Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

C57BL/6 and AKR mice were treated with hamster erythrocytes (HRBC) in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and the development of delayed hypersensitivity and antibody production were examined. 1) Delayed hypersensitivity against HRBC antigen, as determined by the peritoneal macrophage disappearance test, was detected in mice sensitized with HRBC in CFA but not in those sensitized with HRBC in IFA. 2) Antibody production against HRBC or hapten TNP after a booster injection of HRBC or trinitrophenylated HRBC (TNP-HRBC) in saline was enhanced by pretreatment with HRBC in CFA or IFA. 3) Delayed hypersensitivity was not detectable after a booster sensitization with HRBC in CFA in mice which had been pretreated with HRBC in IFA 2 weeks earlier. In the mice treated with both HRBC in IFA (day ?21) and in CFA (day ?7), however, an enhanced antibody production against HRBC or TNP was detected after an intravenous injection with HRBC or TNP-HRBC in saline (day 0). These results suggest that sensitized effector lymphocytes in delayed hypersensitivity and helper cells in antibody production may be derived from the same pool of unprimed T cells. The pool of unprimed T cells with a capacity to differentiate into either type of primed T cells may be exhausted after pretreatment with the antigen in IFA, and the primed helper T cells may not be able to differentiate into sensitized lymphocytes even after sensitization with the antigen in CFA, which favors development of delayed hypersensitivity in normal controls.     

Systemic treatment with interleukin-4 induces regression of pulmonary metastases in a murine renal cell carcinoma model

Advanced metastatic renal cell carcinoma has been shown to be responsive to immunotherapy but the response rate is still limited. We have investigated the therapeutic potential of systemic interleukin-4 (IL-4) administration for the treatment of pulmonary metastases in the murine Renca renal adenocarcinoma model. Renca cells were injected iv in Balb/c mice to induce multiple pulmonary tumor nodules. From Day 5, Renca-bearing mice were treated with two daily injections of recombinant murine IL-4 for 5 consecutive days. IL-4 treatment induced a significant reduction in the number of lung metastases in a dose-dependent manner and significantly augmented the survival of treated animals. Immunohistochemistry studies, performed on lung sections, showed macrophage and CD8⁺ T cell infiltration in the tumor nodules 1 day after the end of IL-4 treatment. The CD8 infiltration increased by Day 7 after IL-4 treatment. Granulocyte infiltration was not detectable. To clarify further the role of the immune system in IL-4 anti-tumor effect, mice were depleted of lymphocyte subpopulations by in vivo injections of specific antibodies prior to treatment with IL-4. Depletion of CD8⁺ T cells or AsGM1⁺ cells abrogated the effect of IL-4 on lung metastases, whereas depletion of CD4⁺ T cells had no impact. These data indicate that CD8⁺ T cells and AsGM1⁺ cells are involved in IL-4-induced regression of established renal cell carcinoma.     

Cytotoxic T lymphocytes generated by short-term in vitro TCR stimulation in the presence of IL-4 are therapeutically effective against B16 melanoma

P14 TCR transgenic CD8+ T cells (LCMV gp33-specific) were activated by antigen in the presence of either IL-2 or IL-2+IL-4 to generate effector cytotoxic T lymphocytes (CTLs). The therapeutic effectiveness of such IL-2- or IL-2+IL-4-grown CTLs was tested in mice that had received intravenous inoculations of B16.gp33 melanoma cells 7 days previously. Administration of P14 CTLs activated by antigen +IL-2+IL-4 was significantly more effective at reducing melanoma colony formation in the lung than those grown in the presence of antigen +IL-2. Highly significant improvement in survival was observed with 80% of B16.gp33-inoculated mice showing long-term survival after therapy with 10×10⁶ antigen +IL-2+IL-4-activated P14 CTLs. Similar therapeutic effectiveness of antigen +IL-2+IL-4-activated P14 CTLs against subcutaneously inoculated B16.gp33 melanoma cells was also found. There was significant reduction in P14 CD8+ T cells in the peripheral blood of B16.gp33-inoculated mice than in mice that did not receive B16.gp33 melanoma cells, indicating possible homing of P14 CD8+ T cells to the site of tumor growth or antigen-induced apoptotic cell death. These results may have implications in tumor therapy using CTLs grown ex vivo, especially during early stages of tumor formation. They also support the concept that the therapeutic effectiveness of CTLs can be governed by the cytokine context in which they are activated.     

