Ability of fixed B-lymphoma cells to present foreign protein antigen fragments and allogenic MHC molecules to a cloned helper-T-cell line

Cloned, L3T4+ T cells have been shown to respond to foreign protein antigens in the context of self-Ia glycoproteins and to non-self Ia glycoproteins. In the case of responses to foreign proteins, fixed antigen-presenting cells can present antigen fragments, but cannot present native proteins. Whether fixed allogenic cells can stimulate has been controversial. We have examined this question using a dual-reactive cloned helper-T-cell line. We find that conditions of fixation that block the presentation of native antigen to this cloned line, but which allow the presentation of antigen fragments, also allow presentation of allogeneic Ia molecules, leading to stimulation of the cloned line. This study also revealed an occult alloreactivity in the cloned T-cell line, which was expressed by fixed, but not by normal, antigen-presenting B lymphoma cells. All of these stimuli proceeded via the same clonotypic receptor, as determined by blocking with anti-T-cell receptor monoclonal antibody. These data suggest that responses to non-self Ia glycoproteins involve direct recognition of the allogeneic Ia molecules and do not require processing and presentation of these antigens by self Ia molecules.     

Ia-specific mixed leukocyte reactive T cell hybridomas: analysis of their specificity by using purified class II MHC molecules in synthetic membrane system

This study was undertaken to determine the nature of the antigens recognized in allogeneic and syngeneic mixed leukocyte reactions (MLR). Specifically, we wished to determine whether Ia antigens alone were recognized by MLR-reactive T cells, or whether the specificity was determined by the corecognition of non-MHC antigens together with syngeneic or allogeneic Ia. To do this we used 11 T cell hybrids that were characterized as being specific for Iad and were tested their capacity to respond to isolated I-Ad or I-Ed that had been incorporated into liposomes and had bound to the surface of glass beads. Of nine alloreactive T cell hybrids (five I-Ad-and four I-Ed-specific), seven were shown to be responsive to the relevant isolated Ia antigen on glass beads. Also, two of two syngeneic I-Ad-specific T cell hybrids responded to I-Ad on the glass beads. One of the two alloreactive T cell hybrids that failed to respond to the relevant Ia antigen on glass beads was shown to be specific for an antigen in fetal calf serum (FCS) that was recognized in the context of the allo-Ia antigen (I-Ed), because when intact accessory cells were used, a response by this hybrid was only observed when FCS was present in the assay culture medium or when the accessory cells were pre-pulsed with FCS. The possible involvement of FCS antigens and non-Ia accessory cell antigens in the stimulation of the nine T cell hybrids that responded to isolated Ia on glass beads was evaluated. T cell hybrids that were grown and were tested in serum free medium were still capable of reacting to Ia on beads. The isolated Ia preparations used were greater than 90% pure, and their capacity to stimulate the T cell hybrids did not correlate with the degree of contamination with non-Ia proteins. We conclude from these studies that the majority of T cells that respond to allogeneic or syngeneic Ia bearing stimulator cells are specific for the Ia antigens themselves, and do not require the co-recognition of other non-Ia antigens; nor is there any requirement for Ia antigen processing for this recognition.     

Astrocytes as antigen-presenting cells. I. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation

Ia antigens seem to control immune responses on at least two levels. First, they influence the antigen recognition repertoire of the T cells. Second, their variable expression on certain antigen-presenting cells is a powerful regulatory mechanism for the local immune reaction. This is particularly important in the central nervous system (CNS) in which no Ia antigens are normally expressed. Recent experiments in this context have shown that astrocytes are able to express Ia antigens during interaction with T cells, and that they function as antigen-presenting cells. The Ia-inducing activity is produced by activated T cells, and can be replaced by immune interferon (IFN-gamma). In this study we report on the functional and kinetic relationship between Ia antigen expression on astrocytes and the immune-specific activation of T cells by astrocytes. Normal resting astrocytes were found to be negative for Ia antigens by immunofluorescence and by biochemical criteria. Moreover, they are only able to stimulate T cells after they have been induced to express Ia antigens by a signal from the T cells, which is probably mediated by IFN-gamma. In conclusion, the immune-specific interaction between astrocytes and T lymphocytes is a sensitively controlled system that might be pivotal to the development of immune responses in the brain. Malfunction of the system could be an important factor in the pathogenesis of aberrant immune reactions in the CNS, e.g., in multiple sclerosis.     

