The cooperative nature of tetraspanin–tetraspanin interactions in membrane organization suggests functional overlap is likely to be important in tetraspanin biology. Previous functional studies of the tetraspanins CD37 and Tssc6 in the immune system found that both CD37 and Tssc6 regulate T cell proliferative responses in vitro. CD37−/− mice also displayed a hyper-stimulatory dendritic cell phenotype and dysregulated humoral responses. In this study, we characterize “double knockout” mice (CD37−/−Tssc6−/−) generated to investigate functional overlap between these tetraspanins. Strong evidence for a cooperative role for these two proteins was identified in cellular immunity, where both in vitro T cell proliferative responses and dendritic cell stimulation capacity are significantly exaggerated in CD37−/−Tssc6−/− mice when compared with single knockout counterparts. Despite these exaggerated cellular responses in vitro, CD37−/−Tssc6−/− mice are not more susceptible to autoimmune induction. However, in vivo responses to pathogens appear poor in CD37−/−Tssc6−/− mice, which showed a reduced ability to produce influenza-specific T cells and displayed a rapid onset hyper-parasitemia when infected with Plasmodium yoelii. Therefore, in the absence of both CD37 and Tssc6, immune function is further altered when compared with CD37−/− or Tssc6−/− mice, demonstrating a complementary role for these two molecules in cellular immunity.

Tetraspanins are a family of four-transmembrane proteins expressed at the cell surface and subcellular compartments (13). Their major role is the spatial organization of proteins in the cell membrane, where tetraspanins direct other proteins into regulated, signal-transducing microdomains known as tetraspanin-enriched microdomains (TEMs) (1) or the “tetraspanin web” (4). The diversity of proteins organized within TEMs is impressive and includes: integrins (5), growth factors (6, 7), metalloproteases (8), signaling molecules (1, 9), and many of the most important immune cell surface molecules, such as CD4, CD8, MHC class I, and MHC class II (2, 3, 9). Within TEMs there exists a hierarchy of molecular interactions. Tetraspanin molecules strongly and directly interact with “partner” molecules. Classical examples of tetraspanin-partner interactions are those between tetraspanins and integrins. The functional evidence that such interactions are important is overwhelming and exemplified by the integrin dysregulation observed in many tetraspanin-deficient cells (1014). A second type of molecular interaction occurs between tetraspanin proteins. Tetraspanin–tetraspanin interactions are weaker than tetraspanin-partner interactions, are stabilized by palmitoylation of conserved membrane-proximal cysteine residues (1), and play a critical role in maintaining tetraspanin microdomain integrity. Interactions between tetraspanins bring into proximity their respective partner molecules, and this allows a third type of interaction to occur within TEMs; “indirect” interactions that can occur between any of the molecules present in the microdomain. These weak interactions can be functionally relevant and the localization of a molecule to a tetraspanin microdomain can influence its function (15).

The functional consequences of molecular organization by tetraspanins in the immune system are not completely understood, and most studies on the role of tetraspanins in the immune system have focused on analyses of tetraspanin-deficient mice (1625). This paper concerns two tetraspanins known to regulate immune function—CD37 (Tspan26) and Tssc6 (Phemx, Tspan32). CD37 and Tssc6 show a similar pattern of tissue expression in mice, restricted to leukocytes and hematopoietic cells, respectively. Moreover, the phenotypes of CD37−/− (16, 20, 23) and Tssc6−/− (21) mice show significant similarities in cellular immunity. Both tetraspanin-deficient mice display a hyper-proliferative T cell phenotype, due, in both cases, to early upregulation of the proliferation-inducing cytokine IL-2. Dendritic cells (DCs) from CD37−/− mice demonstrated an enhanced capacity to present Ag in vitro (20), whereas the Ag-presenting capacity of Tssc6−/− DCs has not been previously described. However, there are differences in phenotype with respect to humoral immunity, as CD37−/− mice show poor T cell-dependent IgG responses (16) and elevated IgA responses (26), whereas Ab responses in Tssc6−/− mice are normal (21).

A major question in tetraspanin biology is the degree to which tetraspanins have unique, specific functions that cannot be replaced by other family members. This may be compared with the extent to which there is functional overlap and compensation between tetraspanins. Given that tetraspanins molecularly associate with one another and exist in the same microdomains, it is not surprising that some functions are shared by tetraspanins. For example, several tetraspanins have been implicated in regulating cancer cell motility (CD9, CD81, CD82, CD151, and CO/029) (1, 9) and also in transducing costimulatory signals in T lymphocytes (CD9, CD53, CD63, CD81, and CD82) (3, 27). One approach toward comparing the degree of functional specificity with functional redundancy in tetraspanin biology is to create mice deficient for multiple tetraspanins. In this study, we present the initial analyses of a CD37−/−Tssc6−/− double knockout mouse, the first mouse deficient in the expression of multiple hematopoietic restricted tetraspanins to be reported. Our analyses show that although CD37 has a unique role in promoting humoral immunity, the exaggerated phenotypes observed in various aspects of the CD37−/−Tssc6−/− cellular immune system demonstrate a complementary role between CD37 and Tssc6 in T cell and DC biology.

CD37−/− and Tssc6−/− mice (16, 21) were backcrossed to a C57BL/6 background for 10 generations. Interbreeding these lines generated CD37−/−Tssc6−/− mice. Tetraspanin-deficient mice were bred at the Burnet Institute (Austin Campus) animal facility under pathogen-free conditions. C57BL/6, DBA/1, OT-I, and OT-II mice were purchased from the Walter and Eliza Hall Institute. In all experiments, age- and sex-matched mice were used between 6 and 12 wk and C57BL/6 mice were used as controls. The relevant institutional animal ethics committees reviewed and approved all animal experimentation.

