Contribution of antigen processing to the recognition of a synthetic peptide antigen by specific T cell hybridomas

Most antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the alpha-helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I-Ad. Hybridoma A.1.1 responds to EYK(EYA)4 as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5 sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the alpha-helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC molecules.     

Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

Single amino acid substitution alters T cell determinant selection during antigen processing of Staphylococcus aureus nuclease.

The effect of amino acid residues outside of T cell determinant regions of Staphylococcus aureus nuclease (Nase) on the activation of T cell hybridomas has been investigated. T cell hybridomas derived from BALB/c mice immunized with Nase were screened against a nested set of overlapping synthetic peptides spanning the entire Nase molecule. Five regions of Nase, encompassing residues 1 to 20, 21 to 40, 61 to 80, 101 to 120, and 112 to 130, were found to be the T cell determinants. Region 61 to 80 is the immunodominant site. Mutants of Nase with a single amino acid substitution outside the defined T cell determinants were tested for their ability to stimulate the T cell hybridomas. The substitution of arginine for glutamic acid at residue 43 markedly reduces the antigenic potency of the protein for I-Ed restricted T cell hybridomas, which recognize Nase peptides comprised of residues 21 to 40 (p21-40) or 112 to 130 (p112-130). In contrast, the stimulatory capacity of this mutant for I-Ad restricted T cell hybridomas remains unchanged. Our results suggest that selective regulation of an immune response may be achieved by appropriately mutagenizing protein Ag.     

Placenta as a selective barrier to cellular traffic

Transplacental passage of cells from mother to fetus during murine pregnancy was examined by using glucose phosphate isomerase (GPI) or fluorescein tagging as markers. Female mice of strains (BALB/cCr X C3H/HeJ)/F1 or (A/J X C57BL/6J)F1 (both Gpi-1a/b) were mated to the corresponding Gpi-la males (BALB/cCr or A/J, respectively). Cells of the liver, blood, and/or spleen in the offspring were typed at days 15, 16, 17, or 18 of gestation, the day of delivery, or 1 day postpartum. Only two of 172 Gpi-1a/a mice obtained from these matings showed evidence of maternal cell trafficking. Sensitivity of the assay was 1% Gpi-1a/a population. Fluorescein-labeled BALB/cCr peripheral red blood cells (RBC) or white blood cells (WBC) were injected i.v. into syngeneically mated BALB/cCr mothers on day 18. After 24 hr, the blood or liver of the neonates was formalin-fixed and examined in the fluorescence-activated cell sorter (FACS). Some RBC crossed the placenta, but WBC were usually not detected in fetal liver in significant numbers. This technique was very sensitive, and we estimated that no more than 225 WBC could enter the fetus via this route. Thus, we conclude that passage of significant numbers of maternal WBC into the fetus is rare, and perhaps this passage is related to a placental abnormality.     

Diverse fine specificity and receptor repertoire of T cells reactive to the major VP1 epitope (VP1230-250) of Theiler's virus: V beta restriction correlates with T cell recognition of the c-terminal residue.

Theiler's murine encephalomyelitis virus induces chronic demyelinating disease in genetically susceptible mice. The histopathological and immunological manifestation of the disease closely resembles human multiple sclerosis, and, thus, this system serves as a relevant infectious model for multiple sclerosis. The pathogenesis of demyelination appears to be mediated by the inflammatory Th1 response to viral epitopes. In this study, T cell repertoire reactive to the major pathogenic VP1 epitope region (VP1233-250) was analyzed. Diverse minimal T cell epitopes were found within this region, and yet close to 50% of the VP1-reactive T cell hybridomas used V beta 16. The majority (8/11) of the V beta 16+ T cells required the C-terminal amino acid residue on the epitope, valine at position 245, and every T cell hybridoma recognizing this C-terminal residue expressed V beta 16. However, the complementarity-determining region 3 sequences of the V beta 16+ T cell hybridomas were markedly heterogeneous. In contrast, such a restriction was not found in the V alpha usage. Only restricted residues at this C-terminal position allowed for T cell activation, suggesting that V beta 16 may recognize this terminal residue. Further functional competition analysis for TCR and MHC class II-contacting residues indicate that many different residues can be involved in the class II and/or TCR binding depending on the T cell population, even if they recognize the identical minimal epitope region. Thus, recognition of the C-terminal residue of a minimal T cell epitope may associate with a particular V beta (but not V alpha) subfamily-specific sequence, resulting in a highly restricted V beta repertoire of the epitope-specific T cells.     

