NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti-4-1BB monoclonal antibodies.

The 4-1BB (CDw137) T-cell molecule is a member of the TNF receptor family and triggering by either 4-1BB ligand or antibody ligation induces T-cell activation and growth. We have recently demonstrated that administration of anti-4-1BB monoclonal antibodies (mAb) induced the regression of established large tumors in several mouse models by activation of T-cell-mediated immunity. Herein we report that selective depletion of natural killer (NK) cells in mice by the anti-AsialoGM1 or anti-NK1.1 antibodies completely abrogated the antitumor effect of anti-4-1BB mAb. However, it is unlikely that NK1. 1 cells are the effectors of the response because P815 cells are resistant to lysis by NK1.1 cells in vitro. Despite the fact that activated NK1.1 cells express 4-1BB on their surface, redirection of NK1.1 cells by anti-4-1BB mAb or by transfection into P815 cells of the 4-1BB natural ligand did not trigger cytolysis. Our results thus gain further insight into the cellular mechanisms of the antitumor effects of anti-4-1BB mAb and implicate an immunoregulatory rather than effector function of 4-1BB molecule on NK1.1 cells.

Use of combinatorial peptide libraries to construct functional mimics of tumor epitopes recognized by MHC class I-restricted cytolytic T lymphocytes.

Identification of cytolytic T lymphocyte (CTL) epitopes presented by major histocompatibility complex (MHC) class I molecules on tumor cells is critical for the design of active immunotherapy. We describe the use of combinatorial peptide libraries with defined amino acids in two MHC anchor positions to search for epitopes that are recognized by H-2Db- and Kb-restricted CTL specific for the mouse lymphoma EL4. An iterative strategy was used for screening libraries in which 16 amino acids were divided into 3 groups and 3 subgroups: alpha (AL, VT, FY); beta (GS, P, DE); gamma (KR, H, NQ). The proportions of each group and subgroup at individual peptide positions were changed in the library synthesis, and the effect of these changes on CTL activity was measured in a sensitive RMA-S cell assay. A single H-2Db epitope mimic was deduced from the original library that contained > 2 x 10(8) potential peptides and was at least 9 logs more potent than the original library. Immunization of syngeneic mice with this peptide elicited CTL that lysed EL4 cells as well as RMA-S cells pulsed with peptides isolated from Db molecules of EL4 cells, indicating functional similarity between the mimicking peptide and the naturally processed CTL epitope. Furthermore, adoptive transfer of such a CTL line had a therapeutic effect in mice with EL4 established as an ascites tumor. Two H-2Kb-restricted epitope mimics of the same tumor were also identified. Our method represents a novel approach for the construction of MHC class I-restricted targets that can serve as immunogens for active immunotherapy of cancer.

B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes.

Immunization of mice with tumors genetically engineered to express the B7 costimulatory molecules amplifies the antitumor immune response mediated by CD8+ cytolytic T lymphocytes (CTL). In this report, we examined the effect of B7-CD28 costimulation on the hierarchy of tumor epitopes. Using a combination of affinity chromatography/reversed-phase high performance liquid chromatography and CTL cloning, we show that major histocompatibility complex (MHC) class I molecules from EL4 lymphoma cells can present at least six distinct CTL epitopes presented by MHC class I molecules. Nevertheless, mice immunized with wild-type B7-negative EL4 cells develop CTL only to one immunodominant epitope. In contrast, immunization with B7-transduced EL4 cells led to not only the amplification of the CTL response to this immunodominant epitope, but also to the recognition of five otherwise silent subdominant epitopes. The adoptive transfer of a CTL clone against such a subdominant epitope cured mice bearing EL4 lymphoma growing as an ascites tumor. The fact that CTL response can be spread to normally silent epitopes as a result of B7-CD28 costimulation suggests a novel approach to manipulate the hierarchy of CTL epitopes and offers an opportunity to explore novel targets for T cell-mediated cancer therapy.

Protective immunity induced by B7/CD28-costimulated gamma delta T cells to the EL-4 lymphoma in allogenic athymic mice.

We previously reported that the murine EL-4 lymphoma (H-2b) transduced with a retrovirus containing the murine B7-1 gene (B7+ EL-4) grew transiently for several weeks and subsequently regressed in allogenic BALB/c (nu/nu) athymic mice (H-2d). We now show that, in contrast, B7+ EL-4 cells grow progressively in several combined immunodeficiency mice, including SCID and NIH III mice, which lack T cells expressing either TCR-alpha beta or -gamma delta. Furthermore, depletion of gamma delta T cells with a specific mAb made possible the progressive growth of B7+ EL-4 cells in 90% of athymic mice while depletion of alpha beta T cells allowed tumor growth in 50% of these mice. Immunization of athymic mice with B7+ EL-4 cells prevented the outgrowth of wild-type B7- EL-4 cells. This protective immunity was abrogated by in vivo treatment with an anti-TCR-gamma delta mAb, further indicating that gamma delta T cells play an important role in tumor rejection by athymic mice. A gamma delta T cell line, Tc1, was established from B7+ EL-4-immunized athymic mice by repeated restimulation in vitro with irradiated B7+ EL-4 cells. When tested against a broad spectrum of target cells, Tc1 lysed several murine lymphoma lines, but did not lyse other tumor lines, suggesting that the Ag recognized by Tc1 has a limited distribution. Our data demonstrate that gamma delta T cells, and, to a less extent, extrathymic alpha beta T cells, mediate an immune response against B7+ EL-4 cells in allogeneic athymic mice.

B7-1/CD80-transduced tumor cells elicit better systemic immunity than wild-type tumor cells admixed with Corynebacterium parvum.

Tumor cells genetically modified by transduction of B7 (B7-1/CD80), a natural ligand for the T-cell costimulatory molecules CD28 and CTLA-4, can elicit potent tumor immunity, and they can be effective for treatment of established cancers in animal models. In this study, three tumor lines, the EL4 lymphoma, the P815 mastocytoma, and the MCA102 sarcoma were transduced with recombinant retrovirus containing the murine B7 gene, and their potency to induce systemic immunity protective against challenge with wild-type tumor was compared to that of the same tumor cells admixed with the commonly used adjuvant Corynebacterium parvum. While admixture of tumor cells with C. parvum resulted in complete regression of tumors in syngeneic mice, it did not induce protective immunity against a subsequent challenge of wild-type cells from any of the 3 tumors tested. In contrast, B7-transduced EL4 and P815 tumors regressed locally and induced a potent systemic immunity to wild-type tumors and a higher level of cytotoxic T-cell activity than did tumor cells admixed with C. parvum. No systemic immunity was induced by B7-transduced nonimmunogenic MCA102 sarcoma cells. Our results demonstrate that immunogenic tumor cells transduced with the B7 gene are superior to tumor cells mixed with C. parvum for the induction of systemic tumor immunity.

Randomized study to evaluate the relation between oral isosorbide dinitrate dosing interval and the development of early tolerance to its effect on left ventricular filling pressure in patients with chronic heart failure.

BACKGROUND: Early development of nitrate tolerance has been shown in patients with chronic congestive heart failure (CHF) receiving continuous nitroglycerin therapy. The influence of dosing interval of oral isosorbide dinitrate (ISDN), the nitrate preparation most widely used for the treatment of CHF, has not been investigated. METHODS AND RESULTS: We performed a prospective, randomized study to evaluate the effect of various regimens of oral ISDN on the development of early tolerance to its effect on left ventricular filling pressure in patients with moderate to severe CHF. Forty-four responders (20% or greater reduction in mean pulmonary artery wedge pressure lasting 1 hour or longer) were divided into four groups of 11 patients each, and randomized to receive their effective ISDN dose (40-120 mg) Q 4 hours, Q 6 hours, Q 8 hours, or t.i.d. (drug given at 0, 6, 12, and 24 hours allowing 12 hours of ISDN washout interval between the third and fourth doses). All groups demonstrated a significant and comparable reduction in LV filling pressure following administration of the first ISDN dose. Early attenuation of hemodynamic response was demonstrated with frequent dosing (Q 4 hours and Q 6 hours) ISDN. Tolerance was with a Q 8-hour regimen as demonstrated by preserved hemodynamic response to each dose. The effect of each dose, however, was short-term, with return of pulmonary artery wedge pressure to baseline level at 2 to 4 hours, resulting in an intermittent effect totaling no longer than 12 hours of the 30-hour study period. The use of a t.i.d. regimen resulted in marked attenuation of response after the third dose with complete restoration of nitrate effect following a 12-hour washout period between the third and fourth doses. ISDN plasma concentration was measured in five patients in each of the Q 4- and Q 8-hour groups. In the Q 4-hour group, plasma levels were significantly higher after administration of the last dose than after the first dose (area under the curve, 242 +/- 216 versus 123 +/- 130 ng/ml, p less than 0.05), and trough levels before administration of the second and the fifth dose (15 +/- 17 and 27 +/- 27 ng/ml, respectively) were both markedly higher than the baseline value of 2 +/- 4 ng/ml. CONCLUSIONS: Our data demonstrate the development of tolerance and early attenuation of effect on left ventricular filling pressure with frequent oral dosing (Q 4 and Q 6 hours) with ISDN in patients with chronic CHF, which may be related to persistently elevated trough blood levels of ISDN. The development of tolerance can be reversed after a washout period of 12 hours and can be prevented with a Q 8-hour administration. These regimens, however, are limited by an inconsistent effect. Although long-term implications of these findings need further evaluation, the present study demonstrates the difficulty of maintaining a persistent ISDN-mediated reduction in left ventricular filling pressure in patients with chronic, moderate to severe CHF. These results suggest the need to use intermittent ISDN therapy allowing a daily nitrate washout interval and the rationale for combined vasodilator therapy in patients with CHF.