In FACS analyses, Abs against the following leukocyte surface markers were used: CD3 (KT31.1), CD4 (GK1.5), CD8α* (YTS169.4), CD11c* (N418), CD19* (1D3), CD28 (37.51), CD40 (FGK45.5), CD80 (16-10A1), CD86 (GL-1), DEC205* (NLDC145), Ly-71 (F4/80), MHC class II (M5/114), and IgM* (II/41). Abs were purified in house and biotinylated or conjugated to FITC, PE, or cyanine 5; those with an asterisk (*) were purchased from BD Pharmingen (San Diego, CA). All profiles were gated on cells that excluded propidium iodide, autofluorescence, and were inclusive of a broad range of forward and side scatter properties.

The 96-well plates were coated with 0.1–10 μg/ml anti-CD3 mAb (KT3.1) overnight at 4°C. CD4+ or CD8+ T cells were purified from lymph node and spleen by Ab-magnetic bead depletion (20) and incubated at 1 × 105 cells/well in the presence or absence of 1 μg/ml anti-CD28 mAbs (37.51). Cells were cultured for 6 d and unstimulated wells were included as a negative control. T cell proliferation was assayed at 24 h intervals by [3H]thymidine incorporation (Amersham, Aylesbury, U.K.).

Splenocytes were plated in 96-well plates at 1 × 105 cells/well and stimulated with 0.1–3.0 μg/ml of the B cell mitogen LPS (Sigma-Aldrich, St. Louis, MO), purified anti-CD40 (FGK 45.5) or the F(ab′)2 region of anti-IgM (Abcam, Cambridge, U.K.). Cells were cultured for 5 d and unstimulated splenocytes were included as a control. B cell proliferation was assayed at 24 h intervals by [3H]thymidine incorporation (Amersham).

Mice were immunized i.p. with 100 μg NP(20)-KLH (4-hydroxy-3-nitrophenylacetyl conjugated to Keyhole limpet hemocyanin–conjugation ratio shown in subscript) (Biosearch Technologies, Novato, CA) precipitated in alum or 50 μg NP(3)-LPS (Biosearch) and after 21 d boosted with the same Ag. Mice were bled retro-orbitally over 35 d and serum used in ELISA. The 96-well immunosorbent plates were coated with 5 μg/ml NP(20)-BSA or NP(3)-BSA (Biosearch) overnight at 4°C. Plates were blocked with 3% BSA/PBS and serum diluted in 1% BSA/PBS. NP-specific Abs were detected using anti-mouse IgG1, IgA, and IgM HRP conjugated Abs (BD Pharmingen). Basal Ig levels were tested by sandwich ELISA. Plates were coated with anti-mouse Ig (H+L) (Chemicon International, Temecula, CA) and naive mouse sera diluted in 1% BSA/PBS. Anti-mouse IgG1, IgG2a+c, IgG2b, IgG3, IgM, IgE, and IgA Abs (BD Pharmingen) detected bound Ab. The 4-hydroxy-3-nitrophenylacetyl peroxidase substrate solution (Invitrogen, Carlsbad, CA) and 0.1 M HCl were used for colorimetric detection and OD measured at 450 nm.

DCs were isolated from spleen by enzymatic digestion and density gradient centrifugation (28), followed by CD11c+ AutoMACS positive selection (Miltenyi Biotec, Bergisch Gladbach, Germany). DCs were then pulsed at 37°C with OVA peptide (SIINFEKL or ISQAVHAAHAEINEAGR) at varying concentrations for 1 h. After repeated washing, 2 × 103 pulsed DCs were coincubated with 2 × 104 Ag-specific T cells derived from either the OT-I or OT-II transgenic mouse lines (29, 30). Unpulsed DCs, T cell only, and DC-only controls were included on all plates. T cell proliferation was assessed at 24 h intervals by [3H]thymidine incorporation (Amersham).

The 96-well plates were coated with 0.5 or 1 μg/ml anti-CD3 mAb (KT3.1) overnight at 4°C. The 1 × 105 total T cells and either 2 × 103 or 1 × 104 DCs per well were coincubated for 3 d with T cell only and DC-only wells included as controls. T cell proliferation was assayed on day 3 by [3H]thymidine incorporation (Amersham).

Arthritis was induced by s.c. base of tail immunization with a 1:1 emulsion of 100 μg bovine collagen type II (Chondrex, Redmond, WA) and CFA containing heat killed Mycobacterium tuberculosis strain H37Ra (2.5 mg/ml) (Sigma-Aldrich) (31, 32). Mice were later boosted at day 21 with 100 μg collagen II emulsified in IFA assay (Sigma-Aldrich) (31). Mice were scored for each paw as follows: 0, normal; 1, minor swelling or redness in a single knuckle or digit; 2, severe swelling and redness, but no loss of joint function; and 3, severe swelling and redness, joint dysfunction and stiffness. Arthritis index was calculated by the sum of the score for each paw and six mice were used in each group. Experimental autoimmune encephalomyelitis (EAE) was induced in mice using a standard protocol (33). A total of 200 μg myelin oligodendrocyte protein peptide (MOG35–55) was dissolved in PBS and emulsified in an equal volume of CFA, containing 1 mg/ml of heat killed M. tuberculosis H37Ra (Sigma-Aldrich). On day 0 (day of disease induction), one s.c. injection was given in each flank per mouse. Pertussis toxin (Sigma-Aldrich) (400 ng/mouse) was injected i.p. on the day of EAE induction, followed 48 h later by a second dose (200 ng/mouse). Animals were evaluated daily for clinical signs of disease, starting from day 1 post immunization using a six-grade clinical scale: 0, normal animal; 0.5, weight loss; 1, inability to elevate the tail above the horizontal level, tail weakness; 2, tail paralysis; 3, tail paralysis/hind limb paresis; 4, hind limb paralysis/forelimb weakness; 5, quadriplegia/moribund; and 6, death from EAE.