Mitogens and T-independent antigens stimulate T lymphocytes to secrete La antigens.

Previous reports from this laboratory suggest that certain I region-associated (Ia) antigens can be detected in normal mouse serum. It was found that, when mitogens are injected into mice, they produce substantial increases (up to 125-fold) in the levels of these Ia antigens in mouse serum. Similar increases were obtained when either T- or B-cell mitogens were injected. Furthermore, in vitro and in vivo studies demonstrated that the mitogens stimulated T cells to secrete Ia antigens. It appears likely, however, that the Ia antigens detected in these studies may differ from the conventional Ia glycoproteins found on the surface of B lymphocytes.All T-independent antigens tested also augmented the concentrations of Ia antigen in serum, the increases depending on the T-independent antigen injected and ranging from 3- to 125-fold. In contrast, T-dependent antigens, unless injected in large amounts, were unable to produce detectable changes in the serum levels of Ia antigen. These data indicate that an inverse relationship exists between the T dependence of an antigen and its ability to stimulate T cells to secrete Ia antigens. On the basis of this conclusion it is proposed that all antigens are T dependent and merely vary in the efficiency with which they activate T cells to release helper factors.     

A comparison of the stimulatory activities of lymphoid dendritic cells and macrophages in T proliferative responses to various antigens

The identities of murine accessory cells and the mechanism by which they process antigen and stimulate T cell proliferation have been examined with cell separation techniques and specific agents to block antigen catabolism. Using preparations of splenic dendritic cells (DC) and macrophages (M phi) with minimal cross-contamination, we found that only DC could induce syngeneic mixed leukocyte reaction (MLR), whereas both DC and M phi could initiate allogeneic MLR. This observation may have significant implications for syngeneic MLR as a manifestation of self Ia recognition, and for the cell type that defines self Ia during ontogeny. DC and M phi could present soluble antigens such as purified protein derivative of tuberculin (PPD) and Salmonella flagellin about equally well to antigen-specific T cell lines. M phi, however, were much more effective than the non-phagocytic DC at inducing T cell proliferation to whole Corynebacterium parvum organisms. These differences could not be attributed to differences in antigen uptake. The results suggest that the bacteria must be ingested and processed by phagocytes before T cell activation. Using the lysosomotropic agent chloroquine to inhibit antigen catabolism in accessory cells, we found that the presentation of large antigens by M phi and DC was abolished by chloroquine treatment, whereas T cell activation by antigens (such as PPD or integral membrane Ia for MLR) that apparently required no processing was relatively insensitive to chloroquine. Thus, in addition to differences between cells, discrete functions within each cell type can also be distinguished.     

Hybrid Ia antigens: Genetic,serologic, and biochemical analyses

Ia specificities 22 and 23 were found to be determinants on hybrid Ia molecules, formed by the noncovalent binding of a 26,000–28,000 dalton beta polypeptide chain (A_e) coded by the I-A subregion and a 32,000–35,000 dalton alpha chain (E_α) coded by the I-E subregion. For expression of Ia. 23 the A_e chain, coded by the I-A subregion, must be derived from the H-2^d haplotype, while A^b, A^s, or A^k can provide the complementing beta chain for the expression of Ia. 22. For expression of Ia. 22 and Ia. 23, most Ia. 7 positive strains can provide the complementing alpha chain (E_α), with the one exception of B 10. PL (E^u), which is Ia. 7 positive but will not complement with A^d to express Ia. 23. Antisera were also produced against hybrid Ia antigens by immunizing with F₁ cells expressing Ia. 22 or Ia. 23 generated by transcomplementation. These antisera detect the same specificities as conventional anti-Ia. 22 and anti-Ia. 23 sera produced against cis-complementing Ia antigens. It is postulated that hybrid Ia determinants are involved in recognition and generation of immune response to antigens under dual Ir gene control. It is also suggested that there are 2 types of Ia specificities: (1) allotypic Ia specificities expressed on the alpha or beta chains (for example, Ia. 7 on the E_α chain) and (2) hybrid Ia specificities, which are unique interaction determinants formed by the association of alpha and beta chains (for example, Ia. 22 and Ia. 23). These interaction gene products may be involved in antigen recognition and presentation.     

