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Summary The mixed lymphocyte reaction (MLR) has been used extensively to measure alloreactive T cells. In clinical practice, a negative MLR of recipient T cells responding to donor cells does not necessarily mean that a donor-specific tolerance has been established. This discrepancy indicates that the presently used methods fail to demonstrate the full repertoire of alloreactive T cells. This could be the result of the fact that some alloreactive T cells do not respond in vitro but will mount a response towards alloantigens in vivo, or that some alloreactive T cells do not respond during the MLR but will respond later if the alloantigen stimulation remains. We therefore examined the non-proliferating T-cell population in a mouse primary alloreactive response. Spleen and lymph node cells derived from C57BL/6J (H-2b) mice were stained with carboxy-fluorescein diacetate succinimidyl ester and injected intravenously into C.B-17 severe combined immunodeficient (SCID) mice (H-2d). The donor cells were recovered 5 d after the injection. The non-proliferating T cells were sorted and were non-reactive to alloantigen stimulation in vitro. Nevertheless, these non-proliferating T cells could proliferate and cause acute graft-versus-host disease after being adoptively transferred to secondary recipients of SCID mice. These results suggest that there exists an alloreactive T-cell population that does not respond to in vitro alloantigen stimulation but can mount a delayed alloresponse in vivo.

Acute graft-versus-host disease (GVHD) involves mainly skin, liver and intestines. Other organs such as heart, muscle and central nervous system are seldom affected, although their parenchymal cells also express alloantigens, such as MHC class I antigens. The mechanism of this selective involvement of distinct organs in acute GVHD is not well understood. We postulated that it might be related to the selective migration of activated alloreactive T cells. Indeed, T cell infiltration, revealed by examination of serial samples using flow cytometry and immunohistology, occurred early and continuously in the target organs such as the liver, but not in a non-target organ, the heart, in a murine acute GVHD model. Since T cell migration is largely controlled by the expression of chemokine and chemokine receptors, we investigated the chemokine spectrum in target/non-target organs of mice with acute GVHD. We found that in the spleen and liver MIP-1alpha, MIP-2 and Mig were the predominant chemokines expressed. In another target organ, the skin, MIP-1alpha, MIP-2, MCP-1 and MCP-3 were all highly expressed. In a non-target organ of acute GVHD, the heart, the predominant chemokines expressed were MCP-1 and MCP-3. This distinct pattern of chemokine expression in these organs may contribute to the preferential recruitment of inflammatory cells into the liver and skin, but not into the heart, in acute GVHD.

L-selectin, LFA-1 and alpha(4) integrins play important roles in the homing of naive T cells into peripheral lymphoid tissues. L-selectin- or LFA-1-deficient lymphocytes cannot effectively home to lymph nodes (LN), and antibody blockade of alpha(4) integrins also hinders lymphocytes homing. The present study was initiated to explore whether it is feasible to ameliorate acute graft-versus-host disease (aGVHD) by modulating the homing process of donor cells in the recipient in a mouse model. Using a fluorescence labeling method, we found that two monoclonal antibodies directed at L-selectin and alpha(4) integrins, respectively, when used in combination, could delay half of the donor C57BL/6J mouse spleen cells homing into the LN of recipient BALB/c mouse 15 h after injection. Spleen cells (1 x 10(7)) derived from C57BL/6J (H-2(b)) mice were injected into each C.B-17 SCID recipient mouse (H-2(d)) with or without prior incubation with 10 microg each of the two antibodies. T cell repopulation in the blood was observed in both groups of mice at a comparable level 14 days after injection of the donor cells. Eight control mice started to show aGVHD signs 7 - 14 days after the injection, and all died by day 31. However, among the ten mice that received the antibody-treated donor cells, two died before day 29, four survived between 36 and 78 days, and the remaining four survived more than 150 days, with two of them aGVHD free. It is apparent that the temporarily reduced lymphocyte homing into LN reduced the alloreactivity of the donor T cells, thus providing a simple way of modifying aGVHD. This novel approach may shed light on the prevention of aGVHD associated with clinical bone marrow transplantation.

The mixed lymphocyte reaction (MLR) has been used extensively to measure alloreactive T cells. In clinical practice, a negative MLR of recipient T cells responding to donor cells does not necessarily mean that a donor-specific tolerance has been established. This discrepancy indicates that the presently used methods fail to demonstrate the full repertoire of alloreactive T cells. This could be the result of the fact that some alloreactive T cells do not respond in vitro but will mount a response towards alloantigens in vivo, or that some alloreactive T cells do not respond during the MLR but will respond later if the alloantigen stimulation remains. We therefore examined the non-proliferating T-cell population in a mouse primary alloreactive response. Spleen and lymph node cells derived from C57BL/6J (H-2(b)) mice were stained with carboxy-fluorescein diacetate succinimidyl ester and injected intravenously into C.B-17 severe combined immunodeficient (SCID) mice (H-2(d)). The donor cells were recovered 5 d after the injection. The non-proliferating T cells were sorted and were non-reactive to alloantigen stimulation in vitro. Nevertheless, these non-proliferating T cells could proliferate and cause acute graft-versus-host disease after being adoptively transferred to secondary recipients of SCID mice. These results suggest that there exists an alloreactive T-cell population that does not respond to in vitro alloantigen stimulation but can mount a delayed alloresponse in vivo.