Induction Of CTL Responses Research Articles

Type 1 and type 2 cytokines in antiviral defense

Ectromelia virus (EV) is a natural mouse pathogen that causes a generalized infection termed mousepox, which, in the genetically resistant C57BL/6 (B6) mouse, is an inapparent disease. In contrast, BALB/c and A strain mice are highly susceptible; one infectious virus particle can result in 100% mortality. The contribution of cytokines in the induction of protective immune responses and recovery from infection with EV in B6, BALB/c and A strain mice have been. In the spleen and lymph node (LN) of resistant B6 mice, IL-2, IFN- γ and TNF- α were induced rapidly with large numbers of cells producing these cytokines. All three cytokines were virtually absent in BALB/c and A strain mice. No significant differences were found in the numbers of IL-4 producing cells in the spleen or LN of both resistant and susceptible mice. IFN- γ-producing cells were detected in the spleen but not in the lymph node whereas IL-2-producing cells were detected only in the lymph node of B6 mice. Despite significant increases in the IFN- γ mRNA levels in the LN of B6 mice, no protein was detected by immunocytochemistry. The mRNA levels of IL-2, TNF- α and IL-12 were also rapidly upregulated in LN of B6 mice. The rapid induction of type I cytokines strongly correlated with a potent antiviral CTL response in B6 mice. The absence of these cytokines also correlated with a complete absence or delayed induction of CTL responses to EV in both the BALB/c and A strain mice. IFN- γ gene knock out mice on a B6 background were as susceptible to EV as the BALB/c and A strain mice.

Internalization of Iscom-Borne Antigens and Presentation under MHC Class I or Class II Restriction

Exogenous nonreplicating antigens (Ag) incorporated into immunostimulating complexes (iscoms) induce CTL responses under MHC class I restriction. A requirement for inducing CTL responses is that the Ag is delivered to the cytosol of antigen-presenting cells (APC), a route restricted to endogenously produced Ag. To investigate the mechanisms by which iscoms elicit MHC class I-restricted responses, the intracellular distribution of influenza virus envelope proteins incorporated in iscoms (flu-iscoms) or in micelles (flu-micelles) was studiedin vitrousing murine peritoneal cells (PEC). Ultrathin sections of cells pulsed with biotinylated flu-iscoms or flu-micelles were analyzed by electron microscopy after detection of the biotin label by reaction with streptavidin–gold. PEC pulsed with flu-iscoms showed a pattern of scattered gold particles distributed in clear and dense vesicles as well as in the intracellular space but not associated with organelles. In cells pulsed with flu-micelles, Ag was also detected in most cellular compartments but at a considerably lower concentration. The intracellular distribution of particulate Ag in iscom or micelle form was confirmed by lysis and differential centrifugation of Ag-pulsed APC. Furthermore, P815 cells pulsed with flu-iscoms were lysed by specific immune effectors showing that the iscom-Ag was processed and presented by class I-expressing APC. Flu-iscoms were internalized about 50-fold more efficiently than ovalbumin iscoms (ova-iscoms) suggesting that the nature of the protein and/or the presence of cellular receptors are important factors influencing the capacity of APC to take up iscom-borne proteins. PEC accounted for the most active internalization of iscom-borne Ag, although splenic dendritic cells and B cells also took up flu-iscoms with remarkable efficiency.

CTL induction using synthetic peptides delivered in emulsions— critical role of the formulation procedure

Emulsions have been used with variable degrees of success to deliver antigen to stimulate immune responses. We have investigated three different ways of incorporating peptide antigen into soybean emulsions to induce CTL responses in mice. Two of these emulsions (oil-in-water, o/w, and water-in-oil-in-water, w/o/w) had peptide incorporated at the formulation stage, while the third had peptide added to a pre-formed o/w emulsion. High levels of CTL activity were induced when peptide was dispersed into the o/w or w/o/w emulsions, in contrast to addition of peptide to the pre-formed o/w emulsion, which did not stimulate a CTL response. Induction of CTL activity was independent of emulsion globule size but was correlated with a negative zeta potential and dispersion of peptide in the oil phase. The ability of peptide in soybean oil emulsion to induce CTL is critically dependent on dispersion of peptide at the time of emulsion formation.