Nature of T-cells resident in spleens of thymectomized,lethally irradiated,bone marrow-reconstituted mice

Adult thymectomized, lethally irradiated, bone marrow-reconstituted (ATxXB) mice that had been weakly primed with SRBC or HRBC between thymectomy and irradiation were shown to retain antigen-specific immunological memories for at least 1–5 months after bone marrow reconstitution. This could be shown by anamnestic antibody response in vivo as well as by proliferative response of the spleen cells to the test antigens in vitro. Spleen cells taken from ATxXB mice showed a reduced but significant proliferative response to nonspecific T-cell mitogens, in particular to Con A, in vitro. Treatment of the donor bone marrow cells used for reconstitution of ATxXB mice with anti-Thy 1.2 sera + C′ did not affect the generation of immunological memories nor the magnitude of the proliferative response of spleen cells to nonspecific T-cell mitogens in vitro, indicating that the cells responsible for such functions were host derived. Finally, the antibody-forming capacity of spleen cells derived from SRBC-primed ATxXB mice to the test antigen in vitro was completely abrogated by exposure to 450 R, whereas the helper function of the same cell suspension remained unaffected even after exposure to 1000 R. Implication of these findings on the nature of T cells resident in spleens of ATxXB mice was discussed.     

Differential expression of asialo GM1 on alloreactive cytotoxic T lymphocytes and lymphokine-activated killer cells

The present study was undertaken to examine the differential expression of asialo GM1 (AsGM1) on the responding cells and effectors of alloreactive cytotoxic T lymphocytes (CTL) and lymphokine-induced activated killers (LAK). It was found that AsGM1 was expressed on the 3-day-cultured LAK effectors. Its expression gradually disappeared to the extent that AsGM1 became undetectable after 5 to 6 days of culturing. In contrast, AsGM1 was detected on 3-day CTL generated in mixed-lymphocyte cultures (bulk cultures); however, the levels of AsGM1 expression remained the same for at least 7 days. When examining the expression of AsGM1 on the responding cells, the reciprocal results were obtained. AsGM1 was expressed the LAK responders, but we were unable to demonstrate AsGM1 on CTL responders. Depletion of AsGM1+ cells from the responding population reduced subsequent CTL responses; however, CTL responses could be restored by adding conditioned media containing both interleukin 2 (IL-2) and other helper-T-cell factors and could not be restored by purified IL-2 alone adding at comparable doses. Reconstituting the AsGM1-depleted responders with Lyt-2-depleted splenocytes also restored the CTL response. Furthermore, depletion of AsGM1 cells from the responding population did not reduce the precursor frequency of allo-CTL, whereas the precursor frequency of LAK cells was reduced 42-fold. These findings show that the reduction of CTL responses after depletion of AsGM1+ cells was not due to the removal of precursors; instead, the defect appeared to be in the helper population. We further found that the helper defect was not due to impaired IL-2 production, because the endogenous production of IL-2 AsGM1-depleted responders was not reduced. Therefore, AsGM1+ cells may play a role in the helper pathway other than IL-2 production.     

Immunological properties of Fc receptor on lymphocytes. 2. Differentiation from FcR- to FcR+ cells and their functional differences in in vitro antibody response.

In in vitro plaque-forming cell (PFC) response to particulate as well as to soluble antigen, the functional difference between Fc receptor-bearing (FcR⁺) and nonbearing (FcR^?) murine splenic lymphocytes was analyzed using the EA rosetting method. In the secondary anti-horse red blood cell (HRBC) response of C3H mice, FcR^? cells showed higher IgM and IgG responses than did FcR⁺ cells. When nylon wool (NW)-purified T cells primed with keyhole limpet hemocyanin (KLH) were fractionated into FcR^? and FcR⁺ T cells, helper activity was proven in the former subset in the cooperation with syngeneic spleen cells primed with dinitrophenylated ascaris extract (DNP-Asc). FcR⁺ T cells showed essentially no helper activity. When FcR^? cells were cultured, neogenesis of FcR⁺ cells was observed on Days 3 to 5. The conversion from FcR^? to FcR⁺ cells was prominent in B cells (40 to 50%), whereas NW-purified nonadherent FcR^? T cells converted poorly (15 to 20%). The converting process was accelerated slightly by mitogens, but was least affected by antigens. To examine the possible contribution of neogeneic FcR⁺ T cells in the helper activity, KLH-primed FcR^? T cells were precultured for 7 days with homologous antigen. The specific helper activity of the cultured T cells proved to be unaffected by the depletion of neogeneic FcR⁺ T cells by EA rosetting. The neogeneic FcR⁺ T cells had no helper activity. It was thus suggested that helper T cells remain in the FcR^? cell fraction and do not convert to the FcR⁺ state during the cooperating process.     

Mechanism for Macrophage Activation against Corynebacterium parvum—Participation of T Cells and Its Lymphokines

It is well known that Corynebacterium parvum activates macrophages to produce tumor necrosis factor (TNF). It is suspected that the activation of macrophages by C. parvum requires T-cell participation. The purpose of this study was to confirm that T cells participate in the activation of macrophages by C. parvum. TNF production in vitro from the spleen cells of BALB/c- + / + mice was abrogated completely by the pre-treatment of spleen cells with anti-Ia antiserum and complement, indicating that Ia+ cells are the source of TNF. TNF production was not elicited at all in BALB/c-nu/nu mice. However, there was an increase in the number of Ia+ cells as well as an increase in the weight of spleen and liver. Supernatant from a culture of spleen cells stimulated with phytohemagglutinin-P (a PHA-induced lymphokine) made it possible for BALB/c-nu/nu mice to produce TNF, associated with an induction of Lyt-1⁺ cells and Lyt-2⁺ cells. However, treatment with the lymphokine did not augment the increases of Ia⁺ cells or liver and spleen weights. These results suggest that increasing the number of Ia⁺ cells is not sufficient to bring about TNF production; Ia⁺ cells must also be stimulated by T cells or T-cell lymphokines in order to produce TNF. These results suggest that T cells play an essential role in the activation of Ia⁺ cells against C. parvum.     

IL-2 limits IL-12 enhanced lymphocyte proliferation during Leishmania amazonensis infection

C3H mice infected with Leishmania amazonensis develop persistent, localized lesions with high parasite loads. During infection, memory/effector CD44^hiCD4⁺ T cells proliferate and produce IL-2, but do not polarize to a known effector phenotype. Previous studies have demonstrated IL-12 is insufficient to skew these antigen-responsive T cells to a functional Th1 response. To determine the mechanism of this IL-12 unresponsiveness, we used an in vitro assay of repeated antigen activation. Memory/effector CD44^hiCD4⁺ T cells did not increase proliferation in response to either IL-2 or IL-12, although these cytokines upregulated CD25 expression. Neutralization of IL-2 enhanced CD4⁺ T cell proliferation in response to IL-12. This cross-regulation of IL-12 responsiveness by IL-2 was confirmed in vivo by treatment with anti-IL-2 antibodies and IL-12 during antigen challenge of previously infected mice. These results suggest that during chronic infection with L. amazonensis, IL-2 plays a dominant, immunosuppressive role independent of identifiable conventional T_reg cells.     

