T Cell Development Is Affected in Mice Reconstituted with rip−/− Precursors.
To investigate the contribution of rip to lymphocyte development, lethally irradiated C57BL/6 mice were reconstituted with embryonic day 14 129/Sv wild-type and rip−/− fetal liver precursor cells. An antibody for the allelic Ly9.1 antigen distinguished donor (129/Sv mice are Ly9.1+) from host cells (B6 mice are Ly9.1−). 6 wk after reconstitution, three recipient mice were killed and the thymus, spleen, and lymph node were analyzed for the presence of Ly9.1+ cells. 10-fold decreases in the number of cells in the lymph nodes and thymus isolated from rip−/− reconstituted mice were observed. In contrast, similar numbers of splenocytes were observed in wild-type and rip−/− reconstituted mice.
In addition to fewer cells in the rip−/− reconstituted thymus, the percentage of wild-type versus rip−/−-derived cells was also different. In rip+/+ reconstituted mice, 97% of the thymus contained Ly9.1+ cells, whereas only 3% of thymocytes in the rip−/− reconstituted mice were donor derived ( A). Although few rip−/− thymocytes were detected in the reconstituted mice, the CD4/CD8 profile did not reveal any developmental changes in rip−/− thymocytes.
Figure 1. Flow cytometric analysis of mice reconstituted with rip+/+ or rip−/− fetal liver precursors. (A) Flow cytometric analysis of thymus. Single cell suspensions of thymocytes were stained for the donor-specific Ly9.1 marker and for CD4 or (more ...)
Interestingly, rip−/− T cells were detected in the periphery. The peripheral lymphoid organs of three wild-type or three rip−/− reconstituted mice were analyzed 12 wk after reconstitution. One representative experiment is shown in . In the cervical lymph nodes of rip−/− reconstituted mice, 17% of the CD3+ cells also stained positive for Ly9.1 and were derived from rip−/− cells ( B). In contrast, mice reconstituted with rip+/+ precursors contained 67% CD3+ Ly9.1+ cells in the cervical lymph nodes ( B). Wild-type reconstituted mice contained 53% Ly9.1+ CD4+ cells and 16% CD8+ T cells in the cervical lymph nodes. In contrast, 2.5% of Ly9.1+ CD4+ cells and 4% of the Ly9.1+ CD8+ cells were detected in the cervical lymph nodes of mice reconstituted with rip−/− precursors. Few peripheral rip−/− T cells were also observed in the spleen ( C) and the inguinal and mesenteric lymph nodes (unpublished data).
Previous studies have implicated Fas in CD4 T cell survival and TNF in CD8 T cell survival (11
). Because rip mediates TNFR1 signaling, we expected the CD8 lineage to be affected by an absence of rip. However, both the CD4 and CD8 T cell lineages were affected by an absence of rip
In contrast to the T cell lineage, rip−/− precursors contributed to the B lymphoid and myeloid cell lineages. Similar numbers of FITC-Ly9.1 and PE-B220+ cells were observed in the spleens of three rip+/+ (36, 38, and 35%) and three rip−/− (29, 35, and 36%) reconstituted mice ( C). Equal numbers of FITC-Ly9.1 and PE–Mac-1+ cells were also observed (unpublished data). Thus, the death domain kinase rip does not appear important in B cell or myeloid lineage development but is required for the normal development of the T lineage.
An Age-dependent Decrease in rip−/− Thymocytes.
The decrease in rip−/− thymocytes and mature T cells in the reconstituted mice suggests that the lack of rip−/− T cells may reflect a deficiency in committed thymocyte precursors. To determine whether equal numbers of rip+/+ and rip−/− thymocytes could be detected at early time points, wild-type and rip−/− reconstituted recombination activating gene 1−/− mice were killed 2 wk after reconstitution and thymi were stained with FITC-Ly9.1 and PE-CD3, and PE-CD4 and PE-CD8. Similar numbers of Ly9.1+ donor-derived thymocytes were observed in mice reconstituted with wild-type (14%) or rip−/− (12%) precursors ( A). Additional reconstituted mice were then killed 6 wk after reconstitution. Although sufficient numbers of Ly9.1+ thymocytes were observed in three rip+/+ reconstituted mice (86, 83, and 87%), only 13% (11, 14, and 13%) of thymocytes were derived from rip−/− precursors ( B). These studies suggest that in the absence of rip, thymocytes either fail to proliferate or undergo cell death.
Figure 2. Flow cytometric analysis of thymocytes from mice reconstituted with rip+/+ and rip−/− precursors 2 and 6 wk after reconstitution. (A) Thymocytes from three wild-type and three rip−/− reconstituted mice 2 and (B) 6 wk after (more ...)
Rip−/− T Cells Respond to Mitogens.
The age-dependent decrease in rip−/−
thymocytes and the lack of mature rip−/−
T cells in the reconstituted mice may reflect the fact that rip, like fadd, mediates proliferative pathways in lymphocytes (34
). To examine this possibility, splenocytes from mice reconstituted with wild-type and rip−/−
precursors were labeled with CFSE and then stimulated in vitro with anti-CD3 antibody or Con A. 4 d later, the cultures were stained with antibodies to Ly9.1, CD4, and CD8. To determine whether rip−/−
T cells proliferate in response to mitogenic stimulation, we gated on the CFSE+
cells. Cells from mice reconstituted with wild-type or rip−/−
cells proliferated in response to Con A or anti-CD3, as evidenced by the decrease in CFSE fluorescence relative to the unstimulated cells ( A). However, rip−/−
T cells consistently exhibit a twofold decrease in the proliferative response when compared with wild-type T cells. This decreased response may reflect the limited numbers of rip−/−
T cells in the splenocyte cultures ( B). In the experiment shown in A, the rip−/−
reconstituted spleen contains 2.8% Ly9.1+
cells and 1% Ly9.1+
cells, whereas wild-type reconstituted spleen contains 18% Ly9.1+
cells and 5.8% Ly9.1+
cells ( B).
Figure 3. Rip−/− T cells respond to polyclonal activators. (A) Splenocytes from mice reconstituted with rip+/+ and rip−/− precursors were labeled with 2.5 μM CFSE and left untreated or stimulated with Con A or plate-bound (more ...)
Although decreases in the rip−/−
T cell proliferative responses were observed, we do not think this difference sufficiently explains the absence of rip−/−
thymocytes and mature T cells in the reconstituted mice. Rip−/−
T cells, like rip−/−
murine embryonic fibroblasts, may fail to survive because they are sensitive to TNF-induced cell death (24
). To test this hypothesis genetically, we examined thymocyte development in neonatal rip−/−
, and rip−/−/tnfr2−/−
An Absence of TNF or TNFR1, but Not TNFR2, Partially Rescues the RIP-associated Lethality.
To determine whether the rip-associated T cell defect(s) were TNF-mediated, we generated rip−/−/tnf−/−, rip−/−/tnfr1−/−, and rip−/−/tnfr2−/− mice. The absence of TNF and TNFR1 improved the survival of rip−/− mice, with rip−/−/tnf−/− mice surviving an average of 5–6 d. Mice deficient for both rip and tnfr1 survived for the longest period, with double-mutant animals surviving an average of 12 d (). The absence of the TNFR2 failed to rescue the rip-associated lethality, as both rip−/− and rip−/−/tnfr2−/− mice died during the perinatal period. These data are consistent with the rip-associated lethality being TNFR1-mediated.
Genetic Analysis of Offspring Obtained from rip+/−, rip+/−/tnf−/−, rip+/−/tnfr1−/−, and rip+/−/tnfr2−/− Heterozygous Matings
mice appear normal at birth and are indistinguishable from littermates. However, by day 2, double-mutant animals are evident because they appear runt-like and cachectic. By day 7, rip−/−/tnfr1−/−
mice only weigh one third of control rip+/+/tnfr1−/−
littermates (unpublished data). Histopathologic examination of the rip−/−
mice failed to reveal the reason for the death of mutant animals. Studies on relA−/−/tnf−/−
, and Ikk-
mice have suggested that the mice are immunocompromised due to a failure to activate NF-κB and as a result die from opportunistic infections (35
). Thus far, gram and silver staining of paraffin-embedded sections of rip−/−/tnfr1−/−
animals has failed to detect any evidence of infection.