Factors leading to the reporting of 'functional' somatic symptoms by general practice attenders.

The aim of this study was to examine the prevalence of 'functional' somatic symptoms in general practice and the factors associated with reporting these symptoms. During a one month period, all attenders aged 16 years and over at a general practice near Leeds were screened for functional somatic symptoms using the Bradford somatic inventory. The general practitioner recorded the patients' personal data and diagnostic assessment. Data were analysed from 670 Europid patients who completed the Bradford somatic inventory at their first attendance during the month. Higher mean numbers of functional somatic symptoms were found in patients with psychiatric and functional syndromes than in patients with organic illness or in well patients. The symptom score on the Bradford somatic inventory was significantly related to five factors: current anxious mood, current depressed mood, sex, chronic physical illness in a parent and a history of depressive illness. Using multiple linear regression analysis, all five factors were found to be independent predictors of symptom scores on the Bradford somatic inventory. This study highlights the multifactorial aetiology of functional somatic symptoms reported by general practice attenders.

HLA-DQw3-related determinants: analysis of subunit and spatial relationships.

More polymorphism exists among DQ region gene products than is suggested by present serologic definitions of these class II molecules. DQ beta polymorphism among haplotypes carrying the DQw3 specificity is considerable. The TA10 specificity is present on one allele of at least three different DQ beta alleles that carry the DQw3 specificity. We have examined a series of monoclonal antibodies directed against different DQ beta alleles carrying the DQw3 specificity to determine subunit and spatial relationship among the epitopes detected by these antibodies. The antibodies were examined by Western blotting and for their ability to inhibit the binding of fluoresceinated antibodies on either TA10+ or TA10- DQw3 haplotypes. Our results reveal that (1) multiple DQw3-related epitopes exist; (2) several anti-DQw3-related antibodies generated against TA10- DQw3 molecules are unable to inhibit the binding of a TA10-specific antibody on a TA10+ haplotype while strongly inhibiting binding of an antibody detecting the reciprocal DQ beta polymorphism on a TA10- DQw3 haplotype; and (3) there is a strong requirement for three-dimensional conformation in the formation of the majority of the epitopes examined here. Analysis of previously published amino acid sequences for the haplotypes investigated here suggest that charge changes at amino acids 45, and 57, respectively, may have a significant effect in changing the spatial relationship between the DQw3-related epitope(s) and other polymorphic determinants on DQ beta chains.

HLA-DQ alpha polymorphisms: oligonucleotide probes characterize the contribution of first and second domains to electrophoretic variants.

Polymorphism is known to exist within the HLA-DQ alpha locus in the human major histocompatibility complex, although such polymorphism may be "silent" in standard HLA typing. However, DQ alpha polymorphism may be functionally significant, either through DQ alpha epitopes functioning directly in the immune response or by affecting tertiary conformation of Ia molecules through differential alpha/beta pairing. We have previously defined a particular DQ alpha polymorphism through reactivity with a monoclonal antibody and restriction fragment length polymorphism pattern. We now characterize this DQ alpha polymorphism through two-dimensional gel electrophoretic analysis and identify a subset of DQ alpha molecules with unique characteristics. Investigation of these allelic variants using synthetic oligonucleotide probe analysis of genomic DNA suggests a localization of the DNA region encoding the DQ alpha 5 epitope and suggests possible evolutionary mechanisms accounting for these unique patterns.

Distribution of "minor" nicks in bacteriophage T5 DNA.

Bacteriophage T5 DNA can be released from the phage particle in such a way that one end of 5 to 10% of the DNA molecules remains attached to either the phage head or tail. Under partial denaturation conditions, the DNA preferentially denatures in the vicinity of a nick so that the nicks can be located relative to the end that remains attached to the phage head or tail. Two classes of nicks were found. "Major" nicks were those found in more than 20% of the molecules and were located at the same points along the DNA molecule as reported by others. "Minor" nicks were found in 5 to 10% of the molecules and often occurred at specific locations near a "major" nick.