Defective helper T-cell activity in LPS-unresponsive C3H/HeJ mice

C3H/HeJ mice, unresponsive to LPS, exhibit a defective ability to mount antibody responses to T-dependent immunogens. The anti-TNP antibody response to TNP-HRBC, a T-dependent immunogen, was found to be lower in these mice as compared to LPS-responsive C3H/HeN mice, whereas the anti-TNP antibody response to TNP-Ficoll, a T-independent immunogen, was of the same magnitude in C3H/HeJ and C3H/HeN mice. An impaired helper activity of C3H/HeJ HRBC-primed spleen cells was demonstrated in a titration assay in which graded numbers of C3H/HeJ or C3H/HeN HRBC-primed spleen cells were added to cultures containing a constant number of unprimed spleen cells from either C3H/HeJ or C3H/HeN mice and the immunogen TNP-HRBC. The reduced helper T-cell activity of C3H/HeJ HRBC-primed spleen cells appears to be independent of macrophage defects, since C3H/HeJ and C3H/HeN macrophages were found equally effective in antigen presentation as evaluated by an in vitro antigen-specific T-cell proliferation assay. The difference in helper T-cell activity between these two substrains probably reflects a lower number and/or proliferation rate of antigen-responsive T cells in C3H/HeJ mice.     

Enhancing effect of IFN-gamma on helper T cell activity and IL 2 production

A single injection of young murine immune interferon (IFN-gamma) in young (3 mo) or old (14 to 24 mo) mice 3 days before carrier-priming significantly enhances helper T cell activity of their spleen cells. Maximal enhancement is attained when IFN-gamma is injected once immediately before priming or for 4 consecutive days from the time of priming. Helper activity for anti-TNP antibody response was titrated in vitro by adding graded numbers of spleen cells from HRBC-primed mice of a given age to cultures containing a constant number of spleen cells from 3-mo-old normal mice and TNP-HRBC. When T cell-enriched spleen cells from HRBC-primed young or old mice, uninjected or injected with IFN-gamma, were separated by nylon wool filtration into passed (Thi) and adherent (Th2) cells, the helper activity of both T cell subpopulations was found to be enhanced by IFN-gamma injection. Helper activity of purified Th1 and Th2 cells was also increased by their in vitro preincubation with IFN-gamma. Furthermore, interleukin 2 (IL 2) production by mitogen-activated spleen cells from young and old mice is enhanced by addition of IFN-gamma to cultures. These data altogether indicate that IFN-gamma plays an important role in immunoregulation of helper T cell activity.     

T-T cell interaction in the induction of delayed-type hypersensitivity (DTH) responses: vaccinia virus-reactive helper T cell activity involved in enhanced in vivo induction of DTH responses and its application to augmentation of tumor-specific DTH responses

The role of antigen-specific helper T cells in augmenting the in vivo development of delayed-type hypersensitivity (DTH) responses was investigated. C3H/HeN mice were inoculated i.p. with vaccinia virus to generate virus-reactive helper T cell activity. These vaccinia virus-primed or unprimed mice were subsequently immunized subcutaneously (s.c.) with either trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self), vaccinia virus-infected spleen cells (virus-self), or cells modified with TNP subsequent to virus infection (virus-self-TNP). Seven days later, these mice were tested for anti-TNP DTH responses either by challenging them directly with TNP-self into footpads or by utilizing a local adoptive transfer system. The results demonstrated that vaccinia virus-primed mice failed to generate significant anti-TNP DTH responses when s.c. immunization was provided by either virus-self or TNP-self alone. In contrast, vaccinia virus-primed mice, but not unprimed mice, could generate augmented anti-TNP DTH responses when immunized with virus-self-TNP. Anti-vaccinia virus-reactive helper activity was successfully transferred into 600 R x-irradiated unprimed syngeneic mice by injecting i.v. spleen cells from virus-primed mice. These helper T cells were found to be antigen specific and were mediated by Thy-1+, Lyt-1+2- cells. DTH effector cells enhanced by helper T cells were also antigen specific and were of the Thy-1+, Lyt-1+2- phenotype. Furthermore, vaccinia virus-reactive helper T cell activity could be applied to augment the induction of tumor-specific DTH responses by immunization with vaccinia virus-infected syngeneic X5563 tumor cells. T-T cell interaction between Lyt-1+ helper T cells and Lyt-1+ DTH effector T cells is discussed in the light of the augmenting mechanism of in vivo anti-tumor-specific immune responses.     

Radiosensitivity of the helper cell function.

The helper function of T cells primed and irradiated in vivo was tested in vitro by the Mishell-Dutton technique. Spleen cells from mice carrier-primed with HRBC and exposed to 50 to 2000 rads of x-radiation were assayed for their ability to help syngeneic normal spleen cells to mount an in vitro anti-hapten antibody response after stimulation with the conjugate TNP-HRBC. The anti-TNP response was evaluated by the Jerne technique. The helper activity was titrated by adding graded numbers of carrier-primed spleen cells to a constant number of normal spleen cells. The slope of the initial linear portion of the response-cell dose titration curve was taken as an estimated of the helper activity and found to decrease with increasing the x-ray dose. The curve describing the remaining helper activity as a function of the radiation dose shows the presence of two components, one radiosensitive, the other, radioresistant. This suggests the existence either of helper cells at different stages of activation or of two cell subpopulations participating in the helper function.     

Activation of idiotype-specific suppressor T cells by injection of antibody carrying a recurrent idiotype

Intravenous injection of spleen cells (SC) coated with an antitrinitrophenyl (anti-TNP) IgM monoclonal antibody, Sp6 (Sp6-SC), which carries a recurrent idiotype, resulted in activation of a Lyt-2-positive population which did adhere to Sp6-coated plates. No effect of Sp6-SC injection could be observed in vivo on an anti-TNP B-cell response when mice were primed with an immunogenic dose of TNP-horse red blood cells (HRBC), but an anti-TNP response was observed when Sp6-SC-injected mice were primed with a subimmunogenic dose of TNP-HRBC. Furthermore, after intravenous (iv) injection of Sp6-SC, it was no longer possible to suppress a primary anti-TNP response by iv injection of TNP-haptenized thymocytes. In vitro analysis showed that the Sp6-induced suppressor T cell (Ts) population had no measurable influence on TNP-specific naive B cells, nor did it suppress TNP-specific helper T cells (THTNP), but it did lead to counterregulation of TNP-specific suppressor T cells (TsTNP). Hence, iv injection of antibody carrying a recurrent idiotype resulted in activation of a Ts population which functioned as inhibitor of suppression, thus displaying a helper effect.     

Helper T cells against tumor-associated antigens (TAA): preferential induction of helper T cell activities involved in anti-TAA cytotoxic T lymphocyte and antibody responses

This study establishes assay systems for helper T cell activities assisting cytotoxic T lymphocyte (CTL) and antibody responses to tumor-associated antigens (TAA) and demonstrates the existence of TAA that induce preferentially anti-TAA CTL helper and B cell helper T cell activities in two syngeneic tumor models. C3H/HeN mice were immunized to the syngeneic X5563 plasmacytoma or MH134 hepatoma. Spleen cells from these mice were tested for anti-TAA helper T cell activity capable of augmenting anti-trinitrophenyl(TNP) CTL and anti-TNP antibody responses from anti-TNP CTL and B cell precursors (responding cells) by stimulation with TNP-modified X5563 or MH134 tumor cells. The results demonstrate that cultures of responding cells plus 85OR X-irradiated tumor-immunized spleen cells (helper cells) failed to enhance anti-TNP CTL or antibody responses when in vitro stimulation was provided by either unmodified tumor cells or TNP-modified syngeneic spleen cells (TNP-self). In contrast, these cultures resulted in appreciable augmentation of anti-TNP CTL or antibody response when stimulated by TNP-modified tumor cells. Such anti-TAA helper activities were revealed to be Lyt-1+2- T cell mediated and TAA specific. Most interestingly, immunization with X5563 tumor cells resulted in anti-TAA helper T cell activity involved in CTL, but not in antibody responses. Conversely, TAA of MH134 tumor cells induced selective generation of anti-TAA helper T cell activity responsible for antibody response. These results indicate that there exists the qualitative TAA-heterogeneity as evidenced by the preferential induction of anti-TAA CTL- and B cell-helper T cell activities. The results are discussed in the light of cellular mechanisms underlying the preferential anti-TAA immune responses, and the interrelationship between various types of cell functions including CTL- and B cell-help.     

