Alloantigen-specific idiotype-bearing receptors on mouse T lymphocytes. I. Specificity characterization and genetic association with the heavy- chain IgG allotype

The present study describes the qualitative reactions of a xenogeneic anti-idiotype (Id) antiserum produced in a mouse-gamma-globulin-tolerant rabbit (5,936) against B6 anti-CBA IgG antibodies. The results showed that such an anti-Id antiserum reacts specifically against anti-H-2k antibodies and against H-2k alloantigen-activated T cells from the following pairs of congenic mice: B10 (H-2b) and B10.D2 (H-2d); and A.BY (H-2b) and A.SW (H-2s), but not against C3H.SW (H-2b) and C3H.OH (H-2o); and BALB/b (H-2b) and BALB/c (H-2d). CB 20 (BALB/c mice with the Ig-1b allotype) anti-CBA T blasts also express idiotypic determinants that react with rabbit 5,936 antiserum. Thus, positive reactions are obtained between rabbit 5,936 anti-Id antiserum and anti-H-2k IgG preparations and T blasts from mice carrying the Ig-1b or Ig-1e allotype, but not from mice carrying the Ig-1a allotype. These reactions are qualitatively independent of the H-2 genotype of the Id-producing mice. Such a finding strongly suggests that the Id-bearing receptor molecules on mouse T cells are coded for by genes that are associated with the Ig heavy-chain-linkage group and not to the mouse histocompatibility complex. Furthermore, the anti-Id antibodies studied react preferentially against anti-H-2k antibodies or T cells with specificity toward the IAk-region-associated serological specificities. Thus, genes associated with the Ig heavy-chain-linkage group seem to be structural genes for at least T-cell receptors with specificity for IA-region-coded membrane antigens.     

T cell development in B cell-deficient mice. II. Serological characterization of suppressor T cell factors (TsF1) produced in normal mice and in mice treated chronically with rabbit anti-mouse IgM antibodies

Serological analysis of idiotypic specificities present in azobenzenearsonate (ABA)-specific first-order suppressor T cell factors (TsF1) from C.AL-20 and BALB/c mice revealed a significant difference between TsF from these two strains of mice. The idiotypic composition of TsF1 from BALB/c mice appears to be more heterogeneous, and at least two different fractions can be readily identified. One bears the characteristic BALB/c-associated CRI(C) (crossreactive idiotype) determinants, and the other is non-CRI(C)-bearing. Analysis of ABA-specific TsF1 from animals lacking B cells uncovered a fundamental change in the expression of their idiotypic specificities. TsF from rabbit anti-mouse IgM (anti-mu)-treated C.AL-20 mice failed to express the characteristic CRI(A) determinants. Instead, they express CRI(C) specificities. Similarly, TsF1 from anti-mu-treated BALB/c mice did not express their characteristic CRI(C) specificities, but rather express CRI(A) determinants. These experiments provide strong evidence that the Igh restriction specificity of TsF is dictated by the particular idiotypic specificities expressed. They also clearly demonstrate that B cells and their products play an important role in establishing the idiotypic composition and repertoire of suppressor T cells.     

Optimization of a self antigen for presentation of multiple epitopes in cancer immunity

T cells recognizing self antigens expressed by cancer cells are prevalent in the immune repertoire. However, activation of these autoreactive T cells is limited by weak signals that are incapable of fully priming naive T cells, creating a state of tolerance or ignorance. Even if T cell activation occurs, immunity can be further restricted by a dominant response directed at only a single epitope. Enhanced antigen presentation of multiple epitopes was investigated as a strategy to overcome these barriers. Specific point mutations that create altered peptide ligands were introduced into the gene encoding a nonimmunogenic tissue self antigen expressed by melanoma, tyrosinase-related protein-1 (Tyrp1). Deficient asparagine-linked glycosylation, which was caused by additional mutations, produced altered protein trafficking and fate that increased antigen processing. Immunization of mice with mutated Tyrp1 DNA elicited cross-reactive CD8(+) T cell responses against multiple nonmutated epitopes of syngeneic Tyrp1 and against melanoma cells. These multi-specific anti-Tyrp1 CD8(+) T cell responses led to rejection of poorly immunogenic melanoma and prolonged survival when immunization was started after tumor challenge. These studies demonstrate how rationally designed DNA vaccines directed against self antigens for enhanced antigen processing and presentation reveal novel self epitopes and elicit multi-specific T cell responses to nonimmunogenic, nonmutated self antigens, enhancing immunity against cancer self antigens.     

