A Murine Xenograft Model for Human T Cell Mediated Graft Versus Host Disease.

Abstract Murine xenograft models of human T cell (HuT) mediated graft-versus-host-disease (GvHD) are of potential value but limited by poor engraftment and low and variable incidence of clinical GvHD even after injection of >108HuT cells. The NOD SCID β2M null mice (β2 mice) lack macrophage activity, T, B and NK cells and represent an improved target for HuT cell expansion and activation compared to other immunodeficient mouse models. To induce GvHD, sublethally irradiated β2 mice were injected intravenously via the tail vein (iv) or retroorbitally (ro) with human peripheral blood mononuclear cells (huPBMC) or purified HuT (98% purity). β2 mice conditioned with 250cGy and injected iv with huPBMC (107T cells;n=4) or HuT (0.5–2x107T cells;n=28) failed to engraft and did not develop GvHD. In contrast, β2 mice conditioned with 250cGy and injected ro with huPBMC (107T cells;n=11) or HuT cells (107;n=14) exhibited 19% HuT engraftment 2–3 weeks post-infusion and developed weight loss (>20%) consistent with lethal GvHD, with an overall survival of 82% and 21%, respectively, at 5 weeks (p=0.006). Addition of IL-2 (3x105 IU IP/TIW) had no effect on T cell expansion or GvHD. FACS analysis demonstrated HuT infiltration in the spleen (46%), liver (60%), lung (49%), kidney (40%), and bone marrow (11%). Histological analysis showed an extensive and diffuse accumulation of immature lymphocytes in the spleen, thymus and lymph nodes, and a perivascular infiltration in the lung, liver, kidney but not in the skin or gut. The immunohistochemestry confirmed that these cells were HuT (human CD45+ and CD3+). Furthermore, we observed a 10–15 fold increase in the expression of T cell activation markers CD25, CD30, and CD69 in both the peripheral blood and tissues, compared with naive T cells or T cells from mice that did not develop GvHD. We also evaluated the levels of various human cytokines in the serum of the β2 mice using a cytometric bead array multiplex assay. On day 10 after the injection of HuT and before the start of any clinical sign of GvHD, mice that went on to develop lethal GvHD had 90 times higher levels of IFNγ in serum (>5000pg/ml) compared to mice that did not develop GVHD (<62 pg/ml) (p=0.003). Interestingly both had nearly identical numbers of HuT/ul in blood (32+39 and 33+41 HuT/ul) on day 10. We also observed a significant increase in human IL-10 levels and TNFα in mice that developed GvHD. Mice that developed lethal GvHD had a 70 fold increase of HuT/ul in the 3rd week (1550 versus 22/ul p<0.003). We improved this model by depletion of murine macrophages using clodronate-containing liposomes (clod) administered iv before the HuT injections. Mice injected with 5x106HuT with clod developed lethal GvHD (3/3) on day 15.7+1.5, with 107HuT (3/3) on day 10.3+5.4 and mice injected with 107HuT without clod on day 13.4+5.4 (8/12)(p<0.05). In contrast, RAG2 γ −/ − mice (RAG2) treated in identical fashion to the β2 mice failed to engraft HuT after both iv and ro injection (350cGy). Both increasing radiation doses (350 to 600cGy) and/or the addition of clod iv resulted in significantly enhanced engraftment of HuT and lethal GvHD. CD4/CD8 ratio of HuT cells expanding in RAG2 mice was <1 in sharp contrast to the β2 mice where the ratio was >2.5. Conclusion: NOD-SCID-β2M null xenotransplant model is uniquely permissive for human T cell expansion after sublethal radiation and may be used as a preclinical platform to study the impact of ex-vivo manipulation and genetic modification of human T cell as GvHD.

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Homeostatic expansion and repertoire regeneration of donor T cells during graft versus host disease is constrained by the host environment

Blood ◽

10.1182/blood-2006-12-061713 ◽

2007 ◽

Vol 109 (12) ◽

pp. 5502-5510 ◽

Cited By ~ 13

Author(s):

Jack Gorski ◽

Xiao Chen ◽

Mariya Gendelman ◽

Maryam Yassai ◽

Ashley Krueger ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Cell Expansion ◽

Host Disease ◽

Host Environment ◽

Graft Versus Host ◽

T Cell Reconstitution ◽

T Cell Expansion ◽

Cell Defect

Abstract Graft versus host disease (GVHD) typically results in impaired T-cell reconstitution characterized by lymphopenia and repertoire skewing. One of the major causes of inadequate T-cell reconstitution is that T-cell survival and expansion in the periphery are impaired. In this report, we have performed adoptive transfer studies to determine whether the quantitative reduction in T-cell numbers is due to an intrinsic T-cell defect or whether the environmental milieu deleteriously affects T-cell expansion. These studies demonstrate that T cells obtained from animals with graft-versus-host disease (GVHD) are capable of significant expansion and renormalization of an inverted CD4/CD8 ratio when they are removed from this environment. Moreover, these cells can generate complex T-cell repertoires early after transplantation and are functionally competent to respond to third-party alloantigens. Our data indicate that T cells from mice undergoing GVHD can respond to homeostatic signals in the periphery and are not intrinsically compromised once they are removed from the GVHD environment. We thereby conclude that the host environment and not an intrinsic T-cell defect is primarily responsible for the lack of effective T-cell expansion and diversification of complex T-cell repertoires that occurs during GVHD.

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Immunohistology and immunocytology of human T-cell chimerism and graft- versus-host disease in SCID mice

Blood ◽

10.1182/blood.v81.12.3440.3440 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3440-3448 ◽

Cited By ~ 54

Author(s):

G Hoffmann-Fezer ◽

C Gall ◽

U Zengerle ◽

B Kranz ◽

S Thierfelder

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocyte ◽

Graft Versus Host Disease ◽

Scid Mice ◽

White Pulp ◽

Activation Markers ◽

Host Disease ◽

Graft Versus Host ◽

Human T Cell

Abstract Surprisingly little graft-versus-host disease (GVHD) has been observed in severe combined immunodeficient (SCID) mice injected intraperitoneally (IP) with human blood lymphocytes (hu-PBL-SCID), which raised the question as to whether GVHD in such a distant species is sporadic or suppressed because of immunologic reasons. After screening for blood T-cell chimerism, we hereby describe generalized lethal xenogeneic human GVHD in unconditioned SCID chimeras, which resembles GVHD in SCID mice injected with allogeneic lymphocytes. We adapted an immunocytochemical slide method for minute cell numbers, which allowed us to follow, by multimarker phenotyping of weekly mouse- tail bleeds, the chimeric status of 100 hu-PBL-SCID injected with 10(7) or 10(8) hu-PBL of Epstein-Barr virus- (EBV-) donors. More than half of the mice showed no or less than 2% T cells. However, 13% to 21% developed substantial blood T-lymphocyte chimerism (10% to 80% human CD+ cells) and high mortality. Immunohistology showed more human CD8+ than CD4+ T cells in the splenic white pulp. The cells developed HLA-DR activation markers and infiltrated the red pulp where human B cells also appeared. Expression of activation and proliferation markers increased within 5 to 6 weeks. Many human CD3+ cells were also found in the portal triads of the liver and in the lung, pancreas, and kidney. The thymus also became heavily infiltrated. The intestines and skin of hu-PBL-SCID were less infiltrated by donor cells than in SCID with allogeneic GVHD. The tongue contained almost no human T cells. Our data show that a relatively low overall incidence of human xenogeneic GVHD, even when high numbers of human PBL are injected, is the consequence of a dichotomy between mice with no or transient T-cell chimerism and a minority of mice with high-blood T-lymphocyte chimerism and GVHD mortality.

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Donor Dual TCR T Cells Preferentially Expand and Mediate Pathologic Alloreactivity in Acute Graft Versus Host Disease

Blood ◽

10.1182/blood.v118.21.1972.1972 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1972-1972

Author(s):

Gerald P. Morris ◽

Geoffrey L Uy ◽

David L Donermeyer ◽

Paul M Allen ◽

John F. DiPersio

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Thymic Selection ◽

Cell Repertoire ◽

Host Disease ◽

Graft Versus Host

Abstract Abstract 1972 The nature of the T cell repertoire mediating pathologic in vivo alloreactivity is an important question for understanding the development of acute graft-versus-host disease (aGvHD) following clinical allogeneic transplantation. We have previously demonstrated that the small proportion of T cells that naturally express 2 T cell receptors (TCR) as a consequence of incomplete TCRa allelic exclusion during thymic development contribute disproportionately to the alloreactive T cell repertoire, both in vitro and in vivo in a mouse model of graft versus host disease (GvHD) (J. Immunol., 182:6639, 2009). Here, we extend these findings to human biology, examining dual TCR T cells from healthy volunteer donors (n = 12) and patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) (n = 19). Peripheral blood was collected at day 30 post-HSCT or at the time of presentation with symptomatic acute GvHD. Dual TCR T cells were measured in peripheral blood by pair-wise staining with 3 commercially-available and 2 novel TCRa mAbs. Dual TCR T cells were consistently and significantly expanded in patients with symptomatic aGvHD, representing 5.3±3.8 % of peripheral T cells, compared to 1.7±0.8 % of T cells in healthy controls (p < 0.005) (Figure 1). There was no correlation between dual TCR T cell frequency and GvHD severity. Furthermore, sequential analysis of peripheral blood in 2 patients demonstrated expansion of dual TCR T cells concurrent with the development of aGvHD (Figure 2). Dual TCR T cells from patients with symptomatic aGvHD demonstrated increased expression of CD69 as compared to T cells expressing a single TCR, indicative of preferential activation of dual TCR T cells during aGvHD. Similarly, dual TCR T cells isolated from patients with symptomatic aGvHD demonstrate increased production of IFN-g ex vivo, indicative of the ability to mediate pathogenic alloreactive responses. Dual TCR T cell clones isolated from healthy donors and patients post-HSCT by single cell FACS sorting demonstrate alloreactive responses against a range of allogeneic cell lines in vitro. We propose that the increased alloreactivity of dual TCR T cells results from the less stringent thymic selection for secondary TCR, and thus provides a link between thymic selection, the TCR repertoire, and alloreactivity. These findings may lead to simple ways of phenotypically identifying specific T cells predisposed to inducing aGvHD for subsequent examination of T cell repertoires and functional studies. Furthermore, these data suggest that dual TCR T cells represent a potential predictive biomarker for aGvHD and a potential target for selective T cell depletion in HSCT. Disclosures: No relevant conflicts of interest to declare.

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Ex vivo Fucosylation Enhances Regulatory T Cell Anti-Graft Versus Host Disease Potency in Mice

Blood ◽

10.1182/blood.v124.21.660.660 ◽

2014 ◽

Vol 124 (21) ◽

pp. 660-660

Author(s):

Simrit Parmar ◽

Xiaoying Liu ◽

Yvon Eric ◽

Patrick zweidler-mcCay ◽

Nina Shah ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Preventive Strategy ◽