In vivo restoration of T cell functions by human IL-1 beta or its 163-171 nonapeptide in immunodepressed mice

The immunorestorative capacities of human (hu) IL-1 beta or its synthetic fragment 163-171 (VQGEESNDK) were assessed in vivo in mice immunodepressed by aging, sublethal irradiation, or both. Subcutaneous administration of hu rIL-1 beta into immunodepressed animals immediately after carrier (horse red blood cells, HRBC) priming could restore to normal levels Th cell activity. This was measured as the ability of spleen cells from HRBC-primed mice to induce a hapten-specific antibody response in spleen cells from nonimmune mice in vitro stimulated with the hapten-carrier conjugate TNP-HRBC. In parallel, the ability of spleen cells from hu rIL-1 beta-treated immunodepressed animals to produce T cell growth factor activity upon in vitro mitogen stimulation was also increased significantly as compared to that of untreated mice and approached that of immunocompetent controls. The immunorestorative activity of hu rIL-1 beta on Th cell activity and T cell growth factor production could be mimicked by the synthetic nonapeptide 163-171 which, at the doses used, produced in most instances even greater effects than the whole protein. Although the optimal immunorestorative doses of the 163-171 peptide were several orders of magnitude higher than those of hu rIL-1 beta, the complete lack of IL-1-like inflammatory and toxic effects suggests that the synthetic hu IL-1 beta fragment may be successfully used as immunomodulating agent in the therapy of T cell immunodeficiencies.     

I-J restriction of T cells that suppress in vivo antibody responses to Thy-1

The contact-sensitizing haptens dinitrophenyl (DNP) and oxazalone (Ox) act as helper determinants for antibody responses to Thy-1 when conjugated to donor thymus cells. The helper effect is transferrable from primed to naive mice with spleen cells, producing specific augmentation of in vivo PFC responses to Thy-1. The helper cells are hapten-specific and require associative recognition of hapten and Thy-1, excluding a role for nonspecific B cell activation. The phenotype of the helper cells is Thy-1+ and Lyt-1+2-. Antigen-specific suppression could be readily generated by using an inoculum of DNP-modified syngeneic RBC. T cells from these suppressed donors (Ts) were shown to abolish the helper effects of TH in adoptive transfer experiments in vivo. These Ts were characterized as Thy-1+ and Lyt-1-2+. A requirement for MHC compatibility at the I-J subregion was necessary between the Ts and the recipient to obtain a transfer of suppression.     

In vitro immune response to the 2,4,6-trinitrophenyl determinant in aged C57BL/6J mice:changes in the humoral immune response to, avidity for the TNP determinant and responsiveness to LPS effect with aging.

An in vitro anti-TNP response of the spleen cells from aged C57BL/6J mice showed approximately 4-fold less PFC than did that from young adult mice. Anti-theta serum-treated young spleen cells gave an anti-TNP response that was definitely greater than the response of the anti-theta serum-treated aged spleen cells in the presence of the exogenous activated thymus cells as helper cells. These results suggest that the deficits in B cells may be partly responsible for the imparied anti-TNP response of the aged spleen cells. To examine further the capacity of stem cells in the bone marrow to generate B cells responsible for anti-TNP response in the spleen, we injected i.v. 1.5 to 2.0 times 10(7) bone marrow cells from young or aged mice into lethally irradiated syngeneic recipients that had previously been thymectomized. Four to 6 weeks later, 10(7) spleen cells from the two groups of these recipient mice were immunized with TNP-SRBC in the presence of the exogenous activated thymus cells and assayed for anti-TNP PFC. The response of the aged marrow-derived B cells was approximately one-half of that of the young marrow-derived B cells.The avidity for TNP determinant of the antibodies produced by the PFC was determined by the plaque-inhibition technique. The avidity of the antibodies produced by the aged mice was approximately 33 times lower than that by the young mice. Anti-TNP response of the young spleen cells were markedly enhanced by the addition of LPS to the cultures, whereas no or little enhancement of the response was induced in the aged spleen cells even in the presence of high concentration of LPS. In contrast, DNA synthesis of both the young and aged spleen cells was comparably stimulated by 1 mug/ml and 10 mug/ml of LPS, however, it was rather less in the aged spleen cells at a concentration of 100 mug/ml. Mechanisms responsible for the changes in avidity and responsiveness to LPS with aging are discussed.     

Evidence for histamine-induced auto-anti-idiotypic antibody immunoregulation in vivo

