Sequential analysis of T cells in the liver during murine listerial infection.

Phenotypes and functions of T cells in the liver were studied after an i.p. inoculation with viable Listeria monocytogenes in mice. T cells in the liver of untreated C3H/HeN mice (C3H; H-2k, Mls-2a) contain Thy-1.2+TCR-alpha beta + cells as a majority and Thy-1.2+TCR-gamma delta + cells and Thy-1.2-TCR-gamma delta + cells as minorities. The liver of untreated C3H mice did not contain T cells expressing V beta 3 and V beta 11, which are potentially autoreactive against self-superantigens of Mls-2a and Dvbl, respectively. On days 3 to 6 after infection, Thy-1.2-CD4lowTCR-alpha beta + T cells or Thy-1.2-TCR-gamma delta + T cells increased significantly in number and proportion in the liver whereas T cells with these phenotypes were hardly detected in the spleen, lymph nodes, peripheral blood, and peritoneal cavity during the course of the infection. The Thy-1.2-CD4lowTCR-alpha beta T cells contained V beta 3 or V beta 11-bearing cells in high frequencies. The potentially autoreactive V beta 3- or V beta 11-bearing T cells disappeared from the liver on day 7 after infection. Furthermore, the V beta 3+ and V beta 11+ cells but not V beta 8+ cells disappeared after culture for 24 h at 37 degrees C. In vitro stimulation of liver T cells using anti-V beta 11 mAb showed no proliferative response. These results suggest that the potentially autoreactive clones with Thy-1.2-CD4low phenotypes, which increased in number after listerial infection, may be anergized after interaction with self-Ag and may be programmed to die. These potentially autoreactive clones induced in the liver of Listeria-infected mice may not be functionally relevant to the host defense against Listeria.     

Phenotypic and functional analysis of the cellular response in regional lymphoid tissue during an acute virus infection

A phenotypic and functional analysis has been made of the cellular response in regional lymphoid tissue of C57BL/6J mice infected with lymphocytic choriomeningitis virus. Massive recruitment of nondividing cells occurred from 3 days after infection, with total numbers of CD8+ T lymphocytes, B220+ B cells, and Thy-1- B220- null cells being high from day 4 to day 6. In contrast, the peak counts for CD4+ T cells were recorded on day 4 and declined dramatically thereafter. Enhanced expression of IL-2R and Ly-24, both of which can be regarded as T cell activation markers, was found for both the CD4+ and the CD8+ subsets, being most prominent for the CD8+ T cells on day 6. Evidence of T cell proliferation was not recognized until days 5 and 6, coincident with enhanced responsiveness of the lymphocytes to rIL-2 and the development of virus-specific cytotoxic activity. Elimination of the CD4+ T cells by treatment of mice with mAb did not modify either the pathogenesis of lymphocytic choriomeningitis, or the expression of activation markers on the CD8+ T cells which are known to be the key effectors in this disease. Thus, the pattern of responsiveness for the CD8+ population is of recruitment to the lymph node, progressive increase in the expression of activation markers and enhanced sensitivity to rIL-2, with late proliferation and generation of cytotoxic activity. This model provides a system for the rigorous in vivo analysis of parameters influencing lymphocyte differentiation and activation in a virus infection.     

Immunologic regulation of experimental cutaneous leishmaniasis. V. Characterization of effector and specific suppressor T cells

