Cyclic variations in cell-mediated immunity to skin allografts detected by the technetium-99m microcytotoxicity assay

The technetium-99m microcytotoxicity assay has been used to detect cell-mediated immunity in CS7BL/6 mice sensitized with A/J skin allografts. Our initial studies of the quantitative in vitro assessment of lymphocyte-mediated cytotoxicity in mice rejecting first-set skin allografts revealed a simple monophasic response peaking at 14 days postgrafting and declining to control levels by 21 days. Subsequent experiments in which the development and persistence of immunity was assessed at daily intervals from 9–21 days postgrafting revealed that the response was considerably more complex. A cyclic rise and fall in killer activity was evident. The first peak occurred 10–13 days after grafting and the second one 3–5 days later. A third peak of cytotoxic activity sometimes could be detected 16–19 days postgrafting. An attempt was made to characterize the phenomenon by studying the cytocidal effects resulting from the admixture of high- and low-responding lymphocyte populations. An intermediate effect generally was observed when lymphocytes with maximal killer activity were combined in equal numbers with those having decreased reactivity. Varying the ratio of high-and low-responding cells resulted in changes in the net killing effect which was consistent with dilution of more reactive lymphocytes with less reactive ones. Mixing lymphocytes from two peak periods produced a maximum killing effect at all effector to target cell multiplicities. Failure to demonstrate modulation of the reactivity of high-responding cells by low-responding ones suggests that these cyclic variations were not mediated by suppressor cells although a role for humoral factors cannot be excluded at the present time. Alternatively, the cyclic pattern may have been due to the specific depletion and subsequent regeneration of cytotoxic lymphocytes in the lymph nodes of sensitized animals.     

Blocking and unblocking of cell-mediated anti-tumor immunity in mice, as detected by the leucocyte adherence inhibition test

Further observations have been made using the in vitro leucocyte adherence inhibition (LAI) test previously described. The test has been slightly modified and shown to give highly reproducible results in detecting cell-mediated immunity in peritoneal cells of mice exposed to tumors, and in demonstrating factors which modify this immunity.Two different methylcholanthrene-induced tumors stimulated specific reactivity in their hosts as shown by the leucocyte adherence inhibition test employing soluble tumor extracts as antigens. When serum from tumor-bearing mice was added to the mixture, reactivity was specifically blocked. Serum from mice after tumor removal lacked the ability to block the leucocyte adherence inhibition reaction, but specifically reversed the effect of blocking serum (unblocking).Thus three important phenomena of tumor immunity, previously discovered by tedious and complex techniques, are now detectable by a brief, simple procedure.     

Analysis of the major histocompatibility complex in Syrian hamsters. III. Cellular and humoral immunity to alloantigens.

Among the genetic loci incorporated into the major histocompatibility complex in every species studied to date have been prominent genes encoding for strong histocompatibility determinants that elicit detectable alloantibody responses and which are the chief antigenic targets of cell-mediated cytotoxicity reactions. The K and D regions of the H-2 complex in the mouse and the A, B, and C regions of the HLA complex in man are representative examples. Syrian hamsters, as described in this report, do not make alloantibodies to antigens of this type and only very poorly do they carry out in vitro cell-mediated cytotoxicity to target cells putatively bearing these antigens. Since hamsters are quite capable of discriminating analogous antigenic differences in xenogeneic species, and xenogeneic sources cannot distinguish immunologically between the antigens encoded by the two hamster major histocompatibility alleles. Hm-1a and Hm-1b, we conclude that the hamster strains we work with are serologically indistinguishable by the methods used here. However, they obviously differ for determinants which elicit T cell-mediated responses, as evidenced by their ability to express acute skin graft rejection, mixed lymphocyte reactivity, graft-vs-host reactions, and cell-mediated cytotoxicity reactions. Such alloreactivity may reflect a mutation at an SD locus, affecting antigenic sites recognized only by T cells, or that the available hamster strains are SD identical, but differ at loci similar to the I region loci in mice. Alternatively, we cannot exclude the possibility that Syrian hamsters somehow fail to express properly the genes coding for SD determinants.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

The development of humoral immunity to tissue-specific tubular basement membrane alloantigens after renal transplantation across the major histocompatibility barrier in rats immunomodulated with blood transfusions and cyclosporin

The development of a humoral immune response to the tubular basement membrane (TBM) alloantigen of Brown-Norway (BN) rat kidneys was studied after transplantation of BN rat kidneys into bilaterally nephrectomized Lewis (LEW) rats. The LEW rat recipients consisted of four groups receiving no form of immunosuppression, pretransplantation cyclosporin alone, or pretransplantation donor-specific or donor-nonspecific transfusions combined with cyclosporin. The latter two regimens induce indefinite allograft survival in the majority of recipients. Circulating antibody to collagenase-solubilized BN rat renal basement membrane (CS-BN-RBM) was present in all four groups of transplant recipients within 1 week after transplantation, and no significant differences in antibody levels were noted between rats receiving no immunosuppression (survival of 1-2 weeks) and the groups of rats who received various immunosuppressive regimens and survived longer. Circulating antibody to BN-CS-RBM continued to increase in quantity in the cyclosporin-treated group until the time of death (2-10 weeks post-transplantation). In the much longer lived combined transfusion and cyclosporin-treated groups, circulating antibody to BN-CS-RBM generally attained a maximum at approximately 2 to 4 months post-transplantation and then plateaued or decreased somewhat before the time of death (3-16 months post-transplantation). No correlation was found between quantity of circulating anti-BN-CS-RBM antibody and post-transplantation survival. Comparative study of the quantity of circulating antibody to BN-CS-RBM (the presumed nephritogenic antigen of experimental tubulointerstitial nephritis in the BN rat) in serum from transplant recipients as compared to serum from BN rats with severe experimental tubulointerstitial nephritis (TIN) (as induced by immunization with heterologous TBM antigens) demonstrated a greater quantity of potentially nephritogenic antibody circulating in transplant recipients than in BN rats with experimental TIN. Histologically, the transplanted kidneys in immunomodulated recipients demonstrated focal chronic interstitial inflammatory infiltrates with tubular atrophy and relative sparing of the glomeruli. The development of immune responses to tissue-specific alloantigens may become of clinical significance as graft-survival times are increased.     

Cooperation between mouse T-cell subpopulations in the cell-mediated response to a natural poxvirus pathogen.

Irradiated CBA anti-DBA/2 cells (10⁶ cells/culture) suppressed the production of effector cells in cultures containing 10⁷ unprimed CBA (responder) and 10⁶ irradiated DBA/2 (stimulator) spleen cells per culture. The suppressive element was cellular and suppression was specific for the stimulating antigen. The suppressive activity resided in the cytotoxic cell population in that both suppressive and cytotoxic activities were found in cells of the same size range, predominantly in T-cells, were produced in response to similar doses of stimulator antigen, and were produced with the same time course following establishment of first sensitization cultures. Eventual suppression correlated with the cytotoxic activity introduced into second sensitization cultures by suppressor cells. The short-term cytotoxic activity and suppressor activity were both highly radioresistant. These studies indicate that the suppressor cells formed in an in vitro mixed lymphocyte culture are cytotoxic to stimulator cells.     

Mucosal and systemic immunity in mice after intranasal immunization with recombinant Lactococcus lactis expressing ORF6 of PRRSV

The purpose of the study was to construct mucosal vaccine of a recombinant Lactococcus lactis expressing PRRSV ORF6 gene and evaluate mucosal and systemic immune response against PRRSV in mice after intranasal immunization. The result show that the vaccine can stimulate mice to produce specific IgG in serum and remarkable special s-IgA in lung lavage fluid, at the same time, the contents of cytokines IL-2 and IFN-γ of the experimental group were significant higher than those of the control group (P < 0.01), however, the contents of cytokines IL-4 was not different to the all groups. In summary, the constructed mucosal vaccine can significantly induce mucosal immune, humoral immunity and cellular immunity involved Th1 type cytokines, which will lay a theoretical foundation on immune mechanism and new efficient vaccines for PRRSV.     

Susceptibility of Ad12-transformed S (+) and S (-) mouse cells to cell-mediated immunity in vitro

Adenovirus type 12 (Ad12)-transformed mouse cells were examined for their susceptibility to cell-mediated immunity in vitro, with respect to the activity of the virus-specific surface (S) antigen in the cells. A transformed cell line, C57AT1, was established from embryonic cells of C57BL/6 mice by Ad12 infection. In fluorescent antibody tests, the transformed cells were positive for the S antigen when the cells were maintained as cultures, whereas when the cells were grown as tumors in animals they became negative for the antigen (referred to as S(+) and S(-) cells, respectively). These S(+) and S(-) cells were subjected to the 51Cr-release test for cell lysis by immune spleen cells (ISC) raised in syngeneic mice by Ad12 infection. When the S(+) cells at various passage levels were exposed to ISC, all of them were lysed extensively and to a similar extent irrespective of their passage history. In contrast, the S(-) cells were consistently refractory to the action of ISC. In addition, the cytotoxic action of ISC was markedly impeded by pretreating the S(+) cells with antiserum to the S antigen, or the ISC with anti-Thy-1,2 serum plus complement. Taken these findings together, the S(+) cells were assumed to be injured by ISC through direct interaction of the S antigen with T-lymphocytes.     

Mucosal immunity in the brushtail possum (Trichosurus vulpecula): detection of antibody in serum and at female reproductive sites after intranasal immunization

Vaccination strategies for the brushtail possum, which rely upon stimulation of mucosal immunity, are being developed for biocontrol purposes. As little is known about how to stimulate possum immune responses via a mucosal site, groups of possums were immunized intranasally with keyhole limpet haemocyanin (KLH) alone or in combination with known or novel mucosal adjuvants. Antigen-specific antibody titres in female reproductive secretions were measured by ELISA and compared with antibody titres in the serum. Antigen-induced lymphocyte proliferative responses were measured as an indicator of cell-mediated responses. Intranasal immunization with KLH alone stimulated a weak serum antibody response that was significantly increased when KLH was given with cholera toxin subunit B (CTB), recombinant possum tumour necrosis factor alpha (TNF alpha) or live Mycobacterium bovis bacillus Calmette Guerin (BCG). Antibody titres in secretions from ovarian follicles and the uterus were very low in animals administered KLH alone. Significantly higher antibody titres to KLH were present in the reproductive secretions of possums immunized with KLH plus CTB, BCG or heat-killed Mycobacterium vaccae. Antibody titres were lower in mucosal secretions than in the serum, but there was a significant correlation between the two. In addition, coadministration of live BCG with KLH produced a strong antigen-specific cell-mediated response to KLH. This study has shown that an immune response to a protein antigen can be stimulated in possums by intranasal immunization and that antigen-specific antibodies can be detected in secretions from the female reproductive tract.     

Neonatal immunization with a Sindbis virus-DNA measles vaccine induces adult-like neutralizing antibodies and cell-mediated immunity in the presence of maternal antibodies

Infants younger than age 9 mo do not respond reliably to the live attenuated measles vaccine due the immaturity of their immune system and the presence of maternal Abs that interfere with successful immunization. We evaluated the immune responses elicited by Sindbis virus replicon-based DNA vaccines encoding measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin and fusion (F, pMSINH-FdU) glycoproteins in neonatal mice born to naive and measles-immune mothers. Despite the presence of high levels of maternal Abs, neonatal immunization with pMSIN-H induced long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs, mainly IgG2a, that also inhibited syncytium formation in CD150(+) B95-8 cells. IgG secreting plasma cells were detected in spleen and bone marrow. Newborns vaccinated with pMSINH-FdU elicited PRN titers that surpassed the protective level (200 mIU/ml) but were short-lived, had low syncytium inhibition capacity, and lacked avidity maturation. This vaccine failed to induce significant PRN titers in the presence of placentally transferred Abs. Both pMSIN-H and pMSINH-FdU elicited strong Th1 type cell-mediated immunity, measured by T cell proliferation and IFN-gamma production, that was unaffected by maternal Abs. Newborns responded to measles DNA vaccines with similar or even higher PRN titers and cell-mediated immunity than adult mice. This study is the first demonstration that a Sindbis virus-based measles DNA vaccine can elicit robust MV immunity in neonates bypassing maternal Abs. Such a vaccine could be followed by the current live attenuated MV vaccine in a heterologous prime-boost to protect against measles early in life.     

Comparison of the protective ability of O and K immunity in mice after immunization withEscherichia coli

The protective abilities ofEscherichia coli O and K immunity against intraperitoneal infection was compared in CBA mice immunized with formalin-killed bacteria. The K13 immunity achieved seemed somewhat more effective than the O6 immunity. This was also reflected in a slightly more rapid clearance of a liveE. coli O6K13 bacteria in K13-immunized animals than in those immunized with O6. However, the protection achieved afterE. coli O6K2 and O2K1 immunization was related more to O immunity than to K immunity.