CD4 cytotoxic T lymphocyte differentiation

In vitro allostimulated CD4+ human lymphocytes were cloned by micromanipulation and expanded for a short time in IL-2 conditioned medium. In the present study we observed that proliferative noncytotoxic cloned cells were able to acquire the specific cytolytic activity under some modification of the cloned cells restimulation cycle. We demonstrated that rIFN-alpha and -gamma are the agents responsible for the acquisition of specific lytic activity.     

IL-10: a novel cytotoxic T cell differentiation factor

A previous report concluded that a new cytokine, designated IL-10, is a growth cofactor for thymocytes, spleen, and lymph node cells. In this report, we have focused on the effects of IL-10 on CD8+ spleen T cells. We first observed that IL-10 enhances the growth of CD8+ T cells to IL-2. We then investigated the effect of murine rIL-10 on the induction of murine effector CTL from CTL precursors (CTL-p) using both bulk and filler cell-free limiting-dilution cultures. IL-10 alone could not induce Con A-activated FACS-sorted CD8+ T cells either to proliferate or to generate effector CTL. In combination with IL-2, however, IL-10 augmented the cytolytic activity of effector CTL generated from Con A-activated spleen CD8+ T cells in bulk cultures incubated for 5 days. In limiting-dilution cultures (using solid-phase anti-CD3 mAb as stimulus), IL-10, in combination with IL-2, substantially increased the CTL-p frequency and augmented the cytolytic activity per clone expanded from one CD8+ T cell when compared with cells cultured in IL-2 alone. Kinetic studies showed that IL-10 is required at both early and late culture stages for optimal generation of effector CTL. The potentiating effects of IL-10 on CTL function were neutralized by an anti-IL-10 mAb. These results indicate that IL-10 has direct effects on mature T cells, and suggest that IL-10 also functions as a cytotoxic T cell differentiation factor, which promotes a higher number of IL-2-activated CTL-p to proliferate and differentiate into effector CTL. In contrast, IL-10 did not enhance significantly the lymphokine-activated killer cell activity of IL-2-grown CD8+ cytotoxic T cells.     

Neurotransmitter suppression of the in vitro generation of a cytotoxic T lymphocyte response against the syngeneic MOPC-315 plasmacytoma

We have previously shown that, as a consequence of low-dose melphalan (l-phenylalanine mustard (l-PAM) therapy, the hitherto immunosuppressed spleen cells from BALB/c mice bearing a large MOPC-315 tumor (in contrast to spleen cells from normal mice) acquire the ability to generate a greatly enhanced anti-MOPC-315 cytotoxic T lymphocyte (CTL) response upon in vitro stimulation with MOPC-315 tumor cells. Here we show that the catecholamines norepinephrine, epinephrine, and isoproterenol suppressed the in vitro generation of anti-MOPC-315 cytotoxicity by spleen cells from mice that had just completed the eradication of a large MOPC-315 tumor following low-dosel-PAM therapy (l-PAM TuB spleen cells), as well as by spleen cells from normal mice. In contrast to the marked suppression obtained with catecholamines, the cholinergic agonist carbachol had no effect on the in vitro generation of splenic anti-MOPC-315 cytotoxicity. The inhibitory effect of the catecholamines was mimicked by the membranepenetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The -adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal orl-PAM TuB spleen cells. Since the curative effectiveness of low-dosel-PAM therapy for MOPC-315 tumor bearers requires the participation of CD8⁺ T cells that exploit a CTL response in tumor eradication, it is conceivable that norepinephrine may reduce the therapeutic outcome of low-dose chemotherapy by inhibiting the acquisition of CTL activity.     

Augmentation of generation of human allospecific cytotoxic T lymphocyte by PPD in in vitro sensitization culture

Summary PPD augmented human lymphocyte blastogeneic response to allogeneic lymphocytes in the mixed lymphocyte reaction (MLR) and generation of human cytotoxic lymphocytes against allogeneic human lymphocytes in in vitro sensitization (IVS) culture. The augmenting effect of PPD in the MLR was unequivocally synergistic at its lower concentrations (0.05 and 0.01 g/ml). The augmentation of MLR was observed following addition of a supernatant of culture medium of lymphocytes which had been precultured with PPD for 24 h then washed free of PPD and recultured without PPD for another 24 h. PHA and Con A, in contrast, suppressed both MLR and the generation of alloreative cytotoxic cells. The alloreactive cytotoxic lymphocytes whose generation was augmented by PPD belonged to the SRBC-rosette forming fraction and passed through a nylon-wool column. The NK cell-like activities of the alloreactive cytotoxic lymphocytes were not augmented by PPD. Analysis of the alloreactive cytotoxic lymphocytes whose generation was augmented by PPD by competitive inhibition assay with unlabeled cells indicated that the same allogeneic lymphocytes used as sensitizing cells in IVS culture inhibited the cytotoxicity, while MOLT-4 cells, which are frequently used as target cells for the human NK-cell assay, did not. When lymphocytes with known HLA-A and HLA-B were used in the IVS culture and the cytotoxicity assay, PPD was found to augment the cytotoxicity only against the target lymphocytes that possessed the same HLA as the sensitizing lymphocytes in IVS.     

Production of a human T lymphocyte chemotactic factor by T cell subpopulations

Concanavalin A-stimulated human peripheral blood mononuclear cells release a lymphocyte chemotactic factor. This lymphocyte chemotactic factor is produced optimally after 24 to 48 hr of culture and is not found before 3 hr of culture, which suggests that the factor is synthesized de novo and is not preformed and secreted after Con A stimulation. This is further supported by experiments showing that the protein synthesis inhibitors cycloheximide and puromycin totally prevent the production of the chemotactic factor. Experiments using cultured and uncultured T lymphocytes as responding cells show that cultured T cells respond more efficiently than uncultured T cells to this factor. Furthermore, the lymphocyte chemotactic factor preferentially stimulates T lymphocyte locomotion as compared to peripheral blood non-T lymphocyte migration. Fractionation of mononuclear cells into glass nonadherent lymphocytes, monocyte-enriched preparations, T lymphocytes, and non-T lymphocytes shows that lymphocyte chemotactic factor is produced by Con A-stimulated, glass nonadherent lymphocytes and T cells but not by monocytes or non-T lymphocytes. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T cell subpopulations shows that the production of T lymphocyte chemotactic factor can be attributed to the Leu-2 suppressor/cytotoxic T cell subset. The generation of a T lymphocyte chemotactic factor by Leu-2 T cells may represent a means of recruiting other T cells to the site of its release.     

Production of specific macrophage-arming factor precedes cytotoxic T lymphocyte activity in vivo during tumor rejection

Summary Recently we published a hypothesis on the immunological events occurring during tumor rejection. One of the implications of this hypothesis is that specific macrophage-arming factor (SMAF) is produced early during the initiation of the immune response, whereas the classical cell-mediated immune response components, such as cytotoxic T lymphocytes (CTL), are produced later, that is, during the amplifier-effector phase. In this paper we establish the kinetics of the induction of (a) lymphocytes producing SMAF and (b) CTL. Groups of DBA/2 mice were injected i.p. once, twice or three times with irradiated and/or non-irradiated syngeneic SL2 tumor cells, the injections being given at intervals of 10 days. After each of these injections the production of SMAF and the expression of CTL activity were established. The results showed that in the peritoneal cavity SMAF-producing lymphocytes appeared earlier than cytotoxic lymphocytes (CTL). In addition, it was shown (a) that SMAF does not interfere with the in vitro cytotoxicity expressed by CTL and (b) that in addition to CTL memory cells, SMAF-producing memory cells were also induced after injection of syngeneic tumor cells. These data support the hypothesis that SMAF is involved in the early phase of the cellular immune response against tumors, whereas CTL are induced later.     

Production of macrophage activating factor by human leukemic T cell lines

Human leukemic T cell lines were tested for their ability to produce a macrophage activating factor. When mouse peritoneal macrophages were cultured for 48 hr in the presence of culture supernatants from cell lines HPB-ALL, CCRF-CEM, or MOLT-4, glucose oxidation via the hexose monophosphate pathway was enhanced by five to seven fold. Culture supernatants from cell line HPB-MLT stimulated the oxidation to a lesser extent. However, cell line CCRF-HSB-2 was essentially inactive as a producer. The active supernatants also stimulated the release of hydrogen peroxide from macrophages, whereas the inactive one did not. Since treatment of the cell lines with 12-o-tetradecanoyl phorbol acetate or phytohemagglutinin had little effect on the production of the factor except HPB-ALL, the cell lines seemed to secrete the factor constitutively. The stimulatory effect was dose-dependent and evident at a concentration as low as a 1/80 dilution. The factor was resistant to heat treatment at 100 C for 20 min, nondialysable and sensitive to protease digestion. The activating factor could be partially purified by anion exchange and gel filtration chromatographies.     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.     

Prostaglandin-mediated regulation of the mixed lymphocyte culture and generation of cytotoxic cells

We examined the role of prostaglandins or prostaglandin-producing cells in the regulation of proliferation and generation of specific cytotoxicity in one-way mixed lymphocyte cultures of mouse spleen cells. Cultures treated with indomethacin or other prostaglandin synthesis inhibitors resulted in enhanced proliferation and cytotoxicity. The level of prostaglandins produced in vitro, as measured by RIA, was 10^?8M and was found to be completely blocked by indomethacin. Adding back 10^?8M PG restored baseline (control) proliferative responses. Kinetics of the enhanced MLC response were unchanged from controls as were the specificities of the cytotoxic cells. Cells from indomethacin-treated cultures were more efficient at killing targets than those from control cultures. Prostaglandins appear to have a preferential effect on the induction of cytotoxic cells.     

The suppressor T cell induced by syngeneic splenic cell antigen down-regulates hapten-specific cytotoxic T cells by elaborating a factor inhibitory for IL2-dependent cell replication

An in vitro study has been made of the mechanism by which a suppressor T cell, that is induced in lymph nodes by a syngeneic splenic cell antigen, prevents generation of cytotoxic T cells specific for hapten-altered self antigens. When popliteal lymph node cells exposed in vivo to syngeneic splenic cells were immunized in vitro with heat-treated syngeneic TNP-coupled thymocytes and excess helper factors, the Ts remained inactive. In this condition the exposed popliteal lymph node cells routinely demonstrated approximately twice the CTL response developed by lymph node cells from normal mice. Nevertheless, when triggered in vitro by splenic antigen on either X-irradiated B or T cells, the exposed but not the normal lymph node cells exhibited reduced hapten-altered self-specific CTL responses. Furthermore, T cells within spleen cell-exposed popliteal lymph node cell populations when reexposed to splenic T cells made a factor that was found to be suppressive of CTL generation by normal lymph node cells in vitro. The nondialyzable T-cell suppressor factor (TsF) did not appear to act on lymph node precursor CTLs, nor on helper T cells but instead acted at the level of utilization of helper factors in the development of CTLs. In an examination of the effect of TsF on cellular replication, TsF was found to be nontoxic for CTLL-20, an IL-2-dependent T cell, and it did not hinder the uptake of IL-2 by receptor blockade of this cell. Nevertheless, the replication of CTLL-20 that is IL-2 driven was diminished in the presence of TsF. Similarly, TsF was found to be inhibitory for T-cell proliferation stimulated by mitogen but had no effect on a B myeloma cell proliferative response. Thus, TsF appears to act as an inhibitor of a T cell's capability to replicate despite the presence of the stimulus for replication, namely, IL-2.     

Ligation of cytotoxic T lymphocyte antigen-4 to T cell receptor inhibits T cell activation and directs differentiation into Foxp3+ regulatory T cells

Cross-linking of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 was engineered to concurrently engage CTLA-4 and cross-link it to the TCR. Cross-linking is expected to be attained via ligation of CTLA-4 first to MHCII and then indirectly to the TCR, generating a CTLA-4-MHCII-TCR trimolecular complex that forms between T cells and antigen-presenting cells during T cell activation. Treating T cells with this bispecific fusion protein inhibited T cell activation. In addition, it induced the production of IL-10 and TGF-β and attenuated AKT and mTOR signaling. Intriguingly, treatment with the bispecific fusion protein also directed early T cell differentiation into Foxp3-positive regulatory T cells (Tregs). This process was dependent on the endogenous production of TGF-β. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. These findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation.     

