Receptors For Alloantigens Research Articles

Functional Characterization of a Continuous Natural Killer Cell Line, KIL, In Vitro and in Adoptive Immunotherapy.

We have recently established a continuous natural killer (NK) cell line, KIL (for Killer Lymphocyte), from a coculture of normal murine whole bone marrow (WBM) and the OP-9 stromal cell line expressing the Notch ligand, Jagged2, in the presence of stem cell factor and IL-7 (Dehart et al. Blood 2005; 105:3521). A clonal prototype, KIL C.2, was derived from a C57BL/6 mouse and has the CD3−NK1.1+CD122+ phenotype, expresses the NKG2D activating receptor, and proliferates extensively upon in vitro stimulation with IL-2 (20ng/ml). We now show that KIL C.2 stimulated with IL-2 for 48 hours produce large quantities of interferon-gamma (10,300 ± 1850 pg/ml) and modest amounts of tumor necrosis factor-alpha (200 ± 60 pg/ml). IL-4 and IL-10 secretion was below background levels. We then evaluated the cytolytic potential of KIL C.2 using a FACS-based flourolysis assay using GFP-labeled target cells. We observed that KIL C.2 potently lyse allogeneic A20 (H2d) lymphoma tumor targets without prior activation. In addition, KIL C.2 is moderately lytic against two syngeneic (H2b) targets: a myeloma cell line, 5T33MM, and BLL C.2, a tumorigenic leukemia/lymphoma cell line that was established from a WBM culture and has the CD19+CD25+CD43+sIg− pre-B-cell phenotype (S.T. and T.M.C., unpublished data). To study the mechanism of cytolysis we performed killing assays in the presence of anti-NKG2D (MI-6) or anti-NK1.1 (PK136) blocking antibodies. Blockade of either NK1.1 or NKG2D reduced cytotoxicity against BLL C.2 targets by 50%. BLL C.2 has the C57BL/6 background, thus does not express H60 but expresses the other NKG2D ligands Rae1 at high levels and Mult1 at moderate levels as determined by quantitative RT-PCR. In contrast, blocking NK1.1 and NKG2D had no effect on cytolysis of A20 suggesting the presence of alternative activation pathways such as those mediated by MHC class I alloantigen receptors. To examine the in vivo tumoricidal potential of KIL C.2, we used a C57BL/6 → BALB/c T-cell depleted (TCD)-WBM transplantation and A20-luciferase tumor model and monitored tumor progression using the Xenogen bioluminescence imaging system. BALB/c mice lethally irradiated and rescued with 2.5 × 107 TCD-WBM along with A20-luciferase inoculation uniformly died from tumor. Mice given TCD-WBM and 1 × 106 donor splenocytes did not develop tumor but died from lethal graft-versus-host disease (GVHD). In contrast, mice given TCD-WBM and two injections of KIL C.2 at doses of 1 × 107 per mouse were protected from tumor progression without developing signs of GVHD. These results show that KIL C.2 is a highly functional NK cell line with the capacity for proliferation, cytokine secretion, and cytotoxicity against syngeneic as well as allogeneic tumors. As such, KIL C.2 provides a useful cell line model for studies of NK cell biology and suggests a potentially effective strategy for NK cell-based immunotherapy.

Leu-3/T4 Expression on Epidermal Langerhans Cells in Normal and Diseased Skin

Recent evidence exists that the expression of the Leu-3/T4 antigen is not restricted to thymus-derived lymphocytes but can also be detected on mononuclear phagocytes and epidermal Langerhans cells (LC). When searching for the presence of Leu-3/T4 antigen-bearing cells in tissue sections of a variety of inflammatory and neoplastic skin disorders, we observed quantitative and qualitative differences in the intensity of anti-Leu-3a labeling of epidermal dendritic cells. Reasoning that Leu-3/T4 expression by these cells might be a dynamic event, we compared the anti-Leu-3a LC staining pattern in clinically normal-appearing skin (CNAS) with the expression of this antigen on epidermal dendritic cells in a variety of skin disorders. For this purpose, 4-microns cryostat sections were exposed to the monoclonal anti-Leu-3a reagent and antibody binding was visualized by a sensitive 4-step immunoperoxidase technique. Within CNAS, Leu-3a+ dendritic epidermal cells were visualized at the threshold of detectability. Immunoelectron microscopic studies confirmed the LC nature of these cells. In sharp contrast to CNAS, strong and prominent anti-Leu-3a LC labeling was almost invariably encountered in biopsy specimens from patients with cutaneous T-cell lymphoma and various inflammatory conditions but not in proliferative disorders of resident skin cells. Whereas in CNAS the density of T6+ epidermal dendritic cells greatly exceeded that of anti-Leu-3a-reactive dendritic cells, these differences were less pronounced in diseased skin. Our results: confirm earlier observations that epidermal LC may bear Leu-3/T4 antigens; and in addition, suggest that the degree of Leu-3/T4 expression is regulated by signals from inflammatory cells. The induction of class II alloantigen receptors on class II alloantigen-bearing LC may represent an important regulation mechanism of antigen-presenting cell function.

