Host Environment Dictates the Outcome Following Transfer of Graft-Versus-Host Reactive Effector/Memory T Cells.

Abstract Donor leucocyte infusions (DLI) given to established mixed chimeras (MC) can eliminate normal and malignant hematopoietic cells without causing graft-versus-host disease (GVHD). Identical DLI transferred immediately following lethal irradiation lead to severe GVHD. In contrast to freshly irradiated mice developing GVHD, established MC receiving DLI show delayed kinetics of effector/memory T cell (TE/M) expansion and a distinct distribution with no accumulation of cells in the gut. Lethal irradiation is associated with tissue injury, altered chemokine expression and cytokine dysregulation, and resolution of these changes is important in limiting GVHD following delayed DLI. In an attempt to define the respective roles of ‘intrinsic’ T cell versus ‘extrinsic’ environmental factors in shaping the character of the GVH response elicited, TE/M developing following GVHD induction were transferred to established MC. Conversely, TE/M developing following delayed DLI to MC were transferred to lethally-irradiated 2° recipients. In the first series of experiments, we transferred 1 x 107 C57BL/6 (B6) splenocytes and 5 x 106 B6 T-cell depleted bone marrow (TCD BM) to lethally-irradiated BALB/c recipients. On day 4 following BMT, when GVH-reactive T cell activation and expansion was maximal, 3 x 106 nylon wool-passaged TE/M derived from recipient spleens (equivalent to 1 recipient spleen to 1 secondary recipient) were transferred to established B6 → BALB/c MC or to lethally irradiated BALB/c 2° recipients. The latter mice received B6 TCD BM to prevent death from aplasia. Secondary established MC recipients were resistant to GVHD whereas the lethally-irradiated 2° recipients developed severe, lethal GVHD (median survival time-MST- mixed chimeras >100d versus irradiated mice 52d, p<0.0001). Histological studies confirmed that 4/5 lethally-irradiated 2° recipients developed colitis versus 0/4 MC. TE/M derived from GVHD mice did not increase donor chimerism in 2° MC recipients. In reciprocal experiments, we transferred 2.5 x 107 B6 CD45.1 splenocytes to established B6 CD45.2 → B6D2F1 MC. This number of splenocytes represents the dose required to convert 100% of MC to full donor chimerism without induction of lethal GVHD. On day 12 post-DLI, when GVH-reactive T-cell expansion was maximal, a cohort of recipient mice was sacrificed and splenocytes harvested. On the basis of 1 recipient spleen to 1 secondary recipient, 2.7 x 107 nylon wool-passaged lymphocytes were transferred to 2° freshly-irradiated B6D2F1 recipients together with B6 TCD BM. In parallel, we depleted host-derived cells by sorting for DLI-derived CD45.1+ lymphocytes and transferred these cells on the basis of 1 recipient to 1 2° recipient (7 x 106 lymphocytes) to a second cohort of lethally-irradiated recipients. After a lag-period of about 20 days, 2° recipients of non-fractionated cells lost >10% weight and developed clinical signs of mild GVHD, but then showed almost complete recovery by day 60 post-BMT. In sharp contrast, after a similar lag period, 2° recipients of CD45.1+-selected lymphocytes developed severe lethal GVHD (MST CD45.1+ cells 55d vs >100d non-fractionated cells, p<0.0005). In conclusion, these findings suggest that the host environment strongly influences the capacity of TE/M to induce GVHD. Furthermore, host-derived cells pre-existing in or developing following delayed DLI, reduce the potential of TE/M to induce GVHD.

