LD Antigens Research Articles

Regulatory T Cells Suppress T Cell Activation at the Pathologic Site of Human Visceral Leishmaniasis

Suppression of T cell response is thought to be involved in the pathogenesis of visceral leishmaniasis (VL). Regulatory T cell (Treg) mediated immune-suppression is reported in animal models of Leishmania infection. However, their precise role among human patients still requires pathologic validation. The present study is aimed at understanding the frequency dynamics and function of Treg cells in the blood and bone marrow (BM) of VL patients. The study included 42 parasitologically confirmed patients, 17 healthy contact and 9 normal bone marrow specimens (NBM). We show i) the selective accumulation of Treg cells at one of the disease inflicted site(s), the BM, ii) their in vitro expansion in response to LD antigen and iii) persistence after successful chemotherapy. Results indicate that the Treg cells isolated from BM produces IL-10 and may inhibit T cell activation in IL-10 dependent manner. Moreover, we observed significantly higher levels of IL-10 among drug unresponsive patients, suggesting their critical role in suppression of immunity among VL patients. Our results suggest that IL-10 plays an important role in suppression of host immunity in human VL and possibly determines the efficacy of chemotherapy.

Lentivirally Transduced Recipient-derived Dendritic Cells Serve to Ex Vivo Expand Functional FcRγ-sufficient Double-negative Regulatory T Cells

αβTCR+CD4-CD8- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress antigen-specific immune responses mediated by CD8+ and CD4+ T cells in mice and humans. In this study, we developed a system to expand DN Treg cells for transplantation therapy that exclusively uses recipient-derived immune cells and confers a high degree of safety as the protocol does not involve the direct injection of lentiviral vectors. Recipient-derived dendritic cells (DCs) were transduced with lentiviral vectors that express major histocompatibility complex class I Ld antigen (LV-Ld), which is expressed by the donor graft but is allogeneic to the graft recipient. LV-Ld-transduced mature DCs (mDCs) were able to expand effectively both FcRγ-/- and FcRγ+/+ DN T cells. After expansion with LV-Ld-transduced mDCs, only the FcRγ+/+ DN Treg cells maintained their ability to suppress CD8+ T cells in vitro. In addition, adoptive transfer of the FcRγ+/+ex vivo expanded DN Treg cells significantly prolonged the survival of Ld+ skin grafts. This study is the first description of successful ex vivo expansion of antigen-specific DN Treg cells using genetically modified syngeneic DCs for adoptive immunotherapy and demonstrates that although FcRγ-/- DN T cells can be expanded, they do not gain regulatory ability.

Immunogenicity of Ld+ transgenic mouse hearts

Background. C57BL/6 mice transfected with the Ld gene coupled to the α-myosin heavy chain promoter result in transgenic mice with Ld antigen expressed only on cardiac tissue. These transgenic animals allow the examination of immune reactivity against cardiac Ld by “self” or by adoptively transferred Ld specific 2C cells, and the response of nontransgenic C57BL/6 mice to the transplanted Ld+ heart. Methods. Naïve cardiac Ld+ transgenic mice were examined for evidence of Ld “autoimmunity.” Forty million fresh 2C cells or 2C cells sensitized in vitro for 7 days against Balb/c (Ld+) + interleukin-2 were also given intravenously to Ld+ transgenic mice. At 5 and 12 days after injection, heart-infiltrating lymphocytes were analyzed by fluorescence-activated cell sorter. The Ld+ transgenic hearts were also transplanted to syngeneic Ld− nontransgenic C57BL/6 to evaluate the heart's immunogenicity. Results. Naïve Ld+ transgenic mice did not exhibit any evidence of lymphocytic infiltration on histologic examination. Adoptive transfer of either fresh or in vitro sensitized 2C cells was also unable to reject the native Ld+ heart in transgenic mice (100% of the mice survived long term [more than 60 days]). Sensitization of the Ld+ transgenic mice with a Balb/c skin graft and interleukin-2 pump infusion (7 days) beginning 1 day before 2C cell injection also did not promote rejection of the native Ld+ heart. However, fluorescence-activated cell sorter analysis did reveal that a significantly greater number of in vitro sensitized 2C cells homed to the Ld+, but not Ld−, heart after both 5 and 12 days (P <.01, P <.001). In contrast, C57BL/6 mice rejected the Ld+ (C57BL/6 background) transgenic heart in a mean survival time of 17 ± 9.7 days (P <.01), whereas a syngeneic C57BL/6 heart transplant was accepted indefinitely. Lymphocytic infiltration consistent with rejection was present in all animals receiving an Ld+ transgenic heart transplant, whereas no infiltrate was present in those receiving a syngeneic C57BL/6 heart transplant. Conclusions. Although the class I Ld transgene is not recognized in its native host, its immunogenicity is shown by the homing of anti-Ld 2C cells to the heart in situ and rejection of Ld+ heart grafts when transplanted into syngeneic C57BL/6 mice. (Surgery 2001;130:217-24.)

