Immunotherapy with autologous tumour antigen-coated microbeads (large multivalent immunogen), IL-2 and GM-CSF in dogs with spontaneous B-cell lymphoma.

Cytotoxic T-lymphocyte responses to subcellular antigens are enhanced when antigens are presented on cell-sized silica microbeads called large multivalent immunogens (LMIs). LMIs prepared with tumour cell membrane fragments have induced partial remissions in humans with melanoma and renal cell carcinoma. The purpose of this phase I study was to evaluate the safety of LMIs, prepared with autologous lymphoma cell membranes, along with subcutaneous interleukin 2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) in dogs with untreated B-cell lymphoma. After lymph node excision and induction chemotherapy, five dogs were vaccinated with three weekly doses of LMI alone; five with LMI and subcutaneous IL-2 and five with LMI, IL-2 and GM-CSF. No significant toxicity was noted, treatment did not adversely affect disease-free interval and half of the dogs showed measurable delayed-type hypersensitivity reactions to intradermal challenge with LMI, suggesting specific cell-mediated immunity.

Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells.

The effects of inflammatory cytokines on naive T cells have been studied using MHC protein/peptide complexes on microspheres, thus avoiding the use of APCs whose functions may be affected by the cytokines. IL-1, but not IL-12, increased proliferation of CD4+ T cells in response to Ag and IL-2, which is consistent with effects on in vivo priming of CD4+ cells. In contrast, proliferation of CD8+ T cells to Ag and IL-2 required IL-12, and IL-12 replaced adjuvant in stimulating an in vivo response to peptide. These results support a model in which distinct inflammatory cytokines act directly on naive CD4+ and CD8+ T cells to provide a third signal, along with Ag and IL-2, to optimally activate differentiation and clonal expansion.

CD8+ memory T cells (CD44high, Ly-6C+) are more sensitive than naive cells to (CD44low, Ly-6C-) to TCR/CD8 signaling in response to antigen.

Memory CD8+ T cells from mice previously primed with alloantigen (alloAg) can respond in vitro to IL-2 and purified class I alloAg presented on microspheres, while no response can be detected using cells from naive mice. Similar results have been obtained using cells from OT-1 mice expressing a transgenic TCR that is specific for OVA(257-264) (SIINFEKL) peptide bound to H-2Kb. A population of resting memory cells (defined on the basis of low forward scatter and CD44high, Ly-6C+, CD25-, CD69-surface phenotype) that is present in the OT-1 mice exhibits a substantially higher sensitivity to Ag-stimulation than do naive cells (CD44low, Ly-6C-) expressing the same TCR. CD44high cells respond vigorously to H-2Kb immobilized on microspheres and pulsed with peptide, while CD44low cells respond weakly and only at high class I density and peptide concentration. The Ag-presenting surface only has ligands for TCR and CD8 (class I and peptide), thus ruling out the possibility that differences are due to ligand binding by other adhesion or costimulatory receptors that are expressed at high levels on the memory cells. Experiments using anti-TCR mAb as the stimulus and coimmobilized non-Ag class I as a ligand for CD8 suggest that the difference between naive and memory cells may be at the level of stimulation through the TCR. Thus, in addition to expressing increased levels of adhesion receptors that may enhance responses to Ag on APCs, memory CD8+ T cells appear to be intrinsically more sensitive than naive cells to stimulation through the TCR/CD8 complex.

Molecular analysis of the immune response to human cytomegalovirus glycoprotein B (gB). II. Low gB-specific T and B cell responses are associated with expression of certain HLA-DR alleles.

Human cytomegalovirus (HCMV) is a common cause of congenital infection leading to birth defects and a leading cause of serious illness in patients with immunodeficiencies. Studies in this laboratory have focused on a molecular analysis of the immune response to glycoprotein B (gB) of HCMV. This protein has been shown to elicit B cell, helper T cell (Th), and cytotoxic T cell responses, suggesting that it may be useful as a subunit HCMV vaccine. However, previous studies showed that although peripheral blood mononuclear cells (PBMC) from all HCMV-seropositive donors proliferate in response to stimulation with whole HCMV, not all donors respond to purified recombinant gB. In the present study, PBMC from HCMV-seropositive donors homozygous for HLA-DR were tested for proliferative responses to whole HCMV and to purified gB expressed in vaccinia virus. PBMC from all donors proliferated in response to HCMV, but those from multiple donors expressing the HLA-DR3Dw3 and -DR4Dw4 specificities, and single donors expressing the -DR15Dw2, -DR13Dw19 and -DR14Dw9 specificities, failed to respond to gB. These results suggested a possible HLA-DR association with low proliferative responses to gB. In further studies, PBMC from donors expressing both putative gB-high responder and low responder HLA-DR alleles were stimulated multiple times with gB to generate gB-specific T cell lines. These cells were then tested for proliferative responses to gB presented by irradiated PBMC sharing only one DR allele with the responder cells. Cells from the gB-specific lines proliferated only when antigen was presented in the context of a responder DR allele but not when presented in the context of a low responder DR allele. Analysis of immune sera revealed that those from donors with PBMC proliferative responses always contained antibodies reactive with B cell epitopes on both the N-terminal gp93 and C-terminal gp55 portions of gB. In contrast, many of the sera from donors with low gB-specific proliferative responses had gp55-specific antibodies but lacked antibodies to gp93. These results suggest that immunogenetic differences in Th responsiveness to gB may lead to lack of antigen-specific help for antibody responses to gp93 in some cases. The prevalence of these low responder HLA alleles in the population, and the central importance of the T cell response to the generation of antibodies suggest that native gB alone may not be an attractive candidate for an HCMV subunit vaccine.

