Cytotoxicity of allogeneic lymphocytes sensitized against H-2 antigens in vitro

A system is described in which C57/Bl lymphocytes can be sensitized in vitro against H-2 alloantigens of DBA/2 fibroblasts. Cytotoxicity of sensitized lymphocytes was measured by ⁵¹Crrelease from DBA/2 mastocytoma cells which were used as sensitive target cells. During the sensitization period one can observe lymphoid blast transformation and proliferation to start from the third day. Optimal cytotoxic effect of sensitized lymphocytes is reached on the fifth day. C57/Bl lymphocytes sensitized on C₃H fibroblasts were found not to be cytotoxic to DBA/ 2 mastocytoma cells.     

Studies on the physiological manifestations of cell-mediated cytotoxicity. I. Early metabilic changes in mouse plasmacytoma cells exposed in vitro to sensitized allogeneic splenocytes.

Early and later biochemical changes occurring in neoplastic target cells interacting with sensitized lymphoid cells were investigated, with emphasis on the detection of very early damage to the target cells. The sensitizing and target cells were a BALB/c plasmacytoma; the effector cells were obtained from the spleens of specifically sensitized and normal C57BL mice. The incorporation of labeled glycine and leucine, uridine, and thymidine into macromolecular entities by the plasmacytoma cells was followed as an indication of ability to take up the precursor and/or biosynthetic capacity. Separation of target and attacking cells was effected in some experiments by use of anti-C57BL lymphocyte serum.Amino acid and uridine incorporation were markedly altered between the second and fourth hour of interaction, and specific release of ⁵¹Cr was seen only after 2 hr. In contrast, thymidine incorporation was inhibited almost immediately after contact; this inhibition was shown to occur in PCT cells incubated at various ratios with sensitized spleen cells.     

Abrogation of neonatally induced permanent transplantation tolerance in mice: quantitative aspects.

Neonatal transplantation tolerance was induced in CBA (H-2k) mice by the intravenous injection of 20 million (CBAxA)F1 spleen cells to the transplantation antigens of the A mouse strain. Those mice which carried an A (H-2a) skin allograft without any sign of rejection for at least 120 days, were considered to be permanently tolerant and were selected for further experiments. Abrogation of permanent transplantation tolerance was achieved by injecting the tolerant mice with different doses (50, 100 and 200 millions, respectively) of normal syngeneic (CBA) lymphoid (spleen) cells. Dynamics of the rejection of the test skin allografts tolerated so far revealed well reproducible dose-response curves. Further groups of tolerant CBA mice were given 10, 50, 100, or 200 million "sensitized" (G + 16) CBA spleen cells: "sensitization" by A-skin allografting was performed 16 days before. The sensitized spleen cells abolished the state of tolerance more vigorously and effectively than the normal CBA spleen cells did. In a third group of experiments, the abrogating capacity of 50 million sensitized CBA spleen cells 16, 120, 240, or 360 days after sensitization was compared. The efficacy of the sensitized cells in abolishing the state of tolerance decreased continuously, but, even 360 days after sensitization a remarkably strong immunologic memory was demonstrable. The excellent quantitative correlations found between the number of the injected lymphoid cells and the dynamics of the abrogation of tolerance offer a highly promising new possibility for studying the immunological activity, the immunologic memory, etc., of the different lymphoid cell (sub)populations in performing the transplantation immune reactions.     

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells: IV. Correlation with H-2 haplotypes

In vitro primary syngeneic sensitization on monolayers of thyroid epithelial cells was performed with 21 inbred strains of mice representing 11 original H-2 haplotypes. Significant differences in the proliferative responses, assessed by thymidine uptake, were found to be related to the major histocompatibility complex haplotype. This result was further confirmed using congenic resistant strains of mice. In comparison with the experimental autoimmune thyroiditis induced by syngeneic thyroglobulin and adjuvant, primary syngeneic sensitization on monolayers of thyroid epithelial cells appeared to be under the same genetic control (H-2^k strains being good responders, while H-2^b mice are poor responders).     