Resistant CBA mice infected with Leishmania tropica promastigotes develop concomitant and convalescent immunity against reinfection. This can be adoptively transferred by splenic and lymph node T cells with a threshold dosage of 1 to 2.5 x 10(7). The effector cells are of Thy-1+, Lyt-1+2- phenotype. The same immune cell population also adoptively transfers specific DTH to L. tropica, which is restricted by the major histocompatibility complex. On the other hand, highly susceptible BALB/c mice infected with L. tropica develop antigen-specific suppressor T (Ts) cells (previously shown to inhibit the induction and expression of DTH), which are capable, on transference, of reversing the healing of lesions induced by prior sublethal irradiation of BALB/c mice. As few as 10(6) of these T cells are effective in abrogating the potent prophylactic effect of 550 rad. The Ts cells are of Thy-1+, Lyt-1+2-, and I-J- phenotype. Sublethally irradiated and infected BALB/c mice produce antibody responses quantitatively and isotypically similar during the critical first 40 days after infection whether or not they are injected with 10(7) Ts cells (nonhealing vs healing). Thus, impairment of DTH and curative immune responses in BALB/c mice cannot be attributed to a helper function of these Thy-1+, Lyt-1+2- cells for the formation of suppressive antibody.     

Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea.

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea induces the rapid infiltration of neutrophils. We investigated whether these cells could influence virus replication. BALB/c mice treated with monoclonal antibody (MAb) RB6-8C5 experienced a profound depletion of neutrophils in the bloodstream, spleen, and cornea. In these animals, virus titers in the eye were significantly higher than those in the immunoglobulin G-treated controls at 3 days postinfection. By day 9, virus was no longer detectable in the controls, whereas titers of 10(3) to 10(6) PFU were still present in the neutrophil-depleted hosts. Furthermore, virus spread more readily to the skin and brains of MAb RB6-8C5-treated animals, rendering them significantly more susceptible to HSV-1-induced blepharitis and encephalitis. Only 25% of the treated animals survived, whereas all of the controls lived. Although MAb RB6-8C5 treatment did not alter the CD4+ T-cell, B-cell, natural killer cell, or macrophage populations, the CD8+ T-cell population was partially reduced. Therefore, the experiments were repeated in severe combined immunodeficiency mice, which lack CD8+ T cells. Again virus growth was found to be significantly elevated in the eyes, trigeminal ganglia, and brains of the MAb RB6-8C5-treated hosts. These results strongly indicate that in both immunocompetent and immunodeficient mice, neutrophils play a significant role in helping to control the replication and spread of HSV-1 after corneal infection.     

Protective effect of N-acetyl chitohexaose on Listeria monocytogenes infection in mice

A water-soluble oligosaccharide, N-acetyl chitohexaose (NACOS-6) was able to enhance the protecting effect of BALB/c male mice against Listeria monocytogenes infection, when administered intraperitoneally 24 hr before the challenge with this microbe. Significant decrease in number of microbes within the peritoneal cavity, spleen, and liver from the mice of NACOS-6-administered group was not observed 1 day after the infection but 4 days after the infection. Administration of NACOS-6 enhanced the delayed-type hypersensitivity response against sheep red blood cells (SRBC) or heat-killed L. monocytogenes. Splenic T lymphocytes from mice administered NACOS-6 released macrophage activating factor (MAF). These results suggested that NACOS-6 was also able to elevate the function of cellular immunity. Macrophages treated with a combination of NACOS-6 and the culture supernatant of splenic T lymphocytes from mice administered NACOS-6, "NACOS-6 sup," were found to exert a fairly strong growth-inhibitory effect on L. monocytogenes. Interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) were able to enhance the growth-inhibitory effect on L. monocytogenes by the NACOS-6-treated macrophages.     

Immunoregulation in experimental disseminated histoplasmosis: flow microfluorometry (FMF) studies of the Thy and Lyt phenotypes of T lymphocytes from infected mice

