Journal: Journal of the American Society of Nephrology : JASN

Article Title: Memory CD4 T Cells Induce Antibody-Mediated Rejection of Renal Allografts

doi: 10.1681/ASN.2015080848

Figure Lengend Snippet: Donor–reactive memory CD4 T cells induce rapid rejection of kidney allografts. B6 mice were injected with 5×106 CD4+CD44hi cells isolated from either A/J skin–sensitized B6 mice (sensitized memory CD4 T cells) or naïve B6 mice (endogenous memory CD4 T cells) and transplanted with A/J renal allografts 2 days later. Groups of recipients injected with sensitized memory CD4 T cells were treated with either anti– IFNγ neutralizing mAb XMG1.2 or control rat IgG (0.5 mg intraperitoneally every other day starting at the time of transplantation). Control B6 mice were transplanted with A/J kidney allografts without CD4 T cell injection (no cells group). (A) Renal allograft survival. *P<0.001 versus recipients injected with endogenous memory CD4 T cells. (B) Serum DSA titers and (C) the frequencies of donor–reactive IFNγ–secreting spleen T cells determined on day 7 post-transplant. Antidonor immune responses in recipients injected with sensitized memory CD4 T cells and treated with control rat IgG were similar to those in untreated recipients containing sensitized memory CD4 T cells (not shown). Graphs represent (A and B) individual animals or (C) means±SDs for 6–13 animals per group. aIFNγ, anti-IFNg antibody; end mem, endogenous memory; rIgG, rat IgG; sens mem, sensitized memory.

Article Snippet: For delayed combined CD4 and CD8 T cell depletion, recipients were treated with anti–CD8 mAbs YTS169 and TIB105 on days 4–6 post-transplant and anti-CD4 mAb (clones GK1.5 and YTS191; 0.2 mg each intraperitoneally; Bio X Cell) on days 5–7 post-transplant.

Techniques: Injection, Isolation, Transplantation Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Memory CD4 T Cells Induce Antibody-Mediated Rejection of Renal Allografts

doi: 10.1681/ASN.2015080848

Figure Lengend Snippet: Renal allografts in recipients containing donor-sensitized memory CD4 T cells have characteristic features of antibody-mediated rejection. Renal allografts were harvested at 7 days post-transplant and analyzed by hematoxylin and eosin staining and immunoperoxidase stains for Mac-2, CD3, C4d, and vWf. The photographs are representative of six to eight animals in each group. end mem, Endogenous memory; sens mem, sensitized memory. Original magnification, ×200 in hematoxylin and eosin, Mac-2, CD3, and C4d; ×400 in vWf.

Article Snippet: For delayed combined CD4 and CD8 T cell depletion, recipients were treated with anti–CD8 mAbs YTS169 and TIB105 on days 4–6 post-transplant and anti-CD4 mAb (clones GK1.5 and YTS191; 0.2 mg each intraperitoneally; Bio X Cell) on days 5–7 post-transplant.

Techniques: Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Memory CD4 T Cells Induce Antibody-Mediated Rejection of Renal Allografts

doi: 10.1681/ASN.2015080848

Figure Lengend Snippet: Alloantibody induced by donor-reactive memory CD4 T cells facilitate vascular injury and platelet aggregation within renal allografts. Renal allografts were harvested at indicated time points after transplantation. Immunohistology for C4d and vWf was scored in a blinded fashion as outlined in Concise Methods. AMR was assessed on evidence of (A) vascular injury and (B) platelet aggregation. aCD8, anti-CD8 antibody; end mem, endogenous memory; sens mem, sensitized memory.

Article Snippet: For delayed combined CD4 and CD8 T cell depletion, recipients were treated with anti–CD8 mAbs YTS169 and TIB105 on days 4–6 post-transplant and anti-CD4 mAb (clones GK1.5 and YTS191; 0.2 mg each intraperitoneally; Bio X Cell) on days 5–7 post-transplant.

