TY - JOUR
AU - Kanagawa,, Osami
AB - Abstract Mice carrying a transgenic TCR with targeted disruption of the TCR α chain (H-Y α–/–) possess CD4+ T cells which express the transgenic TCR β without the α chain. These mice developed the murine acquired immunodeficiency syndrome (MAIDS) after infection with LP-BM5 retroviruses, a process which requires CD4+ T cells. These cells are negative for TCR δ chain and pre-TCR α chain expression, and thus express a unique surface receptor with the TCR β chain as a component. The cells respond to MAIDS virus-associated superantigen and concanavalin A, but not to protein antigens such as ovalbumin. Thus, this novel surface receptor appears to play an important role in the pathogenesis of MAIDS. α chain deficient, murine acquired immunodeficiency syndrome, receptor, T cell CFA complete Freund's adjuvant, Con A concanavalin A, MAIDS murine acquired immunodeficiency syndrome, OVA ovalbumin, PE phycoerythrin, pTα pre-TCR α, SP single positive Introduction Infection of susceptible strains of mice by the retroviral mixture LP-BM5 results in the syndrome known as murine acquired immunodeficiency syndrome (MAIDS) (1–3). The cardinal features include a severe immunodeficiency, hypergammaglobulinemia and a progressive lymphoproliferation. Previous studies have demonstrated that disease progression requires the presence of CD4+ T cells (4,5) and mature B cells (6). The functions of both T and B lymphocytes are profoundly affected in this disease with an inability to respond to subsequent stimulation in vitro and in vivo. The critical events in the pathogenesis of this disease have yet to be defined. The role of a viral superantigen has been implicated, as virally transformed B cell lines have been shown to elicit T cell expansion of specific Vβ-bearing populations (7–9). Characterization of the MAIDS superantigen and its relationship to disease progression are areas of continued investigation. Previous studies by our laboratory have examined the development of MAIDS in mice bearing a transgenic TCR to characterize the fate of T cells of a defined antigen specificity (10). A transgenic mouse strain which expresses a Db class I MHC-restricted TCR specific for the male antigen (H-Y) was used for these studies (11). This line was chosen because T cells expressing this transgenic TCR fail to respond to the MAIDS-associated superantigen. Analysis of the T cells from infected H-Y TCR transgenic mice revealed the loss of high level expression of the idiotype-positive transgene-derived αβ TCR. Expression of the transgenic Vβ chain remained unchanged, however; suggesting that there was expression of endogenous TCR α chains paired with the transgenic TCR β chain. In an attempt to remove the influence of this effect, we utilized mice carrying the same TCR transgene with a targeted disruption of the endogenous TCR α chain, (H-Y α–/–) (12). Characterization of this mouse line revealed the presence of a large number of CD4+ T cells which express a novel receptor and are responsive to the MAIDS superantigen. This unique T cell population may play an important a role in the pathogenesis of MAIDS. Methods Animals Mice expressing the transgenic TCR for the male antigen, H-Y (11), and mice with homozygous disruption of the α chain of the TCR gene (TCR α–/–) (12) and of RAG I gene (RAG I–/–) mice (13) have been previously described. The H-Y TCR transgene was introduced into the α–/– mouse by standard backcrossing (H-Y α–/–). Subsequent progeny were determined to be homozygous (α–/–) by breeding them to known (α–/–) mice and demonstrating that all those progeny who lacked the H-Y TCR transgene were deficient in αβ T cells. RAG I–/– mice were bred with H-Y TCR transgenic mice to obtain H-Y TCR transgene on a RAG I–/– background. H-Y RAG I–/– mice were defined as those expressing the H-Y receptor (stained by an anti-idiotype antibody, MR14.1) and did not possess any mature B cells (stained by an anti-B220 antibody, RA3.6). Mice were maintained in the Washington University School of Medicine animal facility for use in the experiments. C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, ME). All of the mice possessed the H-2b background, which positively selects for the H-Y receptor (14). All experiments using mice expressing the H-Y TCR transgene were performed on female mice. Cell lines and virus preparation The MAIDS virus-transformed B cell lymphomas, B6-1710 and B6-1153 (7), and the γδ TCR-expressing T cell hybridoma, KN6, were maintained in vitro in 10% FCS in DMEM. LP-BM5 viral stock used for infections was obtained by culturing infected SC-1 cells in 10% FCS DMEM (15). Confluent culture supernatants were filtered and stored at –70°C as viral stocks. Infection in vivo with MAIDS virus Mice were infected with 0.5 ml of viral stock injected i.p. and were examined weekly for the development of lymphadenopathy. A clearly affected animal with visible lymph node enlargement was sacrificed for subsequent analysis. Surface immunofluorescence and flow cytometry Cell-surface immunofluorescence analysis of T cell populations was performed as described previously (17). In brief, 1×106 cells were incubated with the appropriate antibody at saturating concentrations at 4°C for 30 min, washed and further incubated with FITC- or phycoerythrin (PE)-conjugated appropriate secondary reagents for an additional 30 min. Control samples were prepared in the same manner, but without primary antibody or with an isotype-matched control. Samples were analyzed by a FACScan analyzer (Becton Dickinson, Mountain View, CA) using the CellQuest program, with calibration utilizing the FACSComp software. Samples were gated on live lymphocyte populations based on forward and side scatter parameters. mAb used in this study include: 500A2 (anti-CD3ε) (18), MR 5-2 (17) (anti-Vβ8.1 and .2), MR14-1 (anti-idiotype for the anti-H-Y TCR, established in this laboratory) (10), 3A10 (anti-TCR δ chain) GK1.5 (anti-CD4) (19) and 53-6.7 (20) (anti-CD8). Establishment of T cell hybridomas T cell hybridomas were established by fusing concanavalin A (Con A)-stimulated (5 μg/ml) lymph node cells from H-Y α–/– mice (4×107 cells) to a TCR– variant of the thymoma, BW5147 (4×107 cells). Fused cells were selected with HAT and resulting hybridoma cells were cloned under limiting dilution conditions as described previously (16). Cloned hybridomas were tested for the expression of TCR β chain and idiotype-positive TCR. Stimulation of T cells in vitro T cell hybridomas (1×105) were stimulated with Con A (10 μg/ml) or B lymphoma cells (1×105) in a final volume of 200 μl of 5% FCS DMEM in flat-bottom microtiter plates. After 24 h of culture at 37°C, culture supernatants were harvested and tested for IL-2 content using the IL-2-dependent cell line CTLL-2. IL-2 units were determined for each culture supernatant as described previously (17). Mice were immunized with 100 μg of ovalbumin (OVA) in complete Freund's adjuvant (CFA; final volume of 100 μl) in the base of the tail. Seven days after immunization, draining lymph node cells (2×105) were stimulated with Con A (5 μg/ml), OVA (1 mg/ml), irradiated (2000 rad) B6-1710 cells (2×104) or media alone (none) in a final volume of 200 μl 5% FCS DMEM in 96-well flat-bottom microtiter plates. The cultures were incubated for 3 days and harvested after a 6 h pulse with [3H]thymidine. Isolation of CD4 and CD8 populations CD4+ and CD8+ T cell populations were isolated by negative selection methods using the MACS magnetic bead system. Cells were labeled with a mixture of biotinylated anti-B220 (21) (RA3.6), anti-γδ TCR(GL3) (22) and anti-NK cells (PK136) (23) together with either GK1.5–biotin (anti-CD4) or LY2–biotin (anti-CD8) (PharMingen, San Diego, CA) and incubated with magnetic beads conjugated to avidin. Cells were then applied to the magnetic column, and the fraction containing the desired CD4+ (in the anti-CD8 antibody treated) or CD8+ (anti-CD4 antibody treated) cells were collected. Fractionated cell populations contained <5% contaminating cells as assessed by surface immunofluorescence. RT-PCR analysis for pre-TCR α (pTα) chain expression Total RNA was extracted from 1×107 cells and isolated by guanidine lysis and CsCl gradient methods (24). cDNA was generated using random hexamer primers and reversed transcribed with Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim, Indianapolis, IN) using the manufacturer's recommended conditions. An equivalent amount of cDNA representing 1×106 cells was amplified using primers specific for pTα and actin. The 5′ and 3′ primers for pTα (5′-CATGCTTCTCCACGAGTG-3′ and 5′-CTATGTCCAAATTCTGTGGGTG-3′) and actin (5′-GTGGGCCGCTCTAGGCACCAA-3′ and 5′-CTCTTTGATGTCACGCACGATTTC-3′) recognized specific 5′ and 3′ regions of each gene, and have been characterized previously. Amplification of the cDNA was performed using Taq polymerase for 35 cycles using the 2400 Gene amp PCR system (Perkin-Elmer Cetus, LOCATION???) in a 50 ml reaction volume containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 and 0.2 mM of dNTP, using the following reaction temperature conditions: denaturation 94°C, annealing 55°C and extension 72°C, each for 1 min. The amplified material was then analyzed on a 1.5% agarose gel, transferred to nitrocellulose membrane and hybridized with 32P-labeled oligonucleotide probe as described (25). Control actin mRNA PCR was carried out as described previously and visualized with EtBr staining. Surface biotinylation and immunoprecipitation Surface biotinylation of fractionated cells was performed as previously described (26) Briefly, 2×107 cells were washed 3 times with HBSS and incubated with sulfo-NHS-LC-biotin (EZ-Link; Pierce, Rockford, IL) at a concentration of 1 mg/ml in PBS at 4°C for 30 min. The cells were then washed twice with DMEM media supplemented with 5% calf serum (Hyclone, Logan, UT). Successful biotinylation was defined as a 3 log increase in fluorescence upon staining with PE-conjugated avidin compared to unbiotinylated control cell samples. Cells were lysed by lysis buffer at 4°C for 45 min and the lysates were spun at 10,000 g to remove cellular debris and nuclei. Lysates were incubated with antibodies specific for the β chain of the TCR, H57-597, (H57) (27) covalently bound to Sepharose (Sigma, St Louis, MO). Sepharose bound proteins were washed extensively with lysis buffer. Immunoprecipitated proteins were boiled in the presence of 5% 2-mercaptoethanol in sample buffer, separated on SDS–PAGE, then transferred to nitrocellulose membrane (BioRad, Hercules, CA). The transferred membrane was blocked with 10% non-fat dry milk and then incubated for 20 min with streptavidin-conjugated horseradish peroxidase (Pierce). The immunoprecipitated cell surface proteins were visualized using the ECL system (Amersham, Piscataway, NJ). Results Susceptibility of H-Y α–/– mice to LP-BM5 Infection of female H-Y α–/– and control C57BL/6 mice results in the development of disease in both groups as manifested by visible lymphadenopathy (Table 1). Non-transgenic TCR α–/– mice, devoid of significant numbers of peripheral CD4+ T cells, are resistant to the MAIDS virus infection. The rate of disease development in the H-Y α–/– and the susceptible C57Bl/6 mouse was