CTL responses induced by a single immunization with peptide encapsulated in biodegradable microparticles

A synthetic peptide representing a measles virus (MV) cytotoxic T cell epitope (CTL) when encapsulated in poly ( d, l-lactide co-glycolide) (PLG) 50:50 microparticles induced a strong CTL response after a single intraperitoneal immunization of mice which was greater than that following administration of the peptide in Freund's complete adjuvant. A 100 μg dose of encapsulated peptide was shown to be more effective for CTL priming than 50 and 25 μg doses. A vaccine formulation prepared by simply mixing empty 50:50 PLG microparticles with the peptide resulted in the induction of CTL responses comparable to those induced by the encapsulated peptide. Moreover, a CTL response against MV-infected target cells was observed. These findings highlight the potential immunostimulatory effect of PLG microparticles for the induction of MV and peptide-specific CTL responses.

The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection.

We have previously described the development of a lipopeptide-based vaccine, Theradigm-HBV, capable of inducing CTL responses in humans. This vaccine incorporates the HLA-A2.1-restricted CTL epitope hepatitis B core Ag 18-27, linked to the universal helper T lymphocyte (HTL) epitope tetanus toxoid (TT) 830-843. Herein, we report the results of a phase I trial designed to examine the effects of Theradigm-HBV dose and regimen on the magnitude and duration of the memory CTL response. A total of four injections of up to 5 mg/dose were found to be a safe and effective means of generating substantial memory CTL responses. Precursor frequency analysis demonstrated CTL responses of similar magnitude to those previously observed in patients who successfully cleared hepatitis B virus infection and to influenza-specific memory CTL responses induced by natural exposure to influenza virus. Theradigm-HBV induced CTL responses that persisted for more than 9 months after the last injection. HTL responses were associated with significant CTL responses, and sustained HTL activity was necessary for development of persistent memory CTL activity. These results represent the first demonstration of the importance of HTL activity for development of long-lived memory CTL responses in humans. In conclusion, our results show that lipopeptides safely induce specific CTL activity in humans of such magnitude and persistence as to be of potential therapeutic significance.

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene.

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.

The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites.

Although plasmid DNA immunization provides an effective means of inducing CTL responses to an expressed Ag, the mechanism by which CTL precursors are activated remains to be established. Insights could be gained by identifying the cells responsible for Ag presentation when DNA is introduced into different tissue sites. By immunizing parent into F1 bone marrow chimeric mice with an influenza nucleoprotein-expressing plasmid, we have demonstrated that the key cells in this presentation process for both gene gun-mediated epidermal injection and needle intramuscular injection of plasmid DNA are bone marrow derived. Furthermore, as assessed by intramuscular injection, coexpression of nucleoprotein with the costimulatory molecule B7-2, or the cytokines granulocyte-macrophage CSF and IL-12, did not convert nonhemopoietic cells into APCs. Thus, for two distinctly different modes of DNA immunization, in one case with or without coexpressed immunostimulatory factors, the APCs were consistently found to be of hemopoietic origin.

Analogues of CTL epitopes with improved MHC class-I binding capacity elicit anti-melanoma CTL recognizing the wild-type epitope.

The MHC class-I binding affinity of an epitope is an important parameter determining the immunogenicity of the peptide-MHC complex. In order to improve the immunogenicity of an epitope derived from melanocyte lineage-specific antigen gp100, we performed amino-acid substitutions within the epitope and assayed both HLA-A*0201 binding and CTL recognition. Anchor replacements towards the HLA-A*0201 peptide-binding motif gave rise to peptides with higher HLA-A*0201 binding capacity compared to the wild-type epitope. In addition, several of the gp100 154-162 epitope-analogues were more efficient at target-cell sensitization for lysis by anti-gp100 154-162 CTL compared to the wild-type epitope. These altered gp100 154-162 epitopes were subsequently tested for their capacity to induce CTL responses in vivo using HLA-A*0201/Kb transgenic mice, and in vitro using HLA-A*0201 + donor-derived lymphocytes. Interestingly, the peptide-specific CTL obtained, which were raised against the different gp100 154-162 epitope-analogues, displayed cross-reactivity with target cells endogenously processing and presenting the native epitope. These data demonstrate that altered epitopes can be exploited to elicit native epitope-reactive CTL. The use of epitope-analogues with improved immunogenicity may contribute to the development of CTL-epitope based vaccines in viral disease and cancer.

