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Physiology of B cells in mice with X-linked immunodeficiency (xid). III. Disappearance of xid B cells in double bone marrow chimeras.

Evidence is presented that B cells from mice with X-linked immunodeficiency (xid) differentiate at a slower rate than normal B cells. This conclusion stems from studies in which (B6 X CBA/J)F1 mice were heavily irradiated (1,000 rads) and reconstituted with a mixture of T-depleted marrow cells taken from (a) nondefective B6 mice (H-2b) and (b) xid CBA/N or nondefective CBA/Ca mice (both H-2k). With transfer of CBA/Ca plus B6 marrow cells, the irradiated recipients become repopulated with B cells derived from both parental marrow sources; except for an early imbalance (probably reflecting Hh resistance), the degree of chimerism remained relatively stable over a period of more than 6 months. Very different results occurred with transfer of a mixture of xid CBA/N and normal B6 marrow. Within the first 2 months after marrow reconstitution, a low but significant proportion of the B cells in both spleen and lymph nodes were of CBA/N origin. Thereafter the proportion of these cells fell progressively, and by 6-9 months virtually all of the B cells were of B6 origin. This gradual decline in CBA/N-derived cells did not apply to other cell types, i.e., T cells or pluripotential stem cells. Analogous results were obtained with transfer of CBA/N vs. CBA/Ca marrow cells into sublethally irradiated (750 rads) (CBA/N X DBA/2)F1 male vs. female mice. For example, CBA/N-marrow derived B cells differentiated effectively and survived for long periods in F1 male mice (xid----xid) but not in F1 female mice (xid----normal). The finding that xid B cells eventually disappear in the presence of normal B cells strengthens the view that xid B cells are an abnormal population not represented in normal mice.

Cytotoxicity Assays. A one-step assay was employed, using Ficoll-gradient-separated cells (5). A mixture of mouse-spleen-absorbed guinea pig serum (final concentration 1:6) and rabbit serum (final concentration 1:60) was used as a source of C'. Cell viability was measured by phase contrast microscopy.
Identity of PFC. The identity of PFC in the spleens of double chimeras was established according to the technique of Miller and Mitchell (9). In brief, aliquots of spleen cells containing a known number of PFC were incubated in the presence of C' with anti-H-2 antibody (either alloantisera or monoclonal antibody) or normal mouse serum (NMS). After this treatment the cells were then assayed for PFC in Cunningham chambers (9).
Purification of T and B Cells. B cell-enriched populations were prepared by treating spleen or lymph node (LN) cells (pooled cervical, inguinal, axillary, and mesenteric nodes) with anti-Thy-l.2 antibody plus C'. T cell-enriched populations were obtained either by passing cells over nylon wool columns (5) or by treating the cells with a cocktail of J1 ld and anti-I-A antibodies. Dead ceils were removed by Ficoli gradient separation before using the cells for cytotoxicity assays (5).

Results
Experimental Approach. In addition to B cells, the T cells and macrophages of CBA/N mice are reported to show subtle abnormalities (1). In this respect, the initial aim of the experiments described below was to investigate whether the B cell defect in CBA/N mice was secondary to a defect in other cell types. If this were the case, the addition of normal (nondefective) lymphohematopoietic cells during early CBA/N B cell differentiation would be expected to abrogate the B cell defect. To investigate this question, double ("tetraparental") bone marrow chimeras were prepared, using a mixture ofxid and normal stem cells. Irradiated (1,000 rads) (BB × CBA/J)F1 mice were reconstituted with a mixture of xid CBA/N (H-2 k) and nondefective B6 (H-2 b) T-depleted marrow cells (CBA/N + B6 --~ F1 chimeras) (Materials and Methods); controls were reconstituted with a mixture of nondefective CBA/Ca (H-2 k) (closely related to CBA/N) and B6 marrow cells (CBA/Ca + B6 --~ F1 chimeras). The origin of the B cells differentiating in the chimeras was established with the aid of RaMig and specific anti-H-2 and anti-I-A reagents.  Type II (T1-2) antigens, e.g. TNP-Ficoll, but react well to TI-1 antigens, e.g. TNP-LPS (1). To examine whether CBA/N B cells would gain responsiveness to TNP-Ficoll as the result of differentiation in the presence of normal lymphohematopoietic cells, the above two groups of double chimeras were immunized with TNP-Ficoli at 6 months postreconstitution.