rapid, with palpable lymphadenopathy developing within 6 weeks of inoculation. This is in contrast to previous studies of the wild-type H-Y transgenic mouse in which disease development was significantly delayed compared to control mice (10). Analysis of the lymph node T cell populations from virus-infected H-Y α–/– mice revealed a drastic increase of both CD4 and CD8 T cells (Table 2). The expansion of the CD4+ population was unexpected, since the transgenic receptor was derived from a CD8+, class I MHC-restricted T cell clone and thus the sole T cell population present in an TCR α–/– background was predicted to be CD8+, expressing the transgenic TCR. Analysis of the T cell subpopulations in the H-Y α–/– mouse The finding of this unexpected CD4+ T cell population in MAIDS virus-infected H-Y α–/– mice prompted us to examine the non-infected mice in detail (Fig. 1A). Both H-Y α–/– and wild-type H-Y TCR transgenic female mice possess increased numbers of CD8 single-positive (SP) thymocytes compared to normal controls. This is due to the preferential development of the transgenic TCR+ T cells to the CD8 SP stage. It should be noted that the number of CD4+ T cells in H-Y α–/– thymus is significantly lower compared to normal and H-Y TCR transgenic mice. Despite this observation, both transgenic mouse lines possess similar numbers of CD4 and CD8 SP populations in the periphery. Further analysis, however, reveals that they differ significantly in the expression of the transgenic αβ TCR in the periphery. The H-Y α–/– mice possess an increased number of CD8 cells expressing the clonotypic TCR. Seventy percent of the peripheral CD8 T cells are positively stained with the anti-idiotype antibody, MR 14-1 (Fig. 1B). In contrast, only 30% of CD8+ T cells in the wild-type H-Y TCR transgenic mice are idiotype-positive (Fig. 1B), consistent with previous observations in this mouse line (14,28). The transgenic Vβ chain, however, is expressed in virtually all of the mature T cells for both mouse lines. The CD4+ T cells in the wild-type H-Y TCR transgenic mice do not express idiotype-positive receptor and are assumed to express the transgenic TCR β chain paired with endogenous TCR α chains. The high percentage of idiotype-negative, Vβ8+, CD4+ cells was unexpected, however, in the H-Y α–/– mouse since functional endogenous TCR α cannot be produced. The H-Y α–/– mice were mated to C57BL/6 α–/– mice to confirm the genotype of these mice. Those progeny positive for expression of the H-Y TCR transgene (five out of 10) possessed CD4 and CD8 SP T cells in the lymph node with numbers comparable to the H-Y α–/– parents. In contrast, all progeny negative for the transgenic TCR (five out of 10) were lacking both CD4 and CD8+ T cells in the periphery (Fig. 2). This confirms the homozygosity for the targeted disruption of TCR α chain in the parental H-Y α–/– mice and thus excludes the possibility of endogenous TCR α chain expression accounting for the idiotype-negative, CD4 SP T cells in this mouse line. Additional studies of the H-Y α–/– mice suggest that the CD4+ T cells in this mouse line also differ functionally from those present in wild-type H-Y TCR transgenic mice. H-Y α–/–, H-Y TCR transgenic mice and control C57Bl/6 mice were immunized with OVA in CFA. Cells from the draining lymph nodes were stimulated with OVA, Con A and the MAIDS virus-transformed B cell line B6-1710 (7). Cells from control C57Bl/6 and the wild-type H-Y TCR transgenic mice exhibited vigorous proliferative responses to all of these stimuli (Fig. 3). The responses to conventional antigens other than H-Y in the wild-type H-Y TCR transgenic mouse line have been previously shown to be mediated by T cells expressing transgenic TCR β chain associating with endogenous TCR α chains (14). The lymph node cells from OVA-immunized H-Y α–/– mice failed to mount proliferative responses to OVA, yet surprisingly their responses to Con A as well as to B6-1710 were comparable to the control C57BL/6 mice (Fig. 3). Thus, functional assays also suggest that the CD4+ T cells in the H-Y α–/– lack endogenous TCR α chain expression and thus are incapable of responding to conventional protein antigens. These cells, however, do possess a receptor that can be stimulated by both Con A and by cells expressing the MAIDS-associated superantigen activity. In order to further characterize the function of this unique T cell population, T cells from H-Y α–/– mice were fused to a TCR– BW5147 thymoma. Both idiotype-positive and -negative hybridomas were generated. Stimulation with Con A elicited IL-2 production in both idiotype-positive and -negative populations (Table 3). Stimulation with plate-bound antibodies to the CD3 complex also gave similar results (data not shown). These hybridomas were subsequently stimulated with a MAIDS virus-transformed B cell line, B6-1710, which has been shown to express superantigen activity. A fraction of the idiotype-negative population responded, producing IL-2. This response was not observed with a similar MAIDS virus-transformed B cell line, B6-1150, which lacks superantigen activity (7). Thus, idiotype-negative T cell hybridomas from H-Y α–/– are capable of responding to MAIDS-associated superantigen, while no response was observed to B6-1710 cells in all of the idiotype-positive T cell hybridomas tested. Extensive analysis T cell hybridomas from three independent fusions have revealed identical results, with the MAIDS-associated superantigen-responsive population always restricted to the idiotype-negative phenotype. These results indicate that the response of lymph node cells from H-Y α–/– mice to B6-1710 