A surrogate hepatitis B virus antigenic epitope represented by a synthetic peptide and an internal image antiidiotype antibody

The use of molecules that represent single, defined epitopes able to substitute for antigen (i.e. surrogate antigens) offers considerable advantages over the use of native antigen for the precise manipulation of the immune response. We have investigated the immunochemical characteristics of two types of surrogate hepatitis B surface antigen (HBsAg) epitopes: (a) linear and cyclical synthetic peptides representing amino acid residues 139-147, a hydrophilic region corresponding to part of the a determinant of the HBsAg, and (b) four monoclonal antiidiotypes raised against anti-HBs mAb, two of which behave as an internal image of an a determinant. Polyclonal anti-HBs antisera bound the monoclonal antiidiotypes with affinities of the order of 10(8)/M, and to the peptides with greater than 10-fold lower affinities. However, the levels of antibody in the polyclonal antisera for the peptides was greater than for the antiidiotypes. In inhibition RIA, the surrogate antigens show concordance in that the internal image antiidiotypes inhibit the binding of both monoclonal and polyclonal anti-HBs to the linear and cyclical 139-147 peptides. These results imply that surrogate antigens could indeed be useful as potential hepatitis vaccines, but while the antiidiotypes may stimulate B cells of higher affinity, they would react with a more restricted range of B cell reactivities than would the peptides. A future HBV vaccine may thus comprise a synthetic peptide such as cyclical 139-147 or a cluster of monoclonal internal image antiidiotypes.     

Rat T cell response to superantigens. I. V beta-restricted clonal deletion of rat T cells differentiating in rat-->mouse chimeras

T cells of mice display V beta-specific reactivity for a spectrum of mouse mammary tumor virus (Mtv) antigens; confrontation with these antigens during ontogeny causes substantial "holes" in the T cell repertoire. Since endogenous Mtv antigens are rare in other species, the question arises whether V beta-specific recognition of Mtv antigens is unique to mice. To examine this question, rat T cells were allowed to differentiate from stem cells in severe combined immunodeficiency (SCID) mice. These rat-->mouse xenochimeras were prepared under a variety of conditions. The results show that rat T cells are strongly reactive to mouse Mtv antigens, both in terms of tolerogenicity and immunogenicity. In fact, the V beta specificity of rat and mouse T cells for Mtv antigens is almost indistinguishable.     

Cross-species transplantation tolerance: rat bone marrow-derived cells can contribute to the ligand for negative selection of mouse T cell receptor V beta in chimeras tolerant to xenogeneic antigens (mouse + rat----mouse)

Mixed xenogeneic bone marrow reconstitution (mouse + rat----mouse) results in stable mixed lymphopoietic chimerism (1-48% rat), long-term survival, and the induction of stable functional donor-specific transplantation tolerance to xenoantigens in vivo. To examine the role of negative selection of potentially xenoreactive T lymphocytes during tolerance induction across a species barrier, mixed xenogeneic chimeras (mouse + rat----mouse) were prepared and analyzed using a mixture of mouse and rat bone marrow cells for relative T cell receptor (TCR)-V beta expression on mouse T cells. In mixed xenogeneic chimeras (B10 mouse + rat----B10 mouse), T cell maturation proceeded normally in the presence of rat bone marrow-derived elements, and functional donor-specific tolerance to rat xenoantigens was present when assessed by mixed lymphocyte reactivity in vitro. V beta 5, which is expressed at high (undeleted) levels in normal B10 mice, was consistently deleted in B10 recipients of Wistar Furth (WF), but not F344 rat bone marrow, whereas the coadministration of either F344 rat or WF rat bone marrow with B10 mouse bone marrow cells resulted in a significant decrease in expression of TCR-V beta 11. Taken together, these data demonstrate for the first time that rat bone marrow-derived cells can contribute in a strain-specific manner to the ligand for negative selection of specific mouse TCR-V beta during tolerance induction across a species barrier.     