Response of Double
Both groups of chimeras gave high anti-TNP responses when splenic PFC were measured on day 6 ( Table I). To establish the identity of the PFC, aliquots of spleen cells pooled from each group were treated in the presence of C' with anti-H-2 k antibody, anti-H-2 b antibody, or NMS, and then assayed for numbers of PFC. In the case of the CBA/Ca + B6 ~ Fl chimeras, PFC numbers relative to the NMS controls were reduced by 75% with anti-H-2 k antibody and by 12% with anti-H-2 b antibody. This finding implied that although the chimeras were reconstituted with equal numbers of CBA/Ca and B6 marrow cells, the majority of the PFC were of CBA/Ca origin. [Possible reasons for this imbalance will be discussed later.] Very different findings were observed with the CBA/N +B6 ---, F~ chimeras. In these chimeras, virtually all of the PFC were of B6 origin (Table  I). Another experiment gave identical results.
At face value, the above data clearly implied that CBA/N-derived B cells in * (B6 x CBA/J)FI mice were exposed to 1,000 rads and injected with a 1:1 mixture (2 x 106 of each) of anti-Thy-l.2 + C'-treated marrow cells taken from B6 and CBA/Ca or CBA/N mice. Chimeras were tested at 6 months post-reconstitution. * Mice immunized with 20 #g TNP-Ficoll given intraperitoneally. Normal CBA/N mice, not tested in this particular experiment, were invariably totally unresponsive to TNP-FicolI. Geometric mean; number in parentheses is the value by which the mean is multiplied or divided to give upper and lower limits of the SE. ! See Materials and Methods for details. Pooled spleen cells from each group were used for PFC identification.
the chimeras remained totally unresponsive to TNP-Ficoil. Nevertheless, it was important to show that CBA/N-derived B cells were indeed present in the chimeras. The chimeras were therefore immunized against TNP-LPS, a known immunogen for CBA/N B cells (see above) . Surprisingly, virtually all of the anti-TNP PFC in the CBA/N + B6 ~ F1 chimeras given TNP-LPS were of B6 origin, i.e., 100% of the PFC were killed with anti-H-2 b antibody plus C' (Table II); this treatment lysed only ~30% of PFC in spleens of the CBA/Ca + B6 ---* F~ chimeras, implying that most of the PFC in these chimeras were of CBA/Ca origin. In a further experiment, the two groups of double chimeras were immunized against horse erythrocytes (HRC), a T-dependent antigen. As for responses to TNP-Ficoll and TNP-LPS, virtually all of the anti-HRC PFC in the CBA/N + B6 ~ F~ chimeras were of B6 origin (compared with <30% B6 PFC in CBA/Ca + B6 ~ Fl chimeras) (data not shown). Two possibilities could account for the above findings: (a) The CBA/N-derived B cells in the chimeras were unresponsive to all three antigens tested, or (b) CBA/N-derived B cells failed to differentiate in the chimeras. To assess this second possibility, the identity of total T and B lymphocytes in the chimeras was tested.
Identity of T and B Cells in Double Chimeras. Spleen and LN cells from the two groups of double chimeras were passed over nylon wool columns or treated with anti-Thy-1 antibody and C' to yield populations enriched for T or B lymphocytes, respectively. The identity of the T and B cells was assessed by cytotoxicity, using anti-H-2 and anti-I-A antibodies (Table III). In the case of the CBA/Ca + B6 ~ F] chimeras, approximately two-thirds of the B (Thy-l-, I-A +) cells were of CBA/Ca origin and one-third of B6 origin. These data correlated closely with the origin of the antigen-specific PFC in these chimeras (Tables I  and II). B cells in the CBA/N + B6 --* F] chimeras, by contrast, were almost entirely of B6 origin. The proportion of CBA/N-derived B cells was very small   ("8%). Indeed, when further chimeras of the same batch were left for another 3 months (9 months post-reconstitution), virtually no CBA/N-derived B cells were detected (Table III). It should be mentioned that CBA/N-derived B cells were readily detected in single chimeras, i.e., irradiated (B6 x CBA/J)F1 mice reconstituted with CBA/N marrow alone (Table III).