cells is mediated by CD4+ T cells expressing a unique surface receptor. Lack of CD4+ TCR α– T cells in H-Y TCR transgenic mice deficient in the RAG I gene In previous studies, the H-Y specific TCR transgene was introduced into the SCID background to eliminate an appearance of endogenous alphα chain (29). These mice were noted to express the idiotype-positive receptors exclusively in a CD8+ T cell population and to lack CD4+ T cells. We confirmed their finding using RAG I gene-deficient H-Y TCR transgenic mice (Fig. 4). In these mice, there are virtually no CD4 SP T cells, with only CD8 SP T cells in the thymus and periphery. This indicates that the generation of the unique CD4+ T cells found in the H-Y α–/– mouse may require either RAG-dependent rearrangement of an endogenous gene or the maturation stage of T cells which are linked to RAG activity in the thymus. Characterization of the receptor expressed on CD4 T cells from H-Y α–/– mice Candidates that could form functional receptor dimers with the transgenic β chain were sought. TCR δ chain expression could not be demonstrated on either CD4+ and CD8+ T cell populations from H-Y α–/– mice using a mAb specific for TCR δ chain (Fig. 5). The expression of pTα (30) was also not found in either population, as the analysis of lymph node mRNA using RT-PCR could not demonstrate message for the pTα chain from H-Y α–/– mice, although message was readily detectable in the thymus (Fig. 6). In an effort to characterize the biochemical nature of this previously undescribed receptor, idiotype-positive, CD8+ T cells and idiotype-negative, CD4+ T cells were isolated from the H-Y α–/– mouse. Each T cell population was surface biotinylated and the solubilized membranes were immunoprecipitated with an antibody specific for the β chain of the TCR followed by SDS–PAGE separation under reducing conditions. The protein species isolated from the idiotype-positive CD8+ population was a dimer consistent with the transgene-derived H-Y antigen-specific αβ receptor. In contrast, the idiotype-negative CD4+ cells possess a receptor species which is distinct from the clonotypic receptor found in the CD8 population (Fig. 6). The molecules immunoprecipitated by anti-TCR β chain antibody contain the band corresponding to the TCR β chain of CD8+ T cells. However, no band on the gel corresponding to TCR α chain was found. Instead, an additional distinct band of ~30 kDa was visible. This small mol. wt protein is not apparent in the idiotype-positive CD8 T cell lysate. Discussion This report characterizes a CD4+ T cell population expressing a novel receptor. This receptor possesses the TCR β chain without the TCR α chain and is responsive to the MAIDS superantigen but is unresponsive to conventional protein antigens such as OVA. The H-Y α–/– mice possess significant numbers of these cells and are highly susceptible to MAIDS development. In contrast, H-Y RAG I–/– mice, lacking this cell population, are resistant to MAIDS development even in the presence of an exogenous source of B cells (data not shown). Since these superantigen reactive T cells are the predominant CD4+ population in this mouse line, they must play an important role in the development of MAIDS which requires functional CD4 cells (4,5). It has previously been shown that TCR α–/– mice possess small numbers of CD4+, CD3+, TCR β+ T cells. The cells were noted to be very low in number in young mice but they gradually increased to 9% of the total lymph node population by 9 months of age (31). In contrast, in the H-Y α–/– mice, although the number of these CD4+ T cells in the thymus is relatively low, far greater numbers are present in the periphery. Additional studies using Vβ-specific antibodies have failed to detect alternative Vβ chain expression indicating that the TCR β chain expression of these T cells is homogeneous (data not shown). Analysis of another class I MHC-restricted TCR transgenic mouse line, 2C (32), in a TCR α–/– background also show a significant CD4+, idiotype-negative population, indicating that this is not a unique phenomenon of the H-Y TCR transgenic mice (data not shown). In addition, in both the H-Y α–/– and H-Y RAG I–/– mouse lines, CD8+ T cells in the male mice bearing the clonotypic receptor are deleted, indicating that normal thymic selection is present in these two mouse lines and that the increased numbers of CD4 cells seen in the H-Y α–/– mouse is not simply due to an abnormally functioning thymus (data not shown). Analysis of the CD8 T cell population also reveals a smaller number of Vβ+/Vα– cells. These cells are unresponsive to the MAIDS-associated superantigen activity expressed on the B6-1710 cell line, possibly due to the lack of the CD4 co-receptor. The nature of this receptor species is also being investigated. A CD4 T cell population bearing the receptor described in this report has yet to be identified in normal mice. One possible explanation is that this novel receptor may only be co-expressed on cells also expressing conventional αβ TCR. This hypothesis is based upon our analysis of mice expressing a transgene-derived TCR (DO11.10) which is expressed on CD4 cells. When these mice are bred on an α–/– background, and compared to mice bearing the same TCR transgene on a RAG II–/– background, virtually all of the CD4 cells in both mouse lines express the clonotypic receptor. However, only the CD4 cells on the α–/– background can be stimulated by the B6-1710 cell line that expresses MAIDS-associated superantigen activity (data not shown). Since both mouse lines possess the same αβ TCR species in the form of the clonotypic receptor, the