Mice were injected i.p. with 5 × 105Plasmodium yoelii yoelii YM-parasitized RBCs (34). Parasitemia was monitored daily from day 3 postinfection microscopically by Giemsa-stained thin blood smears. The presence of parasitemia was determined by counting 500 cells per slide. Mice were culled after reaching 70% parasitemia.

Mice were anesthetized with methoxyfluorane and then intranasally infected with a nonlethal challenge of 104.5 PFU of A/HKx31 (H3N2, X31) influenza virus diluted in 25 μl sterile PBS. Virus-specific CD8+ T cells were identified using MHC class I/peptide tetrameric complexes of the H-2Db gp and peptides derived from the nucleoprotein (ASNENMETM) (35) and acid polymerase (PA) (SSLENFRAYV) (36) referred to as DbNP366 and DbPA224, respectively. Recombinant H-2Db molecules with a BirA biotinylation motif substituted for the C-terminal transmembrane domain were refolded with human β2-microglobulin plus the appropriate viral peptide, biotinylated with BirA, and complexed at a 4:1 molar ratio with neutravidin-PE (Molecular Probes, Eugene, OR). Lymphocytes were stained for 1 h at room temperature with the tetrameric complexes in PBS-BSA-azide and then were stained with CD8α-FITC for 30 min on ice, washed twice, and analyzed by flow cytometry.

In all quantitative assays, data points represent the average of multiple replicates within a single representative experiment and error bars represent the SEM. Significance was tested by Student t test in comparison with CD37−/−, Tssc6−/−, and wild-type (WT) controls. The log-rank test was used to compare survival curves during malaria challenge in comparison with CD37−/−, Tssc6−/−, and WT controls. The χ2 analyses were used to compare the incidence of high parasitemia (>60%) in the same assay. The asterisk (*) denotes a p value <0.05 when compared with WT controls; ** denotes a p value <0.05 when compared with both CD37−/− and Tssc6−/− controls.

To determine whether CD37 and Tssc6 have a collaborative role in development, we assessed the numbers and types of cells present in the lymphoid organs of CD37−/−Tssc6−/− mice. Normal numbers of erythroid, lymphoid, and myeloid cells were found in all CD37−/−Tssc6−/− tissues tested (Table I). A peripheral blood examination of CD37−/−Tssc6−/− mice in comparison with WT controls provided no evidence of anemia, lymphopenia, or thrombocytopenia. Flow cytometric analysis was used to dissect major cell populations in thymus, bone marrow, spleen, peritoneal exudate, peripheral blood, and lymph node (Fig. 1, Table I). In CD37−/−Tssc6−/− mice, all major T cell, B cell, granulocyte, macrophage, NK, and DC populations were present and in normal numbers.

Table I.
Frequency of lymphocytes in CD37−/−Tssc6−/− mice
Genotype
   Tissue/Lymphocyte SubsetWTCD37−/−Tssc6−/−
Peripheral blooda   
 Hematocrit (%) 47 ± 3 46 ± 3 
 WBC count (×106/ml) 7 ± 2 7 ± 2 
 Neutrophils 0.7 ± 0.6 0.6 ± 0.2 
 Lymphocytes 5 ± 2 6 ± 2 
 Monocytes 0.1 ± 0.1 0.2 ± 0.1 
 Eosinophils 0.1 ± 0.1 0.2 ± 0.2 
 Platelets (×106/ml) 958 ± 212 1014 ± 227 
 Bone marrow (×106/femur × 2) 29 ± 5 26 ± 6 
 Spleen (×106/ml) 53 ± 10 55 ± 10 
 Thymus (×106/ml) 191 ± 42 204 ± 130 
 Lymph nodes (×106/inguinal nodes) 4 ± 1 4 ± 2 
Thymusb   
 CD4CD8 2 ± 0 2 ± 0 
 CD4+CD8+ 87 ± 1 88 ± 1 
 CD4+ 8 ± 1 7 ± 2 
 CD8+ 3 ± 0 3 ± 1 
Bone marrowb   
 B220+IgM 42 ± 6 46 
 B220lowIgM+ 28 ± 4 31 
 B220highIgM+ 14 ± 2 10 
Spleenb   
 B220+IgM+ 47 ± 5 50 ± 8 
 CD4+ 15 ± 3 14 ± 3 
 CD8+ 9 ± 3 8 ± 3 
Lymph nodeb   
 B220+IgM+ 12 ± 2 17 ± 3 
 CD4+ 34 ± 4 34 ± 2 
 CD8+ 24 ± 1 21 ± 2 
Genotype
   Tissue/Lymphocyte SubsetWTCD37−/−Tssc6−/−
Peripheral blooda   
 Hematocrit (%) 47 ± 3 46 ± 3 
 WBC count (×106/ml) 7 ± 2 7 ± 2 
 Neutrophils 0.7 ± 0.6 0.6 ± 0.2 
 Lymphocytes 5 ± 2 6 ± 2 
 Monocytes 0.1 ± 0.1 0.2 ± 0.1 
 Eosinophils 0.1 ± 0.1 0.2 ± 0.2 
 Platelets (×106/ml) 958 ± 212 1014 ± 227 
 Bone marrow (×106/femur × 2) 29 ± 5 26 ± 6 
 Spleen (×106/ml) 53 ± 10 55 ± 10 
 Thymus (×106/ml) 191 ± 42 204 ± 130 
 Lymph nodes (×106/inguinal nodes) 4 ± 1 4 ± 2 
Thymusb   
 CD4CD8 2 ± 0 2 ± 0 
 CD4+CD8+ 87 ± 1 88 ± 1 
 CD4+ 8 ± 1 7 ± 2 
 CD8+ 3 ± 0 3 ± 1 
Bone marrowb   
 B220+IgM 42 ± 6 46 
 B220lowIgM+ 28 ± 4 31 
 B220highIgM+ 14 ± 2 10 
Spleenb   
 B220+IgM+ 47 ± 5 50 ± 8 
 CD4+ 15 ± 3 14 ± 3 
 CD8+ 9 ± 3 8 ± 3 
Lymph nodeb   
 B220+IgM+ 12 ± 2 17 ± 3 
 CD4+ 34 ± 4 34 ± 2 
 CD8+ 24 ± 1 21 ± 2 
a

Values (± SD) represent mean cell numbers from 5–11 mice of each genotype, except thymus cellularities, which are derived from 3 mice of each genotype.

b

Values (± SD) represent mean percentage of lymphocytes that express the indicated cell surface marker. Values are derived from three to five animals of each genotype, except CD37−/−Tssc6−/− bone marrow where the mean of two animals is shown.

FIGURE 1.

Normal cell surface immunophenotype in CD37−/−Tssc6−/− leukocytes. Cell surface marker expression of WT and CD37−/−Tssc6−/− leukocytes was assessed by flow cytometry. A, Splenocytes were purified and stained for the B cell and T cell markers CD19 and CD3. B, Bone marrow was assessed for CD45R (B220) and IgM expression in developing B cells. C, CD4+ and CD8+ T cell subsets were compared in CD3+ spleen. D, Splenic DCs were purified, gated on CD11c+ expression and CD4+, CD8+, and CD4CD8 (DN) subsets compared. E, DCs from inguinal lymph nodes were purified, gated on CD11c+ expression and DEC205CD8 (1), DEC205intCD8lo (2), DEC205hiCD8int (3), and DEC205intCD8hi (4) subsets.

FIGURE 1.

Normal cell surface immunophenotype in CD37−/−Tssc6−/− leukocytes. Cell surface marker expression of WT and CD37−/−Tssc6−/− leukocytes was assessed by flow cytometry. A, Splenocytes were purified and stained for the B cell and T cell markers CD19 and CD3. B, Bone marrow was assessed for CD45R (B220) and IgM expression in developing B cells. C, CD4+ and CD8+ T cell subsets were compared in CD3+ spleen. D, Splenic DCs were purified, gated on CD11c+ expression and CD4+, CD8+, and CD4CD8 (DN) subsets compared. E, DCs from inguinal lymph nodes were purified, gated on CD11c+ expression and DEC205CD8 (1), DEC205intCD8lo (2), DEC205hiCD8int (3), and DEC205intCD8hi (4) subsets.

Close modal

To investigate a cooperative role for CD37 and Tssc6 in T cell function, we assessed CD37−/−Tssc6−/− and WT T cell proliferative responses to TCR cross-linking and costimulation, in comparison with responses from CD37−/− and Tssc6−/− single knockout T cells (Fig. 2). We confirm earlier findings that demonstrated a T cell hyper-proliferative defect in both CD37−/− and Tssc6−/− T cells (21, 23). Although, more striking was the finding that CD4+ and CD8+ T cells from CD37−/−Tssc6−/− mice are hyper-proliferative to stimulation and this defect is significantly exaggerated in comparison with the hyper-proliferative CD37 and Tssc6 “single knockout” T cells (Fig. 2). This exaggerated hyper-proliferative response was evident in both CD4+ and CD8+ CD37−/−Tssc6−/− T cells when stimulated with anti-CD3 and in CD4+ T cells when stimulated in the presence of costimulation (anti-CD3 and anti-CD28 mAbs).

FIGURE 2.

Exaggerated in vitro hyper-proliferation in CD37−/−Tssc6−/− T cells. Purified CD4+ or CD8+ T cells from WT, CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice were stimulated over 3 d with titrated concentrations of surface adsorbed anti-CD3 mAbs, in the presence or absence of soluble anti-CD28 mAbs (1 μg/ml). A, CD4+ T cells (anti-CD3), (B) CD4+ T cells (anti-CD3+anti-CD28), (C) CD8+ T cells (anti-CD3), (D) CD8+ T cells (anti-CD3+anti-CD28). Data points (mean + SEM) represent [3H]thymidine incorporation across quadruplicate wells. *p < 0.05 when compared with WT controls; **p < 0.05 when compared with both CD37−/− and Tssc6−/− controls.

FIGURE 2.

Exaggerated in vitro hyper-proliferation in CD37−/−Tssc6−/− T cells. Purified CD4+ or CD8+ T cells from WT, CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice were stimulated over 3 d with titrated concentrations of surface adsorbed anti-CD3 mAbs, in the presence or absence of soluble anti-CD28 mAbs (1 μg/ml). A, CD4+ T cells (anti-CD3), (B) CD4+ T cells (anti-CD3+anti-CD28), (C) CD8+ T cells (anti-CD3), (D) CD8+ T cells (anti-CD3+anti-CD28). Data points (mean + SEM) represent [3H]thymidine incorporation across quadruplicate wells. *p < 0.05 when compared with WT controls; **p < 0.05 when compared with both CD37−/− and Tssc6−/− controls.