Constitutive production of a factor supporting B lymphocyte differentiation by a T cell hybridoma

To investigate the role of soluble T cell products during B cell differentiation more fully, we have produced T cell hybridomas by the fusion of normal helper T cells with the T cell lymphoma BW5147. In this report we describe the production of one such hybrid, 14G3, the subclone 14G3.1F2, and the functional activity of the constitutive product. The hybrid supernatant acts exclusively in antigen-nonspecific, but antigen-dependent, promotion of B cell differentiation. It is optimally effective in the presence of small amounts of EL4 supernatant. It does not itself contain any detectable IL 2 or BCGF or interferon activity, however. 14G3.1F2 activity is probably an important component of the conventional TRF preparations produced by mixed lymphocyte populations, and will be useful in further dissection of the contributions of different soluble T cell products to B cell differentiation.     

Participation of lymphocyte activating factor (Interleukin 1) in the induction of cytotoxic T cell responses

The initiation of murine alloantigen-specific cytotoxic T cell responses is macrophage dependent. The requirement for macrophages can be replaced in macrophage-depleted cytotoxic T lymphocyte assay by a soluble factor released from phorbol myristic acetate-stimulated mouse peritoneal adherent cells or P388D1 macrophage tumor cells. On the basis of the cell of origin, m.w. (15,000), and elution profiles on DEAE cellulose and polyacrylamide gels, this macrophage-replacing factor for T cell-mediated cytotoxicity was identical to lymphocyte activating factor (LAF). The stimulatory effect of LAF was totally dependent on the presence of the priming alloantigen. The results presented in this report support the hypothesis that macrophage-mediated antigen-induced T cell activation is dependent on two signals, antigen and LAF.     

Production of factors regulating macrophage motility in the early stages of mixed lymphocyte culture

The macrophage migration stimulation factor (MSF) is produced during the first hours of mixed lymphocyte culture (MLC) in the H-2 system. The activity of MSF is characterized by sharp peaks of migration stimulation in the culture fluids of allogeneic MLC. The peaks appeared every 5 hours. After 12--16 hours in the culture fluids of allogeneic MLC, there has been demonstrated macrophage migration inhibition factor whose activity rose by 48 hours. Optimal concentrations were revealed for stimulation and inhibition of macrophage migrations on the dilution of 1--2-day MLC culture fluids. The existence of the biorhythmical mechanism that controls the macrophage mobility through the balance of soluble mediators with alternative activity is suggested.     

Heterogeneous mechanisms of human cytotoxic T lymphocyte generation. I. Differential helper cell requirement for the generation of cytotoxic effector cells from CD8+ precursor subpopulations.

The subpopulations of CD8+ T cells defined by CD45RA Ag expression have been hypothesized to represent cells varying in their relative maturation along a common, activation-dependent differentiation pathway. Previous studies have shown that both the CD8+CD45RA+ and CD8+CD45RA- subsets contain precursor cells capable of developing into alloreactive CTL. In the current study, we have examined the mechanisms involved in the generation of CTL effector cells from these two CD8+ subsets. Purified CD8+CD45RA+ or CD8+CD45RA- cells were stimulated with allogeneic non-T cells, either alone or in the presence of CD4+ Th cells. Although the generation of CTL from CD8+CD45RA- precursor cells consistently required the presence of CD4+ Th cells, cytotoxic effector cells could be generated from CD8+CD45RA+ precursor cells in the absence of CD4+ cells. Several lines of evidence indicated that the helper cell-independent generation of cytotoxic effector cells from CD8+CD45RA+ precursors resulted from the unique ability of this subset to produce and use IL-2 in an autocrine fashion: 1) exogenous IL-2 could replace the effects of CD4+ helper cells for either CD8+ subset; 2) the helper cell-independent functional maturation of CD8+CD45RA+ cells could be blocked by anti-CD25 or anti-IL-2 antibodies; and 3) CD8+CD45RA+ cells produced IL-2 after activation with allogeneic cells. The finding that precursors for helper cell-independent CTL generation are restricted to the CD8+CD45RA+ subset suggests that this capability may vary as a function of the maturation of CD8+ cells.     

Homologous human macrophage hybridomas that produce a novel cytotoxic factor in their culture supernatants

Homologous human macrophage hybridoma cell lines were obtained by somatic cell fusion between peripheral blood monocyte-derived macrophages and a subclone of the myelomonocytic cell line, U937-F9. The hybridoma cell lines grown in vitro for more than a year were confirmed by manifestations of phagocytosis, adherence, nonspecific esterase, acid phosphatase, chromosome numbers and other cell surface antigens. Cell surface antigens on hybridomas were detected by flow cytometry analysis with monoclonal antibodies. With interclonal differences, a typical phenotype of hybridoma cells was CDw14+, OKM5+, Mac-1+ (equivalent to OKM1 and Mol), OKT9+, HLA-DR- and CD20+. After stimulation with lipopolysaccharide and calcium ionophore A23187, culture supernatants of clones c18A and c29A showed cytotoxic activity against human melanoma A375 Met-Mix and other cell lines which were resistant to the tumor necrosis factor, lymphotoxin and interleukin 1. This cytotoxic factor was found to be distinct from the tumor necrosis factor, lymphotoxin and interleukin 1 using the anti-tumor necrosis factor, anti-lymphotoxin and anti-interleukin 1 antisera.     

Inhibition of generation of cytotoxic T lymphocyte activity by a CCL19/macrophage inflammatory protein (MIP)-3beta antagonist

Chemokines constitute a group of over 40 secreted peptides that are important for the control of leukocyte migration both during homeostasis and inflammation. Recent studies have implicated the ligands CCL19 and CCL21 and their receptor, CCR7, in the specific migration of na?ve lymphocytes and mature dendritic cells to secondary lymphoid organs during immune homeostasis. However, the role that these molecules play during immune priming is not well understood. In this study, using CCL19((8-83)), a novel N-terminal truncation mutant, we have investigated the role of CCL19 in a primary allogeneic immune response, a response of particular relevance to transplant rejection. This antagonist specifically inhibited wild type CCL19-induced chemotaxis and intracellular calcium mobilization without affecting that of CCL21. The treatment of mice with CCL19((8-83)) did not globally inhibit the recruitment of cells into lymph nodes; however, it inhibited the generation of cytotoxic T lymphocytes toward allogeneic dendritic cells. This is the first evidence that CCL19 plays a role in immune priming.     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Monoclonal antibody against IFN-gamma inhibits Moloney murine sarcoma virus-specific cytotoxic T lymphocyte differentiation

The role of autochthonous IFN- production was evaluated in immune reactions to Moloney murine sarcoma virus (M-MSV)-induced tumors which are characterized by spontaneous regression mainly caused by virus-specific CTL activity. A functional IFN- depletion, induced by repeated administration of mAb anti-IFN- at the site of virus inoculation, prevented tumor regression in M-MSV-injected mice. Moreover, this antibody inhibited in vitro both proliferation and differentiation of M-MSV-specific T lymphocytes obtained in bulk cultures, but not growth and lytic activity of the already differentiated virus-specific CTL clone CHM-14 stimulated with rIL-2 and relevant tumor Ag. In addition, in mice receiving mAb treatment the frequency of M-MSV-specific CTL precursors, evaluated by means of limiting dilution analysis, was strongly reduced in comparison with that of control mice injected only with virus. Because CTL secrete IFN- following antigenic stimulation, the possibility that non-T effector cells recruited by this lymphokine might mediate tumor regression was also considered. Adoptive immunotherapy experiments, performed in T cell-deficient (Tx + BM) and in sublethally irradiated mice, demonstrated that transfer of CHM-14 CTL clone, which secretes IFN-, was able to counteract M-MSV tumor growth despite the local mAb anti-IFN- treatment which may have prevented host cell recruitment. Moreover, repeated local rIFN- inoculations in Tx + BM mice did not counteract M-MSV tumor progression, thus confirming that other IFN- properties such as non-T cell recruitment, antiviral or anti-proliferative IFN- activities have little or no relevance when M-MSV-specific CTL are lacking. On the whole, these results indicate that in M-MSV-injected mice, tumor enhancement after mAb anti-IFN- treatment is principally caused by impaired differentiation of virus-specific CTL precursors.