T cell responses to the human papillomavirus type 16 E7 protein in mice of different haplotypes.

The response of murine T cells to the E7 molecule of human papillomavirus type 16 (HPV-16) was studied using eight different mouse strains of six distinct H-2 haplotypes. HPV-16 E7 protein was prepared as a fusion protein with glutathione-S-transferase, purified by affinity chromatography and used for immunization. Cells from the lymph nodes were cultured with whole fusion protein, glutathione-S-transferase or HPV-16 E7 protein synthetic peptides. All the mouse strains tested, with the exception of BALB/c, recognized the E7 molecule, as evidenced by a proliferative response to at least two of the peptides. The profile of responses to peptides varied between and within a strain, but five distinct immunodominant regions could be identified. These regions were defined on the basis of a reaction to one or more peptides in a given part of the E7 molecule by at least four strains. The five regions were encompassed by amino acid residues 1 to 9, 17 to 32, 42 to 59, 62 to 77 and 87 to 98. The findings suggest that in an outbred population, such as man, the E7 molecule of HPV-16 would be recognized by a large proportion of the population. However, the poor response of two mouse strains [B10.RIII (71NS) and BALB/c] could also have a corollary in man.

Erythrocyte-reactive T-cell lines and clones from normal mice recognize serum-derived antigens.

T-cell clones and cell lines which apparently respond to autologous (syngeneic) erythrocytes have been generated from the spleens of normal mice. The response showed considerable cross-reactivity with red blood cells (RBC) from other species, including chicken, and was 'heteroclitic' in that reactivity against some species of RBC, notably rat and monkey, was greater than to mouse. The clones were Thy-1+ L3T4+ Lyt-2- and recognized antigen in association with I-Ak or I-Ek. At least three specificities were identified on the basis of reactivity to crude lysates of mouse and rat RBC. One of the putative clones, M0.5/1/D2, showed a change in reactivity during culture, proliferating strongly against antigen-presenting cells (APC) without added erythrocytes. Analysis of the clones and lines using an I-Ak I-Ek expressing hybridoma, HB-98, has indicated that only one, M5/1/F5, was likely to be erythrocyte-specific; the remainder were responding to antigens present in foetal calf serum (FCS). The data demonstrate that apparent erythrocyte specificity can be a result of serum components being presented to T lymphocytes via red blood cells.

Isolation of a human T cell line specific for a streptococcal cell surface antigen.

A streptococcal cell surface antigen of Mr 185,000 (SAI/II) expressed by Streptococcus mutans has previously been well characterised. A T cell line specific for native SAI/II has been isolated from peripheral blood mononuclear cells (PBMC) of a naturally sensitised normal individual. This line has been maintained in culture for several months and was shown to be highly specific, not only for different preparations of native antigen but also for recombinant SAI/II protein. It did not respond to a homologous antigen SpaA (Mr 210,000), extracted from Strep. sobrinus. The phenotype of the line was CD3+ CD4+ CD8- TcR alpha beta +. HLA typing and inhibition studies showed that the response was restricted by both DR and DP encoded class II.

The comparative frequency of human T cells responding to a native antigen and a synthetic peptide derived from Streptococcus mutans.

The frequency of human peripheral blood T cells responding to a 21-residue synthetic peptide (SP 21) derived from the sequence of a 3.8-kDa streptococcal antigen was estimated by limiting dilution analysis and compared with the frequency of cells responding to the native, cross-reactive 185-kDa streptococcal antigen. Frequency estimates were made by measuring both [3H]thymidine incorporation and IL 2 production in the same cell cultures. The results provided frequency estimates for SP 21-reactive cells of between 1:42 147 and 1:306 110, with a mean of 1:160 617 by [3H]thymidine incorporation, and 1:139 893 to 1:241 315 (mean 1:165 315) using the IL 2 assay. With the native 185-kDa streptococcal antigen, frequency estimates were between 1:38 393 and 1:86 142 (mean 1:169 934) according to the proliferative response and 1:22 462 and 1:100 400 (mean 1:61 189) by the IL 2 assay.

Human T cell responses to beta-galactosidase.

