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The Journal of Immunology, 2003, 171: 6502-6509.
Copyright © 2003 by The American Association of Immunologists

Alloreactive CD4 T Cell Activation In Vivo: An Autonomous Function of the Indirect Pathway of Alloantigen Presentation1

Amy J. Reed*, Hooman Noorchashm*, Susan Y. Rostami*, Yasaman Zarrabi*, Alison R. Perate*, Arjun N. Jeganathan*, Andrew J. Caton{dagger} and Ali Naji2,*

* Harrison Department of Surgical Research, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and {dagger} The Wistar Institute, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Activation of alloreactive CD4 T cells occurs via the direct and indirect pathways of alloantigen presentation. A novel TCR/alloantigen transgenic system was designed that permitted in vivo visualization of CD4 T cell priming through these pathways. When both pathways of alloantigen presentation were intact, CD4 T cell activation in response to cardiac allografts was rapid and systemic by day 4 after transplantation, in contrast to that seen in response to skin allografts, which was delayed until 10–12 days after transplantation. Despite this systemic CD4 T cell activation in response to cardiac allografts, there was a paucity of activated graft-infiltrating CD4 T cells at 4 days posttransplantation. This finding suggests that the initial priming of alloimmune CD4 T cell responses occurs within draining lymphoid organs. Furthermore, alloantigens derived from cardiac allografts failed to promote thymic negative selection of developing thymocytes expressing the alloreactive TCR clonotype. In the absence of a functional direct pathway, the kinetics of activation, anatomic localization, and effector function of alloreactive CD4 T cells remained unchanged. Overall, the present study defines the anatomic and temporal characteristics of CD4 T cell alloimmune responses and demonstrates that CD4 T cell priming via the indirect pathway proceeds optimally in the absence of the direct pathway of alloantigen presentation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Activation of alloreactive CD4 T lymphocytes is essential for the initiation of allograft rejection (1, 2). CD4 T cell clonotypes participating in alloimmune responses are recruited via two distinct pathways of allorecognition: the direct pathway, driven by graft-derived APCs; and the indirect pathway, mediated by host APCs (1, 3, 4, 5). CD4 T cell activation via both these pathways has been implicated in the progression of alloimmune responses. CD4 T cell activation via the direct pathway of allorecognition has been demonstrated both by in vivo and in vitro mixed lymphocyte assays as well as experimental allograft rejection models (1, 6, 7, 8, 9). Activation of CD4 T cells via the indirect pathway has also been inferred from several studies demonstrating: 1) accelerated rejection of skin allografts in recipients immunized with donor allopeptides (10); 2) in vitro CD4 T cell reactivity in response to the immunodominant donor allopeptide in human recipients of cardiac allografts (11); and 3) rejection of allografts deficient in MHC class II expression (1, 12). Although these studies highlight the importance of indirect alloantigen presentation, the anatomic sites, kinetics of priming, and effector function of alloreactive CD4 T cells in vivo remain incompletely defined. In this regard, the limitation has been the inability to track the activation of naive alloreactive CD4 T cells at a clonotypic level in vivo.

In the present study, we developed an experimental model wherein activation of a clonotypically identifiable CD4 T cell population could be traced in vivo. To this end, the TS1 TCR-transgenic (Tg)3 mouse strain was used (13). This transgene encodes a MHC class II-restricted CD4 TCR clonotype specific for the MHC class II molecule, I-Ed determinant of HA, termed S1 (13, 14). The TS1 Tg mouse was used in conjunction with a novel HA Tg mouse strain, the HACII mouse, in which HA protein expression was specifically targeted to APCs expressing MHC class II (15). By using HACII mice as organ donors, hemagglutinin (HA) was used as a surrogate alloantigen to characterize the dynamics of alloreactive CD4 T cell activation in vivo after organ transplantation. This novel system has allowed four main issues to be addressed: 1) characterization of the fidelity of the TS1/HACII system for the study of in vivo alloimmune CD4 T cell responses to transplanted organs; 2) delineation of the kinetics and anatomic localization of CD4 T cell priming elicited by immediately vascularized cardiac vs delayed vascularized skin allografts; 3) determination of the impact of organ transplantation upon thymic selection of developing alloreactive CD4 T cell clonotypes; and 4) determination of the extent of in vivo CD4 T cell priming via the indirect pathway, independent of direct alloantigen presentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Mice