T cell development in B cell-deficient mice. V. Stopping anti-mu treatment results in Igh-restricted expansion of the T suppressor cell repertoire concomitant with the development of normal immunoglobulin levels

B cell-deficient (anti-mu-treated) mice have proven to be a valuable tool with which to examine the influence of Ig idiotypic determinants upon the development of the Ts repertoire. We have previously reported that ABA-specific Ts repertoires matured in normal and Ig-deficient environments differ from one another in their composition, and consequently, their functionally expressed Igh restrictions. The present report characterizes the impact of natural development of mature B cell activity upon the composition of the Ts repertoire. After stopping anti-mu treatment of C.AL-20 mice, ABA-specific Ts repertoires undergo a defined expansion shown by their acquisition of an additional Ts network that displays Igh restrictions characteristic of normal C.AL-20 mice. This Igh-1d-restricted repertoire can be readily shown within 2 wk of major increases in surface Ig spleen cells and total serum Ig levels in these mice. At the same time, the original Ts restriction specificity (Igh-1a-restricted) generated in the Ig-deficient environment of anti-mu. C.AL-20 mice, is not lost for at least 20 wk. The resulting dual Ts repertoire, characterized by expression of parallel, idiotypically restricted Ts networks, is demonstrable for at least 13 wk. These findings favor an important role for Ig determinants in determining the makeup of the T cell repertoire, and ultimately, the composition of immunologic networks as a whole.     

Antigen requirements for induction of B-memory cells: studies with dinitrophenyl coupled to T-dependent and T-independent carriers

Mice were primed with dinitrophenyl (DNP) (trinitrophenyl, TNP) coupled to thymus-independent (TI) or thymus-dependent (TD) carriers. B cells from these mice were transferred to irradiated recipients with T cells from keyhole limpet hemocyanin (KLH)-primed mice. After secondary immunization with DNP-KLH a significant DNP-specific IgG memory response was produced only by mice which received B cells which had been primed with TD antigens. TI antigens were unable to induce differentiation of B-cell precursors to IgG producing memory B cells but they did not suppress the induction of B-memory cells by TD antigens. The results indicate that TI antigens fail to activate a cell type which is required for the induction of memory B cells.     

The B cell repertoire revealed by major histocompatibility complex-specific helper T cells. I. Frequencies of a genetically defined V region marker among mitogen- and T helper cell-reactive B lymphocytes in normal and immunized mice

The aim of the present work was to analyze the frequencies of a genetically defined variable (V) region marker in the B cell subset sensitive to T cell help. To this end we used an alloreactive T cell line that has the property of inducing B cells of the appropriate haplotype to exponential growth and polyclonal antibody synthesis. The frequency obtained with this helper line was also directly compared to that obtained with lipopolysaccharide (LPS). We found that in normal BALB/c mice the frequency of M460-positive clonotypes was respectively, 1/100 and 1/1,000 among the T helper- and LPS-sensitive B cell subsets. In mice immunized with antiidiotype coupled to a thymus-dependent antigen, the differences in the numbers of idiotype-positive precursors were even more accentuated, i.e. 1/20 in the B cell subset triggered by T helper cells and 1/800 in those cells responsive to LPS. The frequencies of the M460 determinant in mice immunized with anti- idiotypes coupled to thymus-independent antigens were not significantly different, in either B cell subset, from those obtained with spleen cells of normal nonimmunized animals. Taken as a whole, our results imply that the V gene repertoire revealed by LPS includes precursor distribution, as this distribution occurs during the early stage of B cell development (potential repertoire), while the repertoire revealed by T helper cells includes the V region distribution of those clones that are selected in the periphery of the functional immune system.     

Role of variable region gene expression and environmental selection in determining the antiphosphorylcholine B cell repertoire

To evaluate the role of environmental selective processes, as opposed to variable region gene expression, in the determination of B cell repertoire expression, we have assessed the phosphorylcholine (PC)- specific repertoire of precursor cells that remain in bone marrow cell populations after the removal of surface immunoglobulin (sIg)-bearing cells. Such cells are assumed to represent a stage in B cell maturation before the expression of sIg, and thus at a time when they have not as yet interfaced with environmental influences that operate through sIg receptors such as antigenic stimulation, tolerance, or antiidiotypic regulation. The repertoire as expressed in these cells, therefore, should reflect the readout of immunoglobulin variable region genes as they are expressed in progenitors to B cells. The results of these studies indicate that, as in mature primary B cell pools of BALB/c mice, the majority of PC-responsive sIg- bone marrow cells are of the T15 clonotype. Thus, environmental selective mechanisms would not appear to be required for the high frequency of B cells of the T15 idiotype in the primary B cell repertoire of BALB/c mice. Analysis of the sIg- bone marrow cells in (CBA/N X BALB/c)F1 male mice demonstrated that the deficit of PC-responsive mature B cells, which is a characteristic of this murine strain, must occur after receptor expression, since a normal frequency of PC-responsive and T15- expressing cells is present in their sIg- bone marrow population. Finally, these same mice were used to obtain bone marrow cell preparations from individual leg bones, so as to permit an analysis of the occurrence of T15+ and T15- clonotypes within individual bone marrow populations. The findings from these studies indicate that T15+ B cells occur as a high frequency event within bone marrow generative cell pools. Furthermore, bone marrow populations that are positive for PC-responsive precursor cells often display multiple copies of such precursor cells that are exclusively either T15+ or T15-. This finding indicates that clonal expansion of cells within the B cell lineage apparently occurs before immunoglobulin receptor acquisition.     

