We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4+ and Vγ6+ γδ T cells (B6.TCR-Vγ4−/−/6−/−), we observed expanded Vγ1+ cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αβ T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4−/−/6−/− mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations.

This article is featured in In This Issue, p.1

B cell differentiation from immature precursors to Ab-producing plasma cells comprises numerous stages (1, 2). Development begins in the bone marrow with common lymphocyte precursors and progresses to immature surface IgM+ B cells, which migrate via the bloodstream to the spleen. Once there, new arrivals differentiate through transitional stages into mature B cells, including B2 follicular (FO), B2 marginal zone (MZ), and B1 B cells. Particularly in the serous cavities, B1 B cells are further divided into B1a and B1b B cells, which differ from one another by their expression of CD5, developmental requirements, and functional roles (3, 4). Some mature B cells recirculate to the bone marrow (5, 6). B cell development is controlled by specific transcription factors (2). Further differentiation also depends on tonic BCR signaling, which is critical for incorporating immature B cells into the peripheral B cell pool (7), as well as on several additional factors and their interplay, including BAFF and NF-κB (2, 8, 9). B cells can develop in the absence of IL-4 (10), but when it is present, this cytokine affects B cell development in bone marrow and periphery (1113), and it enhances CD23 and MHC class II expression (11, 14, 15), and suppresses CD5 expression by B cells (16). During B cell differentiation, B cell tolerance is established at several distinct checkpoints, including one in the bone marrow (central tolerance, BCR selection and editing) (17, 18), another during transition (more BCR selection and competition for BAFF) (17, 19), and a third during Ag activation in the germinal center, where B cells undergo somatic mutation as well as positive and negative selection (20, 21).

In contrast to the well-studied role of T cells as B cell helpers during the immune response and the differentiation of mature B cells into specific Ab-producing cells or memory cells (2229), their role during preimmune B cell development is unclear. Alternatively, NKT cells have been implicated in peripheral B cell homeostasis, especially regarding MZ B cells (30), and recent studies of hematopoietic transplantation in humans and humanized mice indicate that αβ T cells play such a role in the setting of transplantation (31, 32). However, studies in mouse strains with impaired TCR signaling suggested that γδ T cells influence Ab production already in nonimmunized mice (3335).

Subsets of murine γδ T cells as defined by their expression of different TCR-Vγ genes develop sequentially in the thymus during ontogeny (36, 37) and segregate to different organs and tissues (38, 39). Vγ1+ and Vγ4+ cells colocalize in the spleen, where they form comparatively large populations, but they are also present in other lymphoid tissues as well as in the lung and the dermal layer of the skin (40, 41). Comparison of these cells in thymus and spleen revealed different gene expression profiles (42, 43), and functional assays showed that they tend to play opposite roles during certain immune responses (44, 45). In particular, some Vγ1+ cells can produce large amounts of IL-4 whereas Vγ4+ cells have the capability of producing IL-17 (39, 46, 47). Additionally, studies of the role of γδ T cells in a tumor model and during West Nile virus infection produced an indication of reciprocal regulatory interactions between these two γδ subsets during the immune response (48, 49), and we recently found in untreated mice genetically deficient in two γδ T cell subsets, including Vγ4+ cells (B6.TCR-Vγ4−/−/6−/−), that the splenic Vγ1+ cell population was substantially altered: in this mouse strain, Vγ1+ cells were expanded, changed in composition, showed signs of activation, and produced more IL-4 upon in vitro stimulation (50). Vγ6+ cells are not present in the spleen of untreated mice but they colocalize with Vγ4+ cells in skin and lung (40, 41, 51), and they are also found in tongue and female reproductive tract (38). However, at the present time, we have no indication of interactions between Vγ1+ and Vγ6+ cells.

Mindful of the functional differences between γδ T cell subsets and their ability to cross-regulate each other, we hypothesized that changes in γδ T cell composition might have effects on other immune cells and the immune responses. Our recent study examining mouse strains with genetic deficiencies in distinct γδ T cell subsets (5254) validates this assumption with regard to serum Ig levels in nonimmunized mice (50). Specifically, we found that mice deficient in Vγ1+ cells (B6.TCR-Vγ1−/−) generally had diminished Ab levels (with the exception of IgE), whereas B6.TCR-Vγ4−/−/6−/− mice had increased Ab levels (with the exception of IgG3 and IgA). This mouse strain also developed autoantibodies. The net effect of γδ T cells assessed in mice deficient in all γδ T cells (B6.TCR-δ−/−) was neutral (for IgM, IgG3, IgG2c, and IgA) or enhancing (for IgG1, IgG2b, and IgE). Several of the effects on the Abs in γδ-deficient mice could be linked to changes in IL-4 production (50). Furthermore, B6.TCR-Vγ4−/−/6−/− mice displayed changes in granulocytes (50) likely to be associated with increased levels of IgE in this mouse strain (55).

Having observed such profound effects of γδ T cell composition on serum Abs in nonimmunized mice, and on IL-4 production (50), we wondered at which stages in B cell development γδ T cells might intervene to effect changes in circulating Abs. In this study, we report that γδ T cells begin to shape preimmune B cell populations during the transitional stage in the spleen, eventually affecting all major populations of mature B cells. Additional data suggest that splenic γδ T cells modulate peripheral B cell populations in part through direct interactions with B cells that migrate through or reside within the MZ.

C57BL/6 mice and γδ T cell–deficient mice of the same genetic background (B6.TCR-δ−/−) were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and bred at National Jewish Health (Denver, CO). TCR-Vγ4−/−/Vγ6−/− mice, which were a gift from Dr. K. Ikuta (Kyoto University, Kyoto, Japan), were then backcrossed onto the C57BL/6 genetic background and re-established after 11 backcross generations. B6.TCR-Vγ1−/− mice were a gift from Dr. Simon Carding (Norwich Medical School, Norwich, U.K.) and distributed by Dr. C. Wayne Smith (Baylor College of Medicine, Houston, TX). B6.TCR-Vγ1tg mice were a gift from Dr. Pablo Pereira (Institut Pasteur, Paris, France). B6.IL-4−/− mice (C57BL/6-Il4tm1Nnt/J) were obtained from The Jackson Laboratory and were a gift from Dr. P. Marrack at National Jewish Health. Double knockout mice were generated by crossing the corresponding mutant strains and selecting double knockout mice in the F2 generation. These mice (TCR-Vγ4−/−/Vγ6−/−/IL-4−/−) were then bred as new homozygous strain. All mice were cared for at National Jewish Health following guidelines for normal and immune-deficient animals, and all experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee.

Cells obtained from single-cell suspensions (2 × 105/well) were stained in 96-well plates (Falcon; BD Biosciences, Franklin Lakes, NJ) for the cell surface markers shown in the figures/tables using the specific mAbs and derivatized reagents listed in Table I. CD93+ cells were detected using mAb AA4.1. Live cells were always gated based on forward and side scatter characteristics (lymphocyte gate) and, unless indicated otherwise, forward scatter height and amplitude, side scatter width and amplitude (to exclude or specifically include cellular conjugates), as well as expression of various B or T cell markers (Table I). All samples were analyzed on an LSR II flow cytometer, counting a minimum of 25,000 events per gated region, and the data were processed using FlowJo 9.5.2 software (FlowJo, Ashland, OR).

Table I.
Anti-mouse Abs and secondary reagents used in flow cytometry
AbCloneDyes/ConjugatesSource
Anti-CD3ε 145-2C11 PE-Cy7 BioLegend 
Anti-CD5 53-7.3 PE BD Biosciences 
Anti-B220 RA3-6B2 Allophycocyanin-Cy7 BioLegend 
Anti-CD19 1D3 Allophycocyanin/biotin eBioscience 
Anti-CD22 OX97 FITC BioLegend 
Anti-CD23 B3B4 PE-Cy7 eBioscience 
Anti-CD25 PC61 PE BD Biosciences 
Anti-CD32 24G2 FITC In-house 
Anti-CD38 90 PE eBioscience 
Anti-CD44 1M7 PE BD Biosciences 
Anti-CD62L MEL-14 PE BD Biosciences 
Anti-CD69 H1.2F3 PE BD Biosciences 
Anti-CD80 16-10A1 PE BioLegend 
Anti-CD86 GL-1 PE BioLegend 
Anti-CD93 AA4.1 Allophycocyanin eBioscience 
Anti-CD40L MR1 PE BD Biosciences 
Anti-ICOS 15F9 PE BD Biosciences 
Anti–MHC class II M5/114 PE eBioscience 
Anti-Igκ 187.1 FITC SouthernBiotech 
Anti-IgM B7.6 FITC/DyLight 488 In-house 
Anti-NK1.1 PKH136 Allophycocyanin eBioscience 
Anti–TCR-β H57-597 PE-Cy5 BD Biosciences 
Anti–TCR-δ eBioGL3 Allophycocyanin eBioscience 
Anti–TCR-δ GL3 FITC/biotin In-house 
Anti–TCR-Vγ1 2.11 
Anti–TCR-Vγ4 UC3 
Peanut agglutinin  FITC Vector Laboratories 
Streptavidin  FITC eBioscience 
Streptavidin  PE-Cy5 
Streptavidin  Allophycocyanin 
Streptavidin  eFluor 450 
AbCloneDyes/ConjugatesSource
Anti-CD3ε 145-2C11 PE-Cy7 BioLegend 
Anti-CD5 53-7.3 PE BD Biosciences 
Anti-B220 RA3-6B2 Allophycocyanin-Cy7 BioLegend 
Anti-CD19 1D3 Allophycocyanin/biotin eBioscience 
Anti-CD22 OX97 FITC BioLegend 
Anti-CD23 B3B4 PE-Cy7 eBioscience 
Anti-CD25 PC61 PE BD Biosciences 
Anti-CD32 24G2 FITC In-house 
Anti-CD38 90 PE eBioscience 
Anti-CD44 1M7 PE BD Biosciences 
Anti-CD62L MEL-14 PE BD Biosciences 
Anti-CD69 H1.2F3 PE BD Biosciences 
Anti-CD80 16-10A1 PE BioLegend 
Anti-CD86 GL-1 PE BioLegend 
Anti-CD93 AA4.1 Allophycocyanin eBioscience 
Anti-CD40L MR1 PE BD Biosciences 
Anti-ICOS 15F9 PE BD Biosciences 
Anti–MHC class II M5/114 PE eBioscience 
Anti-Igκ 187.1 FITC SouthernBiotech 
Anti-IgM B7.6 FITC/DyLight 488 In-house 
Anti-NK1.1 PKH136 Allophycocyanin eBioscience 
Anti–TCR-β H57-597 PE-Cy5 BD Biosciences 
Anti–TCR-δ eBioGL3 Allophycocyanin eBioscience 
Anti–TCR-δ GL3 FITC/biotin In-house 
Anti–TCR-Vγ1 2.11 
Anti–TCR-Vγ4 UC3 
Peanut agglutinin  FITC Vector Laboratories 
Streptavidin  FITC eBioscience 
Streptavidin  PE-Cy5 
Streptavidin  Allophycocyanin 
Streptavidin  eFluor 450 

Throughout this study, we use the nomenclature for murine TCR-Vγ genes introduced by Heilig and Tonegawa (56).

Suspensions of splenocytes were prepared by mechanical dispersion, treated with Gey’s solution for lysis of RBCs, and passed through nylon wool columns to obtain T lymphocyte–enriched cell preparations, as previously described (57). Enriched cells were then incubated with biotinylated anti-TCR Abs (mAb GL3, anti–TCR-δ or mAb 2.11, anti–TCR-Vγ1) for 15 min at 4°C, washed and incubated with streptavidin-conjugated magnetic beads (streptavidin microbeads; Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4°C and passed through magnetic columns to purify total γδ T cells, as previously described in detail (58). This produced cell populations containing >85% viable γδ T cells as determined by dye exclusion and staining with specific anti-TCR mAbs. These cells were used for cell transfer and coculture experiments. CD8+ and CD8 Vγ1 subpopulations were sorted using a Sony/iCyt Synergy FACS based on their distinctive phenotypes (CD3+TCR-βTCR-δ+TCR-Vγ1+CD8+ and CD3+TCR-βTCR-δ+TCR-Vγ1+CD8, respectively).

High protein–binding Microlon ELISA plates were coated with either 3 μg/ml polyclonal goat anti-mouse Ig(H+L) or goat anti-mouse IgG1 (SouthernBiotech) in 1× PBS overnight at 4°C. Plates were blocked with Iscove's complete tumor medium for 30 min at room temperature. Splenocytes (5.0 × 105) were added to the first well of a row and titrated in serial 2-fold dilutions in Iscove's complete tumor medium. After 7 h, plates were washed three times with 0.05% Triton X-100 in 1× PBS. Biotinylated goat anti-mouse–detecting Abs were then applied at 0.5 ng/ml in blocking buffer and allowed to incubate overnight at 4°C. Biotinylated anti-Igκ and anti-IgG were paired with goat anti-mouse Ig(H+L)–coating Ab to determine total Ig- and IgG-producing cells, respectively. For detecting IgG1-producing cells, biotinylated anti-IgG was used to pair with anti-IgG1–coating Ab. Plates were washed in 1× PBS, and streptavidin–alkaline phosphatase (BioLegend) was applied at a dilution of 1:2000 in blocking buffer. After washing, plates were developed in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 10 mM MgCl2 with 1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Pierce) for 2 h at 37°C. Plates were scanned into TIFF images for blinded counting.

For in vivo cell transfer, magnetic bead–purified cells were washed in PBS, resuspended to a concentration of 2.5 × 107 cells/ml in PBS, and 5 × 106 cells/mouse were injected in 200 μl PBS via the tail vein of the transfer recipient.

For coculture experiments, MZ B-rich B cells were purified by labeling splenocytes from B6.TCR-Vγ1−/− mice with anti-CD43–conjugated beads, followed by magnetic separation. The flow-through was collected and contained >90% viable B220+CD43 B cells. These purified B cells at 2 × 106/ml in culture medium were incubated with or without the addition of total Vγ1+ γδ T cells (1 × 106 cells/ml), or with CD8+ or CD8 fractions of Vγ1+ cells (0.5 × 106 cells/ml). Cells were collected after 60 h of cell culture, stained with the indicated Abs, and analyzed by flow cytometry.

We followed the protocol described by Barral et al. (59), with minor modifications. Briefly, mice were injected via the tail vein with an Ab specific for the pan-lymphocytic marker CD45 (mAb clone 104, anti-CD45.2 conjugated with PE or Pacific Blue), at 2 μg Ab/mouse in 200 μl PBS, euthanized after 20 min of in vivo incubation, and single-cell suspensions of spleen cells were prepared after first perfusing the spleens with PBS to wash out unbound Abs. Splenocytes were then stained with specific Abs to identify lymphocyte subsets as indicated in the figures and analyzed cytofluorimetrically.

Data are presented as means ± SD. The unpaired t test was used for two-group comparisons, and ANOVA was used for analysis of differences in three or more groups. A p value <0.05 was considered statistically significant.

We previously reported that nonimmunized mice deficient in individual γδ T cell subsets have changed levels of serum Igs, and one strain developed autoantibodies (50). The latter strain, which is deficient in Vγ4+ and Vγ6+ γδ Τ cells (B6.TCR-Vγ4−/−/6−/−), has much elevated serum levels of IgE and IL-4 and T cells (both αβ T cells and residual γδ T cells) that secrete larger quantities of this cytokine (50). IL-4, originally termed B cell stimulatory factor-1, acts on resting B cells, drives their maturation (60), and, when overexpressed, can promote autoimmunity and allergic-like inflammatory disease (61). These observations, which uncovered an altered regulatory environment for B cells in partially γδ-deficient mice, led us to examine the B cells themselves.

Comparing wild-type (wt) and B6.TCR-Vγ4−/−6−/− adult peripheral B cell populations, we found several differences in lymph nodes, blood, and peritoneal cavity (Fig. 1, Table II). Total B cells (IgM+B220+) in the lymph nodes of all mice, and in the peritoneal cavity of the older mutant mice, were dramatically decreased, mainly because of decreases in B2 B cells (IgM+B220+CD23+CD43). This was unexpected given that IL-4 (50) and BAFF (not shown), which are elevated in these mice, promote B cell growth (8, 60). Even more surprising, the removal of IL-4 in B6.TCR-Vγ4−/−/6−/−/IL-4−/− mice restored peripheral B cell numbers (Table II). Although numbers of B1 B cells (IgM+B220+CD23CD43+) were not substantially changed, their phenotype was altered in blood and the peritoneal cavity (Fig. 1, Table II). B1 B cells have been divided into two subsets based on the expression of the inhibitory receptor CD5 (16). In B6.TCR-Vγ4−/−/6−/− mice, cells expressing CD5 at high levels (B1a B cells) were much diminished in numbers and relative frequency, whereas cells expressing CD5 at low levels (B1b B cells) were increased. Given that IL-4 inhibits CD5 expression (16), this change was predictable. Moreover, the restored composition of B1 B cells in B6.TCR-Vγ4−/−/6−/−/IL-4−/− mice confirmed the inhibitory role of IL-4 (Table II). The changes were quite stable and were seen in mice between the ages of 4 and 20 wk (Fig. 1F, 1K). Subsequently, we also examined mice deficient in Vγ1+ γδ T cells (B6.TCR-Vγ1−/−) and in all γδ T cells (B6.TCR-δ−/−) (Table II). In lymph nodes and peritoneal cavity, B6.TCR-δ−/− mice had normal or somewhat enlarged B cell populations, including both B2 and B1 B cells, with a normal B1a/B1b B cell ratio (peritoneal cavity), and similar results were obtained with B6.TCR-Vγ1−/− mice, although peritoneal B1a B cells were increased in these mice, presumably due to the retention of IL-4–suppressive (Vγ4+) and absence of IL-4–producing (Vγ1+) γδ T cells (50). Collectively, the data show that the particular γδ deficiency in B6.TCR-Vγ4−/−/6−/− mice has a large effect on peripheral B cells whereas the absence of Vγ1+ γδ T cells, or of all γδ T cells, affects peripheral B cell populations more subtly (but see distinct effects with splenic B cells, below).

FIGURE 1.

Influence of γδ T cells on peripheral mature B cell populations. (AK) Comparison of B cell populations in female C57BL/6 (B6) and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice. Total B cells, B1 and B2 B cells, and B1a and B1b B cells were identified using the indicated markers. (A, D, and G) Representative staining profiles of individual 8-wk-old mice. (AC) Lymph node B cells: (B) and (C) show total numbers and relative frequencies of inguinal lymph node B cells (both sides pooled), respectively, comparing mice of different ages. The frequency of total B cells was calculated relative to total lymphocytes, and the frequencies of B1 and B2 B cells relative to total B cells were calculated. (D–F) Blood B cells: (E) and (F) show relative frequencies (total B, B1, and B2) and B1a/B1b ratios, respectively, comparing mice of different ages. (G–K) Peritoneal cavity B cells: (H) and (I) show total numbers and relative frequencies, respectively, comparing mice of different ages; (J) and (K) show total numbers of B1a and B1b cells and their ratio, respectively, comparing mice of different ages; n ≥ 4 mice per group. For clarity, only comparisons where no significant differences were found are marked; all others are significant at a p value of <0.05 or less.

FIGURE 1.

