A Role for Regulatory Cells in Tolerance Induction in Recipients of Haploidentical Combined Non-Myeloablative Bone Marrow and Kidney Transplantation?.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3255-3255
Author(s):  
Giovanna Andreola ◽  
Meredith Chittenden ◽  
Juanita Shaffer ◽  
A. Benedict Cosimi ◽  
Tatsuo Kawai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Conditioning Regimen ◽  
Tolerance Induction ◽  
In Vitro Assays ◽  
Mixed Chimerism ◽  
Post Transplant ◽  
Hla Dr

Abstract Following an in vivo T cell depleting non-myeloablative conditioning regimen, 5 patients, aged 22–49, received combined kidney and bone marrow transplantation from a haploidentical related donor. Rituximab was included in the conditioning for patients 4 and 5. All patients developed initial mixed chimerism but lost it by day 21; no patient developed GVHD. Four patients discontinued immunosuppression from 240 to 422 days after BMT and have remained off immunosuppression for 9 to 52 months with no evidence of allograft rejection. Flow cytometry was used to assess lymphocyte subsets recovering after transplant. CD3 counts recovered slowly, exceeding 500 cells/μl at days +271, +365, +640 and +450. While memory CD45RO+ cells were most prevalent among CD4+ cells, naïve-type CD4+CD45RA+ cells, presumably arising from the recipient thymus, ranged from 8% to 56% at the time when total CD4 counts recovered to >100 cells/μl (days +165, +21, +352, +240). Notably, a very high proportion of initially recovering T cells were CD3+CD4+ expressing CD25 in all patients as early as day 7 and persisted over 1 year in 2 patients. At approximately day +120 and +365, we further characterized these cells for CD127, FOXP3, CD45RO, CD45RA, HLA-DR and CD62L expression. At Day +120, all 4 patients showed increased frequencies (10.7±4.6%) of CD25+CD127-FOXP3+ regulatory T cells (Treg) within the CD4 population compared to healthy subjects (3.8±0.4%). Expression of CD45RO, CD45RA, CD62L and HLA-DR was variable. By 1 year post-transplant, frequencies of Treg had decreased to levels similar to those in normal subjects. In vitro assays for CD8 and CD4 T cell-mediated alloreactivity (CML/MLR) showed development of long-lasting donor-specific unresponsiveness by 3 months after transplant in Patients 2, 4 and 5, and by 9 months in Patient 1. Responses to 3rd party recovered in all patients after a period of unresponsiveness. In Patient 1, in whom anti-donor CML reactivity declined gradually to become unresponsive by 9 months, depletion of CD4+CD25+ cells revealed a residual anti-donor CML and MLR response at 1year but not at 18 months. In 2 other patients, depletion of CD4+CD25+ cells did not reveal an anti-donor response at time points analyzed from day +122 to 2 years. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, loss of killing activity against donor RTEC was observed post-transplant. The high percentage of Treg recovering early after transplant suggests that they may play a role in initial tolerance induction. This regulatory mechanism may be followed by later deletion of donor-reactive T cells. The variable ability to detect regulation of anti-donor reactivity may reflect the strength of the initial response, as patients with weak pre-transplant anti-donor responses and rapid post-transplant development of donor unresponsiveness did not reveal anti-donor response when Treg were depleted. In addition, infiltration of Treg at the graft site, not revealed by the assays described, might be responsible for tolerance in these patients.

