REVIEW

Revisiting and revising suppressor T cells Barry R. Bloom, Padmini Salgame and Betty Diamond A great deal of experimental evidence supports the phenomenon of immunological suppression. The molecular mechanisms to explain the phenomenology have, however, remained controversial. In this review, the data are reinterpreted in light of the recent advances in the understanding of T-cell subsets, the crossregulatory properties of lymphokines and the differential presentation capacities of different antigen-presenting cell types. No problem is of greater fundamental importance to immunology than tolerance, yet no realm of immunology has less credibility than that of suppressor T (Ts) cells. Reasons are neither hard to discern nor, in fact, wholly unjustified. Merely contemplating one of the hypothetical conceptualizations of the suppressor cascade of the mouse (Fig. 1) is, to say the least, a daunting exercise. Four major difficulties are encountered. First, one is confronted by a multifarious array of T-cell players, that include both CD4 + and CD8 + T cells, and a plethora of soluble antigen-specific and nonspecific factors that comprise a functionally unique network. The cascade involves antigen-specific, I-Jrestricted, CD4 + suppressor-inducers (T s 1), CD8 + antiidiotype-specific cells (Ts2), followed by CD8 + antigenspecific, effector cells (Ts3) whose suppression is not restricted by the major histocompatibility complex (MHC) L,2.Some of these cells are reported to be uniquely capable of binding directly to immobilized antigen in the absence of MHC molecules. Connectivity in this cellular cascade is mediated by a series of soluble factors (TsFs): TsF1 is idiotypic, antigen-specific, and, curiously, immunoglobulin heavy chain variable region (Ig VH) restricted; TsF2 is anti-idiotypic and requires delivery by an armed macrophage; and TsF3 acts totally nonspecifically. TsF1 and TsF2 require antigen-presenting cells (APCs), but TsF3 does not. The second problem is the mystery of I-J, which in congenic mouse strains represents a genetic restriction element for suppression, mapping between E(x and EIB.L From DNA sequence data of that region of the MHC, no sufficiently large uncharted portion of chromosome 17 is available to encode a unique 1-J polypeptide 4. Nor, after prodigious effort, has there been any molecular characterization of an I-J molecule that discriminates between the appropriate congenic strains. Third, and perhaps even more devastating, are molecular studies of antigen specific, murine T-T suppressor hybridomas. These studies revealed that half had deleted T-cell receptor (TCR) 6 genes and the remainder had unrearranged TCR (x or 13genes, and hence would be incapable of producing a functional receptor protein 5. Fourth, of the soluble TsFs described as having extraordinary properties, for example specificity for antigen and I-J or Ig Vn, until recently no two had similar properties, and over a period of 20 years not one had been convincingly characterized at a molecular level.

In a recent review of T-cell suppression in infectious diseases, and ot peripheral tolerance in general% we found the evidence so compelling that much of the phenomenology attributed to T s cells must, in fact, be valid, but were forced to conclude that many of the interpretations were not. We wish to challenge some of those interpretations and attempt to formulate a new framework for understanding T-cell suppression.

Functional and phenotypic subdivisions The minimal hypothesis, we believe, that is required to explain T-cell suppression in particular, and most phenomena in cellular immunology in general, would hold that: (1) essentially all T cells have specific recognition and nonspecific effector activity; (2) specificity resides only at the level of the TCR; (3) only two levels of specificity are possible in cellular immune responses recognition of antigen plus MHC and an idiotype-antiidiotypic network of TCRs; (4) most, if not all, effector functions of T cells are regulated by the patterns of lymphokines produced (we consider molecules released by cytotoxic T cells (CTL) required for target cell lysis, such as performs, as lymphokines); and (.5) functional T-cell subsets exist in mice and humans, and can be defined by the pattern of lymphokines produced. It took a great leap of faith to believe that surface antigens on T cells would be predictive of function, yet it is remarkable how well the surface markers on T cells have correlated with T-cell functions-. An important recent advance, derived from studies in the mouse, demonstrated that functional subsets of cells with otherwise indistinguishable surface phenotypes could be defined by the patterns of lymphokines produced ~,'. That has allowed the delineation of subsets of CD4 ~ T cells in the mouse: THI cells, involved in delayed-type hypersensitivity (DTH), secrete interleukin 2 (IL-2) and gammainterferon (IFN-~,), while TH2 cells, involved in activating B cells, produce IL-4. The generality of this thesis and its relevance to humans has been problematic, we believe, until recently. Most T-cell lines and chines from normal donors have characteristics of T,, cells, producing a mixture of virtually all possible lymphokines, and do not fit into the functional TH1 or TH2 subsets of mice. Yet, recent studies from several laboratories studying individuals whose immune system is actively engaged by antigen, for example by allergy or infection, suggest that functional subsets o~c CD4 + T cells do exist in man. The

1992, Elsevier Science Publishers [td, L:K.

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Fig. 1. Classical suppressor cell cascade, modified from Klein2. Ag: antigen; APC: antigen-presenting cell; ld: idiotype; FPC: factor-presenting cell; T~F: T-suppressor-cell factor; T~: suppressor T cell; T-acc: T acceptor factor; DTH: delayed-type hypersensitivity. work of Romagnani and coworkers '° is particularly relevant, since they showed that T-cell clones from normal and atopic individuals responsive to tuberculin purified protein derivative (PPD), or to parasite antigens, could be classified into functional subsets of CD4 + T-cell clones analogous to those in the mouse. Similar results have been seen in studies of strongly lepromin-positive contacts or patients, showing that CD4 + T-cell clones produce predominantly IFN-y, while CD4 + tetanustoxoid-specific clones produce IL-4 and 1L-5, but little IFN-y (Refs 11,12). These will be referred to as type 1 and type 2 CD4 + T cells, respectively, for convenience. This analysis has been extended to CD8 + T-cell clones which, we believe, can similarly be divided into functional subsets based on their patterns of lymphokine production II. The lymphokine patterns of a series of lepromin-specific CD8 + Ts-cell clones derived from lesions and blood of immunologically unresponsive lepromatous leprosy patients were compared with patterns from alloreactive HLA-B27-specific human CD8 + CTL clones. The CD8 + CTL produced 1FN-y and not IL-4. All the human CD8 + T-cell clones expressed ~p TCR receptors, all were genetically restricted through HLA-DQ l-~, and, in contrast to the CD8 + CTL, all have been found to produce essentially the type 2 pattern of lymphokines, in particular IL-4, with little or no IFN-y. Consequently, we have designated these as type 1 and type 2 CD8 + T cells, respectively. Using monoclonal anti-IL-4 antibodies, Salgame et al. '~ have shown that IL-4 produced by the CD8 + T-cell clones following receptor stimulation is a necessary condition, in vitro, for suppression of proliferation of type 1 CD4 + T-cell clones. Preliminary data have indicated that IL-4 may be necessary but is not a sufficient condition. Interestingly, we derived a single CD4 + T-cell clone from a lepromatous leprosy patient and, upon stimulation with antigen, like CD8 + T cells, it produced IL-4 and suppression. Thus, the pattern of lymphokines produced was more predictive of function than was surface phenotype. It is, therefore, appropriate to refer to these functional T-cell subsets as type I and type 2, rather than TH1, TH2 or T(:TL, or Ts, both to reveal our ignorance of their full functional capabilities in vivo, and to distinguish between helper cells for antibody formation and helper cells or effector cells for cell-mediated immunity.

