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We report that I-Ab-restricted T cell clones, elicited by influenza infection of C57BL/10 mice and specific for the hemagglutinin peptide HA1 186-205, express class II. They respond to peptide stimulation by IL release (IL-3 or IFN-gamma) without a requirement for APC but do not proliferate. Moreover, surface expression of class II requires de novo synthesis in the presence of the stimulatory peptide and is inhibited by coculture with TCR-specific Ab, or brefeldin A or cycloheximide. Clonotypic specificity of peptide induction was confirmed by failure of other allele specific peptides to enhance class II expression. Addition of the viral peptide to T cells induced homotypic adhesion, which provides a physical basis for stabilization of class II-peptide complexes at the cell surface. Extinction of class II expression was evident in the corresponding T cell hybridomas, which might account for the failure to report class II expression by murine T cells. Control studies indicated that class II was not passively acquired from APC by demonstrating 1) failure of processed Ag to induce class II expression, 2) allo-class II (Ak) was not acquired by coculture with peptide and semisyngeneic (H-2 b/k) APC, 3) absence of class II expression by a NP peptide-specific Th2 clone under identical culture conditions, and most significantly, 4) reverse-transcriptase PCR amplification and surface expression of class II using highly purified preparations of FACS-selected CD4+ class II- cells cocultured with the stimulatory peptide.

A functional analysis was undertaken of the effects of mutating single amino acid residues in the alpha chain of the I-Ak molecule (to alanine; residues 50-79) on the ability of I-Ak transfectants to process and present influenza haemagglutinin to CD4+ T cell clones specific for two major antigenic sites of the HA1 subunit. In each instance, T cells were insensitive to a majority of substitutions in Ak with the exception of a few critical residues that differed for individual T cell clones. But more significantly, the failure of T cell clones to respond to mutant influenza viruses, containing drift substitutions within a T cell recognition site, in association with wild type I-Ak, could be reversed by single substitutions in Ak alpha. A T cell clone specific for HA1 120-139 failed to respond to a laboratory mutant virus (HA1 135 Gly----Arg) whereas optimal responses were observed with a mutant Ak transfectant (Ak alpha 56 Arg----Ala). Similarly, mutant transfectant 62 (Ak alpha 62 Gly----Ala) was able to present a natural variant virus A/TEX/77 to a T cell clone specific for HA1 48-67. We propose that Ak alpha 56 and Ak alpha 62 increase the affinity of association of mutant HA1 peptides for class II and therefore confer T cell recognition of variant viruses.

A panel of H-2k class II-restricted T helper (Th) clones were established from individual CBA mice intranasally infected with A/X31 influenza virus. In a study of their fine specificity, it was revealed that recognition of hemagglutinin (HA) by certain Th clones was dependent on conformational features of the molecule. Five HA-specific Th clones failed to recognize X31 mutant viruses R19 and R20 each with a His to Arg substitution at position 17 of HA1. This amino acid substitution affects the conformational stability of the molecule. Each of the 5 clones responded to purified HA but failed to recognize tryptic fragments of HA consisting of HA1 residues 28-328 (tops) and the remainder of the virus including residues 1-27 of HA1 (aggregate). A further 20 HA-specific clones responded to R19 and R20 mutants and to HA1 tops. The R19 and R20 negative clones also showed diminished proliferative responses to pH 5-treated X31. Low pH treatment results in an irreversIble conformational change which affects the integrity of the globular head region of the HA trimer. In addition, four of the five clones failed to recognize variant virus Eng-72 which has Arg to Gly substitution at position 208 in the interface antibody-binding region. This region is antigenically and structurally altered in tops and pH 5-treated X31. The results suggest that recognition of HA by certain Th clones is dependent on the three-dimensional structure of the molecule and that Th cells may recognize conformational determinants in addition to primary structure.

In a recent study, we reported extensive diversity in the Iak-restricted T cell repertoire for the hemagglutinin molecule (HA) of influenza A viruses (H3 subtype). Synthetic peptides identified six nonoverlapping epitopes on the HA1 subunit, and CD4+ T cell clones, specific for these regions, discriminated between natural variant viruses that had accumulated amino acid substitutions during antigenic drift. Here, we demonstrate similar specificity and diversity for the Iad haplotype and have identified multiple T cell epitopes within the sequences HA1 56-76, 71-91, 81-97, 177-199, 186-205, and 206-227. These also include recognition sites for neutralizing antibodies and correlations can be made between antigenic drift substitutions in H3 subtype viruses and the specificity of individual CD4+ clones for mutant HA. Moreover, these peptides appear not to exhibit structural homology and fail to compete for Ag presentation, indicating heterogeneity in peptide-Ia interaction. To explain the observation that CD4+ T cells, from two major haplotypes, recognize antibody binding regions of the HA molecule, we propose that surface Ig receptors of the Ag-specific B memory cell exert a direct effect on the processing of HA peptides and subsequent selection of the class II-restricted T cell memory repertoire after natural infection.