Techniques: Transplantation Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Memory CD4 T Cells Induce Antibody-Mediated Rejection of Renal Allografts

doi: 10.1681/ASN.2015080848

Figure Lengend Snippet: Effector T cell depletion does not prevent renal allograft rejection induced by donor–reactive memory CD4 T cells. B6 mice injected with A/J–reactive memory CD4 T cells were treated with a cocktail of anti–mouse CD8 depletion mAb before transplantation, treated with anti–mouse CD8 and anti–mouse CD4 depletion mAbs starting on day 4 post-transplant, or left untreated. The control group received no memory CD4 T cells (no cells group). (A) The frequencies of donor–reactive IFNγ–secreting spleen T cells on day 7 post-transplant. Graphs represent means±SDs for 7–12 animals per group. (B) Serum DSA titers determined on day 14 post-transplant for the aCD4 + aCD8 delayed rejection group (open green squares) and day 7 post-transplant for all remaining groups. Graphs represent individual animals. (C) Renal allograft survival. (D) Renal allografts were harvested from CD8 T cell–depleted recipients containing memory CD4 T cells at the time of rejection (days 6–9) and analyzed by hematoxylin and eosin staining and immunohistochemistry. The photographs are representative of at least six grafts per group. Endogenous memory; sens mem, sensitized memory. Original magnification, ×200. aCD4, anti-CD4 antibody; aCD8, anti-CD8 antibody.

Article Snippet: For delayed combined CD4 and CD8 T cell depletion, recipients were treated with anti–CD8 mAbs YTS169 and TIB105 on days 4–6 post-transplant and anti-CD4 mAb (clones GK1.5 and YTS191; 0.2 mg each intraperitoneally; Bio X Cell) on days 5–7 post-transplant.

Techniques: Injection, Transplantation Assay, Staining, Immunohistochemistry

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Memory CD4 T Cells Induce Antibody-Mediated Rejection of Renal Allografts

doi: 10.1681/ASN.2015080848

Figure Lengend Snippet: Renal allograft rejection induced by memory CD4 T cells is prevented by B cell depletion. (A) B6.huCD20 mice were either injected with Rtx (0.5 mg intraperitoneally on 2 consecutive days) or left untreated. Percentages and numbers of B220+ cells were determined in peripheral blood and spleen by flow cytometry 48 hours after last Rtx injection. (B) Renal allograft survival. B6.huCD20 mice were injected with donor–reactive memory CD4 T cells, transplanted with A/J renal allografts, and either treated with Rtx (0.5 mg intraperitoneally on days −1 and 0 and every 5 days throughout the duration of the experiment) or left untreated. Control B6.huCD20 mice received A/J kidney transplants without memory CD4 T cell transfer (white circles). *P=0.003 for comparison between Rtx-treated and untreated recipients injected with memory CD4 T cells. (C) Serum DSA titers at rejection or on day 30 post-transplant if rejection did not occur earlier. (D) The frequencies of donor–reactive IFNγ–secreting spleen cells determined on day 7 or 30 post-transplant (n=3–5 animals per group). (E) Histologic analysis of renal allografts harvested from Rtx–treated B6.huCD20 recipients containing memory CD4 T cells at day 30 post-transplant. The photographs are representative of three animals per group. sens mem, Sensitized memory. Original magnification, ×200 in hematoxylin and eosin, Mac-2, CD3, and C4d; ×400 in vWf.

Article Snippet: For delayed combined CD4 and CD8 T cell depletion, recipients were treated with anti–CD8 mAbs YTS169 and TIB105 on days 4–6 post-transplant and anti-CD4 mAb (clones GK1.5 and YTS191; 0.2 mg each intraperitoneally; Bio X Cell) on days 5–7 post-transplant.