B7-negative versus B7-positive P815 tumor: differential requirements for priming of an antitumor immune response in lymph nodes.

Efficient T cell activation requires two synergistic but distinct signals derived from antigenic peptides presented by the MHC and from costimulatory molecules, particularly those belonging to the B7 family. Lack of B7-CD28 interaction may cause unresponsiveness of T cells to subsequent exposure to Ag. Nevertheless, immunization by some B7- tumors induces an antitumor immune response. We found that the immune response against two B7- tumors, the mouse P815 mastocytoma and the E7C3 melanoma, requires host-derived B7, since blockage of the B7-CD28 interaction facilitates tumor growth and eliminates an antitumor response. B7 costimulation is provided in the regional, tumor-draining lymph nodes for the induction of a primary CTL response against both B7+ tumor and B7- tumor. However, the induction of a CTL response to B7+ tumors and its clonal expansion may occur at tumor sites in addition to secondary lymphoid organs so as to generate more effective tumor immunity.

Intranasal immunization with CTL epitope peptides from HIV-1 or ovalbumin and the mucosal adjuvant cholera toxin induces peptide-specific CTLs and protection against tumor development in vivo.

To evaluate the ability of mucosal immunization protocols using peptide immunogens to induce CTL responses, BALB/c and C57BL/6 mice were immunized intranasally (i.n.) with peptides corresponding to a known CTL epitope in HIV-1 glycoprotein 120 or OVA, respectively, and the mucosal adjuvant cholera toxin (CT). Intranasal immunization of BALB/c mice with a 10- or 15-amino acid peptide corresponding to a CTL determinant in HIV-1 glycoprotein 120 and CT induced peptide-specific CTLs in spleen cells that persisted through 35 days after the last immunization. Intranasal immunization of C57BL/6 mice with the octameric OVA peptide and CT produced similar results with detectable peptide-specific CTL in both the cervical lymph node and spleen. To test whether CTL induced by i.n. immunization with OVA peptide and CT were functional in vivo, groups of C57BL/6 mice were injected with E.G7-OVA tumor cells that express the OVA protein and monitored for tumor growth. Animals immunized i.n. with OVA and CT were protected against tumor development as efficiently as animals immunized by the potent CTL induction protocol of i.v. injection with OVA-pulsed dendritic cells. Intranasal immunization with peptides corresponding to known CTL epitopes and CT provides a noninvasive route of immunization for the induction of CTL responses in vivo.

Making the most of mucin: a novel target for tumor immunotherapy.

Throughout this brief review I have emphasized the unique biochemical and immunological properties of MUC-1 mucin that make this tumor-associated antigen a novel, exciting and tangible target for tumor immunotherapy. The tandemly repeating nature of the antigenic epitopes in the mucin polypeptide chain, the under-glycosylation of these epitopes, and the expression of the molecule at the cell surface, are all central to the immune recognition of this antigen and must be acknowledged in the design of MUC-1 vaccines. There are considerable difficulties associated with such a design. Short, immunogenic peptides that associate with MHC class I molecules on the cell surface to induce CTL responses are not useful here; MUC-1 must be expressed at the cell surface and this introduces the numerous problems associated with gene transfer. Furthermore, generating under-glycosylated mucin molecules that resemble tumor-associated antigen is not trivial. Competitive inhibition of glycosylation with PhGalNAc is often incomplete, and merely increasing inhibitor concentration results in cell toxicity. But treating tumors is also not trivial. MUC-1 mucin has many characteristics of the ideal tumor-associated antigen, and our understanding of the unique mechanism of mucin recognition on tumors and the appropriate vaccine designs to target this antigen is now advanced. Transgenic mouse and non-human primate models provide excellent preclinical models to test immunotherapeutic and vaccine strategies, and in vitro studies and early-stage clinical trials in humans provide considerable cause for optimism. The next few years in this field should certainly be productive.