CD4 cells on the α–/– background must possess an additional receptor which can account for this difference in responsiveness to the B6-1710 cell line. We propose that this additional receptor is analogous to the receptor described in this report. Such a receptor bearing a β chain in the absence of an α chain could potentially go undetected, especially if it is normally expressed at levels significantly lower than a coexisting αβ TCR. Thus, only in a mouse deficient in the α chain expression would such an alternative receptor become apparent. Previous characterization of CD4+ T cells in TCR α–/– mice revealed that they can expand in response to external stimuli, viral infections and also respond to superantigens (33,34). They also appear to mediate the development of autoimmune disease in MRL/lpr mice (35,36). However, no responses to conventional antigens in an MHC-restricted fashion have been demonstrated in these T cells (31). Our functional characterization of CD4+ T cells in H-Y α–/– support the observations of these previous studies. We have shown that these T cells are capable of responding to Con A or the MAIDS-associated superantigen activity expressed on the transformed B cell line B6-1710, yet they fail to mount responses to the soluble antigen, OVA. The response to conventional antigens by T cells from H-Y transgenic mice on a wild-type background has been shown to be mediated by a surface receptor composed of transgene-derived TCR β chain associating with endogenous TCR α chain (14). Thus, the lack of responses to conventional antigens in the H-Y α–/– mice clearly indicate that the CD4+ T cells are not expressing αβ receptors and are similar to those found in TCR α–/– mice without transgenic TCR. Our comparison of the H-Y TCR transgenic mice in TCR α–/– and RAG I–/– background also confirms the earlier observations in the SCID background (29). Expression of the TCR β chain transgene in RAG-deficient mice failed to generate CD4+ T cells (37), generating only CD8+ T cells. The male mice with this genotype have a small thymus (2–3×106 cells in contrast to 1–2×108 thymocytes in male mice) with very few CD4/CD8 double-positive and SP cells, demonstrating that the self-reactive T cells are negatively selected in the thymus. However, TCR α–/– mice, with functional RAG genes, resulted in a significant CD4+ T lymphocyte population in the periphery (13). Thus it seems that the establishment of CD4+ TCR α–β+ T cells requires not only the expression of TCR β chain but also RAG gene activity. This indicates the molecule associating with the TCR β chain in CD4+ T cells in TCR α–/– mice may require RAG gene-dependent rearrangement or, alternatively, the maturation pathway of these cells may be linked to RAG gene activity in the developing thymocytes. At present the precise mechanism by which this CD4+ T cell population is generated or how this population is increased in H-Y α–/– mice is not known. However, it is possible that this particular Vβ8.2 chain from the H-Y-specific CD8+ T cell clone may associate well with this yet unknown molecule to facilitate the generation of this unique CD4+ T cell population. The exact nature of the surface receptor expressed on these CD4+ T cells is not known. We did not find TCR δ chain or pTα chain expression in the T cells from these mice. A recent report has suggested that a isoform of pTα is expressed in peripheral CD4+ T cells, which has been named pTαb (38). Analysis using RT-PCR techniques revealed the presence of a 300 bp product representing this new isoform, a result from an alternatively spliced mRNA message from the originally described pTα (pTαa). pTαa, in contrast, is characterized by a larger RT-PCR product which is 600 bp in length (25,30). Our analysis for pTα message (Fig. 6) reveals the presence of the smaller species in the thymus, as has been previously observed (25,30). However, we fail to identify detectable message in the CD4+, TCR β+ population from the H-Y α–/– mice despite the use of the same primer pair used in the previous studies (30,38). Thus, it would appear that this molecule is not expressed in the CD4+ cells in the H-Y α–/– mouse line and is not the protein responsible for the formation of the α–β+ receptor observed in this report. Immunopecipitation of this receptor using antibodies specific to a common determinant of the TCR β chain reveals an absence of a molecular species corresponding to TCR α chain, confirming that these T cells express TCR β chain without any previously known associating molecules. The presence of T cells expressing ββ homodimers has been reported previously (39). However, these ββ TCR+ cells express neither CD3 nor CD4 molecules, and fail to respond to any TCR-mediated stimulation including Con A and superantigens. It is therefore unlikely that CD4+ T cells in the H-Y α–/– mice are expressing transgene-derived ββ homodimers. It should be noted that the unique 30 kDa protein was co-precipitated from idiotype-negative CD4+ T cell lysate and this band is absent from lysates from idiotype-positive T cells. This indicates that these cells possess a receptor which contains a β chain paired with a protein species which is distinct from the α chain of an αβ TCR as it is of a smaller mol. wt (30 kDa), distinguishing it also from a possible ββ homodimer. We are currently purifying this protein using a large-scale culture of T cell lines and hybridomas for protein sequencing. Although the CD4 T cells of the H-Y α–/– mouse possess a Vβ8.2 TCR, it is highly responsive to the superantigen activity expressed on B6-1710 cells. Initial reports characterizing this MAIDS-associated