Cellular basis of allograft rejection.

In summary, we currently hypothesize that systemic immunization within the lymphoid tissue is amplified by a sequence of specific and non-specific immunological interactions which lead to enrichment of specifically sensitized T-cells at the graft site. Nascent sensitized cells enter the graft via non-specific attractants generated by the healing response. The interaction of alloantigen with responder T-cells bearing specific alloantigen receptor results in production of lymphokines which increase blood flow and vascular permeability, and which are chemotactic for circulating lymphocytes. These events and other non-specific events recruit circulating cells regardless of the specificity of their sensitization. The state of activation of these cells may control their presence within the circulation. Cells which are more activated are released more readily from the lymphoid depots into the circulation so that relatively more specifically sensitized (though not necessarily mature cytotoxic cells) cells would enter the circulation and be recruited to the graft. At the allograft, there is an escalating sequence of lymphokine production and non-specific recruitment. II-1 has been demonstrated to be capable of mediating such recruitment. A second phase of amplication occurs at the graft site where precytotoxic (x-ray-sensitive) cells mature and expand into cytotoxic (x-ray-resistant) cells. These cells may be important effector cells of allograft rejection in concert with many unsensitized cells which can participate in non-specific attacks on graft viability. The essential point made from the studies summarized here is the consideration that trafficking patterns of lymphocytes and recruitment of lymphocytes and other inflammatory cells are in essential component to the local mechanism of graft rejection. The relative importance of specificity of response has not been defined.

SUPPRESSOR CELLS IN TRANSPLANTATION TOLERANCE

In order to differentiate between donor (chimeric F1) cells and host cells as being responsible for suppression in transplantation tolerance (TT), cells from tolerant donors were first subject to negative selection through F1 hybrid intermediate host animals. This revealed that the suppressor cells was neither completely removed from the lymph nor recoverable from the lymphoid tissues of the filter rat when highly suppressive inocula were used, suggesting that suppression did not directly depend on cells with receptors for alloantigens. The phenotype of donor and host cells in the recirculating pool was studied with fluorescent antisera and showed that both host cells and chimeric F1 cells were in the thoracic duct lymph of tolerant rats and were capable of recirculation. T and B lymphocytes of both types were present but the IgG-positive, presumptive memory B cells were highly enriched in those rapidly recirculating cells, obtained by filtration of tolerant inocula through irradiated intermediate hosts. These cells were also highly enriched for suppressor function. Methods which selectively depleted either the chimeric T cells or the B cells were applied to tolerant inocula and on adoptive transfer of these inocula, suppression was eliminated only when chimeric T cells were eliminated. This strict dependence of suppression of F1 hybrid T cells is interpreted as evidence that these cells probably suppress directly, via an anti-idiotypic mechanism, the alloreactive cells bearing idiotype-positive major histocompatibility complex receptors.

In vitro analysis of allogeneic lymphocyte interaction. V. Identification and characterization of two components of allogeneic effect factor, one of which displays H-2-restricted helper activity and the other, T cell-growth factor activity.

