In vitro growth of cytotoxic human lymphocytes. I. Growth of cells sensitized in vitro to alloantigens.

Human lymphocytes sensitized to allogeneic determinants in vitro were continuously cultured on medium prepared from phytohemagglutinin-(PHA-P) stimulated human mixed lymphocyte cultures. With such human-conditioned medium (HCM), human peripheral lymphoid cells could be grown in culture for over 90 days with a doulbing time of about 54 hr. Cytotoxic lymphocytes could be grown in culture for this time with no loss of cytotoxicity or change in cytotoxic specificity.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells: II. Sensitization against melanoma-associated antigens

Peripheral blood lymphoid cells from patients with malignant melanoma can be sensitized on allogeneic or autochthonous melanoma monolayers. Peak cytotoxicity occurred after 5 days of sensitization. Sensitization appeared to be directed against melanoma-associated antigens, as judged by the pattern of cytotoxic reactivity. Sensitized cells were cytotoxic against autochthonous or allogeneic melanoma cells, but not against autochthonous fibroblasts or allogeneic tumor cells of different histologic types. Sensitization of responder lymphoid cells from melanoma patients on allogeneic melanoma cells usually resulted in more pronounced cytotoxicity against autochthonous melanoma target cells than did sensitization on autochthonous melanoma monolayers. These results indicate that cell cultures of human malignant melanoma contain tumor-associated antigens which can sensitize human peripheral blood lymphoid cells in vitro. These results also support the concept that there are cross-reactive tumor-associated antigens in human malignant melanomas.     

In vitro sensitization of human lymphocytes against histiocytic lymphoma cell lines. I. Primary sensitization of lymphocyte subpopulations.

Peripheral blood lymphocytes from normal donors expressed spontaneous cytotoxic activity against human diffuse histiocytic lymphoma cell lines. In the unfractionated state, they could not be further sensitized in vitro against these cell lines. By applying cell separation techniques before culture, subpopulations of lymphocytes were obtained which could be sensitized in vitro and manifested cytotoxic activity against human histiocytic lymphoma cells. Three methods of separation were found effective: E rosette enrichment; elimination of Fc receptor positive cells; and removal of nylon wool adherent cells. Under these conditions, cross-reactive cytotoxicity was observed against non-neoplastic lymphoblastoid cell lines, but not against normal lymphocytes.     

Generation and characterization of purified adherent lymphokine-activated killer cells in mice

During the incubation of murine spleen, lymph node, or bone marrow cells with IL-2 (1000 U/ml) a small percentage of cells became adherent to the surface of plastic tissue culture flasks. After removal of the non-adherent lymphoid cells, plastic adherent lymphokine-activated killer (LAK) cells could be efficiently expanded in the presence of IL-2. Plastic adherent-derived A-LAK cells were characterized by high rates of proliferation and their cytotoxic activity was more than 10 fold higher than LAK cells generated in the bulk (unfractionated) spleen cell cultures. A-LAK cells could be continuously generated from the non-adherent cell population. Using multiple transfers (every 1 to 2 days) of non-adherent LAK cells into new flasks, new rounds of plastic adherent cells were generated with high expansion capability and high levels of cytotoxic activity. Morphologically, A-LAK cells were large granular lymphocyte and phenotypically expressed markers characteristic of NK cells (asialo GM1+, NK1.1+, Qa5+, Ly-6.2+, Thy-1.2+, but negative for Lyt-2.2 and L3T4). A-LAK cells generated from mice of different strains expressing low and high levels of NK cell activity were equally highly cytotoxic. However, A-LAK cells obtained from nude or beige mice had relatively lower levels of cytotoxicity. Stimulation of NK cell activity by poly I:C or inhibition by in vivo or in vitro treatment with anti-asialo GM1 serum did not affect the generation of A-LAK cells. A-LAK cells derived from spleen or bone marrow of C57BL/6 or nude mice treated with anti-asialo GM1 serum were found to be asialo GM1+ suggesting that A-LAK cell could be generated from the asialo GM1- precursor cells. Expansion of plastic adherent A-LAK cells in the presence of IL-2 could provide large numbers of highly purified cytotoxic A-LAK cells suitable for cancer immunotherapy.     

In vitro growth of murine T cells. I. Production of factors necessary for T cell growth.