TS1 Tg BALB/c mice (13) and HACII Tg BALB/c mice (15) were bred and maintained in a specific pathogen-free barrier facility at The Children’s Hospital of Philadelphia. The transgenic status of the mice was determined by PCR amplification of tail DNA as previously described (13, 16). BALB/c, BALB/c SCID, C57BL/6, and C57BL/6 MHC class II-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

Flow cytometry

Cells (1–5 x 106) were stained for flow cytometry in 96-well plates and read on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed using CELLQuest software (BD Biosciences). Abs used for staining were: anti-I-Ad-FITC/PE (BD PharMingen, San Diego, CA), anti-CD4-APC and -PE (BD PharMingen), anti-CD8-FITC (BD PharMingen), anti-CD44-PE (BD PharMingen), anti-CD5-PE, 6.5-biotin (12), and anti-HA-biotin (a HA-specific IgG Ab-producing hybridoma isolated from an influenza-immunized BALB/c mouse (B62-82)). Streptavidin-Red 670 (Life Technologies, Gaithersburg, MD) was used to detect 6.5-biotin. For phenotypic analysis of HA and MHC class II expression, 100,000 live, lymphoid events were collected per sample. For analysis of CD4 T cell activation in response to HACII allografts, between 500,000 and 2,000,000 live, lymphoid events were collected per sample.

In vitro proliferation assay

Pooled cervical, axillary, inguinal, and brachial lymph node (LN) cells (5 x 104) from TS1 mice were cultured with either BALB/c splenocytes (5 x 105) and titrated doses of S1 peptide as previously described (14) or HACII splenocytes (5 x 105) in the absence of added S1 peptide. Briefly, after 48 h, cultures were pulsed with 0.5 µCi/well of [3H]dThd for 16h and then harvested.

CFSE labeling and adoptive transfer

LN cells were isolated and labeled with CFSE (Molecular Probes, Eugene, OR) as previously described (17). Briefly, LN single-cell suspensions were prepared in serum-free supplemented medium and incubated with 5 µM CFSE at 1 x 107 cells/ml for 5 min. An equal volume of FCS was then added, and cells were washed with supplemented medium. Adoptive transfer recipients were injected i.v. with 10 x 106 labeled cells (for in vivo activation studies) or 5 x 106 labeled cells (for tracing activation in response to HACII allografts). Cardiac and skin allograft recipients were grafted the day after adoptive transfer. Recipients were sacrificed at the indicated times, and lymphoid organs were harvested and stained for flow cytometry.

Skin and cardiac transplantation

All operations were performed under general anesthesia as prescribed by the Institutional Animal Care and Use Committee guidelines. For cardiac transplantation, an end-to-side anastomosis of donor and recipient aortas was performed. A venous anastomosis was also performed in an end-to-side fashion between the donor pulmonary artery and the inferior vena cava of the recipient. All anastomoses were done using 7-0 prolene sutures. For skin grafting, a square full thickness graft was harvested and stripped of the panniculus carnosus. The grafts were transplanted onto recipient mice. All allografts were monitored daily by visual and manual inspection.

HA-specific Ab ELISA

Virus-specific ELISAs were conducted as previously described (18). Briefly, 96-well flat-bottom microtiter Immunlon 1B plates (Dynex Technologies, Chantilly, VA) were coated overnight with 50 µl of purified PR8 virus (1000 hemagglutinating U/ml). Plates were washed and blocked for 1 h with 100 µl of PBS plus 1% BSA and incubated with serum samples (diluted 1/100 in PBS plus 1% BSA) for 90 min at room temperature. Plates were washed, and bound Ab was detected using alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL). Plates were developed using p-nitrophenyl phosphate, and optical densities were read at 405 nm using a microplate reader.

Bone marrow (BM) chimeras

TS1 BM chimeras were generated by injecting 10 x 106 T cell- and B cell-depleted BM cells from TS1 mice i.v. into BALB/c SCID recipients. T and B cell depletion was conducted using the VarioMACS system (Miltenyi Biotec, Sunnyvale, CA). After reconstitution, all mice were housed in a pathogen-free animal facility at the University of Pennsylvania Medical Center (Philadelphia, PA) for 10 days, at which point the experimental mice received HACII heart grafts. Mice were sacrificed 2 wk later.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Expression of HA by APC in HACII Tg mice

HACII mice express HA under the control of a MHC class II promoter that has been previously shown to direct expression of transgenes to MHC class II-bearing APCs (19). Using a HA-specific Ab, the surface expression of whole HA protein on MHC class II+ cells in HACII mice was assessed by flow cytometry (Fig. 1A). In both the BM and spleen of HACII mice, HA was expressed on the surface of MHC class II+ but not MHC class II- cells. No staining was seen on cells from non-Tg littermates. In vitro analyses revealed whole HA being expressed by both B cells and CD11c+ dendritic cells (data not shown).



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  		FIGURE 1. A, Expression of surface HA in HACII mice. BM cells and splenocytes from HACII mice and non-Tg littermates were stained with Abs to HA and MHC class II and analyzed by flow cytometry. B and C, T cell responses to HA as an alloantigen. B, In vitro proliferation assay measuring [3H]dThd incorporation of TS1 LN cells cultured with either titrated doses of S1 + non-Tg splenocytes (as a source of APCs; ——) or splenocytes from HACII mice without the addition of exogenous S1 (red bar). T cells cultured with HACII APCs alone proliferated similarly to those cultured with 1 µM S1 peptide and non-Tg APCs. C, In vivo proliferation of CD4 TS1 T cells to HACII APCs. LN cells from either TS1 or non-Tg mice were labeled with CFSE and adoptively transferred (10 x 106 cells/mouse) into the recipients indicated. Recipient spleens were harvested 62 h later and stained with anti-CD4. R1, CD4 CFSE-labeled T cells that divided; R2, CD4 CFSE-labeled T cells that had not divided. Percentages indicate the fraction of CFSE-labeled cells that divided (% = number of cells in R1/number of cells in R1 + R2) x 100).

 
We next evaluated the extent to which APCs from HACII mice could present HA-derived peptides via MHC class II to TS1 CD4 T cells. The TS1 Tg encodes an {alpha}{beta} TCR specific for the major I-Ed peptide determinant of HA (termed S1) (14, 15). The use of this TCR Tg strain is particularly attractive given the existence of a TCR clonotype-specific mAb (termed 6.5) (14). As a first step in determining whether this system was applicable for use in the study of alloimmune CD4 T cell responses, we characterized the ability of HACII APCs to present the S1 peptide via I-Ed leading to specific activation of TS1 CD4 T cells in vitro and in vivo. In Fig. 1B, proliferation of TS1 LN CD4 T cells cultured in vitro with varying concentrations of S1 peptide (and BALB/c APCs) was compared with TS1 LN CD4 T cells stimulated directly by HACII APCs (i.e., in the absence of exogenously added peptide). TS1 CD4 T cell proliferation to HACII APCs was comparable with that seen in culture with 1 µM S1 peptide. Thus, targeting HA to MHC class II+ cells in HACII mice leads to a high degree of in vitro immunogenicity for MHC class II-restricted HA-specific CD4 T cells, reminiscent of that observed in primary mixed lymphocyte cultures.

A pattern similar to that seen in vitro was observed when TS1 CD4 T cells were CFSE labeled and adoptively transferred into HACII stimulator mice (Fig. 1C). The TS1 CD4 T cells had undergone several rounds of division in vivo when analyzed by flow cytometry 4 days after adoptive transfer. By contrast, TS1 CD4 T cells transferred into non-Tg BALB/c counterparts remained undivided. The kinetics of TS1 CD4 T cell reactivity in response to HA+ I-Ed-bearing APCs is reminiscent of that seen in the setting of alloreactive CD4 T cells stimulated with polymorphic histocompatibility alloantigens both in vivo and in vitro (6, 7, 8). Therefore, targeting expression of HA to MHC class II+ cells results in HA acting as a surrogate alloantigen when used in the context of TS1 CD4 T cells.

TS1 CD4 T cell activation after transplantation of HACII organs

First we assessed the ability of the HA-reactive TS1 Tg CD4 T cells to mediate the rejection of HACII+ cardiac and skin grafts. BALB/c recipient mice that received adoptively transferred TS1 CD4 T cells promptly rejected HACII cardiac (median survival time, 19 days; n = 4) and skin grafts (median survival time, 21 days; n = 11). It is important to highlight that the described rejection took place in response to a single surrogate alloantigenic disparity (i.e., HA), because both the TS1 and HACII transgenes have been backcrossed onto the BALB/c background for >10 generations. BALB/c mice that did not receive TS1 LN cells failed to reject HACII cardiac and skin allografts. This finding is likely attributable to a low frequency of HA-reactive T cells in the preimmune BALB/c repertoire and indicates that the rejection of HACII allografts is, indeed, HA-specific using the TS1/HACII transgenic system.

We next sought to follow the fate of TS1-alloreactive CD4 T cells in response to donor organs from HACII mice. To this end, CFSE-labeled TS1 LN cells were adoptively transferred into recipient BALB/c mice, which were subsequently transplanted with cardiac or skin allografts from HACII or non-Tg littermate donors. At several time points after transplantation, the kinetics of activation and the extent of division of the adoptively transferred HA-reactive CD4 T cells were assessed in secondary lymphoid organs of the recipient mice. Transplantation of skin and cardiac allografts from non-Tg donors did not induce division of the adoptively transferred CD4 T cells (Fig. 2B; data not shown). In contrast, HACII cardiac allografts elicited a strong TS1 CD4 T cell response that was detectable as early as 3 days after transplantation (Fig. 2A). This CD4 T cell division became clearly systemic and was detectable in all secondary lymphoid organs by day 4 after transplantation (Fig. 2A). This impressively global response is likely attributable to the immediately vascularized nature of the cardiac allografts, which could provide all secondary lymphoid organs ready access to HA epitopes. This could be accounted for either by HA proteins/peptides being directly shed into the circulation or by HACII APCs migrating out of the allograft and emigrating into the draining lymphoid organs (20, 21). In contrast to the vascularized cardiac allografts, HACII skin grafts, which undergo a delayed vascularization process, elicited a CD4 T cell response that did not develop until days 10–12 posttransplantation and was predominantly confined to the draining LN (Fig. 2B). Occasional evidence of CD4 T cell division in nondraining lymphoid organs was observed and correlated with the size of the skin graft. Importantly, the dividing CD4 T cells in the case of both HACII skin and cardiac allografts were 6.5+, indicating that the CD4 T cell division was alloantigen specific (Fig. 2C).



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  		FIGURE 2. Alloreactive CD4 T cell responses to HACII allografts. Division of alloreactive HA-specific T cells in vivo to HA-bearing or non-Tg cardiac (A) or to HA-bearing skin (B) allografts. CFSE-labeled TS1 LN cells (5 x 106) were transferred into non-Tg BALB/c mice the day before transplant. Recipients of cardiac allografts were sacrificed on days 2, 3, and 4 after transplant. Recipients of skin allografts were sacrificed 12 days after transplant. Recipient spleen, para-aortic (for cardiac allografts), cervical, axillary/inguinal (Ax/Ing; ispilateral and contralateral (Contra) LN relative to skin allografts), and mesenteric LN were harvested separately, stained for CD4 and 6.5, and analyzed by flow cytometry. C, 6.5 expression on CD4 T cells activated by HACII cardiac allograft. Rectangle, CD4+ gate which was then analyzed for 6.5 vs CFSE expression. D, CD4 vs CFSE expression on heart infiltrating lymphocytes (shown in rectangular gate). FL4-H, fluorescence.