DQ beta sequences in HLA-DR4 haplotypes.

A cDNA clone encoding the entire mature DQ beta chain was isolated and sequenced from the DR4-Dw14 homozygous cell line, LS40. The sequence was compared with published DQ beta sequences from cells expressing DR4-DQw3, and found to be identical. The lack of DQ beta sequence polymorphism within this serotype (to date, DQ beta sequences have been derived from five cell lines comprising three Dw subtypes) adds to the data that suggest a recent evolutionary divergence of Dw subtypes within DR4-DQw3.

Evolutionary and genetic implications of sequence variation in two nonallelic HLA-DR beta-chain cDNA sequences.

Most HLA haplotypes carry two expressed DR beta-chain genes; in the DR4 haplotype, the polymorphic locus has been called DR beta 1 and the apparently nonpolymorphic locus has been called DR beta 2. We have isolated nearly full-length DR beta-chain cDNA clones representing each of these two loci from a cell line homozygous for DR4 and Dw4. The clones have been sequenced and the sequences compared with published DR beta cDNA sequences derived from other haplotypes. A comparison of our sequences with other published cDNA sequences did not allow assignment of these other sequences to either the beta 1 or beta 2 locus. Comparison of our DR4 beta 1 sequence with DR beta 1 sequences isolated from other DR4-positive cells suggests that the alleles of DR4 beta 1 may have recently diverged from a common ancestor. The apparent lack of polymorphism of DR beta 2 may in part be a reflection of this recent divergence.

Sequence polymorphism of HLA DR beta 1 alleles relating to T-cell-recognized determinants.

HLA class II molecules are a highly polymorphic family of dimeric cell-surface proteins primarily involved in regulating T-cell responses to extrinsic antigens. To define regions of class II molecules involved in T-cell recognition, we have now compared sequences of three HLA DR beta cDNA clones obtained from cells that all express the same serologically defined determinants but differ in terms of T-cell-recognized specificities. The comparisons indicate that very few (one to four) nucleotides differ between what are almost certainly alleles of the DR beta 1 locus. All differences were in the first domain of the molecule and all localized to a region from amino acids 71-86. Because all differences were found only in this region of the molecule, and because DR alpha-chains seem to be relatively non-polymorphic, these positions in the DR beta-chain must have a major role in influencing T-cell recognition of the DR molecule.

A helper factor needed for the generation of mouse cytolytic T lymphocytes is made by tumor cell lines, cloned T cells, and spleen cells exposed to a variety of stimuli.

A helper factor termed cytolytic T lymphocyte helper factor (CHF) that is needed for the generation of allospecific mouse cytolytic T lymphocytes (CTL) in vitro was produced by mouse spleen cells 3 to 4 days after the time when interleukin 2 (IL 2) had reached its maximal production. These kinetics were observed by stimulation of immune spleen cells with allogeneic tumor or spleen cells, with Sendai or influenza viral peptides, with virus infected cells, or with concanavalin A (Con A). CHF produced by rat spleen cells was able to help in the generation of mouse CTL, indicating that this cytokine was not restricted genetically. CHF could also be made by WEHI-3 and EL4 cell lines, as well as cloned cytolytic and helper T cells. The production of CHF by WEHI-3 cells argues that CHF is not IL 2. In addition, if CHF was not present early in the in vitro stimulation no CTL were generated, suggesting that CHF participated in the activation of CTL precursors. The addition of IL 2-containing conditioned medium to the CHF assay resulted in no substantial CTL generation, although significant cellular proliferation was observed. In contrast, CHF-containing conditioned medium allowed the generation of CTL in the absence of the same level of proliferation.

Interleukin 3 augments the murine primary cytolytic T lymphocyte response to allogeneic tumor cells.

The primary anti-H-2k allospecific cytolytic T lymphocyte (CTL) response by BALB/c (H-2d) spleen cells in vitro to x-irradiated RDM4 (H-2k) tumor cells is weak. This response has been shown to be augmented by CTL helper factor (CHF), a factor present in supernatants of spleen cells cultured with Sendai virus (SC-CM). Conditioned medium from WEHI-3 cells (WEHI-CM) also contains activity that augments the BALB/c anti- RDM4 CTL response. Attempts to separate the CHF activity from interleukin 3 (IL 3), also present in WEHI-CM, were unsuccessful. Purified IL 3 was then tested, and was found to increase the BALB/c anti- RDM4 CTL response by five- to 10-fold. IL 3 is apparently the only material in WEHI-CM that is active in this assay. The response is apparently a classical CTL response because: 1) the effector cells are sensitive to monoclonal anti-Thy-1.2 antibody plus C; 2) the response is dependent on antigen stimulation, and it peaks on day 5 or 6 of culture; and 3) the effector cells are specific for H-2k targets. IL 3 must be added very early during the in vitro culture period for maximal augmentation of the response, consistent with possible action of IL 3 as a differentiation factor.