Immunization with Leishmania-specific T cell not B cell lines or hybridomas can modulate the response of susceptible mice infected with viable parasites

Monoclonal antibodies (mAb), T cell lines, and T or B cell hybridomas were prepared from BALB/c, CBA, or E1 mice infected with Leishmania mexicana. Various mAb were produced which inhibited the growth and motility of parasites in vitro. T cell lines (hybridomas) were screened for their ability to release interleukin 2 on specific antigen exposure. Passive transfer of mAb or T cell lines to infected adult mice caused little perturbation of parasite growth. Recipient naive mice were immunized with purified Ig or irradiated cells from these sources and were subsequently infected with viable parasites. Only preimmunization with T cell lines (hybridomas) led to exacerbation of parasite growth, although enzyme-linked immunosorbent assays could detect the production of anti-idiotype antibodies in mAb (B cell hybridoma)-immunized mice. Either nylon wool-purified T cells or serum Ig from T cell-immunized mice could be used to immunize further naive recipients for protection against parasite growth. These data have implications for the development of anti-idiotype vaccines for Leishmania antigens.     

Unusually diverse T cell response to a repeating tripeptide epitope.

The immune system utilizes a diverse T cell repertoire for the recognition of foreign antigens in the context of self MHC gene products. We have examined the potential diversity of the T cell response directed to a immunodominant repeating tripeptide epitope (EYA)5. This peptide represents one of the two T cell epitopes on the synthetic alpha-helical polypeptide antigen Poly 18, Poly EYK(EYA)5 in H-2d mice and does not require antigen processing prior to presentation to Poly 18-specific T cell hybridomas. The T cell response directed to the repeating tripeptide epitope (EYA)5 is extremely heterogenous even though the epitope has a relatively simple amino acid sequence. We have analyzed the fine specificity of 21 randomly chosen Poly 18-reactive, (EYA)5-specific and H-2d-restricted T cell hybridomas derived from H-2d, H-2bxd, and H-2b----H-2bxd Poly 18-responding mice to determine the number of unique antigen reactivity patterns represented by this T cell population. We used alanine- and/or lysine-substituted (EYA)5 peptides and a panel of haplotype-varied splenocytes and observed a great deal of microheterogeneity in response. We find that 13 of the 21 hybridomas have a distinct fine antigen specificity and T cell receptors. The binding of (EYA)5 to the antigen-binding groove of I-Ad appears to generate a highly diversified T cell response. Therefore, (EYA)5-I-Ad complex allows the activation of unrelated T cell clonotypes with the same overall antigen specificity and MHC restriction, but with distinct microheterogeneity in response and receptor usage.     

Complete dissection of the Hb(64-76) determinant using T helper 1, T helper 2 clones, and T cell hybridomas.