 
We next analyzed the composition of the graft infiltrating lymphocytes isolated from the HACII heart grafts transplanted into BALB/c recipients harboring adoptively transferred TS1 CD4 T cells. Despite the presence of a vigorous and systemic CD4 T cell response in secondary lymphoid organs at day 4 posttransplantation, very few, if any, 6.5+ CD4 T cells were recovered from the allografts. Notwithstanding, the majority of the TS1 CD4 T cells found within the HACII allografts were in an undivided state (Fig. 2D) despite the presence of systemic CD4 T cell activation. This latter finding suggests that the initial priming of CD4 T cells after transplantation of an immediately vascularized organ occurs in the host secondary lymphoid organs and not within the allograft itself. This conclusion is supported by the inability of mice lacking secondary lymphoid organs to reject cardiac allografts (22). Furthermore, an elegant series of experiments by Barker and Billingham (23) have demonstrated that interruption of afferent lymphatics prevents allograft rejection. These phenomena are attributable to insufficient alloreactive T cell priming in the draining lymphoid organs of the recipient.

In the above experiments, the CD4 T cell response to HA as an alloantigen was targeted against a single epitope, in contrast to the polymorphic nature of histocompatibility Ags targeted in the setting of allotransplantation. Thus, we sought to determine the characteristics of HA-reactive CD4 T cell activation when the HA Ag was nested within a full MHC Ag disparity. To this end, cardiac allografts from BALB/c x C57BL/6 (H-2d x b) HACII Tg mice were transplanted into wild-type BALB/c (H-2d/d) recipients, which had received adoptively transferred CFSE-labeled TS1 CD4 T cells. In Fig. 3, we quantified the percentage of divided TS1 CD4 T cells in mice that had received either H-2d x b HACII or H-2d HACII cardiac allografts. The response of the adoptively transferred TS1 CD4 T cells to the HA alloantigen by day 4 was accelerated when nested within a polyclonal T cell response against the H-2d x b donor haplotype, as compared with that seen in response to HA as a single surrogate alloantigen (Fig. 3). Specifically, in the case of the polyclonal alloimmune response (i.e., H-2d x b HACII donors) we observed that between 62.9% (in the spleen) and 26.6% (in axillary/inguinal LN) of the HA-reactive CD4 T cells had divided, whereas between 38.6% (in the spleen) and 11.3% (in axillary/inguinal LN) of HA-reactive T cells had divided in response to H-2d HACII grafts. Despite the occurrence of a polyclonal T cell response, however, HA-reactive CD4 T cells did not divide in wild-type BALB/c recipients of H-2d x b non-Tg hearts (data not shown), demonstrating the HA specificity of the TS1 CD4 T cell response measured in response to H-2d x b HACII hearts. This finding corroborates the validity of using HA as a surrogate alloantigen in conjunction with TS1 CD4 T cells.



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  		FIGURE 3. Kinetics of T cell division in individual lymphoid compartments on days 2, 3, and 4 after transplant of HACII BALB/c (H2d) (A) and HACII BALB/c x C57BL/6 (H2d x b) (B) cardiac grafts. S, Spleen; P, para-aortic; C, cervical; A, axillary/inguinal; M, mesenteric LN. Columns, Percent of CFSE-labeled CD4 T cells in each division peak relative to the number of total CFSE-positive CD4 T cells per organ analyzed. Italicized numbers, total percent of CD4 CFSE-labeled T cells that underwent division in each organ. SDs are indicated. Between three and six mice were analyzed per group.

 
The host Ab response to donor alloantigen(s) was also characterized using ELISA to measure HA-specific IgG in mice that received HACII grafts (Fig. 4). Because HA is a glycoprotein expressed on the surface of influenza virus A/PR/8/34 (24), a virus-based ELISA could be used to detect HA-specific Ab in the serum of the recipient mice. In the case of cardiac allografts, the anti-HA IgG response emerged at ~7 days after transplantation (Fig. 4A), whereas the response to skin allografts was not detected until day 12 and did not reach its peak until day 17 (Fig. 4B). That this Ab response to the HA alloantigen is class switched to IgG suggests that the HA-specific B cells have interacted with alloantigen (HA)-specific CD4 T cells. In this context, it may be important to consider that B cells are potent APCs in activating CD4 T cells in vivo (25, 26). Thus, B cells may participate as important APCs in the indirect pathway of allorecognition given: 1) their sizable in vivo frequency in secondary lymphoid organs; 2) ability for specific Ag uptake, via membrane Ig, and processing/presentation via the MHC class II pathway; and 3) ability to clonotypically expand after Ag-driven activation. Therefore, our laboratory is currently investigating the importance of cognate T-B collaboration via MHC class II for the progression of the CD4 T cell alloimmune responses in vivo.



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  		FIGURE 4. B cell IgG response to HA as an alloantigen. Serum from BALB/c recipients of HACII H2d cardiac (A) or skin (B) allografts were serially bled after transplant. HA-specific Abs were assessed by ELISA on influenza virus. Columns, Serum levels of HA-specific IgG at the time points indicated with SDs shown. Between 3 and 10 mice were analyzed per time point.

 
Impact of transplantation on the thymic selection of developing alloreactive CD4 T cell clonotypes

As demonstrated above, cardiac allografts induce a rapid and global CD4 T cell response. This characteristic is likely to be a reflection of the systemic distribution of alloantigenic epitopes. Thus, we sought to determine whether peripheral cardiac allografts influence the development/selection of the alloantigen-specific T cell repertoire at the thymic level. Importantly, HA-specific TS1 thymocytes have been shown to be susceptible to both deletion and anergy induction in the presence of HA as a systemic Ag (16, 27). Thus, to determine whether cardiac allografts influence the thymic selection of developing allospecific (HA-specific) CD4 T cells, we reconstituted BALB/c SCID mice with BM from TS1 Tg donors and 10–12 days later transplanted these recipients with HACII cardiac allografts. Two weeks after cardiac transplantation, 6.5+ CD4 T cells were similarly present in the thymi of mice that had received HACII cardiac allografts and those that had not (Fig. 5A). Thus, there was no evidence of thymic deletion of the developing 6.5+ CD4 single-positive (SP) thymocytes. However, the 6.5+ CD4 SP thymocytes in mice receiving HACII cardiac allografts were CD5high (Fig. 5A), indicating an increased Ag-mediated (i.e., the S1 epitope of HA) TCR signal during thymic selection in the presence of a peripheral cardiac allograft as a source of cognate Ag (28). However, this signal was clearly below the threshold for deletion and differentiation into CD25+ anergic cells, given that CD25 was not up-regulated on the developing CD4 SP thymocytes (data not shown). Moreover, in recipients of HACII heart allografts, 6.5+ CD4 T cells were expanded in the periphery relative to 6.5- T cells. Consistent with their activation by the HACII Tg cardiac allografts, these T cells were CD44high (Fig. 5B). Thus, thymic regulation did not occur despite the presence of the peripheral HACII heart graft and clear evidence of HA-specific CD4 T cell activation in the peripheral lymphoid organs of the bone marrow chimeric recipient mice. These findings suggest that peripheral allografts do not centrally tolerize alloreactive CD4 T cells and are consistent with a recent study indicating that thymic regulation of developing thymocytes occurs primarily on the basis of ectopic expression of peripheral proteins by thymic stromal cells and is, thus, self-contained (29).



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  		FIGURE 5. Influence of peripheral cardiac allografts on thymic (A) and peripheral (B) alloreactive T cell repertoire formation. BALB/c SCID mice reconstituted with TS1 BM received HACII cardiac allografts 10 days later. Control mice did not receive a graft. Two weeks after transplantation, mice were sacrificed and their thymi (A) and spleens (B) were stained for CD4, CD8, 6.5, CD5, and CD44 and examined by flow cytometry. Mice that had received HACII heart grafts (HACII graft; red lines) are compared with those that did not (No graft; blue lines).

 
TS1 CD4 T Cell activation in the absence of the direct pathway of allorecognition

Thus far, we have used organs from BALB/c HACII Tg mice transplanted into BALB/c recipients that were previously inoculated with CFSE-labeled TS1 CD4 T cells. In this setting, the HA peptides from the graft-derived APCs (i.e., passenger leukocytes) are presented by the direct pathway to the TS1 CD4 T cells. In addition, HA protein can also be shed from the surface of HACII APCs, processed, and presented by recipient APCs via the indirect pathway. Therefore, a unique attribute of this system is that a single TCR clonotype can be used to trace the activation of alloreactive CD4 T cells via either the direct or the indirect pathway depending on whether I-Ed is expressed by donor or recipient APCs, respectively. The division history presented in Fig. 2 was the result of TS1 CD4 T cell activation when HA peptide was presented by both the direct and indirect pathways of allorecognition. In view of the potency of the direct pathway of alloantigen presentation, we sought to assess the degree to which the observed systemic response to HACII+ cardiac allografts was due to direct presentation of the HA to TS1 CD4 T cells. To this end, HACII+ MHC class II-/- (i.e., I-A{beta}-/-, I-Enull) mice were generated and used as donors (Fig. 6A). APCs in HACII+ MHC class II-/- mice lack MHC class II expression but continue to express HA on their surface at a level comparable with that seen in MHC class II-sufficient HACII Tg mice (Fig. 6B). However, due to their MHC class II deficiency, the APCs of these mice are incapable of direct presentation of the HA site 1 peptide to TS1 CD4 T cells. Cardiac allografts from these HACII+ MHC class II-/- mice were transplanted into wild-type BALB/c mice harboring adoptively transferred TS1 CD4 T cells. As shown in Fig. 6, C and D, abrogation of the direct pathway of HA alloantigen presentation had little impact on the kinetics or extent of TS1 CD4 T cell division in response to cardiac allografts. This result indicates that the indirect pathway of alloantigen presentation promotes optimal CD4 T cell activation and is autonomous of the direct pathway of allorecognition. This latter conclusion challenges the proposition that indirect alloantigen presentation requires alloreactive T cell priming via the direct pathway to proceed (30). The latter conclusions have been based on results from experimental systems in which adoptively transferred T cells are subject to homeostatic expansion in the lymphopenic hosts used. This feature likely introduces a confounding element to the readout, in that the laws governing CD4 T cell activation/tolerance change in lymphopenic recipients (31). Indeed, transplantation models in which the recipient is not T cell lymphopenic but lacks a functionally competent indirect pathway have indicated that indirect allorecognition may be dominant in promoting the rejection of cardiac allografts (32). In line with this contention, the present study definitively demonstrated that CD4 T cell activation via the indirect pathway of allorecognition occurs in a vigorously autonomous fashion and does not require the presence of alloantigen presentation via the direct pathway.



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  		FIGURE 6. A, Strategy for the generation of HACII MHC class II-/- mice. Experimental design for the generation of HACII MHC class II-/- mice from HACII MHC class II-sufficient and MHC class II-/- mice. B, Flow cytometric analysis of splenocytes from non-Tg, HACII, and HACII MHC class II-/- mice for expression of B220, MHC class II, and HA demonstrating HA expression in the absence of MHC class II. C, Division of alloreactive HA-specific T cells in vivo to cardiac allografts from HACII MHC class II-/- mice 5 days posttransplantation. CFSE-labeled TS1 LN cells (5 x 106) were transferred into non-Tg BALB/c mice the day before transplant. Recipient spleen, para-aortic, cervical, axillary/inguinal (Ax./Ing.), and mesenteric LN were harvested separately, stained for CD4, and analyzed by flow cytometry. FL4-H and FL1-H, fluorescence. D, Magnitude of T cell division in individual lymphoid compartments 4 days after transplantation of HACII MHC class II-/- cardiac grafts. S, Spleen; P, para-aortic; C, cervical; A, axillary/inguinal; M, mesenteric LN. Values are the percent of CFSE-labeled CD4 T cells in each division peak relative to the number of total CFSE-positive CD4 T cells. Italicized numbers, total percent of CD4 CFSE-labeled T cells that underwent division. Between three and six mice were analyzed per time point.

 


   Acknowledgments
 
We thank Howard K. Song and Siri A. Greeley for their invaluable input.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants DK49814 and DK54215. Back

2 Address correspondence and reprint requests to Dr. Ali Naji, 4 Silverstein Pavilion, Hospital of the University of Pennsylvania, Philadelphia, PA 19104. E-mail address: ali.naji{at}uphs.upenn.edu Back

3 Abbreviations used in this paper: Tg, transgenic; HA, hemagglutinin; BM, bone marrow; LN, lymph node; SP, single-positive. Back

Received for publication July 9, 2003. Accepted for publication October 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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