A fine specificity analysis of influenza hemagglutinin-specific IAk-restricted T cell clones using natural virus variants of the H3N2 subtype, monoclonal antibody-selected variants and a synthetic peptide corresponding to a variable region of the HA1 polypeptide has provided insight on the structural basis for T cell recognition. A glycine to arginine substitution at HA1 135 abrogates recognition by a panel of T cell clones which, according to their reactivity for natural virus variants, have different antigenic specificities: three clones recognize a synthetic peptide (HA1 residues 118-138) but fail to recognize the monoclonal antibody-selected mutant (Gly135/Arg). There is no correlation, however, between differences in T cell specificity for the natural virus variants and HA1 amino acid sequences in this region. Two further clones have a reduced proliferative response to mutant recognize a completely different spectrum of natural variants, and only one of these clones recognizes the synthetic peptide. We speculate that influenza hemagglutinin employs a common strategy during antigenic drift to evade antibody recognition and effective processing/presentation to subtype-specific T cell clones.

We have previously demonstrated diversity in the specificity of murine, H-2k class II-restricted, T cell clones for the hemagglutinin (HA) molecule of H3N2 influenza viruses and have mapped two T cell determinants, defined by synthetic peptides, to residues 48-68 and 118-138 of HA1. In this study we examine the nature of the determinant recognized by six distinct P48-68-specific T cell clones by using a panel of truncated synthetic peptides and substituted peptide analogs. From the peptides tested, the shortest recognized were the decapeptides, P53-62 and P54-63, which suggests that the determinant was formed from the 9 amino acids within the sequence 54-62. Asn54 was critical for recognition since P49-68 (54S) was not recognized by the T cell clones. Furthermore this peptide analog was capable of competing with P48-68 for Ag presentation, thereby suggesting that residue 54 is not involved in Ia interaction and may therefore be important for TCR interaction. Residue substitutions at position 63 also affected T cell recognition, but in a more heterogeneous fashion. Peptide analogs or mutant viruses with a single amino acid substitution at position 63 (Asp to Asn or Tyr) reduced the responses of the T cell clones to variable extents, suggesting that Asp63 may form part of overlapping T cell determinants. However since the truncated peptide P53-62 was weakly recognized, then Asp63 may not form part of the TCR or Ia interaction site, but may affect recognition through a steric or charge effect when substituted by Asn or Tyr. Ag competition experiments with the two unrelated HA peptides, P48-68 and P118-138, recognized by distinct T cell clones in the context of the same restriction element (I-Ak), showed that the peptides did not compete for Ag presentation to the relevant T cell clones, whereas a structural analog of P48-68 was a potent inhibitor. This finding is discussed in relation to the nature of the binding site for peptide Ag on the class II molecule.

The humoral response to hapten-protein conjugates is an invaluable model for dissecting the cellular elements of lymphocyte cooperation, and the Mitchison secondary adoptive transfer system provides convincing evidence of cooperation between hapten-specific B cells and carrier-specific T cells in the production of anti-hapten antibody. Recently, attention has focused on the role of suppressor T cells in the regulation of antibody production. Several workers have shown that carrier-priming may, in some instances, suppress a subsequent hapten antibody response, both in vivo and in vitro. This effect is attributed to a suppressor T-cell population, generated during the initial phase of the immune response. Gershon and co-workers have postulated that such suppressor T cells function in a feedback regulatory loop to limit the duration of an immune response. We have examined the suppressive effect of carrier immunization in a secondary anti-hapten response in vivo and demonstrate a cyclic change in susceptibility of memory B cells to T-help and suppression. Such variation presents a severe restriction to any model of feedback control by suppressor T cells.

Conditions are described for the induction of autoimmunity to Thy-1 and a large panel of monoclonal CBA and AKR autoantibodies has been characterized. These reveal a hitherto unrecognized complexity at the Thy-1 locus and evidence for intragenic control. Epitopes recognized by the autoantibodies differed in species and tissue distribution from alloantigenic determinants but their specificity was confirmed by transfection studies with the cloned Thy-1b gene. The quantitative and qualitative differences in epitope expression between Thy-1a and Thy-1b alleles were inherited as single Mendelian traits and in the absence of recombinants, suggesting translational control.

NIM-M8 is a monoclonal IgM antibody, specific for the LWab antigen as shown by its reaction with red cells of all donors except those lacking LWa, LWb and LWab. Indirect immunofluorescent staining and cell sorter analyses have shown that LWab is present on a subpopulation of human lymphocytes. Cell fractionation studies indicate that subsets of both B and T cells express LWab and it may, therefore, provide a further marker for heterogeneity in these lymphocyte populations.