Structural requirements for synthetic immunogens to induce measles virus specific CTL responses

We have studied the immunogenicity of a synthetic peptide representing a cytotoxic T cell epitope (CTL) from the nucleoprotein of measles virus (MV). For the induction of peptide and MV-specific CTL responses after subcutaneous immunization, covalent linkage of the CTL epitope to a T-helper epitope was required. The presence of two copies of the T-helper epitope at the amino terminus of the CTL epitope (TT-CTL) resulted in the induction of strong CTL responses after administration in saline. In contrast, a chimeric peptide with one copy of the T-helper epitope at the amino terminus of the CTL epitope (T-CTL) was weakly immunogenic when given in saline. Analysis of the structure of the TT-CTL chimeric peptide by CD spectroscopy revealed an α-helical conformation, as compared to the random coil conformation favored by the T-CTL chimeric peptide. In addition, the CD spectra of the TT-CTL peptide in the presence of small unilamellar vesicules (SUV) revealed an increased helicity, as compared to the spectra of the T-CTL chimera in the presence of SUV. This suggests that the amphipathic character of the TT-CTL chimeric construct favors its interaction with the cell membrane of antigen presenting cells, therefore, facilitating its cytosolic delivery for class I presentation. These findings highlight the importance of antigen structure for the in vivo induction of CTL responses and may have implications for the design of synthetic peptide vaccines.

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying viral CD8+ T cell epitopes.

Exogenous Ags enter the endosomal pathway and are presented to CD4+ T cells in association with class II molecules whereas endogenously synthesized Ags, such as viral proteins, are presented to CD8+ T cells in association with MHC class I molecules. Therefore, most CTL activation strategies use live vectors although an alternative possibility could be to deliver the epitope into the cytosol by targeting it to an invasive nonreplicative vector. The adenylate cyclase toxin of Bordetella pertussis is able to invade a large number of eukaryotic cells and to deliver its catalytic domain to the cytosol of the cells. In the present study, we have tested the in vivo immunogenicity of recombinant adenylate cyclase toxins expressing CTL epitopes either from the nucleoprotein of lymphocytic choriomeningitis virus or from the V3 region of HIV-1 gp120. BALB/c mice immunized with these toxins developed high specific CTL responses that were shown to be mediated by class I-restricted CD8+ CTL. The induction of CTL responses by recombinant toxins did not require CD4+ T cells and the cytotoxic activity persisted 2 mo after immunization. The activation of CTL responses by the recombinant adenylate cyclase toxin required the full-length invasive activity of the toxin but did not depend upon its catalytic adenylate cyclase activity as demonstrated with a genetically inactivated recombinant toxin expressing the lymphocytic choriomeningitis virus epitope. This genetically detoxified invasive toxin represents, therefore, an attractive new vector for CTL activation.

Induction of measles virus‐specific cytotoxic T‐cell responses after intranasal immunization with synthetic peptides

We have investigated the structural requirements for the induction of cytotoxic T-cell responses (CTL) in vivo after intranasal immunization with an immunodominant CTL epitope from the nucleoprotein of measles virus (MV). For the induction of CTL responses, covalent linkage of the CTL epitope to a helper T-cell epitope was required and the orientation of the epitopes influenced the immunogenicity of the CTL epitope. The presence of two copies as compared with one copy of a T-helper epitope, rendered the CTL epitope more immunogenic and resulted in the in vivo induction of MV-specific CTLs without the need for an adjuvant. The role of CTL responses to this epitope in protection after intranasal administration was evaluated in a mouse model against challenge with a neuroadapted strain of MV. Although a decreased mortality in the peptide immunized compared with that in unimmunized mice was observed, the protection achieved was not significant. These findings highlight the importance of the rational design of synthetic immunogens for the induction of CTL responses and the potential of the intranasal route for immunization.