Close modal

To investigate a cooperative role for CD37 and Tssc6 in Ag presentation and DC function, we tested the ability of CD37−/−Tssc6−/− and WT DC to present peptide to Ag-specific class I (Fig. 3A, 3B) and class II (Fig. 3C, 3D) MHC-restricted T cells, in comparison with DCs isolated from CD37−/− and Tssc6−/− single knockout mice. The data confirms our recent reports that CD37−/− DCs display a hyper-stimulatory phenotype in vitro, due to enhanced MHC/peptide presentation (20). In this study, we found a similar but less prominent hyper-stimulatory phenotype in Tssc6−/− DCs (Fig. 3A–D). Furthermore, CD37−/−Tssc6−/− DCs were significantly more hyper-stimulatory than DCs isolated from either single knockout mouse. CD37 is thought to regulate MHC/peptide presentation independent of costimulatory signals (20), whereas the mechanism by which Tssc6 regulates Ag presentation by DCs remains unknown. Therefore, to further understand the role of CD37 and Tssc6 in DC function, we assessed the costimulatory capacity of CD37−/−Tssc6−/− DCs. Because splenic DCs spontaneously upregulate the expression of costimulatory molecules in tissue culture (20), we compared the basal expression and the increase in expression of CD40, CD80, CD86, and MHC class II over a period of 24 h in vitro. We found no difference in basal expression of costimulatory molecules or in the rate of upregulation of these molecules between WT and CD37/Tssc6 knockout DC (data not shown). To confirm this finding, we also performed costimulation assays, where WT T cells were stimulated with anti-CD3 mAbs and costimulation provided by coincubated syngeneic WT or tetraspanin knockout DC. We found there was no difference in costimulatory capacity between WT and CD37−/−Tssc6−/− DCs (Fig. 3E, 3F).

FIGURE 3.

Exaggerated in vitro stimulation but normal costimulation by CD37−/−Tssc6−/− DCs. Splenic DCs isolated from WT, CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice were (A) pulsed with 0.01 μg SIINFEKL and coincubated with OT-I T cells, (B) pulsed with titrated doses of SIINFEKL and coincubated with OT-I T cells (day 2), (C) pulsed with 25 μg Helper peptide and coincubated with OT-II T cells, or (D) pulsed with titrated doses of Helper peptide and coincubated with OT-II T cells (day 3). Purified WT T cells were stimulated in vitro by (E) 0.5 μg/ml or (F) 1.0 μg/ml surface adsorbed anti-CD3 mAbs and purified naive DC from either WT or knockout mice provided costimulatory signals. Data points (mean + SEM) represent [3H]thymidine incorporation across triplicate wells. *p < 0.05 when compared with WT controls; **p < 0.05 when compared with both CD37−/− and Tssc6−/− controls.

FIGURE 3.

Exaggerated in vitro stimulation but normal costimulation by CD37−/−Tssc6−/− DCs. Splenic DCs isolated from WT, CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice were (A) pulsed with 0.01 μg SIINFEKL and coincubated with OT-I T cells, (B) pulsed with titrated doses of SIINFEKL and coincubated with OT-I T cells (day 2), (C) pulsed with 25 μg Helper peptide and coincubated with OT-II T cells, or (D) pulsed with titrated doses of Helper peptide and coincubated with OT-II T cells (day 3). Purified WT T cells were stimulated in vitro by (E) 0.5 μg/ml or (F) 1.0 μg/ml surface adsorbed anti-CD3 mAbs and purified naive DC from either WT or knockout mice provided costimulatory signals. Data points (mean + SEM) represent [3H]thymidine incorporation across triplicate wells. *p < 0.05 when compared with WT controls; **p < 0.05 when compared with both CD37−/− and Tssc6−/− controls.

Close modal

To assess a cooperative role for CD37 and Tssc6 in humoral immunity, we first measured CD37−/−Tssc6−/− and WT B cell proliferative responses (Fig. 4). CD37−/−Tssc6−/− B cells, stimulated with the B cell mitogen LPS or by cross-linking the BCR via anti-IgM mAbs in the presence or absence of costimulation (anti-CD40 mAbs), proliferated normally in response to all stimulants (Fig. 4). We next measured serum Ab in CD37−/−Tssc6−/− and WT mice in comparison with single knockout controls (Fig. 5). In this study, we observed an identical pattern across both CD37−/− and CD37−/−Tssc6−/− genotypes. Basal serum Ig levels in naive CD37−/−Tssc6−/− mice, like CD37−/− mice (16), display a striking deficit in IgG1 (Fig. 5A). Ab responses were normal in all tetraspanin-deficient mice immunized with the T cell-independent Ag NP-LPS (Fig. 5B). However, CD37−/−Tssc6−/− mice, like CD37−/− mice (16, 21), displayed IgG1 responses to NP-KLH that were statistically poorer than that of WT mice (Fig. 5C). CD37−/− mice display elevated serum IgA responses to NP-KLH (26), although Tssc6−/− responses were untested. In this study, we demonstrate that IgA production in Tssc6−/− mice is normal and CD37−/−Tssc6−/− IgA production is similar to that of CD37−/− mice (Fig. 5D). Therefore, the altered IgA production in response to NP-KLH immunization is CD37−/− specific. Taken together, the impaired humoral response of CD37−/−Tssc6−/− mice appears largely dependent upon CD37 deficiency.

FIGURE 4.

Normal in vitro proliferation of CD37−/−Tssc6−/− B cells. Splenocytes from WT, CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice were isolated and stimulated with (A) titrated doses of LPS, (B) 2 μg LPS over 4 d, (C) titrated doses of anti-IgM [F(ab′)2] or (D) varying doses of anti-IgM [F(ab′)2] in the presence of anti-CD40. B cell proliferative responses were measured at 2 d unless otherwise stated. Data points (mean + SEM) represent the [3H]thymidine incorporation across quadruplicate wells. *p < 0.05 when compared with WT controls; **p < 0.05 when compared with both CD37−/− and Tssc6−/− controls.

FIGURE 4.