Adoptive Therapy ◽

Host Disease ◽

Graft Versus Host

Abstract Emerging preclinical data indicate a potential therapeutic use of regulatory T cells (Tregs) to suppress or eliminate graft versus host disease (GVHD). Adoptive therapy with Tregs has been examined as a prophylactic strategy for GVHD. Thus far, 1:1 ratio of Tregs to conventional T cells (Tcons) has been required to prevent GVHD, and such high targeted cell doses are hard to achieve in a clinical setting due to variability in Treg frequency and expansion potential. As a result, novel strategies are needed to generate clinically-effective Treg products. Additionally, since the ability of Tregs to enter inflamed tissues has been shown to be critically dependent on their ability to bind E- and P-selectins, we sought to exploit this pathway to improve Treg homing. Incubation of culture expanded Cord Blood (CB) Tregswith fucosyltransferase-VI (FTVI) increased the degree of fucosylation from 8% to 64%. Importantly, fucosylated Tregs are able to suppress T cell proliferation in an in vitro allogeneic mixed lymphocyte assay and show preferential increase in the E-selectin binding ability when compared to untreated Tregs. We injected 10e6 Tregs vs. 10e6 fucosylated Tregs followed by 10e7 peripheral blood mononuclear cells in the sublethally irradiated NOD/SCID gamma null (NSG) xenogenic GVHD mouse model, and recipients of untreated Tregs showed weight loss as early as day 6 whereas fucosylated Treg recipients retained weight until day 12 (Fig 1). Fucosylated Tregs were detected in the peripheral blood of mice until day 31 as opposed to day 12 for untreated Tregs. As compared to recipients of untreated Tregs, lower numbers of GVH-inducing allogeneic T-cells were detected in the secondary lymphoid organs in the fucosylated Treg recipients (Table 1). The overall survival of fucosylated Treg recipients was significantly superior to that of untreated Treg recipients (Fig 2). We conclude that prophylactic treatment with fucosylated Tregs prevents GVHD and improves survival in a xenogenic mouse model at doses that are i) at less than 1:1 ratio with conventional T-cells (Tcon) and ii) are lower as compared to untreated Tregs. We believe that by overcoming the 1:1 dose requirement of Tregs: Tcons, we will be able to effectively translate Treg based adoptive therapy into the clinic for the prevention of GVHD. Establishing fucosylated Tregs as a preventive strategy for GVHD will result in a major breakthrough in the field of stem cell transplantation eliminating a major life threatening outcome of the therapy. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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Immunohistology and immunocytology of human T-cell chimerism and graft- versus-host disease in SCID mice

Blood ◽

10.1182/blood.v81.12.3440.bloodjournal81123440 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3440-3448 ◽

Cited By ~ 1

Author(s):

G Hoffmann-Fezer ◽

C Gall ◽

U Zengerle ◽

B Kranz ◽

S Thierfelder

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocyte ◽

Graft Versus Host Disease ◽

Scid Mice ◽

White Pulp ◽

Activation Markers ◽

Host Disease ◽

Graft Versus Host ◽

Human T Cell

Surprisingly little graft-versus-host disease (GVHD) has been observed in severe combined immunodeficient (SCID) mice injected intraperitoneally (IP) with human blood lymphocytes (hu-PBL-SCID), which raised the question as to whether GVHD in such a distant species is sporadic or suppressed because of immunologic reasons. After screening for blood T-cell chimerism, we hereby describe generalized lethal xenogeneic human GVHD in unconditioned SCID chimeras, which resembles GVHD in SCID mice injected with allogeneic lymphocytes. We adapted an immunocytochemical slide method for minute cell numbers, which allowed us to follow, by multimarker phenotyping of weekly mouse- tail bleeds, the chimeric status of 100 hu-PBL-SCID injected with 10(7) or 10(8) hu-PBL of Epstein-Barr virus- (EBV-) donors. More than half of the mice showed no or less than 2% T cells. However, 13% to 21% developed substantial blood T-lymphocyte chimerism (10% to 80% human CD+ cells) and high mortality. Immunohistology showed more human CD8+ than CD4+ T cells in the splenic white pulp. The cells developed HLA-DR activation markers and infiltrated the red pulp where human B cells also appeared. Expression of activation and proliferation markers increased within 5 to 6 weeks. Many human CD3+ cells were also found in the portal triads of the liver and in the lung, pancreas, and kidney. The thymus also became heavily infiltrated. The intestines and skin of hu-PBL-SCID were less infiltrated by donor cells than in SCID with allogeneic GVHD. The tongue contained almost no human T cells. Our data show that a relatively low overall incidence of human xenogeneic GVHD, even when high numbers of human PBL are injected, is the consequence of a dichotomy between mice with no or transient T-cell chimerism and a minority of mice with high-blood T-lymphocyte chimerism and GVHD mortality.

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