Induction of HIV-specific cytotoxic T lymphocytes in vivo with hybrid HIV-1 V3:Ty-virus-like particles.

Abstract In general, it has proven difficult to induce CTL responses using simple proteins or peptides without resorting to specialized adjuvants. In this study we show that particulate polymeric Ag in the form of hybrid Ty virus-like particles carrying the V3 region of HIV-1 gp120/160 envelope protein (V3:Ty-VLP) induce V3-specific CTL in BALB/c mice in the absence of adjuvant or lipid vehicle. In vitro restimulation of splenocytes with V3 peptide was necessary in order to generate effector CTL. Th cell activation was not required for this in vitro restimulation phase. The CTL induced by the V3:Ty-VLP were CD8+ve, H-2d-restricted, and HIV-1 isolate-specific (IIIB or MN). Co-administration of IIIB V3:Ty-VLP and MN V3:Ty-VLP primed both IIIB and MN V3-specific CTL. However, only IIIB V3-specific CTL were primed by hybrid Ty-VLP carrying IIIB, MN, and RF V3 loop sequences on the same particle indicating that there is intra- but not intermolecular competition between CTL epitopes. In direct comparisons, V3:Ty-VLP were substantially more potent than rgp120. Rgp160 and a 40mer IIIB V3 peptide both failed to prime V3-specific CTL. These data suggest that the particulate nature of hybrid Ty-VLP facilitates uptake into APC with subsequent access to the MHC class I processing pathway and that they may be useful vaccine vehicles for inducing cytolytic immunity against HIV-1 and other intracellular pathogens.

SUPPRESSION OF ALLOGRAFT RESPONSES BY COMBINING DONOR ALLOANTIGEN-SPECIFIC INTRAVENOUS PRESENSITIZATION WITH SUBOPTIMAL DOSES OF FK506

C57BL/6 (B6) mice were injected i.v. with class I H-2-disparate B10.QBR spleen cells (10(7)/mouse). This regimen, termed "donor alloantigen-specific i.v. presensitization" (DSP), induced almost complete elimination of anti-B10.QBR mixed lymphocyte reaction/IL-2 production but did not affect the generation of CTL responses. Repeated (5 or 11 times) administration in vivo (over 7 or 18 days) of FK506 at suboptimal doses (0.75-1.0 mg/kg/day) failed to eliminate the capacities to exhibit MLR/IL-2 production and to generate CTL responses. Prolongation of skin graft survival was not induced by either of a single DSP or FK treatment (0.75-1.0 mg/kg/day, 11 times during 18 days) alone, but by the combination of these. Such combined treatment also resulted in almost complete reduction of CTL responses before (5 rounds of FK injection) or after (11 rounds of FK injection) recipients were engrafted with B10.QBR skin grafts. Under conditions in which lymphoid cells from mice receiving both treatments failed to generate CTL responses, the addition of recombinant IL-2 to cultures restored the CTL generation, suggesting that CTL precursors themselves are not attenuated by the combined treatment. Both prolongation of graft survival and suppression of CTL responses were obtained when the administration of FK506 was started before but not after DSP. Prolongation of graft survival could also be obtained in class I and II MHC-disparate combinations when the combined treatment was performed in a particular protocol. These results indicate that (1) DSP alone fails to prolong graft survival in class I and class I and II MHC-disparate combinations; (2) such failure is ascribed to the induction of CTL responses by CTL precursors and CTL helpers, both of which are DSP-resistant; (3) the administration of suboptimal doses of FK506 is not sufficient for the suppression of CTL responses, but is effective selectively for suppressing DSP-resistant CTL helpers; and (4) the combination of DSP with FK506 treatment in an appropriate protocol can thus prolong graft survival through the suppression of CTL-involved as well as CTL-independent graft rejection pathways.

In vivo cytotoxic T lymphocyte induction with soluble proteins administered in liposomes.

