Differential effect of cryopreservation on natural killer cell and lymphokine-activated killer cell activities

The use of lymphokine-activated killer (LAK) cell therapy in delayed treatment requires the use of cryopreserved effector cells. The purpose of this study was to determine the optimal cryopreservation protocol for the maintenance of cytotoxic activity in mononuclear cells (MNCs). MNCs were cryopreserved with dimethyl sulfoxide or 1,2-propanediol before and after 3 days of culture with recombinant interleukin 2. The effects of cryopreservation on cell recovery, LAK cell and natural killer (NK) cell cytotoxic activities, and surface antigen markers were studied. Recovery of nonactivated MNCs was higher with 1,2-propanediol than with dimethyl sulfoxide (p < 0.05). Cytotoxic activities, measured with a 51Cr release assay, significantly decreased after thawing, on both activated cells (76.3%; range, 35.8-92.2%) and fresh cells (54.6%; range, 17.5-75.4%). A 6-day kinetic test was used to compare the cytotoxic activity of cryopreserved and fresh cells. The results showed different patterns for NK cells (cryopreserved cells had lower levels of activity than fresh cells) and LAK cells (cryopreserved cells had higher levels of activity than fresh cells). Phenotype changes of effector cells in culture, with and without cryopreservation, were monitored by flow cytometry using monoclonal antibodies. These results were compared with changes in the cytotoxicity of cells with and without cryopreservation. After thawing, there was a decrease in MNCs expressing CD14 and CD56. Recovery of the CD56 marker correlates with increased cytotoxic activity. Despite some loss of NK cell activity, it is concluded that MNCs may be successfully cryopreserved before their use in immunotherapeutic treatment.     

Induction of lymphokine-activated killer and natural killer cell activities from cryopreserved lymphocytes

Lymphokine-activated killer (LAK) and natural killer (NK) cells were studied for their capacity to retain cytotoxicity after cryopreservation. LAK cells were generated by a 4-day culture of lymphocytes with recombinant interleukin-2 (rIL-2). Cytotoxicity was measured by 51Cr-release assay at effector:target ratios of 10:1 to 80:1. Cryopreserved LAK cells retained 58.8 to 87.4 percent of cytotoxicity, as compared with that in fresh control cells. Cryopreserved NK cell activity against K562 and Molt-4 targets was 45.7 to 67.9 percent of the respective values of the fresh control cells. The responsiveness of NK cells to polyinosinic-polycytidilic acid (poly I:C), interferon-alpha (IFN-alpha), or rIL-2 remained intact. Activated NK cell activity after poly I:C or IFN-alpha stimulation and that after rIL-2 were, respectively, comparable to and higher than the endogenous NK cell activity of the fresh cells. The composition of lymphocyte subsets as determined by flow cytometry using monoclonal antibodies did not change after cryopreservation, indicating that cellular loss of the given subsets did not occur during the procedure. The retention of substantial levels of cytotoxicity in cryopreserved LAK and NK cells may make them promising candidates as cytotoxic effector cells.     

The role of natural killer cells and lymphokine activated killer cells in the pathogenesis of hepatic injury in hepatitis A [corrected]

In order to clarify the mechanism of liver damage in hepatitis A, we studied the role of Natural Killer (NK) cells and Lymphokine Activated Killer (LAK) cells in non-specific immunological reactions using hepatitis A (HAV) infected cells (JTC-12.P3 cell) using the 51Cr release assay. No significant difference in specific cytotoxicity was observed between uninfected cells (JTC) and HAV-infected cells (JTC-HAV) to fresh and Poly I:Cor rIL-2 pretreated peripheral blood mononuclear cells (PBMC) from healthy donors or patients with acute hepatitis A. But in an experiment employing fresh and rIL-2 pretreated PBMC from cynomolgus monkey as effectors, a significant difference in NK and LAK sensitivity between JTC and JTC-HAV was noticed. Cytotoxicity assay was then carried out using as effectors fresh or rIL-2 pretreated cells of B, NK and T cell fractions obtained after separation of PBMC from monkey by the E-rosette formation method. Both fresh and rIL-2 pretreated B/NK cells showed significantly higher cytotoxicity for JTC-HAV than JTC, but neither fresh or rIL-2 pretreated T cells showed cytotoxicity for JTC-HAV or JTC. These results imply that both NK and LAK cells may play a part in the mechanism of hepatocellular injury.     

