American Journal of Transplantation 2015; 15: 234–241 Wiley Periodicals Inc.
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Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13051
Brief Communication
Serine Protease Inhibitor-6 Differentially Affects the Survival of Effector and Memory Alloreactive CD8-T Cells J. Azzi*, S. Ohori, C. Ting, M. Uehara, R. Abdoli, B. D. Smith, K. Safa, Z. Solhjou, P. Lukyanchykov, J. Patel, M. McGrath and R. Abdi* Transplantation Research Center, Renal Division, Brigham and Women’s Hospital and Children’s Hospital, Harvard Medical School, Boston, MA Corresponding authors: Jamil Azzi, [email protected] and Reza Abdi, [email protected] The clonal expansion of effector T cells and subsequent generation of memory T cells are critical in determining the outcome of transplantation. While cytotoxic T lymphocytes induce direct cytolysis of target cells through secretion of Granzyme-B (GrB), they also express cytoplasmic serine protease inhibitor-6 (Spi6) to protect themselves from GrB that has leaked from granules. Here, we studied the role of GrB/Spi6 axis in determining clonal expansion of alloreactive CD8-T cells and subsequent generation of memory CD8-T cells in transplantation. CD8-T cells from Spi6/ mice underwent more GrB mediated apoptosis upon alloantigen stimulation in vitro and in vivo following adoptive transfer into an allogeneic host. Interestingly, while OT1.Spi6/ CD8 T cells showed significantly lower clonal expansion following skin transplants from OVA mice, there was no difference in the size of the effector memory CD8-T cells long after transplantation. Furthermore, lack of Spi6 resulted in a decrease of short-lived-effector-CD8-cells but did not impact the pool of memory-precursor-effector-CD8-cells. Similar results were found in heart transplant models. Our findings suggest that the final alloreactive CD8-memory-pool-size is independent from the initial clonal-proliferation as memory precursors express low levels of GrB and therefore are independent of Spi6 for survival. These data advance our understanding of memory T cells generation in transplantation and provide basis for Spi6 based strategies to target effector T cells. Abbreviations: CTLs, cytotoxic T lymphocytes; GrB, Granzyme-B; MPEC, memory-precursor-effector-CD8cells; SLEC, short-lived-effector-CD8-cells; Spi6, serine protease inhibitor-6 Received 23 January 2014, revised 07 August 2014 and accepted for publication 25 August 2014
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Introduction Memory T cells present in human transplant recipients can resist the effects of conventional immunosuppressive drugs and costimulatory blocking agents, thereby impeding our efforts to achieve transplantation tolerance (1). The mechanisms controlling the emergence and survival of memory T cells are still poorly understood. Hence, there is an urgent need for new approaches to target memory T cells with the major goal of achieving long-term allograft survival and transplantation tolerance. Upon exposure to a foreign antigen, combined TCR stimulation and costimulatory signaling trigger T cell proliferation during the clonal expansion phase. This leads to the differentiation of na€ıve CD8 T cells into effector cytotoxic T lymphocytes (CTLs) (2). CTLs will induce direct cytolysis of target cells through either Granzyme B (GrB)/ perforin mediated mechanisms or through the engagement of Fas/Fas ligand (3,4). Perforin is exocytosed first, facilitating the entry of GrB into target cells, to trigger apoptosis through caspase-dependent and -independent mechanisms (5,6). The clonal expansion is then followed by the contraction phase of CTLs with elimination of 90–95% of effector T cells through programmed cell death (PCD) (2). However, those cells that survive the contraction phase subsequently develop into memory CD8 T cells (2,7,8). The memory phase can extend for the lifetime of the host, providing long-term immunity (2). While 5–10% of the effector cells survive and differentiate into memory CD8 T cells, it was believed that the size of the memory pool at the end of the contraction phase is proportional to the initial clonal expansion size (9,10). While CD8 T cells produce large amounts of GrB upon activation, they also up-regulate endogenous inhibitors of GrB in both the mouse and human (11–13). Human proteinase inhibitor (PI)9 belongs to the OVA family of intracellular serine protease inhibitors (Spi), or serpins, which inactivate proteases by acting as suicide substrates (14). PI9 is also found in the cytoplasm of CTLs to protect from GrB that has leaked from granules (15). The murine homologue of PI9 is Spi6 (16). We have shown recently that Spi6/GrB axis is also important in regulatory
Spi6 and CD8 Memory T cells
T cell homeostasis (17). Most of the published literature has studied the role of Spi6 in CD8 T cells homeostasis in the context of viral infections. No data exist on the role of Spi6 in CD8 effector and memory T cells homeostasis in the context of transplantation. Here, we hypothesized that the lack of Spi6 in CD8 T cells will induce GrB mediated apoptosis upon allostimulation, reducing the size of the clonal burst of CTLs and its contribution to the size of the memory pool at the end of the contraction phase in the context of transplantation.
Materials and Methods Mice B6 (H-2b) and BALB/c (H-2d) were purchased from the Jackson Laboratory (Bar Harbor, ME). Spi6/C57BL/6 (18), C57BL/6-Tg(CAG-OVA)916Jen/J transgenic mice expressing the membrane bound chicken ovalbumin on all cell surface and OT-I TCR transgenic mice (C57BL/6 background) express a transgenic TCR that recognizes the 8-mer SIINFEKL peptide derived from residues 257–264 of ovalbumin and OT1.Spi6/ mice were maintained in our animal facility. All animals were used at 6–10 weeks of age (20–25 g) and were housed in accordance with institutional and National Institutes of Health guidelines. The Harvard Medical School Animal Management Committee approved all animal experiments.
Flow cytometric analysis Anti-mouse antibodies against CD8, CD44, CD62L, KLRG1, CD127(IL7R), CCR7, Annexin-V and GrB were purchased from BD Biosciences (San Jose, CA). Cells recovered from spleens and peripheral lymphoid tissues were analyzed by flow cytometry with a FACS Canto-II flow-cytometer (BD Biosciences) and analyzed using FlowJo software version 9.3.2 (Treestar, Ashland, OR).
Dendritic cells and CD8 T cells isolation Splenic dendritic cells (DC) were purified from na€ıve C57BL/6 mice using N418 magnetic beads and MidiMACS columns or AutoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s protocol. The DCs were then incubated in a CO2 incubator with the SIINFEKL peptide overnight. Splenic CD8 na€ıve T cells were purified by magnetic isolation using CD8aþ T Cell Isolation Kit (Miltenyi Biotec) following the manufacturer’s protocol.
MLR assays 105 BALB/c DCs were incubated and 2 105 WT C57BL/6 or Spi6/ CD8 T cells responders were added to each well in a 96-well round bottom plate and incubated at 378C for 72 h. We then pulsed the cultures with 1 mCi of triturated thymidine (3H) and determined the incorporation efficiency, which was expressed as the mean counts per minute of 3H uptake by triplicate cultures plus the standard deviation. For the antigen-specific assay, 105 C57BL/6 DCs pulsed with the SIINFEKL peptide overnight were incubated and 2 105 OT1 or OT1.Spi6/ CD8 T cells responders. We also measured cell proliferation using the CFSE dilution method as described later.
Graft versus host disease model: BALB/c hosts underwent total body irradiation (9 Gy) at 85 rad/min and received 2 106 bone marrow cells and 2 106 CD8 T cells derived from WT or Spi6/ mice via tail vein injection. Splenocytes were harvested from recipient mice on day 10 as indicated for flow cytometry analysis.
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Skin transplantation Full-thickness trunk skin grafts (1 cm2) harvested from OVA donors were transplanted onto the flank of OT1 and OT1.Spi6/ recipient mice, sutured with 6.0 silk and secured with dry gauze and a bandage for 7 days.
Results CD8 T cells from Spi6/ and OT1.Spi6/ mice undergo less proliferation and more apoptosis upon stimulation in vitro compared to WT CD8 T cells CD8 T cells are known to proliferate and produce GrB upon allostimulation. While we showed previously that Spi6 protects GrB-producing regulatory T cells from selfinflicted damage during proliferation upon allostimulation, here, we tested the role of Spi6 in CD8 T cell proliferation and survival. We first examined the response of CD8 T cells to alloantigens polyclonally in vitro. CD8 T cells isolated from na€ıve C57BL/6 and Spi6/ splenocytes were stimulated in vitro with DCs isolated from BALB/c mice and cell proliferation was assessed by thymidine incorporation at 3 days poststimulation. Spi6/ CD8 T cells proliferated significantly less than WT CD8 T cells (Figure 1A). To test the antigen-specific response of CD8 T cells lacking Spi6, we generated OT1.Spi6/ mice. CD8 T cells isolated from OT1 transgenic mice express a TCR specific for the SIINFEKL peptide of ovalbumin presented on H-2K^b (MHC class I). CD8 T cells isolated from na€ıve OT1.WT splenocytes and OT1.Spi6/ were stimulated for 3 days in vitro with C57BL/6 DCs pulsed with the SIINFEKL peptide overnight. OT1.Spi6/ CD8 T cells proliferated less than OT1.WT at day 3 as measured by thymidine incorporation (Figure 1B) and CFSE dilution (48.33 0.88% vs. 53.37 1.21%, respectively, p ¼ 0.02) (Figure 1C). Furthermore, we analyzed the rate of apoptosis/necrosis of CD8þ Spi6/ T cells compared to OT1.WT cells by flow cytometry. CD8 T cells lacking Spi6 went more into apoptosis/necrosis as measured by flow cytometry analysis of Annexin-V and 7 AAD expression at day 3 (36.17 0.66% vs. 26.4 0.54%, respectively, p ¼ 0.0002) (Figure 1D). We investigated next whether the absence of Spi6 reduces CD8 T cell proliferation as a result of increased apoptosis. GrB mediated killing is known to be caspase dependent, therefore we repeated the previous experiment in the presence of a caspase inhibitor (Z-VAD(OMe)-FMK; Santa Cruz, Dallas, TX), at a concentration of 50 mM (19). As shown in Figure 1D, inhibiting caspases improved OT1.Spi6/ CD8 T cells proliferation, as measured by Ki67 expression using flow cytometry analysis at day 3. Spi6/ CD8 T cells undergo more apoptosis in vivo upon adoptive transfer into allogeneic host compared to WT CD8 T cells To study the role of Spi6 in CD8 survival in vivo, two groups of lethally irradiated BALB/c mice were reconstituted with 2 106 bone marrow cells and also received 2 106 CD8 T 235
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cells isolated from Spi6/ or WT splenocytes. Splenocytes were recovered from mice at day 10 after adoptive transfer to examine the rate of apoptosis in GrB producing and non-producing CD8 T cells. While both WT and Spi6/ CD8 T cells showed similar level of GrB expression (25.78 0.31% vs. 28.30 3.25%, respectively, p ¼ 0.2) (Figure 2A), lack of Spi6 increased apoptosis in GrBþSpi6/ CD8 T cells compared to GrBþWT CD8 T cells, as measured by Annexin-V, a marker of apoptosis (59.35 8.04% vs. 32.60 8.08%, respectively, p ¼ 0.03) (Figure 2B). Both WT and Spi6/ GrBþ CD8 T cells expressed significantly higher levels of Annexin-V compared to both WT and Spi6/ GrB CD8 T cells (Figure 2C). However, no difference in Annexin-V expression was observed between GrBSpi6/ CD8 and GrBWT CD8 T cells (1.68 0.25% vs. 1.28 0.41%, respectively, p ¼ 0.2) indicating that in the absence of GrB, CD8 T cell apoptosis was unaffected by the presence or absence of Spi6. CD8 T cells from OT1.Spi6/ showed significantly lower clonal burst in response to OVA skin transplant compared to WT CD8 T cells To study the role of Spi6 in the homeostasis of CD8 effector memory T cells generated in response to a specific antigen in the context of transplantation, we used the OT1 transgenic mouse model that express a TCR specific for the SIINFEKL peptide of ovalbumin presented on H-2K^b (MHC class I). OT1.WT and OT1.Spi6/ mice received skin transplant from OVA mice that are transgenic mice expressing the membrane bound chicken ovalbumin on all cell surface. Blood samples were collected from recipients at days 7, 14 and 40 after skin transplantation to analyze for CD8 effector memory T cells (CD8þCD44highCD62llowCCR7low) and CD8 central memory
Figure 1: Continued.
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Figure 1: CD8 T cells from Spi6/ and OT1.Spi6/ mice undergo less proliferation and more apoptosis upon stimulation in vitro compared to WT CD8 T cells. (A) CD8þ T cells from C57BL/6 and Spi6/ (C57BL/6 background) were stimulated in vitro with fully allogeneic BALB/c DC. The graph displays CD8 T cell proliferation as measured by thymidine incorporation. CD8 Spi6/ T cells proliferated less than WT at day 3 poststimulation. (B) CD8þ T cells from OT1 and OT1.Spi6/ were stimulated in vitro with DC pulsed with OVA peptide. The graph displays CD8 T cell proliferation as measured by thymidine incorporation. CD8 OT1.Spi6/ T cells proliferated less than WT at day 3 poststimulation. (C) Representative plots demonstrating OT1 CD8 T cell proliferation in response to OVAloaded fully allogeneic DCs, as measured by CFSE dilution. In the absence of Spi6, decreased proliferation was observed at day 3. (D) Representative examples of apoptosis analysis by flow cytometry on OT1 and OT1.Spi6/ CD8 T cells 3 days after stimulation with DCs pulsed with OVA peptides. OT1.Spi6/ CD8 T cells underwent more apoptosis/necrosis as measured by Annexin-V and 7 AAD expression compared to OT1 CD8 T cells. (E) Adding caspase inhibitor at 50 mM improved OT1.Spi6/ CD8 T cells proliferation as measured by Ki67 expression ( p < 0.05; n ¼ 3–5 mice; data representative of two separate experiments).
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T cells (CD8þCD44highCD62lhighCCR7high). As shown in Figure 3A and B, OT1.Spi6/ recipient of OVA skin generated less effector T cells over time compared to OT1.WT recipients. At day 40, the end of the contraction phase, no difference in the effector memory frequency was observed between the two groups (Figure 3A and B). Similarly, no difference was noted in central memory T cells between OT1.Spi6/ mice recipient of OVA skin and OT1.WT recipients (1.505 0.2361% vs. 1.597 0.44%, respectively, p ¼ 0.8). Those remaining CD8þ T cells are considered to be precursors for effector and memory cells (20,21).
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Granzyme B CD8 T cells from Spi6 showed significantly lower clonal burst in response to BALB/c heart allograft compared to WT CD8 T cells To study the role of Spi6 in the homeostasis of CD8 effector memory T cells generated in a fully mismatched heart transplant model, we transplanted BALB/c hearts into C57BL/6 or Spi6/ mice. Mice were sacrificed at days 7 and 40 after heart transplantation to analyze splenic CD8 effector T cells (CD8þCD44highCD62llowCCR7low) at day 7 and CD8 effector memory T cells (CD8þ CD44highCD62llowCCR7low) at day 40. We first found splenic Spi6/ CD8 T cells producing GrB went more into apoptosis/necrosis (CD8þGrBþAnnexinþ7AADþ) compared to WT as measured by flow cytometry (Figure S1A). Furthermore, Spi6/ recipients of BALB/c hearts generated less effector T cells at day 7 compared to WT recipients, as shown in Figure S1B. At day 40, the end of the contraction phase, no difference in the effector memory cells was observed between the two groups (Figure S1B). Similarly, no difference was noted in central memory T cells (data not shown). As shown in Figure S1C, no difference in heart allograft survival was observed between WT and Spi6/ recipient of BALB/c hearts. Spi6 differentially affects the short-lived-effectorCD8-cells and the memory-precursor-effector-CD8cells CD8 effector T cells are divided into those that are destined to survive and become long-lived memory CD8 T cells (memory-precursor-effector-CD8-cells [MPEC]) and those that are not (short-lived-effector-CD8-cells [SLEC]) (22). We examined the role of Spi6 in the generation of antigenspecific MPEC and SLEC in a skin transplant model. OT1.WT and OT1.Spi6/ received OVA skin and peripheral blood was collected from those mice at day 14 posttransplantation. We analyzed the peripheral blood by flow cytometry for the generation of SLEC (CD8þKLRG1highIL7Rlow) and MPEC (CD8þKLRG1lowIL7Rhigh) by flow cytometry. As shown in Figure 4A, the percentage of SLEC in OT1.Spi6/ recipients was significantly less as compared to OT1.WT recipients (1.67 0.64% vs. 6.92 1.8%, respectively, p ¼ 0.01) (Figure 4B). No difference was observed in the ratio of MPEC between the OT1.WT and OT1.Spi6/ recipients at day 14 (58.83 3.66% vs. 56.03 5.41%, respectively, American Journal of Transplantation 2015; 15: 234–241
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Figure 2: Spi6/ CD8 T cells undergo more apoptosis in vivo upon adoptive transfer into allogeneic host compared to WT CD8 T cells. Lack of Spi6 induces CD8 T cell apoptosis and less proliferation in response to alloantigens in vivo. Spi6/ CD8 T cells were adoptively transferred into irradiated BALB/c mice reconstituted with 2 106 bone marrow cells. (A) Representative examples of Granzyme B expression by CD8 Spi6/ or WT at day 10 postadoptive transfer. (B) Representative examples of apoptosis analysis by flow cytometry of WT and Spi6/ CD8 T cells 10 days postadoptive transfer. (C) The graph displays the percentage of apoptosis (Annexin-V expression) of GrBþ and GrB WT and Spi6/ CD8 T cells at day 10 postadoptive transfer ( p < 0.05; n ¼ 3–5 mice/group).
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Figure 3: OT1.Spi6/ CD8 T cells showed significantly lower clonal burst in response to OVA skin transplant compared to WT CD8 T cells. OT1 and OT1.Spi6/ received skin transplant from OVA mice. (A) Representative examples of the peripheral blood analysis of CD8 effector memory T cells (CD8þCD44highCD62llowCCR7low) and CD8 central memory T cells (CD8þCD44highCD62lhighCCR7high) over time. (B) The graph displays the percentage of effector memory T cells in the peripheral blood of OT1 and OT1.Spi6/ recipient of OVA skin over time ( p < 0.05; n ¼ 3–5 mice/group).
p ¼ 0.3) (Figure 4C). Interestingly, SLEC showed significantly higher expression of GrB compared to MPEC in OT1 recipients as measured by its mean fluorescence intensity (MFI) by flow cytometry (60.70 1.49% vs. 46.48 1.92%, respectively, p ¼ 0.001) (Figure 5A). Furthermore, effector cells are known to increase their expression of IL-7 receptor (IL7R) to differentiate into long-lived memory cells (23). We therefore measured the expression of GrB in IL7Rhigh and IL7Rlow CD8 T cells. GrB expression is significantly lower in CD8þIL7Rhigh compared to CD8þIL7Rlow (49.35 1.926% vs. 61.25 1.55%, respectively, p ¼ 0.002) (Figure 5B). Interestingly, IL7R expression was significantly less on KLRG1 OT1.Spi6/ CD8 T cells compared to WT as measured by MFI (19.78 3.64% vs. 31.08 2.01%, respectively, p ¼ 0.017) (Figure 4A).
Discussion While memory T cell homeostasis remains poorly understood, it is widely accepted that the size of memory CD8 T cell population is determined by the size of the initial clonal burst after antigen encounter (9,10). We showed here that the lack of Spi6 in CD8 T cells induced apoptosis of GrB producing CTLs and reduced the clonal burst upon 238
exposure to alloantigens in a graft versus host disease (GVHD), skin and heart transplant models. However, no decrease in memory CD8 T cell pool was observed at the end of the contraction phase. We found that the reduction in Spi6/ CD8 T cell proliferation is due to increased GrB mediated apoptosis as by inhibiting GrB and caspase activity, we rescued Spi6/ allostimulated CD8 T cells from apoptosis and improved their proliferation in vitro. Furthermore, no difference in apoptosis was observed between GrBSpi6/ CD8 and GrBWT CD8 T cells in the GVHD model, indicating that in the absence of GrB, CD8 apoptosis was unaffected by the presence or absence of Spi6. While this observation explains the reduction in the clonal burst observed across the different animal models, it was interesting to see that there was no increased heart allograft survival in Spi6/ recipients. This lack of graft prolongation is not unexpected in light of the redundancy of the immune system in inducing allograft rejection, including through mechanisms independent of CD8 T cells. While CTLs use granzyme/perforin pathways to induce allogeneic target cell death, prior studies have shown no difference in fully mismatch cardiac allograft survival in perforin or American Journal of Transplantation 2015; 15: 234–241
Spi6 and CD8 Memory T cells
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Figure 4: Spi6 differentially affects the short-lived-effectorCD8-cells (SLEC) and the memory-precursor-effector-CD8cells (MPEC). OT1 and OT1.Spi6/ received skin transplant from OVA mice. (A) Representative examples of the peripheral blood analysis of the SLEC (CD8þKLRG1highIL7Rlow) and MPEC (CD8þKLRG1lowIL7Rhigh) at day 14 postskin transplantation. (B) The graph displays the percentage of SLEC in OT1 and OT1.Spi6/ at day 14. (C) The graph displays the percentage of MPEC in OT1 and OT1.Spi6/ mice at day 14 ( p < 0.05; n ¼ 3–5 mice/group).
granzyme KO recipients compared with WT counterparts (24,25). In addition, we showed previously that Spi6/ recipients have a regulatory T cell defect that can accelerate rejection and oppose the reduction in CD8 effector T cells (17). In this study, transplanting Spi6/ mice with skin or heart allografts results in a significantly diminished clonal burst, as Spi6 deficiency makes the majority of GrB producing CTLs susceptible to PCD. Interestingly, contrary to what we expected, we found that Spi6 does not play the same role in determining the memory pool size that forms after the rejection of the skin or heart transplant. CD8 effector T cells have been recently shown to either overcome the contraction phase and survive to become long-lived memory CD8 T cells called MPEC or become short-lived-effector-cells called SLEC that go into apoptosis (22). This is in agreement with the linear differentiation model of memory T cell development by predicting that memory T cells are derived from CTLs at some point during American Journal of Transplantation 2015; 15: 234–241
0 Granzyme B
Figure 5: Memory precursor cells express less Granzyme B compared to effector cells. (A) Representative example of SLEC and MPEC expression of Granzyme B by flow cytometry at day 14 postadoptive transfer. The graph displays the mean fluorescence intensity (MFI) between MPEC and SLEC at day 14. SLEC had higher expression of Granzyme B compared to MPEC. (B) Representative example of IL7Rhigh and IL7Rlow CD8 T cells expression of Granzyme B by flow cytometry at day 14 postadoptive transfer. The graph displays the MFI between IL7Rhigh and IL7Rlow CD8 T at day 14. IL7Rlow had higher expression of Granzyme B compared to IL7Rhigh ( p < 0.05; n ¼ 3–5 mice/group).
their development (26,27). We show here that Spi6 protects SLEC that express high level of GrB but is not completely required to protect MPEC as they express low level of GrB. The development of memory precursors from effector T cells also suggests that this is due to the enrichment of the IL7R and the down-regulation of GrB to escape from PCD. Finally, our data show that Spi6 deficiency leads to a reduction of the IL7R expression in memory precursors. Whether this will affect the function of memory cells and the strength of the secondary memory responses should be addressed in future studies. Human histological studies have shown an abundance of GrA, GrB and perforin in many types of acutely rejecting allografts which speaks to the clinical relevance of this axis in transplantation (28). However, animal studies using perforin and granzyme KOs have shown mixed results in 239
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different models. While no significant role was found in fully mismatch acute rejection models, both appear to be important in chronic rejection as their absence results in reduced allograft vasculopathy in mouse models of heart transplant rejection (29). On the other hand, GrB was shown to be involved in the immune suppressive function of regulatory T cells through contact mediated killing of effector CD4 T cells. GrB producing regulatory T cells have also been shown to be important in mediating tolerance in a skin allograft model (30). Furthermore, our data demonstrating the role of GrB in self-inflicted damage of effector and regulatory T cells adds another layer of complexity to this fascinating pathway. The involvement of GrB in those opposing forces explains the complexity and mixed results obtained in different animal models. In summary, we show here that the development of memory CD8 T cells to alloantigens is independent from the size of the clonal burst of CTLs and that the differential requirement of Spi6 gives a molecular explanation for this dichotomy. These data advance our understanding of memory T cells generation in transplantation and provide basis for Spi6 based strategies to target effector T cells.
Acknowledgments This work is supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number RO1AI091930 (RA), the 2011 Pfizer Advancing Research in Transplantation Science Award (RA) and by 2013 American Heart Association FTF Transition Award (JA).
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
References 1. Valujskikh A, Li XC. Frontiers in nephrology: T cell memory as a barrier to transplant tolerance. J Am Soc Nephrol 2007; 18: 2252– 2261. 2. Sprent J, Surh CD. T cell memory. Annu Rev Immunol 2002; 20: 551–579. 3. Kagi D, Seiler P, Pavlovic J, et al. The roles of perforin- and Fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses. Eur J Immunol 1995; 25: 3256–3262. 4. Kagi D, Vignaux F, Ledermann B, et al. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 1994; 265: 528–530. 5. Podack ER, Young JD, Cohn ZA. Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules. Proc Natl Acad Sci USA 1985; 82: 8629–8633.
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6. Kagi D, Ledermann B, Burki K, et al. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 1994; 369: 31–37. 7. Lakkis FG, Sayegh MH. Memory T cells: A hurdle to immunologic tolerance. J Am Soc Nephrol 2003; 14: 2402–2410. 8. Surh CD, Sprent J. Regulation of na€ıve and memory T-cell homeostasis. Microb Infect/Institut Pasteur 2002; 4: 51–56. 9. Hou S, Hyland L, Ryan KW, Portner A, Doherty PC. Virus-specific CD8þ T-cell memory determined by clonal burst size. Nature 1994; 369: 652–654. 10. Murali-Krishna K, Altman JD, Suresh M, et al. Counting antigenspecific CD8 T cells: A reevaluation of bystander activation during viral infection. Immunity 1998; 8: 177–187. 11. Sun J, Ooms L, Bird CH, Sutton VR, Trapani JA, Bird PI. A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9). J Biol Chem 1997; 272: 15434–15441. 12. Sprecher CA, Morgenstern KA, Mathewes S, et al. Molecular cloning, expression, and partial characterization of two novel members of the ovalbumin family of serine proteinase inhibitors. J Biol Chem 1995; 270: 29854–29861. 13. Phillips T, Opferman JT, Shah R, Liu N, Froelich CJ, AshtonRickardt PG. A role for the granzyme B inhibitor serine protease inhibitor 6 in CD8þ memory cell homeostasis. J Immunol 2004; 173: 3801–3809. 14. Bird PI. Serpins and regulation of cell death. Results Prob Cell Differ 1998; 24: 63–89. 15. Hirst CE, Buzza MS, Bird CH, et al. The intracellular granzyme B inhibitor, proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation, and its overexpression enhances CTL potency. J Immunol 2003; 170: 805–815. 16. Kaiserman D, Knaggs S, Scarff KL, et al. Comparison of human chromosome 6p25 with mouse chromosome 13 reveals a greatly expanded ov-serpin gene repertoire in the mouse. Genomics 2002; 79: 349–362. 17. Azzi J, Skartsis N, Mounayar M, et al. Serine protease inhibitor 6 plays a critical role in protecting murine granzyme B-producing regulatory T cells. J Immunol 2013; 191: 2319–2327. 18. Zhang M, Park SM, Wang Y, et al. Serine protease inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity of cytotoxic granules. Immunity 2006; 24: 451–461. 19. Warny M, Kelly CP. Monocytic cell necrosis is mediated by potassium depletion and caspase-like proteases. Am J Physiol 1999; 276: C717–C724. 20. Harty JT, Badovinac VP. Shaping and reshaping CD8þ T-cell memory. Nat Rev Immunol 2008; 8: 107–119. 21. Williams MA, Holmes BJ, Sun JC, Bevan MJ. Developing and maintaining protective CD8þ memory T cells. Immunol Rev 2006; 211: 146–153. 22. Joshi NS, Kaech SM. Effector CD8 T cell development: A balancing act between memory cell potential and terminal differentiation. J Immunol 2008; 180: 1309–1315. 23. Mazzucchelli R, Durum SK. Interleukin-7 receptor expression: Intelligent design. Nat Rev Immunol 2007; 7: 144–154. 24. Schulz M, Schuurman HJ, Joergensen J, et al. Acute rejection of vascular heart allografts by perforin-deficient mice. Eur J Immunol 1995; 25: 474–480. 25. Halloran PF, Urmson J, Ramassar V, et al. Lesions of T-cellmediated kidney allograft rejection in mice do not require perforin or granzymes A and B. Am J Transplant 2004; 4: 705–712. 26. Hu H, Huston G, Duso D, Lepak N, Roman E, Swain SL. CD4(þ) T cell effectors can become memory cells with high efficiency and without further division. Nat Immunol 2001; 2: 705–710.