Supernatants of murine lymphoid cultures stimulated with Concanavalin A contain factor(s) capable of sustaining continuous growth of murine lymphoid cells in vitro. Methods for the optimal generation of these growth factors (GM) have been defined, including number of cells required, optimal concentration of Concanavalin A, incubation time, and strain differences in GM production. With GM, cells have been grown for over 15 10-fold generations over a 2-month period. The growing cells express the theta surface marker and do not express surface Ia antigens.     

Methods for amplifying the induction and expression of cytotoxic response in vitro to syngeneic and autologous freshly-isolated solid tumors of mice

Summary Spontaneously arising tumors are frequently poorly immunogenic and exhibit a limited capacity to induce cytotoxic effector lymphocytes. In the present study, various approaches have been used to amplify the induction and expression of cytotoxic responses in vitro toward freshly isolated, autologous, and syngeneic solid neoplasms of spontaneous origin in mice. Cytotoxic lymphocytes were generated in one-way mixed lymphocyte-tumor cell cultures (MLTC) consisting of splenocytes or lymph node cells from normal and from tumor-bearing mice co-cultured with inactivated tumor cells. Optimal culture conditions have been established for the number of responder (R) cells, the method of inactivation of the stimulating (S) tumor cells, the responder/stimulator (R/S) cell ratio, and the duration of sensitization. Under optimal sensitization conditions only weak cytotoxic responses, as measured by the ⁵¹Cr-release assay, were generated. The antitumor cytotoxic activity could be augmented 2- to 12-fold by using each of the following procedures: (a) addition of crude or of partially purified interleukin-2 (IL-2) to the sensitization cultures; (b) depletion of nylon-adherent cells from the responding cell population; (c) enrichment of large lymphoblasts from the sensitized effector cell population by Percoll density gradient; and (d) treatment of mice donating the responder lymphocytes with low doses of either cyclophosphamide, adriamycin, or indomethacin. Although the highly reactive effector cells generated under the improved conditions also reacted appreciably with unrelated tumor target cells, only low levels of cytotoxicity could be demonstrated against normal target cells. The antitumor cytotoxic cells in sensitized splenocyte cultures were exclusively Thy1⁺, Lyt1^–2⁺, whereas in lymph node cell cultures some cytotoxicity was also exerted by Thy1⁺, Lyt1⁺2⁺ cells.     

In vitro elicitation of cytotoxic response against a nonimmunogenic murine tumor by allosensitization

Summary The murine lymphoma (thymoma) PIR-2 of C57BL/6 origin, primarily induced in our laboratory by fractionated X-ray irradiation, has been shown to be nonimmunogenic by its failure to immunize syngeneic mice in vivo or to evoke a cytotoxic response in primary mixed lymphocyte-tumor cell cultures (MLTC) in vitro. We were able, however, to demonstrate the existence of anti-PIR-2 cytotoxic cells among allogeneic-primed C57BL/6 responding lymphocytes using the technique of limiting dilution cultures (LDC). The frequency of anti-PIR-2 cytotoxic cells among C57BL/6 lymphocytes sensitized against BALB/c splenocytes in mixed leukocyte culture (MLC) was 1/20 to 1/40, and the cytotoxic activity of positive LDC wells against PIR-2 reached 60% as determined by a 4-h ⁵¹Cr-release assay. The frequency of anti-PIR-2 cytotoxic cells could be increased two- to 10-fold (up to 1/4) by removing nylon-wool-adherent cells from the primed cell population and/or by enriching the primed lymphoblast population on a Percoll density gradient. Anti-PIR-2 cytotoxic cells were found to be Thy1⁺; Lytl^–2⁺ cells. Clones isolated from the LDC wells manifested strong cytotoxic activity toward PIR-2 cells and the stimulating BALB/c splenocytes but not against other H-2^b tumor lines or C57BL/6 splenocytes. We suggest that the procedure of allostimulation in MLC-LDC is an effective in vitro means of generating highly reactive cytotoxic cells against poorly immunogenic neoplasms.     

Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro

An in vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in vivo rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4 °C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement.Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and ⁸⁹Sr both are able to prevent rejection of marrow allografts in vitro. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with ⁸⁹Sr were effectively cytotoxic but lost practically all of their cytotoxicity afer incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in vivo during marrow allograft rejections.     

Continuously proliferating T killer cells specific for H-2b targets: selection and characterization.

BALB/c (H-2d) thymus-derived lymphocytes sensitized to C57BL/6 (H-2b) alloantigens have been propagated in vitro for over 9 months. These T lymphocytes are specifically cytotoxic to H-2b target cells but are stimulated to proliferate by both H-2b and H-2k spleen cells. This indicates that for these selected cells the antigen requirements for cell proliferation are different from those for cell-mediated cytotoxicity. If not continuously stimulated with allogeneic spleen cells, the cytotoxic cultures fail to divide and rapidly lose their cytotoxic activity. Allogeneic erythrocytes do not stimulate cell proliferation in "quiescent" cell cultures and allogeneic tumor cells do so only in the presence of spleen cells. However, "quiescent" cell cultures display cytotoxicity in the presence of phytohemagglutinin A as do cell cultures which have lost their cytotoxic activity although they proliferate upon allogeneic stimulation. The significance of these findings is discussed.     

Effect of Vibrio cholerae neuraminidase on the generation of cell-mediated cytotoxicity in vitro.

Vibrio cholerae neuraminidase (VCN))12.5 units/2 X 10(6) cells/ml) continuously present for a standard 5-day MLC will significant (p less than 0.02) increase the cytotoxic activity generated by a given number of responding spleen cells without reducing the specificity. Heat-inactiviated VCN produced no such augmentation. This augmented cytotoxicity could be reproduced by preincubating (1 hr) the responding spleen cells with VCN (25 units/2.5 X 10(6) cells/ml) before addition of stimulating spleen cells. Preincubating the stimulator spleen cells with VCN had no effect. VCN preincubation of target cells or presensitized effector cells produced no augmentation. The addition of soluble VCN to the killing assay also did not increase cytotoxicity. Thus, VCN acts only during the generation of specifically sensitized cytotoxic T cells. When the effect of VCN on MLC reactivity, cell recovery and total cytotoxicity (lytic units/10(6) cells) were compared, it became apparent that VCN increases the proliferation of responder cells after stimulation resulting in both an increased number of cells and also an increase in the proportion of specifically sensitized cytotoxic cells in the culture. VCN treatment of responder cell membrane apparently permits a more ready response to allogenic antigens in culture facilitating both increased proliferation and the increased development of specific cytotoxic killers.     

Induction of LAK-like cells in the peritoneal cavity of mice by inactivated Candida albicans

We have investigated the effect of multiple administrations of inactivated Candida albicans (CA) cells on induction of non-MHC-restricted antitumor cytotoxic responses both in normal and congenitally athymic (nude) mice. Intraperitoneal inoculation of CD2F1 mice with five doses of 2 x 10(7) CA cells over a 2-week interval was associated with the induction of peritoneal exudate cells (PEC) that mediated natural killer cell activity. These cells, in contrast to those elicited by a single dose of CA, killed both NK-sensitive and NK-resistant tumor target cells in vitro. This broad-spectrum, antitumor cytotoxicity peaked 1 day after the last injection of CA, and decreased to control values within 6 (NK-resistant) or 14 (NK-sensitive target cells) days. Cytotoxicity could be recalled to a high level by a boosting injection of CA or a major mannoprotein-soluble antigen (MP) from the Candida cell wall, given 30 days after multiple CA treatment. Upon a 24-hr in vitro incubation, CA-induced peritoneal immunoeffectors lost their killing activity unless human recombinant interleukin-2 (rIL-2) was added to cultures. The non-MHC-restricted cytotoxic PEC activity induced by CA was mainly associated with nonadherent, nonphagocytic large granular lymphocytes (LGL) which exhibited the following phenotypes: (i) asialo GM1+, Lyt 2.2-, and partially Thy 1.2+ (effectors active against NK-sensitive targets) and (ii) asialo GM1+, Lyt 2.2-, and Thy 1.2+ (effectors active against NK-resistant targets). Nude mice also responded to multiple CA inoculations by displaying high cytotoxic activity against NK-sensitive targets and significant cytotoxicity against NK-resistant targets. This cytotoxicity could be recalled on Day +30, and the cytotoxic effectors involved were highly sensitive to anti-asialo GM1 plus complement treatment. Overall, the results add further experimental evidence to the wide range of immunomodulatory properties possessed by C. albicans, and demonstrate that the majority of antitumor cytotoxic activity induced by fungal cells was due to lymphokine-activated killer (LAK)-like effectors.     

Synergy between subpopulations of normal mouse spleen cells in the in vitro generation of cell-mediated cytotoxicity specific for "modified self" antigens.

Responding lymphoid cells cultured in vitro with irradiated trinotrophenyl (TNP)-modified syngeneic spleen cells develop direct cell-mediated cytotoxicity which is specific for target cells bearing both the TNP moiety and histocompatibility determinants of the modified sensitizing cell. Two subpopulations of normal mouse spleen cells have been shown to synergize in the in vitro generation of specific cell-mediated cytotoxicity to these "modified self" antigens. The synergizing populations are nylon wool column-adherent and column-nonadherent fractions of normal mouse spleen. When mixtures of these two cell populations are cultured in vitro with irradiated TNP-modified syngeneic spleen cells, greater cytotoxicity is generated in the two populations sensitized separately. The synergizing cell in the column-adherent population is resistant to lysis by rabbit anti-mouse brain serum, is distinct from the cytotoxic effector T lymphocyte, and is unresponsive to phytohemagglutinin; its synergizing function could not be replaced by peritoneal cells. These results suggest that it is a non-T cell which may be distinct from the macrophage.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

Cytotoxicity of allogeneic lymphocytes sensitized against H-2 antigens in vitro

A system is described in which C57/Bl lymphocytes can be sensitized in vitro against H-2 alloantigens of DBA/2 fibroblasts. Cytotoxicity of sensitized lymphocytes was measured by ⁵¹Crrelease from DBA/2 mastocytoma cells which were used as sensitive target cells. During the sensitization period one can observe lymphoid blast transformation and proliferation to start from the third day. Optimal cytotoxic effect of sensitized lymphocytes is reached on the fifth day. C57/Bl lymphocytes sensitized on C₃H fibroblasts were found not to be cytotoxic to DBA/ 2 mastocytoma cells.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

Killer cells of feline leukemia virus- and feline sarcoma virus-infected transformed cells: the role of NK, ADCC, and in vitro generated cytotoxic cells

Feline white blood cells (WBC) manifested a primary in vitro mixed lymphocyte tumor cell culture (MLTC) proliferative response to feline leukemia virus-feline sarcoma virus (FeLV-FeSV)-infected transformed target cells, which reached a peak at Day 15. Furthermore, primary in vitro MLTC cultures generated cytotoxic killer cells capable of killing a variety of targets in non-major histocompatibility gene complex restricted fashion, and effector cells were capable of killing targets introduced repeatedly into cultures over a 49-day period. The presence of feline fibrosarcoma (f-sarc) stimulators was the primary driving force for proliferation and generation of killing because exogenous IL-2 conditioned medium did not appreciably increase the yield of killer cells generated in vitro. WBC cultured without f-sarc stimulators with or without IL-2 supplementation also generated killer (K) cells but at a low level. The killer cell population was composed of approximately 50% lymphocytes and 50% monocytes. Cats had K cells functional in antibody-dependent cell-mediated cytotoxicity against FeLV-coated chicken red blood cells but not against any FeLV-FeSV-infected transformed targets tested. Natural killer (NK) cell activity to any targets tested was not found. Although no evidence was found for K or NK cell activity against FeLV-FeSV-infected transformed cells, feline WBC were readily able to generate killer cells in vitro and it is probable that this cell-mediated immune potential is functionally important in vivo.     

In vitro sensitization and expansion with viable tumor cells and interleukin 2 in the generation of specific therapeutic effector cells

We have investigated the efficacy and immunologic characteristics of immune effector cells generated from cultures containing large numbers of viable tumor cells and interleukin 2 (IL 2) in the adoptive immunotherapy of experimentally induced pulmonary metastases from the newly developed, weakly immunogenic MCA 105 sarcoma in mice. The current culture conditions allowed increases of either normal or MCA 105 immune spleen cells up to 94-fold in 15 days. The in vitro expanded normal and MCA 105 immune cells displayed nonspecific in vitro cytotoxicity against several syngeneic tumor targets. However, therapeutically effective cells could only be obtained from cultures initiated with MCA 105 immune spleen cells. Immunotherapy with expanded immune effector cells could lead to the reduction of established 3 day pulmonary metastases, prolongation of survival, and cure of tumor in the majority of animals. The generation and proliferation of therapeutic effector cells in vitro depended on the presence in cultures of specific tumor stimulator cells as well as the presence of IL 2. Although immunotherapy with either fresh noncultured or secondarily in vitro-sensitized (IVS) MCA 105 immune spleen cells was immunologically specific, the efficacy of the adoptive cellular therapy with cultured but not fresh immune cells could be improved by the administration to tumor-bearing hosts of exogenous IL 2. In addition to numerical expansion, the IVS immune cells, on a per cell basis, afforded an eightfold to 10-fold increase in therapeutic efficacy when compared with fresh noncultured MCA 105 immune cells. Our results indicate that the current culture procedure induced in vitro antigenic stimulation and expansion of tumor-specific immune effector cells that was otherwise not possible by conventional mixed lymphocyte-tumor cultures.     

Generation of suppressor lymphocytes during sensitization in culture against a syngeneic tumor: affinity chromatography on insolubilized histamine

We investigated the effect of depletion of histamine-binding lymphoid cells on immunological properties of lymphocytes sensitized in culture against tumor cells. C57BL/6 spleen cells that were sensitized in vitro on monolayers of the syngeneic Lewis lung carcinoma (3LL) became cytotoxic to the tumor cells in vitro after 3 to 5 days of sensitization. Sensitized cells harvested after 4 days of sensitization occasionally enhanced tumor growth in vivo. Fractionation of the sensitized lymphocytes over insolubilized histamine-rabbit serum albumin-Sepharose (HRS) columns decreased or abolished the enhancing activity in vivo and specifically increased the in vitro cytotoxic activity of the depleted lymphocytes. A similar increase in the cytotoxic activity of HRS-fractionated cells was observed in an allogeneic combination of C57BL spleen cells sensitized against C3H fibroblasts. The effect of HRS chromatography on the in vitro cytotoxic activity increased with prolonged incubation of the depleted effector cells with the target cells.     

Natural cell-mediated cytotoxicity in vitro and inhibition of tumor growth in vivo by murine lymphoid cells cultured with T cell growth factor (TCGF)

Summary Lymphoid cells obtained from the spleen, thymus, bone marrow, peripheral blood, and peritoneal exudate of normal mice (BALB/c, BALB/c nude, C57BL/6, C3H) and from spleens of mice bearing a transplantable lung carcinoma or primary mammary carcinoma were expanded in culture for 1–9 months, with an increase in cell number of 10⁵- to 10⁶-fold per month, in crude or lectin-depleted medium containing T cell growth factor (TCGF). All these cultured lymphoid cell (CLC) lines exhibited strong cytotoxic activity in vitro (assessed by ⁵¹Cr-release assays) toward a variety of freshly harvested and cultured syngeneic, allogeneic, and xenogeneic tumor target cells, both lymphoid and solid (including metastatic growths) in origin. Extensive killing was observed against tumor targets that were resistant to lysis by natural killer (NK) cells as well as to NK-sensitive tumor lines. Low levels of cytotoxic reactivity were also demonstrated against fresh and cultured normal lymphoid cells. The CLC had some characteristics of NK cells but also expressed some typical T cell markers. In local Winn-type neutralization assays, CLC delayed or completely inhibited the growth of lymphomas and carcinomas in syngeneic and allogeneic recipients. In mice with metastatic growth of a second-generation transplant of mammary carcinoma, CLC were shown to have some therapeutic effect when administered IV 1 day after cyclophosphamide. No significant beneficial action of IV administered CLC was observed in the absence of chemotherapy in mice implanted with a lung carcinoma. The possibilities of employing TCGF-propagated cytotoxic effector cells in adoptive immunotherapy of human malignancies are discussed.     

Virus specificity of human influenza virus-immune cytotoxic T cells.

The virus specificity of human in vitro cytotoxic T cell responses to influenza virus was studied with the use of peripheral blood mononuclear leukocytes from normal adult volunteers. Previous natural exposure of these donors to a variety of type A influenza viruses was documented by HI antibody titers. Cells sensitized in vitro with A/HK or A/PR8 were cytotoxic for autologous target cells infected with A/HK, A/PR8, or A/JAP 305 type A influenza viruses, but not for B/HK-infected or uninfected cells. B/HK-sensitized effector cells lysed target cells infected with B/HK but not targets infected with type A viruses. A/HK- and A/PR8-immune effector populations were shown to recognize cross-reactive antigens on A/HK- and A/PR8-infected target cells by cold target competition. Influenza-immune effector cells were cytotoxic for virus-infected autologous targets but much less so for virus-infected allogeneic targets. This self-restriction suggested that the cytotoxicity was largely T cell-mediated and was confirmed by cell separation analysis. Thus, the human secondary cytotoxic T cell response in vitro to influenza viruses is predominantly directed against cross-reactive determinants on cells infected with serologically distinct type A influenza viruses.