The products of transferred H-2 genes express determinants that restrict hapten-specific cytotoxic T cells

Cell lines into which cloned H-2 genes had been introduced (i.e., transformants) were used to correlate the genes and their products that are capable of functioning as H-2 restriction elements for hapten-self-(AED and TNP) specific cytotoxic T cells (CTL). These transformants provided a unique system in which major histocompatibility restricted (MHC) T cell recognition could be examined by using cells that express only H-2Ld or only H-2Dd gene products. BALB/c (H-2d) anti AED-self CTL lysed both the H-2Ld and Dd transformants, but not parental, i.e., untransformed, cells. The AED-self lysis of the Ld and Dd transformants was shown to be specifically inhibited by anti-H-2Ld and anti H-2Dd monoclonal antibody, respectively. In contrast to these results, BALB/c anti TNP-self CTL were found to lyse readily the Dd but not Ld transformed lines, supporting reports indicating that H-2Ld-restricted TNP-self CTL could not be detected. The results of this study thus demonstrate that the cell surface products encoded by these transferred MHC class I genes contain self determinants recognized by CTL.     

Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(alpha 1) and C1(alpha 2) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2Ld has been replaced by that of H-2Dd. Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2Dd replaced by that of H-2Ld) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2Ld and H-2Dd or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2Ld and H-2Dd to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2Ld substituted for that of H-2Dd), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2Ld or anti-H-2Dd N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of beta 2M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2Dd and anti-H-2Ld CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2Ld-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.     

Molecular localization of allogeneic and self determinants recognized by bulk and clonal populations of cytotoxic T cells

The localization of MHC-encoded determinants recognized by hapten- and allo-specific cytotoxic T cells was analyzed with the use of cell lines expressing recombinant H-2Dd and H-2Ld MHC products. Bulk cultures of CTL against TNP-self, FITC-self, and AED-self recognized self determinants associated with the N/C1 domains of both Dd and Ld products. A number of allo- and hapten-specific CTL clones were also tested for recognition of the recombinant MHC products. The allo clones specific for Ld or Dd antigens recognized the respective N/C1-associated determinants. In addition, all clones generated against H-2q and known to cross-react with H-2Dd antigens recognized determinants associated with the N/C1-associated Dd determinants. Thus, some of the results obtained with CTL parallel, whereas others contrast with, those findings obtained with monoclonal anti-H-2 antibodies. Similar to the observations made with the monoclonal antibodies, no determinant as defined by T cells has been found to be lost as a result of the interaction between the N/C1 and C2/M domains of the Ld and Dd proteins. Nor did our studies detect the presence of new antigens resulting from the interaction of these gene products. However, the present T cell findings continue to contrast previous results demonstrating that antibody interaction with class I products includes recognition of C2/M-associated epitopes.     

Tumor rejection mediated by transfection with allogeneic class I histocompatibility gene

Non-self class I histocompatibility Ag can act as strong alloantigens and be recognized as distinct targets by CTL. To study the possibility of using allograft rejection to generate tumor-specific immunity, we have introduced an allogeneic class I histocompatibility gene, the H-2Kb gene, into a k haplotype tumor, K36.16, by DNA-mediated gene transfer. The K36.16 tumor grows readily and does not confer protective immunity in AKR mice. A total of 37 H-2Kb-transfected K36.16 clones (Kb/K36.16) was isolated and studied individually. The Kb/K36.16 clones were found to differ significantly in the amount of the exogenous H-2Kb antigens expressed on their cell surface. Moreover, as a result of the transfection, the level of expression of the endogenous H-2Dk Ag was also altered when compared to that of the parental K36.16 tumor cells. All the Kb/K36.16 clones that were positive for the H-2Kb Ag were rejected by the semisyngeneic AKR mice. Moreover, some of these Kb/K36.16 clones were also rejected by syngeneic (AKR x C57BL/10)F1 mice. In consequence of immunization with the Kb/K36.16 clones, the AKR and F1 mice were able to survive a subsequent challenge of the wild-type, unmodified, parental K36.16 tumor cells. More importantly, some of these Kb/K36.16 clones demonstrated an active and specific immunotherapeutic effect, and they were able to eradicate the growth of the parental K36.16 tumor cells in AKR mice. This observation therefore reinforces the feasibility of using DNA-mediated gene transfer as a molecular approach to abrogate tumor growth.     

Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2.

One model to explain the high frequency of alloreactive T cells proposes that allogeneic MHC molecules are recognized together with host cell-derived peptides. A model system was developed to investigate the relevance of this mechanism by expression of H-2Dd or H-2Ld in 174xCEM.T2 (T2) cells. This human cell line contains a mutation in its Ag-processing pathway that should restrict the association of endogenous peptides with cell surface class I molecules. CTL generated by stimulating C57BL/6 (H-2b) responder cells with H-2Dd or H-2Ld transfectants of the human B cell line C1R or the murine T cell lymphoma EL4 were assayed for their ability to recognize alloantigenic determinants on these transfectants. The major fraction of the H-2Dd-specific allogeneic CTL response, generated in a MLC or under clonal limiting dilution conditions, was composed of T cells that recognized H-2Dd expressed on C1R or EL4 cells, but failed to recognize this molecule on T2 cells. Clonal analysis indicated that approximately one-third of these CTL recognized determinants that were unique to H-2Dd expressed on C1R stimulator cells whereas the remainder recognized determinants that were also found on EL4 transfectants. Less than 10% of H-2Dd-reactive CTL recognized the T2 transfectant, and these clones also killed C1R-Dd and EL4-Dd. This result suggests that the great majority of H-2Dd-specific alloreactive CTL recognize determinants that are formed by a complex of H-2Dd with endogenous peptides that are absent or significantly reduced in T2 cells. Based on recognition of human or murine transfectants, these CTL exhibit some level of specificity for the structure or composition of the bound peptides. Examination of allogeneic CTL specific for H-2Ld revealed populations similar to those described for H-2Dd. In addition, a major new population was present that recognized determinants shared between C1R-Ld and T2-Ld but not present on EL4-Ld. These results are consistent with the idea that the alloreactive response to H-2Ld is also largely dependent on the presence of bound peptide. However, they also may indicate that the H-2Ld molecule expressed on T2 cells is occupied by one or more peptides that are shared with other human, but not murine, cells. The significance of these results to current models of alloreactivity is discussed.     

Immune recognition of AIDS virus antigens by human and murine cytotoxic T lymphocytes

The CTL response to HIV was analyzed in humans and in mice. By using a novel and strictly autologous lymphocyte culture system, human CTL lines were established with PBL from seropositive asymptomatic donors and from patients suffering from AIDS or presenting AIDS-related complex. CTL from HLA-A2 donors recognize and kill murine P815 mastocytoma cells doubly transfected with the human HLA-A2 gene and the HIV env gene; they also kill HLA-compatible human macrophages infected with HIV. CTL specific for the HIV env Ag were also generated in BALB/c mice by immunization with syngeneic murine cells transfected with the HIV env gene. Human and murine HIV-immune CTL populations belong to the CD8 subset of T lymphocytes and are restricted by class I HLA or H-2 transplantation Ag, respectively, in the recognition of HIV env Ag. The two different experimental systems presented here can be used to study CD8 lymphocyte immunity against HIV. The murine model of CTL immunity offers the additional advantage of avoiding the manipulation of infectious virus isolates.     

Chronic relapsing experimental allergic encephalomyelitis. Cytotoxicity effected by a class II restricted T cell line specific for an encephalitogenic epitope

We have examined the possibility that Ag-specific CTL responses may play a role in the pathogenesis of CREAE by using an effector T cell line (LN400) specifically reactive to the SJL encephalitogenic epitope defined by myelin basic protein MBP residues(90-101). The LN400 cell line was capable of adoptively transferring CREAE to naive SJL mice and proliferated specifically to synthetic peptides corresponding to MBP residues(90-101) and an N-acetylated analogue of this epitope, as well as MBP. Moreover, the cell line generated Ag-specific CTL responses only against syngeneic targets that had been pulsed with these Ag. Targets pulsed with irrelevant Ag were not lysed. These CTL responses were MHC restricted to H-2s and were inhibited if targets were preincubated with mAb specific for relevant class II Ag. No inhibition was seen if targets were preincubated with mAb specific for class I Ag, indicating that the CTL responses generated by this L3T4+ Lyt-2.2- cell lines were class II restricted. Studies designed to detect nonspecific CTL through a bystander mechanism failed to demonstrate significant lysis of bystander targets by this Ag-specific cell line. These findings have relevance in defining potential mechanisms of disease induction in this model autoimmune disease.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

Resistance to cell-mediated cytotoxicity is correlated with reduction of H-2K gene products in AKR leukemia

AKR leukemia cell lines differing in the amount of H-2K and H-2D antigens expressed on the cell surface were used to assess cell-mediated immune responses in syngeneic mice against Gross/AKR murine leukemia virus (MuLV)-induced tumors. Leukemic cells with reduced expression of H-2K^k antigens were inactive as inducers of Gross-MuLV/H-2^k-specific cytotoxic T lymphocytes (CTL) and resistant to lysis by CTL raised against H-2K^k positive AKR leukemia cells. H-2K^k positive leukemias induced cytotoxic effectors, which upon restimulation in vitro, lysed the stimulating and other H-2K^k positive leukemia cells. In antibody inhibition experiments, T-cell-mediated cytotoxicity to these leukemias could only be inhibited by antisera and monoclonal antibodies specific for the H-2K^k antigens. Due to this specific role of H-2K^k antigens in T-cell cytotoxicity to Gross/AKR MuLV-induced tumors, reduced expression of H-2K^k antigens on spontaneous AKR leukemic cells could have important implications for surveillance of these neoplastic cells.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MuLV murine leukemia virus     