We have generated cloned Th1 cells, Th2 cells, and T cell hybridomas specific for the single immunogenic peptide from the beta-chain of murine hemoglobin (Hb(64-76)). The availability of these various types of T cells provided us an unique opportunity to examine and dissect the T cell response to an immunogenic peptide. A panel of altered Hb peptides was made by replacing each amino acid in the Hb peptide (positions 64-76) with a conservative amino acid substitution or an alanine. Although none of the eleven T cell clones and hybridomas tested exhibited the same pattern of reactivity to the substituted Hb peptides, some general features were identified for all T cell responses. The primary T cell contact residue of Hb(64-76) was shown to be asparagine 72. For every Hb(64-76) specific T cell, no activation was observed using a peptide containing the conservative substitution of a glutamine for the asparagine at position 72. The flanking glutamic acid at position 73 was also required for a proliferative response for all of the Th1 and Th2 clones. The Th subtypes were not grossly unique in their responses to the substituted Hb peptides, but exhibited minor differences in fine specificity with the Th1 cells identifying more critical amino acids then did the Th2 cells. For the Th1 cells and also the T cell hybridomas, the phenylalanine at position 71 was critical for a T cell response. Analysis of peptide affinity for IEk molecules indicated that position 71 played a role in peptide binding to MHC. Secondary T cell contact residues, which were important for many but not all of the T cells, were identified at positions 69, 70, and 76. Overall T cell responses were minimally affected by changes in the amino acid residues at positions 64-68, 74, and 75. We have also demonstrated that cloned Th1 cells, Th2 cells and T hybridomas can be generated against the same Hb(64-76) determinant.     

Antibody response to simian virus 40 tumor antigen in nude mice reconstituted with T cells.

Athymic BALB/c nude mice (nu/nu) fail to generate circulating antibodies to simian virus 40 (SV40) tumor (T) antigen when immunized with SV40-transformed mouse cells or with T antigen positive somatic cell hybrids derived from SV40-transformed human and normal mouse parental cells. However, normal BALB/c mice readily produce antibodies to SV40 T antigen. When nude mice were reconstituted with normal syngeneic T lymphocytes from spleen or thymus source, the humoral immune responsiveness to SV40 T antigen was restored.     

Immunotherapy of murine sarcomas with auto-anti-idiotypic monoclonal antibodies which bind to tumor-specific T cells

Hybridomas producing monoclonal antibodies (mAb) were obtained from BALB/c mice immunized against either of two transplanted, chemically induced syngeneic sarcomas, MCA-1490 or MCA-1511. Two mAb, 4.72 and 5.96, were obtained, one from each immunization. They were found to have apparent anti-idiotypic specificity in that they, when injected s.c., primed naive BALB/c mice for delayed-type hypersensitivity that was specific for the immunizing tumor and required homology at genes linked to the Igh-1 allotype locus. Neither mAb bound tumor antigen. When mice with established transplants of MCA-1490 or MCA-1511 were treated by repeated i.p. injections of the appropriate anti-idiotypic mAb (4.72 and 5.96, respectively), a significant reduction in tumor growth was observed in those mice that had received the appropriate mAb. The idiotope defined by mAb 4.72 was expressed by T cells in mice responding to MCA-1490. mAb 4.72 bound to T cell suppressor factors that were specific for MCA-1490 and were derived from T cell hybridomas or sera of mice bearing MCA-1490. mAb 4.72 also bound to cells from lymph nodes draining the area of a growing MCA-1490 tumor. It was used, in combination with cell sorting, to establish a T cell line, which mediated delayed-type hypersensitivity to MCA-1490 and inhibited the outgrowth of MCA-1490 in BALB/c mice. Thus, mAb specific for idiotopes on T cells responding to syngeneic tumor antigen had both direct immunotherapeutic activity and could be used to establish cultures of tumor-reactive T cells.     

An IL 2-secreting T cell hybridoma that responds to a self class I histocompatibility antigen in the H-2D region

