beta-estradiol suppresses T cell-mediated delayed-type hypersensitivity through suppression of antigen-presenting cell function and Th1 induction

BACKGROUND: Although an immunomodulatory role for estrogens has long been demonstrated by experimental and clinical observations, the mechanism by which estrogens exert their effect on T cells has not been clearly defined. METHODS: In this study we analyzed the effects of beta-estradiol (E2), at its contraceptive dose, on the delayed-type hypersensitivity (DTH) to purified protein derivatives (PPD) and associated immune response in female mice. RESULTS: E2 treatment decreased PPD-specific DTH response, which coincided with a decrease in the leukocytes numbers in the draining lymph nodes (DLN) and spleen compared with control mice. E2 treatment also suppressed the in vitro PPD-specific proliferative response of DLN and spleen cells from PPD-primed mice. The analysis of production and gene expression of cytokines by DLN cells demonstrated that E2 treatment suppressed IL-2 and IFN-gamma production in response to PPD in vitro. In contrast, IL-4 and IL-10 gene expression by DLN cells of E2-treated mice, taken 24 h after in vivo restimulation of mice with PPD, was enhanced. Furthermore, we found that spleen APC from E2-treated mice failed to induce optimum proliferation of the PPD-primed T cells in response to PPD in vitro. The impaired APC function by E2 was not due to induction of suppressor cell activity because addition of the normal spleen APC to APC from E2-treated mice restored the proliferative response of the PPD-primed T cells in response to PPD. CONCLUSION: Our results suggest that the E2-mediated inhibition of DTH reaction is due to a combination of the down regulation of APC function and deviation of the immune response from Th1-type to Th2-type.     

In vivo treatment with interferon causes augmentation of IL-2 induced lymphokine-activated killer cells in the organs of mice.

Interferon-alpha (IFN-alpha) has been shown to synergize with IL-2 in the regression of a variety of established murine tumours and studies are underway to explore this combination in patients with advanced cancers as well. To understand the mechanism of synergy we have studied lymphokine-activated killer (LAK) cell activity in various compartments of mice in response to IFN-alpha and IL-2 administration. The effects of IFN-gamma, TNF-alpha and IL-4 were also examined. C57BL/6 mice were injected intraperitoneally with HBSS, IL-2 alone, IFN-alpha alone or both, two times a day for 7 days. On days 4 and 8, LAK activity was tested in a 4-h chromium release in cells obtained from lungs, spleen, and liver using fresh MCA-102 tumour cells as targets. The cells from control mice failed to lyse the MCA-102 target. IL-2 caused the generation of LAK activity and an increase in total cell yield in all the organs after 3 days of injection. IFN-alpha failed to generate LAK activity but when administered along with IL-2, caused synergistic enhancement of LAK lysis of MCA-102 target cells. Cell yield in this group was lower as compared with the IL-2-treated group. LAK activity tested after 7 days of IL-2 therapy was significantly decreased compared with that observed after 3 days. However, activity remained at as high a level after 7 days of therapy as after 3 days of therapy in animals treated with IFN-alpha and IL-2. FACS analysis revealed that asialo GM-1+ (ASGM-1) and NK1.1+ cells were increased in number in IL-2 and IL-2 plus IFN-alpha-treated spleen; however, the number of these cells was similar in both groups. In the liver, ASGM-1+ cells were higher in the IL-2 plus IFN-alpha group than in the group treated with IL-2 alone. By in vitro depletion utilizing antibody and Rbc' experiments, it was clear that both ASGM-1+ and NK1.1+ cells from the spleen mediated most of the cytotoxicity of MCA-102 targets. Pre-treatment irradiation (5 Gy) of mice completely abrogated the capability of IL-2 or IL-2 plus IFN-alpha to generate LAK activity. IFN-gamma also had a stimulatory effect on IL-2 induction of LAK activity. Tumour necrosis factor-alpha (TNF-alpha) and IL-4 failed to generate LAK activity and, in combination with IL-2, no additional stimulatory effect was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhancement of a delayed hypersensitivity reaction to a contact allergen, by the systemic administration of interleukin-2.