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. VII. Idiotype-specific suppression of plaque-forming cell responses

The ability of suppressor cells induced by the intravenous administration of 4-hydro-3-nitrophenyl acetyl (NP)-modified syngeneic cells to reduce an idiotypic B cell response was studied in both an in vivo and an in vitro system. Idiotype-positive B cells were assayed by the ability of guinea pig anti-idiotypic antiserum to specifically inhibit idiotype-positive plaque formation. It was found that up to 57% of the PFC response in vivo and 100% of the PFC response in vitro was inhibitable with antiidiotypic antiserum. The expression of these idiotype-positive B cells could be suppressed by the transfer of spleen cells form mice treated 7 d previously with NP coupled syngeneic cels. T cells are both required and sufficient for the transfer of idiotype specific suppression. The induction of these idiotype-specific T suppressor cells directly with antigen suggests that recognition of unique determinants on cell surfaces is important for regulation of lymphoid cell interactions. The role of idiotype-specific suppressor cells in the network of lymphoid interactions is discussed.     

FUNCTIONAL CHARACTERISTICS OF PEYER''S PATCH LYMPHOID CELLS : I. INDUCTION OF HUMORAL ANTIBODY AND CELL-MEDIATED ALLOGRAFT REACTIONS

Peyer's patches from normal mice contain antigen-sensitive B and T cells, but lack the accessory adherent cell type(s) required both for the induction of humoral immune responses and for the induction of allograft reactions against cell surface alloantigens. Immune responsiveness can be restored to cultures of Peyer's patch cells by the addition of either APEC or ME. Peyer's patch B cells can be specifically induced by antigen to synthesize humoral antibody. Peyer's patch T cells can cooperate in B-cell induction and can be induced to mediate an allograft reaction against an allogeneic stimulus. Peyer's patch lymphoid aggregates appear to be a storehouse of antigen-sensitive cells sequestered in such a way as to lack an accessory cell type or factor required for induction,     

Mitogen-reactive B cell subpopulations selectively express different sets of V regions

The experiments presented here were designed to investigate whether the idiotypic repertoire is equally distributed among B cells subpopulations as defined by mitogen reactivity. To this end we used lipopolysaccharides (LPS) and Nocardia delipidated cell mitogens (NDCM), which are two mitogens that have been described to act on different B cell subsets. The repertoire can be defined in quantitative terms as the frequency of B cells that are precursors for clones secreting immunoglobulin with a given specificity or with a determinate idiotype. We determined, therefore, the absolute frequency of LPS- and NDCM-sensitive B lymphocytes secreting immunoglobulin molecules that bear three idiotopes originally found on a monoclonal anti-beta galactosidase antibody. Because the frequencies of B cells carrying one of these idiotypes are dramatically different in the LPS- and NDCM- sensitive B cells subsets, we conclude that the idiotypic repertoire is not randomly distributed among mitogen-reactive B cell subpopulations.     

APRIL modulates B and T cell immunity

The TNF-like ligands APRIL and BLyS are close relatives and share the capacity to bind the receptors TACI and BCMA. BLyS has been shown to play an important role in B cell homeostasis and autoimmunity, but the biological role of APRIL remains less well defined. Analysis of T cells revealed an activation-dependent increase in APRIL mRNA expression. We therefore generated mice expressing APRIL as a transgene in T cells. These mice appeared normal and showed no signs of B cell hyperplasia. Transgenic T cells revealed a greatly enhanced survival in vitro as well as enhanced survival of staphylococcal enterotoxin B-reactive CD4+ T cells in vivo, which both directly correlate with elevated Bcl-2 levels. Analysis of humoral responses to T cell-dependent antigens in the transgenic mice indicated that APRIL affects only IgM but not IgG responses. In contrast, T cell-independent type 2 (TI-2) humoral response was enhanced in APRIL transgenic mice. As TACI was previously reported to be indispensable for TI-2 antibody formation, these results suggest a role for APRIL/TACI interactions in the generation of this response. Taken together, our data indicate that APRIL is involved in the induction and/or maintenance of T and B cell responses.     