Leukocytic Hypersensitivity in Experimental Group A Streptococcal Infections

Skin hypersensitivity to streptococcal antigens was demonstrated in 15 New Zealand red rabbits that were sensitized by intracutaneous injections with group A streptococci, and leukocytic hypersensitivity, as determined by in vitro inhibition of leukocyte migration, was demonstrated in 11 of the rabbits. The skin hypersensitivity persisted for at least 8 weeks, whereas the leukocytic hypersensitivity generally waned rather rapidly. The leukocytic hypersensitivity reappeared in the infected rabbits that had developed this hypersensitivity. However, it did not reappear on reimmunization with living streptococci of the type originally employed, whereas it did reappear with living but not heat-killed streptococci of another M type.     

Efficient Electrotransformation System and Gene Targeting in Pyogenic Streptococci

Hemolytic streptococci are lacking in natural competence for uptake of DNA, and existing electrotransformation methods are still ineffective for most strains. By optimizing biological and electric parameters of electroporation, we established a simple, efficient, and reproducible transformation method for streptococcal cells. The major factor was an increase in the electric field strength. All tested streptococci (6 group A strains and one group C strain) were successfully transformed, and the maximal efficiency was higher than 1×10⁷ transformants per μg of plasmid DNA. Targeted inactivation of the chromosomal genes of group A and C streptococci was achieved, using the electrotransformation method. The slo ^- or sagB ^- mutants constructed by the gene-targeting showed elevated competence for electrotransformation. Availability of the electrotransfer system for cloning and analysis of streptococcal genes is discussed.     

Properties of TAS-1D3, a Tuberculin-Active Substance from BCG,in Regard to Delayed Hypersensitivity

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.     

Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus colubriformis: tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions, and in vitro lymphocyte transformations during primary infections

The kinetics of the lymphocyte responses of Trichostrongylus colubriformis-infected and normal guinea pigs were measured by the in vivo uptake of tritiated thymidine either as dpm ³H/mg tissue or as the percentage change in [³H] -labeled lymphoblasts in autoradiographs of tissue impression smears and sections. The lymphoid response was predominantly a local one centering on the infected area of the small intestine. The greatest lymphocyte reactions as assessed by counts of labeled lymphoblasts occurred in the Peyer's patches and mesenteric lymph nodes where the peak responses took place 11 and 6 days after infection, respectively. The local nature of the responses was exemplified by the fact that the mesenteric lymph nodes of the anterior small intestine showed a peak response on the sixth day but the response from the posterior small intestine peaked 7 days later. A similar but less dramatic relationship existed among the Peyer's patches. In addition no labeled lymphoblast response was elicited in the inguinal lymph nodes or cecal lymphoid patches throughout the infection and the first increased responsiveness of the spleen did not take place until after Day 13, by which time the lymphoid proliferations associated with the infected intestine had subsided. Initially, the spleen showed a marked depletion of labeled blast cells during the first 7 days of the infection. This was taken as indicating at the time the infection was being established the export of cells capable of transformation in response to parasite antigen. This was supported by the observation that large numbers of phytohemagglutinin responsive lymphocytes were found in the peripheral circulation at this time. The in vitro responsiveness of peripheral lymphocytes to T. colubriformis antigen was also studied. Positive lymphocyte transformations first occurred 6 days after infection but thereafter declined to the normal level by Day 13; the peak transformation ratio was found 25 days after infection but by Day 38 it had declined to a low but persistently positive level. There was a correlation between the circulation of specifically sensitized cells, probably of thymic origin, IgE antibody titers, and the development of positive dermal delayed hypersensitivity reactions in infected guinea pigs, suggesting a close relationship among these three immunological phenomena.All lymphoblast responses in Peyer's patches, mesenteric lymph nodes, and lamina propria of the intestine were completed before the immune elimination of the parasite commenced 10 days after infection. During the first 10 days of infection specifically sensitized lymphocytes appeared and disappeared from the circulation. The loss of circulating sensitized lymphocytes at the time immune elimination of the parasite was taking place in the gut suggested that the sensitized cells were “homing-in” on the local area of infection. After the immune elimination of the parasite had commenced, the level of sensitized lymphocytes and IgE antibodies then increased rapidly in the blood. Evidence from the kinetics of the hemagglutinating antibodies indicated that stage specific antigens occur in T. colubriformis.     