A self-reactive T cell hybridoma that secretes IL-2 in response to H-2d haplotype cells resulted from a fusion of BALB/cBy lymph node cells with the AKR thymoma BW5147. The lymph node cells used had been enriched for cells reactive to (TG)-A--L, but neither this antigen nor fetal calf serum were required for stimulation of the hybridoma designated 3DT52.5. The gene product responsible for stimulation mapped to the H-2D region. Allogeneic cells of the b, f, k, q, and s haplotypes failed to stimulate. Not all H-2d haplotype cells were effective stimulators of 3DT52.5. Peritoneal cells and splenic B cells were much more stimulatory than splenic T cells. Most tumor cell lines of H-2d derivation and of B cell or macrophage/monocyte lineage were stimulatory, whereas H-2d T cell lines were not. The capacity to stimulate 3DT52.5 did not correlate with the ability to stimulate I region-restricted hybridomas, or with the ability to be induced to stimulate such hybridomas. Stimulatory cell lines did not apparently produce a soluble factor required for stimulation, and negative cell lines were not inhibitory. The monoclonal antibody 27-11-13, which reacts with H-2D of the b, d, and q haplotypes, inhibited stimulation of 3DT52.5 but did not inhibit stimulation of the sibling hybridoma 3DT18.11, which responds to (TG)-A--L plus I-Ad. Conversely, the monoclonal anti-I-Ad antibody MK-D6 inhibited stimulation of 3DT18.11 but not 3DT52.5. Although it is clear that 3DT52.5 recognizes a class I antigen coded for in the H-2D region, the precise molecular nature of the antigen is unknown. The structure of the antigen receptor on this hybridoma may prove to be of interest when it can be compared with receptors found on T cell hybridomas restricted by class II histocompatibility antigens.     

Characterization of the fine specificity and antigen markers associated with the receptor of autoreactive T-cell hybridomas

In this study we attempted to define the determinants on Ia molecules recognized by autoreactive hybridomas obtained from (C57BL/6 X BALB/c)F1 mice. The epitopes recognized by the T cells were characterized (a) using stimulating cells from various congenic and H-2 recombinant inbred strains and (b) by inhibition of activation with anti-Ia antibodies. Our hybridomas were strictly autoreactive and did not exhibit any alloreactivity, as is often observed for such cells. Our results show that more epitopes than previously believed are recognized by autoreactive T cells. One T-cell hybridoma (QW27.1) is unique in that it recognizes a hybrid F1 Ia determinant. Antigenic markers associated with the receptor of the T-cell hybridomas were studied with monoclonal antibodies (mAbs) specific for L3T4 and a V beta "idiotype". The results indicate that all Lyt 1.2 autoreactive T cells express L3T4 antigen in association with their receptor. One clone (QW64.14) expresses the V beta idiotype recognized by F23.1 monoclonal antibody. Moreover, this clone is activated by F23.1, linked to Sepharose 4B beads, which was believed previously to activate only Lyt 2+, L3T4 T cells. The supernatant of one clone (QW17.5) helps B cells to differentiate into antibody-producing cells without requiring direct contact with the autoreactive clone.     

Nucleotide sequence analysis of the BALB/c murine sarcoma virus transforming gene.

We determined the nucleotide sequence of the v-H-ras-related oncogene of BALB/c murine sarcoma virus. This oncogene contains an open reading frame of 189 amino acids that initiates and terminates entirely within the mouse cell-derived ras sequence. The protein encoded by this open reading frame matches the sequence predicted for the T24 human bladder carcinoma oncogene product, p21, in all but two positions. The presence of a lysine residue in position 12 of BALB/c murine sarcoma virus p21 likely accounts for its oncogenic properties.     

Molecular evolution of catalytic antibodies in autoimmune mice.

Catalytic Abs (catAbs) preferentially evolved in autoimmune MRL/MPJ-lpr/lpr (MRL/lpr) mice upon immunization with the phosphonate transition-state analogue (TSA), but this did not happen in normal BALB/c mice. The majority of the catAbs from MRL/lpr mice were from several independent clones of the same family. Most of them had a lysine at position 95 in the heavy chain (H95), which is at the junctional region. This residue, which interacts with the phosphonate moiety of the TSA and presumably is involved in the catalytic activity, was not changed even after expansive evolution following multiple mutations. By contrast, the majority that arose from BALB/c mice were the non-catAbs, which were quite different in the sequence from the catAbs from MRL/lpr mice, but they were clonally related to one another, so most of them were originated from a single clone. In the MRL/lpr mice, the catalytic subsets that existed in the initial repertoire were effectively captured by the phosphonyl oxygens in the TSA by interacting with the lysine at H95. In the BALB/c mice, however, another noncatalytic subset with only the binding capability directed to a moiety other than the phosphonate moiety was alternatively evolved, because of the lowest abundance or elimination of the catalytic subsets.     

Prethymic T cell precursors express receptors for antigen

An anti-idiotype serum raised in BALB/c mice against syngeneic lymph node T cells from 2,4-dinitrofluorobenzene (DNFB)-sensitized mice was used to study the early expression of antigen receptors on developing T cells. Normal BALB/c bone marrow cells were treated with either anti-Thy-1.2 plus complement or anti-Thy-1.2 and anti-idiotype plus complement before use in the reconstitution of lethally irradiated syngeneic mice. Five weeks after reconstitution, recipient mice were assayed for both contact sensitivity (CS) and in vitro proliferative responses to DNFB. Mice reconstituted with bone marrow cells treated with both anti-Thy and anti-idiotype sera showed a significant decrease in reactivity to DNFB in both assay systems when compared with mice reconstituted with marrow treated with anti-Thy only. CS response to the noncross-reacting hapten oxazolone was identical in both recipient groups. Bone marrow mixing experiments showed no evidence of anti-idiotype-induced suppressor cells in these experiments. These data provide strong evidence that at least some T cell precursors express receptors for antigen prethymically.     

Elucidation and role of critical residues of immunodominant peptide associated with T cell-mediated parasitic disease.

Granulomatous inflammation in schistosomiasis is strictly dependent on CD4+ Th lymphocytes sensitized to egg Ags, but its intensity is genetically regulated. C3H and CBA (H-2k) are strains of mice that develop large granulomas; they also strongly respond to the major egg Ag Sm-p40. We now show that the immunodominant epitope recognized by CD4+ Th cells from infected H-2k mice is confined to 13-mer peptide 234-246 (PKSDNQIKAVPAS), which elicits an I-Ak-restricted Th1-type response. Using a panel of alanine-monosubstituted peptides, we identified Asp237 as the main contact residue with I-Ak. On the other hand, three TCR contact residues were essential to stimulate epitope-specific T cell hybridomas: for two hybridomas these were Asn238, Gln239, and Lys241; and for one, Asn238, Lys241, and Pro244. In one instance, alanine substitution for Gln239 generated an antagonist that blocked subsequent stimulation with wild-type peptide. Most importantly, replacement of Asn238, Gln239, or Lys241 caused a profound loss of polyclonal CD4+ T cell reactivity from schistosome-infected mice. This study identifies the critical residues of immunodominant peptide 234-246 involved in the T cell response against the Sm-p40 egg Ag and suggests that suitable altered peptides may be capable of precipitating its down-regulation.     

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.     

Elimination of an immunodominant CD4+ T cell epitope in human IFN-beta does not result in an in vivo response directed at the subdominant epitope

The BALB/cByJ mouse strain displays an immunodominant T cell response directed at the same CD4(+) T cell epitope peptide region in human IFN-beta, as detected in a human population-based assay. BALB/cByJ mice also recognize a second region of the protein with a lesser magnitude proliferative response. Critical residue testing of the immunodominant peptide showed that both BALB/cByJ mice and the human population response were dependent on an isoleucine residue at position 129. A variant IFN-beta molecule was constructed containing the single amino acid modification, I129V, in the immunodominant epitope. The variant displayed 100% of control antiproliferation activity. Mice immunized with unmodified IFN-beta responded weakly in vitro to the I129V variant. However, BALB/cByJ mice immunized with the I129V variant were unable to respond to either the I129V variant or the unmodified IFN-beta molecule by either T cell proliferation or Ag-specific IgG1 Ab production. This demonstrates that a single amino acid change in an immunodominant epitope can eliminate an immune response to an otherwise intact therapeutic protein. The elimination of the immunodominant epitope response also eliminated the response to the subdominant epitope in the protein. Modifying functionally immunodominant T cell epitopes within proteins may obviate the need for additional subdominant epitope modifications.     

