Modulation of the Functions of Dengue Virus-Specific Human CD8+ Cytotoxic T Cell Clone by IL-2, IL-7 and IFNγ

Lymphokines play an important role in immune responses to viruses by modulating functions of T lymphocytes. In the present study, we examined the effects of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), and interferon gamma (IFNγ) on proliferation, cytotoxic activity and lymphokine production of a dengue virus-specific CD8⁺ human cytotoxic T lymphocyte (CTL) clone. IL-2 and IL-7 induced proliferation of the CD8+ CTL clone in the presence or absence of specific antigen, while IFNγ suppressed proliferation of the clone. IL-7 and IFNγ augmented dengue virus-specific cytotoxic activity without inducing non-specific cytotoxic activity, and IL-2 induced non-specific cytotoxic activity. IL-2 induced IFNγ production by the CD8+ CTL clone. IL-4 and IL-6 did not modulate the functions of the CD8+ CTL clone in these experimental conditions.

These results suggest that functions of dengue virus-specific CD8+ CTL are modulated by IL-2, IL-7 and IFNγ, and that IL-7 is a lymphokine useful to induce growth and to maintain specific cytotoxic activity of CD8+ CTL clones in vitro.     

Characterization of CD28CD4 T cells in living kidney transplant patients with long-term allograft acceptance

CD28⁻CD4⁺ T-cell subpopulation is expanded in kidney allograft patients with long graft survival. To seek for the roles of CD28⁻CD4⁺ T cells in the long-term acceptance of kidney allografts, we characterized this population by analyzing cell surface molecules, TCR V_β repertoire, mixed lymphocyte reaction (MLR), and cytokine production. The number of CD28⁻CD4⁺ T cells increased correlatively with time after transplantation in this group of patients. The CD28⁻CD4⁺ T cells did not express detectable levels of CD25, CD69, V24, or CTLA-4 but expressed heterogeneous amounts of CD45 RA on the surface. Freshly sorted CD28⁻CD4⁺ T cells revealed a restricted V_β repertoire, whereas the V_β usage of CD28⁺CD4⁺ T cells from the same patients was much diversified. Expression levels of TGF-β and IFNγ gene were significantly higher in the CD28⁻ CD4⁺ T cells than in the CD28⁺CD4⁺ T cells from the kidney allograft patients. These findings suggest that an oligoclonal CD28⁻ CD4⁺ T-cell population is continuously activated in patients with long allograft survival, which may be linked with the long-term acceptance.     

异体富血小板血浆免疫原性的小型猪动物实验研究

目的评价异体富血小板血浆(PRP)在小型猪体内产生的免疫原性反应。 方法无菌条件下经颈动脉取1只雄性巴马小型猪全血,第1次以1 000×g力离心15 min分离红细胞,第2次以3 000×g力离心10 min获取PRP;剩余9只雄性巴马小型猪,抽签法选取3只,取其耳缘静脉血各1.0 mL。后9只小型猪双侧股部分别注射激活的异体PRP 0.5 mL,按抽签法分为1、2、4周组,每组3只,于异体PRP注射后1、2、4周分别取对应组各小型猪耳缘静脉血1.0 mL,之后处死该小型猪,取注射部位组织。流式细胞仪检测比较CD4^＋/CD8^＋T淋巴细胞亚群比例,生物显微镜下观察组织形态学变化及免疫组织化学分析局部组织免疫原性反应。对数据行单因素方差分析和Dunnett-t检验。 结果小型猪肌肉注射异体PRP前,CD4^＋/CD8^＋T淋巴细胞亚群比例为0.75±0.04,异体PRP注射后1、2、4周CD4^＋/CD8^＋T淋巴细胞亚群比例分别为0.96±0.02、0.85±0.02、0.79±0.02,比较差异有统计学意义(F＝37.970,P<0.001)。异体PRP注射后1、2周CD4^＋/CD8^＋T淋巴细胞亚群比例与参照组比较,差异均有统计学意义(P<0.001、＝0.004);异体PRP注射后4周CD4^＋/CD8^＋T淋巴细胞亚群比例与参照组比较,差异无统计学意义(P＝0.201)。病理检查可见,异体PRP注射部位的局部组织于注射后1周有轻微炎性细胞浸润和白细胞介素-2(IL-2)的表达,注射后2、4周炎性细胞浸润逐渐消失,IL-2表达转为阴性。 结论异体PRP注射仅在小型猪体内产生轻微的局部和全身免疫原性反应,且在注射后4周该免疫原性反应基本消失。     

Pregnancy-induced expansion of regulatory T-lymphocytes may mediate protection to multiple sclerosis activity

The role of activated CD8⁺ T cells in shaping the dynamics of in vivo antigen presentation and immune responses is a subject receiving more attention. We studied whether cytotoxic T lymphocyte (CTL) would limit antibody responses by targeting antigen-specific B cells. A modified in vivo CTL assay was developed and used herein to demonstrate cytotoxicity in vivo, and to show that antigen-specific B cells that process exogenous antigen and present peptide in association with MHC class I can be the targets of CD8⁺ T cells. B cells from C57BL/6 mice immunized with ovalbumin (OVA)/alum were pulsed with OVA in vitro, and transferred into C57BL/6 recipient mice that had been immunized with vaccinia virus expressing SIINFEKL minigene to generate CD8⁺ CTL against K^b/SIINFEKL. OVA-pulsed B220⁺ B cells from OVA-immunized mice were killed to a greater extent than B220⁺ B cells from naïve mice (28 ± 20% versus 12 ± 16%, p = 0.0042). However, mice receiving vaccinia-SIINFEKL and generating CTL, did not appear to target endogenous B cells, since both primary and secondary antibody responses to OVA were unaffected. Our findings indicate that CTL responses to the protein antigen do not interfere with endogenous B cell responses, even though exogenous B cells expressing the CTL epitope can be efficiently lysed.     

In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naïve repertoire

Innovations in gene discovery and the analysis of gene expression are facilitating the identification of a growing number of antigens that could potentially be targeted for immunotherapy of tumors. Methods to reliably generate antigen-specific T-cell responses in vitro would be useful not only to screen candidate antigens for immunogenicity prior to embarking on in vivo vaccination trials, but also to generate T-cell lines or clones that could be used directly for adoptive immunotherapy approaches. Although many techniques have proven successful for expanding ex vivo effector cells from antigen-specific memory CD8⁺ cells that have been primed in vivo, methods to reliably generate high-avidity CTL clones from the naïve repertoire have not been well described. Various methods for the induction and expansion of antigen-specific CD8⁺ CTL clones from healthy A2⁺ donors were compared, using WT1 as a model tumor-associated antigen for which there is a low frequency of precursor T cells in naïve individuals. In contrast to the well-studied Melan-A/MART-1 (Melan-A) A2-restricted response, for which the CD8⁺ T-cell precursor frequency in the naïve repertoire is unusually high, successful expansion of WT1-specific CD8⁺ T cells appeared to be more dependent upon cell culture conditions. In particular, primary stimulation with autologous peptide-loaded monocyte-derived DC generated in 48 h (DC2d) was more effective in expanding WT1-reactive populations of CTL than stimulation with DC generated using the more standard week-long protocol (DC7d). Adding supplemental IL-7 2 to 3 days after initiation of a stimulation cycle expanded antigen-specific cells within CTL lines more efficiently than including the cytokine from the beginning of the cycle. Following primary stimulation with peptide-loaded mature DC, subsequent restimulation with peptide-loaded PBMC as the stimulators was more effective at expanding antigen-specific cells than repeated stimulation with mature DC. Using these techniques, high-avidity CTL clones specific for an A0201-restricted epitope of WT1 have been generated from nearly all normal A2⁺ donors tested. Such clones have been demonstrated to be capable of recognizing and lysing leukemic cells, and will soon be tested for therapeutic activity in clinical trials of adoptive immunotherapy in patients with relapsed leukemia after transplantation.     