Differential lack of class I H-2d antigen expression by sublines of the BALB/c S49 T cell lymphoma

Five different sublines of the BALB/c murine S49.1 T cell lymphoma were found to exhibit distinct patterns of absence of detectable H-2d class I major histocompatibility antigen expression. The results were demonstrated and verified by a) the generation of H-2Kd-, H-2Dd,Ld-, and H-2Ld-specific cytotoxic T lymphocytes that were assayed on S49.1 target cell lines, b) antibody-mediated cytotoxicity with the use of anti-H-2d monoclonal reagents, and c) flow microfluorometry. The five lines investigated were S49.1, T-25, T-25ADH, Thy-1-, and 100/0. None of these lines expressed detectable levels of Ld. S49.1 expressed both Kd and Dd, T-25 and T-25ADH expressed Dd but not Kd or Ld, Thy-1- expressed Kd but not Dd or Ld, and 100.0 did not express any detectable amounts of Kd, Dd, or Ld. These results indicate that K and D (and L) antigens can be expressed independently of each other and suggest that expression of class I antigens is controlled in a locus-specific manner.     

Abelson virus-transformed lymphocytes: null cells that modulate H-2.

A-MuLV-transformed lymphoid cells from Balb/c mice had the properties of null lymphocytes. They did not secrete Ig and all but one did not have detectable cell-associated Ig; one line synthesized, but did not secrete, the mu chain of IgM. The cells expressed H-2D and H-2K, but not H-21 histocompatibility antigens or theta-antigen; they had Fc receptors. Most cell lines grew to form donor cell tumors after inoculation into (Balb/c X C57B1/6)F1 mice. The tumor cells have more H-2Dd than cells passaged in vitro. Cell lines carried in vitro progressively lost H-2Dd. A line in which 5-30% of the cells were lysable by anti-H-2Dd was cloned; all eleven clones had H-2Dd (13-69% lysable) demonstrating that H-2 modulates in vitro. A clone with little H-2Dd (10-15% lysable) was tumorigenic even after treatment with anti-H-2Dd sera; at least 50% of the tumor cells were lysed by anti-H-2Dd. Thus A-MuLV-transformed lymphocytes modulate H-2 in vivo to higher levels and in vitro to lower levels.     

Genetic control of the induction of cytolytic T lymphocyte responses to AKR/Gross viral leukemias. II. Negative control by the Fv-1 locus in AKR mice of responder H-2b haplotype

To assess whether the presence of a responder H-2b haplotype would be sufficient to allow mice of nonresponder "high leukemic" phenotype to generate syngeneic anti-AKR/Gross virus cytolytic T lymphocytes (CTL), the AKR.H-2b strain was examined. Although capable of mounting vigorous apparent anti-minor histocompatibility-specific CTL responses, AKR.H-2b mice failed to produce anti-viral CTL after a variety of stimulation protocols. In contrast, the "doubly congenic" AKR.H-2b:Fv-1b strain was able to respond with substantial levels of H-2-restricted anti-AKR/Gross virus CTL activity. These results indicated that Fv-1n alleles could exert negative epistatic control over responder H-2b-encoded gene(s). Because the B6.Fv-1n congenic was also able to generate anti-viral CTL indistinguishable from the prototype B6 strain, however, it was apparent that other genes of AKR background were required for the Fv-1n-mediated inhibition in AKR.H-2b mice. The mechanism by which Fv-1 intereacted with other genes to override positive H-2b control appeared to be related to the expression of the CTL-defined, virus-associated antigens by normal AKR.H-2b cells. Thus, AKR.H-2b spleen cells but not thymus cells were able to stimulate the production of B6 anti-AKR/Gross virus CTL and were recognized as target cells by such anti-viral CTL. In contrast, both spleen cells and thymocytes from AKR.H-2b:Fv-1b mice were negative when tested as stimulator or target cells in these assays. In addition, AKR.H-2b but not AKR.H-2b:Fv-1b spleen cells were shown to display serologically defined gp70 determinants and the Gross cell surface antigen. Taking these data together, it appeared that the inhibition of anti-viral CTL responsiveness might be due to tolerance induced by the cell surface expression of virus-associated antigens by normal AKR.H-2b cells. Widespread display of viral antigens, in turn, may have been due to the permissive effects of Fv-1n on the spread of the early arising N-ecotropic, endogenous AKR leukemia virus controlled by other background genes. In this context, the implications of the multi-gene control of anti-AKR/Gross virus CTL production are discussed with respect to the induction of spontaneous leukemia in the high incidence AKR strain.     