The immunopharmacological effects of interleukin-2 (IL-2) on the sensitization and effector phases of the delayed-type hypersensitivity (DTH) reaction were studied using contact sensitivity to the haptenizing agent dinitrochlorobenzene (DNCB). When administered at the time of priming to DNCB, IL-2 had no effect on the subsequent magnitude of the response. Interleukin-2 was, however, able to increase the magnitude of the response when given at the time of secondary challenge; the degree of change was directly related to the dose of IL-2. The proportions of T cells in the draining lymph node and spleen of IL-2-treated animals decreased by approximately one-third, but there was no alteration to the balance between CD4+ and CD8+ T cells. The results suggest that the increase in DTH observed was due to a pharmacological effect rather than to an increase in T-cell number.     

Up-regulation of IL-2 induced lymphokine activated killer cell activity by cisplatin and FK-565: involvement of calcium ion.

As reported earlier, the IL-2 induced lymphokine activated killer (LAK) activity is significantly up-regulated in the presence of cisplatin/FK-565. Based on these observations, we have investigated whether calcium is involved in the generation of LAK activity by IL-2 alone or along with CP/FK-565. We have shown that treatment of PBMC with IL-2 for four days caused an increase in intracellular free calcium and in ATP levels, which were further significantly enhanced when LAK cells were generated in the presence of CP/FK-565. Depletion of calcium resulted in decreased cytotoxic activity. Addition of tumor cells to LAK cells, generated in the presence of IL-2 alone or along with CP/FK-565 caused an instant rise in intracellular free calcium which was significantly decreased when an increase in intracellular free calcium was observed in calcium-free, EGTA-containing buffer. These data suggest that calcium is required for the activation and manifestation of lytic activity by LAK cells. Further, we observed that the increase in intracellular free calcium is not associated with the blastogenic response of peripheral blood mononuclear cells in response to treatment of IL-2 alone or together with CP/FK-565.     

Immunopharmacological actions of an extract isolated from inflamed skin of rabbits inoculated with Vaccinia virus (Neurotropin)--II. Restorative effect on immune responses through the recovery of IL-2 production in aging mice

To examine the immunopharmacological actions of an extract isolated from inflamed skin of rabbits inoculated with Vaccinia virus (Neurotropin), its effect on the immune responses in aging BALB/c mice was examined. Neurotropin clearly restored the decreasing T-cell-dependent immune responses such as delayed-type hypersensitivity (DTH) response and plaque-forming cells (PFC) response to sheep red blood cells (SRBC) when administered i.p. from 13 months old (mo) to 16 mo. However, Neurotropin administration from 2 to 5 mo had no effect on the immune responses of young animals. Neurotropin administration from 13 to 16 mo restored not only the T-cell proliferation of spleen cells induced by concanavalin A (Con A) and phytohemagglutinin (PHA), but also the interleukin-2 (IL-2) production by spleen cells activated with Con A. However, Neurotropin did not affect the responsiveness of Con A-activated spleen cells to exogenous recombinant IL-2. An absence of suppressor cells capable of inhibiting the IL-2 production in the spleens was confirmed in the 16 mo mice. Neurotropin administration also restored IL-1 production by peritoneal macrophages stimulated with lipopolysaccharide (LPS). These results suggest that long-term administration of Neurotropin restores the decreasing T-cell-dependent immune responses through the recovery of IL-2 and in part IL-1 production, but not the responsiveness to IL-2 in aging BALB/c mice.     

The effect of MS14 on innate and cellular immune responses in BALB/c mice

MS14 is an Iranian natural preparation of herbal-marine source with no obvious toxicity in oral administration, which possesses anti-inflammatory and immunomodulatory effects. In this study, the effect of oral administration of MS14 on nitric oxide (NO) production of peritoneal macrophages and lymphocyte Th1 cytokines and delayed-type hypersensitivity (DTH) test in BALB/c mice were investigated. Peritoneal macrophages were cultured and NO production was measured by Griess method. Viability of macrophages was assayed by MTT (3-(4,5-dimethy-2-lthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) test. Interleukin-2 (IL-2) and INFγ levels in supernatant of spleen lymphocytes culture were assayed by enzyme-linked immunosorbent assay kit. For DTH test the mice were immunized with sheep red blood cell and DTH was measured 24?h after the last immunization of mice. NO production of macrophages has been diminished significantly in MS14 treated group (about 40%) at the presence or absence of stimulators. Macrophage viability had no significant alteration after MS14 administration. However, interferon-γ production of lymphocytes was significantly decreased in MS14 group both at the absence or presence of concanavalin A (ConA; about 50%); IL-2 production declined about 20% at the presence of ConA. In comparison with the control group, MS14 had no statistically significant effect on DTH test. The results have pointed that MS14 may have immunomodulatory potentials at least through its decreasing effect on NO production of macrophages and level of Th1 cytokine pattern.     