Diterpene, 16-phyllocladanol enhances Th1 polarization induced by LPS-primed DC, but not TNF-alpha-primed DC

16-Phyllocladanol is diterpene isolated form the heartwood of Cryptomeria japonica. We demonstrate that the effect of 16-phyllocladanol on the phenotypic and functional maturation of human monocytes-derived DC in vitro. Human monocytes were exposed to 16-phyllocladanol alone, or in combination with LPS and thereafter co-cultured with naïve T cells. The expression levels of CD83 and HLA-DR on LPS-primed DC were enhanced by 16-phyllocladanol. 16-Phyllocladanol dose-dependently augmented the T cell stimulatory capacity in an allo MLR to LPS-primed DC and the production of IL-12p70 by LPS-primed DC. The cytokine production by 16-phyllocladanol-primed DC was not inhibited by anti-TLR2 and 4 mAbs. IFN-γ secretion from naïve T cells co-cultured with DC differentiated with LPS was also augmented by 16-phyllocladanol. These results suggest that the enhancement of Th1 cells polarization to LPS-primed DC induced by 16-phyllocladanol via the activation of IL-12p70 and independent on TLR2 or TLR4.     

Antigen-specific suppression of the plaque-forming cell response induced polyclonally by lipopolysaccharides

Horse erythrocytes (HRBC) were added with LPS in mouse spleen cell cultures, and the effects of HRBC on the LPS-induced polyclonal PFC response were investigated by enumerating total IgM-secreting PFC, anti-HRBC PFC, and PFC against sheep erythrocytes (SRBC). The addition of HRBC influenced the frequencies of anti-HRBC PFC in the total IgM-secreting PFC, but did not influence those of anti-SRBC PFC. The augmentation of the frequencies of anti-HRBC PFC occurred only when an appropriate dose of HRBC was added in the cultures containing T cells. Higher doses of HRBC decreased the frequencies of anti-HRBC PFC whether T cells were present or absent. The degree of reduction of the frequencies of anti-HRBC PFC was dependent on the dose of HRBC, but independent of the dose of LPS. The addition of HRBC at 1 day after LPS stimulation also decreased the frequency of anti-HRBC PFC, though the addition of 2 or 3 days hardly suppressed it. These results suggest that the antigen-specific augmentation occurs via helper T cells, and the suppression is ascribed to the direct action of antigen on the antigen-specific B cells.     

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.     

Stimulation of T-cell activation by UV-treated, antigen-pulsed macrophages: evidence for a requirement for antigen processing and interleukin 1 secretion

The nature of the defect(s) in the ability of UV-treated guinea pig macrophages to stimulate the proliferative response of guinea pig T cells to soluble protein antigens was investigated. T cells proliferated vigorously when cultured with peritoneal exudate cells (PEC) which had been pulsed with soluble protein antigens, but failed to proliferate when cultured with soluble antigen or with antigen-pulsed, UV-treated PEC. UV-treated macrophages were unable to secrete interleukin 1 (IL-1). Addition of IL-1 partially restored the T-cell proliferative response stimulated by antigen-pulsed, UV-treated PEC. However, IL-1 was able to restore such a response only when the PEC were pulsed with antigen before being exposed to UV. Similar results were obtained when antigen-pulsed PEC were used to stimulate T cells to secrete interleukin 2 (IL-2). These results demonstrate that UV-treated macrophages are defective both in their ability to properly process and present antigen for T-cell recognition and in their ability to secrete IL-1.     

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the α_s1-casein-specific proliferation and IFN-γ productivity of CD⁴⁺ T cells, did not affect the proliferative response of CD8⁺ T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8⁺ T cells, we established α_s1-casein-specific CD8⁺ T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8⁺ T cells in oral tolerance is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed. Project supported by the National Natural Science Foundation of China (Grant No. 39230320).     

