T-helper function of parent leads to F1 chimeras. Presence of a separate T-cell subgroup able to stimulate allogeneic B cells but not syngeneic B cells.

Parent leads to F1 chimeras were prepared by reconstituting sublethally irradiated H-2 heterozygous mice with marrow cells from one parental strain. Purified parental strain T cells prepared from unprimed chimeras were exposed to sheep erythrocytes in heavily irradiated mice of each of the two parental strains and recovered from thoracic duct lymph of the recipients at either day 1 or day 5 posttransfer. The lymphoborne cells were then tested for their capacity to collaborate in vivo with B cells of the two parental strains. From this approach it was concluded that parent leads to F1 chimera T cells contain two discrete subgroups of T-helper cells, one specific for self H-2 determinants and the other restricted to H-2 determinants of the opposite parental strain. The restrictions mapped to the K-end of the H- 2 complex.

30-60% with spleen and/or LN cells removed at 1-18 mo postreconstitution; the proportion of host cells was slightly higher with B6 ~ F1 chimeras, presumably because of the strong Hh barrier in this situation. Both types of chimeras survived in excellent health and had normal sized spleen and LN. Chimeras were used as T-cell donors at 4-12-too reconstitution.
LN-cell suspensions from the chimeras were passed through nylon-wool columns and treated with anti-host alloantiserum and complement to leave a purified population of T cells of donor parental strain origin (Materials and Methods); these cells gave no detectable response to host-type determinants in mixed-lymphocyte culture (data not shown). For detecting subgroups of T-helper cells, purified chimera T cells were subjected to positive and negative selection to SRC in irradiated parental strain mice. For reasons discussed elsewhere (9), selection to SRC in vivo appears to reflect H-2-restricted contact oft cells with antigen presented by radioresistant macrophages (or related cells) of the host. The responding T cells initially become sequestered in the spleen and at this stage (day 1-2 posttransfer) are absent from thoracic duct lymph (negative selection). After extensive proliferation, the responding cells then reenter the circulation in large numbers, e.g., at day 5-6 posttransfer (positive selection). Nonresponsive T cells (e.g., cells restricted to H-2 determinants of the opposite parent) fail to undergo either negative or positive selection. Hence, the lymph at day 1-2 posttransfer contains only nonresponsive T cells, whereas at day 5-6 the clonally expanded responding T cells far outnumber other cells.
T-B collaboration was measured in vivo by transferring T plus B cells (anti-Thy 1.2-serum-treated SRC-primed spleen) and SRC into irradiated (CBA × B6)Fa mice and measuring splenic PFC 1 wk later.
Helper function of T cells from primed chimeras. CBA T cells were purified from CBA F1 chimeras primed with SRC 3 mo previously. As shown in Table I, these cells showed apparent unrestricted T-helper function, i.e., they collaborated as effectively with allogenic B6 B cells as with CBA and (CBA × B6)F1 B cells.

Helper Function of Unprimed Chimera T Cells Positively Selected to SRC in Irradiated Parental
Strain Mice. Purified CBA T cells from unprimed CBA ~ F1 chimeras were transferred intravenously into heavily irradiated (900 rads) CBA, B6, or (CBA × B6)F1 mice, together with 0.5 ml of 25% SRC (Materials and Methods). The donor CBA cells were recovered from thoracic duct lymph of the recipients 5-6 d later, i.e., during the stage of positive selection. (Testing with appropriate alloantiserum showed that >90% of the lymph-borne cells were indeed of donor origin.) For measuring T-helper function, the lymph-borne cells were transferred with B cells derived from mice of the B 10 congenic lines (these mice have identical genetic backgrounds and differ only at the H-2 complex).
As shown in Table II Table IV shows a similar experiment with B6 T cells derived from unprimed B6 --~ F1 chimeras. The results closely resemble those obtained above with CBA chimera T cells. Thus, selection in irradiated B6 mice stimulated helper cells which collaborated well with B10 B cells but not with B10.BR or B10.A(4R) B cells. The reverse applied to cells selected in irradiated CBA mice. Because positively selected T-cell populations are contaminated with small numbers (5-10%) of radioresistant host cells it could be argued that these latter cells accounted for the collaboration observed with H-2-different B cells in the above experiments. To exclude this possibility, the B6 chimera T cells positively selected to SRC in irradiated CBA mice (Table IV) were treated with B6 anti-CBA H-2 serum both before and after selection. As shown in Table IV, this additional treatment with anti-host alloantiserum did not diminish collaboration with B 10.BR B cells.

Helper Function of Unprimed Chimera T Cells Negatively Selected to SRC in Irradiated
Parental Strain Mice. The simplest explanation for the above findings is that H-2restricted T cells in parent ~ F1 chimeras exist as a dichotomous population before first contact with antigen. If so, negative selection of unprimed chimera T cells to   Table II,     Using this technique, the present studies indicate that homozygous cells from parent ---) F1 chimeras contain two discrete subgroups of H-2 restricted T cells. Stimulation of a subgroup restricted to self H-2 determinants occurred when T cells from unprimed chimeras were positively selected to SRC in syngeneic irradiated mice. Selection in mice of the opposite parental strain revealed a second subgroup restricted to allo H-2 determinants. Functionally, these two T-cell subgroups appear to be identical to those observed previously in normal heterozygous mice (9). In both situations the restrictions map to the K, I-A, region of the H-2 complex. Analogous experiments with double (tetraparental) chimeras have given similar results (unpublished data).

Selection to antigen in irradiated mice provides a useful tool for isolating H-2 restricted T-helper cells. Based on previous findings with heterozygous T cells, selection appears to he controlled by H-2 determinants on host macrophages or related cells (8). From a variety of approaches it has been concluded that H-2 restriction affects T-helper cells in vivo at two different levels: first, during T-cell activation (T-macrophage interaction) and second, during T-B collaboration. Expo-
Before discussing the significance of these findings, it is first necessary to consider * B6 T cells were purified from unprimed B6---* (CBA X B6)F~ chimeras (see footnote to Table IV) and transferred intravenously in a dose of l0 s viable cells into irradiated (900 rads 6 h before) B6 mice together with 0.5 ml of 50% SRC (group A) or without SRC (group B). Thoracic duct fistulas were inserted in the recipients (two mice per group) l d later and the lymph-borne cells were collected for the following 24 h. Each group yielded ~ 3 × 107 lymph-borne cells. These cells were transferred intravenously with SRC (0.5 ml of 25%) into irradiated (900 rads 6 h before) (CBA × B6)F1 mice. 6 d later the cells were recovered from the spleens of the second hosts and used as helper cells in a dose of 106 viable cells. Testing with appropriate alloantiserum showed that >90% of the cells from the spleen were Thy 1.2-positive cells of donor B6 origin. :~' § The same as for Tables I and IV. H The same as for Table I (13). Similarly, normal T cells lyse haptenated or virus-infected target cells across MHC barriers in some situations (14; J. Bennink and P. Doherty, unpublished data) but not in others (15)(16)(17)(18), Despite this controversy, in our hands normal and chimeric T cells clearly behave differently in the response to SRC in vivo. With the same strain combinations used in the present study, we have found no evidence that normal homozygous T cells depleted of alloreactivity by an acute procedure can collaborate with H-2-different B cells (3). This applies even with prior exposure of unprimed cells to antigen in irradiated F1 mice (19). From such findings one is thus led to the conclusion that, at least for the response to SRC in vivo, normal homozygous T cells behave as a single population restricted to self H-2 determinants. From the work of Zinkernagel et al. (5) and Fink and Bevan (20) for T-killer cells, it seems highly probable that H-2 restriction is imposed in the thymus. With respect to T-helper function, two pieces of evidence provide strong, if indirect, support for this viewpoint. First, induction of long-term tolerance to MHC determinants by a process which does not involve differentiation in a foreign thymus, i.e. classic neonatal tolerance, does not abrogate restriction to self H-2 determinants (21,22). Second, F1 T cells differentiating from stem cells in irradiated parental strain mice (F1 ~ parent chimeras) are restricted to H-2 determinants of the parental strain used for reconstitution (23).
It appears therefore that the capacity for T cells to collaborate with B cells bearing a particular set of H-2 determinants depends upon the T ceils encountering these H-2 determinants in the thymus (presumably on epithelial cells) during early differentiation. Thus, differentiation of strain a or (a × b)Fl stem cells in a strain a thymus produces an anti-a population of T cells which interacts only with target cells bearing strain a H-2 determinants. By the same token, strain a or (a × b)Fa stem cells processed in an (a X b)Fl thymus generate a mixture of anti-a and anti-b cells. As mentioned earlier, H-2 determinants (presumably I-A determinants) restrict the function of these cells both during T-macrophage interactions and during T-B collaboration (4,8,9,24). It seems very likely that the restriction(s) applying first in the thymus and second on macrophages and B cells are controlled by the same H-2 gene products. Although appealing from the point of view of symmetry, this notion remains to be proved.
A key assumption in the above model is that H-2 restriction is imposed before contact with antigen. Hence, the dichotomy ofT cells in parent ~ Fa chimeras should be apparent before antigen-priming. Perhaps the best support for this notion comes from the finding that negative selection of unprimed parent ~ Fa chimera T cells to antigen in syngeneic irradiated mice leaves a T-cell subgroup restricted to B cells of the opposite parental strain (Table V); analogous data have been reported for unprimed heterozygous T cells (12,25). These findings, unlike data based on positive selection, would seem to exclude the possibility that contact with H-2-associated antigen imposes restriction on uncommitted virgin precursor cells, i.e., cells with specificity for both parental haplotypes.
Katz et al. (26) have recently reported that T cells from parent ~ F1 chimeras do not collaborate with B cells of the opposite parental strain. It is important to emphasize that this finding was based on studies with single chimeras, i.e., F1 mice totally repopulated with marrow cells of one parental strain. The essential difference between these mice and the parent ~ sublethally irradiated F~ chimeras used in the present study is that the antigen-presenting cells in single chimeras are virtually all of donor strain origin (J. S. unpublished data). Hence, priming single chimeras with antigen in situ would be expected to stimulate only one of the two subgroups of T cells, i.e., the subgroup restricted to self H-2 determinants. According to this viewpoint, both T-cell subgroups would be stimulated if unprimed cells were activated to antigen in the presence of normal heterozygous macrophages, i.e., by transfer to normal irradiated Fa mice. Indeed, both Waldmann et al. (22) and ourselves (J. S. unpublished) have shown that single chimera T cells primed under these circumstances give excellent responses with B cells of the opposite parental strain. Analogous findings have been reported for T killer cells from single chimeras (5).
Finally, it should be stressed that although the role of the thymus in determining H-2 restriction is clearly important, it may not necessarily be absolute. Indeed, as mentioned earlier, there are an increasing number of reports that in certain situations T cells do interact with target cells bearing MHC determinants not encountered in the thymus (13,14,(27)(28)(29). It is perhaps tempting to dismiss these anomalous findings as having little physiological significance. Conversely, one could argue that those groups who have failed to see interaction with MHC determinants not encountered in the thymus have simply failed to push their systems hard enough (29).

Summary
Parent ~ F1 chimeras were prepared by reconstituting sublethally irradiated H-2 heterozygous mice with marrow cells from one parental strain. Purified parental strain T cells prepared from unprimed chimeras were exposed to sheep erythrocytes in heavily irradiated mice of each of the two parental strains and recovered from thoracic duct lymph of the recipients at either day 1 or day 5 posttransfer. The lymphborne cells were then tested for their capacity to collaborate in vivo with B cells of the two parental strains. From this approach it was concluded that parent ---* F1 chimera T cells contain two discrete subgroups of T-helper cells, one specific for self H-2 determinants and the other restricted to H-2 determinants of the opposite parental strain. The restrictions mapped to the K-end of the H-2 complex.