In marked contrast to B cells, a high proportion (~50%) of the T cells in CBA/N + B6 ---* Fx chimeras were of CBA/N (H-2 k) origin. Indeed, the ratio of CBA:B6 T cells was indistinguishable in the two groups of double chimeras.
[  Table IV can be summarized as follows.
At 1.5 wk post-reconstitution, the spleens of both types of chimeras consisted largely of Ig-I-A-"null" cells, the vast majority of which were of CBA origin (see below).
At 3.5 wk, Thy-1 ÷, Ig +, and I-A + cells were detectable in the spleen, but only in small numbers. Typing with anti-I-A antibodies indicated that I-A k cells were more common than I-A b cells in both groups of chimeras.
At 7.5 wk, typical T and B cells were conspicuous in both spleen and LN. In the case of the CBA/Ca + B6 ~ F1 chimeras, B (Thy-l-, I-A +) cells were derived from both parental strains, with only a slight preponderance of CBA/Ca-derived cells (compare with the origin of the null cells at 1.5 wk). In the CBA/N + B6 F1 chimeras, B6-derived B cells clearly predominated. Significantly, however, in contrast to the long-term reconstituted chimeras considered earlier (Table  III)  CBA/N + B6 ~ F1 chimeras had fallen to very low levels (<5% I-Ak+). The proportion of CBA/N-derived T cells in these chimeras, by contrast, was very high, in fact higher than at 7.5 wk. In the case of the CBA/Ca + B6 ~ F~ chimeras, the origin of the T and B cells at 20 wk was similar to 7.5 wk except for a relative increase in B6-derived B cells. The significant finding in the above experiment was that CBA/N-derived B cells did make an appearance in CBA/N + B6 ~ F~ chimeras, but that this appearance was only transient. It should be mentioned that results virtually identical to those shown in Table IV were obtained in an additional experiment with a quite separate batch of chimeras (data not shown).
Before proceeding, comment should be on the conspicuous paucity of B6derived null cells in the double chimeras assayed at 1.5 wk post-reconstitution (Table IV). In all probability, this was a manifestation of Hh resistance, B6 (D b) cells being particularly susceptible to this reaction (11). To seek support for this explanation, a 50:50 mixture of CBA/N and B6 marrow cells (2 × 106 of each) were transferred to Ft mice conditioned by split dose irradiation, i.e., 600 rads followed 2 wk later by 1,000 rads. This procedure is known to abolish Hh resistance (11). When spleens from the chimeras were removed 1 wk later, the ratio of B6:CBA cells was now close to 1:1 (data not shown). This compared with a ratio of about 1:15 when control mice received a single dose of 1,000 rads. Although these data strongly suggest that Hh resistance was responsible for the early paucity of B6-derived cells in the spleen, it might be asked why these cells rebounded in proportion at later stages (see Table IV). The likely answer is that the effector cells for Hh resistance are short-lived cells that reside largely in the spleen, but not in other organs (11). Donor B6 marrow cells seeding the marrow cavity of the recipients fail to encounter Hh resistance: the descendants of these cells then appear in the spleen once Hh resistance in this organ has waned.
The irradiated hosts in the preceeding experiments were all (B6 × CBA/J)F~ mice. To determine whether CBA/N-derived B cells would fail to thrive in a different F1 combination, (AKR X DBA/2)F1 (H-2 k X H-2 a) mice were used as recipients of a mixture of BALB/c (H-2 d) and CBA/N or CBA/Ca (H-2 k) marrow. As shown in Table V, control single chimeras prepared by injecting either CBA/Ca or CBA/N marrow cells alone were near totally repopulated with donorderived T and B cells when the chimeras were typed 4 months later. The splenic B cells from CBA/Ca + BALB/c ~ F~ chimeras were derived from both parental strains with an unexpected preponderance of BALB/c (I-Aa)-derived cells. ~20% of the B cells were CBA/Ca (I-Ak)-derived. In CBA/N + BALB/c ~ F~ chimeras, by contrast, CBA/N (I-Ak)-derived B cells were completely undetectable. CBA/N-derived T cells, however, were conspicuous and indeed outnumbered the BALB/c-derived T cells.
Double Chimeras Prepared with Sublethal Irradiation. A dose of irradiation in the range of 750 rads destroys the vast majority of mature T and B cells, but spares small numbers of CFU/s. Transfer of parental strain marrow cells to these mice thus produces a form of double chimera in which the lymphohematopoietic system is derived from both donor and host (12). Table VI shows the effects of transferring CBA/Ca vs. CBA/N marrow cells to sublethally irradiated (CBA/N × DBA/2)FI mice; the recipients were either female (normal, nondefective) or

TABLW V Paucity of CBA/N-derived B Cells but Not T Cells in Irradiated (AKR/J × DBA/2)F1 Mice Reconstituted with CBA/N Plus BALB/C Marrow Cells 4 Months Previously
Chimeras tested* Cells tested  Table 111; SD are shown in parentheses (except for LN T where pooled cells were used--see Table llI). * F~ mice were exposed to 750 rads and then injected intravenously with 2 × 106 T cell-depleted CBA/Ca or CBA/N male marrow cells. Chimeras were assayed at 4 months post-reconstitution. *! As for Tables III, V.

male (xid). T and B cells were typed at 4 months post-reconstitution. The results can be summarized as follows. In the case of B cells, the proportion of host (H-2d-bearing) cells in CBA/Ca
Fl ~ chimeras (Group 1) and CBA/N ~ F1 ~ chimeras (Group 2) was quite similar (34% vs. 25%). Note that in these two situations, the donor and host were either both nondefective (normal) (Group 1) or both defective (xid) (Group 2). Very different results were seen in the other two situations. With transfer of nondefective marrow to xid recipients (CBA/Ca ---* F~ c~, Group 3), host cells were virtually undetectable (1%). In marked contrast, host cells reached 74% in the reciprocal situation, i.e., with transfer of xid marrow to nondefective recipi-ents (CBA/N ---* F1 9, Group 4). In the case of T cells, there was no marked variation in the proportion of host cells among the four groups (20-50%).
These results indicate that normal B cells displace xid B cells, irrespective of whether the xid B cells originate from the donor marrow or from the FI host. These data with sublethally irradiated chimeras thus corroborate the earlier findings with xid B cell growth in double chimeras given lethal irradiation (Tables  III-V).
Pluripotential Stem Cells in Long-term CBA/N + B6 ~ Fl Double Chimeras. The finding that CBA/N-marrow-derived B cells were virtually absent in long-term reconstituted double chimeras given 1,000 rads (Table III) raised the question of whether CBA/N-derived CFU/s would survive in these chimeras. To examine this question, marrow cells were taken from CBA/N + B6 ~ (B6 x CBA/J)FI chimeras at 5 months post-reconstitution; the spleens of these chimeras contained ~:5% I-Ak-bearing cells. As controls, marrow cells were also taken from CBA/Ca + B6 ~ F1 chimeras; these chimeras contained >60% I-Ak-bearing cells in the spleen. Marrow cells (4 x 106 anti-Thy-l-treated cells) from these two groups of chimeras (3 donors/group) were transferred to normal (B6 X CBA/J)Fl mice, pretreated with split dose irradiation (600 rads plus 1,000 rads) to overcome Hh resistance. Spleens were removed from the recipients 7 d later and typed with anti-H-2 antibody; the spleens at this stage were large (~ 10 s cells) and consisted mostly of primitive hematopoietic cells. As shown in Table VII, the proportion of B6-derived cells, i.e., cells lysed by anti-H-2 b antibody, was very similar in the two groups (---40%). As controls, the anti-H-2 b antibody lysed <2% of spleen cells from irradiated FI mice given normal (nonchimeric) CBA/N marrow 7 d  * Marrow cells were taken from the chimeras at 6 months post-reconstitution; the chimeras were from the same batch used in Table IV. Marrow cells pooled from 3 chimeras/group were treated with anti-Thy-1 antibody + C' and transferred intravenously in a dose of 4 X 10 6 viable cells/ mouse. * Mice were first exposed to 600 rads, left for 10 d and then exposed to 1,000 rads; marrow cells were transferred 4 h after the second dose of irradiation. Arithmetic mean of 3 mice/group (+ SD).
