Virus-specific CD8+ effector T cells (eCTL) are enriched in the lungs of mice with primary influenza pneumonia, though later detection of memory T cells (mCTL) in the mediastinal lymph nodes (MLN) or spleen by peptide-based staining protocols is at the limits of flow cytometric analysis. Respiratory challenge with an H3N2 virus months after H1N1 priming induces a massive recall response, which reduces virus titers 2-3 days earlier than in nave controls. Influenza-specific mCTL produce interferon-gamma within 6 hr, but still take 4-5 days to localize to the infected respiratory tract. The delay reflects that the recall response develops first in the MLN, which contains relatively few mCTL. The response to a subdominant epitope is less obvious after secondary challenge.

No abstract

The virus-specific CD8 ؉ T cell response has been analyzed through the development, effector, and recovery phases of primary and secondary inf luenza pneumonia. Apparently, most, if not all, memory T cells expressing clonotypic receptors that bind a tetrameric complex of inf luenza nucleoprotein (NP) 366 -374 peptide؉H-2D b (NPP) are induced to divide during the course of this localized respiratory infection. The replicative phase of the recall response ends about the time that virus can no longer be recovered from the lung, whereas some primary CD8 ؉ NPP ؉ T cells may proliferate for a few more days. The greatly expanded population of CD8 ؉ NPP ؉ memory T cells in the lymphoid tissue of secondarily challenged mice declines progressively in mean prevalence over the ensuing 100 days, despite the fact that at least some of these lymphocytes continue to cycle. The recall of cell-mediated immunity thus is characterized by massive proliferation of the antigen-specific CD8 ؉ set, whereas the extent of lymphocyte turnover in the absence of cognate peptide is variable, at a low level, and can be inf luenced by intercurrent infection.The quantitation of cell-mediated immunity has been revolutionized by the recent development of tetrameric complexes of MHC class I glycoprotein ϩ peptide (tetramers) for the direct staining and subsequent flow cytometric analysis of virusspecific CD8 ϩ T cells (1). The results with the tetramers (2-4) suggest that the magnitude of both the acute response and long-term CD8 ϩ T cell memory has been grossly underestimated by the earlier, microcloning limiting dilution analysis, which required cytotoxic T lymphocyte precursors (CTLp) to undergo 10-15 cycles of replication before reading out as CTL effectors in a 51 Cr release assay (4-7). However, it is still unclear whether limiting dilution analysis or tetramer staining offers a better estimate of the functional T cell memory that can be recalled after a repeat exposure to antigen (4). If, as suggested by earlier studies by using ionizing radiation (8), memory CTLp must divide further before mounting an effective secondary response, simply measuring the numbers of tetramer ϩ CD8 ϩ T cells might be illusory. Mice that first are immunized with an H1N1 influenza A virus and then challenged intranasally (i.n.) with an H3N2 virus that shares the immunodominant nucleoprotein ) epitope show a massive increase (3) in the numbers of virus-specific CD8 ϩ T cells detected by direct staining with a tetrameric complex of H-2D b ϩNP 366 -374 (NPP). The present analysis defines the extent of lymphocyte proliferation for CD8 ϩ NPP ϩ T cells recovered from the lymphoid tissue and the site of pathology in the lung, following both primary and secondary challenge with the HKx31 influenza A virus. The prevalence, and further turnover, of these virus-specific CD8 ϩ T cells then is followed into long-term memory.

Inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome) is characterized by failure of immunoglobulin isotype switching and severe defects of cell-mediated immunity. To test the potential for gene transfer therapy to correct this disorder, we transduced murine bone marrow or thymic cells with a retroviral vector containing the cDNA for the murine CD40 ligand (CD40L) and injected them into CD40L-/- mice. Even low-level, constitutive expression of the transgene stimulated humoral and cellular immune functions in these mice. With extended follow-up, however, 12 of 19 treated mice developed T-lymphoproliferative disorders, ranging from polyclonal increases of lymphoblasts to overt monoclonal T-lymphoblastic lymphomas that involved multiple organs. Our findings show that constitutive (rather than tightly regulated), low-level expression of CD40L can produce abnormal proliferative responses in developing T lymphocytes, apparently through aberrant interaction between CD40L+ and TCRalphabeta+CD40+ thymocytes. Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades.