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We have used a panel of monoclonal antibodies in a study of the expression of multiple tubulins in Physarum polycephalum. Three anti-beta-tubulin monoclonal antibodies, DM1B, DM3B3 and KMX-1 all reacted with the beta 1-tubulin isotypes expressed in both myxamoebae and plasmodia. However, these antibodies showed a spectrum of reduced reactivity with the plasmodial beta 2-tubulin isotype - the competence of recognition of this isotype was graded DM1B greater than KMX-1 greater than DM3B3. The anti-alpha-tubulin monoclonal antibody, YOL 1/34 defined the full complement of Physarum alpha-tubulin isotypes, whilst the anti-alpha-tubulin monoclonal antibody, KMP-1 showed a remarkably high degree of isotype specificity. KMP-1 recognises all of the myxamoebal alpha 1-tubulin isotypes but only recognises 3 out of the 4 alpha 1-tubulin isotypes expressed in the plasmodium (which normally focus in the same 2D gel spot). KMP-1 does not recognise the plasmodial specific alpha 2-tubulin isotype. This monoclonal antibody reveals a new level of complexity amongst the tubulin isotypes expressed in Physarum and suggests that monoclonal antibodies are valuable probes for individual members of multi-tubulin families.

SummarySurviving infection represents a balance between the proinflammatory responses needed to eliminate the pathogen, and anti-inflammatory signals limiting damage to the host. IL-10 is a potent immunosuppressive cytokine whose impact is determined by the timing and localization of release. We show that NK cells rapidly express IL-10 during acute infection with diverse rapidly disseminating pathogens. The proinflammatory cytokine IL-12 was necessary and sufficient for NK cell induction of IL-10. NK cells from mice with systemic parasitic infection inhibited dendritic cell release of IL-12 in an IL-10-dependent manner, and NK cell depletion resulted in elevated serum IL-12. These data suggest an innate, negative feedback loop in which IL-12 limits its own production by eliciting IL-10 from NK cells. In contrast to disseminating pathogens, locally restricted infections did not elicit NK cell IL-10. Thus systemic infections uniquely engage NK cells in an IL-10-mediated immunoregulatory circuit that functions to alleviate inflammation.

With age, T-cell generation from the thymus is much reduced, yet a substantial naïve T-cell pool is maintained even in aged animals, suggesting that naïve T cells either persist longer or turn over faster to maintain T-cell homeostasis. We found that with age, naïve CD4 T cells became progressively longer-lived. Their longer lifespan did not depend on recognition of self-peptide/class II. Newly generated naïve T cells derived from aged stem cells had a shorter lifespan, like that of young naïve T cells. Conversely, naïve CD4 T cells derived from middle-aged thymectomized mice were longer-lived in vivo, and their development of functional defects was accelerated. These observations suggest that naïve T cells develop their longer lifespan during their sojourn in the periphery. Increased longevity of naïve CD4 T cells correlated well with reduced expression of proapoptotic molecule Bim. We suggest that the intrinsic increase in longevity helps maintain naïve T-cell homeostasis but facilitates the development of functional defects in mice.

There are well-characterized age-related changes in the peripheral repertoire of CD8 T cells characterized by reductions in the ratio of naive:memory T cells and the development of large clonal expansions in the memory pool. In addition, the TCR repertoire of naive T cells is reduced with aging. Because a diverse repertoire of naive T cells is essential for a vigorous response to new infections and vaccinations, there is much interest in understanding the mechanisms responsible for declining repertoire diversity. It has been proposed that one reason for declining repertoire diversity in the naive T cell pool is an increasing dependence on homeostatic proliferation in the absence of new thymic emigrants for maintenance of the naive peripheral pool. In this study, we have analyzed the naive CD8 T cell repertoire in young and aged mice by DNA spectratype and sequence analysis. Our data show that naive T cells from aged mice have perturbed spectratype profiles compared with the normally Gaussian spectratype profiles characteristic of naive CD8 T cells from young mice. In addition, DNA sequence analysis formally demonstrated a loss of diversity associated with skewed spectratype profiles. Unexpectedly, we found multiple repeats of the same sequence in naive T cells from aged but not young mice, consistent with clonal expansions previously described only in the memory T cell pool. Clonal expansions among naive T cells suggests dysregulation in the normal homeostatic proliferative mechanisms that operate in young mice to maintain diversity in the naive T cell repertoire.

Yersinia pestis causes pneumonic plague, an exceptionally virulent disease for which we lack a safe and effective vaccine. Antibodies specific for the Y. pestis F1 and LcrV proteins can protect mice against pulmonary Y. pestis infection. We demonstrate that neutralizing tumor necrosis factor-alpha (TNFalpha) and gamma-interferon (IFNgamma) abrogates this protection at sub-optimal levels of F1- or LcrV-specific antibody, but not at optimal levels. Moreover, we demonstrate that endogenous TNFalpha and IFNgamma confer measurable protection in the complete absence of protective antibodies. These findings indicate that antibodies and cytokines independently protect against pneumonic plague and suggest that surrogate assays for plague vaccine efficacy should consider both the level of vaccine-induced antibody and the capacity of vaccine recipients to produce TNFalpha and IFNgamma upon exposure to Y. pestis.

CD8+ T cells are a major source of IFN-γ, a key effector cytokine in immune responses against many viruses and protozoa. Although the transcription factor T-bet is required for IFN-γ expression in CD4+ T cells, it is reportedly dispensable in CD8+ T cells, where the transcription factor Eomesodermin is thought to be sufficient. The diverse functions of IFN-γ are mediated through the IFN-γR and STAT1. In CD4+ T cells, STAT1 appears to be critical for the activation of T-bet and IFN-γ, suggesting an IFN-γ-dependent positive feedback loop. However, STAT1 can also be activated by other cytokines, including IL-27. In the present study we show that, in contrast to in vitro conditions and the prevailing paradigm, T-bet is critical for the in vivo IFN-γ production by CD8+ T cells upon infection of mice with diverse pathogens. Whereas IFN-γR signals are dispensable for the T-bet-dependent IFN-γ production, direct IL-27Rα signals are critical.

Mice lacking functional CD1d genes were used to study mechanisms of resistance to the protozoan parasite Toxoplasma gondii. Wild-type (WT) BALB/c mice, CD1d-deficient BALB/c mice, and WT C57BL/6 mice all survived an acute oral infection with a low dose of mildly virulent strain ME49 T. gondii cysts. In contrast, most CD1d-deficient C57BL/6 mice died within 2 wk of infection. Despite having parasite burdens that were only slightly higher than WT mice, CD1d-deficient C57BL/6 mice displayed greater weight loss and intestinal pathology. In C57BL/6 mice, CD4+ cells can cause intestinal pathology during T. gondii infection. Compared with WT mice, infected CD1d-deficient C57BL/6 mice had higher frequencies and numbers of activated (CD44high) CD4+ cells in mesenteric lymph nodes. Depletion of CD4+ cells from CD1d-deficient mice reduced weight loss and prolonged survival, demonstrating a functional role for CD4+ cells in their increased susceptibility to T. gondii infection. CD1d-deficient mice are deficient in Vα14+ T cells, a major population of NKT cells. Involvement of these cells in resistance to T. gondii was investigated using gene-targeted Jα18-deficient C57BL/6 mice, which are deficient in Vα14+ T cells. These mice did not succumb to acute infection, but experienced greater weight loss and more deaths than B6 mice during chronic infection, indicating that Vα14+ cells contribute to resistance to T. gondii. The data identify CD4+ cells as a significant component of the marked susceptibility to T. gondii infection observed in CD1d-deficient C57BL/6mice, and establish T. gondii as a valuable tool for deciphering CD1d-dependent protective mechanisms.

Prior studies have demonstrated that B cells are important components of protection in vaccinated mice challenged intraperitoneally with a highly virulent type I strain of Toxoplasma gondii parasites. However, it is not known whether B cells are required for vaccinated mice to successfully resist a more physiologically relevant challenge infection with a mildly virulent type II strain of T. gondii. To investigate that question, we vaccinated B cell-deficient C57BL/6 (μMT) mice with an attenuated strain of T. gondii and challenged them with a potentially lethal oral dose of type II T. gondii cysts. Vaccinated μMT mice survived the challenge as well as vaccinated B6 controls, controlled parasites equally well in critical tissues, produced equivalent levels of mRNA for several type 1 cytokines, and exhibited comparably mild histopathology. Thus, a vaccine can protect against infection with a mildly virulent type II strain of T. gondii in the absence of a B cell-dependent immune response.

Fibrinogen-like protein 2 (Fgl2, fibroleukin) is a leukocyte product that exhibits significant homology to secreted proteins of diverse function, including growth factors, lectins, and components of extracellular matrix. Prior studies found that Fgl2 is IFNγ-inducible, possesses direct coagulant activity, and inhibits T cell proliferation and dendritic cell maturationin vitro. Here, we demonstrate that Fgl2 expression is up-regulated during type 1 immunityin vivoand establish that such up-regulation is IFNγ-, signal transducer and activation of transcription protein 1-, and IFN response factor 1-dependent. To investigate functional roles for Fgl2 during type 1 immunity, we generated Fgl2-deficient mice. Those animals are born at predicted Mendelian frequencies, appear overtly healthy, and contain normal numbers and frequencies of lymphoid cells. Although Fgl2 is IFNγ-inducible and putatively regulates T cell activation/proliferation, we demonstrate that Fgl2-deficient and control mice exhibit similar degrees of T cell expansion, immunopathology, and/or pathogen burdens during protozoan (Toxoplasma gondii), bacterial (Yersinia enterocolitica, Listeria monocytogenes, andMycobacterium tuberculosis), and viral (murine γ-herpesvirus-68 and Sendai) infections. Fgl2-deficient mice also reject allografts with similar kinetics as control mice. Moreover, despite prior reports that Fgl2 functions as a procoagulant enzyme, we demonstrate that Fgl2-deficient and control mice produce similar levels of fibrin, a product of the coagulation cascade, duringT. gondiiinfection and allograft rejection. Together, our findings suggest that Fgl2, although highly conserved and IFNγ-inducible, is not a critical mediator of either type 1 immunity or immune-associated coagulant activity.

It is often stated that impaired immune functions in the aged underlie their greater susceptibility to infections. Indeed, in many experimental settings, T-cell responses in aged mice have been shown to be deficient compared with those from young adults. Nonetheless, there are very few examples where a greater susceptibility to infection in aged mice has been demonstrated to result from impaired T-cell function. The clinical importance of understanding the basis for increased susceptibility to infection that accompanies advanced age dictates a need for experimental models with which to study the effect that aging has on immunological resistance to infection. This study was undertaken to investigate whether aged mice were less resistant than young adult control mice to infection with the fungus Cryptococcus neoformans . After a primary intravenous challenge with yeast, aged mice died sooner and developed higher organ burdens of yeast than did young adults. Deficient in vitro responses were observed in T cells from aged mice; however, greater susceptibility to intravenous infection appeared not to result from less effective T-cell-dependent resistance in vivo. In fact, T-cell-replete aged mice were more susceptible to intravenous cryptococcal infection than were T-cell-depleted young adults. Furthermore, aged mice were as resistant to primary pulmonary challenge with Cryptococcus as were young adults. Similarly, vaccinated aged mice were as resistant to rechallenge as were young adult counterparts. Therefore, despite demonstrably deficient in vitro responses of T cells from aged mice, their T-cell-dependent resistance to C. neoformans is as effective as that of young adults.

The early induction of interleukin (IL)-12 is a critical event in determining the development of both innate resistance and adaptive immunity to many intracellular pathogens. Previous in vitro studies have suggested that the macrophage (MPhi) is a major source of the initial IL-12 produced upon microbial stimulation and that this response promotes the differentiation of protective T helper cell 1 (Th1) CD4+ lymphocytes from precursors that are primed on antigen-bearing dendritic cells (DC). Here, we demonstrate by immunolocalization experiments and flow cytometric analysis that, contrary to expectation, DC and not MPhi are the initial cells to synthesize IL-12 in the spleens of mice exposed in vivo to an extract of Toxoplasma gondii or to lipopolysaccharide, two well characterized microbial stimulants of the cytokine. Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand. IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells. The capacity of splenic DC but not MPhi to synthesize de novo high levels of IL-12 within hours of exposure to microbial products in vivo, as well as the ability of the same stimuli to induce migration of DC to the T cell areas, argues that DC function simultaneously as both antigen-presenting cells and IL-12 producing accessory cells in the initiation of cell-mediated immunity to intracellular pathogens. This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.

The ts-4 strain of Toxoplasma gondii is a temperature-sensitive mutant that fails to grow at 40 degrees C in vitro. Unlike mildly virulent cyst-forming strains, which can cause fatal chronic infections in certain mouse strains, ts-4 has been widely used to vaccinate mice against virulent T. gondii and is a valuable tool with which to investigate mechanisms of acquired resistance to this parasite. In this report, the basis for the avirulence of ts-4 is analyzed. It is shown that ts-4 is able to persist long-term in vivo in mildly immunocompromised mice, which rules out an intrinsic growth defect as a reason for avirulence. ts-4 does not induce body temperatures in mice as high as that needed to kill it in vitro. Moreover, the mild fevers elicited in resistant B6 mice are also seen in susceptible C57BL/6 scid/scid mice. However, ts-4 elicits strong preimmune defenses, dependent on gamma interferon, which are needed by mice to survive acute infection. Furthermore, CD4+ and CD8+ T-cell-dependent acquired immunity is essential for long-term survival of ts-4-infected mice.

Studies were performed to determine whether resistance to acute Toxoplasma gondii infection in mice depends on a mechanism involving CR3, the type 3 complement receptor. Nineteen of 22 mice (86%) given multiple injections of the anti-CR3 monoclonal antibody, 5C6, prior to and after intraperitoneal inoculation of cysts of the ordinarily mildly virulent ME49 strain of T. gondii died within 8 to 12 days, whereas control antibody-treated mice survived. All (five of five) anti-CR3-treated BALB/c mice infected via the natural peroral route died within 8 days of infection. Flow cytometric analysis of cells recovered from peritoneal lavages of anti-CR3-treated T. gondii-infected mice revealed that the percentage of Thy-1+ CD4- CD8- cells was reduced to about 50% of that of control antibody-treated mice and to about 20% of the number of such cells in controls. The numbers of macrophages, polymorphonuclear leukocytes, and lymphocytes recovered from the peritoneal cavities of T. gondii-infected mice were all reduced in anti-CR3-treated mice to about 40% of those of controls. In addition, anti-CR3-treated mice had less than 25% of the induced NK cell activity of the controls, and gamma interferon was reduced to undetectable levels. Thus, the rapid death of anti-CR3-treated mice was probably caused by impaired preimmune defenses. Histological examination of anti-CR3-treated T. gondii-infected mice revealed extensive liver pathology compared with that of infected mice given a control antibody or uninfected mice given anti-CR3. The inflammation, degeneration, and necrosis in most of the anti-CR3-treated mice were severe enough to account for the observed mortalities.

Although naive C.B-17 and BALB/cBy mice die of meningoencephalitis within 5 weeks of intravenous infection with an opportunistic strain of Cryptococcus neoformans, immunized mice express an acquired, CD4+ T-cell-dependent immunity and survive an intravenous infection. Infusion of lymphocytes from immune mice into severe combined immunodeficiency (SCID) mice renders these mice more resistant to cryptococcal brain infection than uninfused controls. We have investigated the role of gamma interferon (IFN-gamma) and tumor necrosis factor (TNF) in acquired resistance to C. neoformans. Neutralization of either IFN-gamma or TNF impaired resistance of immune BALB/cBy or C.B-17 mice to cryptococci. At 10 days postinfection, there were approximately 10 times as many yeast cells in the brains of mice treated with either anticytokine antibody as in the brains of mice treated with control antibody. Simultaneous neutralization of IFN-gamma and TNF further exacerbated infection. Neutralization of IFN-gamma or TNF also impaired resistance in immune lymphocyte-infused SCID mice, resulting in significantly higher yeast burdens in brains of cytokine-neutralized mice than in brains of controls. Concurrent neutralization of IFN-gamma and TNF rendered SCID recipients of immune cells equivalent to uninfused SCID mice with respect both to brain yeast burdens at 10 days and to survival. Anti-TNF treatment alone also curtailed survival. Histological examination of the brains of cytokine-neutralized mice revealed deficiencies in ability to focus inflammatory cells at brain lesions. These data demonstrate that both IFN-gamma and TNF are important mediators of acquired resistance to cryptococcal meningoencephalitis.

A murine model of Toxoplasma gondii infection was used to investigate whether gamma delta T cells are required to resist primary acute toxoplasmosis. The surface phenotype of peritoneal exudate and spleen cells obtained from T. gondii-infected mice was examined using anti-gamma delta T cell-specific reagents and flow cytometry. Survival of mice depleted of gamma delta T cells was also followed during the acute phase of toxoplasmosis. Numbers of gamma delta T cells did not increase in spleen or peritoneal exudates of mice infected with T. gondii. Moreover, infected mice depleted of gamma delta T cells survived as long as untreated infected mice. These results indicate that gamma delta T cells do not play an important role in host defense against T. gondii infection in mice.

Influenza causes >250,000 deaths annually in the industrialized world, and bacterial infections frequently cause secondary illnesses during influenza outbreaks, including pneumonia, bronchitis, sinusitis, and otitis media. In this study, we demonstrate that cross-reactive immunity to mismatched influenza strains can reduce susceptibility to secondary bacterial infections, even though this fails to prevent influenza infection. Specifically, infecting mice with H3N2 influenza before challenging with mismatched H1N1 influenza reduces susceptibility to either Gram-positive Streptococcus pneumoniae or Gram-negative Klebsiella pneumoniae. Vaccinating mice with the highly conserved nucleoprotein of influenza also reduces H1N1-induced susceptibility to lethal bacterial infections. Both T cells and Abs contribute to defense against influenza-induced bacterial diseases; influenza cross-reactive T cells reduce viral titers, whereas Abs to nucleoprotein suppress induction of inflammation in the lung. These findings suggest that nonneutralizing influenza vaccines that fail to prevent influenza infection may nevertheless protect the public from secondary bacterial diseases when neutralizing vaccines are not available.

In mice infected sublethally with Listeria monocytogenes , fibrin is deposited at low levels within hepatic tissue, where it functions protectively by limiting bacterial growth and suppressing hemorrhagic pathology. Here we demonstrate that mice infected with lethal doses of L. monocytogenes produce higher levels of fibrin and display evidence of systemic coagulopathy (i.e., thrombocytopenia, fibrinogen depletion, and elevated levels of thrombin-antithrombin complexes). When the hepatic bacterial burden exceeds 1 × 10 6 CFU, levels of hepatic fibrin correlate with the bacterial burden, which also correlates with levels of hepatic mRNA encoding the hemostatic enzyme factor XI (FXI). Gene-targeted FXI-deficient mice show significantly improved survival upon challenge with high doses of L. monocytogenes and also display reduced levels of hepatic fibrin, decreased evidence of coagulopathy, and diminished cytokine production (interleukin-6 [IL-6] and IL-10). While fibrin limits the bacterial burden during sublethal listeriosis in wild-type mice, FXI-deficient mice display a significantly improved capacity to restrain the bacterial burden during lethal listeriosis despite their reduced fibrin levels. They also show less evidence of hepatic necrosis. In conjunction with suboptimal antibiotic therapy, FXI-specific monoclonal antibody 14E11 improves survival when administered therapeutically to wild-type mice challenged with high doses of L. monocytogenes. Together, these findings demonstrate the utility of murine listeriosis as a model for dissecting qualitative differences between protective and pathological host responses and reveal novel roles for FXI in exacerbating inflammation and pathogen burden during a lethal bacterial infection.

It is well established that resistance to acute primary Toxoplasma gondii infection is mediated by a gamma interferon (IFN-gamma)-dependent mechanism. The present in vivo experiments were undertaken to investigate the cellular basis for this resistance. We show here that immunocompetent T. gondii-infected C57BL/6 (B6) mice treated with anti-IFN-gamma or with anti-Thy-1 or anti-asialo-GM1 antibodies die sooner than infected mice treated with antibodies that deplete both CD4+ and CD8+ T lymphocytes. Thy-1+ CD4- CD8- cells accumulated in the peritoneal cavities of B6 mice during the early stages of an intraperitoneal infection but did not accumulate in sham-infected control mice, and substantial numbers of Thy-1+ CD4- CD8- cells were recovered from the peritoneal cavities of infected B6 mice treated with antibodies that depleted CD4+ and CD8+ lymphocytes. Depletion of Thy-1+ cells reduced IFN-gamma to undetectable levels, whereas depletion of CD4+ and CD8+ cells did not reduce IFN-gamma levels. Thus T. gondii infection in immunocompetent B6 mice elicits Thy-1+ CD4- CD8- cells which either produce protective IFN-gamma themselves or control its production by other cells. It is likely that the function of these Thy-1+ CD4- CD8- cells is to control T. gondii tachyzoites during the early stages of primary infection before specific CD4(+)- and/or CD8(+)-dependent immunity develops.

Rejection of H-Y-bearing primary skin grafts and generation of H-Y-specific cytolytic T cells by female mice requires the participation of both CD4+ and CD8+ T lymphocytes. Studies were conducted to investigate long-term tolerance of H-Y antigen induced in female mice by transiently depleting them of CD4+ and/or CD8+ T cells and, at the same time, giving them an injection of male lymphoid cells. We confirmed that after recovery of CD4+ to normal levels, female mice that had been transiently depleted of CD4+ cells and concurrently given an injection of male spleen cells were unable to generate H-Y-specific cytolytic T cells. Tolerance was also manifest by greatly extended survival (probably permanent in most cases) of male skin grafts. Further investigations revealed that female mice transiently depleted of CD8+ cells, and concurrently given an injection of male spleen cells, were similarly tolerant of H-Y antigen later when numbers of CD8+ T cells returned to normal. Moreover, small numbers of male cells were detectable in spleen and lymph nodes of tolerant females long after they had been given an injection of male cells and depleted of either CD4+ or CD8+ T cells, whereas no male cells were detected in (nontolerant) females given male cells and control antibodies. These findings show that tolerance of the relatively weak transplantation antigen, H-Y, can be achieved simply by giving male antigen-bearing spleen cells to the host while it is transiently depleted of a type of cell it needs in order to reject those cells, thus allowing the male cells to persist in the host. Furthermore, depletion of helper cells is not obligatory to achieve tolerance. It has been hypothesized that tolerance of H-Y antigen in females given male lymphoid cells while temporarily depleted of CD4+ lymphocytes results from unresponsiveness (anergy) induced in H-Y-specific CD8+ cells that are exposed to H-Y antigen in the absence of help from CD4+ cells. Interpretations of our findings are discussed in relation to this hypothesis.

Lymphodeficient scid/scid (SCID) mice died from acute infection with a strain of Toxoplasma gondii that causes chronic infection with mild symptoms in immunocompetent non-SCID mice. However, most SCID mice reconstituted with spleen cells from immunocompetent mice 1 month prior to T. gondii infection survived in good health after a transient period during which they appeared ill. Unreconstituted SCID mice given sulfadiazine in their drinking water from day 10 of Toxoplasma infection onward survived the acute phase of infection and lived for many weeks without overt symptoms. Histological examination revealed Toxoplasma cysts in their brains. However, if sulfadiazine was withdrawn from the drinking water of these chronically infected SCID mice, the mice died within 1 week with large numbers of trophozoites throughout their brains. These findings establish SCID mice as a potentially useful resource with which to study various aspects of immunological control of T. gondii infection during either its acute or chronic phase. Furthermore, the ability to produce chronic infections with avirulent T. gondii in SCID mice and to cause acute relapsing infections at will suggests that SCID mice may be helpful in evaluating potential therapies for acute and chronic T. gondii infections in immunocompromised patients.

The involvement of tumor necrosis factor (TNF) in resistance to Toxoplasma gondii infection was examined by means of experiments in which mice were treated with anti-TNF antibodies prior to infection with ME49, a low-virulence Toxoplasma strain. In (BALB/cBy x C57BL/6J)F1 (CB6F1) mice, which are highly resistant to intraperitoneal (i.p.) infection with T. gondii ME49, 10(4) neutralizing units of anti-TNF caused a significant increase in trophozoite numbers in the peritoneal cavities of infected mice and transient signs of illness but no deaths. i.p. infection of anti-TNF-treated C57BL/6J (B6) mice, which are more susceptible to T. gondii and develop a chronic progressive toxoplasmosis, resulted in death for some of the mice. If the mice were infected perorally, however, and treated with anti-TNF, mortality was extensive in B6 mice but not in CB6F1 mice. Although it was not detected in their sera, TNF was found in the peritoneal fluids of i.p.-infected CB6F1 and B6 mice. Endogenously produced TNF thus appears to be an important mediator of resistance to T. gondii.

Vaccinating with live, conditionally attenuated, pigmentation (Pgm)-deficient Yersinia pestis primes T cells that protect mice against pneumonic plague. However, Pgm-deficient strains are not considered safe for human use because they retain substantial virulence in animal models. Y. pestis strains engineered to express Escherichia coli LpxL are avirulent owing to constitutive production of lipopolysaccharide with increased Toll-like receptor 4-activating ability. We generated an LpxL-expressing Pgm-deficient strain (D27-pLpxL) and demonstrate here that this avirulent strain retains the capacity to prime protective T cells. Compared with unvaccinated controls, mice immunized intranasally with live D27-pLpxL exhibit a decreased bacterial burden and increased survival when challenged intranasally with virulent Y. pestis . T cells provide a substantial degree of this protection, as vaccine efficacy is maintained in B-cell-deficient μMT mice unless those animals are depleted of CD4 and CD8 T cells at the time of challenge. Upon challenge with Y. pestis , pulmonary T-cell numbers decline in naive mice, whereas immunized mice show increased numbers of CD44 high CD43 high effector T cells and T cells primed to produce tumor necrosis factor alpha and gamma interferon; neutralizing these cytokines at the time of challenge abrogates protection. Immunization does not prevent dissemination of Y. pestis from the lung but limits bacterial growth and pathology in visceral tissue, apparently by facilitating formation of granuloma-like structures. This study describes a new model for studying T-cell-mediated protection against pneumonic plague and demonstrates the capacity for live, highly attenuated, Y. pestis vaccine strains to prime protective memory T-cell responses safely.

Natural regulatory T cells constitutively express the IL-2Rα chain (CD25) on their surface. Consequently, administration of anti-CD25 antibodies is a commonly utilized technique to deplete regulatory T cell populations in vivo. However, activated effector T cells may also transiently express CD25, and are thus also potential targets for anti-CD25 antibodies. In this study utilizing Toxoplasma gondii as a model pro-inflammatory infection we have examined the capacity of anti-CD25 antibodies to target effector T cell populations during an inflammatory episode, to determine to what extent that this may modulate the outcome of disease. Anti-CD25 antibody treated C57BL/6 mice displayed significantly reduced CD4+ T cell IFN-γ production during acute T. gondii infection and exhibited reduced weight loss and liver pathology during early acute infection; aspects of infection previously associated with effector CD4+ T cell responses. In agreement, anti-CD25 antibody administration impaired parasite control and caused mice to succumb to infection during late acute/early chronic stages of infection with elevated tissue parasite burdens. In contrast, anti-CD25 antibody treatment of mice with established chronic infections did not markedly affect brain parasite burdens, suggesting that protective T cell populations do not express CD25 during chronic stages of T. gondii infection. In summary, we have demonstrated that anti-CD25 antibodies may directly abrogate effector T cell responses during an inflammatory episode, highlighting important limitations of the use of anti-CD25 antibody administration to examine regulatory T cell function during inflammatory settings.

The hypothesis was investigated that suppressor cells may be responsible for maintenance of immunologic tolerance of a minor H3 antigen in mice that express the antigen naturally. Lymphoid cell populations from B6.C-H-24c (HW54) mice, a congenic-resistant strain histoincompatible with H-24b-expressing C57BL/6 (B6) mice only with respect to the H-24 locus, were examined in cell-transfer experiments to see if they contained naturally arising H-24c-specific suppressor cells. The H-24 antigen was chosen for these studies because, unlike most other minor and major histocompatibility (H) antigens, it is not detectable on mature lymphoid cells by any of several functional criteria. Thus transfer of HW54 lymphoid cells to B6 hosts could be done without the complication of inducing hyporesponsiveness de novo in the host, as occurs with other minor H antigens that are expressed on lymphocytes. B6 hosts were given HW54 skin grafts along with HW54 lymphoid cells to assess their tolerance of the H-24c-encoded antigen. The hosts were either (1) normal, nonimmune B6 mice; (2) B6 mice rendered immunodeficient by thymectomy and irradiation (TXB) and repopulated with H-24c-immune B6 lymphocytes; or (3) TXB B6 hosts repopulated with nonimmune B6 lymphocytes. In each case it was found that the additionally infused HW54 lymphoid cells did not suppress the ability of these hosts to reject HW54 skin grafts. In other words, HW54 lymphoid cells appear not to possess suppressive activity specific for the H-24c antigen that might maintain antigen-specific natural tolerance. Additional experiments were performed to determine whether HW54 lymphoid cells can inhibit the ability of sublethally irradiated B6 mice to regain the capacity to reject HW54 skin. The purpose of these experiments was to determine whether there are naturally occurring HW54 lymphoid cells that can suppress the development of lymphocytes potentially reactive to H-24c antigen that presumably emerge in B6 mice following sublethal irradiation. Such suppressor cells would be candidate initiators of self-tolerance. Here, too, no evidence for such suppressor cells was obtained.

Summary. Background: Inflammatory cytokines potently impact hemostatic pathways during infection, but the tissue-specific regulation of coagulation and fibrinolysis complicates studies of the underlying mechanisms. Methods and Results: Here, we describe assays that quantitatively measuring prothrombinase (PTase), protein C-ase (PCase) and plasminogen activator (PA) activities in situ, thereby facilitating studies of tissue-specific hemostasis. Using these assays, we investigate the mechanisms regulating hepatic fibrin deposition during murine toxoplasmosis and the means by which interferon-gamma (IFN-γ) suppresses infection-stimulated fibrin deposition. We demonstrate that Toxoplasma infection upregulates hepatic PTase, PCase, and PA activity. Wild type and gene-targeted IFN-γ-deficient mice exhibit similar levels of infection-stimulated PTase activity. By contrast, IFN-γ-deficiency is associated with increased PCase activity and reduced PA activity during infection. Parallel analyses of hepatic gene expression reveal that IFN-γ-deficiency is associated with increased expression of thrombomodulin (TM), a key component of the PCase, increased expression of thrombin-activatable fibrinolysis inhibitor (TAFI), a PC substrate, and reduced expression of urokinase PA (u-PA). Conclusions: These findings suggest that IFN-γ suppresses infection-stimulated hepatic fibrin deposition by suppressing TM-mediated activation of TAFI, thereby destabilizing fibrin deposits, and concomitantly increasing hepatic u-PA activity, thereby promoting fibrinolysis. We anticipate that further application of these in situ assays will improve our understanding of tissue-specific hemostasis, its regulation by cytokines, and its dysregulation during coagulopathy.