before, and 84% of spleen cells from F1 mice given normal B6 marrow cells. These data imply that the early hematopoietic cells developing in the spleens of both groups of irradiated mice given chimera marrow cells were derived in approximately equal proportions from CBA and B6 stem cells in the donor marrow. In contrast to B cells, there was thus no evidence that CBA/N-derived CFU/s become outnumbered by normal CFU/s during long-term residence in the chimeras.

Discussion
The results in this paper demonstrate that xid B cells differentiating from stem cells in bone marrow chimeras gradually disappear when the chimeras harbor stem cells/B cells from nondefective mice. xid B cells are detectable within the first 1-2 months of marrow reconstitution, but then eventually become almost totally replaced by normal B cells, both in spleen and LN. This replacement of xid cells by normal cells does not apply to T cells or CFU/s. On a priori grounds, one might argue that the gradual disappearance of xid B cells in chimeras is a reflection of chronic alloaggression, mediated either by the irradiated host or by cells differentiating from the nondefective marrow inoculure. Rejection by the host would seem to be ruled out by the finding that xid B cells differentiate effectively in CBA/N -o F1 single chimeras (mice reconstituted with CBA/N marrow alone, Tables III and V). Alloaggression mediated by cells derived from the nondefective marrow also seems most unlikely. To sustain this argument one would have to postulate a novel form of ailoaggression that (a) escapes tolerance induction (the marrow cells were rigorously T-depleted before transfer), (b) is unidirectional (anti-xid but not vice versa), and (c) is directed selectively to xid B cells and not other xid-marrow-derived cells, i.e., T cells or CFU/s. Perhaps the most convincing evidence against alloaggression is provided by the data on the growth of xid B cells in sublethaily irradiated CBA/N (male) ---* (CBA/N × DBA/2)FI male vs. female mice (Table VI). Here it was found that CBA/N-derived B cells differentiated well in the male Fl recipients (xid ~ xid) but not in the female recipients (xid --o normal). Interpreting this finding in terms of alloaggression is virtually impossible. [A response to a Y-chromosome controlled antigen is ruled out by the fact that CBA/Ca male B cells grew well in F1 female recipients.] If alloaggression can be disregarded, the simplest interpretation of the data is that the rate of differentiation of xid B cells is slightly slower than for normal B cells. A precedent for this notion stems from findings of Kincade et al. (13). This group has reported that, unlike normal B cells, xid B cells are unable to form colonies in agar. These workers observed that young heterozygous female mice raised between xid and normal mice contained about one-half the number of colony-forming B cells as homozygous normal mice, implying that the heterozygotes are X-chromosome mosaics containing a roughly 1 : 1 ratio of defective and normal B cells. Interestingly, the reduction in colony-forming B cells was less apparent in older heterozygotes, suggesting that the xid B cells in these mice gradually become replaced by normal B cells. More recently, Nahm et al. (14) used X-chromosome linked alloenzymes to study mosaicism in xid-heterozygous females. These workers observed that, in marked contrast to other cell types, spleen cells enriched for B cells were "nearly devoid" of the isoenzyme encoded by the X-chromosome bearing the xid gene. This suggested a virtual absence of xid B cells in the heterozygotes. Unfortunately, the age of the mice examined in this elegant study was not stated. If old mice were used, the data would be fully consistent with the data of Kincade et al. (13) mentioned above and also with the findings in the present paper.
