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Immortalized T cells such as T cell hybridomas, transfectomas, and transductants are useful tools to study tri-molecular complexes consisting of peptide, MHC, and T cell receptor (TCR) molecules. These cells have been utilized for antigen discovery studies for decades due to simplicity and rapidness of growing cells. However, responsiveness to antigen stimulation is typically less sensitive compared to primary T cells, resulting in occasional false negative outcomes especially for TCRs having low affinity to a peptide-MHC complex (pMHC). To overcome this obstacle, we genetically engineered T cell hybridomas to express additional CD3 molecules as well as CD4 with two amino acid substitutions that increase affinity to MHC class II molecules. The manipulated T cell hybridomas that were further transduced with retroviral vectors encoding TCRs of interest responded to cognate antigens more robustly than non-manipulated cells without evoking non-antigen specific reactivity. Of importance, the manipulation with CD3 and mutated human CD4 expression was effective in increasing responsiveness of T cell hybridomas to a wide variety of TCR, peptide, and MHC combinations across class II genetic loci (i.e. HLA-DR, HLA-DQ, HLA-DP, and murine H2-IA) and species (i.e. both humans and mice), and thus will be useful to identify antigen specificity of T cells., (Copyright © 2018. Published by Elsevier B.V.)

B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.

T cells bearing receptors made up of α and β chains (TCRs) usually react with peptides bound to major histocompatibility complex proteins (MHC). This bias could be imposed by positive selection, the phenomenon that selects thymocytes to mature into T cells only if the TCRs they bear react with low but appreciable affinity with MHC + peptide combinations in the thymus cortex. However, it is also possible that the polypeptides of TCRs themselves do not have random specificities but rather are biased toward reaction with MHC. Evolution would therefore have selected for a collection of TCR variable elements that are prone to react with MHC. If this were to be so, positive selection would act on thymocytes bearing a pre biased collection of TCRs to pick out those that react to some extent, but not too well, with self MHC + self-peptides. A problem with studies of this evolutionary idea is the fact that there are many TCR variable elements and that these differ considerably in the amino acids with which they contact MHC. However, recent experiments by our group and others suggest that one group of TCR variable elements, those related to the mouse Vβ8 family, has amino acids in their CDR2 regions that consistently bind a particular site on an MHC α-helix. Other groups of variable elements may use different patterns of amino acids to achieve the same goal. Mutation of these amino acids reduces the ability of T cells and thymocytes to react with MHC. These amino acids are present in the variable regions of distantly related species such as sharks and human. Overall the data indicate that TCR elements have indeed been selected by evolution to react with MHC proteins. Many mysteries about TCRs remain to be solved, including the nature of auto-recognition, the basis of MHC allele specificity, and the very nature and complexity of TCRs on mature T cells., (© 2012 John Wiley & Sons A/S.)

Major histocompatibility complex class I (MHCI) and MHCII proteins differ in structure and sequence. To understand how T cell receptors (TCRs) can use the same set of variable regions to bind both proteins, we have presented a comparison of a single TCR bound to both MHCI and MHCII ligands. The TCR adopts similar orientations on both ligands with TCR amino acids thought to be evolutionarily conserved for MHC interaction occupying similar positions on the MHCI and MHCII helices. However, the TCR antigen-binding loops use different conformations when interacting with each ligand. Most importantly, we observed alternate TCR core conformations. When bound to MHCI, but not MHCII, Vα disengages from the Jα β strand, switching Vα's position relative to Vβ. In several other structures, either Vα or Vβ undergoes this same modification. Thus, both TCR V-domains can switch among alternate conformations, perhaps extending their ability to react with different MHC-peptide ligands., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Methods to induce antigen-specific immune responses in mice using insect cells infected with recombinant baculoviruses are described in this unit. Although this vaccine strategy has been used to generate both antibody and T cell responses, it has been more thoroughly characterized for the peptide-specific cytotoxic T cell responses. Nonspecific responses to the vaccine vehicle are controlled for by vaccinating with insect cells infected with baculoviruses encoding irrelevant antigens or no antigen. The baculovirus-infected insect cells alone are an effective immune adjuvant to elicit antigen-specific T cells. Overall, immune responses generated using this approach are similar to those generated by more conventional vaccine strategies., (Copyright 2009 by John Wiley & Sons, Inc.)