Immunology Today

There are, clearly, mechanisms described other than suppression that can induce peripheral tolerance in vitro, particularly clonal anergy 14 and the veto phenomenon L~,16,as well as the activation of idiotype- or antigen specific CTL '7-'9, but it is more difficult to establish the role of these mechanisms in vivo. In the case of the type 2 CD8 + T cells in leprosy, it has been established that they are the predominant lymphocyte subset in lepromatous lesions; when isolated and cloned from lesions they suppress antigen-stimulated proliferation of type 1 CD4 + T-cell clones. Reverse transcription and polymerase chain reaction (PCR) amplification of mRNAs from lepromatous lesions reveal that IL-4 predominates 2°. Suppression induced by these leprosy-specific CD8 + T s cells can be distinguished from the other mechanisms for peripheral tolerance 21. These suppressor cells do not function as CTL nor do they serve as 'veto' cells, presenting antigen in the absence of co-stimulatory signal or directly inducing cell death, but they do negate appropriate stimulatory signals. When antigen-specific CD4 + T-cell clones are exposed to CD8 + Ts-cell clones and antigen, they become anergic and remain unable to respond to antigen plus APC over at least a 10 day period, even though they continue to proliferate normally to IL-2 (Ref. 21). Thus, while the mechanism of inducing anergy may be distinct, we believe that the outcome in vivo is likely to be the same. The suppressed CD4 + T cells become anergic, and, while expressing normal levels of TCR, they can no longer respond to antigen. A new model of suppression Based on the assumption that functional subsets of T cells exist in mice and humans, as outlined above, we believe that much of the information that led to the formulation of the T-suppressor cascade (Fig. 1) can be reinterpreted in a way that is both simpler and more consistent with the generally accepted paradigms of T-cell function and recognition, and ultimately testable in experimental and, perhaps, disease systems. The model can be envisaged as follows: (1) The Tsl or T-suppressor-inducer cell, generally characterized as an antigen-specific CD4 + T cell, is not a specialized suppressor cell, but a T H cell against which an anti-idiotypic response is directed. Indeed, Tsl cells in the mouse have been distinguished from T H cells, as far as

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Fig. 2. Model for T-cell suppression involvmg fimctional C1)4 ÷and (.l)S ~ T-cell subsets, di//i'n'Jltial .m/&,cn presentation and differing patterns o/lymphokine pmd.ctio~t. we can discern, only bv their secretion of TsFl (see below). Otherwise they produce normal lymphokines such as IL-2 and IL-4. (2) T~2 cells are anti-idiotypic CD4 +, or in some cases CD8 +, T cells, that simply suppress or kill T H cells. (3) Ts3 effector suppressor cells, previously described as antigen-specific CD8 + T cells, correspond to the type 2 CD8 + T s cells found ill leprosy, which exert their suppression by production of a particular pattern of lymphokmes. (4) TsF 1, the factor released by T s 1 cells, is shed TCR molecules, either o~ chains or, more rarely, [3 chains released intact from the cell. These polypeptides induce the anti-idiotypic 'Ts2" cells. This is consistent with the recent convergence of fndings by several laboratories that TsF1 contains TCR o~ chains -'2,23, and that some C1)4 + T-cell clones shed their TCR in culture -'4. The general point is that these suppressor factors do not m themselves have specific effector function, but serve merely as antigen from which idiotypic epitopes are derived. (5) 'fhe lg VH region restriction on TsFI can be explained by the fact that antigen presentation by B cells is several orders of magnitude more effective than that of other cells-". B cells with specificity for TCR idiotype would be m a position to bind and present the minute quantities of soluble TCR chains available in viw). In many systems, B and T cells express common idiotypic and anti-idiotypic determinants, providing a mechanism for B cells to bind and subsequently present TCRs. Note also that B ceils have a predilection for presenting antigen to type 2 or CD4 + TH2 cells >. (6) TsF2, which has not been well characterized but appears to require presentation by a cyclophosphamideresistant host cell, could be a shed anti-idiotypic receptor that may require chistering or multivalency by a host cell, perhaps a macrophage, in order to induce clonal anergy in the target T cell. (7) TsF3, generally agreed to be a factor lacking in specificity for antigen that acts to effect suppression, is merely one or more suppressive lymphokine(s) produced by antigen-stimulated type 2 cells, which suppresses proliferation of type 1 cells. There is abundant evidence that

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