed suppression and the role of the I region in Ts recognition of antigen.     

Suppression of delayed-type hypersensitivity to third party "bystander" alloantigens by antigen-specific suppressor T cells

Delayed-type hypersensitivity (DTH) against alloantigens can be induced by sc immunization with allogeneic cells. The induction of DTH can be suppressed by iv preimmunization of the mice with similar allogeneic spleen cells, provided the cells are irradiated before injection. This suppression is mediated by T cells. The suppressor activity can be induced not only by H-2-and non-H-2-coded antigens, but also by H-2 subregion-coded antigens. Suppression induced by K, I, or D subregion-coded antigens is specific for that particular subregion as well as for its haplotype. I-J-coded alloantigens were found to not be necessary for the induction of antigen-specific suppressor T cells. After restimulation of suppressor T cells by the "specific" alloantigens, the DTH to simultaneously administered third-party alloantigens becomes suppressed as well. This nonspecific suppression of DTH to third party "bystander" alloantigens also occurs when the specific and the third-party antigens are presented on separate cells, provided that both cell types are administered together at the same site. The simultaneous presentation of both sets of alloantigens during the induction phase of DTH only is sufficient to prevent the normal development of DTH to the third-party antigens.     

Synergism of T lymphocyte subsets in the response to Mls-locus coded antigens during graft-versus-host reaction

After transplantation of lymphoid cells into lethally irradiated (semi)allogeneic mice specific anti-host directed effector T cells are generated. This can be demonstrated using a delayed type hypersensitivity (DTH) assay. In H-2 compatible combinations, Mls-locus antigens, but no other minor histocompatibility antigens, can induce the generation of such effector T cells. This paper shows that maximal anti-host DTH responses are obtained when the lymphoid cells transplanted constitute of a mixture of long-lived, recirculating T2 cells and short-lived, sessile T1 cells. It was demonstrated that anti-Mls locus-directed DTH effector T cells are the progeny of T2 cells, and that T1 cells amplify this response. The latter, however, are by themselves incapable of displaying anti-Mls DTH reactivity. The T1 cells were found to be of the Lyt-1+2+ phenotype, and the T2 cells of the Lyt-1+2- phenotype. The same Lyt phenotypes were found for T1 and T2 cells synergizing in the GvH reaction against H-2 alloantigens.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. III. Single histocompatibility antigens dominate the male antigen

Immunization of mice with multiple non-H-2 histocompatibility antigens results in the generation of cytolytic T lymphocytes that are specific for a limited number of immunodominant antigens. The experiments presented in this communication were designed to reveal immunodominance in pairwise combinations of autosomal and sex-linked non-H-2 histocompatibility (H) antigens. Priming and boosting responders with the male antigen, H-Y, paired with the H-4.2, H-7.1, or H-3.1 antigens, resulted in the generation of cytolytic T cells specific for the autosomal H antigens but not the H-Y antigen. Furthermore, co-immunization and boosting of C57BL/6 female responder spleen cells with BALB.B male cells resulted in the generation of cytolytic T cells specific for the BALB.B immunodominant antigens but not H-Y. No dominance was observed in H-4-plus H-7-incompatible combinations. Co-immunization of three different H-3 congenic strains with H-3.1 plus H-Y demonstrated that an efficient anti-H-3.1 T cell response is required for observing H-3.1 immunodominance over H-Y. Co-expression of H-3.1 and H-Y on the same priming and boosting cells was required for immunodominance. In fact, immunization with H-3.1 and H-Y presented on different cells resulted in normal generation of H-Y-specific cytolytic T cells, but no generation of H-3.1-specific cytolytic T cells resulted unless H-Y-specific cells were stimulated in the mixed lymphocyte cultures. These observations suggest that in vitro T cell responses to paired, non-H-2 H antigens may be independent, competitive, or synergistic, depending on the identity of the antigens and the priming and boosting conditions.     

Restriction in adoptive transfer of resistance to Listeria monocytogenes. I. Influence of non-H-2 loci

In vivo adoptive transfer of T-cell-mediated immunity to the facultative intracellular bacterium Listeria monocytogenes is restricted, not only by the H-2 haplotype of the mice, but also by incompatibilities at non-H-2 loci. Thus, transfer between H-2 identical strains of mice with different background genes was reproducibly and significantly less efficient than transfer between completely syngeneic mice, although the restriction was less marked than that across the H-2 barrier. Restriction also occurred when parental cells were injected into semisyngeneic F1 hybrids and when cells from F1 hybrids were injected into parental strains. Using congenic strains of mice differing only at defined minor histocompatibility antigens, it was found that, of those loci available for study, antigens arising from the H-4 and H-8 loci strongly restricted transfer, whereas those specified by H-1, H-3, and H-7 did not.     

The role of H-2 and non-H-2 antigens and genes in the rejection of murine cardiac allografts

Genes of the major histocompatibility complex (MHC) in the mouse (H-2 complex) have been shown to be an important factor in determining the immune responsiveness of various strains of mice to isolated antigens (e. g., lysozyme). The role of MHC genes in controlling the responsiveness of mice to multiple alloantigens is less well-defined, and although non-MHC genes have been shown to be important in determining responsiveness in some systems (e. g., haptens), they have not been demonstrated as yet to influence the rejection of vascularized organ allografts. In this study, the responsiveness of mice to vascularized cardiac allografts transplanted across well-defined major (H-2) and minor (non-H-2) histocompatibility barriers was investigated using congenic mice in 32 different donor/recipient combinations. The results show that both H-2 and non-H-2 gene products can act as target alloantigens for rejection. At the responder level, they may interact to effect responsiveness of a recipient strain to multiple alloantigens. In no case in this study has any one gene or group of genes been found to confer universal high or low responder status.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.     

Suppression of antigraft immunity by preimmunization. III. Characterization of suppressor T cells involved in suppression of the efferent phase of DTH against alloantigens

Suppressor T (Ts) cells that can suppress delayed type hypersensitivity (DTH) against histocompatibility (H) antigens can be isolated from spleen and lymph nodes a few days after i.v. immunization of mice with irradiated allogeneic spleen cells. In this paper we investigated the suppression of the efferent phase of DTH to characterize the Ts cells involved, and to compare them with the afferent phase Ts cells that have been characterized in a previous paper of this series. The DTH against third party alloantigens that were not used for the i.v. suppressive immunization could be suppressed by presenting the third party alloantigens together with the original alloantigens in the challenge inoculum for eliciting the DTH reaction. Thus the ultimate suppressive effect by the Ts cells that are active during the efferent phase of DTH is nonspecific. This non-specific suppression of DTH to alloantigens has previously been found for the afferent phase Ts cells as well. For suppression of the efferent phase of DTH to alloantigens, a population of Lyt-1+2+ Ts cells appeared to be essential, just like in the suppression of the afferent phase of DTH to alloantigens. We did not find evidence for the involvement of cyclophosphamide-sensitive auxiliary Ts cells in suppression of the efferent phase of DTH. Also no evidence was found for H-2 or Igh-restricted activation and function of the Ts cells that were active during afferent and efferent phases of the DTH response to H antigens. In view of these similarities between afferent phase and efferent phase Ts cells we conclude that there are no arguments as yet to suppose that there is more than one type of T cells involved in the suppression of the afferent and efferent limb of DTH against H antigens.     

Primary and secondary delayed-type hypersensitivity to minor histocompatibility antigens in the mouse.

Immunization of mice with viable allogeneic H-2-compatible spleen cells can induce a persistent state of delayed-type hypersensitivity (DTH) to these alloantigens, as measured with the footpad swelling test. Boosting of such mice, 2–4 months after priming, induced a typical secondary-type DTH reactivity. The capacity of secondary DTH to non-H-2 alloantigens could be adoptively transferred from primed mice into irradiated syngeneic hosts by means of nylon wool-nonadherent, Thy-1.2⁺ spleen cells. Vinblastine treatment of the donor mice did not affect the adoptive DTH responsiveness. These results suggest that a population of long-lived T memory cells contributes to secondary-type DTH responsiveness to non-H-2 alloantigens. The phenomenon of persistent DTH is discussed in the light of these results. The hypothesis is put forward that persistent DTH is dependent on the continuous antigen-driven differentiation of long-lived, recirculating T memory cells into nonrecirculating, functionally short-lived DTH effector cells.     