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The MEK Inhibitor Trametinib Enhances Diverse T Cell Reconstitution with Suppressing Xenogeneic Graft-Versus-Host-Disease

Blood ◽

10.1182/blood-2019-128913 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1929-1929

Author(s):

Hidekazu Itamura ◽

Hiroyuki Muranushi ◽

Takero Shindo ◽

Kazutaka Kitaura ◽

Seiji Okada ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Mek Inhibitor ◽

Effector Memory ◽

Graft Versus Host ◽

T Cell Reconstitution ◽

Tcr Repertoire ◽

Repertoire Diversity ◽

Equity Ownership

Introduction: Early immune reconstitution without severe graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We showed that MEK inhibitors suppress GVHD but retain antiviral immunity and graft-versus-tumor (GVT) effects (Shindo, Blood2013; Itamura, Shindo, JCI Insight2016). Furthermore, we have shown that they attenuate graft rejection but spare thymic function following rat lung transplantation (Takahagi, Shindo, Am J Respir Cell Mol Biol2019). Here we analyzed their effects on human polyclonal T cell reconstitution in xenogeneic transplant by evaluating T-cell receptor (TCR) repertoire diversity. Methods: As a xenogeneic GVHD model, human PBMCs were infused to NOD/Scid/JAK3null mice, immunodeficient mice lacking T/B/NK cells, after total body irradiation. Vehicle, tacrolimus, or the MEK inhibitor trametinib was administered from day 0 through 28 or day 15 through 28. Human TCR repertoire diversity was evaluated by an adapter ligation PCR method with next generation sequencing (Shindo, Oncoimmunol2018) in the liver, lung, and spleen. The assignment and frequencies of TCRαV/J clones were determined at the single-cell level. Their diversity and clonality were evaluated by Inv. Simpson's index 1/λ. Results: Trametinib prolonged their survival compared with vehicle (median survival: 88 vs 46 days, p<0.05). It enhanced engraftment of human leukocytes in peripheral blood (human CD45+cells: 11.0 vs 2.5%), but prevented their infiltration into the lung (human CD45+cells on day 60: 1.5 vs 6.5%). Treatment with vehicle resulted in skewed TCR repertoire with limited clones in the spleen, liver and lung. Interestingly, expansion of one specific clone (TRAV20/J10) was commonly observed, which might reflect the GVHD-inducing pathological clone (Fig. 1: 3D graphs show the frequencies of TCRαV/J clones). However, trametinib enabled diverse and polyclonal T cell engraftment without the TRAV20/J10 clone. While CD4+and CD8+T cells within injected human PBMCs mainly consisted of naïve (CD45RA+CD27+) and central memory (CD45RA-CD27+) T cells, infiltrating T cells in each organ showed effector memory (CD45RA-CD27-) T cell phenotype. Of note, CD8+T cells in the bone marrow, spleen, and lung of trametinib-treated recipients showed reduced effector memory T cells (CD45RA-CD27-) compared with vehicle-treated mice at day 28 (bone marrow 21.7 vs 74.7%, p<0.01; spleen 66.3 vs 88.7%, p<0.05; lung 33.0 vs 72.5%, p<0.05), which indicating that MEK inhibition suppresses functional differentiation of human T cells in vivo. Furthermore, trametinib treatment from day 14 to 28 still ameliorated clinical GVHD score, and maintained polyclonal T cell repertoire. Conclusions:GVHD can be characterized with skewed TCR repertoire diversity and expansion of pathological T cell clones in the target tissues. Trametinib suppresses GVHD but maintains polyclonal T cell reconstitution, even in established GVHD. These results explain the facts that MEK inhibitors separate GVHD from GVT effects/antimicrobial immunity. Furthermore, MEK inhibition enhances immune reconstitution after allo-HSCT, which would avoid post-transplant complications. Disclosures Shindo: Novartis: Research Funding. Kitaura:Repertoire Genesis Inc.: Employment. Okada:Bristol-Myers Squibb: Research Funding; Japan Agency for Medical Research and Development: Research Funding. Shin-I:BITS Co., Ltd: Equity Ownership. Suzuki:Repertoire Genesis Inc.: Equity Ownership. Takaori-Kondo:Celgene: Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ono: Research Funding; Takeda: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Janssen: Honoraria; Pfizer: Honoraria. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

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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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Dendritic cell–activated CD44hiCD8+ T cells are defective in mediating acute graft-versus-host disease but retain graft-versus-leukemia activity

Blood ◽

10.1182/blood-2003-09-3135 ◽

2004 ◽

Vol 103 (10) ◽

pp. 3970-3978 ◽

Cited By ~ 76

Author(s):

Yi Zhang ◽

Gerard Joe ◽

Jiang Zhu ◽

Richard Carroll ◽

Bruce Levine ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Dendritic Cell ◽

Graft Versus Host Disease ◽

T Cell Subsets ◽

Effector Memory ◽

Antitumor Effects ◽

Host Disease ◽

Graft Versus Host

Abstract Graft versus host disease (GVHD) is triggered by host antigen-presenting cells (APCs) that activate donor T cells to proliferate and differentiate, but which APC-activated donor T-cell subsets mediate GVHD versus beneficial antitumor effects is not known. Using a CD8+ T cell–dependent mouse model of human GVHD, we found that host dendritic cell (DC)–induced CD44hiCD8+ effector/memory T cells were functionally defective in inducing GVHD, whereas CD44loCD8+ naive phenotype T cells were extremely potent GVHD inducers. Depletion of CD44loCD8+ T cells from host DC-stimulated T cells before transplantation prevented GVHD without impairing their antitumor activity in vivo. Compared with CD44loCD8+ T cells, CD44hiCD8+ T cells expressed high levels of Fas and were efficiently deleted in vivo following transplantation. These results suggest that ex vivo allogeneic DC stimulation of donor CD8+ T cells may be useful for the prevention of GVHD and for optimizing antitumor therapies in vivo.

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In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets

Blood ◽

10.1182/blood-2005-02-0509 ◽

2005 ◽

Vol 106 (3) ◽

pp. 1113-1122 ◽

Cited By ~ 229

Author(s):

Andreas Beilhack ◽

Stephan Schulz ◽

Jeanette Baker ◽

Georg F. Beilhack ◽

Courtney B. Wieland ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Lymphoid Organs ◽

T Cell Subsets ◽

Effector Memory ◽

Host Disease ◽

Graft Versus Host ◽

Secondary Lymphoid Organs

AbstractGraft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (TEM) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.

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Impaired Effector Memory T-Cell Regulation Facilitates Graft Versus Host Disease in CCR7-Deficient Bone Marrow Transplant Chimeras

Transplantation Journal ◽

10.1097/tp.0b013e3181b241df ◽

2009 ◽

Vol 88 (5) ◽

pp. 631-639 ◽

Cited By ~ 5

Author(s):

Saskia C. A. de Jager ◽

Kirsten Canté-Barrett ◽

Ilze Bot ◽

Cathrine Husberg ◽

Gijs H. van Puijvelde ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Bone Marrow Transplant ◽

Graft Versus Host Disease ◽

Marrow Transplant ◽

Effector Memory ◽

Cell Regulation ◽

Graft Versus Host ◽

Memory T Cell ◽

T Cell Regulation

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Proliferation, Expansion, Effector Differentiation and Survival of GVH-Reactive T Cells Following Delayed DLI to Mixed Chimeras.

Blood ◽

10.1182/blood.v104.11.594.594 ◽

2004 ◽

Vol 104 (11) ◽

pp. 594-594

Author(s):

Ronjon Chakraverty ◽

Jennifer Buchli ◽

Guiling Zhao ◽

Megan Sykes

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Expansion ◽

T Cell Responses ◽

Cd8 T Cell ◽

Cell Responses ◽

Mixed Chimeras ◽

Kinetics Of ◽

Lethal Irradiation

Abstract Donor leucocyte infusions (DLI) given to established mixed chimeras (MC) can eliminate normal and malignant hematopoietic cells without causing graft-versus-host disease (GVHD). DLI given immediately following lethal irradiation lead to severe GVHD. We examined the proliferation, expansion, differentiation and survival of GVH-reactive T cells following delayed DLI and compared the outcomes to those observed when identical DLI were administered early following lethal irradiation (TBI). MC recipients were prepared by TBI of BALB/c mice and reconstitution with mixed BALB/c and CD45.2 C57BL/6 (B6) T-cell depleted bone marrow (TCD BM). 10 weeks later, we transferred 1 x 107 CD45.1 B6 and 5 x 106 2C transgenic (tg) splenocytes. CD8+ T cells from 2C tg mice bear TCR specific for recipient class I MHC Ld. Polyclonal DLI-derived CD4+/CD8+ T cell responses were monitored by gating on CD45.1+ events and clonal 2C CD8+ T cell responses tracked using a clonotypic marker. For comparison, identical DLI together with TCD BM was administered to freshly irradiated BALB/c or B6 CD45.2 syngeneic recipients. MC recipients of delayed DLI developed a GVH reaction, as indicated by increases in donor chimerism, but no GVHD. In contrast, allogeneic recipients receiving DLI immediately following TBI developed lethal GVHD (median survival 32d vs. >100d post-delayed DLI, p<0.0001). By day 6, donor CD4+/CD8+ T cells had undergone almost equivalent proliferation as monitored by CFSE-dilution in MC and TBI allogeneic recipients. However, the kinetics and distribution of donor T cell expansion were distinct. Following delayed DLI, marked expansion of donor CD4+ cells (peak day 10) preceded expansion of CD8+ cells (peak day 13) in the spleen, with less accumulation in the lymph nodes, BM, liver and lung, and no accumulation in the gut. Histology revealed transient, mild lymphocytic infiltrates within the lung/liver but no evidence of colitis. In contrast, the kinetics of donor CD4+/CD8+ T cell expansion were more rapid in freshly irradiated recipients with CD4+/CD8+ responses peaking on day 4–7. The distribution was also different with major increases in donor CD4+/CD8+ numbers in the gut but less accumulation in the spleen. Histology confirmed severe colitis. The kinetics of proliferation, expansion and distribution of tg 2C CD8+ T cells showed a similar pattern to the polyclonal donor CD8+ T cell population. Following delayed DLI, 2C CD8+ T cells acquired a memory/activation phenotype (CD44hi, CD45RBlo, CD62Llo, CD49d+, CD27+) with similar kinetics to those observed in TBI mice developing GVHD. Despite the marked differences in trafficking to the gut, 2C CD8+ T cells in both groups of allogeneic recipients expressed equivalent levels of the gut homing receptor, α4β7. However, we also observed important differences: 1) IL-2Rαexpression was absent on 2C CD8+ T cells following delayed DLI, but was expressed at high levels (>50% by day +3) in freshly irradiated DLI recipients; 2) 2C CD8+ T cells showed greater reductions in expression of IL-7Rαfollowing delayed DLI; and 3) high rates of 2C CD8+ T cell apoptosis, as indicated by annexin V staining, were observed following delayed DLI with absolute numbers up to 6-fold greater than in TBI recipients. Thus, in contrast to responses in freshly irradiated mice that develop GVHD, GVH reactions induced by delayed DLI are characterized by delayed kinetics, a distinct distribution, marked apoptosis and reduced expression of cytokine receptors important for CD8+ T-cell survival.

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Clonal Expansion of Graft-Versus-Host Reactive CD8+ T Cells Is Dissociated from Full Effector Differentiation Following Delayed DLI.

Blood ◽

10.1182/blood.v110.11.2170.2170 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2170-2170

Author(s):

Ronjon Chakraverty ◽

Barry Flutter ◽

Hyeon-Seok Eom ◽

Farnaz Fallah-Arani ◽

Guiling Zhao ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Population ◽

Cd8 T Cells ◽

Granzyme B ◽

Cd8 T Cell ◽

Host Environment ◽

Graft Versus Host ◽

Donor T Cells ◽

Ifn Γ

Abstract Extrinsic factors within the host environment are crucial in determining recruitment of graft-versus-host (GVH)-reactive T cells to peripheral tissues and the capacity of these cells to induce graft-versus-host disease (GVHD). In this study, we have examined how the host environment influences graft-versus-leukemia (GVL) activity. Transfer of small numbers of allogeneic T cells to freshly irradiated (TBI allo) mice induces both GVL and GVHD, whereas transfer of much higher numbers of T cells to established (>8 weeks) mixed chimeras (MC) can induce GVL without GVHD. Using an EL4 tumor protection assay and low doses of B10.A splenocytes (3 × 10e6, a dose 1 log lower than normally transferred to MC to induce GVL), we observed that tumor-free survival of recipient mice was greater following transfer to TBI allo B6 recipients than in B6 + B10.A → B6 MC. To determine the mechanisms for this disparity, we used a BALB/c recipient/B6 donor strain combination, in which we could track the distribution of donor T cells within secondary lymphoid organs and bone marrow (BM) following transfer to TBI allo or MC recipients. Despite similar expansions within the spleen, there was a significant delay in the accumulation of polyclonal donor T cells (B6 CD45.1) and transgenic 2C GVH-reactive CD8+ T-cells (bearing TCR specific for recipient antigen) in the BM of MC compared to TBI allo mice. Moreover, in vivo cytotoxicity of host B cell targets occurred rapidly and was virtually complete in TBI allo recipients, but was absent in MC even at late time points. To evaluate the acquisition of effector functions in a clonal GVH-reactive CD8+ T cell population, we sorted 2C T cells at intervals following transfer and performed quantitative RT-PCR of molecules linked to effector differentiation. Strikingly, transcription of IFN-γ, granzyme B and TNF-α was significantly higher in cells derived from TBI allo hosts compared to MC. Similar differences in IFN-γ and granzyme B protein expression were confirmed in the polyclonal donor CD8+ T cell population. Since, full GVL activity might also depend upon the survival of GVH-reactive CTL, we also examined the viability of donor T cells during the initial response in both environments. We observed higher rates of sustained 2C CD8+ T cell apoptosis (as indicated by annexin V staining) following T cell transfer to MC. Furthermore, we also detected lower expression of common γ chain cytokine receptors that mediate responsiveness to IL-2, IL-7 and IL-15, upon donor T cells from MC. However, following secondary co-transfer to syngeneic recipients for 21 days, memory phenotype polyclonal donor CD8+ T cells derived from established MC (CD45.1+) were recovered to a greater extent than T cells initially derived from TBI allo mice (Thy1.1+), arguing against any intrinsic defect in the viability of GVH-reactive T cell populations emerging in the former setting. Indeed, when co-cultured in the presence of individual cytokines, MC-derived CD8+ T cells maintained viability to a greater (IL-2, IL-7) or equivalent (IL-15) extent as TBI allo-derived cells. Taken together, these data suggest that disparity in GVL activity following to TBI allo recipients and MC can be explained by differences in the effector function and survival of anti-host CTL in quiescent MC environment. Reduced GVL activity of donor T cells on a per-cell basis in MC can be compensated for by transferring greater numbers of cells.

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Bone Marrow T Cells Are More Effective Than Peripheral T Cells in Inducing Chimeric Conversion Following MHC-Mismatched Nonmyeloablative Bone Marrow Transplantation

Blood ◽

10.1182/blood.v112.11.4593.4593 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4593-4593

Author(s):

Seok-Goo Cho ◽

Hyunsil Park ◽

Min Jung Park ◽

Ho-Youn Kim ◽

Jong-Wook Lee ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

T Cell ◽

Marrow Transplantation ◽

Memory T Cells ◽

Mixed Chimerism ◽

Graft Versus Host ◽

Peripheral T Cells ◽

Mixed Chimeras

Abstract Background & Objectives: Recently, T cells in BM have attracted renewed interest because they are now known to have different surface phenotypes, subsets, and activation states from those in the periphery. Memory T cells undergo extensive migration from the blood to the BM and vice versa. The BM plays an important role in preferential homing and extensive proliferation of memory T cells, and contributes considerably to the longlived memory T cell pool. BM T cells are more activated than their splenic counterparts and have a higher rate of local proliferation. Although BM-T (NK1.1– CD4+ or CD8+) cells did not induce lethal GVH disease, even at high cell numbers, BM-T cells mediated vigorous graft-versus-tumor activity and facilitated engraftment of hematopoietic progenitor cells. These studies suggested that BM-T cells could be a useful cellular source for adoptive immunotherapy following ABMT, instead of peripheral T cells. Non-myeloablative bone marrow transplantation (NMT) and allogeneic mixed chimerism can provide an environment adequate for diminishing susceptibility to DLI-mediated GVHD and an immunological platform for DLI in both mouse and human models. In patients treated with DLI, a successful GVL effect is often associated with conversion to complete donor chimerism, supporting the concept of a graft-versus-host (GVH) response as part of the GVL effect. Thus, a quiet chimeric conversion following DLI is desirable to reach an optimal DLI-mediated GVL effect, without the occurrence of GVHD. Although in a mouse model, the administration of non-tolerant donor spleen cells to established mixed chimeras has been shown to convert mixed hematopoietic chimerism to full donor chimerism, without the concomitant development of GVHD, DLI in humans frequently results in serious GVHD and life-threatening complications. However, the use of BM-T cells, as compared with spleen T cells (SP-T), as the DLI source has not been investigated in allogeneic mixed chimerism prepared with NMT. In this study, we evaluated the beneficial alloreactivity of DLI using cryopreserved BM-T cells, a by-product obtained during the T cell depletion (TCD) procedure in BM grafting, to effectively induce chimeric conversion without the occurrence of GVHD in MHC-mismatched NMT. Methods: Cells were prepared using established procedures. During the T cell depletion (TCD) procedure in BM grafting, BM-T cells were obtained as a by-product and then cryopreserved for subsequent DLI using BM-T cells 21 days after the bone marrow transplant. Results: The administration of 5–10 × 105 BM-T (Thy1.2+) cells in mixed chimeras resulted in complete chimeric conversion, with self-limited graft-versus-host disease (GVHD) and no pathological changes. However, the administration of 5–10 × 105 SP-T (Thy1.2+) cells resulted in persistent mixed chimerism, with pathological GVHD signs in the liver and intestine. Conclusion: Our results suggest that DLI using BM-T cells, even in small numbers, could be more potent for inducing chimeric conversion in mixed chimerism than DLI using SP-T cells. Further study is needed to determine whether cryopreserved BM-T cells are an effective cell source for DLI to consolidate donor-dominant chimerism in clinical practice, without concerns about GVHD.

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Expression of the Butyrate/Niacin Receptor, GPR109a on T Cells Plays an Important Role in a Mouse Model of Graft Versus Host Disease

Blood ◽

10.1182/blood-2018-99-118783 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 61-61 ◽

Cited By ~ 2

Author(s):

Melissa D Docampo ◽

Christoph K. Stein-Thoeringer ◽

Amina Lazrak ◽

Marina D Burgos da Silva ◽

Justin Cross ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Effector Memory ◽

Knock Out ◽

Graft Versus Host

Abstract INTRODUCTION: The intestinal microbiota is essential for the fermentation of fibers into the short-chain fatty acids (SCFA) butyrate, acetate and propionate. SCFA can bind to G-protein-coupled receptors GPR41, GPR43 and GPR109a to activate downstream anti-inflammatory signaling pathways. In colitis or graft versus host disease (GVHD), GPR43 signaling has been reported as an important regulator of intestinal homeostasis by increasing the pool of regulatory T cells. In contrast to GPR43, which binds preferentially propionate and acetate, GPR109a is the major receptor for butyrate. We and others have demonstrated that butyrate can ameliorate gastrointestinal injury during GVHD through enterocyte protection. Therefore, we hypothesized that GPR109a plays an important role in the pathophysiology of intestinal GVHD, focusing specifically on alloreactive T cells. METHODS AND RESULTS: Using mouse models of GVHD, we examined the role of the butyrate/niacin receptor, GPR109a in allogeneic hematopoietic cell transplantation (allo-HCT). First, we studied whether a genetic knock-out (KO) of GPR109a in transplant recipient mice affected GVHD, but GPR109a-KO recipient mice did not exhibit increased mortality from GVHD compared to wild type (WT) mice. We next investigated the role of GPR109a in the donor compartment by transplanting either BM or T cells from WT or GPR109a-KO mice into major MHC mismatched BALB/c host mice. Mice transplanted with B6 BM, with T cells from a GPR109a-KO mouse into BALB/c hosts displayed a lower incidence of lethal GVHD (Fig. 1A). To determine whether the attenuation of GVHD is intrinsic to GPR109a-KO T cells, we established BM chimeras and performed a secondary transplant by transplanting B6 BM + (B6 à Ly5.1) or (GPR109a à Ly5.1) T cells into BALB/c hosts. We observed the same improvement in survival in mice that received GPR109a-KO T cells. This indicates an intrinsic role for GPR109a specifically in the donor hematopoietic compartment. Having identified a T-cell specific requirement for GPR109a we next examined expression of GPR109a on WT T cells in vitro at baseline and following stimulation with CD3/28 and found GPR109a significantly upregulated on both CD4+ and CD8+ T cells after 72 hours of stimulation (Fig 1B). At steady state in vivo, we observed the same numbers and percentages of splenic effector memory, central memory, and naïve CD4+ T cells as well as regulatory T cells in WT B6 mice and GPR109a-KO mice, suggesting normal T cell development in the knockout mice. In an in vitro mixed lymphocyte reaction (MLR), GPR109a-KO CD4+ T cells become activated, proliferate, polarize and secrete cytokine (specifically IFNg) to the same level as WT CD4+ T cells, suggesting normal functional capacity. However, after allo-HCT in mice we observed significantly fewer CD4+ and CD8+ T cells, and specifically fewer effector memory CD4+ T cells (Fig. C), in the small and large intestines of mice that received GPR109a-KO T cells at day 7 post transplant. In contrast, we found significantly more regulatory T cells in the intestines (Fig. 1D) and the spleen of GPR1091-KO T cell recipients, while numbers and percentages of polarized Th1 and Th17 T cells were similar between the two groups. We further 16S rRNA sequenced the gut microbiota of mice at day 7 after transplant and observed an increased relative abundance of bacteria from the genus Clostridium (Fig. 1D) along with an increased concentration of cecal butyrate as measured by GC-MS (Fig. 1E). In a preliminary graft versus tumor (GVT) experiment, we found that mice that received A20 tumor cells and GPR109a-KO T cells exhibited increased survival compared to mice that received A20 tumor cells and WT T cells. These preliminary findings suggest that GPR109a-KO T cells maintain their graft versus tumor response while causing less GVHD, and exclude a defective functional capacity. CONCLUSIONS: We report a novel role of the butyrate/niacin receptor GPR109a on donor T cells in allo-HCT as a genetic knock-out on T cells attenuates lethal GVHD. As these T cells are tested as functionally intact, we propose that the reduction in overall T cells of KO T cell recipients may underlie the attenuation in GVHD. Furthermore, such a reduction in allograft-induced gut injury is accompanied by maintenance of the gut commensal Clostridium and butyrate production, which is known to protect the intestinal epithelium and increases the regulatory T cell pool. Disclosures No relevant conflicts of interest to declare.

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Allospecific Effector Memory T Cells Are Able to Mediate Second-Set Skin Graft Rejection but Unable to Induce Graft-Versus-Host Disease.