An allogeneic effect factor (AEF) derived from mixed lymphocyte reaction (MLR) cultures of alloactivated A.SW (H-2s) responder T cells and irradiated A/WySn (H-2a) stimulator spleen cells helps an in vitro primary anti-erythrocyte plaque-forming cell PFC response of BALB/c nude spleen cels and also A/WySn but not A.SW T cell-depleted spleen cells. AEF activity is adsorbed by anti-Ik and anti-I-Ak but not by anti-I-Jk, anti-I-ECk, and anti-Is. Gel filtration of ACA 54 resolves AEF into two main components that which appear in the 50,000- to 70,000-mol wt (component I) and 30,000- to 35,000-mol wt (component II) regions, respectively. Component I has a mol wt of 68,000, elutes from DEAE-Sephacel at 0.05-0.1 M NaCl, and has an isoelectric point (pI) of 5.8. It helps A/WySn but not A.SW B cells and, therefore, is H-2 restricted. Component II is not H-2 restricted, because it helps both A.SW and A/WySn B cells. It also stimulates (a) the growth of a long-term cytotoxic cell line in vitro, (b) Con A-induced thymocyte mitogenesis, and (c) the generation of cytotoxic T cells. The latter three properties of component II are not shared by component I. In addition, component II elutes from DEAE-Sephacel at 0.15-0.2 M NaCl and has a pI of 4.3 and 4.9. Ia determinants and Ig VH, CH, L-chain, and idiotypic determinants are not present on either component I or component II. The properties of component II are identical to that of a T cell growth factor produced by Con A-stimulated spleen cells. It is suggested that the H-2-restricted component I of AEF might be an MLR-activated responder T cell-derived Ia alloantigen receptor.

Idiotypic determinants in alloantigen receptors. I. Role in a leucocyte migration inhibition reaction.

An anti-idiotypic serum was prepared by injecting BALB anti-AKR serum into (BALB X AKR)F1 mice. Pretreatment of BALB anti-AKR immune cells with this anti-idiotypic serum plus complement abrogated a leucocyte migration inhibition reaction (LMIR) against AKR extracts but not against purified protein derivative By itself, the serum induced LMIR in immune cells but not in normal cells. The reaction was strain-specific and anti-Thy 1,2 plus complement sensitive. These results indicate that alloantigen receptors, able to trigger in LMIR in immune cells, have idiotypic determinants similar to those of circulating antibodies of the same specificity. Thus the LMIR is a good model for the study of receptors cell-mediated immune reactions of primed cells.

Representation of heavy but not light chain Ig idiotypes on T cell receptors for alloantigens.

We have analyzed idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants by the use of different anti-idiotypic antibodies. Such antisera were produced in (Lewis X DA) F1 rats against Lewis anti-DA alloantibodies (= B cell product) and Lewis T lymphocyte receptors with the same specificity. We found that B lymphocytes bear unique idiotypic determinants which are not present on the corresponding T lymphocytes. T cell unique (not shared by B lymphocytes) idiotypes were so far not detected. T cells idiotypic determinants which are present on heavy but not light chains of the corresponding alloantibodies.

Alloantigen receptors on activated T cells in mice. I. Binding of alloantigens and anti-idiotypic antibodies to the same receptors.

B6 alloantigens in supernates from one-way mixed lymphocyte reaction (MLR) cultures of AKR T cells against B6 lymph node cells rebound specifically to (B6)AKR-T-cell blasts after overnight incubation and recovery of these blasts from trypsin treatment. A similar specificity was observed with the binding of SJL alloantigens to (SJL)AKR-T-cell blasts. In both cases, the corresponding alloantigens were rebound several-fold more efficiently than control alloantigens. In a different assay system, T-cell receptors were studied with anti-idiotypic sera. These antisera were raised by repeated injection of purified (B6)AKR-T-cell blasts into (AKR X B6)F1 hybrid mice. These F1a(AKRaB6) sera reacted with the majority of (B6)AKR-T and (B6)(AKR X SJL)F1-T-cell blasts. They also reacted with a lower, though sizable, number of (SJL)AKR-T- and (SJL)(AKR X B6)F1-T-cell blasts. No reaction was observed with (B6)SJL-T, concanavalin A-activated AKR T-cell blasts, normal B6, (AKR X B6)F1, and (AKR X SJL)F1 T cells. Normal AKR T cells were positive only minimally above background. F1a(AKRaB6) sera could be made specific for (B6)AKR-T and (B6)(AKR X SJL)F1-T-cell blasts by absorption of contaminating antibodies with (SJL)AKR-T-cell blasts. Finally, it was shown by competition experiments that the receptors on MLR-activated T-cell blasts that bind alloantigens were the same as those binding anti-idiotypic antibodies. In addition, it was found that at least a fraction of alloantibodies share common idiotypic determinants with receptors on MLR-activated T-cell blasts.