Restriction molecules involved in the interaction of T cells with allogeneic antigen-presenting cells

The proliferative responses of T cells, depleted of alloreactive cells, were tested upon stimulation by antigens presented on allogeneic antigen-presenting cells (APC). Restriction molecules involved in these responses were identified by inhibition of T cell proliferation with monoclonal antibodies against A(A alpha A beta) and E(E alpha E beta) molecules of the APC. The responses to all three antigens tested [Poly(Glu40Ala60) (GA), lactate dehydrogenase B (LDHB), and poly(Glu51, Lys34, Tyr15) (GLT)] were A plus E restricted when the allogeneic APC expressed both molecules, and only A restricted when the APC did not express cell surface E molecules. In contrast, when T cells and APC are syngeneic, the same antigens are recognized only in the context of either A molecules (GA and LDHB) or E molecules (GLT). The data indicate that the immune response gene control of these responses is not associated with either a failure of antigen presentation, or the lack of certain T cell specificities from the germ line repertoire, but probably with selective somatic elimination (tolerance) of certain clones from the T cell repertoire.     

B-cell tolerance. II. Trinitrophenyl human gamma globulin-induced tolerance in adult and neonatal murine B cells responsive to thymus- dependent and independent forms of the same hapten

Neonatal splenic B cells which are responsive to thymus-dependent antigens (TD) are exquisitely susceptible to induction of tolerance (1,2). This state of tolerance is not mediated by suppressor T cells and is not a result of suboptimal macrophage function (1 and footnote one). In adult mice, induction of B-cell tolerance is not achieved with doses of antigen 1,000-fold higher (1) than those required to produce the same degree of unresponsiveness in neonates. In contrast to these results, studies with T-independent (TI) antigens indicate that neonatal and adult splenic B cells are equally susceptible to tolerance induction (3,4). However, such studies have not ascertained whether the neonate is more resistant to tolerance induction or the adult is hypersusceptible, i.e., does the induction of tolerance in cells responsive to TI antigens resemble that of adult or neonatal cells responsive to TD antigens? The answer is pertinent to determining the relative maturity of the B cells which can be tolerized or respond to TI or TD antigens. We report here the direct comparison of tolerogen sensitivity of adult and neonatal TD and TI responses by inducing tolerance in vitro with trinitophenyl human gamma globulin (TNP(17)HgG) and assaying unresponsiveness with TD and TI forms of the TNP determinant.     

Multiple low-dose streptozotocin-induced diabetes in the mouse. Evidence for stimulation of a cytotoxic cellular immune response against an insulin-producing beta cell line

Mice were examined for the presence of splenocytes specifically cytotoxic for a rat insulinoma cell line (RIN) during the induction of diabetes by streptozotocin (SZ) in multiple low doses (Multi-Strep). Cytotoxicity was quantitated by the release of 51Cr from damaged cells. A low but statistically significant level of cytolysis (5%) by splenocytes was first detectable on day 8 after the first dose of SZ. The cytotoxicity reached a maximum of approximately 9% on day 10 and slowly decreased thereafter, becoming undetectable 42 d after SZ was first given. The time course of the in vitro cytotoxic response correlated with the degree of insulitis demonstrable in the pancreata of the Multi-Strep mice. The degree of cytotoxicity after Multi-Strep was related to the number of effector splenocytes to which the target RIN cells were exposed and was comparable to that detectable after immunization by intraperitoneal injection of RIN cells in normal mice. The cytotoxicity was specific for insulin-producing cells; syngeneic, allogeneic, and xenogeneic lymphocytes and lymphoblasts, 3T3 cells, and a human keratinocyte cell line were not specifically lysed by the splenocytes of the Multi-Strep mice. This phenomenon was limited to the Multi-Strep mice. Splenocytes from mice made diabetic by a single, high dose of SZ exhibited a very low level of cytotoxicity against the RIN cells. The cytotoxic response was also quantitated in splenocytes from control and Multi-Strep mice (10 d after the first dose of SZ) before and after culture with mitomycin-treated RIN cells in the presence of T cell growth factor (TCGF). The cytotoxicity of the Multi-Strep splenocytes was enhanced more than fivefold after such culture, suggesting the proliferation of an effector cell that could be stimulated and supported in vitro by TCGF. These results support the hypothesis that cell-mediated anti-beta cell autoimmunity may play a role in the destruction of the beta cells in this animal model. The stimulation of this response by TCGF may provide a tool by which enough cytotoxic effector cells could be obtained to establish their possible direct pathogenetic role in the induction of insulin-dependent diabetes. In addition, such cells will be a valuable tool to define the specific beta-cell antigens that may direct the highly selective cell-mediated destruction of these cells in experimental models and, perhaps, in human insulin-dependent diabetes mellitus.     