Influence of γδ T cells on peripheral mature B cell populations. (AK) Comparison of B cell populations in female C57BL/6 (B6) and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice. Total B cells, B1 and B2 B cells, and B1a and B1b B cells were identified using the indicated markers. (A, D, and G) Representative staining profiles of individual 8-wk-old mice. (AC) Lymph node B cells: (B) and (C) show total numbers and relative frequencies of inguinal lymph node B cells (both sides pooled), respectively, comparing mice of different ages. The frequency of total B cells was calculated relative to total lymphocytes, and the frequencies of B1 and B2 B cells relative to total B cells were calculated. (D–F) Blood B cells: (E) and (F) show relative frequencies (total B, B1, and B2) and B1a/B1b ratios, respectively, comparing mice of different ages. (G–K) Peritoneal cavity B cells: (H) and (I) show total numbers and relative frequencies, respectively, comparing mice of different ages; (J) and (K) show total numbers of B1a and B1b cells and their ratio, respectively, comparing mice of different ages; n ≥ 4 mice per group. For clarity, only comparisons where no significant differences were found are marked; all others are significant at a p value of <0.05 or less.

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Table II.
Peripheral B cells in genetically γδ T cell–deficient mice
Percentage (Absolute Cell Number)B6 (n = 8)δ−/− (n = 4)Vγ1−/− (n = 5)Vγ4−/−/6−/− (n = 8)Vγ4−/−/6−/−/IL4−/− (n = 7)
Lymph node      
 B/lymph (%) 23.9 ± 9.8 24.9 ± 0.9 15.0 ± 2.2 8.5 ± 3.0§ 27.5 ± 7.9 
 (B cells [×106]) (1.9 ± 0.7) (2.9 ± 0.3)* (1.6 ± 0.5) (0.4 ± 0.2)*** (2.8 ± 1.0) 
 B2 B/B (%) 94.0 ± 1.6 92.9 ± 0.9 62.3 ± 6.2*** 79.9 ± 4.5*** 91.5 ± 2.3 
 (B2 B cells [×106]) (1.7 ± 0.6) (2.7 ± 0.3)* (1.0 ± 0.4)*** (0.3 ± 0.1)*** (2.6 ± 0.8) 
 B1 B/B (%) 2.1 ± 0.6 3.0 ± 0.4 6.3 ± 1.9*** 13.5 ± 4.8*** 3.3 ± 0.8 
 (B1 B cells [×103]) (54.7 ± 39.6) (92.9 ± 21.6) (100.2 ± 47.5) (43.8 ± 22.5) (97.2 ± 52.2) 
Peritoneal cavity      
 B/live (%) 25.1 ± 1.6 21.4 ± 4.0 32.0 ± 7.5* 13.3 ± 5.3*** 26.0 ± 9.5 
 (B cells [×103]) (394.0 ± 148.6) (530.2 ± 31.8)* (1027.5 ± 465.3)*** (227.2 ± 153.3) (566.7 ± 243.8) 
 B2 B/B (%) 18.5 ± 4.1 20.7 ± 7.0 8.2 ± 2.4*** 21.9 ± 7.3 21.8 ± 9.1 
 (B2 B cells [×103]) (57.4 ± 17.6) (54.3 ± 4.8) (84.6 ± 40.2) (41.0 ± 25.9) (122.5 ± 87.4) 
 B1 B/B (%) 67.5 ± 5.6 66.2 ± 7.6 70.8 ± 2.7 60.8 ± 6.8 57.8 ± 17.9 
 (B1 B cells [×103]) (269.5 ± 112.2) (346.5 ± 31.2) (727.4 ± 334.2)** (143.7 ± 104.84) (384.1 ± 176.0) 
 B1a B/B1 B (%) 83.5 ± 2.3 83.8 ± 2.8 88.0 ± 1.8 20.5 ± 7.1*** 74.8 ± 6.1* 
 (B1a B cells [×103]) (226.7 ± 98.0) (290.9 ± 35.7) (642.8 ± 305.9)** (34.4 ± 32.9)*** (273.9 ± 137.3) 
 B1b B/B1 B (%) 16.2 ± 2.3 15.9 ± 2.7 11.5 ± 1.7 79.1 ± 6.8*** 25.0 ± 6.2* 
 (B1b B cells [×103]) (42.0 ± 14.7) (54.3 ± 4.8) (81.5 ± 31.6)* (109.0 ± 73.8)* (109.8 ± 57.4)* 
 B1a/B1b 5.3 ± 0.9 5.4 ± 1.0 7.8 ± 1.4** 0.3 ± 0.1*** 3.2 ± 1.1** 
Blood      
 B/lymph (%) 50.9 ± 10.4 66.9 ± 4.1* ND 48.1 ± 5.3 68.1 ± 11.0 
 B2 B/B (%) 83.1 ± 3.2 85.3 ± 1.9 ND 63.1 ± 2.1*** 89.6 ± 2.3 
 B1 B/B (%) 2.2 ± 0.6 2.0 ± 0.4 ND 3.7 ± 0.2*** 1.4 ± 0.5 
 B1a B/B1 B (%) 73.3 ± 6.5 70.5 ± 7.1 ND 33.6 ± 7.0*** 67.8 ± 3.5 
 B1b B/B1 B (%) 26.0 ± 6.6 29.5 ± 7.1 ND 65.4 ± 7.0*** 32.3 ± 3.5 
 B1a/B1b 3.0 ± 0.9 2.5 ± 0.8 ND 0.5 ± 0.2*** 2.1 ± 0.3 
Spleen      
 Total B cells      
  B/lymph (%) 58.0 ± 3.0 52.5 ± 8.7 38.3 ± 10.6** 29.3 ± 1.9*** 52.0 ± 2.7* 
  (B cells) (45.3 ± 10.2) (40.1 ± 8.7) (41.1 ± 9.8) (11.2 ± 2.4)*** (45.6 ± 21.3) 
 Mature B cells      
  Mature B/lymph (%) 46.8 ± 2.0 43.9 ± 9.7 32.3 ± 8.8** 25.7 ± 1.8*** 46.9 ± 4.5 
  (Mature B cells) (36.3 ± 6.7) (33.6 ± 9.3) (34.6 ± 8.2) (9.7 ± 1.8)*** (41.4 ± 20.8) 
  MZ B/mature B (%) 4.3 ± 0.8 3.8 ± 1.1 9.5 ± 1.2*** 0.6 ± 0.2*** 4.7 ± 1.8 
  (MZ B) (1.6 ± 0.4) (1.3 ± 0.6) (3.3 ± 0.9)*** (0.1 ± 0.0)*** (1.7 ± 0.2) 
  FO B/mature B (%) 84.0 ± 1.4 81.2 ± 3.6 74.9 ± 9.2* 62.7 ± 6.5*** 80.0 ± 2.2 
  (FO B) (30.5 ± 5.9) (27.4 ± 8.1) (25.9 ± 7.2) (6.1 ± 1.5)*** (32.9 ± 15.8) 
  New B/mature B (%) 8.3 ± 1.3 11.2 ± 4.4 10.5 ± 4.0 32.2 ± 8.9*** 11.4 ± 3.6 
  (New B) (3.0 ± 0.6) (3.6 ± 0.9) (4.8 ± 3.3) (3.1 ± 0.9) (5.3 ± 4.3) 
 Immature B cells      
  Immature B/lymph (%) 9.5 ± 1.1 8.6 ± 2.4 6.1 ± 2.2** 3.9 ± 0.4*** 6.1 ± 0.9*** 
  (Immature B) (8.1 ± 1.2) (3.4 ± 0.9) (2.6 ± 1.3) (2.1 ± 1.1)*** (5.7 ± 1.0)* 
  T1/immature (%) 27.4 ± 1.2 25.4 ± 1.0 27.6 ± 0.9 48.0 ± 1.2*** 25.8 ± 3.3*** 
  (T1) (2.1 ± 0.2) (1.5 ± 0.3) (2.0 ± 0.3) (1.0 ± 0.1)*** (1.6 ± 0.5) 
  T2 + T3/immature (%) 59.9 ± 1.5 59.8 ± 1.6 61.1 ± 1.2 33.4 ± 1.1*** 55.9 ± 3.5 
  (T2 + T3) (5.0 ± 0.8) (3.3 ± 0.6)** (4.3 ± 0.5) (0.7 ± 0.1)*** (3.0 ± 0.5) 
 B2 cells      
  B2/B (%) 76.9 ± 3.0 78.9 ± 0.4 32.5 ± 2.8*** 58.7 ± 5.2*** 80.2 ± 0.5 
  (B2) (36.5 ± 10.4) (31.6 ± 8.0) (13.2 ± 3.6)*** (5.9 ± 1.1)*** (37.8 ± 18.3) 
 B1 cells      
  B1/B (%) 2.1 ± 0.3 2.6 ± 0.9 2.8 ± 0.7 10.8 ± 3.3*** 3.2 ± 1.9 
  (B1) (0.8 ± 0.2) (1.2 ± 0.6) (1.1 ± 0.4) (0.9 ± 0.3) (1.3 ± 1.2) 
Percentage (Absolute Cell Number)B6 (n = 8)δ−/− (n = 4)Vγ1−/− (n = 5)Vγ4−/−/6−/− (n = 8)Vγ4−/−/6−/−/IL4−/− (n = 7)
Lymph node      
 B/lymph (%) 23.9 ± 9.8 24.9 ± 0.9 15.0 ± 2.2 8.5 ± 3.0§ 27.5 ± 7.9 
 (B cells [×106]) (1.9 ± 0.7) (2.9 ± 0.3)* (1.6 ± 0.5) (0.4 ± 0.2)*** (2.8 ± 1.0) 
 B2 B/B (%) 94.0 ± 1.6 92.9 ± 0.9 62.3 ± 6.2*** 79.9 ± 4.5*** 91.5 ± 2.3 
 (B2 B cells [×106]) (1.7 ± 0.6) (2.7 ± 0.3)* (1.0 ± 0.4)*** (0.3 ± 0.1)*** (2.6 ± 0.8) 
 B1 B/B (%) 2.1 ± 0.6 3.0 ± 0.4 6.3 ± 1.9*** 13.5 ± 4.8*** 3.3 ± 0.8 
 (B1 B cells [×103]) (54.7 ± 39.6) (92.9 ± 21.6) (100.2 ± 47.5) (43.8 ± 22.5) (97.2 ± 52.2) 
Peritoneal cavity      
 B/live (%) 25.1 ± 1.6 21.4 ± 4.0 32.0 ± 7.5* 13.3 ± 5.3*** 26.0 ± 9.5 
 (B cells [×103]) (394.0 ± 148.6) (530.2 ± 31.8)* (1027.5 ± 465.3)*** (227.2 ± 153.3) (566.7 ± 243.8) 
 B2 B/B (%) 18.5 ± 4.1 20.7 ± 7.0 8.2 ± 2.4*** 21.9 ± 7.3 21.8 ± 9.1 
 (B2 B cells [×103]) (57.4 ± 17.6) (54.3 ± 4.8) (84.6 ± 40.2) (41.0 ± 25.9) (122.5 ± 87.4) 
 B1 B/B (%) 67.5 ± 5.6 66.2 ± 7.6 70.8 ± 2.7 60.8 ± 6.8 57.8 ± 17.9 
 (B1 B cells [×103]) (269.5 ± 112.2) (346.5 ± 31.2) (727.4 ± 334.2)** (143.7 ± 104.84) (384.1 ± 176.0) 
 B1a B/B1 B (%) 83.5 ± 2.3 83.8 ± 2.8 88.0 ± 1.8 20.5 ± 7.1*** 74.8 ± 6.1* 
 (B1a B cells [×103]) (226.7 ± 98.0) (290.9 ± 35.7) (642.8 ± 305.9)** (34.4 ± 32.9)*** (273.9 ± 137.3) 
 B1b B/B1 B (%) 16.2 ± 2.3 15.9 ± 2.7 11.5 ± 1.7 79.1 ± 6.8*** 25.0 ± 6.2* 
 (B1b B cells [×103]) (42.0 ± 14.7) (54.3 ± 4.8) (81.5 ± 31.6)* (109.0 ± 73.8)* (109.8 ± 57.4)* 
 B1a/B1b 5.3 ± 0.9 5.4 ± 1.0 7.8 ± 1.4** 0.3 ± 0.1*** 3.2 ± 1.1** 
Blood      
 B/lymph (%) 50.9 ± 10.4 66.9 ± 4.1* ND 48.1 ± 5.3 68.1 ± 11.0 
 B2 B/B (%) 83.1 ± 3.2 85.3 ± 1.9 ND 63.1 ± 2.1*** 89.6 ± 2.3 
 B1 B/B (%) 2.2 ± 0.6 2.0 ± 0.4 ND 3.7 ± 0.2*** 1.4 ± 0.5 
 B1a B/B1 B (%) 73.3 ± 6.5 70.5 ± 7.1 ND 33.6 ± 7.0*** 67.8 ± 3.5 
 B1b B/B1 B (%) 26.0 ± 6.6 29.5 ± 7.1 ND 65.4 ± 7.0*** 32.3 ± 3.5 
 B1a/B1b 3.0 ± 0.9 2.5 ± 0.8 ND 0.5 ± 0.2*** 2.1 ± 0.3 
Spleen      
 Total B cells      
  B/lymph (%) 58.0 ± 3.0 52.5 ± 8.7 38.3 ± 10.6** 29.3 ± 1.9*** 52.0 ± 2.7* 
  (B cells) (45.3 ± 10.2) (40.1 ± 8.7) (41.1 ± 9.8) (11.2 ± 2.4)*** (45.6 ± 21.3) 
 Mature B cells      
  Mature B/lymph (%) 46.8 ± 2.0 43.9 ± 9.7 32.3 ± 8.8** 25.7 ± 1.8*** 46.9 ± 4.5 
  (Mature B cells) (36.3 ± 6.7) (33.6 ± 9.3) (34.6 ± 8.2) (9.7 ± 1.8)*** (41.4 ± 20.8) 
  MZ B/mature B (%) 4.3 ± 0.8 3.8 ± 1.1 9.5 ± 1.2*** 0.6 ± 0.2*** 4.7 ± 1.8 
  (MZ B) (1.6 ± 0.4) (1.3 ± 0.6) (3.3 ± 0.9)*** (0.1 ± 0.0)*** (1.7 ± 0.2) 
  FO B/mature B (%) 84.0 ± 1.4 81.2 ± 3.6 74.9 ± 9.2* 62.7 ± 6.5*** 80.0 ± 2.2 
  (FO B) (30.5 ± 5.9) (27.4 ± 8.1) (25.9 ± 7.2) (6.1 ± 1.5)*** (32.9 ± 15.8) 
  New B/mature B (%) 8.3 ± 1.3 11.2 ± 4.4 10.5 ± 4.0 32.2 ± 8.9*** 11.4 ± 3.6 
  (New B) (3.0 ± 0.6) (3.6 ± 0.9) (4.8 ± 3.3) (3.1 ± 0.9) (5.3 ± 4.3) 
 Immature B cells      
  Immature B/lymph (%) 9.5 ± 1.1 8.6 ± 2.4 6.1 ± 2.2** 3.9 ± 0.4*** 6.1 ± 0.9*** 
  (Immature B) (8.1 ± 1.2) (3.4 ± 0.9) (2.6 ± 1.3) (2.1 ± 1.1)*** (5.7 ± 1.0)* 
  T1/immature (%) 27.4 ± 1.2 25.4 ± 1.0 27.6 ± 0.9 48.0 ± 1.2*** 25.8 ± 3.3*** 
  (T1) (2.1 ± 0.2) (1.5 ± 0.3) (2.0 ± 0.3) (1.0 ± 0.1)*** (1.6 ± 0.5) 
  T2 + T3/immature (%) 59.9 ± 1.5 59.8 ± 1.6 61.1 ± 1.2 33.4 ± 1.1*** 55.9 ± 3.5 
  (T2 + T3) (5.0 ± 0.8) (3.3 ± 0.6)** (4.3 ± 0.5) (0.7 ± 0.1)*** (3.0 ± 0.5) 
 B2 cells      
  B2/B (%) 76.9 ± 3.0 78.9 ± 0.4 32.5 ± 2.8*** 58.7 ± 5.2*** 80.2 ± 0.5 
  (B2) (36.5 ± 10.4) (31.6 ± 8.0) (13.2 ± 3.6)*** (5.9 ± 1.1)*** (37.8 ± 18.3) 
 B1 cells      
  B1/B (%) 2.1 ± 0.3 2.6 ± 0.9 2.8 ± 0.7 10.8 ± 3.3*** 3.2 ± 1.9 
  (B1) (0.8 ± 0.2) (1.2 ± 0.6) (1.1 ± 0.4) (0.9 ± 0.3) (1.3 ± 1.2) 