Techniques: Injection, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: p40 homodimers bridge ischemic tissue inflammation and heterologous alloimmunity in mice via IL-15 transpresentation

doi: 10.1172/JCI172760

Figure Lengend Snippet: ( A ) C57BL/6 ( n = 5–6 per group) mice received A/J cardiac allografts subjected to 0.5 or 8 hours of CIS. Recipients of 8-hour-CIS allografts were treated with 200 μg control rat IgG or anti-CD4 mAb on days –3, –2, and –1 before transplant. Grafts were harvested on day 2, infiltrating CD8 + T cells were enriched by negative selection, and total RNA was isolated and tested for expression of the indicated cytokine receptor mRNAs by quantitative PCR. Results shown indicate relative expression by infiltrating CD8 + T cells versus expression in purified splenic CD8 + T cells from naive A/J mice. * P < 0.05 as determined by Kruskal-Wallis test. ( B ) C57BL/6 mice ( n = 5–7 per group) received A/J cardiac allografts subjected to 8 hours of CIS and were treated with 100 μg BrdU and either 100 μg control rat IgG, anti-CD25 mAb, or anti-CD122 mAb i.p. on days 0 and 1. On day 2, allografts were harvested and digested, and cell suspensions were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD4 + and CD8 + T cells. ** P < 0.01 as determined by the Kruskal-Wallis test. ( C ) C57BL/6 mice ( n = 5 per group) received A/J cardiac allografts subjected to 0.5 hours of CIS. The indicated recipients were treated with 100 μg BrdU and 200 μg control rat IgG or anti-CD122 mAb i.p. on days 0 and 1 and with 2 μg recombinant p40HDs i.v. on day 1. On day 2, allografts were harvested and digested, and cell suspensions were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD8 + T cells. * P < 0.05 as determined by Mann-Whitney nonparametric test.

Article Snippet: CD4 + T cell depletion in graft recipients was performed using a 1:1 cocktail of anti-CD4 mAb (catalog BE0119 and BE0003-1, clone YTS191 and GK1.5, Bio X Cell) and 0.2 mg given i.p. on days –3, –2, and –1 before transplant.

Techniques: Control, Selection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Purification, Staining, Flow Cytometry, Recombinant, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: p40 homodimers bridge ischemic tissue inflammation and heterologous alloimmunity in mice via IL-15 transpresentation

doi: 10.1172/JCI172760

Figure Lengend Snippet: ( A ) Groups of BALB/c mice ( n = 4–6 per group) received cardiac allografts subjected to 8 hours of CIS from either C57BL/6 mice or B6.129X1-IL15ra tm1Ama /J mice and were injected with 100 μg BrdU i.p. on days 0 and 1. On day 2 after transplant, allografts were harvested and digested, and aliquots of single-cell suspensions were stained with antibody and analyzed by flow cytometry to quantitate the infiltration and proliferation of memory CD4 + and CD8 + T cells. * P < 0.05 as determined by the Mann-Whitney nonparametric test. ( B ) Allografts were harvested 48 hours after transplant, and total RNA was isolated from heart graft homogenates from each recipient and analyzed by quantitative reverse transcriptase PCR for expression of the indicated inflammatory mediator gene. Data indicate relative RNA expression of each test mediator versus expression in hearts from non-transplanted C57BL/6 mice. * P < 0.05 vs. RNA expression of C57BL/6 allografts subjected to 8 hours of CIS, as determined by the Mann-Whitney nonparametric test. ( C ) Survival of C57BL/6 or B6.129X1-IL15ra tm1Ama /J allografts subjected to 8 hours of CIS in BALB/c recipients conditioned with 250 mg CTLA-4Ig i.p. on days 0 and 1. Graft survival was monitored daily by abdominal palpation, and rejection was confirmed visually by laparotomy. ** P < 0.01 vs. survival of C57BL/6 allografts in CTLA-4–treated BALB/c recipients, as determined by the log-rank (Mantel-Cox) test.

Article Snippet: CD4 + T cell depletion in graft recipients was performed using a 1:1 cocktail of anti-CD4 mAb (catalog BE0119 and BE0003-1, clone YTS191 and GK1.5, Bio X Cell) and 0.2 mg given i.p. on days –3, –2, and –1 before transplant.