Pentostatin Facilitates Fully MHC-Disparate Murine Mini-Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3539-3539
Author(s):  
Jacopo Mariotti ◽  
Kaitlyn Ryan ◽  
Paul Massey ◽  
Nicole Buxhoeveden ◽  
Jason Foley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Suppression ◽  
Graft Rejection ◽  
Mixed Chimerism ◽  
Post Transplant

Abstract Abstract 3539 Poster Board III-476 Pentostatin has been utilized clinically in combination with irradiation for host conditioning prior to reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, murine models utilizing pentostatin to facilitate engraftment across fully MHC-disparate barriers have not been developed. To address this deficit in murine modeling, we first compared the immunosuppressive and immunodepleting effects of pentostatin (P) plus cyclophosphamide (C) to a regimen of fludarabine (F) plus (C) that we previously described. Cohorts of mice (n=5-10) received a three-day regimen consisting of P alone (1 mg/kg/d), F alone (100 mg/kg/d), C alone (50 mg/kg/d), or combination PC or FC. Combination PC or FC were each more effective at depleting and suppressing splenic T cells than either agent alone (depletion was quantified by flow cytometry; suppression was quantified by cytokine secretion after co-stimulation). The PC and FC regimens were similar in terms of yielding only modest myeloid suppression. However, the PC regimen was more potent in terms of depleting host CD4+ T cells (p<0.01) and CD8+ T cells (p<0.01), and suppressing their function (cytokine values are pg/ml/0.5×106 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-g (13±5 vs. 48±12), IL-2 (59±44 vs. 258±32), IL-4 (34±10 vs. 104±12), and IL-10 (15±3 vs. 34±5). Next, we evaluated whether T cells harvested from PC-treated and FC-treated hosts were also differentially immune suppressed in terms of capacity to mediate an alloreactive host-versus-graft rejection response (HVGR) in vivo when transferred to a secondary host. BALB/c hosts were lethally irradiated (1050 cGy; day -2), reconstituted with host-type T cells from PC- or FC-treated recipients (day -1; 0.1 × 106 T cells transferred), and challenged with fully allogeneic transplant (B6 donor bone marrow, 10 × 106 cells; day 0). In vivo HVGR was quantified on day 7 post-BMT by cytokine capture flow cytometry: absolute number of host CD4+ T cells secreting IFN-g in an allospecific manner was ([x 106/spleen]) 0.02 ± 0.008 in recipients of PC-treated T cells and 1.55 ± 0.39 in recipients of FC-treated cells (p<0.001). Similar results were obtained for allospecific host CD8+ T cells (p<0.001). Our second objective was to characterize the host immune barrier for engraftment after PC treatment. BALB/c mice were treated for 3 days with PC and transplanted with TCD B6 bone marrow. Surprisingly, such PC-treated recipients developed alloreactive T cells in vivo and ultimately rejected the graft. Because the PC-treated hosts were heavily immune depleted at the time of transplantation, we reasoned that failure to engraft might be due to host immune T cell reconstitution after PC therapy. In an experiment performed to characterize the duration of PC-induced immune depletion and suppression, we found that although immune depletion was prolonged, immune suppression was relatively transient. To develop a more immune suppressive regimen, we extended the C therapy to 14 days (50 mg/Kg) and provided a longer interval of pentostatin therapy (administered on days 1, 4, 8, and 12). This 14-day PC regimen yielded CD4+ and CD8+ T cell depletion similar to recipients of a lethal dose of TBI, more durable immune depletion, but again failed to achieve durable immune suppression, therefore resulting in HVGR and ultimate graft rejection. Finally, through intensification of C therapy (to 100 mg/Kg for 14 days), we were identified a PC regimen that was both highly immune depleting and achieved prolonged immune suppression, as defined by host inability to recover T cell IFN-g secretion for a full 14-day period after completion of PC therapy. Finally, our third objective was to determine with this optimized PC regimen might permit the engraftment of MHC disparate, TCD murine allografts. Indeed, using a BALB/c-into-B6 model, we found that mixed chimerism was achieved by day 30 and remained relatively stable through day 90 post-transplant (percent donor chimerism at days 30, 60, and 90 post-transplant were 28 ± 8, 23 ± 9, and 21 ± 7 percent, respectively). At day 90, mixed chimerism in myeloid, T, and B cell subsets was observed in the blood, spleen, and bone marrow compartments. Pentostatin therefore synergizes with cyclophosphamide to deplete, suppress, and limit immune reconstitution of host T cells, thereby allowing engraftment of T cell-depleted allografts across MHC barriers. Disclosures: No relevant conflicts of interest to declare.

