The non–Fc-binding anti-CD3 Ab [anti-CD3F(ab′)2] can induce graft acceptance depending on the therapeutic window in a rodent heart transplant model. The delayed protocol allows for early graft infiltration of lymphocytes, which may behave in an inhibitory manner. We investigated the most effective protocol for anti-CD3F(ab′)2 in sensitized conditions to confirm the evidence for clinical application. C57BL/6 mice were sensitized with BALB/c tail skin grafts and transplanted with BALB/c heart grafts at 8–12 wk after sensitization. Fifty micrograms of anti-CD3F(ab′)2 was administered daily for 5 consecutive days on days 1–5 (day 1 protocol) or days 3–7 (delayed protocol). In nonsensitized mice, the delayed protocol significantly prolonged graft survival after transplantation from BALB/c to naive B6 (median survival time [MST], >100 d). In contrast, the delayed protocol was unable to prevent graft rejection in sensitized mice (MST, 5 d). A significantly increased percentage of granzyme B+ CD8+ T cells was observed in the graft on day 3 posttransplantation in sensitized conditions. Further, the day 1 protocol significantly prolonged graft survival (MST, 18 d), even in sensitized conditions. Day 1 treatment significantly increased the percentage of Foxp3+CD25+CD4+ T cells and phenotypically changed CD8+ T cells in the graft (i.e., caused a significant increase in the proportion of Ly108+TCF1highPD-1+CD8+ T cells). In conclusion, different timings of delayed anti-CD3F(ab′)2 treatment promoted allograft preservation in association with phenotypic changes in CD4+ and CD8+ T cells in the graft under sensitized conditions.

Lifelong immunosuppressive therapy after solid organ transplantation contributes to an increased incidence of various diseases, including malignant diseases, metabolic diseases, infectious diseases, and cardiovascular diseases, in comparison with the general population (1). The induction of transplant tolerance is an ideal treatment to overcome the problems associated with immunosuppressive treatment.

Induction therapy using anti-CD3 Abs is a potential approach to achieve transplant tolerance. Decades ago, anti-CD3 therapy was used to treat intractable graft rejection (2); however, severe infusion reactions involving anti-CD3 Abs have hampered organ transplantation. The development of Fc-nonbinding anti-CD3 Abs to resolve the infusion reaction has emerged. Notably, Fc-nonbinding anti-CD3 Ab has a unique effect; that is, the administration of anti-CD3 treatments after the onset of diabetes (3, 4) or autoimmune diseases (5) has benefits. In addition, the delayed timing of Fc-nonbinding anti-CD3 Ab treatment after transplantation successfully promoted long-term graft acceptance through an increased lymphocyte subset of intragraft Foxp3+CD4+ T cells and anergic CD8+ T cells producing TGF-β (6, 7). The key determinant of successful induction of tolerance by delayed protocol of Fc-nonbinding anti-CD3 treatment seems to be the allowance of early entry of lymphocytes infiltrating into the graft (6, 7). Indeed, an immunological feature of graft-infiltrating lymphocytes (GILs) in the early posttransplantation period differed from that of alloreactive lymphocytes, leading to graft rejection. Schenk et al. (8) reported that activated CD8+ graft-infiltrating T cells with a memory phenotype were observed until 3 d after murine cardiac transplantation. Consistently, we recently reported that GILs consisting of CD8+ T cells with the memory phenotype could not be involved in graft rejection until 3 d posttransplantation (9), suggesting that these early GILs (before 3 d posttransplantation) may not contain enough alloreactive T cells.

Recently, a phase 2 clinical study of type 1 diabetes demonstrated both the efficacy and the safety of an Fc-engineered humanized anti-CD3 Ab (10). This promising trial using Fc-engineered anti-CD3 therapy provides us with a clinical use of a delayed treatment strategy allowing early GILs to promote transplant tolerance. However, previous efforts to achieve transplant tolerance have been struggling for decades in the clinical setting. The treatment protocols used to induce tolerance in rodent models could not be adopted in clinical organ transplantation, probably because of the resistance of alloreactive memory T cells. The existence of pretransplant alloreactive memory T cells and sensitization was associated with poor clinical outcomes (11, 12). Furthermore, memory T cells, such as homeostatic proliferation-driven residual lymphocyte subsets, prevent the induction of tolerance by resistance to costimulatory blockade after lymphoablation therapy (13, 14). Overcoming graft rejection involving memory T cell subsets activated by donor Ags is crucial for the successful induction of tolerance. In particular, the first entry allowance of GILs in the delayed protocol of Fc-nonbinding CD3 treatment may determine the graft fate in a sensitized setting. In this study, we examined the efficacy of delayed Fc-nonbinding anti-CD3 treatment in a sensitized model and the features of alloreactive memory T cells containing first-entry GILs as a treatment target.