B cells in lymph nodes, peritoneal cavity, blood, and spleen were identified based on their surface phenotype as described in Figs. 1 and 3 and enumerated in individual mice. Absolute numbers (in parentheses) and relative frequencies are shown; n = 4–8 mice per group.

*

p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Influence of γδ T cells on splenic B cell populations. (AM) Comparison of B cell populations in 8-wk-old female C57BL/6 (B6) (black columns), B6.TCR-δ−/−−/−) (open columns), B6.TCR-Vγ1−/− (Vγ1−/−) (dark gray columns), and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) (light gray columns) mice. Mature and immature B cells, B1 and B2 B cells, MZ B cells, FO B cells, new B cells, and GC B cells were identified using the indicated markers. (A, D, G, H, and K) Representative staining profiles of individual mice. (A–C) Relative frequencies (compared with total B cells) and absolute numbers of mature (m) and immature (imm) B cells; (B) and (C) also show numbers and frequencies (compared with total splenic lymphocytes) of total B cells. (D–F) Relative frequencies (compared with total B cells) and absolute numbers of B1 and B2 cells. (G–J) Relative frequencies (compared with total B cells) and absolute numbers of MZ B cells, FO B cells, and new B cells B1; (G) and (H) show two different ways of identifying MZ B cells, based on expression of CD21 in combination with CD23 or CD1d. The counts of MZ B cells in (I) and (J) are based on the method shown in (H). (K–M) Relative frequencies (compared with total B cells) and absolute numbers of GC B cells; n = 5–8 mice per group. For visibility, only significant differences between wt and the γδ-deficient mice are marked. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Influence of γδ T cells on splenic B cell populations. (AM) Comparison of B cell populations in 8-wk-old female C57BL/6 (B6) (black columns), B6.TCR-δ−/−−/−) (open columns), B6.TCR-Vγ1−/− (Vγ1−/−) (dark gray columns), and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) (light gray columns) mice. Mature and immature B cells, B1 and B2 B cells, MZ B cells, FO B cells, new B cells, and GC B cells were identified using the indicated markers. (A, D, G, H, and K) Representative staining profiles of individual mice. (A–C) Relative frequencies (compared with total B cells) and absolute numbers of mature (m) and immature (imm) B cells; (B) and (C) also show numbers and frequencies (compared with total splenic lymphocytes) of total B cells. (D–F) Relative frequencies (compared with total B cells) and absolute numbers of B1 and B2 cells. (G–J) Relative frequencies (compared with total B cells) and absolute numbers of MZ B cells, FO B cells, and new B cells B1; (G) and (H) show two different ways of identifying MZ B cells, based on expression of CD21 in combination with CD23 or CD1d. The counts of MZ B cells in (I) and (J) are based on the method shown in (H). (K–M) Relative frequencies (compared with total B cells) and absolute numbers of GC B cells; n = 5–8 mice per group. For visibility, only significant differences between wt and the γδ-deficient mice are marked. *p < 0.05, **p < 0.01, ***p < 0.001.