Techniques: Injection, Staining, Flow Cytometry, MANN-WHITNEY, Isolation, Reverse Transcription, Expressing, RNA Expression

Journal: Cancer Medicine

Article Title: Schedule-dependent therapeutic efficacy of L19mTNF-α and melphalan combined with gemcitabine

doi: 10.1002/cam4.89

Figure Lengend Snippet: Administration schedules of gemcitabine (G) and L19mTNF-α/melphalan (L-M) as single or combined treatments

Article Snippet: Cytofluorimetric analysis was done by direct staining for CD4 fluorescein isothiocyanate (FITC-conjugated YTS 191.1.2 mAb; Immunotools, GmbH, Germany) or CD8 (PE-conjugated YTS 169.4 mAb; Immunotools) and was always >95%.

Techniques: In Vivo

Journal: Cancer Medicine

Article Title: Schedule-dependent therapeutic efficacy of L19mTNF-α and melphalan combined with gemcitabine

doi: 10.1002/cam4.89

Figure Lengend Snippet: Immune population involved in WEHI-164 and K7M2 tumor eradication in in vivo -depleted mice. Tumor-free survival curves (%) versus time (days) of the WEHI-164 and K7M2 tumor-bearing mice subjected to G-G treatment, in vivo depleted with antibodies direct with CD4 + or CD8 + T cells as described in the Materials and Methods section. The tumor-free survival of groups of five WEHI-164 (A) and K7M2 (B) tumor-bearing mice subjected to G-G treatment (open squares), co-CD4-depleted (black triangles), co-CD8-depleted (black asterisks), or untreated tumor-bearing mice (black diamonds) is indicated. Specific cytolytic activity of the immune splenocytes after WEHI-164 and K7M2 tumor cure and persistence of antitumor memory. Specific lysis (%) of WEHI-164 (C) and K7M2 (D) cells by different E:T ratio of splenocytes from WEHI-164- and K7M2-cured mice at 6 months after G-G (open squares) or L-M-G-G (black diamonds) treatment and after s.c. tumor challenge. The specific lysis is totally inhibited by anti-MHC class I antibodies (open diamonds) and unaffected by anti-MHC class II antibodies (black asterisks). Results are representative of three independent 51 Chromium-release experiments with similar results. (E) The ability of WEHI-164- and K7M2-cured mice subjected to G-G and L-M-G-G treatments to reject the first tumor challenge at different times post cure. The number of challenged mice is indicated in round brackets.

Article Snippet: Cytofluorimetric analysis was done by direct staining for CD4 fluorescein isothiocyanate (FITC-conjugated YTS 191.1.2 mAb; Immunotools, GmbH, Germany) or CD8 (PE-conjugated YTS 169.4 mAb; Immunotools) and was always >95%.

Techniques: In Vivo, Activity Assay, Lysis

Journal: Cancer Medicine

Article Title: Schedule-dependent therapeutic efficacy of L19mTNF-α and melphalan combined with gemcitabine

doi: 10.1002/cam4.89

Figure Lengend Snippet: Immunohistochemical assessment of tumor infiltrates. Immunohistochemical assessment of CD4 + T cells, CD8 + T cells, and Gr-1 + CD11b + MDSCs in untreated or treated WEHI-164 (A–C) and K7M2 (D–F) tumor-bearing mice 3 days after the conclusion of all therapeutic protocols. Untreated group of mice received PBS only. Results are expressed as cell number (mean ± SD) per high-magnification microscopic field (HMMF). Data are representative of at least three mice per each treatment group. *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05.

Article Snippet: Cytofluorimetric analysis was done by direct staining for CD4 fluorescein isothiocyanate (FITC-conjugated YTS 191.1.2 mAb; Immunotools, GmbH, Germany) or CD8 (PE-conjugated YTS 169.4 mAb; Immunotools) and was always >95%.

Techniques: Immunohistochemical staining

Journal: Cancer Medicine

Article Title: Schedule-dependent therapeutic efficacy of L19mTNF-α and melphalan combined with gemcitabine

doi: 10.1002/cam4.89

Figure Lengend Snippet: Flow-cytometric assessment of regulatory T cells

Article Snippet: Cytofluorimetric analysis was done by direct staining for CD4 fluorescein isothiocyanate (FITC-conjugated YTS 191.1.2 mAb; Immunotools, GmbH, Germany) or CD8 (PE-conjugated YTS 169.4 mAb; Immunotools) and was always >95%.

Techniques: