Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity.

We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

Enhancement of tumor lysate- and peptide-pulsed dendritic cell-based vaccines by the addition of foreign helper protein.

We have evaluated whether the addition of a foreign helper protein, keyhole limpet hemocyanin (KLH), can augment the efficacy of tumor lysate-pulsed dendritic cells and peptide-pulsed DC immunizations in vivo. Besides being used as a "surrogate antigen" in approaches to measure immunological response in cancer patients, KLH is also an immunogenic carrier protein to elicit T-cell help. Using the D5 subline of B16 melanoma, we demonstrate that DCs pulsed with both KLH and tumor lysate mediate enhanced immune priming and rejection of established metastases in vivo, which is dependent on host-derived T cells. Interleukin 2 augments the enhancement afforded by KLH, as measured by cure rates and overall survival, in the absence of autoimmune depigmentation. KLH added to DC immunizations markedly enhances tumor-specific T cell production of IFN-gamma. D5 melanoma exposed to similar levels of IFN-gamma results in substantial expression of MHC class I molecules. DCs pulsed with KLH and mouse tyrosinase-related protein-2 peptide results in enhanced reduction of B16 melanoma metastases; the effect is most pronounced in a setting where tyrosinase-related protein-2 peptide-pulsed DCs alone are completely ineffective. Collectively, these findings demonstrate that KLH addition to tumor antigen-pulsed DC immunizations can augment IFN-gamma production and enhance in vivo antitumor activity.

T cell-dependent antitumor immunity mediated by secondary lymphoid tissue chemokine: augmentation of dendritic cell-based immunotherapy.

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that is selective in its recruitment of naive T cells and dendritic cells (DCs). In the lymph node, SLC is believed to play an important role in the initiation of an immune response by colocalizing naive T cells with DC-presenting antigen. Here, we used SLC as a treatment for tumors established from the poorly immunogenic B16 melanoma. Intratumoral injections of SLC inhibited tumor growth in a CD8+, T cell-dependent manner. SLC elicited a substantial infiltration of DCs and T cells into the tumor, coincident with the antitumor response. We next used SLC gene-modified DCs as a treatment of established tumors. Intratumoral injections of SLC-expressing DCs resulted in tumor growth inhibition that was significantly better than either control DCs or SLC alone. Distal site immunization of tumor-bearing mice with SLC gene-modified DCs pulsed with tumor lysate elicited an antitumor response whereas control DCs did not. We also found that s.c. injection of lysate-pulsed DCs expressing SLC promoted the migration of T cells to the immunization site. This report demonstrates that SLC can both induce antitumor responses and enhance the antitumor immunity elicited by DCs.

Analysis of testes and semen from rabbits treated by intravenous injection with a retroviral vector encoding the human factor VIII gene: no evidence of germ line transduction.

In a phase 1 clinical trial, we are evaluating a murine leukemia virus (MuLV)-based retroviral vector encoding the human factor VIII gene [hFVIII(V)], administered intravenously, as a therapy for hemophilia A. Preclinical biolocalization studies in adult rabbits revealed vector-specific PCR signals in testis tissue at low levels. In follow-up animal studies we used PCR to (1) estimate the frequency with which a given cell in testis tissue is transduced, and (2) determine whether a positive PCR signal could be detected in semen samples from animals treated with hFVIII(V). Using the 99% confidence bound, results indicate that the probability that a given cell within the testis was transduced is less than 1/709,000 (97 days after treatment). This probability decreased with time after hFVIII(V) administration. Moreover, the rate of provector sequence detection in semen samples collected weekly throughout two cycles of spermatogenesis was 3/4281 reactions (0.07%), which is lower than the rate of false positives (1/800, 0.125%) observed for control animals. Using PCR assays with single-copy sensitivity, we have shown that the small number of transduced cells present in testis tissue does not give rise to detectable transduced cells in semen.

Induction of Th1 response by dendritic cells pulsed with autologous melanoma apoptotic bodies.

BACKGROUND: We hypothesize that dendritic cells (DCs) can process antigens from autologous melanoma apoptotic bodies (MABs) and induce effector T cells in melanoma patients. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained from three stage IV melanoma patients and adherent cells were cultured in complete medium (CM) containing GM-CSF (800 U/ml) and IL-4 (1000 U/ml) for 7 days. Autologous MABs from melanoma cells following actinomycin D treatment (0.5 microgram/ml) for 24 hours, were added to 72 hour DC culture. Autologous effector T cells were cultured in CM containing 60 IU/ml of IL-2 and were stimulated by MAB-pulsed DCs three times at a weekly interval. Effector T cells were harvested at the end of third cycle of DC stimulation. RESULTS: Using ELISPOT, IFN-gamma production by effector T cells stimulated by MAB-pulsed DCs was significantly higher than that by T cells without DC stimulation. Microscopy demonstrated phagocytosis of MABs by DCs. CONCLUSIONS: MAB-pulsed DCs are capable of stimulating Th1-directed autologous effector T cells. Pulsing DCs with autologous MABs may be a novel approach in future DC-based immunotherapeutic trials.

Immunologic and virologic effects of subcutaneous interleukin 2 in combination with antiretroviral therapy: A randomized controlled trial.

CONTEXT: While interleukin 2 (IL-2) is capable of inducing a marked expansion of the CD4 T-lymphocyte pool, limited data exist on whether IL-2 treatment can add significantly to the immunologic and virologic effects of potent antiretroviral therapy (ART). OBJECTIVE: To determine the rate and magnitude of CD4 cell recovery and viral suppression when using a combination therapy of IL-2 and ART compared with ART alone. DESIGN AND SETTING: Randomized, controlled multicenter trial conducted from April 1996 through April 1998 at 8 clinical sites in the United States. PATIENTS: Eighty-two adult outpatients who were infected with human immunodeficiency virus (HIV) and had baseline CD4 cell counts of 200 x 10(6)/L to 500 x 10(6)/L and baseline RNA levels of fewer than 10,000 copies/mL were randomized; 78 completed the study. INTERVENTIONS: Thirty-nine patients were randomly assigned to receive a combination therapy of subcutaneous IL-2 (administered in 5-day courses every 8 weeks at a starting dosage of 7.5 mIU twice per day) and ART; 43 were to receive ART therapy alone. MAIN OUTCOME MEASURES: Interleukin 2 safety and differential effects on CD4 cell counts, CD4 cell percentages, and plasma HIV RNA levels. RESULTS: The mean (SD) percentage increase in CD4 cell counts at 1 year for patients who received IL-2 was 112% (113%) compared with 18% (35%) in recipients of ART alone (P<.001). Both groups had mean (SD) increases in CD4 cell percentage: from 20.4% (6.3%) to 32.3% (12.4%) for the combination therapy group compared with 20.4% (5.1%) to 23.0% (7.2%) for recipients of ART alone (P<.001). Using a sensitive viral RNA assay, mean viral load changes were -0.28 and 0.09 log(10) copies for IL-2 recipients and control patients, respectively (P=.03). Twenty (67%) of 30 evaluable patients receiving IL-2 achieved final viral loads of fewer than 50 copies/mL compared with 13 (36%) of 36 control patients (P=.02). Toxic effects were common among patients who received IL-2 and were managed with antipyretics, hydration, rest, and dosage reduction as needed. CONCLUSIONS: Intermittent therapy with IL-2 and ART produced a substantially greater increase in CD4 cells and was associated with a larger decrease in viral load than ART alone. Clinical end-point trials will be necessary to determine whether the enhanced viral suppression and CD4 cell increases associated with IL-2 therapy will translate into improved clinical outcomes. JAMA. 2000;284:183-189

Potentiation of immunologic responsiveness to dendritic cell-based tumor vaccines by recombinant interleukin-2.

PURPOSE: Dendritic cells (DC) can elicit potent immune responses to tumors through their capacity to efficiently process and present tumor-associated antigens. In a variety of animal tumor models, vaccines based on tumor lysate-pulsed DC (TP-DC) have been shown to effectively immunize against lethal tumor challenges as well as to treat established growing tumors at skin and organ sites. The antitumor effects elicited by TP-DC-based vaccines in vivo have been shown to be mediated by tumor-specific proliferative, cytotoxic, and cytokine-secreting host-derived T cells. Because of the critical involvement of T cells in the antitumor immune response, we have been investigating whether the systemic administration of recombinant interleukin (IL)-2 can enhance the therapeutic efficacy of TP-DC-based tumor vaccines. MATERIALS AND METHODS: Immunization with TP-DC plus IL-2 administration was evaluated to determine if this combination could enhance protective immunity toward a weakly immunogenic sarcoma (MCA-207) and a poorly immunogenic subline (D5) of the B16 melanoma and mediate therapeutic rejection of established tumors in C57BL/6 (B6) mouse models. RESULTS: We have demonstrated in our murine models that the addition of IL-2 at relatively nontoxic doses can markedly augment the antitumor activity of TP-DC-based tumor vaccine therapies against both a weakly immunogenic sarcoma and a poorly immunogenic melanoma. Animals treated with the combination exhibited significantly greater protection from tumor-cell challenge, significantly greater regression of established tumors, and significantly longer mean survival time than with either TP-DC or IL-2 therapy alone. The mechanism operative in vivo appears to involve the enhancement of immune T-cell function. CONCLUSION: These preclinical studies demonstrate the potential of this novel treatment strategy and support the rationale for planned phase I/II clinical trials of TP-DC-based vaccines plus IL-2 in patients with advanced melanoma and colorectal cancer.