Sortase A Induces Th17-Mediated and Antibody-Independent Immunity to Heterologous Serotypes of Group A Streptococci

Group A streptococci (GAS) are associated with a variety of mucosal and invasive human infections. Recurrent infections by highly heterologous serotypes indicate that cross-serotype immunity is critical for prevention of GAS infections; however, mechanisms underlying serotype-independent protection are poorly understood. Here we report that intranasal vaccination of mice with Sortase A (SrtA), a conserved cell wall bound protein, reduced colonization of nasal-associated lymphoid tissue (NALT) by heterologous serotypes of GAS. Vaccination significantly increased CD4⁺ IL-17A⁺ cells in NALT and depletion of IL-17A by neutralizing antibody prevented GAS clearance from NALT which was dependent on immunization with SrtA. Vaccination also induced high levels of SrtA-specific antibodies; however, immunized, B cell-deficient mice cleared streptococcal challenges as efficiently as wild type mice, indicating that the cross-serotype protection is Th17-biased and antibody-independent. Furthermore, efficient GAS clearance from NALT was associated with a rapid neutrophil influx into NALT of immunized mice. These results suggest that serotype independent immune protection against GAS mucosal infection can be achieved by intranasal vaccination with SrtA and enhanced neutrophil function is critical for anti-GAS defense and might be a target for prevention of GAS infections.     

The effect of heat-killed streptococci on the survival of heart grafts in inbred strains of mice.

Studies with inbred strains of mice revealed that exposure to type 12, Group A, beta-hemolytic streptococci affects the host response to heart grafts implanted in the ear. Intraperitoneal injection of streptococci 10 days before grafting led to curtailed survival of syngrafts without altering the normal rejection time of allografts. Similar sensitization, combined with local injection of streptococci into the graft site at the time of grafting, was followed by rapid rejection of both syngrafts and allografts. The time interval between exposure and grafting was critical. Injection with streptococci 5 days before grafting led to a prolonged survival of allografts and no demonstrable effect on syngrafts. In contrast, injection of streptococci 15 days before grafting did not alter survival of either type of graft. The data indicate heart grafts implanted in the ear may serve as a useful model for the study of the host responses to streptococcal antigens.     

In vitro sensitization to transplantation antigens. VII. Enhanced graft-vs-host activity of in vitro allosensitized lymphoid cells

We have previously demonstrated that C57BL/6J lymphoid cells sensitized in vitro to C3H/He transplantation antigens, present on macrophage monolayers, can transfer an accelerated C3H allograft response to recipient C57 mice. The present report indicates that C57 lymphoid cells sensitized to C3H alloantigens, present on macrophage monolayers, can also mediate a graft-versus-host (GVH) reaction in (C3H × C57) F₁ newborn mice. This GVH reaction is of greater magnitude than that produced by noncultured C57 cells. The magnitude of the augmented GVH reaction produced by cultured C57 cells is dependent on the source of lymphoid cells: lymph node, spleen, and bone marrow cells are consistently more active than cultured thymus cells—the reduced capability of cultured thymus cells to mediate the GVH reaction parallels their reduced ability to transfer allograft immunity. To test whether monolayers, other than macrophages, can sensitize lymphoid cells in vitro we incubated C57 lymphoid cells on C3H-derived L cells. Lymph node cells incubated with L cells demonstrate an increased GVH reaction in newborn mice. The in vitro sensitization of spleen and bone marrow cells on L cells is less consistent. Thymus cannot be sensitized by L cells. Monolayers of L cells are therefore not as efficient a sensitizing source as macrophages.     

Changes in lymphocyte populations in protein—Calorie-deficient mice

Changes in lymphocyte subpopulations induced by postweaning malnutrition were studied in C57BL/6 mice kept on a protein-restricted diet (D), by weekly assessment of the “homing” properties and the response to mitogens of thymus and spleen lymphocytes during the first 2 months of diet. Cell loss in the lymphoid organs during the early phase of protein restriction was mainly due to depletion of nonrecirculating cells. This resulted in relative enrichment of medullary cells in the thymus and T₂ cells in the periphery as shown by the rise in the percentage migration of D lymphocytes to the lymph nodes as well as in their response to optimal doses of PHA and Con A and PHA:Con A response ratio. Reversion of the distribution pattern of D lymphocytes, with depressed homing to the lymph nodes and decrease in the response to mitogens, was observed concomitantly with a second phase of partial recovery in the whole-body weight and cell content of the thymus and spleen. The [³H]thymidine uptake by D spleen cells stimulated with supraoptimal doses of mitogen was significantly increased during the whole length of the experiment. The suppression of DNA synthesis induced by high doses of mitogen reappeared after short-term nutritional rehabilitation.     

Endothelium-lymphocyte interactions in vitro. II. Adherence of allergized lymphocytes.

Peripheral blood lymphocytes from skin graft-sensitized pigs will adhere in vitro to fresh donor-type large vessel endothelium, but do not spread out or migrate. Similar cells will however spread out on and migrate through monolayers of cultured donor-type aortic endothelium to a significantly greater extent than nonallergized lymphocytes. Cells sensitized in mixed lymphocyte culture at first exhibit a nonspecific increase in adherence and migration correlated with increased thymidine uptake, but after more prolonged incubation adherence becomes specific for stimulator-type endothelium. It is suggested that lymphocyte infiltration of an allograft in the presence of circulating sensitized cells involves a combination of nonspecific lymphocyte adhesion to endothelium, antigenic stimulation of “primed” cells to increased motility, endothelial penetration and lymphokine production, and soluble-factor-mediated stimulation of migration by nonsensitized cells.     

Ascaris suum: Immunosuppression in mice during acute infection

Mice inoculated by stomach intubation with 10,000 embryonated Ascaris suum eggs, 4, 11, or 21 days before an intraperitoneal (ip) immunization with 2 × 10⁸ sheep erythrocytes (SRBC) had reduced numbers of direct (IgM) splenic hemolytic plaques measured at 4 days after immunization and only a marginal reduction in indirect plaques (IgG) measured at 9 days after immunization. Lower dosages of Ascaris eggs or simultaneous inoculation of Ascaris eggs and SRBC did not suppress antibody responses to SRBC. No reduction in a secondary antibody response to SRBC injected 4 days after Ascaris inoculation was observed. IgM and IgG hemagglutinin titers, as distinguished by 2-mercaptoethanol sensitivity, were suppressed in mice injected ip with 10⁸ SRBC 10 days following inoculation of 10,000 Ascaris eggs, but titers in both Ig classes were similar in infected and control mice injected with 2 × 10⁹ SRBC. At Day 20, antibody titers following ip injection of 1.0 or 100 μg of ovalbumin in alum were reduced in mice infected with 10,000 Ascaris eggs 4 days before antigen injection.Contact hypersensitivity to oxazalone was not altered in mice sensitized at 5 or 14 days after inoculation of 10,000 Ascaris eggs. The delayed hypersensitivity response, measured by footpad swelling, to an optimum intravenous sensitizing dosage of SRBC was inhibited in mice sensitized 10 days after Ascaris infection, but not inhibited in mice sensitized at 21 or 32 days after infection. In contrast, the delayed hypersensitivity response to subcutaneous sensitization with SRBC 10 days after Ascaris infection was not altered.     

Streptococcal immunotherapy of a chemically induced murine fibrosarcoma: Effect of dose,route, sham surgery,and splenectomy on adjuvant action

Summary Adjuvant immunotherapy with hemolytic streptococci abates the growth of a syngeneic methylcholanthrene (MCA)-induced fibrosarcoma propagated in C3H/HeJ mice. Interperitoneal (IP) administration of streptococci either prior to or after tumor inoculation reduced neoplastic outgrowth. While subcutaneous (SC) administration of streptococci prior to tumor inoculation did not influence tumor outgrowth, SC treatment afterwards was effective. Thus, therapeutic effects of streptococcal vaccine depend upon the route and/or timing of administration.Surgery performed shortly before tumor inoculation abrogated host response to streptococci. On the other hand, surgery performed 3 days after tumor inoculation did not alter the adjuvant action of streptococcal vaccines. The failure of attempts to achieve an immunotherapeutic effect in splenectomized hosts suggests that the spleen was essential for the action of the streptococcal vaccine. Present address: Department of Surgery, The University of Texas Medical School at Houston, 6431 Fannin, Room 6240, Houston, Texas 77030, USA     

Permanent survival of fully MHC-mismatched islet allografts by targeting a single chemokine receptor pathway

Chemokine receptor blockade can diminish the recruitment of host effector cells and prolong allograft survival, but little is known of the role of chemokine receptors in promoting host sensitization. We engrafted fully allogeneic islets into streptozotocin-treated normal mice or mice with the autosomal recessive paucity of lymph node T cell (plt) mutation; the latter lack secondary lymphoid expression of the CCR7 ligands, secondary lymphoid organ chemokine (CCL21) and EBV-induced molecule-1 ligand chemokine (CCL19). plt mice showed permanent survival of islets engrafted under the kidney capsule, whereas controls rejected islet allografts in 12 days (p < 0.001), and consistent with this, plt mice had normal allogeneic T cell responses, but deficient migration of donor dendritic cell to draining lymph nodes. Peritransplant i.v. injection of donor splenocytes caused plt recipients to reject their allografts by 12 days, and sensitization at 60 days posttransplant of plt mice with well-functioning allografts restored acute rejection. Finally, islet allografts transplanted intrahepatically in plt mice were rejected approximately 12 days posttransplant, like controls, as were primarily revascularized cardiac allografts. These data show that the chemokine-directed homing of donor dendritic cell to secondary lymphoid tissues is essential for host sensitization and allograft rejection. Interruption of such homing can prevent T cell priming and islet allograft rejection despite normal T and B cell functions of the recipient, with potential clinical implications.     

Relationship of Footpad Sensitivity to Purified Protein Derivatives and Resistance to Airborne Infection with Mycobacterium tuberculosis of Mice Vaccinated with Mycobacterial Cell Walls

Footpads of mice sensitized by oil-treated Bacillus Calmette-Guérin (BCG) cell walls given either intravenously, subcutaneously, intradermally, intraperitoneally, or intramuscularly became swollen and reddened after injection of purified protein derivative (PPD). This reaction, greatest after intradermal and subcutaneous sensitization, generally reached a maximum about 24 hr after challenge and was still marked at 48 hr. The histological response was characterized by infiltration with both polymorphonuclear and mononuclear cells. The proportion of mononuclear cells increased with time and they predominated at 48 hr. The footpad reaction could be detected as early as 1 week after sensitization and persisted for at least 37 weeks. Footpad sensitivity to PPD and acquired resistance to airborne infection with Mycobacterium tuberculosis H37Rv were correlated in that (i) both reached a peak approximately 1 month after sensitization of the mouse, and (ii) cell walls treated with NaOH or given without oil neither protected mice against challenge infection nor sensitized them to PPD. Although, as we reported previously, mice vaccinated subcutaneously or intradermally exhibited little or no enhanced resistance to experimental infection, mice given oil-treated cell walls by these routes were highly sensitive to footpad inoculation of PPD. Therefore, the footpad test cannot be used to determine immunity of the mouse to pulmonary infection with tubercle bacilli.     