Activation of antigen-specific suppressor T cells by B cells from mice immunized with type III pneumococcal polysaccharide

The transfer of B lymphocytes from mice immunized with type III pneumococcal polysaccharide (SSS-III) results in antigen-specific suppression of the antibody response of recipients immunized with SSS-III. Such suppression shares many features associated with low-dose paralysis, a phenomenon mediated by suppressor T cells; it reaches maximal levels 3 d after the transfer of viable or irradiated immune B cells and can be eliminated by the depletion of SSS-III-binding cells from spleen cell suspensions before transfer. In a two-step cell transfer experiment, purified T lymphocytes, isolated from recipients previously given immune B cells, caused suppression upon transfer to other mice immunized with SSS-III. Also, B-cell-induced suppression could be abrogated in a competitive manner by the infusion of amplifier T lymphocytes, as was previously demonstrated in the case of low-dose paralysis. These findings suggest that B cell surface components, presumably the idiotypic determinants of cell-associated antibody specific for SSS-III, are instrumental in activating suppressor T cells involved in regulating the magnitude of the antibody response to SSS-III.     

Immunogenicity and tolerogenicity of self-major histocompatibility complex peptides.

Mechanisms involved in self-antigen processing and presentation are crucial in understanding the induction of self-tolerance in the thymus. We examined the immunogenicity of determinants from major histocompatibility complex (MHC) molecules that are expressed in the thymus and have tested peptides derived from the polymorphic regions of class I and class II molecules. We found that two peptides corresponding to NH2 termini of the class II alpha and beta chains (Ak alpha 1-18 and Ak beta 1-16) could bind to self-Ak molecules with high affinity and, surprisingly, were immunogenic in that they could elicit strong proliferative T cell responses in B10.A mice (Ak, Ek). Neonatal injection of peptide Ak beta 1-16 resulted in complete unresponsiveness to this peptide at 8 wk of age showing that these T cells were susceptible to tolerance induction. We have also tested certain class I MHC peptides and showed that some can interact efficiently with class II MHC peptides to induce an autoreactive T cell proliferative response. Among these class I peptides is one (Dd 61-85) that has the capacity to bind to self-Ia without being immunogenic, and therefore represents an MHC determinant that had induced thymic self-tolerance. We conclude that some self-MHC molecules can be processed into peptides that can be presented in the context of intact class II molecules at the surface of antigen-presenting cells. Autoreactive T cells recognizing optimally processed self-peptide/MHC complexes are eliminated during development, whereas other potentially autoreactive T cells escape clonal inactivation or deletion. Incomplete tolerance to self-antigens enriches the T cell repertoire despite the fact that such T cells may eventually become involved in autoimmune disease.     

Xenogeneic proliferation and lymphokine production are dependent on CD4+ helper T cells and self antigen-presenting cells in the mouse

We studied proliferation and interleukin 2 production by B6 mouse spleen cells in response to stimulation by irradiated cynomolgus monkey spleen cells and compared the results with responses against whole MHC-disparate allogeneic controls (BALB/c). We found that (a) primary xenogeneic helper responses were absent, whereas primary allogeneic responses were brisk, (b) secondary xenogeneic helper responses were dependent on CD4+ T cells and responder antigen-presenting cells (APCs), whereas allogeneic responses could be mediated by either CD4+ or CD8+ T cells independently and were primarily dependent on the presence of stimulator APCs, and (c) secondary xenogeneic helper responses were blocked by an antibody directed against responder class II MHC molecules. These results suggest that mouse helper T cells recognize disparate xenoantigens as processed peptides in association with self class II MHC molecules, similar to the recognition of nominal antigens and unlike direct allo-recognition.