Characterization of novel antimicrobial peptoids.

Peptoids differ from peptides in that peptoids are composed of N-substituted rather than alpha-carbon-substituted glycine units. In this paper we report the in vitro and in vivo antibacterial activities of several antibacterial peptoids discovered by screening combinatorial chemistry libraries for bacterial growth inhibition. In vitro, the peptoid CHIR29498 and some of its analogues were active in the range of 3 to 12 microg/ml against a panel of gram-positive and gram-negative bacteria which included isolates which were resistant to known antibiotics. Peptoid antimicrobial activity against Staphylococcus aureus was rapid, bactericidal, and independent of protein synthesis. beta-Galactosidase and propidium iodide leakage assays indicated that the membrane is the most likely target of activity. Positional isomers of an active peptoid were also active, consistent with a mode of action, such as membrane disruption, that does not require a specific fit between the molecule and its target. In vivo, CHIR29498 protected S. aureus-infected mice in a simple infection model.

A randomized trial of high- versus low-dose subcutaneous interleukin-2 outpatient therapy for early human immunodeficiency virus type 1 infection.

Forty-nine outpatients infected with human immunodeficiency virus with baseline CD4 cell counts >/=500/mm3, who were on stable antiretroviral therapy, were randomized to receive 5-day cycles of either low-dose (1.5 million IU [MIU] twice a day) or high-dose (7.5 MIU twice a day) subcutaneous (sc) interleukin (IL)-2 every 4 or every 8 weeks. High-dose recipients experienced mean slopes of +116.1 cells/month and +2.7 %/month in CD4 cells and percents, respectively, whereas low-dose recipients displayed mean slopes of +26.7 and +1.3% in the same parameters. At month 6, high-dose recipients achieved a 94.8% increase in mean CD4 cells over baseline compared with a 19.0% increase in low-dose recipients. While high-dose recipients encountered more constitutional side effects, these were generally not dose-limiting. High-dose scIL-2 therapy in outpatients with early HIV-1 infection was well tolerated and induced dramatic, sustained rises in CD4 cells.

Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines.

We have reported previously that murine bone marrow-derived dendritic cells (DC) pulsed with whole tumor lysates can mediate potent antitumor immune responses both in vitro and in vivo. Because successful therapy was dependent on host immune T cells, we have now evaluated whether the systemic administration of the T cell stimulatory/growth promoting cytokine interleukin-2 (IL-2) could enhance tumor lysate-pulsed DC-based immunizations to further promote protective immunity toward, and therapeutic rejection of, syngeneic murine tumors. In three separate approaches using a weakly immunogenic sarcoma (MCA-207), the systemic administration of nontoxic doses of recombinant IL-2 (20,000 and 40,000 IU/dose) was capable of mediating significant increases in the potency of DC-based immunizations. IL-2 could augment the efficacy of tumor lysate-pulsed DC to induce protective immunity to lethal tumor challenge as well as enhance splenic cytotoxic T lymphocyte activity and interferon-gamma production in these treated mice. Moreover, treatment with the combination of tumor lysate-pulsed DC and IL-2 could also mediate regressions of established pulmonary 3-day micrometastases and 7-day macrometastases as well as established 14- and 28-day s.c. tumors, leading to either significant cure rates or prolongation in overall survival. Collectively, these findings show that nontoxic doses of recombinant IL-2 can potentiate the antitumor effects of tumor lysate-pulsed DC in vivo and provide preclinical rationale for the use of IL-2 in DC-based vaccine strategies in patients with advanced cancer.

Activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation.

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.

The circulating common gamma chain (CD132) in inflammatory bowel disease.