Cell surface expression and alloantigenic function of a human class I MHC heavy chain gene (HLA-B7) in transgenic mice

We have introduced the gene encoding the heavy chain of the human MHC class I Ag HLA-B7 into transgenic mice. The gene was shown to be expressed at both the RNA and protein level. Cell surface HLA-B7 was detected on whole spleen cells by immunoprecipitation and on purified T cells by flow cytometry (FACS). Normal mice immunized with H-2-syngeneic B7-transgenic spleen cells generated CTL capable of killing transgenic cells and B7-expressing human JY cells. Anti-HLA mAb blocked the killing of JY cells. These results indicate that the human class I Ag HLA-B7 can be expressed at the surface of transgenic spleen cells in the absence of human beta 2-microglobulin, and that a significant fraction exists in a form recognizable by nontransgenic CTL as a major histocompatibility Ag unrestricted by H-2.     

Rat cytotoxic T lymphocytes against human T-lymphotropic virus type 1-infected cells recognize gag gene and env gene encoded antigens

T cell immune responses in syngeneic WKA/H rats were analyzed by using lymphoid cell lines, TARS-1, TART-1, and TARL-2, infected with human T-lymphotropic virus type 1 (HTLV-1). Spleen cells of rats in which these cell lines had been rejected were sensitized in vitro with the same cell lines, and cells cytotoxic to these HTLV-1+ cell lines, and cells cytotoxic to these HTLV-1+ cell lines were generated. The effector cells were CTL of the CD5+ CD8+ phenotype and showed restriction of MHC class I Ag. Direct tests as well as cold target cell inhibition tests with an array of cell populations showed that these CTL reacted only with syngeneic HTLV-1+ cell lines. When xenogeneic HTLV-1+ cell lines were similarly utilized for in vitro sensitization, rat CTL specific for syngeneic HTLV-1+ cells were generated. They were not, however, reactive with xenogeneic HTLV-1+ cells used for sensitization. Syngeneic rat cells selectively expressing gag, env, or pX gene coded Ag were prepared by infection of recombinant vaccinia viruses. In cold target cell inhibition tests of anti-HTLV-1 CTL with thus prepared cells, cytotoxicity against the syngeneic HTLV-1+ cells line, TARS-1, was inhibited by syngeneic cells expressing gag gene or env gene coded Ag. Inhibition was, however, more consistent and more dominant by cells with gag gene than those with env gene. Syngeneic cells with pX gene and MHC class I incompatible cells with gag, env, or pX gene did not inhibit cytotoxicity.     

Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.     

Cytotoxic T lymphocyte response to minor H-43a alloantigen in H-43b mice. Privileged H-2Kb restriction to the response is not due to immunodominance or epistatic effect but due to Ir gene function of H-2Kb itself

Previous study demonstrated that anti-H-43a cytotoxic T lymphocyte (CTL) response of H-43b CWB (H-2b) stain carrying non-major histocompatability complex (MHC) genes of C3H and F1 strains raised by crossing CWB with various H-43b strains was restricted exclusively by self H-2Kb (Kb). In the present study, newly produced C3W strain (H-2k, H-43b), which is H-43-congenic to C3H/HeN (H-2k, H-43a), was used as H-43b mice, and possibility of immunodominance of Kb was examined. No anti-H-43a CTL response could be induced in C3W strain and F1 strains raised by crossing C3W with other H-43b strains not carrying Kb. Thus, the possibility of immunodominance of Kb over the other MHC class I alleles could not be supported. We also examined possibility of epistatic effect of I region genes and non-MHC genes on the Kb restriction. (C3W x C57BL/6)F1(I-Ak/b) and (C3W x B6.CH-2bm12)F1(I-Ak/bm12)mice showed equally anti-H-43a CTL response restricted exclusively by self Kb, and (C3W x B10.MBR)F1(Ik/k) mice also showed anti-H-43a CTL response restricted solely by self Kb. Cold target competition experiments demonstrated that H-43b C57BL/10 or A.BY mice, which do not have non-MHC genes of C3H mounted anti-H-43a CTL response restricted solely by self Kb. Thus, no relation of I region genes or non-MHC genes to the Kb restriction was shown. All the results indicate that H-43b mouse strains, including F1, can not achieve anti-H-43a CTL response unless they carry Kb allele. Notably, (C3W x C57BL/6)F1 mice mounted self Kb-restricted anti-H-43a CTL response, whereas (C3W x B6.CH-2bm1)F1 mice carrying mutated Kb could not mount anti-H-43a CTL response at all. These findings indicate strongly that Kb itself is classical Ir gene of anti-H-43a CTL response and directs self Kb restriction of the response.