Overcoming Rejection of Bone Marrow Allografts by Treg Cells in a Stringent Mouse Model for T Cell Mediated Rejection: Tolerance Induction by Third Party “Off-the-Shelf” Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 576-576
Author(s):  
David Steiner ◽  
Noga Brunicki ◽  
Esther Bachar-Lustig ◽  
Yair Reisner
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Graft Rejection ◽  
Ex Vivo ◽  
Tolerance Induction ◽  
Treg Cells ◽  
Third Party ◽  
Donor Type

Abstract Recent reports have shown that donor or host CD4+CD25+ Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. More recent data suggests that in the context of T cell depleted BM allografting, engraftment was only mildly improved by Treg cells alone, or by Rapamycin (RAPA) alone, but it was markedly enhanced by using Treg cells in conjunction with RAPA. These studies were carried out in a mouse model specifically designed to measure T cell mediated graft rejection. In this model, lethally irradiated (11Gy) C3H mice were infused with 1x104 purified host type T cells (HTC) and were transplanted one day later with 2x106 BM cells from Balb-Nude donors, which are markedly depleted of T cells and do not induce GVHD. Rejection mediated by the HTC is manifested by severe aplasia and lethality within 21 days posttransplant. In 10 independent experiments none of the mice in the irradiation control survived (0/62), the majority of the mice receiving BM survived (58/63) while marked rejection, associated with poor survival (2/62) was found in the group receiving purified HTC prior to the BM transplant. In the present study we further tested in this model whether third party Treg cells could be used instead of donor or host Treg cells to overcome rejection of BM allografts. We initially tested freshly isolated lymph node CD4+CD25+ cells. C3H (H2k) recipients received BM from Balb- Nude (H2d) donors and the Treg cells were obtained from Balb/c or FVB (H2q) donors. As in our previous study, while none of the recipients survived upon treatment with RAPA alone, using third party or donor type Treg cells in conjunction with RAPA led to survival of 9 of 13 and 7 of 10 mice respectively. Thus, the third party fresh Treg cells were as effective as the donor type cells in preventing graft rejection (P&gt;0.05). Considering the low levels of CD4+CD25+ cells in peripheral blood or spleen, new strategies for growing these cells ex-vivo have been developed. Although, Treg cells exhibit low proliferative potential in-vitro upon TCR stimulation, the feasibility of growing mouse or human regulatory cells has been demonstrated mainly using the combination of TCR stimulation (either with an anti-TCR antibody or with allogeneic stimulator cells), costimulatory signals and high doses of IL-2. When tested in the same model, Treg cells ex-vivo expanded by stimulation against 4th party allogeneic cells, exhibited effective enhancement of engraftment of Balb-Nude BM. Thus, in four independent experiments, when assessing treatment with expanded Treg cells, of third party or donor type origin, the survival rate was 19 of 35 (54%) and 25 of 40 (62%) mice, respectively. Again, in both instances the marked potential of Treg cells to overcome T cell mediated rejection was exhibited only when co-administered with RAPA. In conclusion, our data strongly indicate that, at least in the bone marrow transplantation setting, third party Treg cells could afford a new viable ‘off-the-shelf’ source for tolerance induction. The use of third party Treg cells in contrast to donor type cells could allow advanced preparation of a large bank of Treg cells, with all the appropriate quality controls required for cell therapy. Further studies with human Treg cells in-vitro are required to ascertain the potential of third party cells as a valuable source for clinical transplantation.

scholarly journals In vitro inhibition of normal human hematopoiesis by marrow CD3+, CD8+, HLA-DR+, HNK1+ lymphocytes