C57BL/6 (B6, H2b), BALB/c (H2d), and C3H/hej (C3H, H2k) males purchased from Japan SLC (Shizuoka, Japan) were maintained in the conventional facilities of the Biochemical Service Unit of Hokkaido University and used at 8–12 wk of age. All experiments were performed in accordance with the institutional animal care and use guidelines of Hokkaido University.

Mouse heart transplantation was performed as described previously (6). In brief, the aorta of the donor mouse was incised for blood removal and perfused with 1 ml of 10% heparinized saline. The pulmonary artery and ascending aorta of the heart were incised, and the heart graft was recovered. The recipient’s abdominal aorta and inferior vena cava were incised and anastomosed with the donor ascending aorta and pulmonary artery, respectively. Vascular anastomosis was performed with running sutures using 10–0 monofilament nylon sutures. Graft rejection was monitored by daily palpation and determined by direct inspection. Full-thickness tail skin allografts (diameter, 1.0 cm) from BALB/c mice were transplanted on the dorsal side of B6 mice with 6–0 nonabsorbable sutures and protected with bandages for 1 wk. Eight weeks later, mice transplanted with skin or heart allografts were used as alloantigen-sensitized recipients.

Anti-mouse non–Fc-binding anti-CD3 Ab [CD3F(ab′)2] fragments (145-2C11; BioXCell, West Lebanon, PA) were used. Fifty micrograms of anti-CD3F(ab′)2 was administered i.v. via the tail vein for 5 consecutive days. Three different protocols were performed as follows: early protocol (administration from 1 d before transplantation [day −1] to 3 d posttransplantation [day 3]), day 1 protocol (administration from day 1 to 3), and delayed protocol (administration from day 3 to 7).

Cardiac grafts were sufficiently flushed with saline solution at the time of removal to exclude blood and circulating lymphocytes within the vessels. Tissues were minced with scissors and digested with collagenase IV (Sigma-Aldrich, St. Louis, MO). A single-cell suspension was isolated from the digested tissue using a 70-μm cell strainer. Lymphocytes were purified from the suspension using a Lympholyte-M (Cedarlane Laboratories, Burlington, ON, Canada).

The following Abs were used: CD3ɛ (145-2C11), CD4 (GK1.5), CD8α (53-6.7), CD25 (PC61), CD44 (IM7), CD62L (MEL-14), EOMES (Dan11 mg), Foxp3 (FJK-16 s), Granzyme B (NGZB), IFN-γ (XMG1.2), PD-1 (J43), T-bet (4B10), TCF1 (S33-96), and Ly108 (330-AJ). Abs were purchased from BD Biosciences (San Diego, CA), BioLegend (San Diego, CA), or Thermo Fisher Scientific (Waltham, MA). For intracellular staining, cell permeabilization was performed with Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) after surface staining and staining with intracellular Abs for 30 min at 4°C, followed by washing with 1× PBS. For intracellular cytokine staining, cells were stimulated with 10 ng/ml phorbol 12-myristate 13-acetate (Sigma, St. Louis, MO), 1 μM ionomycin, and 2 μl/2 ml brefeldin A (BD Pharmingen, San Diego, CA) for 4 h, followed by permeabilization with Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ) and staining with Abs at 4°C for 45 min. A FACS Canto II flow cytometer (BD Biosciences) was used as the analyzer. The data were analyzed using the FlowJo software program (Tree Star, Ashland, OR).

The IFN-γ ELISPOT assay was performed according to the protocol of BD Biosciences. Splenocytes (5 × 105) obtained from recipient mice were cocultured with 30 Gy irradiated splenocytes (1.0 × 106) obtained from the donor and a third-party mouse strain in RPMI 1640 with 10% FCS, penicillin (100 U/l), streptomycin (100 µg/ml), and 2-ME (50 µg/ml) at 37°C and under 5% CO2 using MultiScreen 96-well plates (Millipore Corporation, Billerica, MA) precoated with an IFN-γ capture Ab (BD Biosciences, Bedford, MA). After 24 h of culture, the plates were washed, spots were detected using an IFN-γ detection Ab (BD Biosciences) and visualized using streptavidin-HRP (BD Biosciences), followed by the addition of AEC substrate solution (BD Biosciences). The spots were counted using an ImmunoScan Elispot reader (Cellular Technology, Cleveland, OH).

The immune responses of T cells obtained from the spleen of nonsensitized or allosensitized B6 mice were evaluated according to the percentages of dividing T cells labeled with CellTrace Violet (Thermo Fisher Scientific, Philadelphia, PA) for 4 d in coculture with 30 Gy irradiated BALB/c splenocytes.