superantigen activity indicated that Vβ5-bearing CD4 T cells were most responsive. The exact nature of the superantigen activity expressed on B6-1710 and that expressed in in vivo virus-infected mice is unknown. There is a clear discrepancy between findings in vitro assays with B6-1710 cells and in vivo T cell analysis in virus-infected mice with regard to TCR Vβ chain usage which has been published previously (40). Recently, we have shown that MAIDS virus infection of B cells both in vitro and in vivo induces endogenous mouse mammary tumor superantigen expression. Thus, it is possible that multiple superantigens are expressed in virus-infected mice and virally transformed B cell lines. Although stimulation of T cells in vitro with B6-1710 may only induce proliferation of T cells with high-affinity TCR, such as Vβ5+ T cells, in the absence of these high-affinity TCR+ T cells in TCR transgenic mice (Vβ8.2 TCR in H-Y TCR transgene), T cells expressing transgenes with different TCR α chain (41) or unique β chain associating molecule can be activated by the same B6-1710 cells. Finally, we do believe that there is heterogeneity in this receptor. Its absence in mice with a RAG I–/– background indicates that the formation of this receptor may require gene rearrangement, possibly resulting in a diverse repertoire of receptors. In addition, since we have generated hybridomas from these mice which are positive for this receptor yet vary in their responsiveness to the cell line B6-1710, this would indicate that the receptors expressed by these hybridoma clones express different receptors of different sensitivity to the MAIDS-associated superantigen activity. In summary, our studies have demonstrated the presence of a novel receptor expressed on CD4+ T cells which plays an important role in the development of a superantigen-mediated disease process. Further investigation is needed to determine whether this receptor is expressed in normal mice, either co-expressed with conventional αβ TCR or on a distinctly different T cell population. Identification and characterization of this receptor will provide useful information concerning its biological significance in the normal host. Table 1. Development of MAIDS in H-Y α–/– micea Mouse . No. of mice affected (6 weeks after infection) . . Experiment I . Experiment II . aC57BL/6, H-Y α–/– and TCR α–/– (five mice each) were infected with LP-BM5 retrovirus mix (0.5 ml/mouse i.p.). Disease development was monitored weekly and disease development was defined as visible lymphoadenopathy. bNo. of mice affected/no. of mice infected. ND, not done. C57BL/6 5/5b 5/5 H-Y α–/– 5/5 5/5 C57Bl/6 α–/– 0/5 ND Mouse . No. of mice affected (6 weeks after infection) . . Experiment I . Experiment II . aC57BL/6, H-Y α–/– and TCR α–/– (five mice each) were infected with LP-BM5 retrovirus mix (0.5 ml/mouse i.p.). Disease development was monitored weekly and disease development was defined as visible lymphoadenopathy. bNo. of mice affected/no. of mice infected. ND, not done. C57BL/6 5/5b 5/5 H-Y α–/– 5/5 5/5 C57Bl/6 α–/– 0/5 ND Open in new tab Table 1. Development of MAIDS in H-Y α–/– micea Mouse . No. of mice affected (6 weeks after infection) . . Experiment I . Experiment II . aC57BL/6, H-Y α–/– and TCR α–/– (five mice each) were infected with LP-BM5 retrovirus mix (0.5 ml/mouse i.p.). Disease development was monitored weekly and disease development was defined as visible lymphoadenopathy. bNo. of mice affected/no. of mice infected. ND, not done. C57BL/6 5/5b 5/5 H-Y α–/– 5/5 5/5 C57Bl/6 α–/– 0/5 ND Mouse . No. of mice affected (6 weeks after infection) . . Experiment I . Experiment II . aC57BL/6, H-Y α–/– and TCR α–/– (five mice each) were infected with LP-BM5 retrovirus mix (0.5 ml/mouse i.p.). Disease development was monitored weekly and disease development was defined as visible lymphoadenopathy. bNo. of mice affected/no. of mice infected. ND, not done. C57BL/6 5/5b 5/5 H-Y α–/– 5/5 5/5 C57Bl/6 α–/– 0/5 ND Open in new tab Table 2. Analysis of lymphoadenopathy and T cell subset distribution in MAIDS virus-infected H-Y α–/– micea Mouse . No. of lymph node cells (×106) . . Total . CD4+ . CD8+ . aC57BL/6 and H-Y α–/– mice were infected with LP-BM5 virus mixture and mice with visible lymph nodes were sacrificed (6 weeks after infection). Lymph nodes (two inguinal and two axillar) were harvested, counted, stained with anti-CD4 and anti-CD8 antibody, and analyzed with a FACScan. Non-infected mice were used as controls. Five mice for each group were analyzed. bCell no./single lymph node ± SD. C57BL/6 2.2 ± 0.4b 0.7 ± 0.1 0.4 ± 0.1 H-Y α–/– 1.5 ± 0.8 0.3 ± 0.1 0.2 ± 0.1 C57BL/6 MAIDS 130.0 ± 30 35.0 ± 10 5.7 ± 2.0 H-Y α–/– MAIDS 90.0 ± 50 20.0 ± 10 4.0 ± 2.0 Mouse . No. of lymph node cells (×106) . . Total . CD4+ . CD8+ . aC57BL/6 and H-Y α–/– mice were infected with LP-BM5 virus mixture and mice with visible lymph nodes were sacrificed (6 weeks after infection). Lymph nodes (two inguinal and two axillar) were harvested, counted, stained with anti-CD4 and anti-CD8 antibody, and analyzed with a FACScan. Non-infected mice were used as controls. Five mice for each group were analyzed. bCell no./single lymph node ± SD. C57BL/6 2.2 ± 0.4b 0.7 ± 0.1 0.4 ± 0.1 H-Y α–/– 1.5 ± 0.8 0.3 ± 0.1 0.2 ± 0.1 C57BL/6 MAIDS 130.0 ± 30 35.0 ± 10 5.7 ± 2.0 H-Y α–/– MAIDS 90.0 ± 50 20.0 ± 10 4.0 ± 2.0 Open in new tab Table 2. Analysis of lymphoadenopathy and T cell subset distribution in MAIDS virus-infected H-Y α–/– micea Mouse . No. of lymph node cells (×106) . . Total . CD4+ . CD8+ . aC57BL/6 and H-Y α–/– mice were infected with LP-BM5 virus mixture and mice