We have generated a sublibrary of cDNA clones that appears to be highly enriched for sequences which are specifically upregulated when IL-7–dependent pro/pre B-I cells are induced by the removal of IL-7 and the addition of CD40-specific mAb and IL-4 to differentiate to B cells with a mature phenotype and Sμ-Sε–switched genotype (14
). Because of the very broad developmental changes that occur during such a stimulation, a very heterogeneous group of genes are expected to be found in the subtracted sublibrary. Genes involved in the changes from pro/pre B-I to mature B cell, genes induced by either the anti-CD40 or the IL-4 signal transduction pathways, genes controlling Ig V-gene hypermutation, and genes involved in Sμ-Sε switching are likely candidates. Indeed, several genes already known to be upregulated when pro/pre B cells develop to mature B cells were found among the selected clones. They are genes critical for the capacity of developing B cells to present antigen, namely those encoding MHC class II proteins (52
), the invariant chain of MHC class II (51
), and the MHC locus–linked H-2M gene (53
); genes related to IL-4 signaling, such as the IL-4 receptor (56
); and, finally, genes related to the anti-CD40 plus IL-4 class-switching process, such as the Iγ1 and Iε germline transcripts (for a review, see reference 54
). In fact, their frequency within the total collection was one in six cDNA sequences in the sublibrary.
One sequence of the remaining clones of the library— and the structure of the corresponding gene—is described in this paper. It was present in surprisingly high frequency among the enriched clones (10 of 120). It was found to encode a novel murine member of the CC or β chemokine family, which we name ABCD-1. We show that this gene is expressed rather selectively by DC and activated B cells, but not by resting B cells, T cells, IL-3–induced macrophages, or IL-2–stimulated NK cells.
While our characterization of the ABCD-1 gene and its protein was in progress, two groups independently described a human chemokine molecule that is structurally and functionally related to ABCD-1 (MDC  and STCP-1 ). The overall amino acid sequence identity between the mouse ABCD-1 and the human protein MDC/STCP-1 is ~64% (similarity 84%). In addition, the genomic organization of the ABCD-1 and STCP-1 genes is very similar; particularly striking is the identity of sequences at the intron–exon boundaries of the mouse and human gene (Fig. B). All of these findings are strong support for the notion that the mouse ABCD-1 gene is the orthologue of the human MDC/STCP-1 gene.
A number of characteristics are the same, or comparable, for the mouse ABCD-1 and human MDC/STCP-1 chemo-kines (references 61
, and this report). All three studies reported that they are expressed at low levels in freshly isolated cells from thymus, LNs, spleen, and lung. In our studies, high levels of ABCD-1 mRNA expression were only seen in DC and activated B lymphocytes, and we have shown that MDC/STCP-1 is expressed by activated human B cells. Hence, the patterns of expression are consistent in mouse and human cells. Further evidence for the close relationship between mouse ABCD-1 and human MDC comes from hybridization experiments with mouse and human probes under low or high stringency, suggesting that in human anti-CD40 plus IL-4–stimulated B cells, the sequences most similar to mouse ABCD-1 are those encoded by MDC/STCP-1 (Fig. D
Finally, the chemotactic properties are similar. Activated mouse T cells of the CD8+ as well as CD4+ phenotype are the best chemoattracted cell populations. Activated human T cells are also chemoattracted by the mouse chemokine (Fig. D). It is generally accepted that stimulation of anti-CD3/anti-CD28–activated CD4+ human T cells by IL-12 plus anti–IL-4 generates Th1-like cells, whereas stimulation with IL-4 plus anti–IL-12 develops Th2-like cells. Our results show that the murine chemokine ABCD-1 acts on both types of Th cells. These results suggest a close relationship between the mouse and the human chemokine. Its role appears to be to attract activated T cells to DC and activated B cells, but not to polarize them or select helper or killer T cell subpopulations.
Other than a low level production of the MIP-1α and -3α chemokines in some malignant B cell lines (63
), expression of chemokines by normal B cells has not previously been described. Our discovery that ABCD-1 and MDC/STCP-1 are produced by mouse and human activated B cells, respectively, provides a missing link in a scenario that attempts to describe the T cell–dependent activation of B cells and the formation of germinal centers. In such a scenario, foreign antigen is first taken up and processed by immature DC, e.g., Langerhans cells in the epidermis. Inflammatory stimuli lead to activation and differentiation of these immature DC to mature professional antigen-presenting DC (65
), which migrate into the spleen and into regional LNs. Antigen-presenting mature DC could do three things. First, they could attract naive T cells by the production of the DC-CK-1 chemokine (25
). Second, they could present antigen in the context of MHC molecules and thus activate the T cells. Third, the production of ABCD-1 by the DC would then keep the T cells attracted to the DC and continuously activated. This is all likely to occur in or near the T cell–rich regions, e.g., in or near the periarteriolar lymphocyte sheath of the spleen. As a result, a focus of antigen-activated T cells could be formed around the ABCD-1–producing antigen-presenting DC.
B cells in B cell–rich follicular regions also have to be activated. Our studies show that T cell–independent as well as T cell–dependent stimulation activates B cells to ABCD-1 production. Once activated, B cells become attractive for Th cells, which migrate from the periarteriolar lymphocyte sheath into the follicular areas of the spleen and begin to interact with activated B cells.
At the same time, the B cells are attracted into the follicular regions by the CXC chemokine BLC (23
), alternatively known as BCA-1 (24
), which is most likely produced by FDC. Thereafter, Th cells follow the activated B cells through chemoattraction via ABCD-1 into the follicle, where T and B cells begin with the formation of a germinal center, i.e., with extensive proliferation, Ig V-region hypermutation, class switching, and antibody secretion.
Such a scenario makes a number of predictions. First, all attracted cells should express chemokine-specific receptors. Hence, activated but not necessarily resting T cells are expected to express ABCD-1 receptors. In humans, MDC and TARC bind to CCR4 and attract cells transfected with CCR4 (66
). On the other side, CCR4 is expressed selectively on Th2 cells (68
). However, we show that Th1 and Th2 cells are attracted by ABCD-1. Hence, ABCD-1 is suspected to have a second receptor expressed on Th1 cells, and perhaps also on Th2 cells. Future desensitization experiments with the CCR4-specific TARC and MDC/STCP-1 chemokines (61
) and with ABCD-1 might clarify how many chemokine receptors function with the different chemokines on the different T cells.
The second prediction is that targeted disruption of the ABCD-1 gene might impede the initiation of T cell– dependent B cell stimulation and the formation of germinal centers, like the targeted disruption of BLR-1, the BLC/ BCA receptor (16
). The targeted disruption of the ABCD-1 gene is under way.
An exciting new development came from the discovery that certain chemokines can function as HIV-suppressive factors. RANTES, MIP-1α, and MIP-1β were shown to be potent inhibitors of viral infection by monocyte/macrophage-tropic HIV strains (71
). Interestingly, Pal and co-workers (72
) have recently identified an HIV-inhibitory activity in the supernatants of some immortalized CD8+
cell lines. When this activity was purified and the protein sequence was determined, the molecule was identical to the MDC/STCP-1 chemokine. Hence, we would expect that ABCD-1, which we show here to chemoattract human T cells, might also act as an HIV-suppressive factor. Such experiments are now in progress. Because of this suggested suppressive effect of a B cell–produced chemokine, administration to HIV-infected individuals of soluble CD40 ligand or anti-CD40 mAb could be an additional way of inhibiting viral spread.