Regulation of the in vitro secondary antibody response: I. Suppression by relatively high,but physiological levels of calcium ion

Secondary antibody responses generated in vitro with spleen cells from mice primed and boosted with SRBC or TNP-KLH antigen were found to be influenced by the amount of Ca²⁺ in the culture medium. Relatively low levels of Ca²⁺ (0.1 mM) were optimally supportive for the generation of PFC in vitro, with higher, more physiological levels of Ca²⁺ (1.0–1.7 mM) suppressing the generation of PFC by as much as 100%. Suppression by high levels of Ca²⁺ was most pronounced when the amount of antigen used to elicit the in vitro antibody response was high, whereas responses generated by lesser amounts of antigen were minimally affected by Ca²⁺ level. Ca²⁺-mediated suppression was localized to an intermediate phase (24–48 hr) of the response. Mitogenic and polyclonal antibody responses were not affected by high levels of Ca²⁺. The effect of Ca²⁺ concentration on the secondary, IgG-producing antibody response may be significant in terms of understanding the various control mechanisms interacting in regulation of IgG synthesis.     

IL-4-transduced tumor cell vaccine induces immunoregulatory type 2 CD8 T lymphocytes that cure lung metastases upon adoptive transfer.

Vaccinations with tumor cells engineered to produce IL-4 prolonged survival and cured 30% of mice bearing pulmonary metastases, an effect abrogated by in vivo depletion of T cells. Vaccination induced type 2 T cell polarization in both CD4 and CD8 T lymphocyte subsets. We focused on the antitumor activity exerted by type 2 CD8+ T cells (Tc2) activated by IL-4 tumor cell vaccination. Tc2 lymphocytes lacked in vitro tumor cytotoxicity, but released IL-4 upon stimulation with tumor cells, as shown by limiting dilution analysis of the frequencies of tumor-specific pCTL and of CD8 cells producing the cytokine. In vivo fresh purified CD8+ T lymphocytes from IL-4-vaccinated mice eliminated 80-100% of lung metastases when transferred into tumor-bearing mice. CD8+ lymphocytes from IL-4-vaccinated IFN-gamma knockout (KO), but not from IL-4 KO, mice cured lung metastases, thus indicating that IL-4 produced by Tc2 cells was instrumental for tumor rejection. The antitumor effect of adoptively transferred Tc2 lymphocytes needed host CD8 T cells and AsGM1 leukocyte populations, and partially granulocytes. These data indicate that Tc2 CD8+ T cells exert immunoregulatory functions and induce tumor rejection through the cooperation of bystander lymphoid effector cells. Tumor eradication is thus not restricted to a type 1 response, but can also be mediated by a type 2 biased T cell response.     

Biological response modifiers (BRM) as antigens

In order to investigate tumoricidal effector cells in therapy by biological response modifiers (BRM) such asPropionibacterium acnes, bacillus Calmette-Guérin (BCG),Streptococcus pyogenes and a protein-bound polysaccharide (PSK), we established T cell lines specific for each BRM from BALB/c mice immunized with the corresponding BRM. These T cell lines proliferated and produced interleukin-2-(IL-2) and/or IL-4, but only in the presence of the relevant BRM and BALB/c spleen cells as the antigen and antigen-presenting cells respectively. Cross-functional experiments indicated that each BRM acts as a nominal antigen, but not as a non-specific immunostimulator. In addition, the T cell lines killed Ia-positive syngeneic B lymphoma cells, but only in the presence of the relevant BRM. These experiments excluded the possibility of cytotoxic effects by each BRM. The T cell lines and clones also killed Ia-negative bystander target cells, but only in the presence of both a relevant antigen and antigen-presenting cells. The T cell clones specific forS. pyogenes orP. acnes tested were Thy1⁺, L3T4⁺ and Lyt2^–. These results indicate that some BRM exert tumoricidal activity by inducing T cells that recognize them as an antigen and kill tumor cells in an antigen-specific manner. The T cells killed tumor targets in either a tumor-necrosis-factor(TNF)-dependent or a TNF-independent manner. The mediator of the latter pathway remains to be elucidated.     