OBJECTIVE: Inflammatory bowel disease (IBD) is characterized by T cell activation. Activated T cells shed interleukin-2 receptors (IL-2R) in a soluble form. A positive correlation between sIL-2Ralpha (CD25) and disease activity is well documented in IBD, whereas IL-2Rgamma (CD132) has not been investigated in this respect. Sera from 42 patients with ulcerative colitis (UC), 34 with Crohn's disease (CD), 31 healthy volunteers, and 12 patients with infectious enterocolitis were obtained. METHODS: Disease activity was scored according to a semiquantitative score for UC and CD. sIL-2R alpha chain and gamma chain were assessed by sandwich ELISA techniques using monoclonal antibodies specific for CD25 and CD132, respectively. RESULTS: The concentration of IL-2Ralpha chain (CD25) was found to be median 3.8 ng/ml in healthy volunteers versus 7.0 ng/ml in UC patients (p < 0.001), and 9.6 ng/ml in CD patients (p < 0.001). With respect to IL-2Rgamma (CD132), significantly higher amounts were found in CD patients: 6.6 ng/ml as compared with healthy controls <1.0 ng/ml (p < 0.004). A Kruskal-Wallis test revealed a significant correlation between alpha chain and disease activity in CD (p < 0.001), and further significantly higher gamma chain levels were found in active CD (p = 0.03). For UC patients, a statistically significant increase of the alpha chain with increasing disease activity (p < 0.01) was observed, whereas no significant changes of the gamma chain levels were found (p > 0.05). A difference of gamma chain levels were found between CD and UC in moderate and severe disease activity (p < 0.05). Further analyses revealed that mesalazine did not influence the IL-2Ralpha or -gamma concentration either in UC or in CD patients. CONCLUSION: An increased circulating level of the soluble common gamma chain (CD132) seems to be found in CD, and an overlap exists between CD and UC.

Systemic cytokine immunotherapy for experimental cytomegalovirus retinitis in mice with retrovirus-induced immunodeficiency.

PURPOSE: To evaluate and compare the in vivo administration of interleukin-2 (IL-2) or interleukin-12 (IL-12) in the immunotherapy of necrotizing retinitis caused by murine cytomegalovirus (MCMV) in mice with a retrovirus-induced immunodeficiency syndrome (MAIDS). METHODS: Adult C57BL/6 mice with MAIDS of 8 weeks' duration were treated with either a single intramuscular injection of polyethylene glycol-modified human recombinant IL-2 (PEG-IL-2) or multiple intramuscular injections of murine recombinant IL-12; untreated mice with MAIDS received phosphate-buffered saline. Two days later, the left eyes of all mice were inoculated with MCMV by subretinal injection and evaluated at day 6 for intraocular MCMV titers or at day 10 for frequency of necrotizing MCMV retinitis. RESULTS: Infectious MCMV was significantly reduced in whole eyes of PEG-IL-2-treated mice with MAIDS (2.8 log10), but not in whole eyes of IL-12-treated animals (4.4 log10) when compared with whole eyes of untreated animals with MAIDS (4.5 log10). Similarly, whereas eyes from approximately 80% of IL-12-treated and untreated mice with MAIDS showed histopathologic features consistent with classic necrotizing MCMV retinitis (full-thickness retinal necrosis associated with virus inclusions and cytomegalocytes), none (0%) of PEG-IL-2-treated animals with MAIDS showed classic MCMV retinitis. Instead, eyes from these animals showed either retinal folding or outer retinal atrophy, a pattern of histopathology similar to that observed in eyes from immunologically normal C57BL/6 mice inoculated subretinally with MCMV. CONCLUSIONS: These results provide proof-of-principle for the hypothesis that systemic cytokine immunotherapy will reduce the frequency of CMV retinitis in a setting of retrovirus-induced immunosuppression. Because of the striking differential effects of IL-2 and IL-12 on MCMV-retinitis in mice with MAIDS, the authors conclude that cytokine immunotherapy for cytomegalovirus-induced retinitis is cytokine-specific, even for such cytokines as IL-2 and IL-12 that have T cell regulation in common.

Immunopharmacology and cytokine production of a low-dose schedule of intraperitoneally administered human recombinant interleukin-2 in patients with advanced epithelial ovarian carcinoma.

We determined in the peritoneal cavity (p.c.) of epithelial ovarian carcinoma patients during a 4-day treatment cycle of low-dose recombinant human interleukin-2 (rIL-2): (a) pharmacokinetics of IL-2, (b) endogenous cytokine production, and (c) numbers and percentages of peritoneal exudate lymphocytes. We administered 6 x 10(5) IU/m2 of rIL-2 (0.03 mg/m2 Proleukin rIL-2) intraperitoneally (i.p.) over 30 min on each of 4 days. One and one-half liters of D5 0.25 NS was injected i.p. before each rIL-2 infusion. Multiple peritoneal fluid samples were obtained from each of four patients on day 1 and day 4 for detection of IL-2, endogenous cytokines, and soluble IL-2 receptor (IL-2R-alpha). IL-2 concentrations in the peritoneal fluid were determined by bioassay and interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-10, transforming growth factor (TGF)-beta 2, and sIL-2R-alpha by enzyme-linked immunosorbent assay. Numbers of cells per microliter and lymphocyte subpopulation percentages after staining with a panel of monoclonal antibodies were determined on day 1, day 4, and subsequent off-treatment days. IL-2 disappearance in the p.c. was well described by a pharmacokinetic model having constant-rate infusion and biexponential disposition. About 90% of the IL-2 disappearance occurred during the beta-phase, during which IL-2 concentrations were sustained at approximately 10-30 ng/ml (day 1 and day 4) and the median t1/2 beta was 21.5 and 9.2 h on days 1 and 4, respectively. In four of four patients, p.c. production of IL-10 was observed on day 1 and day 4 (maximum 387 pg/ml). Maximum levels of IFN-gamma and sIL-2R-alpha were observed on day 4. (IFN-gamma 217 pg/ml; sIL2-R-alpha: 3486 U/ml). No increases in TNF-alpha or TGF-beta 2 were observed. Large increases in p.c. CD3+, CD4+, CD8+, CD16+, and CD56+ cells were observed. We conclude that biologically active levels of IL-2 are generated in p.c. fluids after i.p. administration of rIL-2 at 0.03 mg/m2.

Daily subcutaneous injection of low-dose interleukin 2 expands natural killer cells in vivo without significant toxicity.

We aimed to determine the toxicity and immunological effects of daily s.c. administered low-dose interleukin (IL) 2. Adult cancer patients received a single daily s.c. injection of IL-2 as outpatients for 90 consecutive days. Cohorts of four to nine patients were treated at escalating IL-2 dose levels until the maximum tolerated dose (MTD) was defined. Peripheral blood mononuclear cell phenotyping, IL-2 serum levels, and the presence of anti-IL-2 antibodies were investigated. Thirty-eight patients were treated at seven IL-2 dose levels ranging from 0.4 to 1.75 million International Units (mIU)/m2 daily. The MTD was 1.25 mIU/m2, with constitutional side effects, vomiting, and hyperglycemia dose limiting. Severe toxicity did not occur at or below the MTD, although mild local skin reaction and mild constitutional side effects were common. Objective tumor regressions were not observed during this Phase I trial. Low-dose IL-2 resulted in natural killer (NK) cell (CD3(-) CD56(+)) expansion at all dose levels. This effect was dose dependent (P < 0.01), ranging from a 154 to 530% increase over baseline. Peak NK levels were achieved at 6-8 weeks and sustained through 12 weeks of therapy. As predicted by in vitro studies of IL-2 receptor structure-activity relationships, the subset of NK cells that constitutively express high-affinity IL-2 receptors (CD3(-)CD56(bright+)) showed more profound dose-dependent expansion, with increases ranging from 368 to 2763% (P = 0.015). NK expansion occurred at peak IL-2 levels <10 pM (2.3 IU/ml). Three patients developed nonneutralizing anti-IL-2 antibodies. Thus, we concluded that selective expansion of NK cells may be achieved in vivo with daily s.c. injections of low-dose IL-2 with minimal toxicity.

IL-8 induces neutrophil chemotaxis predominantly via type I IL-8 receptors.

IL-8 is a potent proinflammatory cytokine that has a key role in the recruitment and activation of neutrophils during inflammation. IL-8 reacts with neutrophils via two distinct types of IL-8-R. Receptor-specific Abs were raised against peptides derived from the first extracellular domain of each IL-8-R. Anti-IL-8-R1 and anti-IL-8-R2 selectively block 125I-IL-8 binding to rIL-8-R type 1 or 2, respectively. The anti-peptide Abs were used to assess the role of each receptor in the chemotactic response of neutrophils to GRO alpha and to IL-8. Anti-IL-8-R2 blocks GRO alpha-induced chemotaxis of neutrophils. Chemotaxis to GRO alpha is not inhibited by anti-IL-8-R1. Thus GRO alpha stimulates chemotaxis exclusively through IL-8-R2 and independently of IL-8-R1. Surprisingly, anti-IL-8-R1 inhibits the majority (78 +/- 3%) of IL-8-induced neutrophil chemotaxis. Only a minor proportion of IL-8-induced chemotaxis (29 +/- 5%) is inhibited by anti-IL-8-R2. These findings indicate that chemotaxis to IL-8 is mediated predominantly by type 1 IL-8-Rs and suggest that IL-8-R1 is an appropriate target for therapeutic strategies to limit neutrophil influx in diseases where neutrophils contribute to pathophysiology.

The use of recombinant human interleukin-2 in treating infectious diseases.

Data from animal models indicate that interleukin-2 is potentially valuable in the treatment of a variety of infectious diseases of viral, fungal, protozoal, bacterial, and mycobacterial origin. The role of interleukin-2 in resistance to infection with human immunodeficiency virus or Mycobacterium leprae (the causative agent of leprosy) has recently been studied in detail. Data from animal models and clinical trials indicate that relatively low doses of interleukin-2 effectively stabilize or reverse the course of these infections. The recent characterization of Th1 and Th2 helper T cells, and their relationship to the control of infectious diseases, are revealing the mechanisms involved in producing disease. Increased understanding of these mechanisms may help extend interleukin-2 therapy to other clinical applications.

T-cell antigen receptors with identical variable regions but different diversity and joining region gene segments have distinct specificities but cross-reactive idiotypes.

The T-cell antigen receptor alpha-chain genes of an alloreactive, H-2Db-specific cytotoxic T-cell clone (3F9) are described. This study and our work on the 3F9 beta-chain genes reveal that the variable region gene segments for the alpha and beta chains expressed in 3F9 are identical to the ones used by a chicken erythrocyte-specific, I-Ab-restricted helper T-cell clone (LB2). These two clones differ, however, in the diversity and joining portions of the alpha and beta chains of their T-cell receptor molecules. The analysis of 3F9 and LB2 with monoclonal antibodies specific for the 3F9 T-cell receptor shows that these two T-cell clones share the same idiotype; however, 3F9 and LB2 do not exhibit any antigen and/or major histocompatibility complex cross-reactivity. This suggests that the diversity and joining regions of the T-cell receptor may play a key role in antigen and/or major histocompatibility complex recognition.

A requirement for physical linkage between determinants recognized by helper molecules and cytotoxic T cell precursors in the induction of cytotoxic T cell responses.

It has long been understood that both antibody and delayed-type hypersensitivity responses are induced through collaborative events in which the determinants recognized by the precursor cells must be physically linked to the determinants recognized by the helper. Although it is clear that the generation of memory cytotoxic T lymphocyte precursors (CTLp) involves linked recognition of determinants, the induction of CTL responses has been viewed as being dependent upon interleukin 2 (IL 2), which could be provided by a helper cell, but independent of requirements for antigen bridging. In this work, we have designed a system that lacks exogenous IL 2 by using as our source of help, antigen-specific helper molecules derived from helper T cells. These soluble helper molecules are uncontaminated by IL 2 and unlike a helper cell, are unable to produce IL 2. Helper molecules specific for chicken red blood cells (Crbc) and for a synthetic polypeptide, poly 18, were tested. Thymocyte responders require a source of help to respond to alloantigens intrinsically expressed on the surface of adherent stimulator cells. To analyze the mechanism whereby the helper molecules acted, we used a system involving recognition of haptenic and carrier determinants that were physically linked by virtue of being located on the same cell surface (intra-structural linkage). Adherent stimulator cells were pulsed with Crbc or poly 18 so that the alloantigens recognized by the thymocyte CTLp (intrinsically expressed class I) were either linked or unlinked to the carrier determinants (Crbc or poly 18) presented by the adherent cells and recognized by the helper molecules. Both types of helper molecule were shown to be antigen-specific in crisscross experiments. The helper molecules specific for Crbc were able to induce the thymocyte CTLp only when both hapten and carrier were present on the same stimulator cell surface. Because we were not able to detect a requirement for H-2-restricted recognition of carrier antigen, this inductive event must be viewed as requiring linked associative recognition of determinants, but being noncognate. In contrast, the helper molecules recognizing poly 18 showed a requirement for both physical linkage of determinants and for H-2 restricted recognition, indicating that the mechanism of induction was cognate in nature. Therefore, we have shown that interactions between CTLp and soluble, antigen-specific, helper cell-derived inductive molecules are similar in nature to those of other T cell precursors and of B cells in the stringent requirement for close physical proximity achieved by linked or cognate recognition of determinants across an antigen bridge.

Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins.

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.