Paraffin sections (thickness, 10 µm) of the cardiac graft tissues were deparaffinized using xylene and dehydrated with ethanol. The sections were then stained with H&E or immunohistochemistry. Heat-induced epitope retrieval was performed using a pressure cooker for 150 s. Nonspecific staining was blocked by incubation in a blocking buffer for 30 min at room temperature. These slides were incubated with the primary Ab, anti-mouse CD3ε Ab (EPR20752; Abcam, Cambridge, UK), at 1/1000 dilution overnight at 4°C in the dark. They were washed and incubated with biotinylated secondary Ab and Dako EnVision+ System-Rabbit HRP (Agilent, Santa Clara, CA) for 45 min at room temperature and visualized with 3,3′-diaminobenzidine substrate (Cell Signaling Technology, Danvers, MA). Positive cells were confirmed at high magnification (×200 or ×400) using a light microscope.

The Kaplan–Meier method was used to assess graft survival, and each group was compared using the log-rank test. Data are shown as the mean ± SD and were analyzed using Prism version 9.0 (GraphPad Software, San Diego, CA). Student t test was used to compare the mean values of the two groups. The p values <0.05 were considered to indicate statistical significance.

To confirm the previous study in C57BL/10 (B10, H2b) to CBA (H2k) heart transplant model using anti-CD3 therapy (6), we used a more stringent heart transplant model with transplantation from BALB/c to B6 mice (Fig. 1A). In the untreated group, all BALB/c heart allografts were promptly rejected in B6 recipient mice (n = 5; median survival time [MST], 7 d; Fig. 1B). The daily i.v. administration of 50 µg of anti-CD3F(ab′)2 for 5 consecutive days from preoperative day 1 to postoperative day 3 (early protocol) prolonged graft survival, but all grafts were eventually rejected (n = 5; MST, 38 d; p = 0.0016 versus untreated; Fig. 1B). In contrast, the daily i.v. administration of 50 µg of anti-CD3F(ab′)2 for 5 consecutive days from postoperative day 3 to day 7 (delayed protocol) promoted long-term graft survival, and four of the five allografts functioned for more than 100 d (n = 5; MST, >100 d; p = 0.0007 versus early protocol; Fig. 1B). We analyzed the characteristics of GILs and splenocytes obtained from recipient mice treated with the early or delayed protocols on day 28 to determine how regulatory T cells or another lymphocyte subset was involved in graft acceptance. There were no significant differences in the percentage and number of Foxp3+CD25+CD4+ T cells in the spleen and graft between the two treatment groups (Fig. 1C). Significantly higher percentages of PD-1+TCF1highCD8+ T cells were observed in the grafts of the delayed group in comparison with the early group (Fig. 1D). We observed a significantly higher percentage of Ly108 cells in this subset in the delayed group (Fig. 1E). In addition, the mice in the delayed group were likely to have reduced IFN-γ production at 28 d posttransplantation (Fig. 1F); however, there was no significant difference between the groups. These findings are consistent with those of a previous study (6). The histological findings of the graft at 100 d posttransplantation showed a small number of CD3ɛ+ GILs in the delayed group (Fig. 1G).

FIGURE 1.

Delayed anti-CD3F(ab′)2 therapy efficiently prolonged graft survival in nonsensitized recipient mice.

(A) Schematic illustration of the treatment protocol. Anti-CD3F(ab′)2 was administered for 5 consecutive days, either on day −1 to 3 (early protocol) or days 3–7 (delayed protocol), to C57BL/6 recipient mice transplanted with BALB/c hearts on day 0. (B) The heart allograft survival rates of mice treated with anti-CD3F(ab′)2 by the early (blue line, n = 5) and delayed (red line, n = 5) protocols and those of untreated mice (dotted line, n = 5). (CF) In the early (blue triangles, n = 5) and delayed (red circles, n = 5) anti-CD3F(ab′)2 treatment groups, lymphocytes obtained from cardiac allografts and spleens were analyzed 28 d after transplantation. The percentage and number of Foxp3+CD25+CD4+ T cells (C) and TCF1highPD-1+CD8+ T cells (D). In the graft, the percentage of Ly108+TCF1highPD-1+CD8+ T cells was significantly higher in the delayed group than in the early group. *p < 0.05 (E). IFN-γ production by splenocytes against donor Ags was assessed by an ELISPOT assay (F). (G) Representative histopathological images (original magnification ×400) of sections of isografts and allografts obtained on day 100 posttransplantation from mice treated with anti-CD3F(ab′)2 according to the delayed protocol. The images were stained with H&E (left column) and biotinylated anti-mouse CD3ɛ Ab (right column). Each experiment was independently performed at least three times independently with three to five mice per group.

FIGURE 1.

Delayed anti-CD3F(ab′)2 therapy efficiently prolonged graft survival in nonsensitized recipient mice.

(A) Schematic illustration of the treatment protocol. Anti-CD3F(ab′)2 was administered for 5 consecutive days, either on day −1 to 3 (early protocol) or days 3–7 (delayed protocol), to C57BL/6 recipient mice transplanted with BALB/c hearts on day 0. (B) The heart allograft survival rates of mice treated with anti-CD3F(ab′)2 by the early (blue line, n = 5) and delayed (red line, n = 5) protocols and those of untreated mice (dotted line, n = 5). (CF) In the early (blue triangles, n = 5) and delayed (red circles, n = 5) anti-CD3F(ab′)2 treatment groups, lymphocytes obtained from cardiac allografts and spleens were analyzed 28 d after transplantation. The percentage and number of Foxp3+CD25+CD4+ T cells (C) and TCF1highPD-1+CD8+ T cells (D). In the graft, the percentage of Ly108+TCF1highPD-1+CD8+ T cells was significantly higher in the delayed group than in the early group. *p < 0.05 (E). IFN-γ production by splenocytes against donor Ags was assessed by an ELISPOT assay (F). (G) Representative histopathological images (original magnification ×400) of sections of isografts and allografts obtained on day 100 posttransplantation from mice treated with anti-CD3F(ab′)2 according to the delayed protocol. The images were stained with H&E (left column) and biotinylated anti-mouse CD3ɛ Ab (right column). Each experiment was independently performed at least three times independently with three to five mice per group.

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We next investigated whether the same therapeutic window of anti-CD3 therapy could prolong graft survival in an allosensitized transplant model. To sensitize the donor Ags, we performed skin or heart transplantation using donor strain grafts (Fig. 2A). The percentages of the CD44highCD62Llow subset (central memory T cells [Tcms]) and CD44highCD62Lhigh subsets (effector memory T cells) in both CD4+ (upper row, Fig. 2B) and CD8+ T cells (lower row, Fig. 2B) in the spleen at 8–12 wk after skin or heart transplantation were significantly increased in comparison with naive mice, except for CD4+ Tcms after heart transplantation (Fig. 2B). Moreover, IFN-γ production was significantly increased in splenocytes obtained from sensitized mice after donor Ag stimulation in comparison with those obtained from naive mice (Fig. 2C). We then grafted BALB/c hearts to B6 mice sensitized with donor strain skin grafts (Fig. 2D). Similar to a previous report (15), BALB/c heart allograft rejection was significantly accelerated in the sensitized group (MST, 5 d; p = 0.0002 versus naive; Fig. 2E). In addition, the histopathological examination of heart graft tissue at 3 d posttransplantation showed a significantly increased number of CD3ɛ+ GILs in sensitized mice (Fig. 2F, 2G). Because graft rejection on day 3 posttransplantation was just initiated, the findings of graft rejection were not clear. However, the histological findings on day 8 showed the appearance of significant graft rejection (Supplemental Fig. 1). These findings indicate that donor skin sensitization changed the distribution of memory subsets of T cells in the spleen and increased the immune response to donor Ags to shorten graft survival. To investigate whether anti-CD3 therapy prolongs graft survival in the sensitized recipient mice, we adopted the anti-CD3 early or delayed protocol in the sensitized recipient mice (Fig. 3A). The early protocol slightly prolonged graft survival in sensitized recipients (n = 5; MST, 10 d; p < 0.01 versus sensitized control; Fig. 3B), whereas the delayed protocol failed to promote graft survival prolongation (n = 5; MST, 5 d; p = 0.313 versus sensitized control). Therefore, the impact of anti-CD3 therapy on sensitized mice was not the same as that observed in naive mice.

FIGURE 2.

Skin sensitization increased memory-phenotype T cells in the spleen and accelerated graft rejection.