Restricted V-(D)-J junctional regions in the T cell response to lambda-repressor. Identification of residues critical for antigen recognition.

The T cell response to lambda-repressor is directed to a 15 amino acid peptide (P12-26) of the protein in A/J mice. Previous studies have demonstrated a preferential use of V alpha 2 and V beta 1 amongst the T cell hybridomas specific for P12-26 in the context of I-Ek. By using the polymerase chain reaction, the sequences of a panel of the T cells using V alpha 2 and V beta 1 were determined. A highly conserved alpha-chain V-J junctional sequence was found in six of the eight T cell hybrids. This consensus alpha-chain VJ sequence may be combined with different members of V alpha 2, indicating a more restricted selection on the junctional region than on the V element in these T cells. In contrast, greater diversities were found on the V-D-J region of beta-chains despite the same V beta 1 and J beta 2.1 were used. However, a highly conserved glutamic acid residue was found at the same position of beta-chains where a similar conservation was identified in cytochrome c-specific T cells. The correlation of the TCR sequence with the fine specificities of these T cells suggests that a single amino acid deletion in the V alpha-J alpha region may reduce the P12-26 response and abolish the recognition of an altered peptide [Phe22] P12-26. In addition, three amino acid difference in the V-D-J region of the beta-chain also determine the P12-26 reactivity. Thus the V(D)J junctional regions of both alpha- and beta-chains may be critical for the recognition of the peptide Ag presented by the specific MHC molecule.     

Antigen-induced acceleration of shedding and replacement of B cell antigen receptors requires mature T cells

To determine which early and intermediate events in the response of antigen-binding B cells to a T-dependent antigen (sheep erythrocytes [SRC]) require T help, the antigen-induced changes in receptor turnover and surface IgD loss in BALB/c athymic nu/nu mice were compared with that of nu/+ littermates and +/+ BALB/c mice. Nonimmune SRC antigen-binding spleen B cells (ABC) from +/+, nu/+, and nu/nu BALB/c mice coexpressed IgM and IgD, and 85 to 95% retained receptors well when incubated for 2.5 hr in 100 micrograms/ml cycloheximide (which prevents receptor replacement). Also they were able to regain their ability to bind antigen by 18 hr after pronase treatment, but not by 2 hr. However, 5 days after in vivo immunization, 1) the proportion of ABC expressing surface IgD declined from around 90% to less than 50% in +/+ mice and nu/+ mice but not in nu/nu mice; 2) substantial recovery of antigen-binding occurred by 2 hr after pronase treatment in +/+ and nu/+ ABC but not in nu/nu ABC; and 3) when spleen cells were incubated in cycloheximide, uncompensated receptor shedding reduced +/+ and nu/+ ABC by around 80% but produced only about a 10% reduction in nu/nu ABC. Thus, although the ABC in nonimmune nu/nu mice appeared normal with respect to their surface Ig turnover and expression, they failed to undergo the normal antigen-induced loss of IgD or acceleration of surface Ig shedding and replacement, suggesting that these intermediate activation events require interaction with mature T cells. To determine whether this interaction had to occur during B cell development, during the development of the immune response, or during receptor shedding or replacement itself, cell transfer experiments were carried our wherein nu/+ T cells were transferred i.v. to nu/nu littermates 1 day before immunization with SRC. In the transfer recipients, pronase-treated day 5 ABC were then able to replace and shed their receptors at the accelerated rate, like ABC from +/+ and nu/+ mice. In contrast, the co-incubation of 5-day immune nu/+ T cells with nu/nu B cells did not alter the rate of shedding or replacement.