Similarity of idiotypic determinants of T-and B-lymphocyte receptors for alloantigens.

Similarity of idiotypic determinants of T-and B-lymphocyte receptors for alloantigens.

Studies on the Product of Antigenic Recognition I.

A product of antigenic recognition (PAR) was produced whenever receptors for alloantigens from T lymphocytes or a principle present in T-cell dependent alloantisera interacted with alloantigen. With two forms of the PAR assay (direct and indirect) the mechanisms underlying these interactions have been analyzed. For the interaction of T-lymphocyte receptors with alloantigen measured with direct PAR assays, the following conclusion emerged: upon confrontation with alloantigen, receptors (if not already present in secreted form) had first to be released from T-cell membranes. Shed T-cell receptors interacted with alloantigens by solubilizing them. Both processes could be prevented by fixing cells with formaldehyde. Release of T-cell receptors was temperature-dependent, solubilization of alloantigens was not. Because in mixed cell cultures receptors had first to be shed, this process was considerably slower and, in concordance with temperature dependence of receptor release, took place only at 37 degrees C. Titration of T lymphocytes with 'bound' receptors by the direct PAR test revealed that in the presence of excess alloantigen 10(2) T cells sufficed to give measurable responses. Supernates of parental strain lymphocytes containing numerous T-cell receptor specificities could be depleted of one of them. Alloantisera raised in presence of T helper cells ('T alloantisera') contained a principle capable of recognizing alloantigens, alloantisera incited in the absence of T helpers ('B alloantisera') did not. The recognizing principle appeared to be IgG. Like T-cell receptors, it was capable of solubilizing alloantigens form target cell membranes. B alloantisera lacked this capacity and their alloantigen-recognizing moiety was found to be monomeric IgM. The mode of interaction of this IgM with alloantigen most likely consisted in fixation to and shielding of antigen.

Studies on the Product of Antigenic Recognition II.

Cell-bound or shed T-lymphocyte receptors for alloantigens and the IgG-associated, alloantigen-recognizing principle in T alloantiserum reacted with antisera raised against those structures to form a product of antigenic recognition (PAR) in which the receptor or its equivalent took the role of ‘antigen’. Formation of PAR was tested for numerous combinations of reactants and in all the process was completed within 1–2 h at 37 or 4 °C. Cell-bound receptors were found to be solubilized by all three forms of antisera, with the exception of an antiserum lacking anti-T receptor activity. The data indicated that PAR might be a complex of antigen and antibody with granulotactic activity whose formation was specific.

Suppression of Antibody Responses in Allogeneic Mice by Products of Lymphoid Tissue

Abstract Mice were irradiated, infused with thymocytes and immunized with a variety of antigens, i.e., sheep or horse red blood cells (SRBC or HRBC), diphtheria toxoid (DT) or bovine γ-globulin (BGG). The spleen cells (T.Spleen cells) were harvested 5 days later and cellfree extracts were prepared. The extracts contained an allogeneic suppressive factor (ASF) that was capable of inhibiting IgM antibody responses of allogeneic or semi-allogeneic unirradiated mice. ASF had to be injected within 24 hr of immunization to be effective and a single injection delayed, rather than abolished, the antibody response at the cellular level. However, daily injections of ASF resulted in persistent suppression of antibody response. ASF activity was antigen nonspecific, i.e., the antigen used to stimulate ASF production did not have to be the same as the antigen used to test for ASF activity. C3H T.Spleen extracts were even immunosuppressive when prepared by exposure to C3BF1 alloantigens only; such extracts suppressed antibody responses of C3BF1 and DBA/2 mice. C3H ASF was removed from extracts after incubation with C3BF1 spleen cells but not after incubation with C3H spleen cells. C3BF1 spleen cells which had been preincubated with C3H ASF were unable to generate antibody-forming cells upon transfer to irradiated C3BF1 host mice. This suggests that the ASF molecule may be or include receptors for alloantigens. The immunogenetic requirements for ASF activity were evaluated by injecting extracts from C3H, C57BL, C3BF1 and BALB/c T.Spleen cells into C3H, CBA, C57BL, BALB/c, DBA/2, A or C3H.A recipient mice. All extracts tested had ASF activity. However, all allogeneic recipients were not suppressed by the extract material. The suppressive activity of ASF seemed to require two (or more) antigenic differences between donors and recipients of extract material, an H-2K or I antigen difference and a second antigen difference, possibly Ig-1. In the limited numbers of strain combinations tested, T.Spleen extracts suppressed IgM antibody response only if exposed to H-2 and Ig-1 antigens, e.g., BALB/c (H-2d, Ig-1a) ASF suppressed A (H-2a, Ig-1e) but not C3H.A (H-2a, Ig-1a) or DBA/2 (H-2d, Ig-1c). Separate ASF molecules may react with separate antigens on the cell surface, i.e., with H-2 and γG2a. Alternatively, one ASF molecule may react with two structurally associated antigens. If the latter is correct, it is conceivable that the β2-microglobulin which is non-covalently linked to the major component of H-2 molecules expresses allotypic antigens coded for by Ig-1 and β2-microglobulin is one of the antigens recognized by ASF.