(A) A schematic illustration of the experiment. C57BL/6 mice were transplanted with BALB/c hearts or tail skins. Eight to twelve weeks later, the splenocytes of the mice were analyzed by flow cytometry and IFN-γ ELISPOT assay. (B) The percentages of CD44highCD62Llow subsets (Tcms) and CD44highCD62Lhigh subsets (effector memory T cells [Tems]) in both CD4+ (upper row) and CD8+ T cells (lower row) in the spleen at 8–12 wk after skin or heart transplantation. *p < 0.05. (C) ELISPOT assay. IFN-γ production against donor Ag was measured in the spleen 8–12 wk after heart or skin transplantation. (D) C57BL/6 mice sensitized with BALB/c skin were transplanted with BALB/c hearts at 8–12 wk after sensitization. (E) The heart allograft survival rates of naive (empty circles, dotted line) and skin-sensitized mice (filled circles, black line; MST, 5 d; p = 0.0002 versus naive group). (F) Histopathological images of allografts in naive (upper) and skin-sensitized mouse (lower) on day 3 posttransplantation. Representative images of sections stained with H&E (original magnification ×200) and biotinylated anti-mouse CD3ɛ Ab (original magnification ×400). (G) On day 3 posttransplantation, a significantly increased number of CD3ɛ+ cells within the allografts of skin-sensitized mice were observed in comparison with the allografts of naive mice (*p = 0.0066). Each experiment was independently performed at least three times with three to five mice per group.

FIGURE 2.

Skin sensitization increased memory-phenotype T cells in the spleen and accelerated graft rejection.

(A) A schematic illustration of the experiment. C57BL/6 mice were transplanted with BALB/c hearts or tail skins. Eight to twelve weeks later, the splenocytes of the mice were analyzed by flow cytometry and IFN-γ ELISPOT assay. (B) The percentages of CD44highCD62Llow subsets (Tcms) and CD44highCD62Lhigh subsets (effector memory T cells [Tems]) in both CD4+ (upper row) and CD8+ T cells (lower row) in the spleen at 8–12 wk after skin or heart transplantation. *p < 0.05. (C) ELISPOT assay. IFN-γ production against donor Ag was measured in the spleen 8–12 wk after heart or skin transplantation. (D) C57BL/6 mice sensitized with BALB/c skin were transplanted with BALB/c hearts at 8–12 wk after sensitization. (E) The heart allograft survival rates of naive (empty circles, dotted line) and skin-sensitized mice (filled circles, black line; MST, 5 d; p = 0.0002 versus naive group). (F) Histopathological images of allografts in naive (upper) and skin-sensitized mouse (lower) on day 3 posttransplantation. Representative images of sections stained with H&E (original magnification ×200) and biotinylated anti-mouse CD3ɛ Ab (original magnification ×400). (G) On day 3 posttransplantation, a significantly increased number of CD3ɛ+ cells within the allografts of skin-sensitized mice were observed in comparison with the allografts of naive mice (*p = 0.0066). Each experiment was independently performed at least three times with three to five mice per group.

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FIGURE 3.

The delayed anti-CD3F(ab′)2 therapy failed to promote graft survival in sensitized transplant model.

(A) Schematic illustration showing that the skin-sensitized C57BL/6 recipient mice were treated with the daily administration of 50 µg of anti-CD3F(ab′)2 for 5 consecutive days on day −1 to 3 (n = 5, early protocol) or days 3–7 (n = 5, delayed protocol) posttransplantation. (B) Heart allograft survival rates in skin-sensitized recipient mice treated with the anti-CD3F(ab′)2 protocols (sensitized + delayed [red line, circles], p = 0.3133 versus sensitized [black line]; sensitized + early [blue line, triangles]: p = 0.0019 versus sensitized).

FIGURE 3.

The delayed anti-CD3F(ab′)2 therapy failed to promote graft survival in sensitized transplant model.

(A) Schematic illustration showing that the skin-sensitized C57BL/6 recipient mice were treated with the daily administration of 50 µg of anti-CD3F(ab′)2 for 5 consecutive days on day −1 to 3 (n = 5, early protocol) or days 3–7 (n = 5, delayed protocol) posttransplantation. (B) Heart allograft survival rates in skin-sensitized recipient mice treated with the anti-CD3F(ab′)2 protocols (sensitized + delayed [red line, circles], p = 0.3133 versus sensitized [black line]; sensitized + early [blue line, triangles]: p = 0.0019 versus sensitized).

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In the allosensitized model, the previous protocols failed to promote long-term graft survival, as described earlier. It is likely that under sensitized conditions, alloreactive T cells are activated and infiltrate the graft earlier after transplantation in comparison with under naive conditions. To investigate the profile of the responding memory T cells, we characterized the population of T cells that proliferated immediately after transplantation. First, we examined the features of T cells obtained from sensitized mice after coculture with irradiated donor-strain splenocytes (Fig. 4A). A significantly higher percentage of proliferated CD8+ T cells was obtained from sensitized mice (n = 3–4; p = 0.0069 versus naive splenocytes; Fig. 4A). Proliferating CD8+ T cells in sensitized splenocytes displayed a specific feature of the TCF1lowPD-1+ phenotype (Fig. 4B). Furthermore, we found that TCF1lowPD-1+ among CD8+ T cells in the heart grafts on day 3 were increased in sensitized mice in comparison with naive mice (Fig. 4C). This subset displayed high expression levels of T-bet and EOMES (Fig. 4D). The production of IFN-γ by CD8+ T cells did not differ between the groups (Fig. 4E). Moreover, significantly more Granzyme B–expressing CD8+ T cells were observed in the grafts (Fig. 4F). These findings suggest that cytotoxic CD8+ T cells infiltrated the grafts earlier in sensitized mice. These CD8+ T cells that infiltrated within 3 d posttransplantation may be cytotoxic and may be capable of mediating allograft destruction.