Although the life history of B cells is still not fully understood, in mice it appears to involve production in the marrow (15), selection and differentiation in the spleen (16,17), followed by entry of a portion of the cells into the recirculating lymphocyte pool (18). Some B ceils are presumed to die rapidly, whereas others survive for many months in interphase. Precisely where in this life history normal B cells have a growth/survival advantage over xid B cells is unclear. The finding that xid B cells do appear within the first few weeks of reconstitution and then disappear progressively suggests that a reduced rate of production ofxid B cells in the marrow is probably not the explanation. 2 Likewise, the kinetics of the appearance and disappearance of xid B cells is against the possibility that xid B cells die more rapidly than normal B cells (see below). Although neither of these possibilities can be ruled out, we lean to the view that xid B cells might have a reduced capacity to turn over (expand) in the secondary lymphoid tissues, perhaps in response to environmental antigens or mitogens (see reference 13). In this respect, it is well documented that xid B cells respond poorly in vitro to certain antigens and mitogens, including anti-Ig and particular extracts of LPS (1,19). Recent studies from this laboratory have shown that most xid B cells do turn over (incorporate tritiated thymidine) at a slow rate in vivo. s Whether the rate of turnover of xid B cells is actually slower than normal B cells, however, is not clear from the available data.
The finding that xid B cells tend to disappear in the presence of normal B cells adds to the accumulating evidence that xid B cells probably cannot be equated with the Lyb 5-subset of immature B cells found in normal mice (see below, and reference 19). In our view, xid B cells are a unique population that is not represented in normal mice. xid B cells certainly resemble normal Lyb 5-B cells in several respects, notably in the capacity to respond only to TI-1 and not T1-2 antigens (1). In other respects, however, xid B cells share some of the properties attributed to mature Lyb 5 + B cells, e.g. a slow turnover (for LN and most splenic B cells), 3 capacity to recirculate 3 and the expression of strong Mls determinants (20). The retarded differentiation of xid B cells (this paper) and the odd T-dependency of xid B cell differentiation (6,21,22), however, sets these cells apart from both the Lyb 5-and Lyb 5 + B subsets of normal mice. Although it is difficult to provide a simple explanation for how the xid gene might control these various departures from normal B cell physiology, a subtle alteration in the biochemistry of xid cells is a logical working hypothesis. Searching for such alterations might be informative.

Summary
Evidence is presented that B cells from mice with X-linked immunodeficiency (xid) differentiate at a slower rate than normal B cells. This conclusion stems from studies in which (B6 x CBA/J)F1 mice were heavily irradiated (1,000 rads) and reconstituted with a mixture of T-depleted marrow cells taken from (a) nondefective B6 mice (H-2 b) and (b) xid CBA/N or nondefective CBA/Ca mice (both H-2k). With transfer of CBA/Ca plus B6 marrow cells, the irradiated recipients become repopulated with B cells derived from both parental marrow sources; except for an early imbalance (probably reflecting Hh resistance), the degree of chimerism remained relatively stable over a period of more than 6 months. Very different results occurred with transfer of a mixture of xid CBA/N and normal B6 marrow. Within the first 2 months after marrow reconstitution, a low but significant proportion of the B cells in both spleen and lymph nodes were of CBA/N origin. Thereafter the proportion of these cells fell progressively, and by 6-9 months virtually all of the B cells were of B6 origin. This gradual decline in CBA/N-derived cells did not apply to other cell types, i.e., T cells or pluripotential stem cells. Analogous results were obtained with transfer of CBA/N vs. CBA/Ca marrow cells into sublethally irradiated (750 rads) (CBA/N x DBA/2)F1 male vs. female mice. For example, CBA/N-marrow derived B cells differentiated effectively and survived for long periods in F1 male mice (xid ~ xid) but not in Fl female mice (xid ~ normal).
The finding that xid B cells eventually disappear in the presence of normal B cells strengthens the view that xid B cells are an abnormal population not represented in normal mice.
Received for publication 7 May 1984.