ABC increase in the spleen and bone marrow of aged mice
With old age, the representations of B cell subsets within the spleen and bone marrow are considerably altered. Recently, Hao, et. al. (2011)
, have shown that B cells in the spleens of aged mice are increasingly comprised of a novel B cell subset bearing little CD21/35 or CD23. Among mature (AA4.1−
) B cells, CD21/35−
“age-associated B cells” (ABC) were increased an average of 5-fold in proportion and number in the spleens of 24 months old C57BL/6 (B6) mice and were 12-fold increased by 27-29 months of age (). Our ABC were comparable in surface phenotype to those described by Hao, et. al. (2011)
; e.g., CD21/35−
, but differed from another CD21/35−
B cell subset that increases in aged mice described by Rubtsov, et. al (2011)
, and also called “ABC”, in that the ABC in our studies were negative for CD11b and CD11c (data not shown).
Age-associated B cells (ABC) accumulate in spleen and bone marrow of aged C57BL/6 mice
Concomitantly, as ABC increased in aged spleens, follicular (FO) B cells were reduced (). Marginal zone (MZ) (CD21/CD35hi CD23−) and B1 (CD43/S7+) B cells were also examined and each represented minor populations of <10% of B cells in our aged B6 mice (; data not shown). ABC constituted, on average, ~20-50% of total mature B cells in the spleens of 24-29 months old B6 mice. ABC were also increased in the blood (data not shown) and in the bone marrow of old mice compared to young controls (). In the bone marrow, ABC levels were variable, but on average, represented ~20-60% of mature B cells in old mice depending upon the age ().
ABC from old mice inhibited IL-7 mediated pro-B cell, but not pre-B cell, growth via TNFα mediated apoptosis
As an initial test of whether B cells with the ABC phenotype could alter B cell precursor functions, we asked whether the expanded ABC seen in aged spleen could affect B cell precursor growth in vitro. In these experiments, splenic ABC from aged mice were cultured in the upper chambers of transwell cultures separated by a semi-permeable membrane from unfractionated bone marrow cells as a source of young adult B cell precursors in the lower chamber. After 3 days of co-culture in the presence of IL-7, the bone marrow cells were harvested and IL-7 mediated B cell precursor growth capacity was measured in IL-7 colony forming assays (IL-7 CFU). IL-7 CFU are generated by both pro-B cells and early pre-B cells; therefore, any effects on IL-7 CFU are targeted at relatively early stages in the development of new B cells. As shown previously, the decline in B cell precursors in aged mice coincides with losses in IL-7 responding B cell precursors (IL-7 CFU) within the bone marrow (Riley et. al., 1991
). As shown in , co-culture of bone marrow cells with splenic ABC from old mice resulted in diminished growth of B cell precursors with the extent of inhibition dependent upon the numbers of ABC employed.
ABC from old mice produce TNFα which induces apoptosis in IL-7 responsive B cell precursors
The capacity of aged ABC to inhibit B cell precursor growth in vitro was prevented by anti-TNFα neutralizing antibodies (). IL-7, which was present in the transwell cultures, was not required to induce ABC-mediated inhibition of B cell precursor growth. Culture of aged ABC, in the absence of added cytokines, for 3 days resulted in supernatants which reduced B cell precursor growth in IL-7 CFU assays (). Inclusion of neutralizing anti-TNFα antibodies prevented the inhibition of IL-7 CFU by aged ABC supernatants ().
ABC from aged mice have been shown previously to express TNFα mRNA (Frasca et al., 2012
); recombinant mouse (rm)TNFα also was a potent inhibitor of IL-7 CFU (). Inhibition of B cell precursor growth by TNFα was primarily via apoptosis since addition of the caspase 3 inhibitor Z-DEVD-FMK reversed TNFα inhibition of IL-7 CFU (). Indeed, inhibition of caspase activation revealed a positive effect of TNFα on IL-7 CFU. Notably, TNFα acts via multiple pathways leading to activation of NFκB, which may promote survival, as well as caspases (Leong and Karsan, 2000
The maximal inhibition of IL-7 CFU from unfractionated bone marrow that was induced by old ABC as well as TNFα was ~50-60% of control values (). Since ~40-50% of IL-7 CFU were relatively resistant to ABC (as well as TNFα) mediated growth inhibition, a subset of B cell precursors was apparently not affected by ABC and TNFα. This likely reflected the generation of IL-7 CFU from both bone marrow pre-B and pro-B cells. Pre-B cells (CD2+
; 7% ± 0.1% SE) in bone marrow of young adult B6 mice are 3-4 fold higher in incidence than are pro-B cells (CD2−
; 2% ± 0.1% SE). However, as shown in , the cloning efficiency of pre-B cells, most of which fail to respond to IL-7 (Hardy et al., 1991
), is about 5-fold lower than for pro-B cells. Therefore, on balance, roughly equal numbers of IL-7 CFU in unfractionated bone marrow derive from pre-B versus pro-B cells.