Normal in vitro proliferation of CD37−/−Tssc6−/− B cells. Splenocytes from WT, CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice were isolated and stimulated with (A) titrated doses of LPS, (B) 2 μg LPS over 4 d, (C) titrated doses of anti-IgM [F(ab′)2] or (D) varying doses of anti-IgM [F(ab′)2] in the presence of anti-CD40. B cell proliferative responses were measured at 2 d unless otherwise stated. Data points (mean + SEM) represent the [3H]thymidine incorporation across quadruplicate wells. *p < 0.05 when compared with WT controls; **p < 0.05 when compared with both CD37−/− and Tssc6−/− controls.

Close modal
FIGURE 5.

CD37−/−Tssc6−/− and CD37−/− mice share a similar dysregulation of humoral immune responses. Serum Ig was assessed in WT, CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice for (A) basal levels of IgG1 in naive mice, (B) NP-specific IgM responses to NP-LPS immunization, (C) NP-specific IgG1, and (D) IgA responses to NP-KLH immunization. Data points represent the mean (± SEM) humoral responses of 6 mice. *p < 0.05 when compared with WT controls; #p < 0.05 comparing CD37−/− and CD37−/−Tssc6−/− mice. The humoral responses of Tssc6 mice were not significantly different from that of WT at any data point.

FIGURE 5.

CD37−/−Tssc6−/− and CD37−/− mice share a similar dysregulation of humoral immune responses. Serum Ig was assessed in WT, CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice for (A) basal levels of IgG1 in naive mice, (B) NP-specific IgM responses to NP-LPS immunization, (C) NP-specific IgG1, and (D) IgA responses to NP-KLH immunization. Data points represent the mean (± SEM) humoral responses of 6 mice. *p < 0.05 when compared with WT controls; #p < 0.05 comparing CD37−/− and CD37−/−Tssc6−/− mice. The humoral responses of Tssc6 mice were not significantly different from that of WT at any data point.

Close modal

Although tetraspanin-deficient mice display a variety of immunological defects, autoimmune disease in these mice remains largely unstudied. Given the hyper-proliferative T cell and hyper-stimulatory DC phenotypes in CD37−/−Tssc6−/− mice, we tested autoimmune induction via collagen-induced arthritis (CIA) and EAE. These models are well characterized and it has been demonstrated that DCs and T cells play an important role in disease induction in both CIA and EAE. We compared the CD37−/−Tssc6−/− mice with single knockout controls (all bred on the C57BL/6 background) with CIA-resistant (C57BL/6) and susceptible controls (DBA-1). We found that absence of CD37, Tssc6, or both CD37 and Tssc6 were not sufficient to convert these mice to a CIA susceptible phenotype (data not shown). Next, we used EAE, an autoimmune model in which C57BL/6 mice are susceptible to disease induction. We found that in the absence of both CD37 and Tssc6, there was a significant change in the kinetics of EAE onset; however, the overall pattern and severity of the disease was unaffected (Fig. 6).

FIGURE 6.

The absence of CD37 and Tssc6 does not confer any long-term increased susceptibility to autoimmune induction. WT and CD37−/−Tssc6−/− mice were monitored over 30 d during EAE induction and scored for tail and hind limb weakness and paralysis. Data points represent the mean (+ SEM) clinical score of six mice/group. *p < 0.05 when compared with WT controls.

FIGURE 6.

The absence of CD37 and Tssc6 does not confer any long-term increased susceptibility to autoimmune induction. WT and CD37−/−Tssc6−/− mice were monitored over 30 d during EAE induction and scored for tail and hind limb weakness and paralysis. Data points represent the mean (+ SEM) clinical score of six mice/group. *p < 0.05 when compared with WT controls.

Close modal

Ag presentation by DCs, and T cell proliferative responses are exaggerated in CD37−/−Tssc6−/− mice, even in comparison with the hyper-stimulatory DC and hyper-proliferative T cells observed in CD37 and TSSC6 single knockout mice (Figs. 2, 3). To analyze the effects that these defects have on in vivo immune responses, we used two infection models. First, we infected the CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− strains with the murine malaria strain P. yoelii NXL17 and monitored blood parasitemia. Tssc6−/− and CD37−/−Tssc6−/− mice were significantly more sensitive to P. yoelii infection than WT mice (p = 0.007 and p = 0.001, respectively), whereas CD37−/− mice survival was similar to WT mice during the first 12 d of infection (Fig. 7A). Early in infection (day 5), CD37−/−Tssc6−/− mice were the only mouse strain to show significantly higher parasitemias than control C57BL/6 mice (p = 0.006; Fig. 7B). Moreover, at this time point, there was clearly an exaggeration in the incidence of mice with high parasitemia (of at least 60%) in the CD37−/−Tssc6−/− mice 7/13 (54%) compared with 1/15 (7%) in the control group, none in the CD37−/− group and 2/8 (25%) in the Tssc6−/− group (Fig. 7B). Interestingly, all tetraspanin-deficient animals eventually succumbed to infection (Fig. 7A). Second we infected WT, CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice via intranasal inoculation with influenza A/HKx31 virus. The generation of Ag-specific CD8+ T cells in response to influenza infection can be detected by tetramer staining for TCR specific to immunodominant viral epitopes. Eight days postinfection, the frequency of Ag-specific CD8+ T cells in the spleen was somewhat lower in CD37−/− and Tssc6−/− mice, although these results were of borderline significance (Fig. 7C). In CD37−/−Tssc6−/− mice, this reduction was most striking, demonstrating that both CD37 and Tssc6 play a complementary role in the development of Ag-specific CD8+ T cells in response to influenza infection.

FIGURE 7.

In vivo pathogenic challenge of CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice. Groups of 8–15 mice were infected i.p. with 500,000 P. yoelii parasites and parasitemia assessed daily. A, Animals were culled when reaching 70% parasitemia; cumulative survival curves are shown. B, Scatter plots showing parasitemia of individual mice of all strains on days 3 and 5. C, Mice were infected intranasally with a nonlethal dose of A/HKx31 influenza virus. After 8 d, lymphoid organs were harvested and virus-specific CD8+ T cells were identified by flow cytometry using the DbNP366 and DbPA224 tetramers. *p < 0.05 when compared with WT controls.

FIGURE 7.

In vivo pathogenic challenge of CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− mice. Groups of 8–15 mice were infected i.p. with 500,000 P. yoelii parasites and parasitemia assessed daily. A, Animals were culled when reaching 70% parasitemia; cumulative survival curves are shown. B, Scatter plots showing parasitemia of individual mice of all strains on days 3 and 5. C, Mice were infected intranasally with a nonlethal dose of A/HKx31 influenza virus. After 8 d, lymphoid organs were harvested and virus-specific CD8+ T cells were identified by flow cytometry using the DbNP366 and DbPA224 tetramers. *p < 0.05 when compared with WT controls.

Close modal

To examine the functional relationship between two molecules that are related to one another in evolution, there are several examples where multiple gene targeting approaches have been an effective tool. In the immune system, the approach was used to demonstrate that the adaptor proteins c-Cbl and Cbl-b work cooperatively to regulate recycling of the TCR complex (37), and that functional redundancy exists between the adhesion molecules E-selectin and P-selectin in mediating leukocyte trans-endothelial migration (38). In mice, the only prior example of tetraspanin functional overlap using a reverse genetics approach is between the closely related tetraspanins CD9 and CD81. Both CD9−/− and CD81−/− female mice have a significant reduction in fertility due to a defect in sperm–egg fusion (3942). Although, only CD9−/−CD81−/− females are completely infertile, indicating a cooperative role for these two molecules in promoting gamete fusion (42). Further evidence of a cooperative role for CD9 and CD81 in cellular fusion is seen in the formation of multinucleated giant cells, in response to inflammation. Osteoclastogenesis is upregulated in CD9 and CD81 deficient mice in response to in vivo and in vitro stimulation. However, only in CD9−/−CD81−/− mice did multinucleated giant cells spontaneously form in the absence of stimulus (43).

Characterization of immune function in CD37- and Tssc6-deficient mice identified similarities between the single knockout phenotypes suggesting these molecules, like CD9 and CD81, may display some degree of functional overlap (16, 21, 23). Due to the lack of Abs to murine CD37 and Tssc6, little is known about the molecular interactions of these tetraspanins. As an alternative approach to investigating the relationship between these molecules, we investigated the immune system of CD37−/−Tssc6−/− mice in comparison with WT, CD37−/−, and Tssc6−/− counterparts. In pathogen-free conditions, the CD37−/−Tssc6−/− mice are developmentally normal and suffer no reduction in fertility. Cell surface immunophenotyping of the “double knockout” leukocytes showed that all major cell types and subpopulations are present and represented in normal numbers (Fig. 1, Table I). It therefore appears unlikely that these tetraspanins play a critical role in immune cell development, either individually or collaboratively.

By contrast, analyses of humoral immune responses in CD37−/−Tssc6−/− mice indicate a unique role for CD37 in B cell biology that shows little overlap with Tssc6. Although CD37 plays no role in regulating signaling through the Ag receptor or CD40, CD37−/− mice made poor IgG1 responses and exaggerated IgA responses to T cell-dependent Ags (16, 26). Conversely, humoral immune responses in Tssc6−/− mice are normal (21). CD37−/−Tssc6−/− mice, like their CD37−/− counterparts make exaggerated IgA responses and poor IgG1 responses (Figs. 4, 5). For both IgG1 and IgA responses, we observed a tendency for the CD37−/−Tssc6−/− mice to be intermediate between CD37−/− and WT. However, this was of marginal significance and the only data point where the CD37−/− and CD37−/−Tssc6−/− mice show a significant difference to one another is at day 28 of the IgG1 response. Clearly, CD37 regulates Ig production in B cells, even in the absence of Tssc6. The absence of Tssc6 only marginally affects the phenotype induced by CD37 deficiency.

In contrast, there was clear evidence of a cooperative role for both CD37 and Tssc6 in aspects of cellular immunity. Certainly, the two molecules collaborate to regulate T cell proliferation, as CD37−/−Tssc6−/− T cells were significantly more hyper-proliferative than their already hyper-proliferative single knockout counterparts. However, these data indicate there are some differences between the requirements of CD4+ and CD8+ T cell lineages for tetraspanin expression, particularly when regulating T cell proliferation in the presence of costimulation. Although CD37−/−Tssc6−/− CD4+ T cell hyper-proliferative responses were exaggerated in both the presence and absence of costimulation, the hyper-proliferative phenotype of CD37−/−Tssc6−/− CD8+ T cells was exaggerated only in the absence of costimulation (Fig. 2). Similarly, differences were also observed (21) (and confirmed in Fig. 2) between Tssc6−/− CD4+ and CD8+ T cells in earlier studies. These data add to growing evidence that tetraspanins play an important role in T cell proliferation. Mice deficient in the tetraspanins CD81, CD37, CD151, Tssc6, and now CD37 and Tssc6 have all demonstrated hyper-proliferative T cell phenotypes in vitro (19, 2123). The mechanism of tetraspanin regulation of T cell division has only been investigated in CD37−/− mice, where it was shown that the kinase activity of CD4/CD8 associated Lck was increased in CD37−/− T cells (23). Thus, in the absence of regulation by CD37, hyper-activated CD4/CD8 associated Lck may inappropriately contribute to TCR signaling. It will be of major interest to determine whether Lck is also dysregulated in T cells deficient in other tetraspanins and whether Lck dysregulation is exaggerated in CD37−/−Tssc6−/− T cells.