Resting and activated T cells display different requirements for CD8 molecules.

Clonotype-positive (1B2+) T cells from 2C T cell receptor (TCR) transgenic mice were used to define the role of CD8 molecules in the induction phase vs. the effector phase of the primary response to class I alloantigens. Three main findings are reported. First, in the presence of exogenous lymphokines, resting CD8+ 2C cells gave strong proliferative responses to two alloantigens, Ld and Kbm11. In the absence of added lymphokines, however, CD8+ 2C cells responded only to Ld and not to Kbm11; Ld stimulated both interleukin 2 (IL-2) and IL-2 receptor (R) synthesis, whereas Kbm11 elicited only IL-2R synthesis. The primary response of CD8+ 2C cells was thus helper-independent (HI) to Ld but helper-dependent (HD) to Kbm11, presumably reflecting that Ld is a stronger antigen than Kbm11. Second, in contrast to CD8+ cells, CD8- 2C cells mounted only an HD and not an HI response to the strong Ld antigen; conversely, selecting for a minor subset of CD8hi cells enabled 2C cells to mount an HI response to the weak Kbm11 antigen. These findings, together with experiments with heterozygous vs. homozygous stimulator cells, suggest that HI and HD responses reflect differences in the overall avidity of T antigen presenting cell (APC) interaction: high-avidity interaction leads to strong intracellular signaling and an HI response, whereas low-avidity interaction causes weak signaling and an HD response; high-avidity T/APC interaction is heavily dependent on CD8 expression. Third, CD8 expression was found to be less important for CTL activity than for primary proliferative responses. Thus, in contrast to HI proliferative responses, CTL responses of 2C cells to Ld were CD8 independent. However, 2C lysis of Ld targets became strongly CD8 dependent in the presence of limiting doses of anti-TCR (1B2) antibody. Collectively, the data suggest that, both for T cell induction and the expression of effector function, CD8 molecules play a decisive role in augmenting TCR-mediated signaling, either by promoting TCR contact with antigen or delivering kinases (p56lck) to the TCR/CD3 complex, or both.

Embryonic Expression of MHC Class I Heavy and Light Chains in Transgenic Mice

Class I membrane glycoproteins encoded by major histocompatibility complex (MHC) genes are not expressed during early stages of development. This regulation is thought to play an important role in maternal tolerance of the fetal allograft. To elucidate the significance of developmental regulation of MHC class I genes, we produced transgenic mice expressing transgenes encoding the class I Ld heavy chain or the light chain, β2-microglobulin, in the developing mouse embryo. These transgenes were driven by the chiken β-actin promoter, which is very active in the developing mouse embryo. The heavy chain and light chain transgenic mice were mated, and the resultant double transgenic offspring expressed Ld antigen in all the tissues examined by immunostaining and Northern blot analysis. The Ld antigen was also detected by immunostaining in the placenta of the double transgenic fetus. The double transgenic fetus was not rejected by the immunocompetent nontransgenic mother. These results suggest that expression of MHC class I antigens in embryos is not sufficient to provoke a maternal immune response.