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Spi6 and CD8 Memory T cells 27. Opferman JT, Ober BT, Ashton-Rickardt PG. Linear differentiation of cytotoxic effectors into memory T lymphocytes. Science 1999; 283: 1745–1748. 28. Choy JC. Granzymes and perforin in solid organ transplant rejection. Cell Death Differ 2010; 17: 567–576. 29. Choy JC, Kerjner A, Wong BW, McManus BM, Granville DJ. Perforin mediates endothelial cell death and resultant transplant vascular disease in cardiac allografts. Am J Pathol 2004; 165: 127–133. 30. Gondek DC, Devries V, Nowak EC, et al. Transplantation survival is maintained by granzyme Bþ regulatory cells and adaptive regulatory T cells. J Immunol 2008; 181: 4752–4760.
Supporting Information Additional Supporting Information may be found in the online version of this article.
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Figure S1: Spi6/ CD8 T cells showed significantly lower clonal burst in response to fully mismatched heart allograft compared to WT CD8 T cells. C57BL/6 or Spi6/ mice received BALB/c hearts. (A) Flow cytometry analysis of splenocytes showed that Spi6/ CD8þGrBþ T cells went more into apoptosis/necrosis as measured by Annexin-V and 7AAD compared to WT T cells. (B) Flow cytometry analysis of splenocytes showed lower percentage of CD8 effector T cells (CD8þCD44highCD62llowCCR7low) at day 7 but no difference in effector memory T cells at day 40 ( p < 0.05; n ¼ 3–5 mice/group). (C) Kaplan–Meier survival graph shows no differences in survival of fully mismatched cardiac allograft in WT versus Spi6/ recipients (MST 7 vs. 7.5, p ¼ ns, n ¼ 4–6 mice/group).
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Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13051
Brief Communication
Serine Protease Inhibitor-6 Differentially Affects the Survival of Effector and Memory Alloreactive CD8-T Cells J. Azzi*, S. Ohori, C. Ting, M. Uehara, R. Abdoli, B. D. Smith, K. Safa, Z. Solhjou, P. Lukyanchykov, J. Patel, M. McGrath and R. Abdi* Transplantation Research Center, Renal Division, Brigham and Women’s Hospital and Children’s Hospital, Harvard Medical School, Boston, MA Corresponding authors: Jamil Azzi, [email protected] and Reza Abdi, [email protected] The clonal expansion of effector T cells and subsequent generation of memory T cells are critical in determining the outcome of transplantation. While cytotoxic T lymphocytes induce direct cytolysis of target cells through secretion of Granzyme-B (GrB), they also express cytoplasmic serine protease inhibitor-6 (Spi6) to protect themselves from GrB that has leaked from granules. Here, we studied the role of GrB/Spi6 axis in determining clonal expansion of alloreactive CD8-T cells and subsequent generation of memory CD8-T cells in transplantation. CD8-T cells from Spi6/ mice underwent more GrB mediated apoptosis upon alloantigen stimulation in vitro and in vivo following adoptive transfer into an allogeneic host. Interestingly, while OT1.Spi6/ CD8 T cells showed significantly lower clonal expansion following skin transplants from OVA mice, there was no difference in the size of the effector memory CD8-T cells long after transplantation. Furthermore, lack of Spi6 resulted in a decrease of short-lived-effector-CD8-cells but did not impact the pool of memory-precursor-effector-CD8-cells. Similar results were found in heart transplant models. Our findings suggest that the final alloreactive CD8-memory-pool-size is independent from the initial clonal-proliferation as memory precursors express low levels of GrB and therefore are independent of Spi6 for survival. These data advance our understanding of memory T cells generation in transplantation and provide basis for Spi6 based strategies to target effector T cells. Abbreviations: CTLs, cytotoxic T lymphocytes; GrB, Granzyme-B; MPEC, memory-precursor-effector-CD8cells; SLEC, short-lived-effector-CD8-cells; Spi6, serine protease inhibitor-6 Received 23 January 2014, revised 07 August 2014 and accepted for publication 25 August 2014
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Introduction Memory T cells present in human transplant recipients can resist the effects of conventional immunosuppressive drugs and costimulatory blocking agents, thereby impeding our efforts to achieve transplantation tolerance (1). The mechanisms controlling the emergence and survival of memory T cells are still poorly understood. Hence, there is an urgent need for new approaches to target memory T cells with the major goal of achieving long-term allograft survival and transplantation tolerance. Upon exposure to a foreign antigen, combined TCR stimulation and costimulatory signaling trigger T cell proliferation during the clonal expansion phase. This leads to the differentiation of na€ıve CD8 T cells into effector cytotoxic T lymphocytes (CTLs) (2). CTLs will induce direct cytolysis of target cells through either Granzyme B (GrB)/ perforin mediated mechanisms or through the engagement of Fas/Fas ligand (3,4). Perforin is exocytosed first, facilitating the entry of GrB into target cells, to trigger apoptosis through caspase-dependent and -independent mechanisms (5,6). The clonal expansion is then followed by the contraction phase of CTLs with elimination of 90–95% of effector T cells through programmed cell death (PCD) (2). However, those cells that survive the contraction phase subsequently develop into memory CD8 T cells (2,7,8). The memory phase can extend for the lifetime of the host, providing long-term immunity (2). While 5–10% of the effector cells survive and differentiate into memory CD8 T cells, it was believed that the size of the memory pool at the end of the contraction phase is proportional to the initial clonal expansion size (9,10). While CD8 T cells produce large amounts of GrB upon activation, they also up-regulate endogenous inhibitors of GrB in both the mouse and human (11–13). Human proteinase inhibitor (PI)9 belongs to the OVA family of intracellular serine protease inhibitors (Spi), or serpins, which inactivate proteases by acting as suicide substrates (14). PI9 is also found in the cytoplasm of CTLs to protect from GrB that has leaked from granules (15). The murine homologue of PI9 is Spi6 (16). We have shown recently that Spi6/GrB axis is also important in regulatory
Spi6 and CD8 Memory T cells
T cell homeostasis (17). Most of the published literature has studied the role of Spi6 in CD8 T cells homeostasis in the context of viral infections. No data exist on the role of Spi6 in CD8 effector and memory T cells homeostasis in the context of transplantation. Here, we hypothesized that the lack of Spi6 in CD8 T cells will induce GrB mediated apoptosis upon allostimulation, reducing the size of the clonal burst of CTLs and its contribution to the size of the memory pool at the end of the contraction phase in the context of transplantation.
Materials and Methods Mice B6 (H-2b) and BALB/c (H-2d) were purchased from the Jackson Laboratory (Bar Harbor, ME). Spi6/C57BL/6 (18), C57BL/6-Tg(CAG-OVA)916Jen/J transgenic mice expressing the membrane bound chicken ovalbumin on all cell surface and OT-I TCR transgenic mice (C57BL/6 background) express a transgenic TCR that recognizes the 8-mer SIINFEKL peptide derived from residues 257–264 of ovalbumin and OT1.Spi6/ mice were maintained in our animal facility. All animals were used at 6–10 weeks of age (20–25 g) and were housed in accordance with institutional and National Institutes of Health guidelines. The Harvard Medical School Animal Management Committee approved all animal experiments.
Flow cytometric analysis Anti-mouse antibodies against CD8, CD44, CD62L, KLRG1, CD127(IL7R), CCR7, Annexin-V and GrB were purchased from BD Biosciences (San Jose, CA). Cells recovered from spleens and peripheral lymphoid tissues were analyzed by flow cytometry with a FACS Canto-II flow-cytometer (BD Biosciences) and analyzed using FlowJo software version 9.3.2 (Treestar, Ashland, OR).
Dendritic cells and CD8 T cells isolation Splenic dendritic cells (DC) were purified from na€ıve C57BL/6 mice using N418 magnetic beads and MidiMACS columns or AutoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s protocol. The DCs were then incubated in a CO2 incubator with the SIINFEKL peptide overnight. Splenic CD8 na€ıve T cells were purified by magnetic isolation using CD8aþ T Cell Isolation Kit (Miltenyi Biotec) following the manufacturer’s protocol.
MLR assays 105 BALB/c DCs were incubated and 2 105 WT C57BL/6 or Spi6/ CD8 T cells responders were added to each well in a 96-well round bottom plate and incubated at 378C for 72 h. We then pulsed the cultures with 1 mCi of triturated thymidine (3H) and determined the incorporation efficiency, which was expressed as the mean counts per minute of 3H uptake by triplicate cultures plus the standard deviation. For the antigen-specific assay, 105 C57BL/6 DCs pulsed with the SIINFEKL peptide overnight were incubated and 2 105 OT1 or OT1.Spi6/ CD8 T cells responders. We also measured cell proliferation using the CFSE dilution method as described later.
Graft versus host disease model: BALB/c hosts underwent total body irradiation (9 Gy) at 85 rad/min and received 2 106 bone marrow cells and 2 106 CD8 T cells derived from WT or Spi6/ mice via tail vein injection. Splenocytes were harvested from recipient mice on day 10 as indicated for flow cytometry analysis.
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Skin transplantation Full-thickness trunk skin grafts (1 cm2) harvested from OVA donors were transplanted onto the flank of OT1 and OT1.Spi6/ recipient mice, sutured with 6.0 silk and secured with dry gauze and a bandage for 7 days.
Results CD8 T cells from Spi6/ and OT1.Spi6/ mice undergo less proliferation and more apoptosis upon stimulation in vitro compared to WT CD8 T cells CD8 T cells are known to proliferate and produce GrB upon allostimulation. While we showed previously that Spi6 protects GrB-producing regulatory T cells from selfinflicted damage during proliferation upon allostimulation, here, we tested the role of Spi6 in CD8 T cell proliferation and survival. We first examined the response of CD8 T cells to alloantigens polyclonally in vitro. CD8 T cells isolated from na€ıve C57BL/6 and Spi6/ splenocytes were stimulated in vitro with DCs isolated from BALB/c mice and cell proliferation was assessed by thymidine incorporation at 3 days poststimulation. Spi6/ CD8 T cells proliferated significantly less than WT CD8 T cells (Figure 1A). To test the antigen-specific response of CD8 T cells lacking Spi6, we generated OT1.Spi6/ mice. CD8 T cells isolated from OT1 transgenic mice express a TCR specific for the SIINFEKL peptide of ovalbumin presented on H-2K^b (MHC class I). CD8 T cells isolated from na€ıve OT1.WT splenocytes and OT1.Spi6/ were stimulated for 3 days in vitro with C57BL/6 DCs pulsed with the SIINFEKL peptide overnight. OT1.Spi6/ CD8 T cells proliferated less than OT1.WT at day 3 as measured by thymidine incorporation (Figure 1B) and CFSE dilution (48.33 0.88% vs. 53.37 1.21%, respectively, p ¼ 0.02) (Figure 1C). Furthermore, we analyzed the rate of apoptosis/necrosis of CD8þ Spi6/ T cells compared to OT1.WT cells by flow cytometry. CD8 T cells lacking Spi6 went more into apoptosis/necrosis as measured by flow cytometry analysis of Annexin-V and 7 AAD expression at day 3 (36.17 0.66% vs. 26.4 0.54%, respectively, p ¼ 0.0002) (Figure 1D). We investigated next whether the absence of Spi6 reduces CD8 T cell proliferation as a result of increased apoptosis. GrB mediated killing is known to be caspase dependent, therefore we repeated the previous experiment in the presence of a caspase inhibitor (Z-VAD(OMe)-FMK; Santa Cruz, Dallas, TX), at a concentration of 50 mM (19). As shown in Figure 1D, inhibiting caspases improved OT1.Spi6/ CD8 T cells proliferation, as measured by Ki67 expression using flow cytometry analysis at day 3. Spi6/ CD8 T cells undergo more apoptosis in vivo upon adoptive transfer into allogeneic host compared to WT CD8 T cells To study the role of Spi6 in CD8 survival in vivo, two groups of lethally irradiated BALB/c mice were reconstituted with 2 106 bone marrow cells and also received 2 106 CD8 T 235
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cells isolated from Spi6/ or WT splenocytes. Splenocytes were recovered from mice at day 10 after adoptive transfer to examine the rate of apoptosis in GrB producing and non-producing CD8 T cells. While both WT and Spi6/ CD8 T cells showed similar level of GrB expression (25.78 0.31% vs. 28.30 3.25%, respectively, p ¼ 0.2) (Figure 2A), lack of Spi6 increased apoptosis in GrBþSpi6/ CD8 T cells compared to GrBþWT CD8 T cells, as measured by Annexin-V, a marker of apoptosis (59.35 8.04% vs. 32.60 8.08%, respectively, p ¼ 0.03) (Figure 2B). Both WT and Spi6/ GrBþ CD8 T cells expressed significantly higher levels of Annexin-V compared to both WT and Spi6/ GrB CD8 T cells (Figure 2C). However, no difference in Annexin-V expression was observed between GrBSpi6/ CD8 and GrBWT CD8 T cells (1.68 0.25% vs. 1.28 0.41%, respectively, p ¼ 0.2) indicating that in the absence of GrB, CD8 T cell apoptosis was unaffected by the presence or absence of Spi6. CD8 T cells from OT1.Spi6/ showed significantly lower clonal burst in response to OVA skin transplant compared to WT CD8 T cells To study the role of Spi6 in the homeostasis of CD8 effector memory T cells generated in response to a specific antigen in the context of transplantation, we used the OT1 transgenic mouse model that express a TCR specific for the SIINFEKL peptide of ovalbumin presented on H-2K^b (MHC class I). OT1.WT and OT1.Spi6/ mice received skin transplant from OVA mice that are transgenic mice expressing the membrane bound chicken ovalbumin on all cell surface. Blood samples were collected from recipients at days 7, 14 and 40 after skin transplantation to analyze for CD8 effector memory T cells (CD8þCD44highCD62llowCCR7low) and CD8 central memory
Figure 1: Continued.
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Figure 1: CD8 T cells from Spi6/ and OT1.Spi6/ mice undergo less proliferation and more apoptosis upon stimulation in vitro compared to WT CD8 T cells. (A) CD8þ T cells from C57BL/6 and Spi6/ (C57BL/6 background) were stimulated in vitro with fully allogeneic BALB/c DC. The graph displays CD8 T cell proliferation as measured by thymidine incorporation. CD8 Spi6/ T cells proliferated less than WT at day 3 poststimulation. (B) CD8þ T cells from OT1 and OT1.Spi6/ were stimulated in vitro with DC pulsed with OVA peptide. The graph displays CD8 T cell proliferation as measured by thymidine incorporation. CD8 OT1.Spi6/ T cells proliferated less than WT at day 3 poststimulation. (C) Representative plots demonstrating OT1 CD8 T cell proliferation in response to OVAloaded fully allogeneic DCs, as measured by CFSE dilution. In the absence of Spi6, decreased proliferation was observed at day 3. (D) Representative examples of apoptosis analysis by flow cytometry on OT1 and OT1.Spi6/ CD8 T cells 3 days after stimulation with DCs pulsed with OVA peptides. OT1.Spi6/ CD8 T cells underwent more apoptosis/necrosis as measured by Annexin-V and 7 AAD expression compared to OT1 CD8 T cells. (E) Adding caspase inhibitor at 50 mM improved OT1.Spi6/ CD8 T cells proliferation as measured by Ki67 expression ( p < 0.05; n ¼ 3–5 mice; data representative of two separate experiments).
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T cells (CD8þCD44highCD62lhighCCR7high). As shown in Figure 3A and B, OT1.Spi6/ recipient of OVA skin generated less effector T cells over time compared to OT1.WT recipients. At day 40, the end of the contraction phase, no difference in the effector memory frequency was observed between the two groups (Figure 3A and B). Similarly, no difference was noted in central memory T cells between OT1.Spi6/ mice recipient of OVA skin and OT1.WT recipients (1.505 0.2361% vs. 1.597 0.44%, respectively, p ¼ 0.8). Those remaining CD8þ T cells are considered to be precursors for effector and memory cells (20,21).
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Granzyme B CD8 T cells from Spi6 showed significantly lower clonal burst in response to BALB/c heart allograft compared to WT CD8 T cells To study the role of Spi6 in the homeostasis of CD8 effector memory T cells generated in a fully mismatched heart transplant model, we transplanted BALB/c hearts into C57BL/6 or Spi6/ mice. Mice were sacrificed at days 7 and 40 after heart transplantation to analyze splenic CD8 effector T cells (CD8þCD44highCD62llowCCR7low) at day 7 and CD8 effector memory T cells (CD8þ CD44highCD62llowCCR7low) at day 40. We first found splenic Spi6/ CD8 T cells producing GrB went more into apoptosis/necrosis (CD8þGrBþAnnexinþ7AADþ) compared to WT as measured by flow cytometry (Figure S1A). Furthermore, Spi6/ recipients of BALB/c hearts generated less effector T cells at day 7 compared to WT recipients, as shown in Figure S1B. At day 40, the end of the contraction phase, no difference in the effector memory cells was observed between the two groups (Figure S1B). Similarly, no difference was noted in central memory T cells (data not shown). As shown in Figure S1C, no difference in heart allograft survival was observed between WT and Spi6/ recipient of BALB/c hearts. Spi6 differentially affects the short-lived-effectorCD8-cells and the memory-precursor-effector-CD8cells CD8 effector T cells are divided into those that are destined to survive and become long-lived memory CD8 T cells (memory-precursor-effector-CD8-cells [MPEC]) and those that are not (short-lived-effector-CD8-cells [SLEC]) (22). We examined the role of Spi6 in the generation of antigenspecific MPEC and SLEC in a skin transplant model. OT1.WT and OT1.Spi6/ received OVA skin and peripheral blood was collected from those mice at day 14 posttransplantation. We analyzed the peripheral blood by flow cytometry for the generation of SLEC (CD8þKLRG1highIL7Rlow) and MPEC (CD8þKLRG1lowIL7Rhigh) by flow cytometry. As shown in Figure 4A, the percentage of SLEC in OT1.Spi6/ recipients was significantly less as compared to OT1.WT recipients (1.67 0.64% vs. 6.92 1.8%, respectively, p ¼ 0.01) (Figure 4B). No difference was observed in the ratio of MPEC between the OT1.WT and OT1.Spi6/ recipients at day 14 (58.83 3.66% vs. 56.03 5.41%, respectively, American Journal of Transplantation 2015; 15: 234–241
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Figure 2: Spi6/ CD8 T cells undergo more apoptosis in vivo upon adoptive transfer into allogeneic host compared to WT CD8 T cells. Lack of Spi6 induces CD8 T cell apoptosis and less proliferation in response to alloantigens in vivo. Spi6/ CD8 T cells were adoptively transferred into irradiated BALB/c mice reconstituted with 2 106 bone marrow cells. (A) Representative examples of Granzyme B expression by CD8 Spi6/ or WT at day 10 postadoptive transfer. (B) Representative examples of apoptosis analysis by flow cytometry of WT and Spi6/ CD8 T cells 10 days postadoptive transfer. (C) The graph displays the percentage of apoptosis (Annexin-V expression) of GrBþ and GrB WT and Spi6/ CD8 T cells at day 10 postadoptive transfer ( p < 0.05; n ¼ 3–5 mice/group).
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Figure 3: OT1.Spi6/ CD8 T cells showed significantly lower clonal burst in response to OVA skin transplant compared to WT CD8 T cells. OT1 and OT1.Spi6/ received skin transplant from OVA mice. (A) Representative examples of the peripheral blood analysis of CD8 effector memory T cells (CD8þCD44highCD62llowCCR7low) and CD8 central memory T cells (CD8þCD44highCD62lhighCCR7high) over time. (B) The graph displays the percentage of effector memory T cells in the peripheral blood of OT1 and OT1.Spi6/ recipient of OVA skin over time ( p < 0.05; n ¼ 3–5 mice/group).
p ¼ 0.3) (Figure 4C). Interestingly, SLEC showed significantly higher expression of GrB compared to MPEC in OT1 recipients as measured by its mean fluorescence intensity (MFI) by flow cytometry (60.70 1.49% vs. 46.48 1.92%, respectively, p ¼ 0.001) (Figure 5A). Furthermore, effector cells are known to increase their expression of IL-7 receptor (IL7R) to differentiate into long-lived memory cells (23). We therefore measured the expression of GrB in IL7Rhigh and IL7Rlow CD8 T cells. GrB expression is significantly lower in CD8þIL7Rhigh compared to CD8þIL7Rlow (49.35 1.926% vs. 61.25 1.55%, respectively, p ¼ 0.002) (Figure 5B). Interestingly, IL7R expression was significantly less on KLRG1 OT1.Spi6/ CD8 T cells compared to WT as measured by MFI (19.78 3.64% vs. 31.08 2.01%, respectively, p ¼ 0.017) (Figure 4A).
Discussion While memory T cell homeostasis remains poorly understood, it is widely accepted that the size of memory CD8 T cell population is determined by the size of the initial clonal burst after antigen encounter (9,10). We showed here that the lack of Spi6 in CD8 T cells induced apoptosis of GrB producing CTLs and reduced the clonal burst upon 238
exposure to alloantigens in a graft versus host disease (GVHD), skin and heart transplant models. However, no decrease in memory CD8 T cell pool was observed at the end of the contraction phase. We found that the reduction in Spi6/ CD8 T cell proliferation is due to increased GrB mediated apoptosis as by inhibiting GrB and caspase activity, we rescued Spi6/ allostimulated CD8 T cells from apoptosis and improved their proliferation in vitro. Furthermore, no difference in apoptosis was observed between GrBSpi6/ CD8 and GrBWT CD8 T cells in the GVHD model, indicating that in the absence of GrB, CD8 apoptosis was unaffected by the presence or absence of Spi6. While this observation explains the reduction in the clonal burst observed across the different animal models, it was interesting to see that there was no increased heart allograft survival in Spi6/ recipients. This lack of graft prolongation is not unexpected in light of the redundancy of the immune system in inducing allograft rejection, including through mechanisms independent of CD8 T cells. While CTLs use granzyme/perforin pathways to induce allogeneic target cell death, prior studies have shown no difference in fully mismatch cardiac allograft survival in perforin or American Journal of Transplantation 2015; 15: 234–241
Spi6 and CD8 Memory T cells
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Figure 4: Spi6 differentially affects the short-lived-effectorCD8-cells (SLEC) and the memory-precursor-effector-CD8cells (MPEC). OT1 and OT1.Spi6/ received skin transplant from OVA mice. (A) Representative examples of the peripheral blood analysis of the SLEC (CD8þKLRG1highIL7Rlow) and MPEC (CD8þKLRG1lowIL7Rhigh) at day 14 postskin transplantation. (B) The graph displays the percentage of SLEC in OT1 and OT1.Spi6/ at day 14. (C) The graph displays the percentage of MPEC in OT1 and OT1.Spi6/ mice at day 14 ( p < 0.05; n ¼ 3–5 mice/group).
granzyme KO recipients compared with WT counterparts (24,25). In addition, we showed previously that Spi6/ recipients have a regulatory T cell defect that can accelerate rejection and oppose the reduction in CD8 effector T cells (17). In this study, transplanting Spi6/ mice with skin or heart allografts results in a significantly diminished clonal burst, as Spi6 deficiency makes the majority of GrB producing CTLs susceptible to PCD. Interestingly, contrary to what we expected, we found that Spi6 does not play the same role in determining the memory pool size that forms after the rejection of the skin or heart transplant. CD8 effector T cells have been recently shown to either overcome the contraction phase and survive to become long-lived memory CD8 T cells called MPEC or become short-lived-effector-cells called SLEC that go into apoptosis (22). This is in agreement with the linear differentiation model of memory T cell development by predicting that memory T cells are derived from CTLs at some point during American Journal of Transplantation 2015; 15: 234–241
0 Granzyme B
Figure 5: Memory precursor cells express less Granzyme B compared to effector cells. (A) Representative example of SLEC and MPEC expression of Granzyme B by flow cytometry at day 14 postadoptive transfer. The graph displays the mean fluorescence intensity (MFI) between MPEC and SLEC at day 14. SLEC had higher expression of Granzyme B compared to MPEC. (B) Representative example of IL7Rhigh and IL7Rlow CD8 T cells expression of Granzyme B by flow cytometry at day 14 postadoptive transfer. The graph displays the MFI between IL7Rhigh and IL7Rlow CD8 T at day 14. IL7Rlow had higher expression of Granzyme B compared to IL7Rhigh ( p < 0.05; n ¼ 3–5 mice/group).
their development (26,27). We show here that Spi6 protects SLEC that express high level of GrB but is not completely required to protect MPEC as they express low level of GrB. The development of memory precursors from effector T cells also suggests that this is due to the enrichment of the IL7R and the down-regulation of GrB to escape from PCD. Finally, our data show that Spi6 deficiency leads to a reduction of the IL7R expression in memory precursors. Whether this will affect the function of memory cells and the strength of the secondary memory responses should be addressed in future studies. Human histological studies have shown an abundance of GrA, GrB and perforin in many types of acutely rejecting allografts which speaks to the clinical relevance of this axis in transplantation (28). However, animal studies using perforin and granzyme KOs have shown mixed results in 239
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different models. While no significant role was found in fully mismatch acute rejection models, both appear to be important in chronic rejection as their absence results in reduced allograft vasculopathy in mouse models of heart transplant rejection (29). On the other hand, GrB was shown to be involved in the immune suppressive function of regulatory T cells through contact mediated killing of effector CD4 T cells. GrB producing regulatory T cells have also been shown to be important in mediating tolerance in a skin allograft model (30). Furthermore, our data demonstrating the role of GrB in self-inflicted damage of effector and regulatory T cells adds another layer of complexity to this fascinating pathway. The involvement of GrB in those opposing forces explains the complexity and mixed results obtained in different animal models. In summary, we show here that the development of memory CD8 T cells to alloantigens is independent from the size of the clonal burst of CTLs and that the differential requirement of Spi6 gives a molecular explanation for this dichotomy. These data advance our understanding of memory T cells generation in transplantation and provide basis for Spi6 based strategies to target effector T cells.
Acknowledgments This work is supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number RO1AI091930 (RA), the 2011 Pfizer Advancing Research in Transplantation Science Award (RA) and by 2013 American Heart Association FTF Transition Award (JA).
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
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Supporting Information Additional Supporting Information may be found in the online version of this article.
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Figure S1: Spi6/ CD8 T cells showed significantly lower clonal burst in response to fully mismatched heart allograft compared to WT CD8 T cells. C57BL/6 or Spi6/ mice received BALB/c hearts. (A) Flow cytometry analysis of splenocytes showed that Spi6/ CD8þGrBþ T cells went more into apoptosis/necrosis as measured by Annexin-V and 7AAD compared to WT T cells. (B) Flow cytometry analysis of splenocytes showed lower percentage of CD8 effector T cells (CD8þCD44highCD62llowCCR7low) at day 7 but no difference in effector memory T cells at day 40 ( p < 0.05; n ¼ 3–5 mice/group). (C) Kaplan–Meier survival graph shows no differences in survival of fully mismatched cardiac allograft in WT versus Spi6/ recipients (MST 7 vs. 7.5, p ¼ ns, n ¼ 4–6 mice/group).
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