T lymphocyte responses to non-H-2 histocompatibility antigens. I. Role of Ly-1+2+ T cells as cytotoxic effectors and requirement for Ly-1+2+ T cells for optimal generation of cytotoxic effectors

Cytotoxic effector T cells specific for non-H-2 histocompatibility (H) antigens were examined for phenotypic expression of lymphocyte differentiation (Ly) antigens. Virtually all H-Y-specific cytotoxic effectors generated in mixed lymphocyte culture were Ly-1+2+ T cells. H-3-specific effectors comprised both Ly-1+2+ and Ly-1-2+ T cells. However, cytotoxic effectors specific for multiple non-H-2 H antigens were predominantly Ly-1-2+ T cells. The optimal generation of H-Y- and H-3-specific effectors required Ly-1+2+ T cells; optimal generation of multiple non-H-2 H antigen-specific effectors required an interaction between Ly-1+2- and Ly-1-2+ T cells. These observations suggest that the identity of the target H antigen in part determines the Ly type of responsive T cells. Our observations suggest that 2 alternative pathways of T cell response exist for non-H-2 H antigens. The first pathway involves an interaction between Ly-1+2- helper T cells and Ly-1-2+ cytotoxic effector precursors. The 2nd pathway simply involves the response of Ly-1+2+ T cells proliferating and generating H antigen-specific cytotoxic effectors.     

Delayed-type hypersensitivity to allogeneic mouse epidermal cell antigens

Footpad swelling response was used to measure the alloantigenicity of epidermal cells (ECs) in delayed-type hypersensitivity (DTH). Strong footpad swelling was oberserved 3 h after the challenge, and it continued for 48 h after the challenge. Genetical incompatibility between the recipients and the ECs was required to induce significant footpad swelling. H-2 or non-H-2 incompatibility between mice and ECs in the sensitization phase sufficed to develop significant footpad swelling. Incompatibility caused by point mutation in the A region induced strong responses when B6. C-H-2 ^bm12 mice were immunized with B6/J ECs, but the disparity in immuno-globulin h (Igh) allotype genes was insufficient. H -Y antigen on ECs could also elicit the DTH response. Semiallogeneic F₁-derived ECs sensitized the parental recipients. The responses were successfully transferred by immune lymph node cells, but not by immune sera. Treatment of these immune lymph node cells with monoclonal antibodies plus complement revealed that the cells responsible for DTH transfer were Lyt-1⁺2^–, Ia^– T cells.Abbreviations used in this paper DNFB 2,4-dinitro-1-fluorobenzene - DTH delayed-type hypersensitivity - ECs epidermal cells - HBSS Hanks' balanced salt solution - MHC major histocompatibility complex - PBS phosphate-buffered saline     

Immunodominance in the immune response to “multiple” histocompatibility antigens

Cytotoxic effector T cells putatively specific for multiple non-H-2 histocompatibility (H) antigens were generated by immunizing and boosting C57BL/6 and B6.C-H-2 ^dmice with BALB.B and BALB/c stimulator cells, respectively. The generated effectors were tested for cell-mediated lympholysis on a panel of targets whose BALB/c-derived non-H-2 H antigens were donated by CXB recombinant inbred mice. The spectrum of reactivity of cytotoxic effector T cells with CXB targets demonstrated that the effectors did not recognize multiple H antigens but rather preferentially recognized a single immunodominant non-H-2 H antigen. The identity of the immunodominant H antigen was determined by the H-2 genotype of the stimulator cells when (B6 × B6.C-H-2 ^d)F ₁ cytotoxic effectors were tested. These observations indicate that despite the fact that responders were challenged with more than 40 individual non-H-2 H antigens, they preferentially responded to a single immunodominant antigen.     

Kidney transplantation between congenic versus standard inbred strains of rats: I The significance ofH-1 and non-H-1 gene differences

Concepts of “antigenic strength” in organ transplantation have been evaluated in relation to orthotopic kidney allografts inH-1 congenic strains of rats. Untreated recipients reject fully allogeneic kidneys possessing singleH-1 differences as acutely as kidneys displaying multiple histocompatibility differences. Heterozygozity for H-1 specificities as well as for H-1 plus non-H-1 specificities (semiallogeneic kidneys) favors long term survival (autoenhancement), especially when the specific immune response genes of the recipient lead to reduced reactivity. In active enhancement, the transplantedH-1 congenic kidneys, devoid of additional weak antigens, retain prolonged functional integrity. Weak non-H-1 antigens substantially influence the successful establishment of specific enhancement in an adverse way, either as additive immunogens or as target sites for the effector arm of the rejection response.     