Prevention of autoimmune insulitis in nonobese diabetic mice by expression of major histocompatibility complex class I Ld molecules.

Nonobese diabetic (NOD) mice spontaneously develop a T-cell-mediated autoimmune disease that is similar in many respects to insulin-dependent diabetes mellitus in humans. NOD mice were shown to express major histocompatibility complex class I Kd and Db antigens. To examine the possible involvement of major histocompatibility complex class I molecules in the development of autoimmune insulitis, we attempted to express a different type of class I molecule in NOD mice by crossing C57BL/6 mice transgenic for the class I Ld gene with NOD mice. The backcross progeny expressed the Ld antigen on the peripheral blood lymphocytes at a level comparable with that of the BALB/c mice. The cell surface expression of endogenous class I and class II antigens on the peripheral blood lymphocytes was not affected. Analysis of these mice revealed that the expression of the class I Ld antigen significantly reduced the incidence of insulitis at 20 weeks of age. In situ hybridization of a biotinylated probe on mouse chromosomes showed that the Ld transgene was located in the E area of chromosome 6 with which no genetic linkage to insulin-dependent diabetes mellitus was demonstrated. These results suggest that the NOD-type class I molecules are involved in the development of insulitis in NOD mice.

H-2 class I Ld antigen positively selects different TCR V beta gene products in CD8+ and CD4+ T cells.

Expression of CD4+ or CD8+ determinants and ten different TCR V beta genes was analysed by two-colour flow cytometry in lymph node cells (LNC) of BALB/c and dm2 (a BALB/c Ld loss mutant) mice. TCR V beta 14 expression of CD8+ T cells and TCR V beta 7 and 14 of CD4+ T Cells were significantly more frequent in BALB/c than in dm2 LNC, whereas no differences were observed in the frequency distribution of TCR V beta+ CD4-CD8- double negative LNC from BALB/c versus dm2 mice. These results represent the first published evidence for positive selection of particular TCR V beta genes by individual MHC class I molecules in non-manipulated mice.

Major histocompatibility complex binding and T cell recognition of a viral nonapeptide containing a minimal tetrapeptide

The primary immune response of cytotoxic T lymphocytes in H-2d and H-2q mice to infection with lymphocytic choriomeningitis virus is directed mostly towards the common major T cell epitope of amino acids 112-132 on the viral nucleoprotein (NP). The molecules responsible for presentation of the T cell epitope NP112-132 are in both haplotypes the MHC class I L antigens (Ld, Lq). Truncations of the amino and carboxy termini of the NP 112-132 sequence revealed the nonapeptide RPQASGVYM (NP118-126) as a most effective peptide antigen, but even the tetrapeptide GVYM was recognized by CTL of both haplotypes in a class I antigen-restricted specificity. When tyrosine (Y) or methionine (M) were substituted with alanine, CTL recognition of the altered nonamer required 10(6) to 10(8) times higher peptide concentrations and in one case (Y----A on Ld) the peptide was not recognized at all. Up-modulation of the expression of Ld and Lq class I antigens as measured by flow cytometry correlated with the ability to present the peptide antigens. The only exception was peptide NP118-126 (M----A), which was recognized by T cells on L-Ld and L-Lq target cells but failed to up-regulate Ld and Lq antigens.

Altered growth of a human neuroendocrine carcinoma line after transfection of a major histocompatibility complex class I gene.

The major histocompatibility complex (MHC) class I molecules are known to serve as recognition elements for cytotoxic T cells in mediating the rejection of transplanted tumors. We demonstrate that MHC molecules may have nonimmune functions in modulating tumor cell growth in addition to their classical role in antitumor immunity. A human neuroendocrine carcinoma cell line, COLO 320, with low levels of endogenous class I expression was transfected with the murine H-2Ld gene. Eleven independent stable clones were established, four containing only pRSV-neo and seven also containing varying copy numbers of the transfected Ld gene. The ability of the different clones to grow as colonies in soft agar correlated strongly with the relative amounts of Ld antigen expression (r = 0.89; P less than 0.001). There was a weaker correlation between increased clonogenic ability and higher levels of Ld mRNA (r = 0.67; P less than 0.05). There was no correlation between clonogenic ability and relative expression of amplified c-myc gene or of integrated pRSV-neo. Furthermore, in nude mice, Ld antigen expression was associated with increased formation of metastatic lung colonies 6 weeks after intravenous injection of 10(5) cells. These observations are consistent with the concept that MHC class I antigens may have a role in modulating the growth potential of certain tumor cells independent of their involvement in immune responses.

Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants.

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(alpha 1) and C1(alpha 2) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2Ld has been replaced by that of H-2Dd. Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2Dd replaced by that of H-2Ld) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2Ld and H-2Dd or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2Ld and H-2Dd to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2Ld substituted for that of H-2Dd), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2Ld or anti-H-2Dd N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of beta 2M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2Dd and anti-H-2Ld CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2Ld-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular studies of murine mutant BALB/c-H-2dm2 define a deletion of several class I genes including the entire H-2Ld gene.

Inbred mouse strains carrying spontaneous mutations in class I genes have been extremely informative in studies of the genetic mechanisms generating polymorphism in the major histocompatibility gene complex. In this report, we determine the molecular basis of the spontaneous loss mutation in BALB/c-H-2dm2 mice, which were previously shown not to express Ld antigens while maintaining normal expression of two other class I antigens, Kd and Dd. We show BALB/c-H-2dm2 mice do not transcribe detectable levels of Ld mRNA, indicating they do not produce a truncated Ld molecule as previously reported. Furthermore, in Southern blot comparisons using a series of low-copy genomic probes, the deletion was found to be approximately 140 kilobases and include the entire Ld gene along with three or more other class I genes mapping between Dd and Ld. These data represent direct genetic evidence for a spontaneous contraction in the genes encoding class I histocompatibility antigens, which in this case probably resulted from the misalignment of the 3' flanking regions of the Dd and Ld genes.

Molecular loss variants of the murine major histocompatibility complex: nonexpression of H-2K antigens associated with marked reduction in H-2K mRNA as determined by oligonucleotide hybridization analysis.

Somatic cell variants of the murine major histocompatibility complex were isolated to study the molecular features required for H-2K gene expression. In vitro selection was performed on a heterozygous [H-2b (Kb,Db) X H-2d (Kd, Dd, Ld)] pre-B lymphoblastoid cell line (R8) for variants that had lost membrane expression of the H-2Kb gene product. Analysis of a number of independently isolated variant cell lines by cytofluorometry with monoclonal antibodies to the Kb, Kd, Db, and Ld antigens revealed a variety of H-2 phenotypes. Variants were classified as either molecular loss for those that had lost K antigen expression only or as haplotype loss for those that no longer expressed the entire H-2b haplotype (i.e., negative for Kb and Db). DNA hybridization analysis with a K gene-specific oligonucleotide indicated that the Kb gene was present in all of the molecular loss variants, suggesting that gene deletion was not responsible for the loss of Kb antigen expression. In contrast, the Kb gene was not detected in haplotype loss variants. For analyzing the mutants at the RNA level, hybridization with H-2-specific synthetic oligonucleotides provided a definitive procedure to identify specific class I gene transcripts in the H-2 heterozygous cell lines. Such analyses were performed on the molecular loss variants and revealed that in a subset of variants (R8.2, R8.96, R8.116, R8.178) there was an absence of Kb mRNA. Kd mRNA was identified for all but one (R8.2) of the Kb mRNA-deficient cell lines, a finding consistent with the serotype assigned to each. Transcription of the linked Db gene was normal. These data demonstrate that a specific alteration for the K gene in several independently selected cell lines gave rise to the altered H-2 phenotype. Such variants offer the potential to analyze the properties of the mammalian genome responsible for controlling expression of individual members of the major histocompatibility complex multigene family.

Slower processing, weaker beta 2-M association, and lower surface expression of H-2Ld are influenced by its amino terminus.