The in vivo induction of a CTL response usually requires that Ag be endogenously synthesized so that appropriate processing can occur. In most of the few examples where successful CTL induction was reported with proteins and peptides, unacceptable adjuvants or means of Ag formulation were used. In the present report, liposomes were used to incorporate the soluble proteins OVA and beta-galactosidase. This simple and convenient to use approach, which requires minimal amounts of Ag, results in priming for a CD8+ CTL response and the establishment of immunologic memory. The liposome approach may not only prove a convenient means of inducing CTL responses in vivo but may also be useful to study the mechanisms of Ag processing.

In vivo induction of cytotoxic T cell response by a free synthetic peptide requires CD4+ T cell help.

Most attempts to induce CTL responses by in vivo priming with free synthetic peptides have been unsuccessful so far. However, two separate studies have recently succeeded in inducing antiviral CTL responses by immunizing mice with unmodified free synthetic peptides derived from nucleoproteins from either lymphocytic choriomeningitis virus or Sendai virus. In the present study, we have analyzed the cellular mechanisms by which the lymphocytic choriomeningitis virus synthetic peptide induced CTL responses. We demonstrated that this peptide, which was previously shown to be recognized by CD8+ T cells, also contains a helper CD4+ T cell epitope. It stimulates in vivo both CD4+ T cell-mediated CTL response. The in vivo elimination of CD4+ T cells by treatment with a mAb was shown to strongly reduce the antipeptide CTL response. This study therefore demonstrates that to be able to induce CTL responses, a peptide has to stimulate both CD4+ and CD8+ T cell subset.

ANALYSIS OF PRIMARY HLA-SPECIFIC CYTOTOXIC T CELL RESPONSE IN GRAFT-DRAINING LYMPH NODES— A TRANSGENIC MOUSE MODEL FOR IN VIVO RECOGNITION OF HUMAN MHC ANTIGENS

In order to characterize primary anti-HLA cytotoxic T cells and especially those involved in graft rejection, we have utilized a transgenic mouse model. Mice (non-transgenic and HLA-transgenic) were grafted with spleen cells originating from H-2-matched transgenic mice expressing HLA-B27 molecules, and cells from graft-draining lymph nodes were tested in CML assay to investigate the primary in vivo induced CTL responses. The results showed that HLA-B27 molecules were able to raise strong primary xenogeneic CTL responses. Results from split-well analysis indicated that although recognition of HLA-B27 by primary CTL induced in nontransgenic recipients is predominantly unrestricted by H-2, a small fraction (ranging from 2% to 27%) of the primary in vivo induced CTL is able to recognize HLA-B27 in an H-2-restricted manner. HLA-specific H-2-restricted CTL had never so far been demonstrated in the primary T cell response. Thus the protocol used in our study for the generation of a primary CTL response seems to provide not only a more appropriate representation of cytotoxic T cells sensitized by a graft, but also to be a more sensitive approach than the usually used in vitro mixed lymphocyte culture.

Presentation of the H-Y antigen on Langerhans' cell-negative corneal grafts downregulates the cytotoxic T cell response and converts responder strain mice into phenotypic nonresponders.

We have used the murine cornea is an allograft model to investigate the relative roles of graft-derived IA+ APC (Langerhans' cells) and host-derived APC during the induction of CTL responses to H-Y. The natural exclusion of LC from the immunizing corneal graft led to a specific state of unresponsiveness to H-Y in responder strain mice, while inclusion of LC resulted in responsiveness. Failure to respond to H-Y could not be attributed to the absence of H-Y or IA antigen expression on the surface of LC-deficient grafts but instead, appeared to be due to active suppression of the T helper cell response during in vivo priming. Reprocessing of the H-Y antigen by host APC did not occur after immunization with H-Y presented on H-2-incompatible grafts unless presented initially by graft-derived LC. H-2 as well as some non-H-2 alloantigens were presented to the host without a requirement for donor-derived LC. Thus there appear to be differential requirements for the processing and presentation of alloantigens.