Assessment of some genetic non-MHC differences with GVHR-induced anti-listeria activity of macrophages

Parental cells A injected into (A × B)F₁ heterozygotes induce a graftversus-host reaction (GVHR) which induces systemic activation of anti-Listeria-bactericidal capacity of host macrophages. When donor lymphocytes differ from parent A with respect to various non-H-2 genetic markers, they may or may not be able to induce a GVHR. Some, but not all, known and some unknown non-H-2 differences can be assessed by this method within nine to 12 days. The method is described and some of the following non-H-2 differences are shown to influence GVHR-induced macrophage activation: male H-Y antigen, H-3 or H-4 (but not H-1 or H-9), and as yet underlined differences that apparently exist between mouse substrains of the same or similar designation, but obtained from different breeding establishments.Abbreviations used in this paper: H histocompatibility - GVHR graft-versus-host reaction - MHC major histocompatibility gene complex - SCRF Scripps Clinic and Research Foundation - J Jackson Laboratory - St Strong Foundation - Cum Cumberland View Farm - Ola Olac - Gh Dr. G. Haughton, Chapel Hill - Sal Dr. M. Cohn, La Jolla.     

Sex-dependent effects of non-H-2 loci on lethal GVH reaction induced across an H-2 barrier

We have studied the influence of DBA/2 non-H-2 antigens on the lethal graft-versus-host reaction (GVHR) developed across an H-2 barrier. (DBA/2 x B10.D2)F₁ x B10.D2 (H-2 ^d) backcross (BC) mice were typed for their allelic constitution at nine genetically independent chromosome markers and used as individual cell donors simultaneously for two to three (DBA/2 X B10.D2)F₁ recipients incompatible for DBA/2 non-H-2 antigens alone and two to three (DBA/2 x B10.BR)F₁ recipients incompatible for DBA/2 non-H-2 antigens and H-2^k. The results showed that, when compared with that developed in a control group incompatible for H-2 ^kalone [B10.D2(B10.D2xB10.BR)F₁], the GVHR mortality seen in the presence of an additional incompatibility for DBA/2 non-H-2 antigens [(DBA/2 X B10.BR)F₁recipients] is significantly delayed but only in female mice. An analysis of individual BC donors indicated that this protective effect of DBA/2 non-H-2 antigens correlates with incompatibility for gene(s) linked to the Pgm-1 chromosome marker. In contrast, incompatibility for gene(s) linked to Mod-1 and Es-3 markers accelerates GVHR mortality, but only in male mice. Finally, the results obtained with (DBA/2 x B10.D2)F₁ and (DBA/2 x B10.BR)F₁ recipients were compared; they showed that the intensity of the GVHR developed by cells from individual BC donors against a given set of DBA/2 non-H-2 antigens correlates well with that developed by the same BC donor against the same set of non-H-2 antigens plus H-2^k. We conclude that certain non-H-2 genes (and antigens) can modulate the intensity of the GVHR developed across an H-2 barrier. The number of such genes is probably great; their effects are strong and complex, and can be sex-dependent.     

Interaction of H-2Db with mutant histocompatibility gene H (KH-11) in the mouse

The detectable presence of H (KH-11)b, a mutant non-H-2 histocompatibility gene, was previously shown to depend upon the simultaneous presence, in the skin-graft donor, of both the mutant gene and the H-2b haplotype. The experiments reported here demonstrate that H-2Db is the essential element of H-2b for this interaction. Of two H-2Db histocompatibility mutations, H-2bm13 can replace H-2Db in this interaction, but H-2bm14 cannot.     

Influence of the H-2 u haplotype on immune function in F1 hybrid mice

Recently we reported that antigen-primed T cells from (H-2 ^u × H-2 ^s)F₁ and (H-2 ^u × H-2 ^q)F₁ mice responded poorly in vitro to antigen in the context of antigen-presenting cells of the non-H-2 ^u parent. It was suggested that this effect might be due to unbalanced expression of parental antigens in the F₁ hybrid with the result that the non-H-2^u A antigens were greatly reduced or absent in these mice. If this were the case, non-H-2^u Ia-A cells might be expected to stimulate a mixed lymphocyte reaction (MLR) when cultured with Fl responder cells. When tested, (SJL × PL)F₁ responder cells reacted strongly to SJL stimulator cells. There was no significant reaction to PL stimulator cells. The use of major histocompatibility complex (MHC) congenic mice showed the stimulatory antigens to be associated with the MHC. The MLR could be blocked significantly by monoclonal A-specific antibody of the appropriate specificity. When a monoclonal antibody reactivewith a private epitope associated with A^s was used to probe for the presence of A^s on the surface of (SJL × PL)F₁ spleen cells, no antigen could be detected, indicating loss or alteration of this antigen. These findings suggest that an alteration of the expression of the parental A^s molecule may be responsible for this phenomenon.Abbreviations used in this paper APC antigen-presenting cells - BSS balanced salt solution - CTL cytotoxic T lymphocyte - IL-2 interleukin-2 - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - T₂ suppressor T lymphocyte     

Immunogenetic analysis of tolerance induction in anti-alloantigen delayed type hypersensitivity responses by portal venous pre-inoculation with allogeneic cells

The present study investigates some of the immunogenetic bases for tolerance of anti-allo-delayed type hypersensitivity (DTH) responses as induced by pre-inoculating allogeneic cells via portal venous (p.v.) route. BALB/c mice were injected with totally allogeneic C57BL/6 or H-2 incompatible BALB.B spleen cells via p.v. route. These mice not only failed to exhibit anti-H-2b DTH responses, but also abrogated the potential to generate H-2b-specific DTH responses as induced by the subsequent immunization with H-2b spleen cells via subcutaneous (s.c.) route. The p.v. presensitization with allogeneic spleen cells differing at either class I or class II of major histocompatibility complex (MHC) resulted in the tolerance induction of DTH responses to the respective allogeneic class I or class II MHC antigens. Moreover, the p.v. administration of the class I-positive allogeneic cell fraction depleted of class II-positive component into recipients differing at both class I and class II was capable of inducing anti-class I DTH tolerance. These results indicate that anti-allo-class I or class II DTH tolerance can be induced independently and that the existence of class II antigens on p.v.-presensitized cells is not necessarily required for the tolerance induction of anti-allo-class I DTH response.     

Specific T-cell-mediated killing of autologous lung tumour cells

The phenomenon that strong syngeneic T-cell-mediated cytotoxicity is observed if killer, stimulator, and target cells share H-2 histocompatibility antigens is called H-2 restriction. Here a syngeneic model system making use of hapten-coupled stimulator and target cells is used to explore whether H-2 restriction is absolute or not. Using TNP-coupled spleen or tumor cells as stimulator or target cells in syngeneic and allogeneic situations, it is shown that neither the induction step nor the effector step of TNP-dependent killing is H-2 restricted. By varying the experimental assay conditions more or less H-2-restricted, TNP-dependent killing can be observed. For instance, suboptimal coupling of TNP to targets may result in H-2-restricted killing. Similarly, the use of spleen cell targets as opposed to spleen blast cells or tumor cells may result in H-2-restricted lysis. In contrast optimal coupling of TNP to sensitive target cells and coupling of TNP to cells with certain H-2 haplotypes may lead to significant TNP-dependent killing which is not H-2 restricted. Since hapten-coupled cells lacking H-2 are neither stimulators nor targets these results suggest that the T-cell receptor recognizes TNP-modified H-2 antigens simply as nonself-H-2. Thus hapten coupling of syngeneic cells appears to lead to a histocompatibility antigen change similar to the situation in an allogeneic cytotoxic reaction. Experiments are presented which support this view showing that TNP-coupled and uncoupled syngeneic or allogeneic stimulator and target cells cross-react. For instance allogeneic sensitization may lead to killing on TNP-coupled targets syngeneic to the effector cells and TNP-coupled stimulator cells syngeneic to the effector cells may induce killing on uncoupled syngeneic targets. TNP-dependent cytotoxicity can therefore be envisaged as a kind of allogeneic reactivity due to modification of H-2 antigens by the TNP coupling. This conclusion may have bearing on other model systems in which syngeneic killing appears to be H-2 restricted. In support of this possibility it is shown that allogeneic sensitization may lead to priming of memory cells able to respond to minor histocompatibility antigens.