Lymphokine-Activated Killer (LAK) Cell Phenomenon in Cluster Headache. "In Vitro" Activation by Recombinant Interleukin-2

Previous studies showed that the Natural Killer (NK) activity of peripheral blood lymphocytes (PBL) from cluster headache (CH) patients is lower than that of controls. This decreased activity seems to be independent of the cluster period. beta-interferon has been shown to be more effective in increasing NK activity when incubated with PBL from CH patients, than with PBL from control donors. Lymphokine-Activated Killer (LAK) cells can be generated by incubation of human PBL in recombinant Interleukin-2 (rIL-2). This phenomenon was studied in 10 CH patients and 8 healthy volunteers. PBL were activated to LAK cells by "in vitro" incubation for 72 hours in Control Medium containing rIL-2 (1000 I.U./ml). A four hour Chromium 51 release was used to measure LAK Cell Killing of K562 target cells. The released radioactivity was measured in a gamma scintillation counter. The CH patients showed a marked increase of LAK generation compared to control subjects. This effect seems to be augmented during the cluster period.     

Antitumor activity of killer cells stimulated with both interleukin-2 and interleukin-12 on mouse glioma cells.

Interleukin-12 (IL-12), originally called natural killer cell stimulatory factor or cytotoxic lymphocyte maturation factor, has potential for use as an immunomodulator in cancer therapy because it significantly retards the growth of some murine tumors. In this study, we analyzed the antitumor effects of lymphocytes stimulated in vitro with both recombinant IL-2 (rIL-2) and rIL-12. When IL-12 was added to mouse splenocytes (SPCs) or human peripheral blood monocytes (PBMCs) incubated with IL-2 for > 4 days, IL-2-induced cytotoxicity against glioma cells was augmented. In contrast, IL-12 inhibited IL-2-induced lymphokine-activated killer (LAK) cell activity when added concurrently to cultures. The concentration of IL-10 induced by IL-12 increased in the supernatant of human PBMCs costimulated with IL-2 and IL-12. Endogenous IL-10 augmented the cytotoxicity of SPCs stimulated with IL-2 or IL-12 or both. However, tumor-bearing mice treated with PBMCs stimulated with both IL-2 and IL-12 did not survive longer than those treated with PBMCs stimulated with IL-2 alone (LAK cells).     

T cell activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6

This study investigates the molecular mechanisms underlying the induction of and protection from T cell activation-associated hepatic injury. When BALB/c mice were given a single intravenous injection of concanavalin A (Con A) (> or = 0.3 mg/mouse), they developed acute hepatic injury as assessed by a striking increase in plasma transaminase levels within 24 h. Histopathologically, only the liver was injured while moderate infiltration of T cells and polymorphonuclear cells occurred in the portal areas and around the central veins. The induction of hepatic injury was dependent on the existence as well as the activation of T cells, as untreated BALB/c nu/nu mice or BALB/c mice pretreated with a T cell-specific immunosuppressive drug, FK506, failed to develop disease. Significant increases in the levels of various cytokines in the plasma were detected before an increase in plasma transaminase levels. Within 1 h after Con A injection, tumor necrosis factor (TNF) levels peaked, this being followed by production of two other inflammatory cytokines, interleukin 6 (IL-6) and IL-1. Passive immunization with anti-TNF but not with anti-IL-1 or anti-IL-6 antibody, conferred significant levels of protection. Moreover, administration of rIL-6 before Con A injection resulted in an IL-6 dose-dependent protection. A single administration of a given dose of rIL-6 completely inhibited the release of transaminases, whereas the same regimen induced only 40-50% inhibition of TNF production. More than 80% inhibition of TNF production required four consecutive rIL-6 injections. These results indicate that: (a) TNFs are critical cytokines for inducing T cell activation-associated (Con A-induced) hepatitis; (b) the induction of hepatitis is almost completely controlled by rIL-6; and (c) rIL-6 exerts its protective effect through multiple mechanisms including the reduction of TNF production.     