The in vivo effects of histamine injection in LAF₁ male mice on the immune reactivity to trinitrophenylated bovine γ-globulin was studied using plaque-forming cell (PFC) responses and their avidity distributions. Splenic anti-trinitrophenyl (anti-TNP) PFC responses of mice treated with histamine (5 × 10^?6 mol or 1 mg, intravenously) were significantly reduced in number and restricted in heterogeneity and characterized by a preferential loss of high-avidity IgG PFCs. The reduced PFC response in histamine-treated mice was dose and time dependent. No evidence of suppressor cell activity in the spleens from histamine-treated mice was demonstrable. Only histamine-treated mice produced a significantly high percentage of anti-idiotype-blocked, hapten-augmentable IgG PFCs, suggesting the presence of auto-anti-idiotypic activity. Immune sera taken from histamine-treated mice caused an inhibition of anti-TNP PFC in vitro. This PFC-inhibiting factor in immune sera of histamine-treated mice was an antibody of the IgG₁ and IgG_2a class, lacked anti-TNP antibody activity, but reacted with anti-TNP antibody of LAF₁ origin. Passive hemagglutination study of this sera showed anti-(anti-TNP F(ab′)₂-IgG) titer. Thus, the results of this study suggest that histamine in combination with antigen induces auto-anti-idiotypic antibody which, in turn, is involved in the normal regulation of the immune response to trinitrophenylated bovine γ-globulin in vivo.     

Suppressor activity in the spleen of neonatal mice.

Helper cells of T-cell origin are required for the in vitro proliferation of low numbers of adult mouse thymus cells in response to allogeneic spleen cells. These helper cells are present in the adult mouse spleen. We have demonstrated that neonatal mouse spleen cells lack the helper activity present in adult spleen cells. We have also shown that this lack of helper activity is the result of active suppression. The suppression is due to a suppressor cell which is present in high quantities in the neonatal spleen and can be eliminated by treatment with anti-θ serum and complement.     

Secretion rate independent evaluation of IgM antibody avidity at the level of single immunocytes.

The hemolytic plaque inhibition assay has been performed on spleen cells from mice immunized with TNP-HRBC to evaluate avidity of anti-TNP IgM antibodies. At different times after immunization direct plaques were inhibited by soluble TNP-EACA, TNP61-BGG, or anti-mu antiserum. Analysis of the inhibition data provided independent estimates of antibody avidity and secretion rate. Avidity was found to increase with time, to reach a maximum when the antibody response attained the peak value, and then to decline as the response was waning. There was a decrease followed by increase of the secretion rate concomitant with the rise and fall of the antibody response and avidity.     

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.     

An analysis of B cell memory. I. Interaction of helper and suppressor effects in the in vitro expression of IgM memory.

The pfc response of Srbc primed IgM memory cells has been characterized by limiting dilution analysis in vitro, in which LPS was used to maximize the response of spleen cells to Srbc. The analysis suggested that, even under these conditions, expression of B cell memory was not directly assayed and cell collaboration effects were still basic to the system.Two types of cells, as defined by function, appeared necessary to elicit optimal clonal proliferation of IgM B memory cells: firstly, helper T cells were essential for B cell proliferation even with LPS present in culture. Under appropriate conditions, helper activity could be provided by normal thymus cells. Secondly, activated T cells were required for the maximal conversion of normal thymus cells to helper cells. A third activity, T cell-dependent suppression, was observed at high cell doses. The implications of these results and the need for a comprehensive analysis of in vitro conditions for each individual type of experiment is discussed.     

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

The production of anti-hapten antibody after immunization with trinitrophenylated (TNP) hamster erythrocytes (HRBC) or sheep erythrocytes (SRBC) was determined in high- and low-responder mouse strains against HRBC antigen. 1) Anti-TNP antibody was detected in sera of high-responder DDD and CF1 mice after primary immunization with TNP-HRBC, but not in those of low-responder C57BL/6 mice. 2) Anti-TNP antibody was detectable in sera of all the strains after primary immunization with TNP-SRBC. 3) Production of anti-TNP antibody was elicited after a booster injection of TNP-HRBC in low-responder C57BL/6 mice pre-sensitized with HRBC in Freund's complete adjuvant. These results suggest that functions of thymus-derived cells specific for HRBC antigen are deficient in low-responder mice.     

LPS-induced autoantibody response. I. Ontogenic development of PFC response to bromelain-treated syngeneic erythrocytes

Normal adult mice have been shown to contain a large number of cells secreting antibodies against bromelain-treated syngeneic erythrocytes (Br.MRBC) and the numbers remarkably increase by the stimulation with LPS. In this report development of the anti-Br.MRBC response during ontogeny was examined and it was shown that on the injection of LPS suckling mice responded little to generate splenic plaque-forming cells (PFC) against Br.MRBC in vivo and in vitro. The responsiveness of suckling mice to produce anti-Br.MRBC was shown to be less developed than the anti-TNP response or the mitotic response to LPS. The low responsiveness of suckling mice was analyzed in terms of suppressor activity in the spleen cell population, proliferative capacities of the precursors of anti-Br.MRBC PFC, and their frequencies in the spleen. In the coculture experiment of suckling and adult spleen cells or culture of anti-brain-associated Thy 1-treated, macrophage-depleted spleen cell population, no evidence was obtained to show that suckling spleen cells contained suppressor cells. Kinetic profiles studied in vitro showed that anti-Br.MRBC PFC in the suckling spleen did not increase during the culture as those in the adult spleen. Studies on the precursor frequencies revealed that spleen cells of 15-day-old mice contained precursors of anti-Br.MRBC PFC amounting to 20.5% of the adult precursors whereas the PFC response in vitro by the former was only 4% of the latter. From these experimental data, it was concluded that the low responsiveness of suckling mice was partly due to the low frequency of the precursors in the spleen and, in addition, to the defective nature of the precursors in proliferating to differentiate into PFC.     

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.     

Effect of bacterial lipopolysaccharides on anti-trinitrophenyl antibody-producing cells. Nonspecific modification of the affinity at the cellular level.

The effect of lipopolysaccharide (LPS) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. Simultaneous injection of bacterial LPS and TNP-coupled sheep red blood cells(SRBC) induced an obvious increase in anti-TNP PFC numbers and heightened the antibody affinity at cellular levels. The higher the doses of LPS, the greater the effects. Concomitant injection of LPS in TNP-coupled homologous mouse red blood cells (MRBC) also elicited good anti-TNP PFC response and slightly heightened the affinity. Priming with LPS and SRBC together 7 days prior to immunization did not enhance the anti-TNP PFC response and it was difficult to alter the affinity. Preinjection with small amounts of TNP-MRBC or -rabbit red blood cells and LPS simultaneously did not induce any significant increase in anti-TNP PFC secondary response after reimmunization with TNP-SRBC, but obviously heightened the antibody affinity. Injection of LPS simultaneously with the secondary immunization was effective for both the anti-TNP PFC response and the alteration of antibody affinity. These results suggest that LPS affects the control mechanisms of anti-TNP antibody affinity via the non-thymus-derived helper cell function, and the adjuvant action and alteration of antibody affinity induced by LPS are regulated by different mechanisms.     

Effect of Bacterial Lipopolysaccharides on Anti-Trinitrophenyl Antibody-Producing Cells

The effect of lipopolysaccharide (LPS) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. Simultaneous injection of bacterial LPS and TNP-coupled sheep red blood cells(SRBC) induced an obvious increase in anti-TNP PFC numbers and heightened the antibody affinity at cellular levels. The higher the doses of LPS, the greater the effects. Concomitant injection of LPS in TNP-coupled homologous mouse red blood cells (MRBC) also elicited good anti-TNP PFC response and slightly heightened the affinity. Priming with LPS and SRBC together 7 days prior to immunization did not enhance the anti-TNP PFC response and it was difficult to alter the affinity. Preinjection with small amounts of TNP-MRBC or -rabbit red blood cells and LPS simultaneously did not induce any significant increase in anti-TNP PFC secondary response after reimmunization with TNP-SRBC, but obviously heightened the antibody affinity. Injection of LPS simultaneously with the secondary immunization was effective for both the anti-TNP PFC response and the alteration of antibody affinity. These results suggest that LPS affects the control mechanisms of anti-TNP antibody affinity via the non-thymus-derived helper cell function, and the adjuvant action and alteration of antibody affinity induced by LPS are regulated by different mechanisms.     

Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2

Antibody production to sheep erythrocytes (SRBC) or hapten-conjugated SRBC (TNP-SRBC) was studied in mice with chronic Trypanosoma cruzi infections. Studies in vivo demonstrated that both IgM and IgG anti-SRBC responses were suppressed during chronic infection. Secondary IgG responses were suppressed regardless of whether the primary immunization was given before or after infection. The ability of cells from infected mice to provide help for antibody production was examined in vitro. Anti-SRBC responses were restored to cultures of whole spleen cells from infected mice by the addition of interleukin 2 (IL 2)-rich supernatants, indicating that these cells were capable of antibody production when sufficient help was provided. T cells from SRBC-primed infected mice were unable to provide significant help to normal B cell/M phi cultures for in vitro anti-TNP or anti-SRBC responses. The percentages of Thy-1+, Lyt-1+, and Lyt-2+ spleen cells were not significantly different between normal and infected mice. Anti-TNP and anti-SRBC responses were restored to cultures that contained T cells from infected mice and normal B cell/M phi by the addition of IL 2-rich spleen cell supernatants. The suppression of in vitro antibody responses in mice with chronic T. cruzi infections was associated with a lack of T cell help, which was provided by exogenous spleen cell supernatant.