Previous studies have shown that mice infected i.v. with 6 X 10(5) yeast phase Histoplasma capsulatum (Hc) develop suppressed immune responses during weeks 1 to 4 of infection but that by weeks 8 to 12 of infection these responses return to normal. In this study total and differential cell counts showed that as early as the third day of infection there was a marked reduction in the number of lymphocytes recovered from the peripheral blood, bone marrow, and thymus of infected animals. Concomitantly, there was an increase in the number of splenic lymphocytes. By day 28 both the total and differential cell counts were similar in both infected and normal animals. Flow microfluorometric (FMF) studies comparing the Thy-1.2, Lyt-1, Lyt-2, and surface immunoglobulin (slg) phenotypes of lymphocytes from normal and infected mice were performed. Between days 5 and 7 the thymocytes from infected mice displayed a higher relative fluorescence intensity (RFI) of the Thy-1.2 marker than normal thymocytes, whereas at day 10, the RFI was less than that of normal thymic lymphocytes. Between days 7 and 10 of infection the RFI of the Lyt-2 marker was less on thymocytes from Hc-infected mice; however, there was no change in the Lyt-1 marker. Examination of these lymphocyte markers in blood, spleen, and mesenteric lymph nodes showed that there were decreases in the RFI of both the Thy-1.2 and Lyt-2 between days 5 and 10 of infection. No changes were observed in the Lyt-1 or slg markers. By day 28 there were no differences between the normal and infected mice with respect to any surface marker in any of the organs studied. In other experiments, the effect of adrenalectomy before infection on these surface markers was studied. Absolute numbers of Thy-1.2+, Lyt-1+, and Lyt-2+ cells were significantly increased in the spleen and significantly decreased in the thymus and peripheral blood of infected mice relative to normal controls. These studies suggest that there is a migration of cells from the thymus, blood, and bone marrow to the spleens of mice with disseminated Hc infection.     

Early influx of Listeria-reactive T lymphocytes in liver of mice genetically resistant to listeriosis

Murine listeriosis is a classical model for investigating mechanisms of cellular immunity, which involves interaction of macrophages and T lymphocytes. The early course of this experimental infection is under control of a limited number of genes in the murine host. In the present study, we asked whether the early efficient control of bacterial growth in the liver of resistant mice is related to the expression of a more rapid specific immune response in this organ than in susceptible mice. Therefore, we compared the frequencies of Listeria monocytogenes-reactive T cells in blood, spleen, and liver of resistant C57BL/6 and susceptible C3H/He Past mice after i.v. injection of a high dose of Listeria (9 x 10(5) CFU). T cells were titrated through their ability to locally transfer a delayed-type hypersensitivity reaction to viable L. monocytogenes, an effector function potentially relevant to the early step of protective mechanisms. We observed (1) a 9- and 4-fold increase by day 1 in the frequency of Listeria-reactive transfer units in the blood of C57BL/6 and C3H mice, respectively, (2) no increase in the number of Listeria-reactive transfer units in the spleen of 2-day infected mice of both strains, and (3) a 90-fold increase, at day 2, in the number of Listeria-reactive transfer units in the liver of resistant C57BL/6 compared with only a 9-fold increase in the liver of susceptible C3H/He. These results suggest that the ability of C57BL/6 mice to control the early bacterial growth (0 to 48 h) in their liver, may be related to a rapid influx of L. monocytogenes-reactive T lymphocytes.     

Clonal analysis of murine graft-vs-host disease. I. Phenotypic and functional analysis of T lymphocyte clones

Acute and chronic graft-vs-host disease (GVHD) due to non-MHC histocompatibility differences differ histopathologically. Acute GVHD is characterized by the cytotoxic destruction of recipient tissues, whereas chronic GVHD is characterized by increased collagen deposition. In an attempt to determine if acute and chronic GVHD represent two phases of the same pathophysiologic process or two distinct processes, the T lymphocytes from the C57BL/6 (B6) recipients of LP spleen cells (non-H-2 GVHD) have been cloned and compared to clones from immune mice (LP anti-B6). Acute GVHD (G) clones were established on day 10-14 posttransplant and chronic GVHD (CG) clones on day 50 from animals with clinical chronic GVHD. Immune (I) clones were established 10 to 14 days after immunization. All I clones exhibited B6-specific blastogenesis and cytotoxicity and had a Thy-1.2+, Lyt-2.2+, L3T4- phenotype. All CG clones were noncytotoxic, had I-Ab-specific blastogenesis, and had a Thy-1.2+, Lyt-2.2-, L3T4+ phenotype. The acute GVHD (G) clones were heterogeneous. Fourteen of 23 clones exhibited B6-specific blastogenesis and had a Lyt-2.2+, L3T4- phenotype (B6-G clones). Seven of 9 B6-G clones were cytotoxic for B6 targets. Nine of 23 G clones exhibited I-Ab-specific blastogenesis, and all but one clone had a Lyt-2.2-, L3T4+ phenotype as the did CG clones. Thus, the principal clonogenic T lymphocytes from mice with acute and chronic GVHD differ in terms of 1) their antigenic specificity, 2) their cytotoxic capacity, and 3) their surface phenotype. The presence of I-Ab-specific T lymphocytes with a phenotype identical to CG clones early after transplantation suggests that the immunologic events that result in chronic GVHD begin soon after transplantation. These results indicate that acute GVHD is due primarily to recipient-specific cytotoxic donor T lymphocytes, whereas chronic GVHD is due to autoreactive helper T lymphocytes.     

