TRACKING ALLOREACTIVE CELL DIVISION IN VIVO¹

Abstract

Background. 

The study of alloimmune responses has been limited by a lack of assays that can track the behavior of alloreactive lymphocytes in vivo. Here we utilize an experimental system that allows the identification and study of alloreactive CD4⁺ lymphocytes responding to major histocompatibility antigens in vivo.

Methods. 

Responder mouse lymphocytes were labeled with a fluorescein-based dye, adoptively transferred into irradiated allogeneic stimulator mice, and recovered at serial time points for analysis by flow cytometry.

Results. 

Discrete generations of CD4⁺ responder lymphocytes proliferating specifically in response to allogeneic MHC class II were distinguished by fluorescein intensity. Successive division of alloreactive CD4⁺ lymphocytes was traced up to six generations after 60 hr.

Conclusions. 

This experimental system provides information on the division kinetics of alloreactive CD4⁺ cells. Other applications include immunophenotyping of alloreactive lymphocyte subsets. Further study of systems such as this will allow the detailed characterization of how alloimmune responses are initiated and proceed in vivo.

Alloimmunity represents a markedly polyclonal response of T lymphocytes against histocompatibility antigens expressed by transplanted tissues. CD4⁺ T lymphocytes play a central role in this response, being absolutely required in certain settings to initiate allograft rejection ⁽¹⁾. The study of the population dynamics of CD4⁺ T lymphocyte responses to alloantigens has been limited by the lack of assays that can follow the division history of proliferating lymphocytes in vivo. Primary mixed lymphocyte reaction (MLR^*) is the most commonly used assay for the study of T lymphocyte proliferation in response to alloantigens. In MLR, lymphocyte proliferation is typically measured by quantitation of tritiated thymidine incorporation, which only gives a measure of DNA synthesis ⁽²⁾. Furthermore, in vitro MLR may not recapitulate cell interactions that are important during the rejection of tissues transplanted into allogeneic hosts. Reagents with properties that allow the analysis of cell homing and division behavior in vivo are available and have been applied for the study of immune effector cell function ^(3-5). In this study, we utilized the intracellular fluorescein-based dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the division kinetics of alloreactive CD4⁺ T lymphocytes in vivo.

C57BL/6 (H2^b), C3H/HeJ (H2^k), and BALB/c (H2^d) mice (6-8 weeks of age) were purchased from The Jackson Laboratories (Bar Harbor, ME). C57BL/6TacfBr-[KO]A^bb mice, which are homozygous for a targeted disruption of the I-A^b gene and have been backcrossed onto the C57BL/6 background for five generations, were purchased from Taconic (Germantown, NY) ⁽⁶⁾. All mice were maintained under pathogen-free conditions at the University of Pennsylvania laboratory animal facility. Responder lymphocytes were isolated from lymph nodes and spleens of C57BL/6 mice and labeled with CFSE (Molecular Probes, Inc., Eugene, OR) as previously described ⁽⁷⁾. CFSE-labeled responder lymphocytes from C57BL/6 mice were adoptively transferred via tail vein injection into whole body irradiated (1800 rad) C3H/HeJ stimulator mice (30 million cells/stimulator). Whole body irradiated C57BL/6 stimulator mice were used concurrently as syngeneic controls. Splenocytes were harvested from the stimulator mice at serial time points after adoptive transfer. These cells were then incubated with appropriate concentrations of phycoerythrin-conjugated anti-CD4 monoclonal antibody (Pharmingen, San Diego, CA) at 4°C for 30 min, washed, and analyzed on a FACScan (Becton Dickinson, Mountain View, CA).

The CFSE intensity of labeled lymphocytes halves sequentially with cell division ⁽⁷⁾. Here, this property allowed the resolution of multiple generations of daughter cells resulting from the proliferation of alloreactive CD4⁺ responder lymphocytes. Figure 1 depicts the CFSE intensity of CD4⁺ C57BL/6 responder lymphocytes recovered from C3H/HeJ (Fig. 1A) and syngeneic C57BL/6 (Fig. 1B) stimulator mice 48 and 60 hr after adoptive transfer. Alloreactive C57BL/6 CD4⁺ responder lymphocytes completed up to four divisions 48 hr after transfer into C3H/HeJ stimulator mice (Fig. 1A, n>15). The division of alloreactive CD4⁺ precursors led to their progeny entering and accumulating in daughter generation peaks D1-D4. After 60 hr, the division of alloreactive C57BL/6 CD4⁺ responder lymphocytes progressed up to the sixth daughter generation. In contrast, only a small subset of CD4⁺ C57BL/6 responder lymphocytes underwent one division after transfer into syngeneic C57BL/6 stimulator mice even after 60 hr (Fig. 1B, n>15).

Figure 1: CFSE intensity of CD4⁺ C57BL/6 responder lymphocytes 48 and 60 hr after adoptive transfer into C3H/HeJ (A) or C57BL/6 (B) mice. The peak of highest CFSE intensity in each histogram represents parent generation (P) CD4⁺ lymphocytes that have not undergone division. Peaks with successively halved CFSE intensity on the log scale represent daughter generations (D1-D6) of alloreactive CD4⁺ lymphocytes that have undergone division. Only a small population of CD4⁺ responder lymphocytes divided 60 hr after transfer into syngeneic C57BL/6 stimulator mice.

A large number of fifth and sixth generation alloreactive progeny were recovered from allogeneic stimulators after 60 hr. This is attributable to at least two factors: (1) the exponential expansion of alloreactive progeny as they successively divided and (2) their sequestration in spleens of stimulator mice after activation. The latter results in enrichment of alloreactive progeny within spleens harvested for FACS analysis. In fact, Sprent et al. ^(8-10) have established that the majority of alloreactive progeny are sequestered in the peripheral lymphoid organs of irradiated hosts before their recirculation and appearance in the thoracic duct lymph.

We next wanted to confirm that the division of transferred responder lymphocytes resulted from direct recognition of allogeneic major histocompatibility complex (MHC) class II expressed by antigen-presenting cells of stimulator origin. To evaluate this, we used stimulator mice that were deficient in MHC class II (MHC class II^−/−). Because of the unavailability of MHC class II^−/− mice on the C3H/HeJ background, we utilized whole body irradiated C57BL/6 MHC class II^−/− and C57BL/6 MHC class II^+/+ stimulator mice that received CFSE-labeled BALB/c responder lymphocytes. Proliferation of CD4⁺ lymphocytes in MHC class II^−/− stimulator mice (Fig. 2A, n>5) was markedly diminished in contrast to the vigorous proliferation again demonstrated by alloreactive CD4⁺ lymphocytes in MHC class II sufficient stimulators (Fig. 2B). This finding confirms that the proliferation of alloreactive CD4⁺ lymphocytes in this system is dependent upon the direct recognition of allogeneic MHC class II of stimulator origin and does not result from their collaboration with antigen-presenting cells of responder origin. This experimental system therefore allows the study of direct allorecognition without the potentially confounding presence of indirect allorecognition. Interestingly, alloreactive CD4⁺ lymphocytes proliferating in response to MHC class II^+/+ mice in this experiment divided up to the sixth daughter generation after 60 hr as had been seen previously (Fig. 1A). However, the frequency of responding CD4⁺ lymphocytes in this strain combination was consistently lower than that seen in the C57BL/6 response against C3H/HeJ stimulators, as indicated by the lower size of daughter generation peaks relative to the parent generation peak.

Figure 2: CFSE intensity of CD4⁺ BALB/c responder lymphocytes 60 hr after adoptive transfer into C57BL/6 MHC Class II^−/− (A) or C57BL/6 MHC Class II^+/+ (B) mice. Proliferation of CD4⁺ responder lymphocytes was sharply reduced after transfer into allogeneic MHC Class II^−/− stimulators compared with MHC Class II^+/+ stimulators, confirming that the proliferation of alloreactive CD4⁺ lymphocytes in this system is largely dependent upon direct recognition of allogeneic MHC class II.

In summary, we have utilized the fluorescein-based dye CFSE to follow CD4⁺ lymphocyte allorecognition of major histocompatibility antigens. Advantages of this experimental system are that CD4⁺ lymphocyte activation and division occur in vivo and that it provides information on the division kinetics of alloreactive CD4⁺ lymphocytes. Further applications of this assay include the determination of the phenotypic composition of alloreactive T lymphocyte subsets, as well as the direct calculation of precursor frequencies of alloreactive lymphocytes. Better understanding of how alloimmune responses are initiated and proceed in in vivo systems such as described here will allow for more efficient design and testing of immunosuppressive regimens.