Blood ◽

10.1182/blood.v114.22.232.232 ◽

2009 ◽

Vol 114 (22) ◽

pp. 232-232

Author(s):

Ping Zhang ◽

Jieying Wu ◽

Divino Deoliveria ◽

Nelson J. Chao ◽

Benny J. Chen

Keyword(s):

T Cells ◽

T Cell ◽

Skin Graft ◽

Graft Versus Host Disease ◽

Graft Rejection ◽

Memory T Cells ◽

Effector Memory ◽

Memory Phenotype ◽

Graft Versus Host ◽

Effector Memory T Cells

Abstract Abstract 232 Several different groups have independently demonstrated that non-allospecific effector memory T cells do not induce graft-versus-host disease (GVHD). Limited data are available regarding the ability of allospecific effector memory T cells to induce GVHD. We first studied this question in the C57BL/6 into BALB/c model. Similar to the data previously published by other groups, purified CD62L- effector memory T cells isolated from donors, who were primed with the host antigens 8 weeks earlier, had decreased ability to induce acute GVHD compared with unseparated and CD62L+ T cells. Similar results were observed when the parous female mice, who were sensitized to the host antigens during pregnancy, were used as memory T cell donors. In order to study this question more definitely and to understand the mechanisms underlying these surprising observations, we further studied the ability of allospecific effector memory T cells to induce GVHD using a novel GVHD model mediated by transgenic TEa T cells. All TEa T cells are CD4+ and recognize the same peptide in the context of I-Ab. This peptide corresponds to positions 52-68 from the alpha-chain of I-E class II molecules and is expressed in all antigen presenting cells from H-2b/I-E+ strains such as CB6F1 mice. To generate memory T cells, naïve TEa cells were first parked in Rag1−/− mice and then immunized with irradiated CB6F1 spleen cells. More than eight weeks later, ∼98% of TEa cells obtained a memory phenotype (CD44high, CD45RB-.CD127+,CD11a bright, FasL bright, Ki67-, CD28-, KLRG-). Of them, about 93% were CD62L-CD44high effector memory T cells and 7% were CD62L+CD44high central memory T cells. These Rag1−/− mice that contained memory phenotype TEa cells rejected CB6F1 skin grafts much faster than naïve TEa mice did (median survival time: 6.5 vs. 13 days, P=0.01), suggesting that the memory phenotype T cells contained in these mice are functional. Moreover, CD62L-CD44high TEa cells purified from these mice mediated faster and stronger in vitro proliferative responses against alloantigens than naïve TEa cells did, further demonstrating that they are true functional effector memory T cells. We next tested the ability of these effector memory TEa cells to induce GVHD. Effector memory TEa cells were obtained after depletion of CD62L+ cells using magnetic beads and the purity was more than 99%. Transplantation of 1′105 TEa naïve T cells together with 1′107 T cell depleted bone marrow cells into lethally irradiated CB6F1 recipients induced lethal GVHD in all recipients and all animals in this group died within 35 days after transplantation. In contrast, none of the effector memory TEa cell recipients developed GVHD and all of them survived more than 100 days post transplantation (P<0.01, compared with naïve T cell control). To understand the mechanisms underlying these observations, we studied the kinetics of TEa proliferative responses upon challenge with alloantigens. The data indicated that effector memory TEa cells reached the peak responses faster than naïve TEa cells did. CFSE tracking experiment further confirmed this observation. Simultaneous staining with anti-Anexin V antibody and 7-AAD demonstrated that effector memory TEa cells undergone apoptosis and died faster than naïve T cells did. In conclusion, these data underscore the fundamental difference of alloresponses mediated by antigen-specific effector memory T cells in graft rejection and GVHD settings. The TEa transgenic T cell skin graft and GVHD models would allow further understanding of the unique alloresponses mediated by allospecific memory T lymphocytes in GVHD. Disclosures: No relevant conflicts of interest to declare.

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