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We next examined immature B cells in the bone marrow. Comparing bone marrow B cells from wt, B6.TCR-δ−/−, B6.TCR-Vγ1−/−, and B6.TCR-Vγ4−/−6−/− mice, we found negligible differences in immature B cell types (Fig. 2A, Table III), including two developmentally late fractions of immature bone marrow B cells (fractions E and E′) that give rise to some of the mature B cells in bone marrow as well as bone marrow emigrants (5). Only one fraction (fraction F), which represents the mature IgD+ B cell population in bone marrow, was drastically reduced in B6.TCR-Vγ4−/−6−/− mice, both in absolute numbers and relative frequency (Fig. 2, Table III). However, this fraction largely consists of recirculating peripheral B cells (5, 6). To determine whether halted maturation in bone marrow or diminished recirculation is responsible for the loss of mature bone marrow B cells in B6.TCR-Vγ4−/−6−/− mice, we examined bone marrow B cells at several ages, with the older mice having a larger peripheral B cell compartment and increased potential for recirculation (Fig. 2B, 2C). At 4 wk of age, fraction F mature bone marrow B cells in wt and B6.TCR-Vγ4−/−6−/− mice were essentially the same, both in absolute numbers and relative frequency, whereas between 8 and 20 wk of age, mature bone marrow B cells mice increased substantially in wt mice but not in B6.TCR-Vγ4−/−6−/− mice. This result is consistent with the interpretation that fewer returning peripheral B cells account for the smaller number of mature B cells in the bone marrow of B6.TCR-Vγ4−/−6−/− mice, and that B cell development during the bone marrow stages is unaffected by γδ T cells.

FIGURE 2.

Immature B cells in bone marrow are not affected by γδ T cells. (A) Comparison of bone marrow B cell populations in 8-wk-old female C57BL/6 (B6), B6.TCR-δ−/−−/−), B6.TCR-Vγ1−/− (Vγ1−/−), and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice (representative examples). Bone marrow immature and mature B cells (Hardy fractions A–F) were identified using the indicated markers whereby fractions A–C were derived from B220+CD43+ and fractions D–F from B220+CD43 cells. Fraction F represents mature B cells within the bone marrow. (B) Comparison of total and mature bone marrow B cell populations (fraction F) in female B6 and Vγ4−/−/6−/− mice at ages 4–20 wk in absolute numbers/mouse (left femur plus tibia) and (C) in frequency relative to total live cells or fractions D–F, respectively. For (B) and (C), n = 4–8 mice per group. Only significant differences between wt and the γδ-deficient mice are marked. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

Immature B cells in bone marrow are not affected by γδ T cells. (A) Comparison of bone marrow B cell populations in 8-wk-old female C57BL/6 (B6), B6.TCR-δ−/−−/−), B6.TCR-Vγ1−/− (Vγ1−/−), and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice (representative examples). Bone marrow immature and mature B cells (Hardy fractions A–F) were identified using the indicated markers whereby fractions A–C were derived from B220+CD43+ and fractions D–F from B220+CD43 cells. Fraction F represents mature B cells within the bone marrow. (B) Comparison of total and mature bone marrow B cell populations (fraction F) in female B6 and Vγ4−/−/6−/− mice at ages 4–20 wk in absolute numbers/mouse (left femur plus tibia) and (C) in frequency relative to total live cells or fractions D–F, respectively. For (B) and (C), n = 4–8 mice per group. Only significant differences between wt and the γδ-deficient mice are marked. *p < 0.05, **p < 0.01, ***p < 0.001.

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Table III.
B cells in bone marrow from genetically γδ T cell–deficient mice
Percentage (Absolute Cell Numbers [×106])B6 (n = 13)δ−/− (n = 6)Vγ1−/− (n = 5)Vγ4−/−/6−/− (n = 4)Vγ4−/−/6−/−/IL4−/− (n = 5)
B cells/live cells 29.0 ± 3.9 29.7 ± 4.0 23.5 ± 3.6* 24.4 ± 1.7** 27.7 ± 3.6 
(B cells) (6.4 ± 2.1) (6.7 ± 2.2) (4.6 ± 1.7) (4.9 ± 0.9) (6.7 ± 2.1) 
Fraction A/(A–C) 51.0 ± 5.1 47.3 ± 6.0 40.5 ± 7.0* 56.1 ± 2.2 53.5 ± 7.0 
(Fraction A) (0.4 ± 0.1) (0.4 ± 0.1) (0.3 ± 0.1)** (0.5 ± 0.2) (0.3 ± 0.1) 
Fraction B/(A–C) 19.3 ± 1.6 20.2 ± 3.0 30.4 ± 9.3 18.2 ± 5.1 18.4 ± 4.0 
(Fraction B) (0.2 ± 0.1) (0.2 ± 0.0) (0.2 ± 0.2) (0.2 ± 0.0) (0.1 ± 0.0) 
Fraction C/(A–C) 26.2 ± 4.3 28.7 ± 3.6 24.2 ± 3.6 25.5 ± 3.5 23.6 ± 5.0 
(Fraction C) (0.2 ± 0.1) (0.2 ± 0.0) (0.2 ± 0.1) (0.3 ± 0.1) (0.1 ± 0.0) 
Fraction D/(D–F) 59.5 ± 4.1 59.7 ± 3.8 47.8 ± 5.5** 72.4 ± 9.8* 64.4 ± 4.7 
(Fraction D) (3.3 ± 1.2) (4.0 ± 1.1) (1.9 ± 0.9)* (3.1 ± 0.6) (3.8 ± 1.2) 
Fraction E/(D–F) 15.9 ± 2.4 14.1 ± 2.0 15.5 ± 1.6 17.7 ± 8.0 14.5 ± 2.5 
(Fraction E) (0.9 ± 0.3) (1.1 ± 0.3) (0.6 ± 0.3) (0.7 ± 0.3) (0.9 ± 0.3) 
Fraction E′/(D–F) 4.2 ± 0.6 4.1 ± 0.6 5.1 ± 0.3** 4.1 ± 0.9 3.4 ± 1.1 
(Fraction E′) (0.2 ± 0.1) (0.2 ± 0.1) (0.2 ± 0.1) (0.2 ± 0.0)* (0.2 ± 0.1) 
Fraction F/(D–F) 16.3 ± 4.7 18.6 ± 2.4 20.9 ± 6.4 2.1 ± 1.5*** 14.4 ± 1.8 
(Fraction F) (0.9 ± 0.4) (1.1 ± 0.3)* (0.8 ± 0.3) (0.1 ± 0.1)*** (0.8 ± 0.2) 
Percentage (Absolute Cell Numbers [×106])B6 (n = 13)δ−/− (n = 6)Vγ1−/− (n = 5)Vγ4−/−/6−/− (n = 4)Vγ4−/−/6−/−/IL4−/− (n = 5)
B cells/live cells 29.0 ± 3.9 29.7 ± 4.0 23.5 ± 3.6* 24.4 ± 1.7** 27.7 ± 3.6 
(B cells) (6.4 ± 2.1) (6.7 ± 2.2) (4.6 ± 1.7) (4.9 ± 0.9) (6.7 ± 2.1) 
Fraction A/(A–C) 51.0 ± 5.1 47.3 ± 6.0 40.5 ± 7.0* 56.1 ± 2.2 53.5 ± 7.0 
(Fraction A) (0.4 ± 0.1) (0.4 ± 0.1) (0.3 ± 0.1)** (0.5 ± 0.2) (0.3 ± 0.1) 
Fraction B/(A–C) 19.3 ± 1.6 20.2 ± 3.0 30.4 ± 9.3 18.2 ± 5.1 18.4 ± 4.0 
(Fraction B) (0.2 ± 0.1) (0.2 ± 0.0) (0.2 ± 0.2) (0.2 ± 0.0) (0.1 ± 0.0) 
Fraction C/(A–C) 26.2 ± 4.3 28.7 ± 3.6 24.2 ± 3.6 25.5 ± 3.5 23.6 ± 5.0 
(Fraction C) (0.2 ± 0.1) (0.2 ± 0.0) (0.2 ± 0.1) (0.3 ± 0.1) (0.1 ± 0.0) 
Fraction D/(D–F) 59.5 ± 4.1 59.7 ± 3.8 47.8 ± 5.5** 72.4 ± 9.8* 64.4 ± 4.7 
(Fraction D) (3.3 ± 1.2) (4.0 ± 1.1) (1.9 ± 0.9)* (3.1 ± 0.6) (3.8 ± 1.2) 
Fraction E/(D–F) 15.9 ± 2.4 14.1 ± 2.0 15.5 ± 1.6 17.7 ± 8.0 14.5 ± 2.5 
(Fraction E) (0.9 ± 0.3) (1.1 ± 0.3) (0.6 ± 0.3) (0.7 ± 0.3) (0.9 ± 0.3) 
Fraction E′/(D–F) 4.2 ± 0.6 4.1 ± 0.6 5.1 ± 0.3** 4.1 ± 0.9 3.4 ± 1.1 
(Fraction E′) (0.2 ± 0.1) (0.2 ± 0.1) (0.2 ± 0.1) (0.2 ± 0.0)* (0.2 ± 0.1) 
Fraction F/(D–F) 16.3 ± 4.7 18.6 ± 2.4 20.9 ± 6.4 2.1 ± 1.5*** 14.4 ± 1.8 
(Fraction F) (0.9 ± 0.4) (1.1 ± 0.3)* (0.8 ± 0.3) (0.1 ± 0.1)*** (0.8 ± 0.2) 

B cell types were identified based on their surface phenotype as in Fig. 2. Following a scheme first described by Hardy et al. (92), we divided B220+CD43+ early stage bone marrow B cells into developmentally consecutive fractions A–C based on their expression of CD24 and BP1. These cells represent pro–B and early pre–B cells. Later stage B220+CD43 B cells, which include those that returned from the periphery, were divided into increasingly mature fractions D (pre–B cells) and E and F (B cells) based on their expression of IgM and IgD. Cells contained within these fractions were enumerated in individual mice. Absolute numbers (in parentheses) and relative frequencies are shown; n = 4–13 mice per group.

*

p < 0.05, **p < 0.01, ***p < 0.001.

Having found substantial changes among mature peripheral but not immature bone marrow B cells, we proceeded to examine the intermediate stages of B cell development in the spleen (Fig. 3, Table II). We divided B220+ splenic B cells into mature CD93 and immature CD93+ cells (Fig. 3A) and further subdivided the mature B cells into FO B cells (CD23+CD21int), MZ B cells (CD23CD21hi or CD1dhiCD21hi), and “new” B cells (CD23CD21) (Fig. 3G–J) (62). We also identified B1 B cells in the spleen among B220+IgM+ cells based on their CD23CD43+ phenotype (Fig. 3D–F), and germinal center (GC) B cells based on their distinctive CD38Fas+PNAhi phenotype (50) (Fig. 3K–M). Comparing the same panel of mice as before for these splenic B cell populations, we found that mice lacking all γδ T cells (B6.TCR-δ−/−) had nearly unaltered B cell populations. In contrast, mature and immature splenic B cell populations in B6.TCR-Vγ4−/−6−/− mice were diminished in numbers and relative frequencies (Fig. 3A–C), specifically B2 B cells. FO B cells were much diminished but MZ B cells were nearly wiped out (Fig. 3G–J). In contrast, numbers of B1 B cells (Fig. 3B, 3E, 3F) were relatively stable, and GC B cells (Fig. 3K–M) in these mice were relatively increased. Again, removing IL-4 (B6.TCR-Vγ4−/−/6−/−/IL-4−/− mice) reversed all of these changes (Table II), suggesting that the elevated IL-4 levels in B6.TCR-Vγ4−/−6−/− mice are responsible for the changes in their splenic B cells. B6.TCR-Vγ1−/− mice, alternatively, showed little changes in numbers of mature and immature splenic B cells, although their MZ and new B cells were significantly increased. Apparent decreases in FO B cells in these mice seem to be merely a function of the lower CD23 expression, and a different gating strategy for FO B cells (based on CD21 and IgM expression) revealed normal FO B cell numbers (Supplemental Fig. 1). Nevertheless, the low CD23 expression in B6.TCR-Vγ1−/− mice is a distinctive trait (Supplemental Fig. 4) likely to have functional consequences in the IgE responses. CD23 is positively regulated by IL-4 (14) and was diminished in B6.TCR-Vγ4−/−/6−/− mice by ablation of IL-4 Supplemental Fig. 4). Furthermore, it was diminished in wt mice by treatment with anti Vγ1 mAbs (Supplemental Fig. 4), partially restored in cultured splenic B cells of B6.TCR-Vγ1−/− mice by adding IL-4 in vitro (Supplemental Fig. 4), and much induced in B cells of B6.TCR-δ−/− mice following transfer of Vγ1+ cells from B6.TCR-Vγ4−/−/6−/− mice (Supplemental Fig. 4). Another distinctive trait of B6.TCR-Vγ1−/− mice is their enlarged population of MZ B cells (Fig. 3G–J, Supplemental Fig. 1), in direct contrast to the diminished MZ B cells in B6.TCR-Vγ4−/−6−/− mice. The mere absence of Vγ1+ γδ T cells in B6.TCR-Vγ1−/− mice does not account for these traits because Vγ1+ cells are also missing in B6.TCR-δ−/− mice, which have normal CD23 expression and numbers of MZ B cells. Instead, they again probably reflect a changed function of the γδ T cells that remain in B6.TCR-Vγ1−/− mice. Collectively, the data revealed that mature B cells in the spleen are sensitive to the influence of γδ T cells, and that much of this influence depends on IL-4.

B cells newly arrived in the spleen can be divided into discrete transitional stages (T1–T3), distinguished by their surface phenotype and functional capability (17, 63). We identified immature B cells in the spleen of the test panel mice as IgM+B220+CD93+ cells, and further divided these cells into T1 (IgMhiCD21−/lo) and T2 plus T3 (IgM+CD21+) transitional subsets (Fig. 4A). Furthermore, we analyzed both subsets for their expression of CD23 and IgD. At 8 wk of age, B6.TCR-δ−/− mice and B6.TCR-Vγ1−/− mice had nearly unchanged numbers of transitional B cells (Fig. 4B), and relative frequencies of the transitional subsets were normal as well (Fig. 4C). In contrast, B6.TCR-Vγ4−/−6−/− mice produced significantly fewer T1 and T2 plus T3 B cells (Fig. 4B, 4C). Removal of IL-4 (B6.TCR-Vγ4−/−/6−/−/IL-4−/− mice) partially restored immature B cells, including T1 and T2 plus T3 transitional subsets (Table II), suggesting that the diminished transitional B cell compartment in B6.TCR-Vγ4−/−6−/− mice is an indirect consequence of the deregulated IL-4 production in these animals (see below). CD23 and IgD expression revealed further differences between the transitional B cells of the test panel mice (Fig. 4D). Hence, in contrast to immature bone marrow B cells, transitional B cells in the spleen were already altered, and thus might represent the earliest stage in B cell development affected by γδ T cells.

FIGURE 4.

Genetic deficiency in γδ T cells alters transitional B cells in the spleen. Comparison of transitional B cell populations in the spleens of 8-wk-old female C57BL/6 (B6), B6.TCR-δ−/−−/−), B6.TCR-Vγ1−/− (Vγ1−/−), and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice. (A) Immature IgM+B220+CD93+ B cells in the spleen were further subdivided into CD21IgMhi (T1) and CD21+IgM+ (T2 plus T3) B cells. The test panel mice were also compared for CD23 and IgD expression in the two subsets of transitional B cells. Representative examples are shown. (B) Absolute numbers of transitional B cells per spleen in 8-wk-old mice and (C) relative frequencies. (D) Expression of CD23 and IgD in transitional B cells of wt and γδ-deficient mice. Profiles representative of at least three independent staining experiments are shown. For (B) and (C), n = 8–14 mice per group. Significant differences between wt and the γδ-deficient mice are marked. ***p < 0.001.

FIGURE 4.

Genetic deficiency in γδ T cells alters transitional B cells in the spleen. Comparison of transitional B cell populations in the spleens of 8-wk-old female C57BL/6 (B6), B6.TCR-δ−/−−/−), B6.TCR-Vγ1−/− (Vγ1−/−), and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice. (A) Immature IgM+B220+CD93+ B cells in the spleen were further subdivided into CD21IgMhi (T1) and CD21+IgM+ (T2 plus T3) B cells. The test panel mice were also compared for CD23 and IgD expression in the two subsets of transitional B cells. Representative examples are shown. (B) Absolute numbers of transitional B cells per spleen in 8-wk-old mice and (C) relative frequencies. (D) Expression of CD23 and IgD in transitional B cells of wt and γδ-deficient mice. Profiles representative of at least three independent staining experiments are shown. For (B) and (C), n = 8–14 mice per group. Significant differences between wt and the γδ-deficient mice are marked. ***p < 0.001.

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Untreated B6.TCR-Vγ4−/−6−/− mice have increased levels of circulating Abs (50) but decreased numbers of mature B cells (the present study). Both of these changes are IL-4–dependent. These mice also exhibit spontaneous GC formation in the spleen (50). Therefore, we compared numbers of Ab-producing B cells in this partially γδ-deficient strain and in untreated wt mice (Fig. 5). Total Ig-producing cells in the spleen of 8- to 12-wk-old B6.TCR-Vγ4−/−6−/− mice were increased >2-fold compared with wt mice, and IgG1-secreting cells were increased >7-fold (Fig. 5A). In contrast, there were no significant increases of Ab-producing B cells in bone marrow. IgG1 surface-positive B cells (B220 and B220+) were substantially increased but not in bone marrow (Fig. 5B, 5D), and IgG1-secreting plasma cells (surface IgMlow, intracellular IgG1+) were increased in the spleen as well (Fig. 5C, 5D). Numbers of such cells in bone marrow were low and difficult to quantitate. Taken together, these data document increased numbers of Ab-producing B cells in the spleen but not in the bone marrow of B6.TCR-Vγ4−/−6−/− mice, and they suggest that the increased levels of circulating Abs in this mutant strain are a result of this cellular change.

FIGURE 5.

Ab-secreting cells (ASCs) are increased in the spleen of Vγ4−/−/6−/− mice. (A) Total Ig-, IgG-, and IgG1-producing cells determined by ELISPOT assay were enumerated in spleen and bone marrow of female C57BL/6 (wt) and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice; n = 4 per group. (B and C) Representative FACS plots showing surface IgG1-expressing cells (sIgG1+ cells) (B) and intracellular IgG1-expressing cells (iIgG1+ cells) (C) in spleen and bone marrow of above mice. (D) Relative frequencies and total numbers in spleen and bone marrow of mice indicated in (A)–(C); n = 4 mice per group. *p < 0.5, **p < 0.01, ***p < 0.001.

FIGURE 5.

Ab-secreting cells (ASCs) are increased in the spleen of Vγ4−/−/6−/− mice. (A) Total Ig-, IgG-, and IgG1-producing cells determined by ELISPOT assay were enumerated in spleen and bone marrow of female C57BL/6 (wt) and B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice; n = 4 per group. (B and C) Representative FACS plots showing surface IgG1-expressing cells (sIgG1+ cells) (B) and intracellular IgG1-expressing cells (iIgG1+ cells) (C) in spleen and bone marrow of above mice. (D) Relative frequencies and total numbers in spleen and bone marrow of mice indicated in (A)–(C); n = 4 mice per group. *p < 0.5, **p < 0.01, ***p < 0.001.

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To address the question of whether γδ T cells themselves modulate the B cell populations, we took advantage of our earlier observation that residual γδ T cells in B6.TCR-Vγ4−/−6−/− mice, which mostly belong to the Vγ1+ subset, are changed (50). Such changes include higher relative frequencies and absolute numbers of IL-4–competent γδ T cells (50), altered TCR-Vδ expression among Vγ1+ γδ T cells (50), as well as a higher frequency of CD8 expression among these cells when IL-4 is present (see Fig. 6E), suggesting IL-4–driven Tc2-like differentiation. We conducted cell transfer experiments with these changed cells, in a manner as previously described (55). Adoptive transfer of B6.TCR-Vγ4−/−6−/−–derived splenic γδ T cells into B6.TCR-δ−/− mice transiently restored splenic Vγ1+ cells in the cell transfer recipients, albeit only up to ∼10% of the population size in wt mice (Supplemental Fig. 2). Still, the transferred γδ T cells selectively reduced MZ B cells in the transfer recipients (Fig. 6A, 6B), replicating the trend seen in nonmanipulated B6.TCR-Vγ4−/−6−/− mice (Fig. 3). The similar cell transfer experiment shown in Supplemental Fig. 2 further extends this finding: in this case, the transferred γδ T cells, which again reduced splenic MZ B cells, did so despite the presence of recipient γδ T cells (recipient, B6.TCR-Vγ1−/−), which were unable to prevent this effect. When we examined transitional B cells in the B6.TCR-δ−/− cell transfer recipients, we found a reduction in CD21 expression (Fig. 6C), also replicating the situation in B6.TCR-Vγ4−/−6−/− mice (Fig. 6D). In all experiments, transferred γδ T cells derived from B6.TCR-Vγ4−/−/6−/−/IL-4−/− mice failed to induce these changes in B cells (Fig. 6, Supplemental Fig. 2), emphasizing the importance of IL-4 in the functional differentiation of the transferred γδ T cells. The dual effect of the transferred γδ T cells on transitional B cells, which must pass through the MZ (17), and on MZ B cells, which shuttle between follicles and the MZ (64), is consistent with interactions between γδ T cells and B cells inside the splenic MZ.

FIGURE 6.

Transferred residual γδ T cells alter splenic B cells in vivo, and cocultured residual γδ T cells selectively diminish MZ B cells in vitro. (A) B6.TCR-δ−/− mice (δ−/−) were transferred with purified splenic γδ T cells from B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) or B6.TCR-Vγ4−/−/Vγ6−/−/ΙL-4−/− (Vγ4−/−/6−/−/IL-4−/−) mice. Ten days after the cell transfer, B cell populations in the spleen were compared as detailed in Fig. 2, using the indicated markers. Data of one representative experiment are shown. (B) Effect of transferred residual γδ T cells on absolute numbers of MZ B cells in the spleen of δ−/− mice; n = 4 mice per group. (C) Effect of transferred residual γδ T cells on CD21 expression in the spleen of δ−/− mice; n = 4 mice per group. (D) CD21 expression by transitional B cells in untreated wt, Vγ4−/−/6−/−, and Vγ4−/−/6−/−/IL-4−/− mice; n = 4 mice per group. **p < 0.01, ***p < 0.001. (E) CD8+ fraction of Vγ1+ γδ T cells in the spleen. Relative frequencies of CD8+ γδ T cells within the splenic Vγ1+ subset of C57BL/6 (wt), B6.TCR-Vγ4−/−/6−/− (Vγ4−/−/6−/−), and B6.TCR-Vγ4−/−/Vγ6−/−/IL-4−/− (Vγ4−/−/6−/−/IL-4−/−) mice are shown; n = 5 mice per group. **p < 0.01, ***p < 0.001. (F) CD43 MZ B cell–rich splenic B cells from B6-TCR-Vγ1−/− (Vγ1−/−) mice were cultured for 60 h alone or in the presence of splenic Vγ1+ γδ T cells from different mouse strains, and subsequently stained to identify MZ B cells. Only Vγ1+ cells from B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice substantially diminished MZ B cells. (G) Same B cells as in (A) were cultured alone or in the presence of CD8+ or CD8 fractions of Vγ1+ cells from Vγ4−/−/6−/− mice and subsequently stained to identify MZ B cells. Only Vγ1+ cells expressing CD8 diminished MZ B cells. Data in (F) and (G) are representative of several similar experiments.

FIGURE 6.

Transferred residual γδ T cells alter splenic B cells in vivo, and cocultured residual γδ T cells selectively diminish MZ B cells in vitro. (A) B6.TCR-δ−/− mice (δ−/−) were transferred with purified splenic γδ T cells from B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) or B6.TCR-Vγ4−/−/Vγ6−/−/ΙL-4−/− (Vγ4−/−/6−/−/IL-4−/−) mice. Ten days after the cell transfer, B cell populations in the spleen were compared as detailed in Fig. 2, using the indicated markers. Data of one representative experiment are shown. (B) Effect of transferred residual γδ T cells on absolute numbers of MZ B cells in the spleen of δ−/− mice; n = 4 mice per group. (C) Effect of transferred residual γδ T cells on CD21 expression in the spleen of δ−/− mice; n = 4 mice per group. (D) CD21 expression by transitional B cells in untreated wt, Vγ4−/−/6−/−, and Vγ4−/−/6−/−/IL-4−/− mice; n = 4 mice per group. **p < 0.01, ***p < 0.001. (E) CD8+ fraction of Vγ1+ γδ T cells in the spleen. Relative frequencies of CD8+ γδ T cells within the splenic Vγ1+ subset of C57BL/6 (wt), B6.TCR-Vγ4−/−/6−/− (Vγ4−/−/6−/−), and B6.TCR-Vγ4−/−/Vγ6−/−/IL-4−/− (Vγ4−/−/6−/−/IL-4−/−) mice are shown; n = 5 mice per group. **p < 0.01, ***p < 0.001. (F) CD43 MZ B cell–rich splenic B cells from B6-TCR-Vγ1−/− (Vγ1−/−) mice were cultured for 60 h alone or in the presence of splenic Vγ1+ γδ T cells from different mouse strains, and subsequently stained to identify MZ B cells. Only Vγ1+ cells from B6.TCR-Vγ4−/−/Vγ6−/− (Vγ4−/−/6−/−) mice substantially diminished MZ B cells. (G) Same B cells as in (A) were cultured alone or in the presence of CD8+ or CD8 fractions of Vγ1+ cells from Vγ4−/−/6−/− mice and subsequently stained to identify MZ B cells. Only Vγ1+ cells expressing CD8 diminished MZ B cells. Data in (F) and (G) are representative of several similar experiments.

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Additionally, we tested for a possible effect of purified Vγ1+ γδ T cells on MZ B cells in vitro. After a culture period of 60 h without any added stimuli or growth factors, enriched CD43 splenic B cells from B6.TCR-Vγ1−/− mice contained ∼12% MZ B cells (Fig. 6F). Coculturing them with Vγ1+ γδ T cells from B6.TCR-Vγ4−/−/6−/− mice selectively diminished MZ B cells, similarly to the cell transfer experiments in vivo and consistent with the trend in B6.TCR-Vγ4−/−/6−/− mice, whereas γδ T cells from wt mice (either Vγ1+ or Vγ4+) or from B6.TCR-Vγ4−/−/6−/−/IL-4−/− mice failed to mediate this effect. Furthermore, to address the potential significance of the enlarged IL-4–dependent CD8+ subpopulation among Vγ1+ cells in B6.TCR-Vγ4−/−/6−/− mice, we compared purified CD8+ and CD8 subfractions of Vγ1+ cells in B6.TCR-Vγ4−/−/6−/− mice for their effect on MZ B cells in the cocultures (Fig. 6G), and we found that only the CD8+Vγ1+ γδ T cells diminished MZ B cells. The effect in vitro, albeit consistent in its selectivity for MZ B cells with the effect in vivo, was comparatively small, which might reflect the importance of colocalization of γδ T cells and MZ B cells in the MZ in vivo. Collectively, the combined results of the cell transfer and coculture experiments suggest that the altered B6.TCR-Vγ4−/−/6−/− γδ T cells themselves are responsible for the changes of peripheral B cells in this strain.

The compartments of the spleen differ in their accessibility to the circulation (65). Recently arrived immature B cells and MZ B cells migrate through or reside within the MZ, a splenic compartment far more accessible to the circulation than the follicles or the periarteriolar sheath (59, 66). Because of technical difficulties in localizing splenic γδ T cells in wt mice by immunohistochemical methods (67), we instead assessed the exposure of splenic γδ T cells to the circulation. We i.v. injected Abs specific for the ubiquitous leukocyte marker CD45 and compared labeling levels of the splenic lymphocyte types at a fixed time point after the injection (Fig. 7A). The broad range of labeling intensity in all cell populations examined likely reflects positional differences of individual cells within a given population. However, overall, the percentage of labeled γδ T cells in normal C57BL/6 mice was higher than that of αβ T cells, indicating that splenic γδ T cells tend to be more exposed to the circulation than do αβ T cells. The percentage of labeled NK1.1+ γδ T cells was higher still and was similar to that of NKT cells. This finding in mice is consistent with histological studies in several other species placing γδ T cells in the red pulp and MZ of the spleen (6872). Among immature B cells, T1 B cells were more exposed than T2 plus T3 B cells, as would be expected (17), and MZ B cells were most exposed among mature B cells, consistent with the literature (59). Although we found some differences in CD45 expression of the splenic cell types examined (based on in vitro Ab staining, see Supplemental Fig. 3B), the differential labeling in vivo did not correlate with these differences, rather reflecting differential circulation exposure than differential CD45 expression. Thus, circulation exposure assessed in the present study and histological findings of others place γδ T cells, early transitional (T1) B cells, and MZ B cells together inside the MZ. Second, we examined γδ T–B cell conjugates in fresh splenocyte preparations. CD93+ immature B cells (Fig. 7B) and MZ B cells (Fig. 7C) were enriched in the conjugates, consistent with the notion of encounters and contact between splenic γδ T cells and B cells that pass through (T1 B cells) or reside within the MZ (MZ B cells). In turn, the γδ T cells making these contacts with B cells seemed to be “aware” of them as they expressed the activation markers CD40L and ICOS at higher levels than did nonconjugated γδ T cells (Fig. 7D).

FIGURE 7.

Localization of γδ T cells and immature B cells in the spleen, propensity to form T–B conjugates, and activation state of γδ T cells in the conjugates are shown. (A) Eight-wk-old female C57BL/6 mice were injected i.v. with dye-conjugated Abs specific for CD45; splenocytes were stained in vitro for subset-specific markers after a 20-min in vivo labeling period and then analyzed cytofluorimetrically. One example representative of six similar experiments is shown. (B) γδ T–B cell conjugates among splenocytes from 8-wk-old female C57BL/6 mice were identified based on their simultaneous expression of γδ T cell (TCR-δ) and B cell markers (CD19), and B cells in the conjugates were compared with nonconjugated B cells for their expression of CD93, a marker of immature B cells, or (C) for their expression of CD23 and CD21 to differentiate new, MZ, and FO B cells. Individual examples representative of four similar experiments are shown. (D) γδ T cells in conjugates with splenic B cells and nonconjugated γδ T cells were compared for their expression of CD40L and ICOS. One experiment representative of four using 8- to 12-wk-old female C57BL/6 mice is shown.

FIGURE 7.

Localization of γδ T cells and immature B cells in the spleen, propensity to form T–B conjugates, and activation state of γδ T cells in the conjugates are shown. (A) Eight-wk-old female C57BL/6 mice were injected i.v. with dye-conjugated Abs specific for CD45; splenocytes were stained in vitro for subset-specific markers after a 20-min in vivo labeling period and then analyzed cytofluorimetrically. One example representative of six similar experiments is shown. (B) γδ T–B cell conjugates among splenocytes from 8-wk-old female C57BL/6 mice were identified based on their simultaneous expression of γδ T cell (TCR-δ) and B cell markers (CD19), and B cells in the conjugates were compared with nonconjugated B cells for their expression of CD93, a marker of immature B cells, or (C) for their expression of CD23 and CD21 to differentiate new, MZ, and FO B cells. Individual examples representative of four similar experiments are shown. (D) γδ T cells in conjugates with splenic B cells and nonconjugated γδ T cells were compared for their expression of CD40L and ICOS. One experiment representative of four using 8- to 12-wk-old female C57BL/6 mice is shown.

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Overall, the present study shows that γδ T cells are capable of modulating preimmune peripheral B cells populations. This work was inspired by our preceding study indicating that γδ T cells strongly affect Ig serum levels and autoantibody development in nonimmunized mice (50). Both studies take advantage of three connected observations: first, that a correlation exists between TCR-Vγ expression by subsets of murine γδ T cells and their function (73); second, that such subsets in isolation tend to have a larger effect on the immune responses than do γδ T cells as a whole (74); and third, that the absence of a subset can lead to functional changes in the remaining γδ T cells (50). These features enable the manipulation of γδ T cell function in vivo, using TCR-Vγ knockout as the approach. Having employed this approach to reveal the γδ influence on Ab production and self-tolerance (50, 55), we now demonstrate effects on B cell homeostasis and get a first glimpse at underlying mechanisms.

In particular, we found in the present study that mice deficient in two γδ T cell subsets (B6.TCR-Vγ4−/−/6−/−), which have normal numbers of immature bone marrow B cells, nevertheless have much reduced numbers of total peripheral B cells. This occurs in the presence of elevated levels of IL-4 (50) and BAFF (data not shown), both of which would be expected to support B cell growth (8, 60). Indeed, Ab-producing B cells are increased, consistent with the increased levels of serum Ig (50). The loss of peripheral B cells seems to take place during development in the spleen because levels of immature B cells in the spleen and derived mature B cell populations were all affected, whereas immature B cells in bone marrow were not. Among mature splenic B cell populations, those that reside in or repeatedly shuttle in and out of the MZ—the MZ B cells (64)—were diminished most. Immature B cells, which also have to pass through the MZ on their way to the white pulp (75, 76), were hit as well. Collectively, these observations implicate the splenic MZ as a critical site of the γδ influence on peripheral B cells.

The splenic MZ is a portal for cells in transit from the bloodstream to the white pulp (65). It also contains various types of resident cells that depend on each other for their localization and function (77). Besides reticular fibroblasts, these include marginal sinus-lining cells as well as several distinct myeloid and lymphoid cell types. Splenic γδ T cells have been localized in the MZ as well, and within the red pulp, in humans, cattle, sheep, camels, and birds (6872). We previously reported that splenic γδ T cells in mice acquire blood-borne Ags (78), consistent with a similar localization of these γδ T cells. The data of the present study further support this notion: 1) splenic γδ T cells in normal mice were stained well by i.v. injected Abs, indicating an exposure to the circulation similar to that of early transitional B cells and MZ B cells; 2) splenic γδ T cells in αβ T cell–deficient mice, where γδ T cells move into the circulation-inaccessible periarteriolar sheath (67), were no longer well stained by i.v. injected Abs (data not shown); 3) immature B cells and MZ B cells were enriched in splenic γδ T–B cell conjugates; and 4) γδ T cells affected MZ B cells far more than did the other mature B cell types. Owing to their precursor progeny relationship, the still substantial but smaller effect of the γδ T cells on other mature B cells likely is a consequence of their interaction with transitional B cells, which must pass through the MZ as well (17).

The cell transfer and coculture data, as well as the data colocalizing B cells and γδ T cells in the MZ, all suggest that action by splenic γδ T cells themselves, either directly or indirectly, is responsible for the diminished peripheral B cells in B6.TCR-Vγ4−/−/6−/− mice. Splenic γδ T cells in these mice are mainly Vγ1+, and they are also altered in composition and function (50), partly under the influence of IL-4, which drives expansions of both NKT-like (50) and CD8+ Tc2-like (7981) Vγ1+ cells (the present study). Indeed, adoptively transferred γδ T cells from the spleen of B6.TCR-Vγ4−/−/6−/− mice selectively diminished MZ B cells in the recipient mice. The transferred γδ T cells also lowered CD21 expression in transitional B cells, consistent with the changed phenotype of these B cells in B6.TCR-Vγ4−/−/6−/− mice. Both of these results support the notion of γδ T–B cell interactions in the MZ, but they do not rule out such interactions elsewhere. The transfer experiments also underscore the role of IL-4 because γδ T cells obtained from B6.TCR-Vγ4−/−/6−/−/IL-4−/− mice failed to affect the B cells. Normal levels of IL-4 seem to be sufficient for some action of Vγ1+ γδ T cells in the spleen because wt C57BL/6 mice had significantly fewer MZ B cells than did B6.TCR-Vγ1−/− mice. However, in B6.TCR-Vγ4−/−/6−/− mice with their hyperplastic Vγ1+ γδ T cell population and increased IL-4 production, the effect is exacerbated, leading to substantial reductions in mature splenic B cells and a near disappearance of MZ B cells. Even cocultured B6.TCR-Vγ4−/−/6−/−–derived Vγ1+ γδ T cells in vitro reproducibly diminished MZ B cells more than other B cells, although the effect was comparatively small. Both direct and indirect mechanisms might contribute to these γδ-dependent changes in peripheral B cells and serum Ig levels. The elevated IL-4 in B6.TCR-Vγ4−/−/6−/− mice potentially could drive premature switching of immature bone marrow B cells (82), but we did not see substantial changes in this study. The loss of mature splenic B cells in B6.TCR-Vγ4−/−/6−/− mice could be the result of accelerated maturation and differentiation into plasma cells, consistent with the increased numbers of Ab-producing cells in the spleen of these mice (this study) as well as the elevated levels of circulating Abs (50). This mechanism seems to be IL-4–dependent because the changes in peripheral B cells are absent when IL-4 is missing. The preferential loss of MZ B cells likely is due to the greater propensity of these cells to form plasma cells (83). Additionally, their colocalization with γδ T cells in the spleen might make these B cells more available for the γδ influence. As well, MZ B cells might be more likely to interact with γδ T cells due to their differential expression of ligands for the γδ TCR such as the molecules CD1d and T-22 (8486). Finally, we cannot exclude a role of changed microbiota (87) in the γδ-deficient mice, but this seems less probable under conditions of transient reconstitution.

Although the massive loss of peripheral B cells in B6.TCR-Vγ4−/−/6−/− mice was the most noticeable γδ effect, several phenotypic changes in B cells can be ascribed to the influence of γδ T cells as well, such as the much diminished expression of the IgE receptor CD23 in B6.TCR-Vγ1−/− mice, the diminished expression of the inhibitory receptor CD5 and FcγRIIB in B6.TCR-Vγ4−/−/6−/− mice, or the increased expression of MHC class II and IL-4Rα in B6.TCR-Vγ4−/−/6−/− mice. All of the listed changes appear to be mediated or at least indirectly connected to γδ-dependent IL-4 (11, 16, 50) and, in the case of B6.TCR-Vγ4−/−/6−/− mice, might be exacerbated by the loss of peripheral B cells, which likely contributes to the increase in available serum IL-4 (50) and BAFF (not shown) in these mice.

The findings of this study raise several new questions. For example, γδ T cells in the MZ might be a differentiated and functionally specialized population similar to the other specialized residents of this site (65), and they might function as part of a cellular network in the MZ. As already mentioned, it appears that MZ γδ T cells also participate in the monitoring of blood-borne Ags (78). Furthermore, our data suggest that the changes in the peripheral B cells of B6.TCR-Vγ4−/−/6−/− mice are directly connected to the elevated serum Ig and the development of autoantibodies in these mice (50). By extension, the observations described in the present study predict that changes in γδ T cell populations due to natural causes (26, 33, 88) might similarly affect how peripheral B cells differentiate, how much Ig is produced, and whether autoantibodies develop. Consistently, impaired TCR signaling, which causes changes in γδ T cell populations, has already been associated with increased IgE production (33, 34, 89). Likewise, changes in size and composition of γδ T cell populations, which have been found in hematopoietic transplantation (90) and in HIV infection (91), might affect T and B cell functions, B cell reconstitution, and humoral immune competence.

We thank Drs. Roberta Pelanda, Raul Torres, and Philippa Marrack and our reviewers for insight, advice, and critical discussion of the data, Dr. C. Wayne Smith for providing mice, and Shirley Sobus and Joshua Loomis for expert help with flow cytometry and microscopy.

This work was supported by National Institutes of Health Grants R21 AI095765 (to W.K.B.) and R21 AI097962 and R01 EY021199 (to R.L.O.).

This article contains supplemental material.

Abbreviations used in this article:

FO

follicular

GC

germinal center

MZ

marginal zone

SLE

systemic lupus erythematosus

wt

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data