Pro-B cell, but not pre-B cell, growth is inhibited by ABC and TNFα
We asked if pre-B cell and pro-B cell IL-7 CFU differed in susceptibility to TNFα. Most (~80%) of pre-B cell derived IL-7 CFU are resistant to TNFα induced apoptosis while, conversely, most (~80%) of pro-B cell derived IL-7 CFU are susceptible to TNFα growth inhibition (). Therefore, as B cell precursors transit the pro-B to pre-B cell transition, their susceptibility to TNFα-mediated apoptosis is reduced. This results from signaling via the pre-B cell receptor in IL-7 responsive early pre-B cells (Ratliff, M., et al., manuscript in preparation).
IL-7 CFU from isolated pro-B cells from RAG-2 knockout mice, which lack development beyond the pro-B cell stage, were susceptible (~75-80%) to inhibition by splenic ABC as well as TNFα (), consistent with wild-type CD2− pro-B cell susceptibility to TNFα ). Importantly, ABC present in the bone marrow of aged mice, like ABC from aged spleen, mediated inhibition of IL-7 CFU from pro-B cells, albeit old bone marrow ABC were slightly, but significantly, less effective than splenic ABC (). Therefore, ABC from aged mice, via TNFα, specifically inhibited the growth of pro-B cells, but not pre-B cells, in vitro.
Inhibition of B cell precursor growth is acquired by ABC with age and is not seen with follicular B cells in old mice
While ABC from aged mice inhibited IL-7 CFU, B cells with the same ABC phenotype from young adult mice did not affect B cell precursor growth in vitro (). Therefore, capacity to inhibit IL-7 CFU was gained in ABC with advanced age. Moreover, FO B cells from either young or aged spleen failed to inhibit B cell precursor growth (). Similar results were also obtained with isolated mature B cells from the bone marrow whose surface phenotype was analogous to that of FO splenic B cells (IgM+ CD21+ CD23+). With these “FO-like” recirculating B cells from aged bone marrow, again no inhibition of B cell precursor growth was observed in co-cultures ().
Inhibition of B cell precursor growth is acquired with old age by ABC and is not seen with follicular (FO) B cells
A subset within splenic and bone marrow B cells produces IL-10 and rescues TNFα induced growth suppression of B cell precursors
We found it surprising that FO B cells from old mice failed to inhibit IL-7 CFU. We have previously reported that mature splenic B cells with an FO phenotype (IgM+
) in old mice produced TNFα in even greater amounts than did ABC (Frasca et al., 2012
). In mixing experiments, FO B cells, from either young or aged mice, prevented old ABC from inhibiting B cell precursor growth in vitro
(). Neutralization of IL-10 resulted in abrogation of the protective effects of young adult FO B cells (). Moreover, when IL-10 was neutralized, FO B cells, as well as FO-like recirculating bone marrow B cells, from aged mice now became inhibitory (). Similarly, FO splenic B cells, as well as FO-like recirculating B cells from the bone marrow, of old mice were capable of protecting B cell precursors from growth inhibition by recombinant (rm)TNFα ().