CD37 and Tssc6 also cooperate in DC biology as CD37−/−Tssc6−/− DCs were significantly more stimulatory then the already hyper-stimulatory DC purified from either single knockout control (Fig. 3). CD37−/− DC have a significantly higher capacity to stimulate Ag-specific T cells than WT controls and we have argued that this is likely to be due to a dysregulation of MHC function (20). The current study shows a similar phenotype in Tssc6−/− DC. As we found no differences in the maturation rate and costimulation capacity between CD37−/−, Tssc6−/−, and CD37−/−Tssc6−/− DC, we suggest that both CD37 and Tssc6 influence DC Ag presentation independent of costimulatory signals, via the regulation of class I and II MHC.

The dysregulated phenotype observed in CD37−/−Tssc6−/− DC and T cells raises the question of whether in vivo cellular responses are normal, and, in particular, whether these mice might be susceptible to autoimmune disease. To test this, we used two well-characterized autoimmune models. Mice of the C57BL/6 background are resistant to CIA (44), and it was clear that the deletion of either or both Cd37 and Tssc6 genes does not render a genetically resistant mouse susceptible (data not shown). By contrast, C57BL/6 mice are highly susceptible to EAE, and although we did observe a significant, earlier induction of disease in CD37−/−Tssc6−/− mice there was no long-term increase in disease severity (Fig. 6B). Moreover, we have not observed any evidence for spontaneous inflammatory or autoimmune disease in 1-y-old mice (data not shown). We conclude that the exaggerated in vitro phenotypes observed do not lead to aggressive immunopathologies in vivo, even in mice lacking both CD37 and Tssc6.

However, the perturbations in DC and T cell biology induced by tetraspanin-deficiency do have significant effects in vivo; they appear to diminish rather than exacerbate in vivo cellular immune responses. Despite the still very limited understanding of immunity to malaria (45), it is understood that immunity to P. yoelii involves multiple arms of the immune response, including inductions of T cell responses by DCs (34). All tetraspanin knockout animals showed an increased susceptibility to malaria infection (Fig. 7A), where significantly more mice in all knockout strains developed hyper-parasitemia in comparison with WT controls. CD37−/−Tssc6−/− and Tssc6−/− mice were less able to control parasitemia than CD37−/− mice, suggesting a stronger, nonredundant role for Tssc6 than CD37 in clearing plasmodia infection. Nonetheless, there was also some evidence for functional cooperation between CD37 and Tssc6 in immunity to plasmodia infection, as at early time points, CD37−/−Tssc6−/− mice were the only strain to show significantly higher incidence of high parasitemia than control mice (Fig. 7B). Similarly, CD8+ T cell responses to influenza were somewhat diminished in CD37−/− and Tssc6−/− mice and this poor response was significantly magnified in CD37−/−Tssc6−/− mice (Fig. 7C). The results from both infection models suggest that together, CD37 and Tssc6 are involved in the control of antipathogen cellular immunity and that their effects may be additive. Whether, poor cellular responses are due primarily to either aberrant DC or T cell function, or a combination of the two remains to be determined. It is difficult to reconcile the exaggerated cellular responses observed in vitro with the poor responses observed in vivo. T cell cytokine production is known to be linked to T cell division (46), so perhaps a hyper-stimulation of T cells, coupled with the hyper-proliferative phenotype, might dysregulate cytokine production and skew, for example, the anti-influenza immune response away from the production of Ag-specific cytotoxic CD8+ T cells. On this point, there is evidence from both P. yoelii and P. chabaudi murine models that IFN-γ producing T cells (Th1 cells) are induced early in the response, and are important in the control of the initial infection, whereas T cells that produce IL-4 (Th2 phenotype) are produced later in the response to infection, once parasitemia subsides (34, 4749). The loss of control of the early infection stages by parasites in tetraspanin-deficient animals is also consistent with a defect in the generation of effective Th1 responses, which was also suggested by our results measuring CD8+ T cell responses to influenza virus. Alternatively, there are several components of an in vivo immune response, such as response to danger signals, and immune cell migration, that are not analyzed in the in vitro assays.

In summary, when analyzing tetraspanin functions in biology the potential for cooperation and functional overlap between these molecules must be considered. We have identified complementary roles for CD37 and Tssc6 in important aspects of cellular immunity and confirmed that Tssc6 has no major role in humoral immunity. Further work will determine the underlying cellular and molecular defects that result in the poor in vivo cellular responses to infection that were particularly observed in CD37−/−Tssc6−/− mice.

We thank Eliada Lazoura, Roza Nastovska, Carmel Daunt, Nick Van de Velde, Dodie Pouniotis, Hilary Vaughan, and Nick Huntington for technical assistance; Julie Toussaint, Josh Lorimer, and Carly Tobias for animal care; David Tarlinton, David Vremec, and Ken Shortman for the gift of Abs; and George Deraos and John Matsoukas for providing the MOG35–55 peptide.

Disclosures The authors have no financial conflicts of interest.

This work was supported by grants from the National Health and Medical Research Council of Australia (to N.H. and M.R.C.), the Anti-Cancer Council of Victoria, and the Association of International Cancer Research. K.G. was supported by Victoria University, and M.K. was supported by the Ministry of Development Secretariat of Research and Technology of Greece and a Du Pré grant from the Multiple Sclerosis International Federation. A.V.S. was supported by the Dutch Cancer Society (Kankerbestrijding Grant 2007-3917) and the Netherlands Organization for Scientific Research - Earth and Life.

Abbreviations used in this paper:

CIA

collagen-induced arthritis

DC

dendritic cell

EAE

experimental autoimmune encephalomyelitis

MOG

myelin oligodendrocyte protein

NP

4-hydroxy-3-nitrophenylacetyl

TEM

tetraspanin-enriched microdomain

WT

wild type.

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