The peripheral blood of most normal individuals has been shown to contain T cells that respond to beta-galactosidase (beta-Gal), presumably as a result of natural priming. Three T cell clones (clones 1,2,4) specific for beta-Gal were isolated from peripheral blood mononuclear cells (PBMC) after pretreatment with leucine methyl ester (LeuOMe); a fourth clone from the same individual was isolated from untreated cells. All four clones were CD4+ CD8- alpha beta TcR+ and clone 1 was additionally shown to be cytotoxic. Epstein-Barr virus (EBV) transformed B cell lines were derived from LeuOMe-treated or untreated PBMC and used to study the efficiency of presentation of beta-Gal to one of the clones. The results indicated that B cells transformed after LeuOMe treatment presented beta-Gal at lower concentrations than untreated controls. beta-Gal would therefore appear to be a highly suitable model antigen for studies of immunoregulation in humans.

Induction of experimental autoimmune thyroiditis in B cell-depleted mice.

The effect of B cell depletion on the induction and severity of murine experimental autoimmune thyroiditis was investigated. Thirteen CBA mice were given repeated intraperitoneal doses of 700 micrograms purified rabbit anti-mouse Ig antibody from 24 hours to 8 weeks after birth. Controls were given normal rabbit IgG (14 mice) or were left uninjected (10 mice). At six weeks all mice received two doses of 70 micrograms murine thyroid extract in complete Freund's adjuvant. Only 2/13 of the anti-Ig treated mice were fully B cell-deficient as determined by serum IgM, spleen cell immunofluorescence and responsiveness to LPS; however, the levels of anti-thyroglobulin autoantibodies were very low in 7/13 mice. The results demonstrate that thyroiditis can be actively induced in the absence of B cells and autoantibodies but that B cells may play a role in increasing disease severity.

Tolerance to minor histocompatibility antigens.

A neonatal tolerance model employing fully allogeneic lymphoid cells as tolerogen was used in an investigation of tolerance to self and donor minor histocompatibility antigens (miHA). Tolerance was assessed by skin grafting and subsequently by the generation of cytotoxic T lymphocytes. Two strain combinations were investigated. In the first, BALB/c-B10, none of the mice became tolerant to H-2d: all gave responses to BALB/c and B10.D2 antigens comparable to uninjected controls. However, tolerance was secured to BALB miHA in the face of reactivity to the original donor cells (i.e., BALB/c), showing that multiple miHA can induce tolerance independently of major histocompatibility complex (MHC) antigens. In the reverse strain combination, in which tolerance to B10 antigens was successfully established in BALB/c recipients, MHC restriction of tolerance to self miHA could not be demonstrated, as mice tolerant to B10 were unresponsive to BALB.B antigens, too. Again, the induction of tolerance to multiple donor miHA proved to be independent of tolerance to donor MHC antigens, and a great deal easier.

Human fetal lymphocytes require T cell growth factors for cytotoxic responses.

Lymphocytes isolated from the blood of 17 human fetuses, varying in gestational age between 16 and 26 weeks, were tested for their capacity to generate specific cytotoxic cells after mixed lymphocyte culture (MLC) with allogeneic cells in the presence or absence of exogenous T cell growth factor (TCGF). Blood cells from seven fetuses, distributed throughout the age range, failed to generate cytotoxic cells even when TCGF was added in MLC, whereas six others gave positive responses but only when exogenous TCGF was present during the sensitisation phase. The maturation induced in the latter was not caused solely by a direct, non-specific effect of the TCGF, for control responder cells incubated with TCGF in the absence of allogeneic stimulator cells always responded less strongly or not at all. Fetal blood lymphocytes from the remaining four fetuses gave significant cytotoxic responses that were not augmented by TCGF. It is concluded that there can be a clear dichotomy between proliferative and cytotoxic responses to alloantigens and that the inability of human fetal blood lymphocytes to mount cytotoxic responses at this stage of development might be due to deficiencies in helper cells, cytotoxic precursors or both. For three fetuses it was possible to study, additionally, cytotoxic responses of spleen, liver and thymus with and without TCGF. None of them made specific responses even when TCGF was added, though cells from two spleens and one thymus responded directly to TCGF. To ascertain whether the absence of cytotoxic responses might have been caused by a failure of blood, spleen, thymus or liver cells to proliferate, their mixed lymphocyte reactivity, as detected by the uptake of 3H-thymidine, was studied without exogenous TCGF. Whereas thymus cells from only one of five fetuses responded, cells from all other tissues (including blood) responded consistently.

Presentation of alloantigens by host cells.

Presentation of alloantigens by host cells has been examined in vivo by means of a murine cell transfer system. Primary (1 degree) hosts were activated by the i.p. administration of allogeneic spleen cells and their spleen or peritoneal cells were transferred into syngeneic secondary (2 degrees) hosts 3 days later. Sensitization of 2 degrees hosts was assessed by their ability to reject donor strain skin grafts prematurely. The transferred cells were routinely depleted of T lymphocytes. We show that (a) 5 X 10(7) spleen and 3 X 10(6) peritoneal cells consistently caused marked accelerated graft rejection; (b) this effect was antigen specific and observable in all strain combinations studied; (c) it was caused by the active sensitization of 2 degrees hosts, but not by contaminating donor strain cells; (d) the cells involved were plastic adherent and viability was not a requirement; and (e) both class I and II, but not minor, histocompatibility antigens played a role in this model. We conclude that presentation of alloantigens by host antigen-presenting cells can be a potent route of allosensitization.

Red cell volume distribution curves and intracellular globin chain precipitation in the alpha-thalassaemic mouse, Hbath-J.

Red cell volume distribution curves were studied in alpha-thalassaemic mice (Hbath-J/+ mice) and normal mice (+/+ mice) of various ages. Individual Hbath-J/+ mice could not be reliably distinguished from their +/+ littermates on the basis of modal cell volume either at birth or during the first 3 weeks of life. However, between the ages of 4 and 30 weeks Hbath-J/+ mice displayed a degree of microcytosis that enabled them to be readily distinguished from their normal littermates using the criterion of modal red cell volume. Preliminary studies of alpha:beta globin chain synthesis ratios given by blood reticulocytes of Hbath-J/+ and +/+ mice after incubation with 3H-leucine for 5 min and 2 h suggest that there is little or no proteolysis of excess beta-chains in the alpha-thalassaemic mouse. Electron microscope studies revealed that the erythroblasts, marrow reticulocytes and circulating red cells of Hbath-J/+ but not +/+ mice contain stellate and branching intracytoplasmic inclusions, presumed to consist of precipitated beta-chains. These inclusions were ultrastructurally similar to the inclusions which have been previously reported in the erythroblasts and marrow reticulocytes of people with various alpha-thalassaemia syndromes. The proportion of erythropoietic cell profiles with inclusions was higher in Hbath-J/+ mice (in which two of the four alpha-globin genes are deleted) than in Thai patients with HbH disease (in whom there is usually a deletion of three of the four alpha-globin genes); this finding is probably related to a relatively low proteolytic capacity in the more mature mouse erythroid cells when compared with human cells. The presence of inclusion-containing red cells (mainly reticulocytes) in the peripheral blood of unsplenectomized Hbath-J/+ animals contrasts with the absence of such cells in unsplenectomized patients with alpha-thalassaemia I trait and HbH disease; this difference seems to be at least partly due to a poorly-developed pitting function in the mouse spleen.

Acquired immunological tolerance of foreign cells is impaired by recombinant interleukin 2 or vitamin A acetate.

The susceptibility of newborn mice to the inception of tolerance after exposure to antigen is associated with their deficiency in the production of endogenous interleukin 2 (IL-2). As further evidence of the complicity of IL-2 in the inception and maintenance of tolerance, it is shown here that a solid and long-lasting state of tolerance induced by the intravenous injection into newborn CBA mice of lymphoid cells from (CBA X C57BL/10ScSn)F1 hybrids can be brought to an end by the administration of exogenous IL-2 or by supplementing an otherwise normal diet with vitamin A acetate, the effect of which is to increase the proportion of the moiety of the T-cell population that produces IL-2. These results indicate that certain nonspecific stimuli can influence whether immunological tolerance is maintained.

Abrogation of resistance to bone marrow transplantation by induction of specific tolerance in natural killer cells?

Hybrid resistance describes the capacity of first generation (F1) hybrids between certain mouse strains to inhibit the growth of tumour or haematopoietic cells of parental origin. The cells that appear to mediate this phenomenon differ from classical T and B lymphocytes in several respects. For example, they are unusually radioresistant, show no immunological memory, are present in thymectomized or congenitally athymic mice, are not functional until about 3 weeks after birth. These characteristics suggest that the effectors are natural killer (NK) cells. Although most of the evidence implicating NK cells in hybrid resistance is circumstantial, the experiments of Warner and Dennert are more direct in that they show that resistance can be restored to mice with a congenital or induced defect in NK activity by the infusion of cells belonging to an NK clone. Conversely, treatment of mice with an antibody to NK cells abrogated hybrid resistance to parental bone marrow grafts. Both NK cells and the effectors of hybrid resistance are generally considered to be nonspecific. We have now investigated this assumption by attempting to prevent hybrid resistance by neonatal tolerance induction with parental strain antigens. Our data indicate that hybrid resistance can be abrogated by this means and that the tolerance is specific and transferable with Thy-1+ spleen cells.

The effect of T lymphocyte depletion on neonatal tolerance induction, graft-vs.-host disease and cellular chimerism.

Treatment with a monoclonal anti-Thy-1 antibody and complement completely prevented C57BL spleen cells from causing graft-vs.-host disease following their inoculation into newborn CBA mice. The proportion of mice that became tolerant to C57BL antigens, as measured by skin grafting, was significantly less compared with mice given (CBA X C57BL)F1 hybrid cells. This was not due to the elimination of T cells, for antibody-treated F1 cells induced tolerance as readily as complement-treated control F1 cells. To investigate whether the apparent superiority of F1 cells over C57BL cells is attributable to differences in the mechanism inducing and maintaining unresponsiveness, two approaches were followed. First, the level of donor cell chimerism in the spleens of tolerant animals was studied. Though no difference between F1 and C57BL cells was uncovered, the presence of T cells in the donor inocula favored the establishment of chimerism. Second, the involvement of suppressor T cells was examined in adoptive transfer experiments. Splenic suppressor T cells were associated with tolerance regardless of how it was elicited. Preliminary results with F1 cells show that the tolerogenic property is not confined to any one cell type. It is proposed that the greater tolerogenicity of F1 cells is brought about by the presence of host-type (self) antigens, which enable the tolerogenic signals to operate without recourse to antigen processing by host cells.

Tolerance, immunocompetence, and secondary disease in fully allogeneic radiation chimeras.

The aim of this study was to ascertain the extent to which secondary disease and mortality in fully allogeneic chimeras (C57BL leads to CBA) is caused (if at all) by a delayed graft-versus-host reaction. Adult CBA males were thymectomized, irradiated, and reconstituted with T-lymphocyte-depleted C57BL or CBA bone marrow cells (BMC), followed three weeks after irradiation by implantation under the kidney capsule of thymic lobes from C57BL or CBA fetal or adult donors. These mice were observed for the development of secondary disease for periods in excess of 250 days, and they were examined at 5 weeks or 4 months for T lymphocyte reactivity and tolerance to alloantigens, using the cell-mediated lympholysis assay (CML). The following results were obtained. First, removal of T lymphocytes with anti-Thy 1 antibody and complement from allogeneic bone marrow did not prevent wasting and eventual death, although it prolonged the lifespan of mice substantially. Second, T lymphocytes generated from bone marrow-derived precursor cells became tolerant of the histocompatibility antigens of the thymus donor strain but remained normally reactive to third-party antigens. Third, allogeneic radiation chimeras did not survive as well as animals reconstituted with syngeneic cells, even when they were demonstrably tolerant in CML. Fourth, C57BL BMC maturing in a CBA host equipped with a C57BL thymus graft did not become tolerant of host antigens, indicating that extra-thymic tolerance does not occur in fully allogeneic--as opposed to semiallogeneic--chimeras. It is argued that the function of B lymphocytes and/or accessory cells is impaired in fully allogeneic radiation chimeras, and that the mortality observed was directly related to the resulting immunodeficiency. The relevance of the results described in this paper to clinical bone marrow transplantation is discussed.

Suppression of natural cell-mediated cytotoxicity in man by maternal and neonatal serum.

the natural cytotoxicity of cells prepared from the blood of human neonates and women at the time of parturition was investigated, using a 4 hr 51Cr release assay and two established cell lines as targets. Although cord cells proved to be cytotoxic, the overall level was distinctly lower than that of normal adult cells. Whereas adult cells from males gave higher levels of cytotoxicity compared with cells from females, this was not the case for cord cells. Cells from women in labour showed even lower cytotoxic values. Neonatal and maternal serum or plasma caused a profound inhibition of the cytotoxicity shown by adult cells when present during the assay or following preincubation of effector cells with serum. Cord cells were not suppressed by either autologous or allogeneic cord sera. The nature of these suppressive factors and their origin and ontogeny remain to be elucidated. It would appear that in the neonate, and possibly also in the fetus, natural cytotoxicity is largely suppressed by serum factors, both in mother and offspring. This could represent yet another example of immunological modulation in pregnancy.