While coagulation often causes pathology during infectious disease, we recently demonstrated that fibrin, a product of the coagulation pathway, performs a critical protective function during acute toxoplasmosis (L. L. Johnson, K. N. Berggren, F. M. Szaba, W. Chen, and S. T. Smiley, J. Exp. Med.197:801-806, 2003). Here, we investigate the mechanisms regulating the formation of this protective fibrin. Through comparisons ofToxoplasma-infected wild-type and cytokine-deficient mice we dissociate, for the first time, the relative fibrin-regulating capacities of pathogen products, host cytokines, and infection-stimulated hemorrhage. Remarkably, neither the pathogen burden nor hemorrhage is a primary regulator of fibrin levels. Rather, two type 1 cytokines exert dominant and counterregulatory roles: tumor necrosis factor alpha (TNF-α), acting via the type 1 TNF-α receptor, promotes fibrin deposition, while gamma interferon (IFN-γ), acting via STAT1 and IFN-γ receptors expressed on radioresistant cells, suppresses fibrin deposition. These findings have important clinical implications, as they establish that cytokines known to regulate pathological coagulation also dictate levels of protective fibrin deposition. We present a novel model depicting mechanisms by which the immune system can destroy infected tissue while independently restraining hemorrhage and promoting tissue repair through the deliberate deposition of protective fibrin.

Bacterial infections are major causes of human mortality. The activation of coagulation pathways leading to the deposition of insoluble fibrin frequently accompanies bacterial infection, and much attention has focused upon the pathological attributes of infection-stimulated fibrin deposition. Nevertheless, here we present conclusive evidence that infection-stimulated fibrin deposition can perform critical protective functions during bacterial infection. Specifically, we demonstrate that coagulation-impaired fibrin(ogen)-deficient mice, in comparison with genetically matched control mice, display increased mortality upon peritoneal infection with the gram-positive facultative intracellular bacteriumListeria monocytogenes. To distinguish effects of fibrinogen from those of fibrin, we treat wild-type mice with warfarin, an anticoagulant that suppresses fibrin formation without impacting fibrinogen levels. Warfarin treatment exacerbates listeriosis, suggesting that fibrin is the key mediator of protection. With regard to the underlying protective mechanisms, we demonstrate that fibrin(ogen) suppresses anemia, reduces hemorrhagic pathology, and limits bacterial growth during listeriosis. Despite confirming a prior report that fibrin(ogen) promotes the peritoneal clearance of the extracellular bacteriumStaphylococcal aureus, we demonstrate that fibrin(ogen) plays little role in controlling peritoneal numbers ofL. monocytogenesbacteria or the dissemination ofL. monocytogenesbacteria from the peritoneal cavity. Rather, fibrin(ogen) primarily limits the growth of these intracellular bacteria within hepatic tissue. While the pathological potential of excessive infection-stimulated fibrin deposition is well appreciated, our findings reveal that fibrin can function protectively, via multiple mechanisms, during bacterial infection.

The expression of IFN-γ is a hallmark of Th1 cells and CD8+ effector T cells and is the signature cytokine of type 1 responses. However, it is not known whether T cells are homogeneous in their capacity to produce IFN-γ, whether this potential varies between tissues, and how it relates to the production of other effector molecules. In the present study we used bicistronic IFN-γ-enhanced yellow fluorescent protein (IFN-γ-eYFP) reporter mice (Yeti) and MHC class I tetramers to directly quantify IFN-γ expression at the single cell level. The eYFP fluorescence of Th1 cells and CD8+ effector T cells was broadly heterogeneous even before cell division and correlated with both the abundance of IFN-γ transcripts and the secretion of IFN-γ upon stimulation. CD4+ and CD8+ T cells of influenza-infected mice revealed a similarly heterogeneous IFN-γ expression, and eYFPhigh cells were only found in the infected lung. Ag-specific T cells were in all examined tissues eYFP+, but also heterogeneous in their reporter fluorescence, and eYFPhigh cells were also restricted to the infected lung. A similar heterogeneity was observed in Toxoplasma gondii-infected animals, but eYFPhigh cells were restricted to different tissues. Highly eYFP fluorescent cells produced elevated levels of proinflammatory cytokines and chemokines in addition to IFN-γ, suggesting their coregulated expression as a functional unit in highly differentiated effector T cells.

ES-62, a filarial nematode-derived anti-inflammatory immunomodulator, was administered to mice in an attempt to prevent pathology associated with Toxoplasma gondii infection. The nematode product was shown to elevate mitogen and T. gondii-specific IL-10 production but was unable to inhibit Th1 responses. Consequently ES-62 could not prevent Th1 generated immunopathology.

Background Ageing has been shown to reduce CD8 T cell repertoire diversity and immune responses against influenza virus infection in mice. In contrast, less is known about the impact of ageing on CD4 T cell repertoire diversity and immune response to influenza virus infection. Results The CD4 T cell response was followed after infection of young and aged C57BL/6 mice with influenza virus using a tetramer specific for an immunodominant MHC class II epitope of the influenza virus nucleoprotein. The appearance of virus-specific CD4 T cells in the lung airways of aged mice was delayed compared to young mice, but the overall peak number and cytokine secretion profile of responding CD4 T cells was not greatly perturbed. In addition, the T cell repertoire of responding cells, determined using T cell receptor Vβ analysis, failed to show the profound effect of age we previously described for CD8 T cells. The reduced impact of age on influenza-specific CD4 T cells was consistent with a reduced effect of age on the overall CD4 compared with the CD8 T cell repertoire in specific pathogen free mice. Aged mice that were thymectomized as young adults showed an enhanced loss of the epitope-specific CD4 T cell response after influenza virus infection compared with age-matched sham-thymectomized mice, suggesting that a reduced repertoire can contribute to impaired responsiveness. Conclusions The diversity of the CD4 T cell repertoire and response to influenza virus is not as profoundly impaired by ageing in C57BL/6 mice as previously shown for CD8 T cells. However, adult thymectomy enhanced the impact of ageing on the response. Understanding the impact of ageing on CD4 T cell responses to influenza virus infection is an important prerequisite for developing better vaccines for the elderly.

Although B cells produce cytokines it is not known whether B cells can differentiate into effector subsets that secrete polarized arrays of cytokines. We have identified two populations of "effector" B cells (Be1 and Be2) that produce distinct patterns of cytokines depending on the cytokine environment in which the cells were stimulated during their primary encounter with antigen and T cells. These effector B cell subsets subsequently regulate the differentiation of naive CD4+ T cells to TH1 and TH2 cells through production of polarizing cytokines such as interleukin 4 and interferon gamma. In addition, Be1 and Be2 cells could be identified in animals that were infected with pathogens that preferentially induce a Type 1 and Type 2 immune response. Together these results suggest that, in addition to their well defined role in antibody production, B cells may regulate immune responses to infectious pathogens through their production of cytokines.