Protective efficacy against group B streptococcal infection in neonatal mice delivered from preimmunized pregnants

Neonatal mice delivered from mothers preimmunized with heated or formalinized whole cell vaccines of type Ia, Ia/c and III/c group B streptococci were infected with each type of bacteria, and then serum antibodies of mothers and neonates who survived the experiments were measured by enzyme-linked immunosorbent assay. The relationship between the protectivity in neonate mice and the antibody titers to the type specific polysaccharide antigens and the protein c antigen of their sera were examined. In the Ia-immunized group which showed high protection against the type Ia infection, anti-Ia IgG antibody titers were low, and anti-protein c IgG antibody was not detected. Type Ia/c and III/c vaccines were highly effective against both type Ia/c and III/c infection, but less effective in type Ia infection. The protein c antigen was identified in both type strains by the double diffusion assay, and the IgG antibodies to the protein c were significantly high in sera of both maternal mice immunized with types Ia/c or III/c organisms and their newborn infants. High titers of the protein c IgG antibody retained 3 to 4 weeks after the last injection of vaccines which corresponded to the period of pregnancy and lactation. Small amounts of IgM antibody to all antigens were detected only in maternal sera. These results suggest that IgG antibodies to the protein c antigen and to the type-specific polysaccharide antigens are equally important protective factors which are transferable from preimmunized mothers to their newborn infants through placenta and/or lactation.     

Long-term-cultured mouse B-lymphocyte line. II. Modulation of Ia-antigen expression on B-lymphocyte line

Mouse B-cell line, established by culturing anti-Thy-1 and complement-treated splenic B cells with concanavalin A-stimulated conditioned medium, expressed immunoglobulins and Ia antigens on its surface. The long-term-cultured B-cell line was split in two and maintained with or without 3300 R X-irradiated T-cell-depleted syngeneic splenic adherent cells (SAC). Interestingly, the B-cell line cultured without SAC lost its Ia antigen but not its Ig expression, whereas the cell line with SAC maintained both Ia and Ig expression. The ability to express Ia antigens was restored by culturing them only in the presence of Ia-positive feeder cells. Neither recombinant interferon-gamma or lectin-stimulated conditioned medium nor cell-free culture supernatant SAC had the ability to restore Ia antigen expression on the B-cell line. Incubation of Ia-negative B-cell line with phorbol esters restored the Ia expression. It is suggested that the expression of Ia antigen on B lymphocytes was controlled differently from that on macrophage lineage. The B-cell line expressing Ia antigens acts as stimulator cells for alloantigen-activated T lymphocytes and as antigen-presenting cells on the KLH-specific Ia-restricted proliferative T-cell clone in the presence of a specific antigen.     

T cell responses to select Ia determinants using the I-A mutant mouse strain B6.C-H-2bm12

The T cell repertoire of B6.C-H-2bm12 mice (an I-A mutant mouse strain) to wild-type Iab antigens was investigated using both secondary proliferative cultures and cloned T cell lines. Because bm12 mice have a gain-loss mutation of their gene encoding the Ia beta-chain polypeptide, bm12 anti-B6 T cell responses are specific for the select component of Iab specificities that was lost as a result of the mutation. Although stimulator cells bearing Iab antigens elicited the strongest responses, Iaq, d, and s antigens also resulted in reproducible stimulations of these bm12 anti-B6-primed T cells. Cloned T cell lines isolated from bm12 anti-b6 cultures revealed similar findings, with most clones recognizing determinants unique for Iab antigens; however, clones showing cross-reactions with Iad and/or q were also selected. Using F1 hybrid responder T cells (mutant x cross-reactive strain), we further dissected this cross-reactivity into several distinct cross-reactive determinants. Because bm12 mice lack the serologically defined Ia differentiation antigen W39, T cell recognition of this determinant was investigated by using bm12 anti-B6-primed cells. Stimulation by Ia.W39+ cells was appreciably better than by Ia.W39- (Xid-defective) cells, suggesting that bm12 T cells recognize an Xid-regulated, W39-like Ia differentiation antigen.     

