Intestinal Intraepithelial Lymphocytes In Mice Research Articles

Osteopontin provides a survival signal for intestinal intraepithelial lymphocytes in mice

Abstract Intraepithelial lymphocytes (IEL) are a diverse immune population residing in between intestinal epithelial cells, which possess important roles during mucosal immune responses. The IEL family comprises cells such as TCRαb+CD4+, TCRαb+CD8+, TCRgd+, TCRab+CD8aa+, iCD8a and iCD3+, among others. Osteopontin (OPN), a pleiotropic cytokine encoded by the Spp-1 gene, is well known for its function in tissue remodeling, modulation of Th1 responses development, maintenance of Th17 function, and homeostasis of NK cells. Herein, we investigated the impact of OPN in the IEL compartment. We compared the cell numbers of IEL, lamina propria (LP) lymphocytes and splenocytes from Spp-1−/− and wild-type (WT) mice. OPN deficiency reduced the number of TCRγδ+, TCRβ+CD4+, TCRβ+CD8α+ and TCRβ+CD4+CD8α+ IEL, but had no effect on LP and spleen lymphocytes. In in vitro studies, IEL from WT mice cultured in the presence of rOPN showed better survival rates than IEL incubated alone. The increased survival was blunted to control levels when CD44, a receptor for OPN, was blocked with anti-CD44 antibody. When total T cells (including Tregs) from WT splenocytes were transferred into Spp-1−/−Rag-2−/− and Rag2−/− mice, we observed that although OPN deficiency did not affect donor-derived IEL reconstitution at day 7 post-transfer, it decreased the number of CD4+ and CD4+CD8α+ IEL at day 28 post-transfer. As expected, Rag-2−/− mice remained healthy throught the experiment; however, Spp-1−/−Rag-2−/− recipient mice presented a remarkably loss of body weight and colon inflammation. Together, our results indicate that OPN is a critical survival signal for IEL via CD44, and that OPN-deficiency promotes development of colitis even in the presence of Tregs.

Effect of Anti-Mouse CD52 Monoclonal Antibody on Mouse Intestinal Intraepithelial Lymphocytes

CD52 monoclonal antibody (mAb) has been used therapeutically in lymphocytic leukemia, autoimmune disease, and organ transplantation. But the effect of CD52 mAb on intestinal intraepithelial lymphocytes (IELs) was unknown. The aim of this study was to assess the influence of anti-mouse CD52 mAb on IELs. Twenty male C57BL/6 mice were randomly assigned to a treatment or a control group. The treatment group received anti-mouse CD52 mAb (20 mug, subcutaneously), whereas the control group received the same volume of phosphate-buffered solution. On the seventh day after treatment, mice were killed, and ileum and colon were obtained for histopathology and immunohistochemistry examination, IELs were isolated for flow cytometric analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis. Furthermore, the intestinal permeability was measured by lactulose-mannitol test. The number and viability of IELs were decreased significantly in treatment compared with the control group. There were significant differences between the two groups considering IELs phenotypes. In addition, the proportion of apoptotic IELs in CD52 mAb treatment group was significantly higher than the control group (19.59%+/-3.13% and 2.02%+/-0.33%, respectively; P<0.05). Furthermore, lactulose-mannitol test showed a marked increase intestinal permeability after CD52 treatment (90.38%+/-5.96% and 46.02%+/-6.40%, respectively; P<0.05). Anti-mouse CD52 mAb could induce more IELs to apoptosis and result in reducing the number of IELs, which may disrupt intestinal barrier function.

Effect of restraint stress on the population of intestinal intraepithelial lymphocytes in mice

The impact of restraint stress on the intestinal immune system, particularly on intestinal intraepithelial lymphocytes (i-IEL), has not been described in detail. Thus, the purpose of this study was to assess the effects of restraint stress, including those produced by increases in glucocorticoids and catecholamines, on the population of i-IEL. Mice were exposed to 1 or 4 h restraint stress for 4 day, and the number of IEL in the mucosa of the proximal small intestine was determined by immunohistochemistry. The effects of restraint were also analyzed in mice submitted to different procedures: adrenalectomy, chemical sympathectomy, and treatment with a glucocorticoid antagonist (RU486), dexamethasone, and epinephrine. The main findings were that: (1) chronic restraint-stress reduced the i-IEl population in the small intestine; (2) adrenalectomy, treatment with RU-486 and chemical sympathectomy decreased the number of γδ, CD4+ and CD8+ T cells in non-stressed groups; (3) dexamethasone reduced the number of γδ and CD8+ T cells, and (4) epinephrine reduced the number of γδ, CD4+ and CD8+ T cells. These results demonstrated that restraint stress decreased the number of i-IEL in the proximal small intestine of mice, mainly by the combined action of higher concentrations of catecholamines and glucocorticoids, and that lower concentrations of glucocorticoids and catecholamines in unstressed mice preserved the population of i-IEL.

Upregulation of ICOS on CD43+ CD4+ murine small intestinal intraepithelial lymphocytes during acute reovirus infection

Murine intestinal intraepithelial lymphocytes (IELs) can be classified according to expression of a CD43 glycoform recognized by the S7 monoclonal antibody. In this study, we examined the response of S7+ and S7− IELs in mice during acute reovirus serotype 3 (Dearing strain) infection, which was confirmed by virus-specific real-time PCR. In vivo proliferation increased significantly for both S7− and S7+ IELs on day 4 post-infection as determined by BrdU incorporation; however, expression of the inducible costimulatory (ICOS) molecule, which peaked on day 7 post-infection, was upregulated on S7+ CD4+ T cells, most of which were CD4+8− IELs. In vitro ICOS stimulation by syngeneic peritoneal macrophages induced IFN-γ secretion from IELs from day 7 infected mice, and was suppressed by treatment with anti-ICOS mAb. Additionally, IFN-γ mRNA increased in CD4+ IELs on day 6 post-infection. These findings indicate that S7− and S7+ IELs are differentially mobilized during the immune response to reovirus infection; that the regulated expression of ICOS is associated with S7+ IELs; and that stimulation of IELs through ICOS enhances IFN-γ synthesis during infection.

Regional variations in the distributions of small intestinal intraepithelial lymphocytes (IELs) in outbred laboratory mice ( Mus musculus domesticus) and the inbred strain of mice established from Japanese fancy mice ( Mus musculus molossinus)

Previously, we reported regional variations in small intestinal IELs of mice. In this study, we examined the regional variations of IELs in outbred laboratory mice (ddY: Mus musculus domesticus) and the inbred strain of mice established from Japanese fancy mice (JF1: Mus musculus molossinus). IELs were isolated from the proximal, middle and distal parts of the small intestine and analyzed by flow cytometry. The percentages of γδ T cells and αβ T cell subset of extrathymic origin were higher in the proximal part while the percentages of αβ T cell subset(s) of thymic origin were higher in the distal part in both ddY and JF1 mice. Such trends in regional variations of IELs were almost the same as those found in the inbred strains of laboratory mice in our previous reports. This strongly suggests that these regional variations of IELs may be common phenomena in Mus musculus species.

Late Postnatal Expansion of Self-reactive CD8αα+ Intestinal Intraepithelial Lymphocytes in Mice*

The intestinal epithelium is unique in that it harbors auto-reactive T cells largely absent from the peripheral TCR repertoire in normal mice. Intestinal intraepithelial lymphocytes (IEL) expressing self-reactive TCR are mostly CD8αα+ cells in adult H-Y TCR RAG−/− male mice homozygous for the restricting MHC I allele, H-2Db. By contrast, in male mice heterozygous for the restricting and non-restricting MHC I allele, H-2Dd (MHC F1, H-2Db/d), IEL are composed of CD8αβ and CD8αα+ T cells. Here we demonstrate that IEL in the immediate postnatal period of MHC homozygous male mice were mostly CD8− T cells, while IEL in MHC F1 male mice were CD8− and CD8αβ+ T cells. Regardless of the MHC I configuration and the ability to support positive selection of CD8αβ+ cells in the thymus, the expansion of CD8αα+ IEL was a late postnatal event that followed a reduction in CD8− IEL. Furthermore, although in vivo treatment with the specific peptide antigen resulted in an earlier accumulation of activated IEL, the expansion of CD8αα+ IEL remained inefficient until late in postnatal life. Finally, as CD8− IEL stimulated with TCR agonists in vitro, acquired expression of CD8αα, we propose that CD8αα+ IEL derive from CD8− IEL intermediates. Whether CD8− IEL are CD8αβ-lineage cells that escape deletion in the thymus or are T cells targeted to the intestine from the thymus because of the early and high level TCR transgene expression in this model, is not clear. The signals required for the expansion of CD8αα+ IEL are however, incomplete in the immediate postnatal intestine. Determining the factors required for the expansion or retention of CD8αα+ IEL bearing high affinity, self-specific TCR will further elucidate the in vivo role of these T cells in intestinal homeostasis and perhaps, autoimmunity.

Antigen feeding increases frequency and antigen-specific proliferation ability of intraepithelial CD4+ T cells in alphabeta T cell receptor transgenic mice.

To study how intestinal intraepithelial lymphocytes (IEL) are affected by orally ingested antigen, the phenotypes and responses of the IEL in mice expressing a transgenic T cell receptor alphabeta (TCR alphabeta) specific for ovalbumin (OVA) were analyzed after feeding OVA. In the OVA-fed mice, the abundance of alphabeta-IEL as a proportion of the total IEL population increased and the frequency of CD4+ cells increased within the TCR alphabeta+ IEL population. CD4(+) IEL from OVA-fed transgenic mice proliferated in vitro more markedly in response to antigen stimulation than IEL from mice fed the control diet. These results indicate that antigen-specific proliferation of CD4+ IEL was amplified as a result of oral administration of antigen.

Total parenteral nutrition-associated changes in mouse intestinal intraepithelial lymphocytes.

Intraepithelial lymphocytes (IEL) play a major role in mucosal defense mechanisms against intraluminal foreign antigens. To address the role luminal nutrients have on the phenotype and function of the IEL, we administered total parenteral nutrition (TPN) to mice, with the absence of enteral intake. We hypothesized that administration of TPN would result in changes in the phenotype and function of the IEL. For this, we utilized a mouse model of TPN. A significant decline in the CD4+ IEL population occurred with TPN. Additionally, the CD8+,CD44+ IEL subset showed a 65% decline (P < 0.05), and the CD4+,CD44+ subset declined by 55% with TPN (P < 0.05). The CD8alphabeta+ population (a marker of thymic-dependence) also declined by 92% (P < 0.01) with TPN. IEL in the TPN group showed a significantly lower degree of in vitro proliferation. In conclusion, the IEL showed significant phenotypic changes with TPN including the loss of the thymic-derived population. Functionally, the IEL showed a significant decline in proliferation. Such changes demonstrate the important role luminal nutrients have on IEL phenotype and function.

Cutting Edge: TCRαβ+ CD8αα+ T Cells Are Found in Intestinal Intraepithelial Lymphocytes of Mice That Lack Classical MHC Class I Molecules

Abstract TCRαβ+ intestinal intraepithelial lymphocytes (IEL) can express either the typical CD8αβ heterodimer or an unusual CD8αα homodimer. Both types of CD8+ IEL require class I molecules for their differentiation, since they are absent in β2m−/− mice. To gain insight into the role of class I molecules in forming TCRαβ+ CD8+ IEL populations, we have analyzed the IEL in mice deficient for either TAP, β2m, CD1, or K and D. We find that K−/−D−/− mice have TCRαβ+ CD8αα+ IEL, although they are deficient for TCRαβ+ CD8αβ+ cells. This indicates that at least some TCRαβ+ CD8αα+ IEL require only nonclassical class I molecules for their development. Surprisingly, the TCRαβ+ CD8αα+ IEL are significantly increased in K−/−D−/− mice, suggesting a complex interaction between CD8+ IEL and class I molecules that might include direct or indirect negative regulation by K and D, as well as positive effects mediated by nonclassical class I molecules.

Diurnal changes in intraepithelial lymphocytes (IELs) in the small intestine of mice.

Diurnal changes in small intestinal intraepithelial lymphocytes (IELs) were examined in 8- to 10-week-old BALB/cA male mice. The ratio of T cell subsets expressing CD8 alpha alpha homodimer/CD8 alpha beta heterodimer was found to be higher in the dark period than that in the light period. Increased expression of Thy-1.2 on gamma delta T cells was also observed in the light period. No significant changes were found in other subsets. This is the first report to document diurnal changes in the small intestinal IELs in mice.

An opposite pattern of selection of a single T cell antigen receptor in the thymus and among intraepithelial lymphocytes.

The differentiation of intestinal intraepithelial lymphocytes (IEL) remains controversial, which may be due in part to the phenotypic complexity of these T cells. We have investigated here the development of IEL in mice on the recombination activating gene (RAG)-2(-/-) background which express a T cell antigen receptor (TCR) transgene specific for an H-Y peptide presented by Db (H-Y/Db x RAG-2(-) mice). In contrast to the thymus, the small intestine in female H-Y/Db x RAG-2(-) mice is severely deficient in the number of IEL; TCR transgene+ CD8alphaalpha and CD8alphabeta are virtually absent. This is similar to the number and phenotype of IEL in transgenic mice that do not express the Db class I molecule, and which therefore fail positive selection. Paradoxically, in male mice, the small intestine contains large numbers of TCR+ IEL that express high levels of CD8alphaalpha homodimers. The IEL isolated from male mice are functional, as they respond upon TCR cross-linking, although they are not autoreactive to stimulator cells from male mice. We hypothesize that the H-Y/Db TCR fails to undergo selection in IEL of female mice due to the reduced avidity of the TCR for major histocompatibility complex peptide in conjunction with the CD8alphaalpha homodimers expressed by many cells in this lineage. By contrast, this reduced TCR/CD8alphaalpha avidity may permit positive rather than negative selection of this TCR in male mice. Therefore, the data presented provide conclusive evidence that a TCR which is positively selected in the thymus will not necessarily be selected in IEL, and furthermore, that the expression of a distinct CD8 isoform by IEL may be a critical determinant of the differential pattern of selection of these T cells.

Hormone Regulation of Immune Homeostasis: Local or Long Distance?

The endocrine system and the immune system consist of dynamic biological processes involved on the one hand in the regulation of a complex array of metabolic and physiologic activities, and on the other hand in protection against infection and disease. Evidence for bidirectional functional involvement of immune–endocrine interactions can be seen at many levels, including codependence during critical stages of development, the complementary use of shared molecular mediators and receptors by both systems, and the integrated participation of the immune system and the endocrine system in resistance or susceptibility to disease. Moreover, recent findings—principally derived from studies of intestinal intraepithelial lymphocytes in mice—suggest that immune–endocrine interactions are essential for the proper development of intestinal T cells, and indicate that this most likely occurs via a local network of hormone synthesis and utilization. In the present article, these findings will be discussed in the context of immune–endocrine collaboration, with particular attention given to the involvement of the thymus in this process, and a hypothesis will be proposed which suggests that the homeostatic balance between health and disease is largely driven by local rather than systemic hormonal regulatory events.

Effects of a Nonapeptide Thymic Hormone on Intestinal Intraepithelial Lymphocytes in Mice Following Administration of 5-Fluorouracil

A significant fraction of murine small intestinal intraepithelial lymphocytes (i-IELs) maturate in local sites outside the thymus. However, there is evidence suggesting that extrathymic differentiation of i-IELs is still influenced by the thymus or thymus-derived factors. Facteur thymique serique (FTS), a nonapeptide thymic hormone, is involved in several aspects of intra- and extrathymic T cell differentiationin vivo.In this study, we investigated the effects of FTS on the kinetics of i-IELs in mice following a single administration of 5-fluorouracil (5-FU). FTS treatment significantly accelerated the recovery in cell number of i-IELs after administration of 5-FU. Flow cytometric analysis revealed that this accelerated recovery was mainly due to a rapid increase in CD8αα+i-IELs. Similar findings were also evident in adult thymectomized (ATX) mice, indicating that FTS treatment caused a rapid recovery of CD8αα+i-IELs following 5-FU administration in the absence of a functional thymus. Furthermore, expression levels of the mRNAs for interleukin-2, interferon-γ, and transforming growth factor β1in the i-IELs were augmented by FTS treatment. Notably, FTS treatment protected mice from 5-FU-induced lethal toxicity, accompanied with an inhibition of the translocation ofEnterobactericeae.These results suggest that FTS has an important function in the extrathymic maturation and activation of i-IELs in the small intestine following 5-FU administration, which may contribute at least partly to the protection against 5-FU-induced lethal toxicity.