Discrimination of suppressor T cells of humoral and cell-mediated immunity by anti-Ly and anti-Ia sera.

Evidence is presented which demonstrates that the T lymphocytes which suppress humoral and cell-mediated immunity in CBA/H mice differ in the cell surface structures they express. The suppressor T cells of delayed-type hypersensitivity are Ly-1⁺, Ly-2^? and Ia^?, whereas the suppressor T cells of antibody formation are Ly-1^?, Ly-2⁺ and Ia⁺.     

Activation and IL-10 production of specific CD4+ T cells are regulated by IL-27 during chronic infection with Plasmodium chabaudi

IL-27, a regulatory cytokine, plays critical roles in the prevention of immunopathology during Plasmodium infection. We examined these roles in the immune responses against Plasmodium chabaudi infection using the Il-27ra^−/− mice. While IL-27 was expressed at high levels during the early phase of the infection, enhanced CD4⁺ T cell function and reduction in parasitemia were observed mainly during the chronic phase in the mutant mice. In mice infected with P. chabaudi and cured with drug, CD4⁺ T cells in the Il-27ra^−/− mice exhibited enhanced CD4⁺ T-cell responses, indicating the inhibitory role of IL-27 on the protective immune responses. To determine the role of IL-27 in detail, we performed CD4⁺ T-cell transfer experiments. The Il-27ra^−/− and Il27p28^−/− mice were first infected with P. chabaudi and then cured using drug treatment. Plasmodium-antigen primed CD4⁺ T cells were prepared from these mice and transferred into the recipient mice, followed by infection with the heterologous parasite P. berghei ANKA. Il-27ra^−/− CD4⁺ T cells in the infected recipient mice did not produce IL-10, indicating that IL-10 production by primed CD4⁺ T cells is IL-27 dependent. Il27p28^−/− CD4⁺ T cells that were primed in the absence of IL-27 exhibited enhanced recall responses during the challenge infection with P. berghei ANKA, implying that IL-27 receptor signaling during the primary infection affects recall responses in the long-term via the regulation of the memory CD4⁺ T cell generation. These features highlighted direct and time-transcending roles of IL-27 in the regulation of immune responses against chronic infection with Plasmodium parasites.     

Effects of antilymphocyte serum on thymic independent immunity. 1. Lack of immunosuppressive action on the antibody response to E. coli lipopolysaccharide

Previous studies have shown that cellular and humoral antibody production to type III pneumococcal polysaccharide (SSS-III) is not appreciably altered in neonatally thymectomized mice and is enhanced in animals which have been treated with ALS. In order to determine what effect ALS has on the response to another antigen which does appear to require helper T cells, immunity to E. coli 055:B5 has been investigated. BALB/c mice were injected i.p. with 0.25 ml of ALS on days ?1, 0, and +1 relative to the day of immunization (d.0) with a killed E. coli bacterial vaccine. Splenic plaque forming cells (PFC) and serum hemolysin and hemagglutinin titers were determined 6 days later using sheep erythrocytes which had been coated with purified E. coli lipopolysaccharide (LPS). Mice treated with ALS or normal heterologous serum and immunized with an optimal immunogenic dose of bacteria (150 × 10⁶) had similar numbers of splenic PFC and serum antibody titers. No significant immunosuppressive effect was noted over a wide range of antigen (0.015–1500 × 10⁶) although dose related variations were seen. In contrast to its effect on the response to SSS-III, no enhancement was noted. ALS treated mice which had been simultaneously immunized with E. coli and sheep RBC had specific depression of the T helper dependent response to SRBC but not to LPS. The lack of immunosuppressive effect on antibody production to E. coli LPS provides strong evidence that ALS preferentially acts on T lymphocytes. It further indicates that enhancement occurs with some but not all T helper independent antigens.