Anti-thymocyte globulin (ATG) differentially depletes naïve and memory T cells and permits memory-type regulatory T cells in nonobese diabetic mice
© Xia et al.; licensee BioMed Central Ltd. 2012
Received: 29 June 2012
Accepted: 24 November 2012
Published: 14 December 2012
ATG has been employed to deplete T cells in several immune-mediated conditions. However, whether ATG administration affects naïve and memory T cell differently is largely unknown.
The context and purpose of the study
In this study, we assessed how murine ATG therapy affected T cell subsets in NOD mice, based on their regulatory and naïve or memory phenotype, as well as its influence on antigen-specific immune responses.
Peripheral blood CD4+ and CD8+ T cells post-ATG therapy declined to their lowest levels at day 3, while CD4+ T cells returned to normal levels more rapidly than CD8+ T cells. ATG therapy failed to eliminate antigen-primed T cells. CD4+ T cell responses post-ATG therapy skewed to T helper type 2 (Th2) and possibly IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) were less sensitive to ATG depletion and remained at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory T cell phenotype was significantly increased in ATG-treated animals.
ATG therapy may modulate antigen-specific immune responses through inducing memory-like regulatory T cells as well as other protective T cells such as Th2 and IL-10-producing Tr1 cells.
Anti-thymocyte globulin (ATG), trade name thymoglobulin®, has been employed for decades as an immune modulator for a variety of clinical indications. It is currently one of the most common immunosuppressive reagents used in allogeneic transplantation [1–3] and more recently, in the treatment of a variety of autoimmune disorders [4–8]. There is a common belief that ATG therapy functions through complement mediated depletion of mature T cells. However, recent data suggests that ATG therapy induces immune modulation beyond that of simple T cell depletion . For example, ATG therapy may facilitate tolerance induction through modulation of dendritic cells (DC), both phenotypically and functionally . Evidence has also shown that ATG therapy may also induce regulatory T cells (Tregs) in vivo [11–13]. However, it remains unclear how ATG therapy affects naive and memory T cells in autoimmune settings such as T1D, although a recent study suggested that ATG therapy effectively eliminates alloantigen specific memory T cells in an allogeneic transplantation mouse model . In the current study, we used both standard NOD mice, as well as a TCR transgenic form of these mice (i.e., NOD.BDC2.5) to investigate changes within immune cell subsets in peripheral blood, spleen and lymph nodes post-ATG therapy, specifically focusing on addressing the questions of how ATG therapy affected naive and memory T cells, including naïve and memory Tregs. These strains were utilized due to their common utilization in studies of murine ATG efficacy for type 1 diabetes, as well as the ability to utilize mice having a defined antigenic specificity. The results demonstrated that ATG therapy differentially depletes T cells from peripheral blood and lymphoid organs. ATG therapy was more efficient in depleting naïve T cells than memory T cells. Tregs appeared resistant to ATG depletion and their frequency remained at increased levels after homeostatic recovery from ATG therapy. It was also noted that proportionately Tregs with memory T cell phenotype were significantly increased post-ATG therapy. Taken collectively, we believe this is a previously unrecognized mechanism whereby ATG therapy differentially affects naïve and memory Tregs.
Female NOD/Ltj were purchased from Jackson Laboratory and housed in specific pathogen free facilities at University of Florida Animal Care Service. The Institutional Animal Care and Use Committee at University of Florida approved all animal procedures (approval ID: 20090279).
Media and reagents
RPMI1640 media with glutamine were purchased from Fisher Scientific (Pittsburgh, PA). Complete culture media were prepared using RPMI1640 plus 10% fetal bovine serum (Thermo Scientific, Waltham, MA) and 1x penicillin and streptomycin (Cellgro, Manassas, VA). Murine ATG was provided by Genzyme (Framingham, MA). Rabbit IgG isotype was purchased from the Jackson Laboratory. The following antibodies were purchased from BD Biosciences: (San Jose, CA): CD4-PerCp (clone RM4-5), CD8-FITC (clone 53–6.7), B220-APC (clone RA3-6B2), CD44-APC (clone IM7), CD11c-APC (clone HL3), CD3-PE (clone 17A2) and CD25-APC (clone PC61). The antibodies of CD62L-APC and –FITC (mEL-14) and CD11b-PE (m1/70) were purchased from eBioscience (San Diego, CA). Gr1 (Ly6G/Ly6C)-APC (clone RB6-8C5) and Foxp3-PE, -FITC (clone MF-14) were purchased from BioLegend (San Diego, CA). CellTrace CFSE kits, and CD3, CD28 antibody-coated Dynabeads were purchased from Invitrogen (Carlsbad, CA). Multiplex bead cytokine assay kits were purchased from Millipore (Billerica, MA). Mouse CD11c beads were purchased from Miltenyi Biotech (Germany). CD4+ T cell negative selection kits were purchased from StemCell Biotechnology Inc. (Vancouver, Canada). KLH was purchased from CalbioChem (San Diego, CA). Bovine serum albumin was purchased from Sigma-Aldrich (St. Louis, MO). Alum adjuvant was purchased from Thermo Scientific (Waltham, MA).
ATG treatment and observation of peripheral blood cell components
6–8 week old NOD mice were treated with two intraperitoneal injections of ATG or isotype IgG (500 ug/mouse), 3 days apart, as previously described [11, 12]. In some experiments, two groups of mice were monitored longitudinally by examining white blood cell lineages using flow cytometry (LSRFortessa, BD) including CD4+, CD8+ T cells, B220+ B cells, Gr-1+ granulocytes as well as CD62L+ naive T cells and CD62-CD44+ memory T cells. The data were analyzed by FCS express De Novo software version 3 (Vancouver, Canada).
Measurement of splenic memory and naive T cells and T cell response to in vitro stimulation post ATG therapy
NOD mice were treated with ATG and isotype IgG as described above. At day 3 or 22, the treated mice were sacrificed. Single cell suspensions of spleen cells were prepared and CD4+, CD8+ T cells as well as CD62L+ naive and CD62-CD44+ memory T cells were examined by flow cytometry (LSRFortessa, BD). A portion of the spleen cells were used for CD4+ T cell isolation using CD4+ T cell negative isolation EasySep kits following the manufacturer’s instructions (StemCell Inc. Canada). Spleen cells (1 × 106) were stimulated with anti-CD3 antibody (3 ug/ml) and in some experiments, purified CD4+ T cells were stimulated with CD3 and CD28 antibody-coated Dynabeads mouse T cell activator (Invitrogen), with splenic DC plus antigens (KLH), or autoantigens (NIT-1 cell lysates) as indicated for 3–4 days, then, 3H-thymidine (1 uCi/well) was added to each well for the final 16 hours. Incorporation of 3H-thymidine was measured by scintillation counting (Wallac Trilux).
Measurement of Foxp3+ Treg cells
Spleen cells were stained with anti-CD4-PerCp and anti-CD25-APC. The cells were then fixed and permeablized and stained with anti-Foxp3-PE following the instructions of the manufacturer (eBioscience). CD4+CD25+Foxp3+ Treg cells were examined by flow cytometry (LSRFortessa, BD) and analyzed by FCS express De Novo Software version 3.
Splenic CD4+ T cell and CD11c+ DC isolation
Spleen cells were freshly prepared. Purified CD4+ T cells were prepared using EasySep CD4+ T cell negative section kit (StemCell Technologies Inc, Vancouver, Canada), and splenic DCs were labeled with anti-CD11c-microbeads, and then isolated by magnetic cell sorting following manufacturer’s instructions (Miltenyi). The purity of CD4+ T cells and CD11c+ DC was approximately 95%.
KLH immunization and recall response assay
NOD mice were treated with ATG or isotype IgG as described above along with simultaneous immunization by intraperitoneal injections of KLH (25 ug/mouse) in adjuvant Alum. The treated mice were sacrificed at day 22 post-ATG therapy and harvested cells were cultured with stimulators as indicated, or medium only for 4 days. Supernatants (50 ul/well) were harvested and stored for later cytokine assay. Then, 3H-thymidine (1 uCi/well) was added to the cultures and incubated for additional 16 hours. 3H-thymidine incorporation was measured as described above.
Autoantigen immunization and recall response in vitro and in vivo
NIT1 cells (NOD insulinoma cell line) were cultured according to the method provided from the vendor (ATCC). NIT1 cells (2 × 107) were suspended into 1 ml PBS. The cell suspension underwent freeze-thaw procedures 4 times to prepare NIT1 cell lysates. NOD mice (6 weeks old) were treated with ATG or isotype IgG as described above along with intraperitoneal injections of 50 μl of NIT1 lysates in 50 μl of Alum.
In vitro antigen recall response assay
A week following the last treatment, all mice were sacrificed and spleen cells prepared and stimulated with NIT1 lysates (10 ul/well), a control antigen KLH (10 ug/ml), or medium for 4 days. Supernatants (50 ul/well) were harvested and stored for later cytokine assay. Then, 3H-thymidine (1 uCi/well) was added to the cultures and incubated for additional 16 hours. 3H-thymidine incorporation was measured by scintillation counting. A portion of the spleen cells from the above mice were used for CD4+ T cell isolation. The isolated CD4+ T cells (2 × 105/well) were stimulated with splenic dendritic cells (2 × 104/well) purified from naive NOD mice in the presence of NIT1 lysates (20 μl/well). The T cell proliferation was measured by 3H-thymidine incorporation assay, as described above.
In vivo antigen recall response assay
In these experiments, we stained a portion of spleen cells prepared above with carboxyfluorescein succinimidyl ester (CFSE) following the instructions from the manufacturer (Invitrogen). Then, we adoptively transferred CFSE-labeled spleen cells (2 × 107/mouse), as prepared above, into 8-week-old NOD mice. Four days later, the mice were sacrificed and pancreatic lymph node cells, as well as cells from inguinal lymph node, were prepared and the CFSE-labeled T cell proliferation (dilution of CFSE) was examined by flow cytometry. In these experiments, we chose to use CFSE-labeled whole spleen cells but not purified CD4+ T cells because the whole spleen cells would be more reflecting the T cell behaviors post-ATG therapy.
Cytokine concentrations in culture supernatants, including IFN-γ, IL-4, IL-5 and IL-10, were measured by multiplex cytokine assay kits using Luminex 100 (Luminex Map Technology) following the instruction from the manufacturer (Millipore).
Data were analyzed using Student t testing. Differences with p<0.05 were considered to be statistically significant.
ATG therapy efficiently depletes T cells from peripheral blood, but is less efficient in depleting T cells from lymphoid organs
ATG therapy differentially depletes naive and memory T cells from the peripheral blood and spleen
ATG therapy does not affect the response of the remaining non-depleted T cells to TCR stimulation, but induces decreased levels of Th1 and enhanced levels of IL-10-producing T cells
CD4+Foxp3+ Tregs are less sensitive to ATG depletion and remain at an increased frequency post CD4+ T cell recovery
ATG therapy drives more CD62L- memory type Tregs
ATG therapy fails to eliminate antigen primed T cell, but skews antigen-specific immune responses to Th2 and/or Tr1 cells
ATG therapy fails to eliminate autoantigen-stimulated T cells
In this study, we questioned how ATG therapy affected naïve and memory T cells including Tregs with naïve or memory phenotypes. Until now, it was unclear to what extent ATG treatment affected naive and memory T cells pools. Resolving this issue is of great significance in managing ATG therapy in autoimmune diseases as well as in allogeneic transplantation. In line with the previous reports , we found that ATG therapy markedly depleted CD4+ and CD8+ T cells from the peripheral blood, and largely spared B cells and granulocytes. By day 3 post-treatment, T cell numbers reached their nadir. By day 22, CD4+ T cells recovered to within normal ranges, but CD8+ T cells remained lower than baseline. It has been suggested that ATG therapy may have differential effects in depleting T cells in peripheral blood and lymphoid organs dependent on dosing . In this study, we tested our treatment protocol with optimal efficacy in preventing or reversing type 1 diabetes (500 μg/mouse × 2 doses 3 days apart) as described in our other reports [11, 12]. To determine the efficiency of our ATG treatment protocol in depleting T cells from the lymphoid organs, we examined splenic CD4+ and CD8+ T cells in both groups (ATG versus isotype IgG) at day 3 post-ATG therapy. We found that the depletion of both CD4+ and CD8+ T cells was less efficient in spleen than from peripheral blood. The similar results were obtained in lymph nodes such as inguinal or pancreatic lymph nodes (data not shown). Of interest, it appears that ATG therapy preferentially depletes CD62L+ naive T cells from the blood because the proportion of CD62L+CD4+ naive T cells was markedly reduced while CD44+CD62L- CD4+ memory T cells as a fraction of total CD4+ T cells were increased. We observed a similar significant trend in the change of naive and memory T cells in spleen as well. Whether this differential depleting effect of ATG exhibits in local lymph nodes, especially in pancreatic lymph nodes is of interest to be further addressed. It is unlikely that the increase of memory T cells at day 3 post-ATG therapy is due to the conversion from naïve T cells [20, 21] because ATG is still depleting T cells during this short period of time post-ATG therapy, and homeostatic proliferation unlikely leads to much in the way of T cell conversion.
The relative resistance of memory T cells to ATG-induced T cell depletion would allow for survival of memory T cells which potentially could lead to the recurrence of allograft rejection or autoimmunity after reconstitution of immune system post-ATG therapy. Consistent with this, we demonstrated that the proliferation of spleen cells from mice receiving ATG and de novo KLH immunization was as high as that of spleen cells from isotype IgG treated animals in KLH recall responses in vitro. We also found that β cell antigen-primed T cells during ATG therapy could survive ATG depletion as well. However, despite unaffected T cell proliferation in response to antigen stimulation post-ATG therapy, the T cell cytokine-producing profile in ATG treated animals indicated that ATG therapy skewed Th2 and possibly IL-10-producing Tr1, and reduced IFN-γ-producing Th1 responses. We had previously shown long-term reversal of diabetes in NOD mice using ATG or ATG in combination with G-CSF [11, 12] and our current findings in this report provide a mechanistic basis for this in the skewing toward Th2 and/or IL-10-producing Tr1 responses under the regimen of ATG. In this study, although we focused our studies on CD4+ T cells, it is also important to study phenotypic and functional alterations of CD8+ T cells by the ATG therapy, given the pathogenic role of CD8+ T cells in type 1 diabetes, which will be addressed in the following future studies. The memory T cell phenotypic characteristics, as well as the functional alterations post-ATG therapy, may allow the modulated antigen-primed T cells to efficiently exert their regulatory functions in the periphery through affecting their migrating and homing capabilities, thereby preventing the recurrence of autoimmunity in autoimmune diseases and allogeneic rejection in allogeneic transplantation. These findings also implicate that ATG therapy plus antigen vaccination could lead to synergistic effect on induction of antigen-specific immune tolerance. Such information would be of great significance for developing antigen-based immunotherapeutic strategy for autoimmune diseases such as type 1 diabetes.
Prior to this effort, several mechanisms underlying ATG immune modulation have been proposed. A common belief is that ATG therapy works by T cell depletion through complement-mediated cell lysis and activation-induced cell death. However, another view regarding ATG therapy is that this agent exerts immunosuppressive function beyond that of simple T cell depletion [7, 9]. ATG therapy may modulate immune response in vivo through inhibiting chemokine-driven T cell chemotaxis . It may also influence the interaction between T cells and endothelial cells through modulating expression of adhesion molecules . Our recent study showed that ATG therapy eliminated certain subset of dendritric cells and induced tolerogenic dendritic cells . In addition, ATG therapy may facilitate tolerance induction through ATG-mediated apoptosis of T cells; because T cell apoptosis induced by anti-CD3 therapy was recently demonstrated to be associated with CD3 antibody therapy-induced immune tolerance . The skewing of antigen-specific Th2 and IL-10-producing regulatory T cells (i.e., Tr1) by ATG therapy demonstrated in the current study suggests that the non-depleted antigen-responding T cells, instead of causing immune attack, may lead to antigen-specific restoration of immune tolerance, which implies that ATG works as immune modulator rather than immune suppressant.
As suggested previously, Foxp3+ Tregs may play a major role in preventing autoimmune diabetes during ATG therapy [11, 12]. However, it is incompletely understood whether ATG therapy depletes Tregs differently than conventional T cells and how ATG affects the distribution of Tregs in different lymphoid tissues. It is also unclear whether ATG therapy affects naïve and memory Tregs differently. In the present study, we demonstrated that ATG therapy was less efficient in depleting CD4+Foxp3+Tregs and as a result, the proportion of CD4+Foxp3+ Tregs in CD4+ T cells was significantly increased in ATG treated animals compared to controls. This increase is even more dramatic in lymph nodes with greater than a doubling in the frequency of Tregs within total CD4+ T cells in ATG treated as compared to isotype IgG treated animals. In some animals, the percentage of Foxp3+ Tregs reaches 30% of total lymph node CD4+ T cells. Unlike equivalent absolute numbers of splenic Tregs in both groups, the absolute number of Tregs in lymph nodes was significantly higher in ATG than in Isotype IgG treated group at 3 days post treatment, suggesting that more Tregs were recruiting to the lymph nodes besides resistance to ATG depletion. The increase of Tregs in lymph nodes may be of great immunological significance for ATG to control local antigen-specific immune responses in the settings of autoimmunity such as type 1 diabetes, as well as in allogeneic transplantation. This increase of Tregs 3 days post-ATG therapy is unlikely due to the preferential proliferation of Tregs in the ATG-therapy induced lymphopenic animals  because the proliferation is limited in this short period of time especially still under active T cell depletion. Of interest, by day 22 post-ATG treatment when CD4+ T cells return to the normal levels, Tregs remained proportionately higher in the ATG group than in control group, which may be attributable to a faster proliferation of Tregs than conventional T cells  because Tregs possess superior capability to utilize IL-2 to conventional T cells . This may also explain why a short-term ATG therapy offers a long-term protection in type 1 diabetes [11, 12] and in allogeneic transplantation [2, 26]. Intriguingly, Tregs with memory T cell phenotype were preferentially preserved in ATG therapy, which suggests that the preserved memory Tregs specific to certain antigens would be more potent in suppressing effector T cells reactive to the same antigens. As suggested recently , the memory Tregs may home to areas with active immunological reaction to quickly exert their regulatory function preferentially to naïve Tregs. This also explains the findings in our recent report that the post-therapy Tregs gain heightened immunosuppressive capacity . There is evidence that the progression of autoimmunity in NOD mice leads to memory-like CD8+ Tregs which can be expanded in vivo by stimulation of nanoparticles coated with MHC-carried autoantigenic peptides. Of note, injection of these nanoparticles not only prevented T1D but also reversed overt diabetes in NOD mice . Thus, the quantitative and qualitative changes of Tregs post ATG therapy may play an important role in suppressing antigen-specific effector T cells. Although Tregs are generally thought to suppress T cell responses in an antigen non-specific manner, emerging evidence shows that antigen-specific Tregs are more potent in suppressing antigen-specific T cell responses [29–31]. Lu, et al. reported recently that ATG therapy indeed induced self-antigen-specific Tregs in vivo that could provide long-term T1D protection in NOD mice . Whether the increased memory Tregs post ATG therapy plus antigen challenge in our experimental settings contain more antigen-specific Tregs needs to be further explored.
ATG therapy preferentially depletes naive T cells, largely spares Tregs and alters T cell cytokine-producing profiles. Thus, ATG therapy may modulate antigen-specific immune responses through inducing memory-like regulatory T cells as well as other protective T cells such as Th2 and IL-10-producing Tr1 cells.
This work was supported in part by Juvenile Diabetes Research Foundation (17-2008-1036 to CQX), Florida State Lung Transplantation Award to CQX, and Natural Science Foundation of China (Grant #81172854 to CQX).
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