T-lymphocyte receptors for alloantigens.

Recent developments in research on T-cell receptors for alloantigens have been reviewed. Recognition of these antigens is T-cell dependent and, when measured in the PAR test, can be shown to take place with receptors present on T cells, with those shed spontaneously from T cells and also with "recognition structures" of as yet unknown origin and structure present in post-transplantation alloantiserum but absent from B-cell induce alloantiserum. All recognizing structures of parent T-cell origin mentioned above also induce formation of anti-T cell receptor antisera when injected into appropriate F1 hybrid animals. The highly specific inhibitory activity of the sera can be demonstrated both in vitro and in vivo, and the most fascinating aspect constitutes cytotoxic elimination of T cells bearing receptors for a given alloantigenic specificity without harming T cells with other alloreceptors. The biochemical nature of T-cell receptors is not known and, depending on the physical form in which it is investigated, high molecular weight as well as considerably lower molecular weight structures have been found. It has, on the other hand, become quite clear that the antigen-binding region of B- and T-cell receptors shows idiotypic similarity and it is, therefore, likely that this part of the receptor molecule of both lymphocyte classes is similar if not identical

T-Lymphocyte Receptors for Alloantigens

T-Lymphocyte Receptors for Alloantigens

Transplantation Tolerance at the T-Lymphocyte Receptor Level II.

CBA T lymphocytes deprived temporarily of receptors for alloantigens A[RS(A)] cultivated <i>in vitro </i>for 30 h with anti-receptor antibody-forming (AxCBA)F<sub>1</sub> spleen cells were capable of resynthesizing RS(A) if primed F<sub>1</sub> cells exceeded parental T cells by a factor of 25 or less, but not if the excess was 50-fold or more. This indicated that resynthesis of CBA T-cell RS(A) was successful if primed F<sub>1</sub> cells formed insufficient amounts of anti-CBA T-cell RS(A) antibody. Abortive or successful receptor resynthesis was measured by two parameters, (a) reappearing RS(A) formed PAR together with A alloantigens of (A×CBA)F<sub>1</sub> spleen cells and (b) budding receptors bound anti-receptor antibody. CBA B lymphocytes did not interfere with these reactions. A search for putative T suppressor cells in the F<sub>1</sub>cell population was unsuccessful. PAR formation and anti-RS antibody consumption by reappearing receptors differed temporally: receptors forming PAR were present after a delay lasting 8 h; receptor structures fixed anti-RS antibody as early as 5 h after being cultivated. With due caution, these results might reflect processes operating in maintenance of transplantation tolerance, suggesting that this condition is a serum-mediated suppression of long duration. The suppression would encompass continued neutralisation of receptors for the alloantigen to be tolerated by anti-T-cell receptor antibody formed by the F<sub>1</sub> chimeric cells within an animal with acquired transplantation tolerance.

Lymphocyte-target cell conjugation: membrane receptors for alloantigens.

