Impaired Th2 Subset Development in the Absence of CD4 Deborah J Fowell, Jeanne Magram, Christoph W Turck, Nigel Killeen, Richard M Locksley  Immunity  Volume 6, Issue 5, Pages 559-569 (May 1997) DOI: 10.1016/S1074-7613(00)80344-1

Figure 1 Impaired Th2 Development in CD4−/− Mice Infected with N. brasiliensis Cohorts of 5 mice were inoculated with N. brasiliensis. After 12 days the mice were killed and analyzed for (A) serum IgE by ELISA, (B) numbers of IL-4–producing cells by ELISPOT assay of dispersed suspensions of lung cells, and (C) numbers of worms in the intestines by direct recovery and counting. WT, wild-type C57BL/6 mice; CD4−/−, mice with a disrupted CD4 gene; CD2−/−, mice with a disrupted CD2 gene; CD4−/− Δcyt, mice with a disrupted CD4 gene reconstituted with a mouse CD4 transgene that lacked the cytoplasmic tail of CD4. Bars represent means and SEM. Results are shown from one of three representative experiments. Immunity 1997 6, 559-569DOI: (10.1016/S1074-7613(00)80344-1)

Figure 2 Reconstitution of Immunity to N. brasiliensis in RAG-1−/− Mice Groups of 5 RAG−/− mice were either left nonreconstituted or reconstituted by intravenous injection with the designated numbers of CD8−, TCR αβ+ T cells from CD4−/−, or wild-type donors and inoculated with larvae of N. brasiliensis after 24 hr. After 12 days the mice were killed and (A) the percentage worm expulsion (number of worms recovered compared to the number in nonreconstituted RAG−/− mice, × 100) and (B) the numbers of IL-4–producing cells in the dispersed lung cell populations as determined using an ELISPOT assay. Immunity 1997 6, 559-569DOI: (10.1016/S1074-7613(00)80344-1)

Figure 3 Peripheral T Cells in LACK TCR Transgenic Mice Peripheral lymph node cells from a BALB/c LACK TCR transgenic mouse demonstrate large numbers of Vβ4+ transgenic T cells in both CD4+ and CD4− populations. Numbers represent percentages of total lymphoid cells. Representative immunofluorescent analysis from more than 100 transgenic mice. Immunity 1997 6, 559-569DOI: (10.1016/S1074-7613(00)80344-1)

Figure 4 Infection of LACK TCR Transgenic Mice (A) Groups of 5 BALB/c LACK TCR tg+ (Tg+) and BALB/c LACK TCR tg− littermates (Tg−), or the same mice crossed to BALB/c IL-12 p40−/− mice (IL-12 p40−/− Tg+ and Tg−, respectively), were infected with 4 × 105 metacyclic promastigotes of L. major, and the course of disease was followed by measuring the size of footpad lesions. Results are from one of three comparable experiments. (B) Groups of 5 BALB/c LACK TCR tg+ (Tg+), tg− littermates (Tg−), and LACK TCR tg+ mice treated with anti-CD4 MAb (Tg+ + anti-CD4 MAb) were inoculated in the hind footpads with 4 × 105 promastigotes and the course of disease followed by measuring the size of the local lesions. Mice treated with anti-CD4 received 500 μg GK1.5 antibody intraperitoneally on the day of infection and 250 μg three times weekly throughout the experiment. Mice treated with anti-CD4 had less than 1.5% CD4+ T cells at the end of the experiment when lymph nodes and spleens were analyzed. Immunity 1997 6, 559-569DOI: (10.1016/S1074-7613(00)80344-1)

Figure 5 Infection of BALB Mice Reconstituted with DN T Cells from LACK TCR Transgenic Mice (A) Groups of 5 BALB H2-Kk×d mice were either nonreconstituted (BALB/c(d×k)) or intravenously reconstituted with 107 purified DN LACK TCR tg+ T cells (BALB/c(d×k) + DN tg+(d×d)) and infected with L. major along with 5 LACK TCR tg+ mice on the BALB/c background (Tg+(d×d)) as a control. The course of disease was followed by measuring the size of the local footpad lesions. (B) Dispersed lymph node cells were collected after 8 weeks of infection from the designated groups of mice in (A) and used to purify total T cells. DN TCR tg+ T cells were further purified from the reconstituted mice by lysis of H2-Kk–bearing cells using anti-H2-Kk MAb and complement. Cells (5 × 105/well) were incubated with 1 μM LACK156–173 peptide and irradiated BALB/c T-depleted spleen cells (2.5 × 106/well) for 48 hr, and the supernatants were quantitated for IL-4 and IFNγ by ELISA. Immunity 1997 6, 559-569DOI: (10.1016/S1074-7613(00)80344-1)

Figure 6 Analysis of DN LACK TCR Transgenic T Cells (A) Analysis of methylation of the CD8 gene. Immunofluorescent cell sorting was used to obtain highly purified populations of DN thymocytes from BALB/c mice (wt Thymus−/−), CD4+ thymocytes from BALB/c mice (wt Thymus CD4+), DN tg+ T cells from lymph nodes of LACK TCR tg+ mice (Tg+ LNC−/−), and CD4+ tg+ T cells from lymph node cells of LACK TCR tg+ mice (Tg+ LNC CD4+). DNA was isolated and digested with either BamHI (B), BamHI followed by MspI (cuts both methylated and unmethylated DNA) (M), or BamHI followed by HpaII (cuts only unmethylated DNA) (H). After electrophoresis and transfer to nitrocellulose, hybridization was performed using a cDNA from the 5′ end of the murine CD8 gene. Size markers are shown at left. (B) TCR-associated kinases in normal CD4+ and DN TCR tg+ T cells. Immunofluorescent cell sorting was used to purify (>99%) BALB/c CD4+ TCR tg+ T cells (CD4+ TCR tg+) and DN TCR+, tg+ cells (DN TCR tg+) from LACK TCR transgenic mice, or αβ TCR+ CD4−/− T cells (CD4−/−). Complement-depletion was used to produce B cell–enriched T-depleted spleen cells (T-depleted SPN). Lanes were blotted with MAb to ZAP-70, stripped, and reprobed using antiserum to Syk. Molecular weight markers are shown at left. Immunity 1997 6, 559-569DOI: (10.1016/S1074-7613(00)80344-1)

Figure 6 Analysis of DN LACK TCR Transgenic T Cells (A) Analysis of methylation of the CD8 gene. Immunofluorescent cell sorting was used to obtain highly purified populations of DN thymocytes from BALB/c mice (wt Thymus−/−), CD4+ thymocytes from BALB/c mice (wt Thymus CD4+), DN tg+ T cells from lymph nodes of LACK TCR tg+ mice (Tg+ LNC−/−), and CD4+ tg+ T cells from lymph node cells of LACK TCR tg+ mice (Tg+ LNC CD4+). DNA was isolated and digested with either BamHI (B), BamHI followed by MspI (cuts both methylated and unmethylated DNA) (M), or BamHI followed by HpaII (cuts only unmethylated DNA) (H). After electrophoresis and transfer to nitrocellulose, hybridization was performed using a cDNA from the 5′ end of the murine CD8 gene. Size markers are shown at left. (B) TCR-associated kinases in normal CD4+ and DN TCR tg+ T cells. Immunofluorescent cell sorting was used to purify (>99%) BALB/c CD4+ TCR tg+ T cells (CD4+ TCR tg+) and DN TCR+, tg+ cells (DN TCR tg+) from LACK TCR transgenic mice, or αβ TCR+ CD4−/− T cells (CD4−/−). Complement-depletion was used to produce B cell–enriched T-depleted spleen cells (T-depleted SPN). Lanes were blotted with MAb to ZAP-70, stripped, and reprobed using antiserum to Syk. Molecular weight markers are shown at left. Immunity 1997 6, 559-569DOI: (10.1016/S1074-7613(00)80344-1)

Figure 7 Priming of DN T Cells for IL-4 Production In Vitro (A) CD4+ and DN LACK TCR tg+ cells were purified by immunofluorescent sorting and incubated using 5 × 105 cells/well with 0.04 μM LACK156–173 peptide and irradiated T-depleted spleen cells (2.5 × 106/well). After 5 days, the cells were washed, counted and restimulated. (Left) 2 × 105 cells/well in the presence of the designated concentrations of LACK156–173 peptide with irradiated T-depleted spleen cells (2.5 × 106/well) were analyzed for cell proliferation using [3H]thymidine incorporation after 72 hr. (Middle and right) Designated numbers of T cells were incubated in the presence of 1 μM LACK156–173 peptide and irradiated T-depleted spleen cells (2.5 × 106/ well), and supernatants were collected after 48 hr and analyzed for IL-4 (middle) or IFNγ (right) using ELISA. Representative results from one of five comparable experiments. (B) Highly purified CD4+ or DN LACK TCR tg+ T cells were incubated as in (A) either in the absence (no IL-4) or presence of exogenous rIL-4 (100 ng/ml) (+IL-4 in 1o). After 5 days, cells were washed extensively and reincubated using 2 × 105 cells/well with 1 μM LACK156–173 peptide and irradiated T-depleted spleen cells (2.5 × 106 cells/well). After an additional 48 hr incubation, the supernatants were collected and analyzed for IL-4 and IFNγ using ELISA. Immunity 1997 6, 559-569DOI: (10.1016/S1074-7613(00)80344-1)

Figure 8 Priming of CD4+ T Cells for IL-4 Production by Antigen-Presenting Cells That Express Mutations That Abolish MHC Class II Binding to CD4 OVA-specific CD4+ TCR tg+ T cells were purified from DO11.10 mice and incubated with transfected L cells that expressed either wild-type I-Ad (wt I-Ad) or I-Ad mutated to abrogate binding to CD4 (mutant I-Ad) in the presence of 1.6 μM OVA323–339 peptide with (+IL-4 in 1o) or without (no IL-4) 100 ng rIL-4. After 7 days, T cells were washed and reincubated with irradiated T-depleted spleen cells expressing wild-type I-Ad with 0.6 μM OVA323–339 peptide. Supernatants were collected after 48 hr and quantitated for IL-4 (left) and IFNγ (right) using ELISA. Data represent one of two comparable experiments. Immunity 1997 6, 559-569DOI: (10.1016/S1074-7613(00)80344-1)