Mitogenic Responsiveness in the Absence of RIP and TNFR1.
Rip-deficient T cells can be detected in the peripheral lymphoid organs of mice reconstituted with rip−/− precursors and in rip−/−/tnfr1−/− neonatal mice. To further examine the contribution of rip to T cell proliferation, we compared the mitogenic response of rip+/+/tnfr1−/− and rip−/−/tnfr1−/− splenic T cells. Consistent with our findings on the reconstituted mice, rip−/−/tnfr1−/− T cells as well as control littermates proliferated when stimulated with Con A or anti-CD3 treatment (). These studies confirm our proliferative studies of rip−/− T cells in the reconstituted mice and fail to reveal a regulatory role for the death domain kinase rip in T cell proliferation.
Figure 4. T cell proliferation in the absence of rip and tnfr1. Splenocytes from rip+/+/tnfr1−/−, rip+/−/tnfr1−/−, and rip−/−/tnfr1−/− neonatal mice were left untreated or stimulated with Con (more ...)
Double Positive (DP) Thymocyte Apoptosis in the Absence of RIP.
To determine whether targeted mutation of rip affects thymocyte survival, we isolated fetal thymus from a rip heterozygous mating and stained the thymocytes with anti-CD4 and -CD8 antibodies and analyzed them by flow cytometry. A decrease in rip−/− DP thymocytes was observed ( A). In contrast to thymus from control littermates, which on average contained 83% CD4 and CD8 DP cells, the rip−/− thymus only contained 18% viable DP thymocytes. Moreover, an average fivefold increase in the DN thymocyte population was also observed.
Figure 5. Rip-deficient DP thymocytes are sensitive to TNF-induced cell death. (A) Thymocytes from rip+/− and rip−/− neonatal mice were stained with PE–anti-CD4, FITC–anti-CD8, and the fluorescent DNA binding dye, LDS-751. (more ...)
Analysis of the fetal thymus from day 18 rip−/− embryos revealed variation in the penetrance of the DP thymocyte phenotype. For example, in four rip heterozygous matings analyzed at embryonic day 18, 2/3, 1/4, 2/2, and 1/3 of the rip−/− mutants were affected (i.e., exhibited >50% loss of DP thymocytes; unpublished data). Where rip−/− DP thymocyte loss is observed, we consistently observe an average of eightfold increase in the percent of apoptotic cells, detected by FITC–annexin V/propidium iodide or 7-AAD staining (unpublished data). Therefore, rip−/− embryos with normal CD4/CD8 profiles are observed, suggesting that thymocyte development/expansion occurs normally in the absence of rip, but that rip appears required for CD4 CD8 DP thymocyte survival.
To test whether the DP thymocyte loss may reflect sensitivity of rip−/− thymocytes to TNF-induced cell death, thymocytes were incubated with mTNF in the presence of cycloheximide for 18 h and the apoptotic cells were detected by FITC–annexin V/PI staining. Wild-type thymocytes treated with TNF were resistant to TNF-induced cell death with 4.5% of thymocyte staining with FITC–annexin V/PI ( B). A 7–10-fold increase in apoptotic thymocytes was observed when rip−/− thymocytes were treated with mTNF. Therefore, rip−/− thymocytes, like the rip−/− murine embryonic fibroblasts, appear sensitive to TNF-induced cell death. Consistent with the TNF sensitivity observed in vitro, DP thymocyte loss was not observed in neonatal rip−/−/tnf−/− mice (unpublished data).
The RIP-associated Thymocyte Apoptosis Is Not Mediated through TNFR1.
To determine whether the rip-associated thymocyte apoptosis was TNFR1-mediated, we examined the CD4/CD8 profiles of the rip+/−/tnfr1−/− and rip−/−/tnfr1−/− neonatal thymus. Dramatic differences in the overall cellularity were observed, suggesting that survival is not mediated through the TNFR1. The rip-deficient thymus consists of 10× fewer thymocytes than rip+/+/tnfr1−/− littermates ( B). Similar to what was observed in the rip−/− thymus, four- to sevenfold increases in the percent of apoptotic cells were also observed in the rip−/−/tnfr1−/− thymocytes by staining with FITC–annexin V/PI or 7-AAD (unpublished data). In addition to the decreased cellularity and increase in apoptotic cells, there were concomitant decreases in the DP thymocyte population (31, 66, 29, and 56% in four age-matched rip−/−/tnfr1−/− mice compared with 84, 83, 86, and 88% in littermate controls). Thus, the absence of RIP resulted in an average 22-fold decrease in the absolute numbers of DP thymocytes.
Figure 6. DP thymocyte apoptosis in the absence of rip and tnfr1. Flow cytometric analysis of thymocytes from rip+/−/tnfr1−/− and rip−/−/tnfr1−/− mice. (A) Thymocytes from day-6 rip+/−/tnfr1−/− (more ...)
There were concomitant increases in the relative percentage of CD4− CD8− DN thymocytes (35, 23, 12, and 17% in rip−/−/tnfr1−/− mice compared with 7, 4, 3, and 8% in control littermates; A). Additional analysis of the DN thymocytes isolated from the rip−/−/tnfr1−/− mice revealed an increase in the CD44− CD25+ DN precursor thymocytes, suggesting that an absence of rip induces a partial thymocyte arrest at the DN3 stage.
Thymocyte Survival in rip−/−/tnfr2−/− Mice.
In the absence of TNFR1, rip−/−
DP thymocytes undergo apoptosis and fail to survive, which suggests that rip−/−
thymocytes may undergo TNFR2-induced cell death. The TNFR2 (p75) has also been implicated in immune homeostasis (for review see reference 8
). To test whether the thymocyte apoptosis observed in the rip−/−/tnfr1−/−
mice is TNFR2-mediated, we examined the neonatal rip+/−/tnfr2−/−
thymus. In contrast to the decreased cellularity observed in the rip−/−
thymus, the neonatal rip−/−/tnfr2−/−
thymus contained similar numbers of total thymocytes as control littermates ( A). Surprisingly, in the absence of rip and TNFR2, no decreases in the relative percentage of DP thymocytes were observed ( B). Similar numbers of DP thymocytes were detected in rip−/−/tnfr2−/−
mice (72, 84, 82, 95, and 92%) as seen in littermate controls (86, 83, 85, 93, 92, and 90%). Consistent with these studies, no significant increase in the percent of apoptotic cells was observed (unpublished data). These studies implicate TNFR2 in DP thymocyte survival and suggest that rip participates in TNFR2 signaling.
Figure 7. DP thymocyte survival in the absence of rip and tnfr2. To determine the contribution of TNFR2 to rip−/− thymocyte apoptosis, thymus was harvested at day 2 from rip−/−/tnfr2−/− mice and control littermates. (more ...)
Thymocyte Apoptosis in the Presence of Nuclear p65.
The nature of the rip-mediated survival signal in rip+/+/tnfr1−/− thymocytes does not appear to involve the activation of p65 subunit of NF-κB. We observed nuclear NF-κB (p65) in untreated rip+/+/tnfr1−/− and rip+/−/tnfr1−/− thymocytes ( A, lanes 2 and 3, respectively) and rip−/−/tnfr1−/− thymocytes ( A, lane 1). Furthermore, an increase in nuclear p65 was not observed when either wild-type or rip−/−/tnfr1−/− thymocytes were treated with mTNF, suggesting that TNFR2 activates other survival pathways ( A, lane 4, rip−/−/tnfr1−/−, 5 rip+/−/tnfr1−/−, and 6 rip+/+/tnfr1−/−). As expected, nuclear translocation of p65 was observed when murine embryonic fibroblasts were treated with mTNF ( A, lane 8). To control for equivalent amounts of nuclear protein, the immunoblots were probed with an antibody to the nuclear corepressor protein, msin3A.
Figure 8. Nuclear NF-κB in rip+/+/tnfr1−/− and rip−/−/tnfr1−/− thymocytes. (A) Nuclear p65 in rip+/+/tnfr1−/−, rip+/−/tnfr1−/−, and rip−/−/tnfr1−/− (more ...)