It is generally accepted that interactions between cytotoxic T-lymphocytes (CTL) and target cells (TC) are involved in allograft rejection. This interaction is a complex process initially involving a distinct and specific CTL-TC binding step which then leads to TC lysis (1–3). Several indirect methods are available for the study of the binding of CTL and TC (4–7). In this paper a new approach to the direct analysis of the binding of CTL and TC is presented.KeywordsTarget CellBinding CapacityLymphoid CellAllograft RejectionThymus CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Studies on antireceptor antibody and on alloantibody formation

The injection of F1 hybrid spleen cells into lethally irradiated parental strain recipients resulted in the formation of antisera containing antireceptor antibody but no alloantibody activity. The results demonstrated radioresistance of the immunogenic property of T-cell receptors for alloantigens and confirmed earlier conclusions on the origin of antireceptor antibody-forming cell populations. The system employed invited investigation into a possible cooperation between F1 B and F1 T lymphocytes in the formation of anti-RS antibodies in unirradiated and in lethally irradiated parental strain hosts. In the absence of F1 T cells, F1 B cells were incapable of forming these antibodies and vice versa. Semiallogeneic synergism in which parental T cells were to cooperate with F1B cells could not be observed. Addition to F1B inoculum of T lymphocytes from syngeneic, from allogeneic, and from semiallogeneic sources before injection into parental recipients also gave results that excluded all but a syngeneic T-B cell collaboration. The production of antibodies to parental T-cell receptors, therefore, seems to require recognition of allogeneic receptor by both T and B lymphocytes.

Spontaneous release of T cell receptors for alloantigens. III. The effects of anti-T cell receptor antiserum, of alloantiserum and of trypsin on T cell receptor release.

Spleen and lymph node T lymphocytes cultivated in vitro spontaneously released receptors for alloantigens. Among the various specificities, those receptors fitting certain alloantigens could be absorbed by formolized cells or proper genotype without influencing receptors for other alloantigens. Shedding of T cell receptors could be inhibited for 8 h by treating cells with antisera directed against T cell receptors (anti-RS antisera). Inhibition experiments indicated that resynthesis of T cell receptors appears to be complete by 8 h. Experiments on prevention of receptor shedding revealed highly specific and presumably cytotoxic elimination of T cells with receptors for given alloantigens. Treatment of cells with alloantiserum obtained by skin graft rejection failed to inhibit spontaneous release of T cell receptors, whereas treatment of lymphocytes with trypsin resulted in an 8-h delay of receptor shedding similar to that after treatment of cells with anti-RS antisera. This inhibition was, however, nonspecific.

Evidence for a B-cell -like helper function in mixed lymphocyte culture between immunocompetent thymus cells.

The immunocompetent subpopulation by mouse thymus cell (TH-2) was isolated by buoyant density centrifugation and by hydrocortisone pretreatment. TH-2 cells undergo a proliferative one-way or two-way mixed lymphocyte culture (MLC) response only when cultured with allogeneic or congenic peripheral lymphoid cells. However, mixtures of allogeneic TH-2 cells alone do not proliferate in either one-way or two-way MLC reactions. Such MLC mixtures are proliferative only if mitomycin-blocked peripheral lymphoid cells are also present in the mixture. The peripheral helper cell has been found to be of low net density, non-adherent, insensitive to anti-thy-1 serum cytotoxicity, but sensitive to the cytotoxic effets of anti-immunoglobulin serume plus complement. The helper effect does not depend on proliferation nor does it appear to involve demonstrable soluble mediators. The nature of failure of MLC between TH-2 subpopulations appears to be dependent on the exppression of some product of the K, I regions of the H-2 locus. Possible mechanisms by which a B-cell-like helper cell triggers TH-2 proliferation are discussed terms of the present knowledge of specific alloantigen receptor on T and B cells, and the immunoglobulin Fc region receptors on T cells.

Spontaneous release of T cell receptors for alloantigens. II. Induction of antibodies to T cell receptors.

Spleen cells from normal mice, when cultivated in vitro, released receptors or recognition structures (RS) for alloantigens into the surrounding medium. The spontaneous shedding of receptors was revealed by their ability to induce the formation of anti-recognition structure(anti-RS) antibodies upon injection into appropriate F1 hybrid recipients. Cell suspensions containing T and B lymphocytes and those containing T lymphocytes were capable of inducing anti-RS antibody formation, whereas suspensions devoid of T cells were incapable of doing so. Receptors shed from such cell suspensions during a 24-hour cultivation period gave exactly the same results. Cell-free culture supernatants, however, incited higher anti-RS antibody titers, presumably because of an accumulation of RS. The capacity of released T cell receptors to recognize alloantigens as determined in the PAR assay and their ability to induce anti-RS antibodies went roughly parallel.