FIGURE 4.

The roles of TCF1lowPD-1+CD8+ T cells in sensitized mice.

(A and B) Proportions of proliferated CD8+ T cells (A) and TCF1lowPD-1+ in the proliferated CD8+ T cells (B) in the spleens of sensitized or naive mice after allostimulation with irradiated donor strain splenocytes. Significantly higher percentages of proliferated CD8+ T cells (A) and TCF1low PD-1+ in the proliferated CD8+ T cells (B) were found in the sensitized group (both experiments were n = 3–4; *p < 0.01, **p < 0.001 versus naive group). (CF) The analysis of graft-infiltrating CD8+ T cells on day 3 after transplantation in naive and sensitized mice. (C) The frequency of TCF1lowPD-1+ in CD8+ T cells within the allograft was higher than that in the naive group (n = 4; p = 0.0954 versus naive group). (D) The subset of TCF1lowPD-1+CD8+ T cells within the allograft displayed high expression levels of T-bet and EOMES. (E and F) Analyses of CD8+ T cells producing IFN-γ (E) and the granzyme B expression in the peripheral blood, spleen, and graft (F) on day 3 after transplantation. *p < 0.05. These data were obtained from three to four independent experiments.

FIGURE 4.

The roles of TCF1lowPD-1+CD8+ T cells in sensitized mice.

(A and B) Proportions of proliferated CD8+ T cells (A) and TCF1lowPD-1+ in the proliferated CD8+ T cells (B) in the spleens of sensitized or naive mice after allostimulation with irradiated donor strain splenocytes. Significantly higher percentages of proliferated CD8+ T cells (A) and TCF1low PD-1+ in the proliferated CD8+ T cells (B) were found in the sensitized group (both experiments were n = 3–4; *p < 0.01, **p < 0.001 versus naive group). (CF) The analysis of graft-infiltrating CD8+ T cells on day 3 after transplantation in naive and sensitized mice. (C) The frequency of TCF1lowPD-1+ in CD8+ T cells within the allograft was higher than that in the naive group (n = 4; p = 0.0954 versus naive group). (D) The subset of TCF1lowPD-1+CD8+ T cells within the allograft displayed high expression levels of T-bet and EOMES. (E and F) Analyses of CD8+ T cells producing IFN-γ (E) and the granzyme B expression in the peripheral blood, spleen, and graft (F) on day 3 after transplantation. *p < 0.05. These data were obtained from three to four independent experiments.

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Next, we sought to optimize the timing of administration to maximize treatment efficacy. Although no effect was observed in the delayed group in the allosensitization model, we examined the importance of changing the timing of administration even after transplantation. We analyzed the efficacy of the initiation of anti-CD3 therapy for promoting graft survival (Fig. 5A). Notably, the administration of anti-CD3F(ab′)2 starting from day 1 (day 1 protocol) significantly prolonged graft survival (n = 5; p = 0.001 versus delayed protocol). When the GILs and splenocytes on day 8 posttransplantation were analyzed, there were no significant differences between the two treatment groups in the percentage of CD3+ cells in the graft (Fig. 5C). The proportion of Foxp3+CD25+CD4+ T cells in the graft significantly increased in the day 1 protocol group in comparison with the delayed protocol group (n = 4; p = 0.042; Fig. 5D). In addition, the proportion of TCF1highPD-1+CD8+ T cells was similar between the two groups (Fig. 5E). Moreover, this cell population was characterized by a significantly higher percentage of Ly108 in the day 1 group (Fig. 5F). Thus, the day 1 protocol changed the GILs, leading to prolonged graft survival in sensitized conditions.

FIGURE 5.

Anti-CD3 therapy starting from day 1 promoted graft survival in sensitized mice in comparison with the delayed protocol.