Applications of the MTT assay to functional studies of mouse intestinal intraepithelial lymphocytes

The MTT [3(4,5-dimethyl-thiazoyl-2-yl)2,5 diphenyl-tetrazolium bromide] and other related colorimetric assays were compared, and applied to functional studies of mouse intestinal intraepithelial lymphocytes and their hybridomas. Functional studies of intestinal lymphocytes included cell adhesion, cytotoxicity, growth inhibition, and proliferation. Our data demonstrate that MTT and related assays are time, labor, and cost saving alternatives to standard radiolabeling assays. The novel application of the MTT method to measure cell-to-cell adhesion and cell-mediated cytotoxicity greatly speed and simplify the determination. The new application of the MTT methods provides a reliable, reproducible, and rapid alternative for the study of intestinal immune cell functions. © 1996 Wiley-Liss, Inc.

Phenotype and TCR gamma delta variable gene repertoire of intestinal intraepithelial lymphocytes in wild mice (Mus musculus domesticus): abundance of V gamma 1 transcripts and extensive delta gene diversity.

In order to study murine intestinal intraepithelial lymphocytes (IEL) independent of factors imparted by conditions of laboratory housing and breeding, and to provide a basis for comparison of IEL studies between inbred and outbred mouse populations, IEL from the domestic house mouse, Mus musculus domesticus, were analyzed by flow cytometric analyses using mAbs to murine lymphocyte markers, and by polymerase chain reaction to study the TCR gamma and delta V gene repertoires. The majority of IEL in wild mice were CD3+, CD8+CD4- T cells. CD4+CD8- also were present in IEL isolates from wild mice, although at low numbers. Among IEL, but not T cells from the spleen or lymph nodes, there was a notable lack of Thy-1 expression, a preponderance of CD8 alpha alpha + T cells, and a relatively high ratio (3:1) of TCR gamma delta + T cells over TCR alpha beta + T cells, suggesting that some IEL in wild mice may develop via an extrathymic pathway similar to that described for laboratory mice. Analyses of the IEL gamma and delta variable genes revealed rearrangements of three of six V region gamma genes (V gamma 1, V gamma 2, and V gamma 5), with an abundance of V gamma 1 transcripts as determined by Northern blot analyses. For the delta gene, rearrangement of five of seven V region elements had occurred (V delta 2, V delta 3, V delta 4, V delta 5, and V delta 6).(ABSTRACT TRUNCATED AT 250 WORDS)

T-Cell Receptor γδ Diversity and Specificity of Intestinal Intraepithelial Lymphocytes: Analysis of IEL-Derived Hybridomas

The phenotype, T-cell antigen receptor (TCR) usage and specificity of intestinal intraepithelial lymphocytes (IEL), and hybridomas derived from IEL have been examined. In young conventionally reared mice, approximately 50% of the IEL express TCR αβ and 50% express TCR γδ, although there is considerable variation between individuals. Here we demonstrate that T-cell hybridomas can be prepared from freshly isolated BALB/c mouse IEL. The expressed TCR of these hybridomas reflects the TCR isotype distribution of the IEL population. Analysis of the γδ TCR expressed by the hybridomas demonstrates that IEL express a greater number of TCR V gene segments than has been reported for γδ T cells in several other epithelial sites. At least five types of γδ TCRs are expressed by a panel of BALB/c IEL hybridomas, although use of the γδ TCR V gene repertoire clearly is not random. Some TCR γδ cells and γδ hybridomas have been reported to recognize purified protein derivative (PPD); however, none of the IEL hybridomas secreted IL-2 in response to PPD. These data suggest that most TCR γδ IEL are not likely to be PPD reactive.

Age-related changes of T cell subsets in intestinal intraepithelial lymphocytes of mice.

Age-related changes in T cell subsets were examined in intestinal intraepithelial lymphocytes (i-IEL), which contain unique T cells differentiating extrathymically. In 2-month-old mice bred under conventional condition, i-IEL consisted of a large number of CD4-CD8 alpha/alpha+ cells bearing either T cell receptor (TcR)alpha/beta or TcR gamma/delta and only a few CD4+CD8 alpha- cells. In aged mice (6 months old and 24 months old), unique CD4+CD8 alpha/alpha+ i-IEL bearing TcR alpha/beta increased in number and conversely the proportion of TcR gamma/delta+ i-IEL was decreased. Such an increase in number of CD4+CD8 alpha/alpha+ cells was detected in i-IEL from aged (14-months old) nude mice, but not in aged (14 months old) germ-free mice, suggesting that a significant fraction of TcR alpha/beta T cells such as CD4+CD8 alpha+ i-IEL can develop along an extrathymic pathway under the influence of intestinal microflora with age.