To determine why Ld antigens are expressed on the cell surface at levels three to four times lower than Dd or Kd antigens, pulse-chase experiments were used to compare their rates of biosynthesis and processing. Electrophoresis on sodium dodecyl sulfate gradient polyacrylamide gels resolved immunoprecipitates of each of these histocompatibility complex class I molecules into a slower and faster species. During the chase period, the faster migrating species appeared to be converted to the slower migrating species in a time-dependent manner. However, the conversion of Ld from the faster to the slower migrating species proceeded significantly more slowly than did the conversion of either Dd or Kd. Endoglycosidase H sensitivity and cell surface radiolabeling were used to determine the glycosylation state and cell location of each species of Ld and Dd. The results from these experiments, along with the pulse-chase studies and cytofluorometric analyses, suggest that Ld possesses a much slower rate of processing from a faster migrating, high mannose-bearing species to a slower migrating, complex oligosaccharide-bearing species found on the cell surface. Analysis of the beta 2-microglobulin (beta 2-m) association confirmed that Ld is associated with less beta 2-m than Dd. To localize the structures on class I molecules influencing their surface expression, rate of processing, and beta 2-m association, the Ddm1 molecule was analyzed. The Ddm1 molecule of the mutant B10.D2-H-2dm1 has previously been shown to be a chimeric Dd (amino-terminal)/Ld (carboxyl-terminal) polypeptide. The surface expression, processing and beta 2-m association of Ddm1 were found to be similar to Dd rather than Ld, suggesting that each of these phenomena are influenced by protein structure in the amino terminus.

Mice coisogenically immunized against H-2 class I antigens on transfected L cells reject transplanted embryonal carcinoma cells.

Evidence is presented of the ability of H-2 class I antigens to function as teratocarcinoma transplantation (Gt) antigens. Coisogenic immunization against H-2 class I antigens expressed on transfected L cells is shown to induce resistance to embryonic carcinoma (EC) cell allografts. The Kb, Db, Dd, and, in appropriate recipients, Ld antigens can function as Gt antigens. The protocol presented may be useful for the molecular identification of other genes encoding histocompatibility antigens.

Molecular localization of allogeneic and self determinants recognized by bulk and clonal populations of cytotoxic T cells.

The localization of MHC-encoded determinants recognized by hapten- and allo-specific cytotoxic T cells was analyzed with the use of cell lines expressing recombinant H-2Dd and H-2Ld MHC products. Bulk cultures of CTL against TNP-self, FITC-self, and AED-self recognized self determinants associated with the N/C1 domains of both Dd and Ld products. A number of allo- and hapten-specific CTL clones were also tested for recognition of the recombinant MHC products. The allo clones specific for Ld or Dd antigens recognized the respective N/C1-associated determinants. In addition, all clones generated against H-2q and known to cross-react with H-2Dd antigens recognized determinants associated with the N/C1-associated Dd determinants. Thus, some of the results obtained with CTL parallel, whereas others contrast with, those findings obtained with monoclonal anti-H-2 antibodies. Similar to the observations made with the monoclonal antibodies, no determinant as defined by T cells has been found to be lost as a result of the interaction between the N/C1 and C2/M domains of the Ld and Dd proteins. Nor did our studies detect the presence of new antigens resulting from the interaction of these gene products. However, the present T cell findings continue to contrast previous results demonstrating that antibody interaction with class I products includes recognition of C2/M-associated epitopes.

Lateral diffusion of wild-type and mutant Ld antigens in L cells.

We have compared the lateral diffusion of intact transmembrane proteins, wild-type H-2Ld antigens, with that of mutants truncated in the cytoplasmic domain. Diffusion coefficients and mobile fractions were similar for all molecules examined, from wild-type Ld antigens with 31 residues on the cytoplasmic side of the plasma membrane to mutants with only four residues in the cytoplasmic domain. This result limits ways in which the lateral diffusion of a major histocompatibility antigen, a transmembrane protein, can be constrained by interactions with other molecules.

Lack of H-2Ld locus products on a BALB/c fibrosarcoma expressing H-2k-like alien antigens.