Splenic FO and recirculating FO-like bone marrow B cells produce IL-10 which prevents ABC and TNFα-mediated inhibition of B cell precursor growth
FO B cells from young IL-10 KO mice were not capable of preventing rmTNFα-mediated inhibition of B cell precursor growth unlike wild-type FO B cells (). These results indicated that some FO B cells in both young and aged mice secreted IL-10 which prevented TNFα mediated apoptosis in B cell precursors. Notably, TNFα inhibition of IL-7 CFU was highly sensitive to the presence of IL-10. As little as a ~1:200 molar ratio of IL-10 to TNFα was sufficient to abrogate the inhibition of CD19+ B lineage IL-7 CFU seen with TNFα (). Since isolated CD19+ bone marrow B cell precursors served as “targets” for both TNFα and IL-10, both the apoptotic effects of TNFα and the protective effects of IL-10 act directly on IL-7 responsive B cell precursors.
ABC mediate decline of pro-B cells in vivo
Based upon the capacity of ABC in old mice to inhibit pro-B cell survival and IL-7 CFU responses in vitro, we reasoned that as inhibitory, pro-inflammatory ABC capable of TNFα production increased in bone marrow, but protective IL-10 producing FO-like B cells decreased, this would be associated with progressive losses of pro-B cells in individual aged mice. This was seen to be the case: there were reductions in bone marrow pro-B cells, both in percentage and in number, which were proportional to the increased ABC to FO-like B cell ratio (). This suggests the hypothesis that as the balance of mature B cells within the bone marrow favors a higher ABC to FO-like B cell ratio, the targets of ABC and TNFα, e.g., pro-B cells, are proportionally reduced.
ABC are associated with loss of pro-B cells in old mice and promote pro-B cell loss upon adoptive transfer
In order to test whether old ABC directly contributed to loss of pro-B cells in vivo, we adoptively transferred ABC from aged B6 mice into RAG-2 KO young adults. RAG-2 mice have elevated levels of pro-B cells within the bone marrow and, as shown above, RAG-2 KO pro-B cells are sensitive to both old ABC and TNFα mediated suppression. When old ABC were transferred into RAG-2 KO mice, pro-B cells were reduced by ~40% when assessed 4 weeks later (). This coincided with the presence of ABC, little or no FO-like B cells, and a very high ABC to FO-like B cell ratio in the bone marrow of these recipients (). It should be noted that within the spleens of these recipients, ABC were readily detected and were equivalent to ~9% of the original ABC inoculums and typical FO B cells were not observed (<1%) (data not shown). These experiments established that upon adoptive transfer, old ABC both populated the spleen and bone marrow and promoted the loss of pro-B cells within the bone marrow in vivo.
When young adult FO B cells were transferred into RAG-2 KO mice, no loss of pro-B cells was observed (). This was so even though low levels of phenotypic ABC were seen within the bone marrow () and ~30% of splenic B cells were now ABC phenotype (data not shown). That ABC were seen upon FO B cell transfer was expected given the derivation of ABC from FO B cells described in vivo by Hao, et. al. (2011)
. Furthermore, that young FO (or ABC) had no effect on pro-B cell levels was consistent with the failure of either young FO or ABC to inhibit IL-7 CFU ().
In contrast to young FO B cells, when old FO B cells were transferred to RAG-2 KO mice, ~25% loss of pro-B cells was observed (). This coincided with detection of ABC, derived from old FO B cells, within the bone marrow (). Unlike young B cells with the ABC phenotype, old ABC can inhibit pro-B cell survival and their presence in these recipients likely explains the reductions seen in pro-B cells.
Our in vitro experiments demonstrated that ABC from old mice, via production of TNFα, promoted apoptosis among pro-B cells and reduced IL-7 CFU responses. However, we also assessed the capacity of old ABC, transferred into young RAG-2 KO mice, to indirectly affect B cell precursors by altering the bone marrow microenvironment. As shown in , bone marrow cells from old ABC→RAG-2 KO recipients, after depletion of any B cells, were capable of suppressing the IL-7 mediated growth of young B cell precursors. ABC could induce this suppressive capacity in bone marrow cells via TNFα. In vitro, mature B cells (ABC:FO-like ratio of 1.6) from the bone marrow of aged mice were cultured with young adult (non-B) bone marrow cells. After co-culture, this bone marrow was now capable of suppressing IL-7 CFU (). As shown, rmTNFα was capable of inducing IL-7 CFU suppressive capacity in bone marrow cells and TNFα was required for the induction of bone marrow suppression by aged B cells in vitro ().