Thymus-independent development and negative selection of T cells expressing T cell receptor alpha/beta in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes.

We demonstrate that mouse intestinal intraepithelial lymphocytes (IEL) can be divided into subsets based on the differential expression of functional T cell receptor alpha/beta (TCR-alpha/beta) signaling complexes. Two subsets, CD4+ 8 alpha + beta - and CD8 alpha + beta -, are refractory to stimulation with anti-TCR-alpha/beta and contain high frequencies of potentially self-reactive cells. In contrast, the CD4+ and CD8 alpha + beta + IEL subsets are responsive to anti-TCR-alpha/beta and depleted of potentially self-reactive cells. The analysis of fetal liver radiation chimeras using adult thymectomized recipients demonstrates that the four TCR-alpha/beta + IEL subsets are generated in normal numbers in the absence of the thymus. Moreover, expression of the major histocompatibility complex class II-encoded I-E molecule and Mls1a in the gut of the athymic host results in the negative selection of potentially self-reactive T cells expressing V beta 11 and V beta 6, respectively, from those IEL subsets that express functional TCR-alpha/beta signaling complexes. Neither the spleen nor the Peyer's patches of athymic recipients contain T cells of donor origin. In contrast, normal numbers of phenotypically and functionally mature CD4+ and CD8 alpha + beta + T cells of donor origin are found in the lamina propria of chimeric animals. The phenotypic analysis of lymphocytes obtained from Ly5 congenic parabionts reveals that peripheral T cells migrate rapidly to the Peyer's patches and lamina propria, but not to the intestinal epithelium. Taken together, these results demonstrate that the intestinal epithelium is a thymus-independent site of T lymphopoiesis, where selection of the T cell repertoire involves the deletion of potentially self-reactive cells in situ. Moreover, the appearance of donor-derived, phenotypically mature T cells, exclusively in the lamina propria of athymic radiation chimeras, suggests that mature IEL expressing functional TCR-alpha/beta migrate to this site.

Identity between the novel integrin β 7 subunit and an antigen found highly expressed on intraepithelial lymphocytes in the small intestine

A cDNA clone encoding the N-terminal sequence of the murine integrin β 7 subunit, a novel member of the leukocyte cell adhesion molecule subset (Leu-CAM), has been isolated. An N-terminal region of 13 contiguous amino acids deduced from the cDNA shows complete identity with the N-terminus of the 120 kDa subunit of the M290 antigen, a surface molecule found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This unexpected result focuses two previously unconnected areas of research and suggests that integrins may have a special role to play in the defence of the gut mucosa.

A new surface antigen on intraepithelial lymphocytes in the intestine

A new surface molecule has been discovered on mouse intestinal intraepithelial lymphocytes (IEL) using a rat anti-mouse IEL monoclonal antibody, M290. It was expressed at high levels on nearly all IEL and on a majority of T cells in the gut lamina propria. M290 stained, with lower intensity, a small minority of T cells in other lymphoid tissues. Expression was biased towards the CD8+ subset. Stimulation of peripheral T cells with mitogens did not induce expression of the new antigen but addition of transforming growth factor beta to stimulated T cells had a marked inductive effect. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of IEL surface components precipitated with M290 showed principal bands at 135, 120, 28 and 24 kDa (reduced) and 135, 100, 24 and 21 kDa (nonreduced). Precipitation with antibodies to integrin subunits showed that the new molecular complex was not a member of the beta 1, beta 2, or beta 3 integrin families although all of these were represented on IEL. A 13-amino acid N-terminal sequence obtained from the 120-kDa beta subunit of the antigen prepared from an M290+ T hybridoma (MTC-1) did not show homology with integrins. Pulse-chase studies using MTC-1 cells showed that the 135-kDa alpha subunit was derived from a 147-kDa precursor. The function of this new molecular complex is not yet known.