The presence of H-2Ld antigens was evaluated in methylcholanthrene-induced BALB/c fibrosarcomas by a variety of approaches. Transplantation experiments showed that BALB/c-H-2dm2 mice, a mutant strain whose cells do not express H-2Ld antigens, after immunization with BALB/c normal tissues developed a resistance to the growth of two tumours (C-3 and GI-17), but not to a third neoplasm, C-1, which is known to have H-2d- as well as H-2k-like alien antigens. In vitro experiments with cytotoxic T lymphocytes generated against Ld antigens confirmed a loss of Ld antigens on C-1 but not on C-3 tumour cells. Serological experiments with an anti-Ld serum again revealed the presence of H-2Ld determinants on C-3 but not on C-1 cells. Biochemical analysis in SDS-PAGE of immunoprecipitates obtained by specific anti-H-2 sera with NP40 lysates of the tumours studied could detect H-2Kd, H-2Dd and H-2Ld antigens in C-3 fibrosarcoma cells whereas Kd and Dd were the only H-2d molecules found in C-1 lysate along with the H-2k-like specificities. The possible genetic mechanisms which may explain this apparent gain and loss modification of the H-2 profile of C-1 are discussed.

Heterogeneity of H-2D Region Associated Genes and Gene Products

Publisher Summary It is clear that the once accepted dogma that there is only one gene product for each H-2K or H-2D region is incorrect, at least for certain haplotypes. In the case of the H-2D region of the d haplotype designated Dd, there is now a preponderence of data demonstrating at least two gene products, Dd and Ld, determined by two separate genes H-2Dd and H-2Ld, respectively. The cell surface expression of Ld antigens appears 2 to 3 times lower than that of Kd and Dd antigens, and H-2L gene products have been proposed to have unique antigenic properties rendering them less polymorphic than H-2K and H-2D gene products. New technical advances promise to provide the tools to address a few important questions. The availability of monoclonal antibodies to H-2K and H-2D antigens has provided a technical stimulus for the study of class I antigens. Monoclonal antibodies have facilitated the serologic and molecular resolution of H-2 antigens and have circumvented several of the caveats encountered using complex allo-antisera. Recombinant DNA technology not only provides precise information on the genetic organization of H-2 genes, but when taken in the context of serologic, structural, and chemical findings, they should also yield invaluable information regarding the genetic evolution of H-2 molecules.

Expression of histoincompatibility antigens during spermatogenesis in the rat.

The expression of the AgB-SB and -LD (Ia-like) antigens on spermatogenic cells has been investigated by using a protein A-containing Staphylococcus Cowan I rosetting assay. The results show that the Ia-like antigens are present on all spermatogenic cells although the relative expression seems to decrease slightly concomitant with the differentiation. The AgB-SD antigens are detected on spermatogenia, leptotene, and zygotene spermatocytes but not on later developmental stages.

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cells: I. Metabolic inactivation impairs both CD and LD antigen signals

The effects of metabolic inactivation of spleen cells on antigen presentation to precursors of alloreactive cytolytic T lymphocytes (T c) were examined. By serological methods, populations inactivated by ultraviolet irradiation, glutaraldehyde fixation or plasma membrane isolation were found to retain normal levels of H-2K/D and Ia antigens. However, comparison of the antigen doses required to stimulate secondary T c responses in mixed leukocyte culture showed that the inactivated preparations were approximately 10-fold less immunogenic than X-irradiated spleen cells. Their total inability to stimulate primary cytolytic responses pointed to at least a 100-fold impairment of immunogenicity for unprimed T c precursors in the case of uv-irradiated and glutaraldehyde-treated stimulator cells, and at least a 10-fold impairment for membrane fragments. Experiments showing that the capacity of cell monolayers to absorb precursor T c from unprimed spleen populations was reduced following uv-irradiation or glutaraldehyde treatment provided direct evidence that this loss of immunogenicity was due in part to suboptimal antigen presentation to precursor T c. It is concluded that, in addition to the traditional view that these treatments damage the “LD” signal to helper T lymphocytes, metabolic inactivation also impairs recognition of “CD” determinants by precursor T c.