T cells compete in the response to antigen in vivo and this competition may drive the affinity maturation of a secondary T cell response. Here we show that high-affinity T cells out-competed lower affinity T cells during a response to antigenic challenge in vivo. Although competition between T cells specific for different peptide-major histocompatibility complexes (MHC) occurred, it was less efficient than competition between T cells of the same peptide-MHC specificity. In addition, high-affinity T cells efficiently induced antigen loss from the surface of antigen-presenting cells. Thus T cells that responded to the same peptide-MHC competed with each other by lowering the amount of ligand with which the cells could react. As a result, the activation of high-affinity cells was favored. This provides a mechanism for the affinity maturation of a secondary T cell response.

In order to track hematopoetic cells of all lineages unambiguously at all stages of development, we have developed C57BL/6 mice that express a transgene coding for green fluorescent protein (GFP) under control of the human ubiquitin C promoter. These mice, called UBI-GFP/BL6, express GFP in all tissues examined, with high levels of GFP expression observed in hematopoetic cells. UBI-GFP/BL6 mice are unique in that B cells, T cells, and dendritic cells have distinct levels of GFP fluorescence. In cell transfer experiments, leukocytes from UBI-GFP/BL6 mice are readily identified by FACS or fluorescence microscopy. We demonstrate that transplanted UBI-GFP/BL6 dendritic cells are easily identified in secondary lymphoid tissues. Direct interactions between individual dendritic cells and multiple naïve CD8+ T cells are observed in lymph nodes within 12 h of cell transfer and require loading of the dendritic cells with the appropriate peptide antigen. Dendritic cells undergo specific morphologic changes following interactions with antigen-specific T cells.

Superantigens combine with MHC class-II molecules to form the ligands that stimulate T cells via the V beta element of the T-cell receptor. Two groups of superantigens have been described so far: first, endogenous murine products that include the Mls determinants, and second, bacterial products such as the Staphylococcal enterotoxins. Here, we review studies that address the interactions between the foreign superantigens and MHC class-II molecules, the mechanism of T-cell stimulation, and the role that tolerance to self-superantigens plays in shaping the T-cell repertoire. We speculate on the possible evolutionary significance of superantigens.

In the presence of the soluble polypeptide antigens ovalbumin and keyhole limpet hemocyanin, purified mouse spleen dendritic cells induce the secretion of IL-2 by antigen-specific T cell hybridomas. This response is H-2 restricted and can be specifically inhibited by monoclonal anti-I-A. These data indicate that dendritic cells can present soluble antigen to H-2-compatible T cells.

To define the antigen-presenting capacity of alveolar macrophages (AM phi), their ability to activate antigen-specific T cells was determined. It was found that AM phi s are at least as potent as spleen cells at presenting alloantigen or soluble protein antigen plus I-region determinant to cloned T-cell hybridomas. Activation of those hybridomas, shown by interleukin 2 (IL-2) secretion, further indicated the presence on AM phi s of functional products of the I-A and I-E subregions. In addition, it was found that AM phi s are at least as potent as spleen cells and peritoneal macrophages at inducing, in a restricted fashion, specific proliferation of primed, propagated lymph node T cells. AM phi s are thus potent accessory cells in vitro, whose in vivo significance in this capacity remains to be defined.

The T1 subpopulation of peripheral T cells was defined in mice by its short half life, insensitivity to anti-thymocyte sera (ATS) in vivo, and slow kinetics of response to antigen. The T2 subpopulation was defined by its long life time, elimination by ATS in vivo, and rapid response to antigen. Mice containing only T1-type T cells were constructed by adult thymectomy (ATx) followed immediately by the elimination of T2 cells by ATS treatment. Immunization of these mice with SRBC led to the production of memory helper cells in the T2 subpopulation. This process depended on the presence of T1 cells and for the most part required SRBC immunization, although a few SRBC-specific T2 cells reappeared in the mice in the absence of antigen. We conclude that T1 cells can give rise to T2 cells in an antigen-driven step and that the two populations correspond to virgin and memory T cells, respectively.

We have investigated the ability of an Ia-, nonantigen-presenting macrophage tumor cell line, P388D, (H-2d), to present antigen to T cell hybridomas after incubation in a lymphokine-containing preparation. P388D, cells were incubated in microtiter wells with various concentrations of Con A-stimulated spleen cell supernatants. Antigen-specific stimulation of H-2d-restricted, KLH-specific T cell hybridomas was observed by P388D1 incubated with SUP.P388D1 cells incubated for 3 days in medium or control SUP did not present antigen. In addition, no stimulation of T hybridomas was seen by P388D1 in the inhibited by the appropriate monoclonal anti-Ia reagents. These results demonstrate that a macrophage tumor cell line can be induced to present antigen and provides for large numbers of readily available, homogeneous macrophages for studying the cellular biochemical requirements for antigen processing and presentation.

Further evidence is presented for two types of helper T cells in the mouse specific for the protein antigen, keyhole limpet hemocyanin (KLH). The first cell helps B cells respond to the trinitrophenyl hapten (TNP) coupled to KLH, is primed by relatively high doses of antigen in vivo, and yet the effector cell is stimulated by very low doses of antigen in vitro. The second cell helps B cells respond to a non-cross-reacting antigen, sheep red blood cells, presumably via production of a nonspecific factor. This cell is primed by relatively low doses of antigen in vivo, but the effector cell requires relatively high doses of antigen in vitro. Thus, the two T cell types are differently sensitive to antigen dose, both in priming and challenge. The properties of T cells responding to KLH by proliferation in vitro were also studied. These cells showed the same antigen-sensitivity in vitro, as cells producing nonspecific B cell-stimulating factors.

A 17-amino acid tryptic peptide of chicken ovalbumin, designated P323-339, that substituted for processed antigen when presented by glutaraldehyde prefixed accessory cells to specific I-restricted T hybridomas was characterized. The peptide antigen could not be demonstrated to have any specific or stable interactions with accessory cell Ia antigens by either direct binding or functional assays for inhibition of specific T cell activation. In addition, the T cell receptor for I-restricted antigen had no affinity for free antigen alone. A rabbit antibody specific for the antigenic peptide inhibited presentation when introduced before but not after binding of the peptide to accessory cells. These results extend our earlier finding that accessory cell-mediated processing of chicken ovalbumin can be completely explained by the fragmentation of the native molecule into smaller m.w. peptides, and suggests that if an antigen/Ia complex is important in T cell activation, it forms significantly only in the presence of the T cell receptor for I-restricted antigen.

Antigen binding was used as a probe in the definition of functional B cell heterogeneity. Unprimed, anti-Thy 1 and complement-treated spleen cells were stained with fluorescent trinitrophenylated bovine serum albumin (FL-TNP-BSA). These cells were sorted, fluorescence negative from fluorescence positive, by using a multiparameter cell sorter and assayed for precursor frequency in antibody responses to TNP by limiting dilution analysis. The cells that bound FL-TNP-BSA were demonstrated to be enriched for antibody-forming precursors to the antigens TNP-lipopolysaccharide (LPS), TNP-sheep red blood cells (SRBC), and TNP- or DNP-Ficoll, whereas the fluorescence negative cells were depleted for these responses. B cells that bound FL-TNP-BSA were then sorted into populations that bound a moderate or high amount of FL-TNP-BSA. The B cells responsive to TNP-LPS and TNP-SRBC were present in both the moderate and high binding populations. In contrast, the B cells responsive to TNP- or DNP-Ficoll were present only in the cells that bound a moderate amount of FL-TNP-BSA. These experiments suggest that there is a population of B cells in adult mouse spleen that binds large amounts of antigen, and that can respond to antigen carried on LPS or SRBC but not carried on Ficoll.

The specificity of antigen recognition by thymus-derived helper cells (T cells) and antibody was examined in mice, heterologous erythrocyte antigens from sheep (SRBC), goat (GRBC), burro (BRBC), chicken (CRBC), and toad (TRBC) being used. Antibody specificity was tested by a number of functional assays: hemagglutination, hemolysis, and immune suppression. The specificity of T cells was determined by titrating their ability to help the in vitro antitrinitrophenol (TNP) responses of mouse spleen cultures immunized with the hapten coupled to the various test erythrocytes as carrier. Anti-SRBC antibody cross-reacted with GRBC, but not with BRBC, CRBC, or TRBC. In contrast, SRBC-primed helper T cells cross-reacted with both GRBC and BRBC, but not with CRBC or TRBC, indicating a difference in the specificity of antigen recognition between the cellular and the humoral immune responses.