Cellular and metabolic requirements for induction of macrophage procoagulant activity by murine hepatitis virus strain 3 in vitro.

The cellular basis for the variation in induction of monocyte procoagulant activity (PCA) by murine hepatitis virus strain 3 (MHV-3) was examined using a set of recombinant inbred strains of mice derived from the resistant (A/J) and susceptible C57B1/6J (B) progenitors. Induction of PCA by MHV-3 required live virus and host protein and RNA synthesis. Absolute restriction for induction of PCA was observed at the level of the macrophage. Peritoneal macrophages from resistant parental A/J and RI strains (AXB5) could not be induced to express PCA when stimulated by MHV-3 alone or in the presence of lymphocytes from susceptible and H-2 compatible RI mice (AXB3) although they did respond to endotoxin (LPS). In contrast, macrophages from both susceptible (AXB3) and semisusceptible (AXB1) RI strains of mice expressed a similar increase in PCA after stimulation with MHV-3 in the absence of lymphocytes. The levels of PCA expressed by macrophages in the presence of Thy-1.2+ lymphocytes correlated with susceptibility to disease. Thy-1.2+ lymphocytes from susceptible RI AXB3 mice could induce levels of PCA in macrophages from semisusceptible RI AXB1 mice equivalent to that seen in cultures of macrophages and lymphocytes from susceptible mice. Further subfractionation of Thy-1.2+ cells demonstrated that L3T4+ cells instructed macrophages to produce PCA. Thy-1.2+ cells from MHV-3 immunized resistant AXB5 mice, but not from non-immunized mice, were able to suppress induction of PCA. This suppressor cell activity could be detected 4 days after immunization, reaching maximal activity at day 7 with significant suppression even at 28 days. The PCA was shown to have direct prothrombin cleaving activity (prothrombinase) by ELISA and immunofluorescence staining using the mAb 3D4.3. These results demonstrate that induction of a unique PCA (prothrombinase) is restricted at the level of the macrophage and define a regulatory role for T lymphocytes in its induction.     

Sendai virus pneumonia: evidence for the early recruitment of gamma delta T cells during the disease course.

We previously reported that gamma delta T cells appeared and could play a protective role early in infections with intracellular bacteria such as Listeria monocytogenes, Mycobacterium bovis BCG, and Salmonella choleraesuis. To extend these findings to virus infection, we examined the developmental sequence of gamma delta T cells in bronchoalveolar lavage during the course of Sendai virus infection in C57BL/6 mice. To produce a natural but nonlethal infection course as far as possible, we used a sublethal dose of a wild-type virus which had not been subjected to serial passages in a chicken embryo, hence retaining full virulence for mice. Virus titers in lungs reached a peak on day 6 and then decreased to an undetectable level by day 10. This time course of virus reproduction was immediately and coincidentally followed by the developmental course of gamma delta T cells, in which the cell number peaked on day 7 and then decreased to a marginal level by day 10. On the other hand, the alpha beta T-cell number continued to increase until day 10 and remained at a high level thereafter. The early-appearing gamma delta T cells were CD4-, CD8-, IL-2R alpha- beta+, CD44+, Mel-14-, and LFA-1 alpha/beta+ in phenotype and used V gamma 1/2 and V gamma 4 and V delta 3, V delta 4, V delta 5, and V delta 6. The gamma delta T cells were responding to macrophages from infected mice when the cells were cultured in vitro. Furthermore, the expression of endogenous heat shock protein (hsp) was infection specific, and its level appeared to correlate with the gamma delta T-cell development. These results suggest that the early recruitment of gamma delta T cells, which proliferate in response to endogenous hsp+ cells, is also characteristic of this virus infection, although this view appears to be contradictory to earlier reports.     