Downregulation of Key Early Events in the Mobilization of Antigen-bearing Dendritic Cells by Leukocyte Immunoglobulin-like Receptor B4 in a Mouse Model of Allergic Pulmonary Inflammation

Leukocyte Immunoglobulin-like Receptor B4 (LILRB4) null mice have an exacerbated T helper cell type 2 (Th2) immune response and pulmonary inflammation compared with Lilrb4^+/+ animals when sensitized intranasally with ovalbumin (OVA) and low-dose lipopolysaccharide (LPS) followed by challenge with OVA. Moreover, OVA-challenged Lilrb4 ^−/− mice exhibit greater migration of antigen (Ag)-bearing dendritic cells (DCs) to lymph nodes and accumulation of interleukin 4- and interleukin 5-producing lymph node lymphocytes. The main objective of this study was to determine how the absence of LILRB4 leads to a greater number of DCs in the lymph nodes of Ag-challenged mice and increased lung Th2 inflammation. Mice were sensitized intranasally with PBS alone or containing OVA and LPS; additional cohorts were subsequently challenged with OVA. Expression of chemokine (C-C motif) ligand 21 (CCL21) in the lung was assessed immunohistologically. OVA ingestion and expression of LILRB4 and chemokine (C-C motif) receptor 7 (CCR7) were quantified by flow cytometry. Inhalation of OVA and LPS induced upregulation of LILRB4 selectively on lung Ag-bearing DCs. After sensitization and challenge, the lung lymphatic vessels of Lilrb4 ^−/− mice expressed more CCL21, a chemokine that directs the migration of DCs from peripheral tissue to draining lymph nodes, compared with Lilrb4^+/+ mice. In addition, lung DCs of challenged Lilrb4 ^−/− mice expressed more CCR7, the CCL21 receptor. The lungs of challenged Lilrb4 ^−/− mice also contained significantly greater numbers of CD4+ cells expressing interleukin-4 or interleukin-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in lymph nodes and the attendant exacerbated Th2 lung pathology. Our data establish a new mechanism by which LILRB4 can downregulate the development of pathologic allergic inflammation: reduced upregulation of key molecules needed for DC migration leading to decreases in Th2 cells in lymph nodes and their target tissue.     

Progressive increase with time after sensitization in the functional affinity of T lymphocytes

The dose response relationship in peritoneal cell migration inhibition, elicited by various concentrations of ABA-Tyr, was studied in guinea pigs sensitized with a standard dose of ABA-Tyr. The reactivity to 2 × 10^?7 or 2 × 10^?8 mole/ml appeared at 2 wk, and remained at the maximum level from 4 wk to 8 mo after sensitization. The inhibition by 2 × 10^?9 mole/ml increased up to 4 mo and 2 × 10^?10 mole/ml was first inhibitory at $\text{6}\text{1}\text{2}$ mo. The change in the slope of the dose response curve was a property of the ABA-specific cells. As there is no B-cell response to ABA-Tyr, the finding shows that the functional affinity of specific T cells progressively increases with time after sensitization.     

Sensitizing effects of chronic exposure and acute inhalation of ovalbumin aerosol on pulmonary C fibers in rats.

The effect of ovalbumin (Ova) sensitization on pulmonary C-fiber sensitivity was investigated. Brown-Norway rats were sensitized by intraperitoneal injection of Ova followed by aerosolized Ova three times per week for 3 wk. Control rats received the vehicle. At the end of the third week, single-unit fiber activities (FA) of pulmonary C fibers were recorded in anesthetized, artificially ventilated rats. Our results showed the following: 1) Ova sensitization induced airway inflammation (infiltration of eosinophils and neutrophils) and airway hyperresponsiveness in rats; 2) baseline FA in sensitized rats was significantly higher than that in control ones; 3) similarly, the pulmonary C-fiber response to right atrial injection of capsaicin was markedly higher in sensitized rats, which were significantly amplified after the acute Ova inhalation challenge; and 4) similar patterns, but smaller magnitudes of the differences in C-fiber responses to adenosine and lung inflation, were also found between sensitized and control rats. In conclusion, Ova sensitization elevated the baseline FA and excitability of pulmonary C fibers, and the hypersensitivity was further potentiated after the acute Ova inhalation challenge in sensitized rats. Chronic allergic inflammatory reactions in the airway probably contributed to the sensitizing effect on these lung afferents.