with visible lymph nodes were sacrificed (6 weeks after infection). Lymph nodes (two inguinal and two axillar) were harvested, counted, stained with anti-CD4 and anti-CD8 antibody, and analyzed with a FACScan. Non-infected mice were used as controls. Five mice for each group were analyzed. bCell no./single lymph node ± SD. C57BL/6 2.2 ± 0.4b 0.7 ± 0.1 0.4 ± 0.1 H-Y α–/– 1.5 ± 0.8 0.3 ± 0.1 0.2 ± 0.1 C57BL/6 MAIDS 130.0 ± 30 35.0 ± 10 5.7 ± 2.0 H-Y α–/– MAIDS 90.0 ± 50 20.0 ± 10 4.0 ± 2.0 Mouse . No. of lymph node cells (×106) . . Total . CD4+ . CD8+ . aC57BL/6 and H-Y α–/– mice were infected with LP-BM5 virus mixture and mice with visible lymph nodes were sacrificed (6 weeks after infection). Lymph nodes (two inguinal and two axillar) were harvested, counted, stained with anti-CD4 and anti-CD8 antibody, and analyzed with a FACScan. Non-infected mice were used as controls. Five mice for each group were analyzed. bCell no./single lymph node ± SD. C57BL/6 2.2 ± 0.4b 0.7 ± 0.1 0.4 ± 0.1 H-Y α–/– 1.5 ± 0.8 0.3 ± 0.1 0.2 ± 0.1 C57BL/6 MAIDS 130.0 ± 30 35.0 ± 10 5.7 ± 2.0 H-Y α–/– MAIDS 90.0 ± 50 20.0 ± 10 4.0 ± 2.0 Open in new tab Table 3. Superantigen reactivity of T cell hybridomas derived from H-Y α–/– lymph node cellsa Hybridomas . No. of clones analyzed . No. of clones responding tob . . . Con A . B6-1710 . B6-1153 . aT cell hybridomas were established by fusing Con A-activated lymph node cells from H-Y α–/– mice to BW5147 cells. Hybridoma clones were separated based on the expression of idiotype-positive TCR (idiotype+ and idiotype–). Each clone was stimulated with Con A, B6-1710 and B6-1153 B lymphoma cell line, and production of IL-2 was measured as described in Methods. bResponsive clones were those producing a minimum of 10 U/ml of IL-2. Negative stimulation never induced more than 0.3 U/ml of IL-2. Idiotype+ 6 6 0 0 Idiotype– 26 26 15 0 Hybridomas . No. of clones analyzed . No. of clones responding tob . . . Con A . B6-1710 . B6-1153 . aT cell hybridomas were established by fusing Con A-activated lymph node cells from H-Y α–/– mice to BW5147 cells. Hybridoma clones were separated based on the expression of idiotype-positive TCR (idiotype+ and idiotype–). Each clone was stimulated with Con A, B6-1710 and B6-1153 B lymphoma cell line, and production of IL-2 was measured as described in Methods. bResponsive clones were those producing a minimum of 10 U/ml of IL-2. Negative stimulation never induced more than 0.3 U/ml of IL-2. Idiotype+ 6 6 0 0 Idiotype– 26 26 15 0 Open in new tab Table 3. Superantigen reactivity of T cell hybridomas derived from H-Y α–/– lymph node cellsa Hybridomas . No. of clones analyzed . No. of clones responding tob . . . Con A . B6-1710 . B6-1153 . aT cell hybridomas were established by fusing Con A-activated lymph node cells from H-Y α–/– mice to BW5147 cells. Hybridoma clones were separated based on the expression of idiotype-positive TCR (idiotype+ and idiotype–). Each clone was stimulated with Con A, B6-1710 and B6-1153 B lymphoma cell line, and production of IL-2 was measured as described in Methods. bResponsive clones were those producing a minimum of 10 U/ml of IL-2. Negative stimulation never induced more than 0.3 U/ml of IL-2. Idiotype+ 6 6 0 0 Idiotype– 26 26 15 0 Hybridomas . No. of clones analyzed . No. of clones responding tob . . . Con A . B6-1710 . B6-1153 . aT cell hybridomas were established by fusing Con A-activated lymph node cells from H-Y α–/– mice to BW5147 cells. Hybridoma clones were separated based on the expression of idiotype-positive TCR (idiotype+ and idiotype–). Each clone was stimulated with Con A, B6-1710 and B6-1153 B lymphoma cell line, and production of IL-2 was measured as described in Methods. bResponsive clones were those producing a minimum of 10 U/ml of IL-2. Negative stimulation never induced more than 0.3 U/ml of IL-2. Idiotype+ 6 6 0 0 Idiotype– 26 26 15 0 Open in new tab Fig. 1. Open in new tabDownload slide Open in new tabDownload slide Analysis of CD4 and CD8+ T cell subsets and TCR expression in H-Y α–/– mice. (A) Thymocytes and lymph node cells from non-transgenic C57BL/6 mice, H-Y TCR transgenic mice and H-Y α–/– mice were stained with PE-labeled anti-CD4 and FITC-labeled anti-CD8 antibody, and analyzed by FACScan as described in Methods. (B) Lymph node cells were stained with either anti-CD4 or anti-CD8 antibody and counterstained with none (Control), anti-Vβ8 antibody or MR14-1, anti-idiotypic antibody. Cells were gated for the expression of CD4 or CD8 and analyzed for the expression of TCR in each subset of lymph node T cells. Fig. 1. Open in new tabDownload slide Open in new tabDownload slide Analysis of CD4 and CD8+ T cell subsets and TCR expression in H-Y α–/– mice. (A) Thymocytes and lymph node cells from non-transgenic C57BL/6 mice, H-Y TCR transgenic mice and H-Y α–/– mice were stained with PE-labeled anti-CD4 and FITC-labeled anti-CD8 antibody, and analyzed by FACScan as described in Methods. (B) Lymph node cells were stained with either anti-CD4 or anti-CD8 antibody and counterstained with none (Control), anti-Vβ8 antibody or MR14-1, anti-idiotypic antibody. Cells were gated for the expression of CD4 or CD8 and analyzed for the expression of TCR in each subset of lymph node T cells. Fig. 2. Open in new tabDownload slide T cell development in progeny of H-Y α–/–×TCR α–/– mice. Lymph node cells from the offspring of a H-Y α–/–×TCR α–/– mating and a normal C57BL/6 mouse were stained for CD4 and CD8 expression, and analyzed by FACScan as described in Fig. 1. Progeny of the H-Y α–/–×TCR α–/– mating were screened for the presence of T lymphocytes and the expression of transgene-derived TCR using an anti-idiotypic antibody. Among 10 mice screened, five mice contained T cells and were all positive for the expression of transgenic TCR (TG positive). Five mice did not possess a detectable number of T cells and were negative for transgenic TCR expression (TG negative). Fig. 2. Open in new tabDownload slide T cell development in progeny of H-Y α–/–×TCR α–/– mice. Lymph node cells from the offspring of a H-Y α–/–×TCR α–/– mating and a normal C57BL/6 mouse were stained for CD4 and CD8 expression, and analyzed by FACScan as described in Fig. 1. Progeny of the H-Y α–/–×TCR α–/– mating were screened for the presence of T lymphocytes and the expression of transgene-derived TCR using an anti-idiotypic antibody. Among 10 mice screened, five mice contained T cells and were all positive for the expression of transgenic TCR (TG positive). Five mice did not possess a detectable number of T cells and were negative for transgenic TCR expression (TG negative). Fig. 3. Open in new tabDownload slide Antigen and superantigen reactivity of lymph node T cells from H-Y α–/– mice. H-Y α–/–, H-Y TCR transgenic and normal C57BL/6 mice were immunized with OVA/CFA at the base of the tail. Seven days after immunization, lymph node cells were stimulated with OVA, Con A, B6-1710 cells or none as described in Methods. Cultures were harvested after 72 h of incubation with a 6 h pulse of [3H]thymidine. Fig. 3. Open in new tabDownload slide Antigen and superantigen reactivity of lymph node T cells from H-Y α–/– mice. H-Y α–/–, H-Y TCR transgenic and normal C57BL/6 mice were immunized with OVA/CFA at the base of the tail. Seven days after immunization, lymph node cells were stimulated with OVA, Con A, B6-1710 cells or none as described in Methods. Cultures were harvested after 72 h of incubation with a 6 h pulse of [3H]thymidine. Fig. 4. Open in new tabDownload slide Comparison of H-Y α–/– mice and H-Y RAG I–/– mice for T cell development. Thymocytes and lymph node cells from H-Y α–/– and H-Y RAG I–/– mice were analyzed for the CD4/CD8+ T cell subset distribution as described in Fig. 1. Fig. 4. Open in new tabDownload slide Comparison of H-Y α–/– mice and H-Y RAG I–/– mice for T cell development. Thymocytes and lymph node cells from H-Y α–/– and H-Y RAG I–/– mice were analyzed for the CD4/CD8+ T cell subset distribution as described in Fig. 1. Fig. 5. Open in new tabDownload slide Lack of TCR δ chain expression in CD4 T cells from H-Y α–/– mice. Lymph node cells from H-Y α–/– mice were stained with either anti-CD4 and anti-CD8 antibody, and counterstained with none (solid line), anti-Vβ8 antibody (fine dotted line), anti-idiotype antibody (dotted line) or anti-TCR δ chain antibody (bold solid line). KN6, a T cell hybridoma expressing γδ TCR, was used as a positive control for the expression of TCR δ chain. Samples were analyzed with FACScan for the expression of TCR in CD4 and CD8+ T cell populations. Fig. 5. Open in new tabDownload slide Lack of TCR δ chain expression in CD4 T cells from H-Y α–/– mice. Lymph node cells from H-Y α–/– mice were stained with either anti-CD4 and anti-CD8 antibody, and counterstained with none (solid line), anti-Vβ8 antibody (fine dotted line), anti-idiotype antibody (dotted line) or anti-TCR δ chain antibody (bold solid line). KN6, a T cell hybridoma expressing γδ TCR, was used as a positive control for the expression of TCR δ chain. Samples were analyzed with FACScan for the expression of TCR in CD4 and CD8+ T cell populations. Fig. 6. Open in new tabDownload slide CD4+ and CD8+ T cells from H-Y α–/– mice were separated using magnetic beads and mRNA was prepared from both populations as well as from control C57BL/6 thymocytes for the RT-PCR analysis of pTα mRNA expression as described in Methods. Actin RT-PCR was run in parallel as a control. Fig. 6. Open in new tabDownload slide CD4+ and CD8+ T cells from H-Y α–/– mice were separated using magnetic beads and mRNA was prepared from both populations as well as from control C57BL/6 thymocytes for the RT-PCR analysis of pTα mRNA expression as described in Methods. Actin RT-PCR was run in parallel as a control. Fig. 7. Open in new tabDownload slide Biochemical analysis of the surface receptor on CD4+ T cells from H-Y α–/– mice. CD4 and CD8+ T cells were prepared using magnetic beads, biotinylated and lysed. Lysates were incubated anti-TCR β chain antibody-coupled Sepharose beads. Materials bound to the beads were separated in SDS–PAGE under reducing condition, transferred to nitrocellulose membrane and visualized using ECL as described in Methods. Fig. 7. Open in new tabDownload slide Biochemical analysis of the surface receptor on CD4+ T cells from H-Y α–/– mice. CD4 and CD8+ T cells were prepared using magnetic beads, biotinylated and lysed. Lysates were incubated anti-TCR β chain antibody-coupled Sepharose beads. Materials bound to the beads were separated in SDS–PAGE under reducing condition, transferred to nitrocellulose membrane and visualized using ECL as described in Methods. Transmitting editor: T. Saito We would like to thank Jonathan Katz and Calvin Williams for their helpful comments in the preparation of this manuscript. 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TI - Unique CD4+ T cells in TCR α chain-deficient class I MHC-restricted TCR transgenic mice: role in a superantigen-mediated disease process
JF - International Immunology
DO - 10.1093/intimm/11.9.1581
DA - 1999-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/unique-cd4-t-cells-in-tcr-chain-deficient-class-i-mhc-restricted-tcr-htZ0xbm00y
SP - 1581
EP - 1590
VL - 11
IS - 9
DP - DeepDyve
ER -