(A) The sensitized C57BL/6 recipient mice were transplanted with BALB/c hearts and treated daily with 50 µg of anti-CD3F(ab′)2 for 5 consecutive days on days 1–5 (n = 5, day 1 protocol) or days 3–7 (n = 5, delayed protocol). (B) Heart allograft survival rates in skin-sensitized recipient mice treated with either the delayed or day 1 protocol (sensitized + day 1 group: p = 0.001 versus sensitized + delayed group). (CE) The analyses of GILs on day 8 after transplantation in sensitized mice treated with anti-CD3F(ab′)2. The number of CD3+ cells in the graft (C). The percentages of Foxp3+CD25+CD4+ T cells (D) and TCF1highPD-1+CD8+ T cells (E). *p < 0.05. (F) The percentage of Ly108+TCF-1highPD-1+CD8+ T cells in the sensitized + day 1 group was significantly higher than that in the sensitized + delayed group. *p < 0.05. Data were obtained from three to four independent experiments.

FIGURE 5.

Anti-CD3 therapy starting from day 1 promoted graft survival in sensitized mice in comparison with the delayed protocol.

(A) The sensitized C57BL/6 recipient mice were transplanted with BALB/c hearts and treated daily with 50 µg of anti-CD3F(ab′)2 for 5 consecutive days on days 1–5 (n = 5, day 1 protocol) or days 3–7 (n = 5, delayed protocol). (B) Heart allograft survival rates in skin-sensitized recipient mice treated with either the delayed or day 1 protocol (sensitized + day 1 group: p = 0.001 versus sensitized + delayed group). (CE) The analyses of GILs on day 8 after transplantation in sensitized mice treated with anti-CD3F(ab′)2. The number of CD3+ cells in the graft (C). The percentages of Foxp3+CD25+CD4+ T cells (D) and TCF1highPD-1+CD8+ T cells (E). *p < 0.05. (F) The percentage of Ly108+TCF-1highPD-1+CD8+ T cells in the sensitized + day 1 group was significantly higher than that in the sensitized + delayed group. *p < 0.05. Data were obtained from three to four independent experiments.

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Anti-CD3F(ab′)2 has been used in autoimmune disease models using NOD mice and has shown potential in reducing the progression of type 1 diabetes in clinical trials (16–20). In a rodent transplant model, the immunological effects of anti-CD3F(ab′)2 have been reported to selectively deplete pathogenically activated CD8+ T cells via the FasL-mediated pathway, which induces long-term graft survival (21, 22). Our current study demonstrated that the delayed anti-CD3F(ab′)2 protocol promoted graft acceptance, even when using a stringent mouse combination (Fig. 1B). Furthermore, in donor-sensitized mice, the reactivity and cytotoxicity of GILs increased within 3 d after transplantation, in comparison with the conventional delayed protocol. However, modifying the delayed timing of anti-CD3F(ab′)2 could prolong graft survival in sensitized conditions, although the efficacy was limited (Fig. 5B).

Previous studies using delayed CD3 treatment have revealed the enrichment of intragraft regulatory CD4+Foxp3+ T cells critical for prolonging graft survival in a heart transplant model (6). Regulatory CD4+ T cells have properties that make them resistant to apoptosis and depletion by Ab therapy (21, 23–25). In regulatory CD4+ T cells, Foxp3 negatively regulated the expression of FasL (26). Furthermore, CD3 therapy has been reported to induce CD8+ and CD4+ anergic T cells to maintain a hyporesponsive state. Anergic CD8+ T cells within the allograft are characterized by the expression of inhibitory receptors and production of TGF-β (22). These lymphocyte subsets within the graft suppress alloreactivity. In addition, anergic CD4+ T cells play an essential role in maintaining transplant tolerance. The mechanisms underlying tolerance induction promoted by delayed CD3 treatment are associated with CTLA-4+Foxp3+ regulatory T cells and anergic CD4+ T cells in a mouse pancreatic islet transplant model (27). Consistent with these findings, our study showed that CD4+CD25+Foxp3+ T cells and CD8+TCF1highPD-1+ T cells were enriched in the delayed treatment group in the naive situation. These data support the idea that anti-CD3 therapy induces a preferential environment in the graft by modulating an immune condition composed of regulatory and anergic T cells, resulting in the promotion of long-term graft survival.

Alloreactive memory T cells represent a barrier to the induction of transplant tolerance, especially in clinical settings (13, 28). Previous studies have attempted to overcome and resolve these problems using a costimulatory blockade (29, 30). In addition, lymphoablation therapy using murine anti-thymocyte globulin was refractory to tolerance induction because of the rapid recovery of pathogenic memory T cells (31). Previous studies have suggested that memory T cells do not require costimulatory signals to activate or clonally expand (32). Although the kinetics of the response of memory T cells have been identified, a clinically applicable strategy is yet to be established. We hypothesized that the unique effect of anti-CD3F(ab′)2 efficiently prevents the memory immune response in organ transplantation. Anti-CD3F(ab′)2 may have a distinctive effect on remodeling T cells into a specific subset. A clinical trial for type 1 diabetes using a humanized CD3 mAb (teplizumab) demonstrated that dysfunctional CD8+ T cells in the peripheral blood of subjects with the best response to treatment were characterized by an increased expression of EOMES, effector molecules, and multiple inhibitory receptors (33).