Role of nominal antigen and Ia antigen in the binding of antigen-specific T lymphocytes to macrophages.

We have previously demonstrated that when primed T lymphocytes were repeatedly incubated on monolayers of antigen-pulsed macrophages (M phi), the cells that failed to adhere to the monolayer demonstrated a marked depletion of their proliferative response that was specific both for the antigen used for pulsing the M phi and for Ia determinants on the M phi. In order to further analyze the contribution of the nominal antigen and Ia antigens to the physical binding of T lymphocytes to M phi, we have attempted to block the absorption of T lymphocytes to M phi with a large excess of soluble antigen and with anti-Ia sera. Our results demonstrate that anti-Ia sera inhibit but that soluble antigen augments the binding of specific T lymphocytes to M phi. The implications of these findings for "dual recognition" and "linked recognition" models of T lymphocyte receptors are discussed.     

Murine thymocyte and splenocyte Ia antigens are indistinguishable by two-dimensional gel electrophoresis

Ia antigens have been found on the surface of B lymphocytes, macrophages, epidermal Langerhans cells and on certain transformed cells. Ia antigens have also been detected on the surface of thymocytes but the biosynthesis of these antigens by thymocytes has been difficult to demonstrate. We describe the labeling of murine thymocytes with 35S-methionine and the subsequent analysis of Ia antigens by two-dimensional polyacrylamide gel electrophoresis. Cell elimination experiments demonstrated that the Ia antigens detected were not of B cell origin and were synthesized by a Thy-1-positive thymocyte. Ia antigens from thymocytes were found to be indistinguishable from spleen Ia preparations. Since T cell I region determinants have been postulated to be involved in cellular recognition phenomena, models addressing this recognition must allow for the observation that T and B cell I region molecules detected by antisera such as A. TH anti-A. TL are indistinguishable by two-dimensional gel analysis and are thus unlikely to be involved in the generation of specificity in recognition.     

Cytofluorometric isolation of I937, an Ia antigen-bearing variant of the Ia-negative human monocytic cell line U937

Cells of the human monocyte cell line U937 are generally considered devoid of any Ia antigens on their surface. In analyzing U937 cells with a large panel of monoclonal anti-human Ia antibodies by flow cytometry, we detected a small number of cells that appeared to react with antibodies to HLA-DR and HLA-DS/DC molecules. These Ia-positive cells were isolated and were cloned, resulting in a human monocyte cell line that expresses high levels of Ia antigens. We analyzed these antigens by one- and two-dimensional polyacrylamide gel electrophoresis, after radiolabeling and immunoprecipitation. Three distinct Ia molecules, alpha 1 beta 1, alpha 1 beta 3 (HLA-DR-like), and alpha 2 beta 2 (HLA-DC/DS-like) are synthesized by I937 cells, alpha 1 beta 3 molecule being the predominant species. The Ia antigen-bearing human monocyte cell line is expected to be useful for studying events involved in antigen presentation.     

Mouse T lymphocytes proliferative responses specific for human MHC products in mouse anti-human xenogeneic MLR

It has been reported that human T cells recognize the polymorphism of murine Ia antigens in the human anti-mouse xenogeneic mixed lymphocyte reactions (MLR). In this study, murine T cell recognition of human Class II antigens of the major histocompatibility complex (MHC) was analyzed in mouse anti-human xenogeneic MLR responses. The xenoreactive murine T cell proliferative response was blocked by adding anti-HLA-DR monoclonal antibody to the xenogeneic MLR culture. The specificity of xenoreactive murine T cells was examined with regard to the secondary and tertiary xenogeneic MLR system. The xenoreactive murine T cells were restimulated by distinct human stimulator cells that had no shared HLA antigens with the stimulator used in the primary MLR. The data presented here show that the murine xenoreactive T cells recognize the shared determinant(s) of HLA-DR antigen on non-T, non-B stimulator cells. The xenoreactive murine T cell proliferative responses were mediated by Thy-1+, Lyt-1+, and Lyt-2- cells. Furthermore, the xenoreactive T cell responses required Ia+ cells, and Ia antigen on accessory cells plays a crucial role in eliciting the xenoreactive responses against human stimulator cells, while Ia+ accessory cells in the responding cell population are not essential for the elicitation of allogeneic MLR responses, as reported previously.     

Comparison of antigens recognized by xenogeneic and allogeneic anti-Ia antibodies: Evidence for two classes of Ia antigens

Previously published data suggest that both xenogeneic and allogeneic anti-Ia sera can recognize carbohydrate-defined antigenic determinants on the surface of lymphocytes. There is also evidence, based on studies with allogeneic anti-Ia sera, that protein-defined Ia antigens exist. In this paper the relationship between these two types of Ia antigen was examined. It was found that in capping studies, the allogeneic anti-Ia serum could cap off the antigens recognized by the xenogeneic antiserum, whereas the xenogeneic antibodies could, at least partially, clear the surface of lymphocytes of Ia antigens detected by the allogeneic antibodies. On the other hand, when immunoprecipitates of radioiodinated cell-surface antigens were examined by SDS-polyacrylamide-gel electrophoresis, it was found that the xenogeneic anti-Ia serum did not immunoprecipitate any labeled material. In contrast, the allogeneic antiserum immunoprecipitated a labeled molecule which corresponded to the protein-defined Ia antigen described by others. Finally, it was shown that serum Ia antigens could be bound by either mouse or rabbit anti-Ia antibody, and this binding blocked any further reactivity with either serum. These results were interpreted as suggesting that two separate classes of Ia antigen molecule appear on the lymphocyte surface-one class has carbohydrate-defined antigenic specificities and the other has protein-defined determinants. Allogeneic anti-Ia sera contain antibodies against both these antigenic systems, whereas xenogeneic sera recognize only the carbohydratedefined series. The genetic implications of this interpretation are discussed.     

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4⁺ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.     

The fine specificity of antigen and Ia determinant recognition by T cell hybridoma clones specific for pigeon cytochrome c

The activation of proliferative T lymphocytes normally involves the simultaneous recognition of a particular foreign antigen and a particular Ia molecule on the surface of antigen-presenting cells, the phenomenon of major histocompatibility complex (MHC) restriction. An analysis of T cell clones specific for pigeon cytochrome c, from B10.A and B10.S(9R) strains of mice, revealed the unusual finding that several of the clones could respond to antigen in association with Ia molecules from either strain. Using these cross-reactive clones, we performed experiments which demonstrated that both the Ia molecule and the T cell receptor contribute to the specificity of antigen recognition; however, MHC-linked low responsiveness to tuna cytochrome c (an immune response gene defect) could not be attributed solely to the efficacy with which the Ia molecules associated with the antigen. These results imply that antigen and Ia molecules are not recognized independently, but must interact at least during the process of T cell activation.     

Direct stimulation of T lymphocytes by antigen-conjugated beads

To examine T lymphocyte recognition of foreign antigen, specific responses to the photoreactive antigen N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) were determined by using an HSAB/I-Ad-reactive murine T cell hybridoma. It was found that covalent coupling of HSAB to aminoethyl polyacrylamide beads at particular densities directly activated the T cells for IL 2 production, and beads conjugated at higher or lower doses of HSAB were nonstimulatory. This stimulation was specific for the phenyl ring composition of HSAB and for HSAB-reactive T cells. In addition, T cell activation by HSAB-coupled beads was specifically inhibited by soluble monomeric HSAB-glycine. These results indicate that HSAB-specific T cells may be directly stimulated by insolubilized HSAB in the absence of Ia antigens, suggesting direct T cell binding of foreign antigen.     

Low-molecular-weight Ia antigens in normal mouse serum

Low-molecular-weight Ia antigens can be detected in mouse serum. A procedure for isolating these antigens from serum in high yield is described. The Ia antigen preparation was found to be rich in carbohydrate, low in protein, and strongly bound to Concanavalin A andLotus lectins. Furthermore, the Ia antigenicity was destroyed by periodate oxidation and neuraminidase treatment, but was unaffected by pronase. These observations strongly suggest that the Ia antigens in serum are oligosaccharide in nature. Such a conclusion implies that at least some of the genes in theI region of theH-2 gene complex code for glycosyl transferase enzymes.     

Demonstration of carbohydrate- and protein determined Ia antigens by monoclonal antibodies

Ten monoclonal alloantibodies were examined by submitting each antibody to five independent tests in order to determine whether they reacted primarily with the glycoprotein or glycolipid class of Ia antigens. The tests employed were as follows: (1) the ability to participate an Ia-like protein from the cell surface as detected by SDS-PAGE; (2) inhibition by protein-Ia extracts free of CHO-Ia; (3) inhibition by CHO-Ia extracts free of protein-Ia; (4) neuraminidase sensitivity of the antigen and (5) inhibition by simple sugars. Using these tests, three of the ten monoclonal antibodies were shown to recognize a CHO-Ia antigen while seven recognized the protein class of Ia antigens. The three CHO-Ia-specific monoclonal antibodies recognized Ia specificities 2, 9 and 17. Monoclonal antibodies recognizing protein-defined Ia.2 and 17 specificities were also characterized. These results imply that some Ia specificities, as defined by genetic testing, can occur both as carbohydrate-defined and protein-defined determinants.--Sugar inhibition studies showed that CHO-Ia.2 has D-glucosamine as its immunodominant sugar while CHO-Ia.17 shows preference for a beta-linked galactose. Furthermore, studies with neuraminidase demonstrated that sialic acid plays a role in the antigenic determinants of CHO-Ia.9 and CHO-Ia.17. Finally, it is noteworthy that CHO-Ia.2, the private specificity of the k haplotype, appears to be expressed only on cells and not in serum. These studies clearly demonstrate the existence of the two Ia antigen classes and emphasize the complexity of the murine I region.     

Soluble factors produced during an immune response regulate Ia antigen expression by murine adenocarcinoma and fibrosarcoma cells

LT-85 is an alveologenic adenocarcinoma of C3Hf/HeN mice. Comparisons of the in vitro and in vivo surface properties of these cells revealed that under normal conditions, they expressed I-A and I-E antigens iv vivo only. By using clonally derived cells, it was established that this phenomenon was not due to the selection of an Ia antigen-positive tumor cell subpopulation, but resulted from phenotypic conversion of Ia antigen-negative tumor cells. These tumor cells and 1053 cells (a fibrosarcoma of C3H/HeN MTV- mice) could, however, be induced to express I-A, I-E, and much higher levels of H-2 antigens in vitro by co-culturing them with spleen cells from LT-85 tumor-bearing C3H/HeN MTV- mice. In vitro induction of Ia and H-2 antigens did not result from contaminating splenocytes or from antigen transfer, because splenocytes from BALB/c (H-2d) mice immunized with A/J (H-2k/d) cells were able to induce the expression of Iak antigens by both tumor cell lines. It was found that this phenomenon was neither H-2-restricted nor antigen-specific. The results clearly indicated, however, that an immune response was required to generate phenotypic conversion of the tumor cells, both in vivo and in vitro. It was further found that soluble, rather than cellular, factors produced during an immune response induced the expression of Ia antigens by LT-85 and 1053 tumor cells. In contrast to what has been reported about the induction of Ia antigens on macrophages and normal epithelial and endothelial cells, the induction of Ia antigens on LT-85 and 1053 cells did not appear to require T cells, and did not involve gamma-interferon. These findings demonstrate that some tumor cells are capable of altering their MHC antigen phenotype in response to factors produced during an immune response in vivo or in vitro. Because of the involvement of Ia antigens in several aspects of immune phenomena, the ability of tumor cells to differentially express Ia antigens in response to environmental factors may have profound effects on host-tumor interactions. Furthermore, the differences seen in the phenotypes of tumor cells grown in vitro and in vivo suggest that in vitro methodologies of tumor cell characterization may not present a complete picture of the natural state of the tumor cell surface.