A modified cytotoxicity assay with high sensitivity

Since adoptive immunotherapy using lymphokine activated killer (LAK) cells and interleukin-2 (IL-2) has been introduced into clinical medicine, there has been a growing interest in cytotoxicity assays. The standard 51chromium release assay has certain disadvantages, in particular limited sensitivity, because of a high, nonspecific background release. We examined the conditions under which tritiated thymidine, which has been used to assess slow macrophage mediated cytolysis, can be utilized to assess the rapid cytotoxic activity of unstimulated PBL and LAK cells. The optimal assay duration for the 3H-thymidine (3H-TdR) release assay is 24 h. Under standard conditions actively proliferating effector cells do incorporate some of the 3H-TdR released by the target cells during this time, leading to false low results. This problem can be abolished by the addition of excessive amounts of cold TdR to the assay medium. We found a good correlation of the results of the TdR release assay and the Cr release assay. Using the very sensitive TdR release assay, unexpected significant cytolysis of the so-called 'NK resistant' Daudi cells by unstimulated PBL is demonstrated. The modified 3H-TdR release assay is well-suited to monitor the immunological effects of immunotherapy, using IL-2 and LAK cells.     

Adoptive immunotherapy in tumor-bearing mice with OK-432-induced killer cells

Murine spleen cells cultured for 3 or more days in medium with streptococcal preparation OK-432 became cytotoxic in vitro against several allogeneic and syngeneic tumor cells. These cytotoxic cells were designated OK-432-induced killer (OIK) cells. This study examined the in vivo antitumor efficacy of OIK cells in adoptive immuno- and immunochemo-therapy in mice bearing syngeneic tumors, such as EL-4 lymphoma, Meth-A fibrosarcoma, and MOPC-31C plasmacytoma. OIK cells neutralized these tumor cells, as shown by Winn-type tests, and the cell transfer prolonged the survival of mice inoculated intraperitoneally (ip) with EL-4 or Meth-A cells. Concomitant administration of OK-432 plus recombinant interleukin 2 (rIL-2) significantly improved the therapeutic efficacy of the transferred OIK cells. In mice inoculated with 1 x 10(4) EL-4 cells, chemoimmunotherapy consisting of ip administration of 200 mg/kg cyclophosphamide on day 3 followed by treatment with OIK cell (1 x 10(7)) transfer and with OK-432 (50 KE/kg) plus rIL-2 (50 units/mouse) 6 hr later and on day 6, prolonged the survival. Therefore, the immunotherapy with OIK-cell transfer followed by administration of OK-432 and rIL-2 may be clinically useful as an adjunct of cytoreductive chemotherapy for cancer.     

Allogeneic cell therapy in murine B-cell leukemia (BCL1): 1. Alloimmune-mediated graft-versus-leukemia (GVL) effects induced by unmodified and in vitro rIL-2-activated bone marrow and lymphocytes from different lymphoid compartments

We have investigated the efficacy of graft-versus-leukemia (GVL) effects induced by cells obtained from different syngeneic and allogeneic lymphoid compartments, by comparing the response to cell therapy with syngeneic (BALB/c x C57BL/6)F1 (H-2d/b) (F1) or allogeneic C57BL/6 (H-2b) (B6) lymphocytes in F1 recipients inoculated with B-cell leukemia (BCL1) of BALB/c (H-2d) origin. Eradication of BCL1 was confirmed in vivo by adoptive transfer of 10(5) spleen cells obtained from treated mice into syngeneic BALB/c recipients. Immunotherapy induced by allogeneic but not syngeneic spleen and lymph node lymphocytes was therapeutically more effective than thymocytes and bone marrow cells (BMC). Alloreactive cells could be further activated in vivo with recombinant human interleukin-2 (rIL-2). The GVL effect of allogeneic lymphocytes was cell-dose-dependent; a heavy leukemia load was more efficiently eradicated after three doses than after a single dose of allogeneic spleen cells (100% versus 23% disease-free survival rate of secondary adoptive recipients respectively). The GVL effect induced by allogeneic spleen cells was preserved after ex vivo exposure of cells to 250 cGy, but not 500 cGy or more. Interestingly, GVL was preserved following administration of ex vivo irradiated (500 cGy) spleen cells when rIL-2 was administered in vivo (p < 0.05). Syngeneic effector cells did not induce GVL, regardless of in vitro and in vivo activation with rIL-2. Our data suggest that allogeneic but not syngeneic (in analogy to autologous) cell therapy may be an effective tool to control residual leukemia following high-dose chemo-radiotherapy. The feasibility of augmenting GVL by successive doses of activated allogeneic donor lymphocytes, partly inactivated in vitro by low-dose ionizing irradiation to prevent severe graft-versus-host disease (GVHD), may lead to safer therapeutic approaches that can be used to reduce the incidence of relapse while avoiding the risk of uncontrolled GVHD.     

重组白细胞介素-10/Fc融合蛋白对内毒素诱导急性肺损伤小鼠的实验干预作用

目的 探讨重组白细胞介素-10(rIL-10)/Fc融合蛋白对内毒素诱导的急性肺损伤(ALl)小鼠炎症调控作用及其机制.方法 向气管内注射脂多糖(LPS)制成ALl动物模型;rIL-10/Fc融合蛋白采用腹腔内给药方式.132只小鼠被随机均分为正常对照组、rIL-10/Fc对照组、ALl模型组、rIL-10/Fc治疗组.每组选择25只小鼠观察24 h存活率;其余用于检测支气管肺泡灌洗液(BALF)中自细胞数量,肿瘤坏死因子-a(TNF-a)和IL-1β水平,以及肺组织髓过氧化物酶(MPO)活性、肺组织湿/干重(W/D)比值;光镜下观察肺组织病理学改变.结果 注射LPS后4 h可引起BALF中TNF-a和IL-1β显著升高(P均＜0.01),rIL-10/Fc治疗组较ALI模型组有所降低,但差异无统计学意义;但在8 h和12 h,rIL-10/Fc融合蛋白能显著抑制BALF中TNF-a产生,在12 h抑制IL-1β产生;并明显改善LPS注射24 h后实验动物的存活率(P＜0.01).rIL-10/Fc对LPS诱导的ALI小鼠BALF中白细胞数量、肺组织MPO活性、肺组织W/D比值无显著改变.注射LPS 24 h后,肺组织出现了明显的炎性改变,但在rlL-10/Fc融合蛋白干预后没有出现显著的差异.结论 rIL-10/Fc融合蛋白能显著抑制LPS诱导的ALI小鼠肺促炎细胞因子产生,改善预后.     

Constitutive expression of bcl-2 in B cells causes a lethal form of lupuslike autoimmune disease after induction of neonatal tolerance to H- 2b alloantigens

The bcl-2 protooncogene has been shown to provide a survival signal to self-reactive B cells, but it fails to override their developmental arrest after encounter with antigen. Furthermore, constitutive expression of bcl-2 in B cells does not promote the development of autoimmune disease in most strains of mice, indicating that signals other than those conferred by bcl-2 are required for long-term survival and differentiation of self-reactive B cells in vivo. To further examine the factors that are required for the pathogenesis of autoimmune disease, we have assessed the effect of bcl-2 overexpression on the development of host-versus-graft disease, a self-limited model of systemic autoimmune disease. In this model, injection of spleen cells from (C57BL/6 x BALB/c)F1 hybrid mice into BALB/c newborn parental mice induces immunological tolerance to donor tissues and activation of autoreactive F1 donor B cells through interactions provided by allogeneic host CD4+ T cells. BALB/c newborns injected with spleen cells from (C57BL/6 x BALB/c)F1 mice expressing a bcl-2 transgene in B cells developed high levels of anti-single-stranded DNA and a wide range of pathogenic autoantibodies that were not or barely detectable in mice injected with nontransgenic spleen cells. In mice injected with transgenic B cells, the levels of pathogenic autoantibodies remained high during the course of the study and were associated with long-term persistence of donor B cells, development of a severe autoimmune disease, and accelerated mortality. These results demonstrate that bcl-2 can provide survival signals for the maintenance and differentiation of autoreactive B cells, and suggest that both increased B cell survival and T cell help play critical roles in the development of certain forms of systemic autoimmune disease.     

Cell therapy with preimmunized effector cells mismatched for minor histocompatible antigens in the treatment of a murine mammary carcinoma

Cell therapy with allogeneic donor cells mismatched for minor histocompatible (MiHC) antigens was applied to a murine mammary carcinoma (4T1) model to test the feasibility of graft versus tumor (GVT) effect against metastatic epithelial tumor cells. BALB/c mice bearing a 4T1 tumor of BALB/c origin were given syngeneic or MiHC-mismatched splenocytes. GVT effects were determined in secondary recipients of adoptively transferred lung cells derived from primary hosts who had previously been inoculated intravenously with 4T1 cells, and injected with one of the following: 1) naive BALB/c splenocytes, 2) naive DBA/2 splenocytes, 3) 4T1-immune DBA/2 splenocytes, or 4) DBA/2 splenocytes immunized with host-derived BABL/c spleen cells. Naive DBA/2 splenocytes inhibited tumor growth only slightly and only slightly prolonged the survival of secondary recipients, in comparison with fully matched tumor/host BALB/c spleen cells. An efficient GVT reaction was demonstrated in vitro and in vivo with MiHC-mismatched DBA/2 splenocytes from mice presensitized by multiple injections of irradiated tumor or BALB/c-derived spleen cells. All 30 mice adoptively inoculated with lung cells from primary hosts that had previously been treated with these presensitized effector cells were tumor free for >250 days. Secondary recipients inoculated with lung cells from mice given naive BALB/c or DBA/2 spleen cells died of metastatic tumors within 33 to 46 days. These results suggest that preimmunized donor cells represent an effective tool against metastatic disease; hence, the next goal should be to control graft-versus-host disease while exploiting the GVT potential.     

NZB cytotoxic lymphocyte responses. Kinetic analyses

Cytotoxic lymphocyte (CTL) responses of unprimed NZB spleen cells peaked on day 4 of culture as did cells from primed NZB or BALB/c mice. In contrast, primary BALB/c and DBA/2 responses peaked on day 6 of culture. Thus, NZB CTL generation was similar to the accelerated in vitro generation of CTL from the spleen cells of alloantigen-primed NZB and BALB/c mice. To evaluate the kinetics of these CTL responses, multiple-time-point analyses were performed during the initial 90 min of the 51Cr-release assays. Analyses were done on days 4 and 6. On day 4, NZB CTL had an initial velocity of lysis slightly greater than that of BALB/c or DBA/2 CTL; however, it was far less than that of secondary NZB and secondary BALB/c CTL. These studies indicate that NZB mice can generate primary CTL responses at an accelerated rate. Such augmented primary responses are unique and may explain recently described abnormal NZB T cell recognition as well as resistance of NZB CTL to suppressor signals.     

Fractionated extract of Astragalus membranaceus, a Chinese medicinal herb, potentiates LAK cell cytotoxicity generated by a low dose of recombinant interleukin-2

Success with rIL-2 immunotherapy of human cancer appears to depend on the administration of high doses which are frequently associated with excessive toxicity. Future use of rIL-2 will require certain modifications based on the use of lower doses of rIL-2 without significant loss of antitumor efficacy. We tested in vitro the possibility of potentiating the activity of rIL-2 in terms of LAK cell generation. We hypothesized that co-incubation of LAK cell precursors with a Chinese herbal extract (F3) of Astragalus membranaceus, (an immune modulator currently under study in our laboratory), along with a low concentration of rIL-2, would generate levels of LAK cell activity equivalent to those generated by high concentrations of rIL-2 alone. We found (1) a 10-fold potentiation of rIL-2 activity manifested by tumor cell-killing activity of 80% resulting from LAK cell generation with F3 plus 100 u/ml of rIL-2 versus 76% generated by 1,000 u/ml of rIL-2 alone; (2) a significant reduction in the number of effector LAK cells required for equicytotoxic reaction following LAK cell generation with F3 plus rIL-2 compared to rIL-2 alone. We conclude that potentiation of antitumor activity mediated by rIL-2 in low concentrations is possible by the concomitant use of another immune modulator such as Astragalus membranaceus.     

Cell-mediated immunity in lumphocytic choriomeningitis. I. The specificity of the cytotoxic T lymphocytes.

The specificity of cytotoxic T lymphocytes (CTL) generated during murine lymphocytic choriomeningitis (LCM) has been investigated. CTL were obtained from the spleens of mice injected i.p. with LCM virus. The cytotoxic activity of the CTL was tested in an in vitro 51Cr cytotoxicity assay using infected macrophages or fibroblasts as target cells. At the peak of the cytotoxic T cell response (7-8 days after infection) the cytotoxic action was restricted to syngeneic virus-infected target cells. Using H-2 recombinant mice the target antigen of the CTL generated could be identified as products coded for by either the H-2 K or H-2 D region of the major histocompatibility complex. I region identity between CTL and infected target cells was insufficient for optimal lysis to occur. During the early phase of LCM virus infection there was a transient phase during which non-infected H-2 histocompatible targets were lysed as efficiently as virus-infected target cells. This finding may suggest, that during the early phase of LCM disease self-reactive cytotoxic T lymphocytes are temporarily present in LCM virus-infected mice.     

弓形虫感染动物模型的建立及其特异性IgG抗体的研究

目的通过弓形虫RH株速殖子不同途径、不同剂量攻击BALB／c小鼠和sD大鼠,以获得有效的特异性IgG抗体,为建立体液免疫应答的动物模型提供一定的实验依据。方法将实验动物分成速殖子1×10。个腹腔注射sD大鼠组、不同剂量速殖子腹腔注射BALB／c小鼠组和速殖子4×10。个灌胃BALB／c小鼠组。每组在不同的时间取血查IgG抗体,各组均设对照鼠。结果腹腔注射SD大鼠组的实验鼠未出现发病征象;其血清IgG抗体含量逐渐上升,其抗体水平明显高于对照鼠（P〈0．01）。腹腔注射BALB／c小鼠组的实验鼠存活不超过8d;灌胃BALB／c小鼠组到第9天时实验鼠有4％死亡,其余均可存活至28d。且这两组实验鼠和对照鼠的IgG抗体水平差异均无统计学意义（P〉0．05）。结论BALB／c小鼠经口或经腹腔感染弓形虫RH株速殖子均未产生特异性IgG抗体,SD大鼠经腹腔感染可产生特异性IgG抗体。SD大鼠适于建立体液免疫应答的动物模型。     

Prior cryopreservation of ex vivo-expanded cord blood cells is not detrimental to engraftment as measured in the NOD-SCID mouse model

Cytokine-mediated expansion has been proposed and successfully used to facilitate engraftment post transplantation. This study examined whether cryopreservation following expansion has a detrimental effect on the ability of cells to engraft, using the NOD-SCID mouse model. Cord blood (CB) CD34(+) cells were incubated for 7 days with stem cell factor (SCF), flt-3 ligand (FL), and megakaryocyte growth and development factor (MGDF). Expanded CD34(+) cells were transplanted into NOD-SCID mice either fresh or following cryopreservation and thawing. After thawing, recovery of nucleated cells was 94%, of CD34 cells was 63%, and of day-14 progenitors was 17%. The loss of day-14 progenitor cells among the thawed expanded cells did not influence the kinetics of human engraftment in the mouse. Bone marrow (BM) of mice transplanted with thawed expanded CD34(+) cells (14 +/- 3.9%) showed significantly higher levels of human engraftment than mice transplanted with fresh expanded CD34(+) cells (1.5 +/- 0.5%, p = 0.0064). Thawed expanded CD34(+) cells had significantly higher SCID Engrafting Potential (SEP) than freshly expanded CD34(+) cells (p < 0.001). Results suggest that prior cryopreservation does not prevent expanded cells engrafting in NOD-SCID mice.     

Correlation between enhancement of graft-versus-leukemia effects following allogeneic bone marrow transplantation by rIL-2 and increased frequency of cytotoxic T-lymphocyte precursors in murine myeloid leukemia

A model of mouse acute myeloid leukemia (mAML) was used to study the effector mechanism mediating the graft-versus-leukemia (GVL) effects in recipients of allogeneic bone marrow cells (BMC). mAML-bearing SJL/J (H-2s) mice were lethally irradiated and then transplanted with a mixture of BMC and spleen cells (SC) derived from normal syngeneic or allogeneic mice. To augment the GVL effect, recipients were injected intraperitoneally with recombinant human interleukin-2 (rIL-2) (1.2 x 10(5) IU) for 3 consecutive days, starting one day post BMC + SC transplantation. Spleen cells from treated recipients were adoptively transferred to untreated secondary SJL/J mice to test for the existence of residual tumor cells. All the secondary recipients of SC from mAML-bearing SJL/J mice rescued with syngeneic (SJL/J) or allogeneic (B10.S) BMC+SC (H-2s) differing at minor antigens of the histocompatibility complex (MiHC) developed leukemia and died. In sharp contrast, none of the secondary recipients of SC obtained from identical mAML-bearing mice rescued with B10.S BMC + SC but activated in vivo with IL-2 developed leukemia. Adoptive recipients of SC obtained from mAML-bearing recipients of major histocompatibility complex (MHC)-disparate (C57BL/6, H-2b) cells remained free of leukemia regardless of the use of rIL-2. In parallel with the in vivo findings, a 4-day in vitro exposure of splenocytes to 6 x 10(3) IU/ml rIL-2 resulted in a 5- to 20-fold increase in the frequency of alloreactive cytotoxic T-lymphocyte (CTL) precursors (CTLp) across MiHC and MHC barriers and a 2- to 6-fold increase in their cytotoxic activity. Our data suggest that augmentation of GVL effects by rIL-2 may be due to CTL activation by rIL-2, not excluding the potential beneficial role of rIL-2-activated allogeneic natural killer cells and MHC non-restricted killer cells. Cumulatively, our results suggest potentially beneficial effects of rIL-2, when used jointly with bone marrow transplantation or allogeneic cell therapy, on eradication of leukemia.     

Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro

Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.     

A modified cytotoxicity assay with high sensitivity

Since adoptive immunotherapy using lymphokine activated killer (LAK) cells and interleukin-2 (IL-2) has been introduced into clinical medicine, there has been a growing interest in cytotoxicity assays. The standard ⁵¹chromium release assay has certain disadvantages, in particular limited sensitivity, because of a high, nonspecific background release. We examined the conditions under which tritiated thymidine, which has been used to assess slow macrophage mediated cytolysis, can be utilized to assess the rapid cytotoxic activity of unstimulated PBL and LAK cells.

The optimal assay duration for the ³H-thymidine (³H-TdR) release assay is 24 h. Under standard conditions actively proliferating effector cells do incorporate some of the ³H-TdR released by the target cells during this time, leading to false low results. This problem can be abolished by the addition of excessive amounts of cold TdR to the assay medium. We found a good correlation of the results of the TdR release assay and the Cr release assay. Using the very sensitive TdR release assay, unexpected significant cytolysis of the so-called ‘NK resistant’ Daudi cells by unstimulated PBL is demonstrated. The modified ³H-TdR release assay is well-suited to monitor the immunological effects of immunotherapy, using IL-2 and LAK cells.