T lymphocytes and gamma interferon (IFN-γ) are known mediators of immune resistance toToxoplasma gondiiinfection, but whether B cells also play an important role is not clear. We have investigated this issue using B-cell-deficient (μMT) mice. If vaccinated with attenuatedT. gondiitachyzoites, μMT mice are susceptible to a challenge intraperitoneal infection with highly virulent tachyzoites that similarly vaccinated B-cell-sufficient mice resist. Susceptibility is evidenced by increased numbers of parasites at the challenge infection site and by extensive mortality. The susceptibility of B-cell-deficient mice does not appear to be caused by deficient T-cell functions or diminished capacity of vaccinated and challenged B-cell-deficient mice to produce IFN-γ. Administration ofToxoplasma-immune serum, but not nonimmune serum, to vaccinated B-cell-deficient mice significantly prolongs their survival after challenge with virulent tachyzoites. Vaccinated mice lacking Fc receptors or the fifth component of complement resist a challenge infection, suggesting that neither Fc-receptor-dependent phagocytosis of antibody-coated tachyzoites nor antibody-dependent cellular cytotoxicity nor antibody-and-complement-dependent lysis of tachyzoites is a crucial mechanism of resistance. However,Toxoplasma-immune serum effectively inhibits the infection of host cells by tachyzoites in vitro. Together, the results support the hypothesis that B cells are required for vaccination-induced resistance to virulent tachyzoites in order to produce antibodies and that antibodies may function protectively in vivo by blocking infection of host cells by tachyzoites.

Although Cryptococcus neoformans causes serious infections in AIDS patients, cryptococcosis in immunologically immature infants, as in immunocompetent adults, is rare. To investigate the resistance of neonates to C. neoformans and to determine whether they could be efficiently vaccinated as neonates against challenge with the yeast as adults, the course of infection was monitored in the lungs of mice infected intranasally with yeast cells. Neonates were less able than adults to reduce yeast burdens less than 24 h postinoculation and less able to control the progressive growth of yeast over several weeks. However, in both neonates and adults, yeasts were substantially eradicated by 6 to 8 weeks after infection. Numbers of all classes of leukocytes recovered from lung lavages of infected neonates and adults were similar. Significant differences appeared only on day 14, when neonates had more neutrophils and adults had significantly more CD4+ CD45RB cells with low fluorescence intensity. When vaccinated neonates were rechallenged after reaching adulthood, they expressed resistance to C. neoformans as effectively as did mice immunized as adults and survived an intravenous challenge that was lethal for unimmunized controls. Thus, exposure of neonatal mice to viable C. neoformans yeast, which persists in the lungs for many weeks, does not result in immunological tolerance to a yeast challenge in adult mice, as predicted by immunological dogma, but instead immunizes them. Therefore, even in immunologically immature individuals, the immune system serves to protect against pathogens rather than simply to distinguish self from nonself.

The role of B cells in immunity to Cryptococcus neoformans was investigated. Genetically targeted, B-cell-deficient mice (mu Mt) examined at various times after intravenous infection with C. neoformans 184 had lung and brain yeast burdens that were equivalent to tissue burdens in control B-cell-sufficient mice. Both B-cell-deficient and B-cell-sufficient control mice were effectively vaccinated by a sublethal intratracheal instillation of strain 184 yeast against a systemic infection with the C. neoformans strain carrying ura5; vaccinated control and vaccinated B-cell-deficient mice had equivalent brain and lung burdens of the ura5 strain 10 days after intravenous rechallenge. Additionally, B-cell-deficient and B-cell-sufficient vaccinated mice survived an intravenous rechallenge with a dose of yeast cells which is normally lethal for unimmunized mice. In further studies of the role of B cells in murine cryptococcosis, SCID mice were reconstituted with lymphocytes from B-cell-deficient and B-cell-sufficient mice. SCID mice reconstituted with lymphocytes from vaccinated B-cell-deficient animals failed to express effective adoptive immunity to C. neoformans brain infection. In contrast, SCID mice reconstituted with lymphocytes from vaccinated B-cell-sufficient mice had 10-fold fewer yeast cells in their brains than did uninfused SCID controls. However, SCID mice given lymphocytes from B-cell-deficient immune donors had fewer yeast cells in their lungs than did uninfused controls. Fewer CD4+ lymphocytes were recovered at 7 and 11 days after infection from the peripheral blood and spleens of SCID mice reconstituted with lymphocyte suspensions from B-cell-deficient animals than from the peripheral blood and spleens of SCID mice reconstituted with suspensions from B-cell-sufficient control donors. These data suggest that B cells can play an important role in host defense against Cryptococcus in the brain under conditions in which T-cell-mediated immunity is impaired.

We have studied the resistance of Toxoplasma gondii-infected mice to subsequent infection with Cryptococcus neoformans. Mice infected with the moderately virulent ME49 strain of T. gondii are resistant to proliferation of yeast cells in their brains after intravenous inoculation of the serotype A C. neoformans strain 184. The resistance serves to limit proliferation of yeast cells that colonize the brain. Maximal levels of resistance correlate not with maximal systemic specific anti-Toxoplasma resistance but rather with high levels of inflammatory response, presumably to parasites released from cysts in the brain. Resistance is localized, as mice infected with ME49 show only limited resistance in their lungs after intratracheal instillation of yeast cells, but there is substantial protection against development of cerebral cryptococcosis.

Mice were exposed to the protozoan parasite Toxoplasma gondii in utero or were infected as neonates in order to identify and characterize resistance mechanisms that function protectively during the first weeks after birth. About one-half of the mice born of mothers fed T. gondii cysts at 11 days of gestation survived to weaning age or beyond. No effect of major histocompatibility complex (MHC) haplotype on early survival was observed in a group of backcross progeny; however, long-term survival was strongly dependent on MHC haplotype. The ability of mice infected as neonates to survive until weaning was found to depend on gamma interferon and on Thy-1+ cells but not on CD4+ or CD8+ cells. Mice that survived to maturity after infection as neonates were slightly more resistant to challenge with virulent T. gondii parasites than were sham-infected controls but were less resistant than were mice infected as adults. Together the results indicate the following. (i) Mice congenitally infected with T. gondii have a gamma interferon-dependent mechanism of early resistance that involves Thy-1+ cells but not CD4+ or CD8+ cells. (ii) This mechanism is not under MHC-linked genetic control. (iii) Mice that exhibit long-term survival after congenital infection acquire a modest degree of protection against reinfection with virulent organisms. (iv) The extent of long-term survival of congenitally infected neonates, like that in mice infected as adults, is influenced by MHC genes, presumably via MHC-restricted CD4+ and/or CD8+ cells.

Mice given a pretransplant i.v. infusion of spleen cells from donors incompatible with the recipients with respect to individual minor H antigens exhibit a delayed rejection of skin grafts from these incompatible donors. Attempts were made to detect suppressor T cells, possibly induced by the i.v. infusion of donor cells, among spleen cells from female mice made hyporesponsive to H-Y antigen by an i.v. infusion of male spleen cells. When transferred to nonimmune immunocompetent mice, lymphoid cells from hyporesponsive females delayed the rejection of male skin grafts only slightly. Similar results were obtained when putative suppressor cells were transferred to immunodeficient hosts having had their potential to reject male skin grafts restored with either nonimmune or specifically immune female lymphoid cells. The observed suppression of graft rejection could be accounted for, to a significant extent, by male spleen cells carried over with the transferred female cells to the assay recipients. Though an active host-derived suppressor mechanisms was not found, transferred spleen and lymph-node cells obtained from females given a prior i.v. infusion of male spleen cells were poor at restoring the capacity to reject male skin grafts to immunodeficient female recipients, whereas equal numbers of lymphoid cells from untreated females were much better. Thus, i.v. infusion of male spleen cells appeared to deplete the peripheral lymphoid tissue of the recipients of responsivity to H-Y antigen.

Aged individuals are more susceptible to certain infections than are young adults. To investigate the relative resistance capabilities of aged and young adult mice, responses that are induced within the first week of a Toxoplasma gondii infection, which are known to be involved in preimmune resistance, were compared in young adult and aged mice. Aged mice did not differ reproducibly from young adults in numbers of induced Thy-1? CD4- CD8- cells or interferon-y levels. Numbers of induced CD4+ and CD8+ T cells, associated with acquired immunity, were as high in aged mice as in young adults. Natural killer cell activity, although induced to a high level, was lower in aged mice. Aged mice thus are capable of inducing a mechanism of preimmune resistance to T. gondii and presumably other infectious agents. Nonetheless, aged mice died within 8-12 days after intraperitoneal or peroral inoculation of 500 T. gondii cysts, whereas young adult mice survived. Causes other than an age- related impairment in preimmune resistance mechanisms are apparently responsible for the increased susceptibility of aged mice to T. gondii infection. It has been known for some time that senescent individuals may be more susceptible to some infections than are young adults (reviewed in Gardner, 1980). Possibly contributing to this increased susceptibility is the decline, with advanced age, in various immune response capabilities measured in vitro and in vivo (Makinodan and Kay, 1980; Thoman and Weigle, 1989). However, it is not often clear to which infectious agents these age-associated deficiencies are relevant or whether they are suf- ficiently severe to cause increased susceptibility to infection. Others have reported, and we confirm in this study, that aged mice inoculated with a large number of Toxoplasma gondii cysts from a mildly virulent strain die in less than 2 wk (Gardner and Remington, 1977; Emmerling et al., 1979), whereas young adults survive the same treatment. However, if given anti-interfer

An expression is derived for the probability that a congenic strain and its inbred partner will differ by multiple linked histocompatibility genes when the number of incompatibilities between the original donor and background strains is known and the number of backcrosses to produce the strain is specified. This probability is shown to be rather large for typical values of the pertinent variables. Potential complications in data interpretation arising from unsuspected multiple incompatibilities between a congenic pair are discussed in relation to antigenic strength, H-locus polymorphism tissue distribution of H antigens, and certain types of experiments in which the cell-mediated lympholysis assay is used.

Male-specific antigen expression by XY teratocarcinomas PCC7 and 7' is demonstrated first by the rejection of tumors by female but not by male mice following challenge with these cell lines. Male-specific antigen expression is confirmed by an indirect method in which females are immunized against H-Y antigen by male skin grafts. A variant of PCC7 lacking male-specific antigen expression is described. Analysis of the karyotype and of the DNA from this variant indicate that the loss of male-specific antigen expression is a result of the loss of the Y chromosome. The ability to recover variants that have lost expression of male-specific antigen opens the possibility of their selection after mutagenesis.

Twenty-five congenic mouse strains differing at distinguishable minor (non-H-2) histocompatibility loci were paired in 71 different combinations. F1 offspring were used as skin-graft donors for more than 4000 recipients to test whether immune responses to parental strain antigens were statistically independent. Thirty-four (48 percent) of the 71 combinations were predicted adequately by an independent response hypothesis. A simple additive model was consistent with 39 (55 percent) of the observed responses, although 18 of these were among those in agreement with the independent hypothesis. A synergistic response faster than that predicted by either the independent or additive response model was seen in 12 (17 percent) of the combinations. The remaining 5 percent were not well described by any of these models. No strain was represented with unusual frequency among those involved in synergistic interactions.

Karyotypically XY individuals of the C57BL/6J-YPOS mouse stock develop as females or hermaphrodites, but never as normal males. The aberrant sexual development results from the interaction of the C57BL/6J genetic background with the M. poschiavinus-derived Y chromosome. XY females from this stock were assayed for H-Y antigen. By the criteria of skin-grafting, the cell-mediated lympholysis test, and the popliteal lymph node assay, these XY females are antigenically indistinguishable from normal C57BL/6 males. Implications for the hypothesis that H-Y antigen induces formation of the mammalian testis are discussed.

A skin grafting protocol is described for finding H-Y antigenic variants. The method is applicable regardless of the location of the structural gene(s) for this antigen (X, Y, or autosomal). Use of this protocol revealed no evidence for H-Y antigenic variation between C57BL/6J and strains 129/J, A.BY/SnJ, C3H.SW/SnJ, and LP/J.

SUMMARYTheT/t–H-2region on mouse chromosome 17 is known from complex natural variants (‘t-haplotypes’) to contain numerous genes, including some affecting the immune system and the development of the embryo. Rapid progress in the isolation of recombinant DNA clones for this 50 megabasepair region is generating the material for its complete molecular anatomy. A crucial step in revealing the biological functions controlled by the region is to obtain mutants in which genes are inactivated individually. We have used a pair of inbred mouse strains and a series of classical breeding schemes that permit the detection of recessive lethal and detrimental mutations in theT/t–H-2region.In this initial phase of our study, 280 gametes mutagenized in the male germ line by ethylnitrosourea (ENU) have yielded eleven independent pre-natal recessive lethal mutations. Four have been mapped againstTmutations and have been shown to complement one another in all pairwise combinations.