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1616-1621 ◽  
Author(s):  
G Vinci ◽  
JP Vernant ◽  
M Nakazawa ◽  
M Zohair ◽  
A Katz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Inhibitory Activity ◽  
Colony Growth ◽  
Class Ii ◽  
Hla Class Ii ◽  
Hla Dr ◽  
In Vitro Inhibition

Abstract We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E- C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+- sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co- culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T- cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.

scholarly journals In vitro inhibition of normal human hematopoiesis by marrow CD3+, CD8+, HLA-DR+, HNK1+ lymphocytes

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1616-1621
Author(s):  
G Vinci ◽  
JP Vernant ◽  
M Nakazawa ◽  
M Zohair ◽  
A Katz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Inhibitory Activity ◽  
Colony Growth ◽  
Class Ii ◽  
Hla Class Ii ◽  
Hla Dr ◽  
In Vitro Inhibition

We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E- C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+- sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co- culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T- cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.

scholarly journals CD28 Costimulation Is Required for In Vivo Induction of Peripheral Tolerance in CD8 T Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melanie S. Vacchio ◽  
Richard J. Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tolerance Induction ◽  
Peripheral Tolerance ◽  
Cd8 Cells ◽  
Costimulatory Signal ◽  
Cd28 Costimulation

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y–specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y–specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Impact of Different T Cell Depletion Protocols on Immune Reconstitution Following Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-44
Author(s):  
Amandine Pradier ◽  
Adrien Petitpas ◽  
Anne-Claire Mamez ◽  
Federica Giannotti ◽  
Sarah Morin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Cell Depletion ◽  
Unrelated Donor ◽  
T Cell Depletion ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact

Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapeutic modality for a variety of hematological malignancies and congenital disorders. One of the major complications of the procedure is graft-versus-host-disease (GVHD) initiated by T cells co-administered with the graft. Removal of donor T cells from the graft is a widely employed and effective strategy to prevent GVHD, although its impact on post-transplant immune reconstitution might significantly affect anti-tumor and anti-infectious responses. Several approaches of T cell depletion (TCD) exist, including in vivo depletion using anti-thymocyte globulin (ATG) and/or post-transplant cyclophosphamide (PTCy) as well as in vitro manipulation of the graft. In this work, we analyzed the impact of different T cell depletion strategies on immune reconstitution after allogeneic HSCT. Methods We retrospectively analysed data from 168 patients transplanted between 2015 and 2019 at Geneva University Hospitals. In our center, several methods for TCD are being used, alone or in combination: 1) In vivo T cell depletion using ATG (ATG-Thymoglobulin 7.5 mg/kg or ATG-Fresenius 25 mg/kg); 2) in vitro partial T cell depletion (pTCD) of the graft obtained through in vitro incubation with alemtuzumab (Campath [Genzyme Corporation, Cambridge, MA]), washed before infusion and administered at day 0, followed on day +1 by an add-back of unmanipulated grafts containing about 100 × 106/kg donor T cells. The procedure is followed by donor lymphocyte infusions at incremental doses starting with 1 × 106 CD3/kg at 3 months to all patients who had received pTCD grafts with RIC in the absence of GVHD; 3) post-transplant cyclophosphamide (PTCy; 50 mg/kg) on days 3 and 4 post-HSCT. Absolute counts of CD3, CD4, CD8, CD19 and NK cells measured by flow cytometry during the first year after allogeneic HSCT were analyzed. Measures obtained from patients with mixed donor chimerism or after therapeutic DLI were excluded from the analysis. Cell numbers during time were compared using mixed-effects linear models depending on the TCD. Multivariable analysis was performed taking into account the impact of clinical factors differing between patients groups (patient's age, donor type and conditioning). Results ATG was administered to 77 (46%) patients, 15 (9%) patients received a pTCD graft and 26 (15%) patients received a combination of both ATG and pTCD graft. 24 (14%) patients were treated with PTCy and 26 (15%) patients received a T replete graft. 60% of patients had a reduced intensity conditioning (RIC). 48 (29%) patients received grafts from a sibling identical donor, 94 (56%) from a matched unrelated donor, 13 (8%) from mismatched unrelated donor and 13 (8%) received haploidentical grafts. TCD protocols had no significant impact on CD3 or CD8 T cell reconstitution during the first year post-HSCT (Figure 1). Conversely, CD4 T cells recovery was affected by the ATG/pTCD combination (coefficient ± SE: -67±28, p=0.019) when compared to the T cell replete group (Figure 1). Analysis of data censored for acute or chronic GVHD requiring treatment or relapse revealed a delay of CD4 T cell reconstitution in the ATG and/or pTCD treated groups on (ATG:-79±27, p=0.004; pTCD:-100±43, p=0.022; ATG/pTCD:-110±33, p&lt;0.001). Interestingly, pTCD alone or in combination with ATG resulted in a better reconstitution of NK cells compared to T replete group (pTCD: 152±45, p&lt;0.001; ATG/pTCD: 94±36, p=0.009; Figure 1). A similar effect of pTCD was also observed for B cells (pTCD: 170±48, p&lt;.001; ATG/pTCD: 127±38, p&lt;.001). The effect of pTCD on NK was confirmed when data were censored for GVHD and relapse (pTCD: 132±60, p=0.028; ATG/pTCD: 106±47, p=0.023) while only ATG/pTCD retained a significant impact on B cells (102±49, p=0.037). The use of PTCy did not affect T, NK or B cell reconstitution when compared to the T cell replete group. Conclusion Our results indicate that all TCD protocols with the only exception of PTCy are associated with a delayed recovery of CD4 T cells whereas pTCD of the graft, alone or in combination with ATG, significantly improves NK and B cell reconstitution. Figure 1 Disclosures No relevant conflicts of interest to declare.

Coexistence of Two Functioning T-Cell Repertoires in Healthy Ex-Thalassemics Bearing a Persistent Mixed Chimerism Years After Bone Marrow Transplantation

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3432-3438 ◽  
Author(s):  
Manuela Battaglia ◽  
Marco Andreani ◽  
Marisa Manna ◽  
Sonia Nesci ◽  
Paola Tonucci ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Mixed Chimerism ◽  
Functional Studies ◽  
Repertoire Analysis ◽  
Donor Cells ◽  
Donor Lymphocytes

Bone marrow transplantation (BMT) from an HLA-identical donor is an established therapy to cure homozygous β-thalassemia. Approximately 10% of thalassemic patients developed a persistent mixed chimerism (PMC) after BMT characterized by stable coexistence of host and donor cells in all hematopoietic compartments. Interestingly, in the erythrocytic lineage, close to normal levels of hemoglobin can be observed in the absence of complete donor engraftment. In the lymphocytic lineage, the striking feature is the coexistence of immune cells. This implies a state of tolerance or anergy, raising the issue of immunocompetence of the host. To understand the state of the T cells in PMC, repertoire analysis and functional studies were performed on cells from 3 ex-thalassemics. Repertoire analysis showed a profound skewing. This was due to an expansion of some T cells and not to a collapse of the repertoire, because phytohemagglutinin stimulation showed the presence of a complex repertoire. The immunocompetence of the chimeric immune systems was further established by showing responses to alloantigens and recall antigens in vitro. Both host and donor lymphocytes were observed in the cultures. These data suggest that the expanded T cells play a role in specific tolerance while allowing a normal immune status in these patients.

Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2084-2093 ◽  
Author(s):  
Alexander D. McLellan ◽  
Michaela Kapp ◽  
Andreas Eggert ◽  
Christian Linden ◽  
Ursula Bommhardt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Antigen Expression ◽  
Cell Receptor ◽  
In Vitro Assays ◽  
Peptide Complexes ◽  
Marginal Zones

Abstract Mouse spleen contains CD4+, CD8α+, and CD4−/CD8α− dendritic cells (DCs) in a 2:1:1 ratio. An analysis of 70 surface and cytoplasmic antigens revealed several differences in antigen expression between the 3 subsets. Notably, the Birbeck granule–associated Langerin antigen, as well as CD103 (the mouse homologue of the rat DC marker OX62), were specifically expressed by the CD8α+ DC subset. All DC types were apparent in the T-cell areas as well as in the splenic marginal zones and showed similar migratory capacity in collagen lattices. The 3 DC subtypes stimulated allogeneic CD4+ T cells comparably. However, CD8α+ DCs were very weak stimulators of resting or activated allogeneic CD8+ T cells, even at high stimulator-to-responder ratios, although this defect could be overcome under optimal DC/T cell ratios and peptide concentrations using CD8+ F5 T-cell receptor (TCR)–transgenic T cells. CD8α− or CD8α+DCs presented alloantigens with the same efficiency for lysis by cytotoxic T lymphocytes (CTLs), and their turnover rate of class I–peptide complexes was similar, thus neither an inability to present, nor rapid loss of antigenic complexes from CD8α DCs was responsible for the low allostimulatory capacity of CD8α+ DCs in vitro. Surprisingly, both CD8α+ DCs and CD4−/CD8− DCs efficiently primed minor histocompatibility (H-Y male antigen) cytotoxicity following intravenous injection, whereas CD4+ DCs were weak inducers of CTLs. Thus, the inability of CD8α+ DCs to stimulate CD8+ T cells is limited to certain in vitro assays that must lack certain enhancing signals present during in vivo interaction between CD8α+ DCs and CD8+ T cells.

Immunoregulatory Effects of Allogeneic Mixed Chimerism Induced by T-Cell Depleted, Nonmyeloablative Bone Marrow Transplantation on Chronic Inflammatory Arthritis and Autoimmunity Developed in Interleukin-1 Receptor Antagonist-Deficient Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1277-1277
Author(s):  
Seok-Goo Cho ◽  
Min-Chung Park ◽  
So-Youn Min ◽  
Young-Gyu Cho ◽  
Seok Lee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Marrow Transplantation ◽  
Inflammatory Arthritis ◽  
Interleukin 1 ◽  
Mixed Chimerism ◽  
Control Groups ◽  
Chronic Inflammatory Arthritis

Abstract Objective: To investigate the immunoregulatory effects of allogeneic mixed chimerism induced by T-cell depleted, nonmyeloablative bone marrow transplantation (TCD-NMT) on chronic inflammatory arthritis and autoimmunity developed in interleukin-1 receptor antagonist-deficient (IL-1Ra−/ −) mice. Methods: IL-1Ra−/ − mice (H-2kd) were treated with anti-asialoGM1 Ab, TBI 500 cGy, and TCD-NMT from C57BL/6 mice (H-2kb). Engraftment and chimerism were evaluated on peripheral blood (PB), lymph node, and spleen by multi-color flow cytometry. The severity of arthritis was evaluated by clinical score and histopathology. IgG1 and IgG2a subtype of anti-type II collagen (CII) were measured in PB samples. After T cells were stimulated with CII, ovalbumin, and phytohemagglutinin, T-cell proliferation response and cytokines production (INF-g, TNF-a, IL-10, and IL-17) in culture supernatant were assayed. Results: All the transplanted IL-1Ra mice showed marked improvement of arthritis within 3 weeks after transplantation as well as successful induction of mixed chimerism. Mice in mixed chimerism showed higher level of anti-CII IgG1 and lower level of anti-CII IgG2a and weaker T cell proliferative response than in control groups, such as no-treatment and conditioning only without BM rescue. In mixed chimera, INF-g, TNF-a and IL-17 production from CII-stimulated T cells was significantly suppressed and IL-10 production was significantly increased as compared to the control groups. Conclusion: These observations indicate that the introduction of allogeneic mixed chimerism has a strong immunoregulatroy potential to correct established chronic inflammatory arthritis and autoimmunity originating from dysregulated proinflammatory cytokine network.

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