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

Powerful immunosuppression mediated by interleukin 2-activated, nonantigen-specific, or H-2-restricted THY-1+ CD8+ cells

Normal spleen cells cultured in high or low concentrations of interleukin (IL) 2 for 3 days contain Thy-1+ CD4- CD8+ cells that powerfully suppress primary but not ongoing or active lymphocyte responses. The precursors of these cells are Thy-1+ AGM-1- and are absent or present in greatly diminished numbers in athymic and scid mice. Suppression is neither antigen nor H-2 restricted and apparently results from reversible inactivation of resting lymphocytes. Comparable Thy-1+ CD8+ suppressor cells were also recovered from normal spleen cells cultured for 3 days with anti-CD3 antibody without added IL-2, indicating that these cells can be activated during the course of immune responses. Such cells may prevent local recruitment/activation of lymphocytes specific for new epitopes that may be expressed sequentially by proliferating tumor cells or infectious organisms.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

Preferential repopulation of the small intestine by gut-derived T cell precursors in the murine system.

The potential of intestinal intraepithelial lymphocyte (IEL) precursors to repopulate the lymphoid components of lethally-irradiated mice was evaluated. Mice injected with total IEL, or IEL depleted of mature T cells, died within 2 weeks post-irradiation. Injection of T cell-depleted Thy-1.1 IEL and Thy-1.2 bone marrow (BM) into lethally-irradiated Thy-1.2 mice resulted in survival rates greater than 90%. The vast majority of thymocytes analysed at 2, 6, and 10 weeks post-treatment were Thy-1.2+. The Thy-1.1+ and Thy-1.2+ cells were detected in the spleen 2 and 6 weeks post-reconstitution. After 10 weeks, the majority of splenic T cells were Thy-1.2+. The majority of Thy-1+ IEL were of the Thy-1.1 subtype at 2 and 6 weeks after reconstitution. After 10 weeks, Thy-1.2+ IEL became the predominant subtype. Flow cytometry (FCM) analyses of Thy-1.1+ IEL showed that Thy-1.1 was co-expressed with CD3, CD4, CD5, CD8, TCR alpha beta and TCR gamma delta T cell markers. These findings indicate that IEL precursors home preferentially to gut epithelia and generate complex IEL phenotypic subsets.     

Role of interleukin-12 in determining differential kinetics of invariant natural killer T cells in response to differential burden of Listeria monocytogenes

Invariant (i) natural killer (NK) T cells are unique T lymphocytes expressing NKR-P1B/C (NK1.1), which recognize glycolipids, notably alpha-galactosylceramide (alpha-GalCer) presented by CD1d. The characteristic phenotype of these iNKT cells undergoes dramatic changes following Listeria monocytogenes infection, and interleukin (IL)-12 is involved in these alterations. Here we show that liver iNKT cells in mice are differentially influenced by the load of infection. Liver alpha-GalCer/CD1d tetramer-reactive (alpha-GalCer/CD1d(+)) T cells expressing NK1.1 became undetectable by day 2 following L. monocytogenes infection and concomitantly cells lacking NK1.1 increased regardless of the severity of infection. Whereas alpha-GalCer/CD1d(+)NK1.1(+) T cells remained virtually undetectable on day 4 following low-dose infection, considerable numbers of these cells were detected in high-dose-infected mice. Whereas numbers of IL-12 producers in the liver on day 4 post infection were comparable in low- and high-dose-infected mice without in vitro restimulation with heat-killed Listeria, those were more prominent in low-dose-infected mice than in high-dose-infected mice after restimulation despite the fact that higher numbers of macrophages and granulocytes infiltrated the liver in high-dose-infected mice than in low-dose-infected mice. Our results indicate that NK1.1 surface expression on iNKT cells is differentially modulated by the burden of infection, and suggest that a high bacterial load probably causes loss of IL-12 production.     

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1⁺ Ly-1⁺2⁺ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia⁺ B lymphocytes were not required, and Ia⁺ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1⁺2⁺ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.     

Enhancement of in vitro tumor-infiltrating lymphocyte cytotoxicity by heteroconjugated antibodies.

Tumor-infiltrating lymphocytes (TIL) were obtained from a mouse melanoma cell line (CL 62) transfected with the gene for the human melanoma Ag p97. TIL were cultured with anti-CD3 antibody and IL-2 for up to 38 days. Flow cytometry identified these TIL as Thy-1.2 + ve/CD4-ve/CD8 + ve cells. A heteroconjugated antibody 500A2 x 96.5, specific for both the CD3 Ag on TIL and the p97 Ag on CL 62 melanoma cells, was prepared using N-succinimidyl-3-(2-pyridyldithio)-propionate as a linking agent. TIL alone demonstrated low levels of cytotoxicity against autologous CL 62 tumor and also against the parental K1735 tumor and an allogeneic murine melanoma (B16). The addition of 500A2 x 96.5 heteroconjugated antibody enhanced TIL-mediated lysis of CL 62 tumor, but not of the K1735 or B16 tumors. This enhanced cytotoxicity was elicited at E:T ratios as low as 0.4:1, and in TIL cultured for 7 to 38 days. These results suggest that hetero-conjugated antibody may enhance the anti-tumor effect of TIL in vivo.     

TNF-TNFR2 interactions are critical for the development of intestinal graft-versus-host disease in MHC class II-disparate (C57BL/6J-->C57BL/6J x bm12)F1 mice

TNF-TNFR2 interactions promote MHC class II-stimulated alloresponses while TNF-TNFR1 interactions promote MHC class I-stimulated alloresponses. The present studies were designed to evaluate whether TNF-TNFR2 interactions were involved in the in vivo generation of CD4(+) T cell-mediated intestinal graft-versus-host disease (GVHD) in the (C57BL/6J (hereafter called B6) --> B6 x B6.C-H-2(bm12) (bm12))F(1) GVHD model. Briefly, 5 x 10(6) splenic CD4(+) T lymphocytes from B6.TNFR2(-/-) or control B6 mice were transferred with 1--2 x 10(6) T cell-depleted B6 bone marrow cells (BMC) to irradiated MHC class II-disparate (bm12 x B6)F(1) mice. Weight loss, intestinal inflammation, and the surface expression of CD45RB (memory marker) on intestinal and splenic lymphocytes were assessed. IL-2 and IFN-alpha mRNA levels in intestinal lymphocytes were assessed by nuclease protection assays. A significant reduction in weight loss and intestinal inflammation was observed in recipients of the TNFR2(-/-)CD4(+) SpC. Similarly, a significant decrease was noted in T cell numbers and in CD45RB(low) (activated/memory) expression on intestinal but not CD4(+) T cells in recipients of TNFR2(-/-)CD4(+) spleen cells. IL-2 and IFN-alpha mRNA levels were reduced in the intestine in the recipients of TNFR2(-/-) splenic CD4(+) T cells. These results indicate that TNF-TNFR2 interactions are important for the development of intestinal inflammation and activation/differentiation of Th1 cytokine responses by intestinal lymphocytes in MHC class II-disparate GVHD while playing an insignificant role in donor T cell activation in the spleen.