Lymphoid tissues of mice infected with murine leukemia virus (Friend) (MuLV-F) were examined for the presence of cellular markers of MuLV-F infection. The Friend virus-associated cell membrane antigen (FVMA) and the virus group-specific antigen (GSA) were detectable on cells from the spleen and, to a lesser degree, on cells from the bone-marrow. In contrast, neither FVMA nor GSA was found in cells from the thymus. Alterations in the B-cell and T-cell spleen populations of MuLV-F-infected mice were then studied. The proportion of Ig-positive cells declined from the initial 45% (in non-infected controls) to about 10% after 2 weeks of infection. A similar decline of theta-positive cells was noted. However, complement-bearing cells (EAC rosettes) declined even more rapidly and became undetectable in the second week after infection. The treatment of spleen cells from MuLV-F-infected mice with anti-FVMA serum plus complement in vitro reduced the number of detectable Ig-positive cells, specifically, whereas the number of theta-positive cells remained unchanged. Furthermore, B and T cells from spleens of infected mice were separated on an affinity column with anti-Ig antibody-coated beads. The initial cell suspension contained about 45% FVMA-positive cells, about 40% Ig-positive cells and about 40% theta-positive cells. Ig+ cells were retained on the column. The theta-positive cell fraction was collected in the eluate and contained very few FVMA-positive cells with some "null" cells. Most of the FVMA-positive cells were retained on the column, which strongly suggested that they were B cells. These results confirm the previous experiments which showed the selective infections of purified splenic B cells by MuLV-F in cultures.

H-2k-restricted T cell clones derived from CBA mice infected with X31 (H3N2) influenza virus, were shown to recognize distinct, nonoverlapping sequences within the HA1 subunit of the viral hemagglutinin (HA) using synthetic peptides. Three I-Ak-restricted T cell sequences were identified within HA1 68-83, 120-139, and 269-288, and two recognition sites presented in the context of the I-Ek molecule were mapped to HA1 sequences 226-245 and 246-265. T cell clones specific for these regions of HA1 demonstrated varying abilities to differentiate between natural variant viruses that had accumulated substitutions within their HA molecules as a result of antigenic drift. Clones that recognized sequences HA1 226-245 and HA1 246-265 failed to discriminate between natural variants and focused on conserved sequences within these epitopes. A majority of T cell clones were sensitive to amino acid substitutions that have featured in antigenic drift occurring within three major antigenic sites of the HA1 subunit; substitutions at HA1 residues 78 (V)/83(K) and 275(D)/278(I) within the HA1 subunit of mutant viruses correlated with a 75% reduction in the proliferative response for T cell clones specific for sequences HA1 68-83 and HA1 269-288, respectively. Furthermore, a clone that recognized HA1 120-139 was nonresponsive to a mutant virus HK/71, implicating amino acids at HA1 position 129(G) and/or 132(Q) within this sequence as crucial for recognition. Our data, together with the previous finding that sequence HA1 53-63 is also a major I-Ak-restricted T cell recognition site, demonstrate a level of diversity in the T cell recognition of influenza HA, within a single mouse haplotype hitherto unrecognized, and imply that the T cell repertoire diversity against foreign antigens may be greater than previously assumed. Furthermore, the frequency at which HA-specific T cells have been identified that focus on amino acids within the HA1 subunit of HA also featuring in antigenic drift, suggests that a failure of MHC class II-restricted T cells to recognize specific epitopes within mutant HA molecules may contribute significantly to the capacity of variant influenza viruses to evade immune recognition.

A new Thy-1 alloantigenic specificity is defined in the mouse. Antigen expression is restricted to 'mature' and memory T cells, with either helper or killer activity.

An extensive analysis of the class II (I-Ad)-restricted T cell repertoire for influenza hemagglutinin (HA) of the H3 subtype, elicited by natural infection, has shown that majority of CD4+ memory T cell clones focus on antibody-binding regions of HA, sites B and E, and are sensitive to the residue substitutions that have occurred in these regions during antigenic drift. The proliferative responses of CD4+ clones to synthetic peptides have identified T cell epitopes within site B, HA1 177-199 and HA1 182-199, and site E. HA1 56-76. The recognition specificity of T cell clones for antibody-selected mutant viruses, with single amino acid substitutions within these recognition sites identified residues 63, 189, 193 and 198 as being important for T cell recognition and thus established that BALB/c, CD4+ T cell clones were sensitive to the same substitutions known to abrogate BALB/c antibody recognition of the native HA. Our findings indicate extensive commonality of the B cell and T cell repertoires for HA, which may be relevant to an understanding of the immune pressures for antigenic drift, and, moreover, suggest that the antigen-specific B memory cell may be instrumental in selection of the peripheral T cell repertoire.

Evidence is presented that T helper cells change their Lyt-1,2 phenotype, from Lyt-1+2- to Lyt-1-2+, during a secondary response. In a secondary adoptive transfer the Lyt phenotype of carrier-primed T cells was established before and after secondary challenge using monoclonal antibodies and both positive and negative selection in the fluorescence-activated cell sorter. In a carrier-primed donor, the T helper cells were Lyt-1+2- whereas 3 days after boosting the majority were Lyt-2+, and resistant to treatment with anti-Lyt-1 and complement. Parking experiments in lethally irradiated recipients confirmed that Lyt-2+ T helper cells were generated from Lyt-2- precursors. In a primary response, both Lyt-2- and Lyt-2+ T helper cells were present 4 to 20 days after immunization. Therefore T helper cells do not exhibit invariant expression of the Lyt-1,2 alloantigens.