Enhancement of thyroid allograft survival following organ culture: II. Induction of Recipient Peripheral Tolerance

Hyperbaric oxygen culture (HOC) prolongs endocrine graft survival and decreases major histocompatibility complex (MHC) class I surface expression. If graft prolongation were the result of passenger cell inactivation and decreased class I expression, then simultaneous transplantation of both a nontreated and a HOC-treated graft should result in rejection of the nontreated graft and acceptance of the HOC-treated graft. Simultaneous transplant of a nontreated and a HOC-treated thyroid allograft beneath the kidney capsule of recipient mice resulted in prolonged survival of both grafts in three strain combinations differing at class I (K^K , D^d, D^b). In vitro analysis of the recipient splenic population revealed the presence of primed donor-specific cytotoxic T cells. These results suggest that recipient tolerance was not because of anergy or clonal deletion. Splenectomy at the time of transplant, revealed that both graft prolongation and the induction of recipient tolerance were dependent on the spleen. Finally, analysis of graft infiltrating cells reveals the presence of CD8⁺ cells but no CD4⁺ cells in tolerant recipients, whereas graft infiltrating cells from rejecting recipients contained both populations. The results suggest that active peripheral tolerance can be generated following transplantation of a HOC-treated allograft and that tolerance results from redirection of the recipients immune response.     

Does the age-related change in CD44-defined T-cell subsets have functional significance for cytotoxic T lymphocyte generation?

CD44 or Pgp-1 is a transmembrane leukocyte adhesion-related glycoprotein which is often expressed in greater density on the membranes of memory T lymphocytes (CD44^hi) compared to naive T cells (CD44^lo). The proportion of Pgp^hi or CD44^hi cells among T cells is increased with advancing age. We examined the relevance of this alteration for the age-related decrease in the generation of allospecific CTL activity. The findings confirm the age-related increase in the frequency of CD44^hi cells in spleens of aged mice of several strains, but also show inter-strain variability in the magnitude of the increase (bm1> C57BL/6> BALB/c). In contrast, we found that after allo-stimulation, the proportion of cells bearing the memory phenotype is decreased in cells from aged mice, particularly within the CD8⁺ T cell subset. To determine if these observations reflected an alteration in the frequency or responsiveness of naive T cells, enriched populations of spleen cells depleted of CD44^hi cells were prepared from spleen cells of young and aged mice, and stimulated in mixed lymphocyte culture. Enrichment for cells expressing the naive phenotype did not restore the ability of T cells from aged mice to generate allospecific CTL. Together, these findings suggest that (1) the age-related increase in frequency of splenic T cells expressing memory phenotype and concordant decrease in phenotypically naive cells, does not explain the age-related decrease in the ability to generate primary allo-CTL, and (2) naive cells from aged mice exhibit intrinsically compromised ability to generate CTL in response to primary alloantigenic stimulation.     

Ethanol impairs major histocompatibility complex (MHC) class II molecule-mediated but not MHC class I molecule-mediated T cell response in alcohol-consuming mice

The goal of this study was to determine whether alcohol affects alloantigen-induced proliferative and cytolytic activity of T cells in mice, and whether the altered immune response was in part due to a defect of IL-2 activity. The ability of spleen cells from individual alcohol-consuming C57BL/6 mice to generate allo-specific mixed lymphocyte response (MLR) and cytotoxic T lymphocyte (CTL) was compared to that of mice fed on an isocaloric maltose diet and regular diet. Allospecific MLR and CTL were generated by sensitizing spleen cells of C57BL/6 mice against spleen cells from BALB/c mice, and the allo-specific CTL activity was determined by the ability of the CTL to kill 51Cr-labeled P815 mastocytoma target cells. Our results showed that the allo-specific MLR of the responder cells from alcohol-consuming mice was significantly reduced (40% reduction, p<0.0 1), and the addition of exogenous interleukin 2 (IL-2) could not reverse the suppression of MLR induced by ethanol. However, our results clearly showed that ethanol has little suppressive effect on allo-reactive CTL of alcohol-consuming mice as compared to the alloreactivity of the control mice (P>0.05). Finally, we also demonstrated that ethanol did not impair the alloantigen-induced IL-2 production in the mixed lymphocyte cultures (P>0.1).     

Soluble HLA class I/CD8 ligation triggers apoptosis in EBV-specific CD8+ cytotoxic T lymphocytes by Fas/Fas-ligand interaction

In the present study, we report that allogeneic soluble HLA class I (sHLA-I) molecules isolated from serum induce apoptosis on EBV-specific CD8⁺ Fas⁺ cytotoxic T lymphocytes (CTL). CTL apoptosis is induced by the binding of sHLA-I molecules to CD8 and its extent depends on the time of incubation with sHLA-I molecules. Apoptosis is triggered by the interaction of Fas⁺ CTL with soluble Fas-ligand, which is released following the binding of sHLA-I antigens to CD8 molecules. These results suggest that sHLA-I molecules may regulate immune responses by inducing apoptosis in virus-specific CTL.     

人脐带源间充质干细胞体内外移植的免疫学特征

背景：部分体外实验证实人脐带源间充质干细胞具有同种异体的免疫耐受特性,然而对其体内研究尚处于异种异体之间,且结果及机制缺少统一性。 目的：培养人脐带源间充质干细胞,通过体内外试验分析其移植免疫学特性。 方法：应用组织块贴壁法提取人脐带源间充质干细胞,通过体外淋巴细胞混合反应检测其同种异体免疫耐受作用;应用流式细胞仪检测体内行人脐带源间充质干细胞移植前后患者血液中CD4、CD8浓度及比值变化,并通过ELISA方法对比体外淋巴细胞混合培养前后血液、培养上清及体内移植前后患者血液中白细胞介素2,10、γ-干扰素的浓度变化。 结果与结论：体内外实验中,混合淋巴细胞反应前后血浆及细胞上清中的细胞因子浓度变化呈相似趋势：白细胞介素10较反应前上升(P < 0.05),γ-干扰素较反应前下降(P < 0.05)。人脐带源间充质干细胞移植后患者T细胞亚群中CD4⁺/CD8⁺比值较移植前轻度下降。说明人脐带源间充质干细胞具有同种异体免疫耐受及免疫调节作用。     

Inhibition of the human allogeneic mixed lymphocyte response by cyclosporin A: relationship with the IL-12 pathway

Interleukin-12 (IL-12) is an important cytokine in the control of cell-mediated immunity. We have previously shown that endogenous IL-12 plays a role in the development of human allogeneic response. In the present study, we investigated the relationship between Cyclosporin A (CsA)-inhibitory effect and IL-12 pathway during human alloreaction in vitro. CsA addition at the sensitizing phase of primary mixed lymphocyte reaction (MLR) resulted in the inhibition of both p40 and p70 IL-12 production in a dose-dependent manner. In contrast, CsA had no effect on IL-12-receptor β1 chain (IL-12 Rβ) expression in T cells induced upon allogeneic activation. Addition of exogenous IL-12 significantly restored CsA-inhibited alloreactive cytotoxic T lymphocyte (CTL) generation and had a marginal effect on T cell proliferative response. The IL-12-induced restoration of CTL generation was IFNγ-mediated, as it was significantly altered when anti-IFNywas added. The restoration of CTL activity by exogenous IL-12 correlated with the capacity of this cytokine to partially restore granzyme B mRNA expression in alloreactive CTL. This study indicates that inhibition of IL-12 production is a novel additional mechanism for the inhibitory effect of CsA on the development of human allogeneic cytotoxic response.     

A family of T-cell receptor molecules expressed on T-cell clones with different specificities for allomajor histocompatibility antigens

A monoclonal antibody, 3D6, identifies a public idiotope or allotope on the human T-cell receptor for antigen, since it not only reacts with the tumor line HPB-ALL, against which it has been raised, but also with 3–13% of peripheral blood T lymphocytes of normal donors. 3D6⁺ cells have been isolated from an allogeneic mixed lymphocyte culture and cloned by limiting dilution. In this way, allospecific clones were obtained both of the T4⁺ T8⁻ and the T4⁻ T8⁺ phenotype, which included proliferative as well as cytotoxic cells. Within a panel of 20 cytotoxic clones, different specificities for both class I and class II MHC antigens were found. The clones were tested for their reactivities with four additional anti-T-cell receptor antibodies raised against HPB-ALL. Two of these, 1C1 and 1C2, reacted with all 3D6⁺ clones. By means of two other antibodies, 2D4 and 65, the 3D6⁺ receptor family could be divided into four structurally distinct subfamilies. Biochemical analysis suggested that the 1C1, 1C2, 2D4, and 3D6 antibodies define epitopes on the beta chain of the receptor. Isoelectric focusing of receptor molecules isolated from cytotoxic clones with different specificites indicated that there are extensive structural differences in both alpha and beta chains of the receptors. No correlation could be found between the antigenic specificity of a clone and the structure of its receptor in this analysis. It is postulated that the 1C1, 1C2, and 3D6 epitopes may be encoded by a particular germline V beta segment, in analogy with similar, previously described findings in both the human and the murine system.     

异基因间充质干细胞在体内免疫调节作用的时间及剂量依赖性

背景：异基因间充质干细胞移植时,发挥作用需要的剂量、持续的时间及是否具有不良反应是目前研究的热点。 目的：观察异基因的骨髓间充质干细胞在体内免疫调节作用的时间、剂量依赖性。 方法：免疫系统正常的BALB/c小鼠模型,制备骨髓来源间充质干细胞,24只BALB/c小鼠随机分为4组,实验组每只小鼠分别通过尾静脉注射0.3 mL细胞悬液,分别含5×10⁵,5×10⁴,5×10³骨髓间充质干细胞,对照组小鼠仅注射0.3 mL生理盐水。进行淋巴细胞增殖检测、混合淋巴细胞反应、间充质干细胞移植对异基因小鼠免疫系统的影响等实验。 结果与结论：不同剂量的C57BL/6骨髓来源间充质干细胞经尾静脉注射给BALB/c受体小鼠后,骨髓间充质干细胞体内对免疫系统的调节呈剂量依赖性。间充质干细胞促进异基因移植物的植入,同时这种免疫下调作用在2周时最强,1个月逐渐减弱,2个月基本消失,提示间充质干细胞的免疫调节作用具有剂量依赖性,并且只能维持一段时间。     

Human suppressor T cells induced in vitro with an autologous renal allograft-derived T cell line. I. Suppressor cell induction, function, and specificity

The functional characteristics of T suppressor (Ts) cells generated from the peripheral blood lymphocytes (PBL) of a kidney transplant recipient who had excellent graft function for 1 year were examined. Ts cells were induced by co-culture of PBL with an autologous alloreactive cytotoxic T lymphocyte (CTL) line (EE-1) previously grown from a routine renal allograft biopsy of this patient performed 10 days posttransplant. The EE-1 line included CD3+ T cells of CD8+ and CD4+ phenotypes with cytotoxic specificity for disparate class 1 (HLA-B8) and class II (HLA-DR1 and 3) antigens of the kidney donor (JC). The EE-1 induced Ts cell lines (designated TsEE) were found to significantly suppress (50%-95%) autologous fresh responder EE-PBL stimulation by donor EBV-transformed cells (JC-EBV) in mixed lymphocyte reaction (MLR) assay. TsEE cells were CD3+ (98%) and predominantly CD8+ (68-80%), showed no cytotoxic activity, and were suppressive only at the early phase of MLR stimulation. In three-party cell test MLR assays, TsEE-mediated suppression appeared restricted to responder cells sharing HLA-B7 with the suppressor line, and was not abrogated by the addition of exogenous interleukin-2 (IL-2). TsEE cells also showed restricted suppression of CTL generation but not mature CTL activity. The restricted suppressor activity of TsEE lines was dependent upon their induction and restimulation with the autologous EE-1 line.     

IL-6 enhances the generation of cytolytic T lymphocytes in the allogeneic mixed leucocyte reaction.

Cytolytic T lymphocytes (CTL) require soluble proteins termed lymphokines to develop lytic activity. In this report we have studied two of the lymphokines involved in the development of CTL during the allogeneic mixed leucocyte reaction (MLR). High doses of dendritic cells induced lytic activity from purified CD8+ cells in both the murine and human MLR. Under these conditions, IL-2 and IL-6 were endogenously produced and secreted. Antibodies to IL-2 or the IL-2 receptor blocked CTL formation; however, anti-IL-6 receptor antibodies only partially inhibited the response while anti-IL-6 antibodies were largely ineffective. When limiting numbers of antigen-presenting cells were used CTL failed to develop, and neither IL-2 nor IL-6 was secreted into the culture supernatant. Although the addition of IL-6 to such cultures was ineffective in generating CTL, the combination of IL-2 and IL-6 resulted in a 4-5-fold increase in lytic activity over that of IL-2 alone. We conclude that in the allogeneic MLR, IL-2 and IL-6 contribute to the generation of lytically active CD8+ cells, and the effect of IL-6 is evident when the dose of antigen-presenting cell is limited.     

自体造血干细胞移植治疗恶性淋巴瘤后大剂量白细胞介素2过继免疫对长期生存的影响

背景：过继免疫治疗是目前肿瘤免疫治疗的热点,白细胞介素2是一种具有多种生物学活性的细胞因子,在机体的抗肿瘤免疫中起到重要作用。 目的：评价比较淋巴瘤自体造血干细胞移植治疗后应用与不应用大剂量白介素2行免疫治疗的临床疗效。 方法：回顾分析30例恶性淋巴瘤患者(治疗组)自体造血干细胞移植后行大剂量白细胞介素2 治疗,与随机挑选30例患者(对照组)自体造血干细胞移植后未行白细胞介素2治疗进行对比,检测两组患者外周血T淋巴细胞亚群,观察两组免疫功能的变化,并对所有患者进行随访观察。 结果与结论：自体造血干细胞移植后白细胞介素2治疗组外周血T淋巴细胞亚群CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺水平明显提升。随访结束时统计复发率：治疗组13.3%,对照组26.7%;中位生存期：治疗组14~98 (42±2)个月,对照组8~78 (28±2)个月。提示恶性淋巴瘤自体造血干细胞移植后行大剂量白细胞介素2治疗能提高患者的免疫功能,减少移植后复发率,并有望延长生存期。     

Dye Dilution Proliferation Assay: Application of the DDPA to Identify Tumor-Specific T Cell Precursor Frequencies in Clinical Trials

A better understanding of immune effector and regulatory pathways has led to innovative, and complex, immunotherapy strategies. CD8⁺ cytolytic T lymphocytes (CTL) provide one common pathway of tumor cell destruction. The peripheral blood CTL compartment typically comprises a minority of anti-tumor CD8⁺ lymphocytes and the determination of their number during clinical trials is the focus of various laboratory methods. We have monitored tumor specific CD8⁺ as well as CD4⁺ lymphocyte precursor frequencies in the peripheral blood using a Dye Dilution Proliferation Assay (DDPA). We summarize our experience applying DDPA in a multi-parameter, antigen-specific assay, detailing some of its complexities and advantages. We provide examples of our clinical trial results showing tumor-specific CD8⁺ and CD4⁺ precursor frequency (PF) data in patients being treated on novel immunotherapy trials.     

T Helper-Independent Activation of Human CD8+ Cells: the Role of CD28 Costimulation

The concept that activation of MHC class I-restricted CD8⁺ cells entirely depends on help from MHC class II-restricted CD4⁺ T cells has recently been supplemented with an alternative model in which CD8⁺ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8⁺ T cells in two different in vitro models. Freshly isolated CD8⁺ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting FcγR⁺ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8⁺ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8⁺ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8⁺ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8⁺ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.     

Adrenocorticotropic hormone as a potential enhancer of t-lymphocyte function in the rat mixed lymphocyte reaction

The effects of adrenocorticotropic hormone (ACTH) (10⁻⁶ to 1 U/ml) on T-lymphocyte proliferation and function were studied in the rat mixed lymphocyte reaction (MLR). ACTH produced a modest increase in lymphocyte proliferation on day 3 in lymph node (LN) cells and on day 5 in spleen cells. In addition, LN MLR cells, activated in the presence of ACTH, showed a higher proliferative response to restimulation on day 5 and on day 11 of the primary culture. Cytotoxic activity and the number of IL-2R⁺ cells were increased in ACTH-treated LN MLR cultures in experiments where control MLR levels were low. ACTH also overcame the generation of low levels of suppressor activity in spleen MLR cells. These findings indicate that ACTH could play a role in increasing the priming of T-lymphocytes and enhancing, in particular, suboptimal primary responses.