In addition, this subset underwent expansion after treatment with teplizumab. This finding supports the idea that anti-CD3 therapy modifies the functional response of CD8+ T cells (33). Therefore, we examined whether the delayed day 3 anti-CD3F(ab′)2 protocol exerted this effect in a sensitized transplant model. We found that the delayed day 3 protocol could not prevent graft rejection and did not exert any beneficial effects in sensitized mice. We also observed a significant difference in the upregulation of the transcription factor, TCF1, in CD8+ T cells between the splenocytes obtained from naive and sensitized mice after Ag stimulation, that is, a significantly lower expression of TCF1 in proliferated CD8+ T cells in the splenocytes from sensitized mice in comparison with those from naive splenocytes (Fig. 4B). In particular, we identified an increased presence of TCF1lowCD8+ T cells in the grafts of the sensitized group. Previous basic (34) and clinical studies have shown that TCF1 is a crucial regulator of CD8+ T cell differentiation.

Furthermore, the silencing of TCF1 determines effector cell differentiation (35). The subset of CD8+ T cells with the low expression of TCF1 is most enriched in effector memory cells (36). CD8+TCF1high T cells terminally differentiate into CD8+TCF1low cells in chronic infection models (37). In addition, the function of CD8+ T cells differs depending on the expression level of TCF1 (36). We found that low expression of TCF1 was correlated with the increased expression of EOMES and T-bet in CD8+ T cells. Such a lymphocyte subset is reportedly associated with effector functions that lead to acute cellular rejection (38, 39). This suggests that CD8+ T cells with the low expression of TCF1 play a key role in alloreactivity and graft rejection in sensitized mice. Furthermore, treatment with selective targeting of these effector T cells together with delayed CD3 treatment may be a promising strategy for inducing graft acceptance.

We demonstrated that the day 1 protocol was more effective for promoting graft survival than the delayed day 3 protocol, as well as the early protocol (from day −1) in sensitized mice. This result (Fig. 5B) was supported by the beneficial effect of anti-CD3F(ab′)2 depending on the onset of the disease (i.e., the therapeutic window was of crucial importance even in the sensitized state) (7). It also suggests that the therapeutic window of Fc-nonbinding CD3 treatment in clinical use needs to be carefully determined.

In naive mice, the effective therapeutic window was thought to be 3 d after heart transplantation, when T cells were primed to the donor Ag but had no aggressive alloimmune response, which would lead to graft rejection. We have previously focused on the function of early GILs in a mouse heart transplant model (9). Until day 3 posttransplantation, GILs were not capable of promoting graft rejection (9). Furthermore, the graft infiltration of CD8+ T cells within 24 h of transplantation has been observed (8). Although these CD8+ T cells can proliferate within the graft, T cells do not mediate graft rejection (40). Meanwhile, in sensitized conditions, the therapeutic window for delayed CD3 treatment needed to be set earlier than day 3. We showed that the day 1 protocol also yielded regulatory T cell–enriched GILs (Fig. 5C). This may be associated with preservation of early GILs. Furthermore, we demonstrated that the day 1 protocol induced remodeling of TCF1high CD8+ T cells, which was similar to data in mice with tolerance induced by the delayed day 3 protocol in naive situations (Figs. 1D and 5D). In addition, Ly108+TCF1highCD8+ T cells were identified as progenitors of exhausted T cells that could not differentiate into effector cells (41). Indeed, TCF1highCD8+ T cells also expressed Ly108 in the day 1 protocol (Fig. 5E). This suggests that the day 1 protocol may shift the GILs to an “exhausted” state.

In conclusion, we demonstrated the different efficacies of the delayed CD3 treatment protocol depending on the timing of treatment in sensitized mice. It is likely that the augmentation of both TCF1highPD-1+Ly108+CD8+ T cells and CD4+ regulatory T cells in the graft may be associated with beneficial effects.

The authors have no financial conflicts of interest.

We thank the staff of the animal facility for maintaining the experimental animals and laboratory members of Gastroenterology Surgery I for technical assistance.

This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI 19K22646).

The online version of this article contains supplemental material.

anti-CD3F(ab′)2

non–Fc-binding anti-CD3 Ab

GIL

graft-infiltrating lymphocyte

MST

median survival time

Tcm

central memory T cell

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Supplementary data