The effect of MS14 on innate and cellular immune responses in BALB/c mice

MS14 is an Iranian natural preparation of herbal—marine source with no obvious toxicity in oral administration, which possesses anti-inflammatory and immunomodulatory effects. In this study, the effect of oral administration of MS14 on nitric oxide (NO) production of peritoneal macrophages and lymphocyte Th1 cytokines and delayed-type hypersensitivity (DTH) test in BALB/c mice were investigated. Peritoneal macrophages were cultured and NO production was measured by Griess method. Viability of macrophages was assayed by MTT (3-(4,5-dimethy-2-lthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) test. Interleukin-2 (IL-2) and INFγ levels in supernatant of spleen lymphocytes culture were assayed by enzyme-linked immunosorbent assay kit. For DTH test the mice were immunized with sheep red blood cell and DTH was measured 24?h after the last immunization of mice. NO production of macrophages has been diminished significantly in MS14 treated group (about 40%) at the presence or absence of stimulators. Macrophage viability had no significant alteration after MS14 administration. However, interferon-γ production of lymphocytes was significantly decreased in MS14 group both at the absence or presence of concanavalin A (ConA; about 50%); IL-2 production declined about 20% at the presence of ConA. In comparison with the control group, MS14 had no statistically significant effect on DTH test. The results have pointed that MS14 may have immunomodulatory potentials at least through its decreasing effect on NO production of macrophages and level of Th1 cytokine pattern.     

IL-2、IL-4和IL-7体外诱导人外周血淋巴因子激活的杀伤细胞效应的比较

比较了淋巴因子ＩＬ－２、ＩＬ－４和ＩＬ－７在体外诱导健康人外周血淋巴因子激活的杀伤 （ＬＡＫ）细胞活性的效应。结果表明，ＩＬ－７可单独，亦可与ＩＬ－２、ＩＬ－４协同诱导ＬＡＫ细胞，而且ＩＬ－７ 诱导ＬＡＫ细胞的效应不被抗ＩＬ－２、抗ＩＬ－４所抑制。ＩＬ－７单独诱导的ＬＡＫ细胞活性高峰迟于ＩＬ－ ２或ＩＬ－４所诱导的活性高峰，且与增殖反应曲线一致。抗ＣＤ８抗体明显抑制ＩＬ－７诱导ＬＡＫ细胞 的效应，而ＩＬ－７诱导ＬＡＫ细胞的效应不能被抗ＮＫＨ－１所抑制。提示：ＩＬ－７ 激活ＬＡＫ细胞的效应 机制不依赖ＩＬ－２和ＩＬ－４，并很可能成为肿瘤过继治疗中的重要淋巴因子。     

Recombinant IL-7 enhances the potency of GM-CSF-secreting tumor cell immunotherapy

IL-7 is known for its role in lymphopoiesis and T-cell homeostasis. In addition, its capacity to augment the immune response to weak or low affinity antigens makes it an ideal candidate to evaluate in combination with a GM-CSF-secreting tumor cell immunotherapy, which has been shown to elicit broad humoral and cellular immune responses. The studies reported here show that IL-7, when combined with a GM-CSF-secreting tumor cell immunotherapy, significantly prolonged the survival of tumor-bearing mice. The enhanced anti-tumor protection correlated with an increased number of activated dendritic cells (DC) and T cells in lymphoid tissues, such as the draining lymph nodes (DLN) and spleen. Moreover, an increased number of activated effector T cells were found in the tumor microenvironment, correlating with a more potent systemic tumor-specific T-cell response than each monotherapy alone. Taken together, these studies demonstrate that IL-7 augments the anti-tumor response of a GM-CSF-secreting tumor cell immunotherapy in preclinical models.     

In vivo boosting of lung natural killer and lymphokine-activated killer cell activity by interleukin-2: comparison of systemic, intrapleural and inhalation routes.

Natural killer (NK) cells are thought to play a role in host defence against malignancy and infection, in immunoregulation and as precursor cells in a generation of lymphokine-activated killer (LAK) cells which can lyse NK-resistant tumour cells. As the lung is a major site for malignancy and infection and as there are large numbers of lymphoid cells including NK cells in the interstitial compartment of the lung, we evaluated the capacity of interleukin-2 (IL-2), a lymphokine capable of augmenting NK activity in vitro, to augment lung NK cell activity in vivo, using different routes of IL-2 administration. We compared both systemic (i.v. and i.p.) and local (intrapleural and inhalation) routes of IL-2 administration (50,000 U/daily for 5 days) using CBA mice, assessing NK and LAK cell activity in the spleen (systemic) and in the lung. The target cells used for these studies were the YAC-1 (NK-sensitive) and P815, NO36 and HA56 (NK-resistant, LAK-sensitive) cell lines. Splenic NK activity was increased by 1.4-1.9-fold for i.v./i.p., respectively, compared with controls with both systemic routes of administration, and lung NK activity was increased 3.2-fold and 3.8-fold (i.v./i.p, respectively, P less than 0.05), to levels which were comparable to systemic (splenic) NK activity following the same therapy. Intrapleural IL-2 administration similarly enhanced lung NK activity (3.3-fold) and splenic NK activity (1.3-fold; P less than 0.05 versus controls for both). Surprisingly, inhaled IL-2 suppressed both splenic and lung NK cell activity (84 +/- 8% and 78 +/- 10% suppression, respectively, P less than 0.05). LAK cell activity was also enhanced in the lung by 1.8-8-fold in response to i.v., i.p. and intrapleural IL-2, whereas inhaled IL-2 was ineffective in generating LAK cell activity. These results suggest that the systemic and intrapleural administration of IL-2 effectively boost pulmonary NK and LAK activity whereas inhalation of IL-2 does not. Thus, in clinical situations where boosting of local lung NK or LAK cell activity is desired, these routes of IL-2 administration may be effective.     

Up-regulation of induction of lymphokine (IL-2)-activated killer (LAK) cell activity by FK-565 and cisplatin

The role of cisplatin and FK-565 in up-regulation of lymphokine-activated killer (LAK) cell induction by IL-2 was examined. Treatment of blood mononuclear cells (MNC) of healthy donors with cisplatin or FK-565 in the presence of IL-2 resulted in a significant increase in LAK activity against natural killer (NK)-resistant Daudi cells as assessed by the 4 h 51Cr release assay. Blood MNC treated with cisplatin alone was not cytotoxic to Daudi cells. However, MNC treated with FK-565 showed some cytotoxicity against Daudi cells. Addition of cisplatin to IL-2-stimulated MNC did not increase proliferation but did enhance cytotoxicity. FK-565 together with IL-2 increased both proliferation and cytotoxicity of blood MNC. These data suggest the potential of cisplatin and FK-565 in LAK adoptive immunotherapy for cancer treatment.     

rIL-4 differentially regulates rIL-2-induced murine NK and LAK killing in CD8+ and CD8- precursor cell subsets

Interleukin 4 (IL-4) and IL-2 have complementary or synergistic roles in many aspects of lymphocyte development. IL-2 supports the induction of cytolytic activity in cytotoxic T lymphocyte (CTL), natural killer (NK), and lymphokine-activated killer (LAK) cells. IL-4 has also been shown to support CTL and LAK in primary murine spleen cell culture. This report demonstrates that IL-4 selectively down-regulates IL-2 inducible murine CD8- precursors of NK cells. For maximal regulatory effect it is necessary to add IL-4 to cultures before 40 h. Enrichment for NK1.1+ cells failed to recover precursor cells which are down-regulated in overnight cultures or can be cultivated in vitro to yield NK cytolytic activity. Furthermore, phenotypic analysis of effector cells demonstrated a marked inhibition of development of NK1.1+ cells in cultures containing IL-4 plus IL-2 versus IL-2 alone. Thus, it appears that IL-4 down-regulates the precursors of murine NK cells by inhibiting proliferation and/or development. In addition, we show that IL-2-induced murine LAK activity mediated by CD8- precursor cells is unaffected by IL-4, while CD8(+)-derived LAK cells are up-regulated by co-culture with IL-4 and IL-2. Analysis of these data relative to reports documenting down-regulation of human LAK by IL-4 suggests that in vitro cultured, IL-2-activated murine NK cells are the correlates to what are commonly described as human LAK cells. The discrepancy may stem from differences in the characteristics of target cells used in the murine versus the human systems. These results clarify the conflicting reports on the effect of IL-4 on killing activity.     

Th1 adjuvant N-acetyl-D-glucosamine polymer up-regulates Th1 immunity but down-regulates Th2 immunity against a mycobacterial protein (MPB-59) in interleukin-10-knockout and wild-type mice.

Treatment of mice with heat-killed (HK) Mycobacterium bovis BCG or 1- to 10-microm chitin particles (nonantigenic N-acetyl-D-glucosamine polymers) is known to induce innate immune responses, including gamma interferon (IFN-gamma) production, which plays a Th1 adjuvant role. However, HK BCG further induces prostaglandin E2-releasing spleen macrophages (Mphi) (PGE2-Mphi), which potentially inhibit Th1 adjuvant activities. We found that chitin particles did not induce PGE2-Mphi formation. To further assess whether chitin has Th1 adjuvant effects, interleukin-10 (IL-10)-knockout (KO) mice and their wild-type (WT, C57BL/6) controls were immunized with a 30-kDa MPB-59 mycobacterial protein mixed with chitin. Immunization with MPB-59 alone induced Th2 responses, characterized by increases in total serum immunoglobulin E (IgE) and specific serum IgG1 levels and spleen Th2 cells producing IL-4, IL-5, and IL-10. No IFN-gamma-producing spleen Th1 cells, specific serum IgG2a, or delayed-type hypersensitivity (DTH) footpad reactions were detected. On the other hand, chitin-MPB-59 immunization significantly increased spleen Th1 responses, DTH reaction, and serum IgG2a levels along with decreases of Th2 responses. The magnitude of these Th1 adjuvant effects was greater in IL-10-KO mice than in WT mice. In contrast, immunization with HK BCG-MPB-59 showed little or no Th1 adjuvant effect. These data indicate that chitin has a unique Th1 adjuvant effect on the development of Th1 immunity against a mycobacterial antigen. IL-10 down-regulates the adjuvant effect of chitin.     

Purified MHC class I molecules inhibit activated NK cells in a cell-free system in vitro

Natural killer cells have been shown to interact with MHC class I molecules via inhibitory receptors. However, it is not known whether the inhibition induced by MHC class I molecules requires other NK cell-target cell interactions. Thus, we examined whether purified MHC class I molecules alone were able to inhibit NK cell function. Purified H-2K(b) and H-2D(b) molecules inhibited the release of IFN-gamma from spleen (H-2(b))-derived lymphokine-activated killer (LAK) cell cultures stimulated by anti-NK1.1 antibody in a concentration-dependent manner. LAK cells generated from newborn mice that express low levels of MHC class I binding Ly49 inhibitory receptors were significantly less sensitive to inhibition by H-2K(b) compared to LAK cells from adult mice. Furthermore, LAK cells generated from spleen cells of Ly49C-transgenic mice were significantly more sensitive to inhibition by H-2K(b) compared to non-transgenic littermates. Taken together, the data indicate that MHC class I induced inhibition of NK cell mediated effector functions, as assessed by IFN-gamma release after NK1.1 triggering, does not require additional cell surface molecules other than MHC class I.     

硒对LAK细胞活性的影响及其机理研究

研究了硒在体外对LAK细胞活性的影响及作用机理,结果证明在LAK细胞的诱导阶段加入10~5～10mol/L亚硒酸纳能增强LAK细胞的杀伤和增殖活性。采用流式细胞仪测Tac(IL—2Rα)表达,Slot-Blot检测硒对LAK细胞的IL—2RαmRNA水平的影响,结果表明硒能增强LAK细胞的Tac抗原表达和IL-2RαmRNA的水平,提示硒可能通过促进Tac的表达增强了LAK细胞对IL—2的敏感性,从而提高了LAK细胞的增殖及杀伤活性。因此硒可望作为一种新型的免疫调节剂用于抗肿瘤治疗。     

精脒和腐胺对人胎儿脾LAK活性的影响

本研究表明,精脒、腐胺本身均不能使人胎脾单个核细胞(FSMC)转化为LAK细胞,但与5u/ml重组白细胞介素2(rIL-2)伍用,可以增强人胎脾LAK细胞活性;精脒,腐胺不能直接刺激细胞增殖,但能协同亚适剂量Con A诱导淋巴细胞增殖;精脒或腐胺本身可刺激FSMC的IL-2受体(IL-2R)的表达,并能协同rIL-2进一步提高人胎脾LAK细胞IL-2R的表达;精脒、腐胺均可使FSMC及胎脾LAK细胞内鸟氨酸脱羧酶(ODC)活性增强,而以腐胺的作用尤为显著。     

Participation of Interleukins in Synergistic Effect of Bu-WSA on Concanavalin A-Induced DNA Synthesis in Mouse Splenic Lymphocytes

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was mitogenic to murine splenic B lymphocytes, but not T lymphocytes. When murine splenic cells were cultured in the presence of Bu-WSA and concanavalin A (Con A) together, [3H]thymidine uptake of the culture cells synergically increased. The mechanism of the synergy of Con A and Bu-WSA and the participation of interleukin (IL) 1 and 2 in the synergy were studied. The proliferation cells in the synergy were Lyt-1+23- lymphocytes. Ia-positive accessory cells were required for the response. When separated cell populations and Marbrook-type culture vessels were used, a mixed cell population of T lymphocytes and B lymphocytes or macrophages (M phi) produced some active factor(s) after co-stimulation by Con A and Bu-WSA, and the factors enhanced DNA synthesis of another Con A-activated T lymphocyte population. Supernatants obtained from the spleen cell cultures or the mixed cell cultures with T lymphocytes and M phi in the presence of Con A and Bu-WSA contained greater amounts of IL-1 and IL-2 than those from cultures containing Con A or Bu-WSA alone. An addition of exogenous IL-1 or IL-2 to spleen cell cultures with Con A resulted in a proliferative response like that obtained through co-stimulation by Con A and Bu-WSA. These results suggest that the synergistic effect of Con A and Bu-WSA on the proliferative response in murine splenic cells is sustained by the enhancement of production of these T-lymphocyte growth factors.     

人白细胞介素4诱导杀伤细胞的研究

人重组白细胞介素４（ｒｈＩＬ－４）在ＰＨＡ协同下，从人外周血单核细胞（ＰＢＭＣ）诱导出明显的ＬＡＫ活性，其对Ｋ５６２、Ｒａｊｉ细胞的杀伤力低于ＩＬ－２诱导者，对ＴＢＬ－Ｅ，ＰＨＡ活化的淋巴母细胞（ＰＨＡ－ｂｌａｓｔｓ）的杀伤力和ＩＬ－２诱导者相似。在ＰＨＡ介导的４小时５１Ｃｒ杀伤试验中，加入ＰＨＡ后，ＩＬ－４－ＬＡＫ对ＰＨＡ－ｂｌａｓｔｓ的杀伤力提高２．３倍，而ＩＬ－２－ＬＡＫ对ＰＨＡ－ｂｌａｓｔｓ的杀伤力无变化，提示ＩＬ－４主要诱导ＣＴＬ样活性，而ＩＬ－２主要诱导ＮＫ样活性，ＩＬ－４诱导效应ＣＴＬ的能力强于ＩＬ－２。我们的实验同时证实，在淋巴细胞活化的早期，ＩＬ－４抑制ＩＬ－２诱导的ＬＡＫ活性，淋巴细胞活化后，ＩＬ－４与ＩＬ－２有协同作用，增强ＩＬ－２诱导的ＬＡＫ活性。     

Cytokine profiles associated with induction of the anticryptococcal cell-mediated immune response.

Previous studies with a murine model have shown that immunization with cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund adjuvant (CFA) induces two populations of anticryptococcal reactive CD4+ T cells. One population (TDH cells) transfers anticryptococcal delayed-type hypersensitivity (DTH), and the other population (Tamp cells) amplifies the anticryptococcal DTH response of given to recipient mice at the time of immunization of the recipient. Treatment of mice with cyclosporin A (CsA) ablates the induction of Tamp cells but not TDH cells. The present study focused on assessing the cytokines produced by spleen cells taken from CsA-treated and control (solvent-treated) mice at days 1, 2, 4, and 6 after immunization. Supernatants from the spleen cells cultured in vitro for 24 or 48 h in medium alone or with CneF, concanavalin A, or phorbol 12-myristate 13-acetate plus calcium ionophore were assessed for the presence of interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-4, IL-5, and tumor necrosis factor. Spleen cells from CneF-CFA-treated mice produced IL-2 and IFN-gamma, but not IL-4 or IL-5, constitutively and in response to CneF, indicating that CneF-CFA induces a Th1 response. Tumor necrosis factor was not produced. Anticryptococcal TDH cells developed in spleens in which there were low levels of IFN-gamma and IL-2 (CsA-treated, immunized mice), whereas anticryptococcal Tamp cells along with TDH cells matured in spleens in which production of IFN-gamma and IL-2 was high (solvent-treated, immunized mice). The data also suggest that IL-2 and IFN-gamma produced by Tamp cells early after adoptive transfer are influential in the development of the amplified anticryptococcal DTH response that has been observed in Tamp cell-recipient mice.