Differentiation of cytolytic T lymphocytes

Immunology Today, vol. 3, .N'o. 7, 1982

183

) Differentiation of cytolytic T lymphocytes H. R o b s o n M a c D o n a l d Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland

Cytolytic T lymphocytes ( CTL) arise during the course of cell-mediated immune responses to allografis and to certain virus-ir~fected cells I. C T L are distinguished from other cytolytic cell types (such as natural killer cells and antibody-dependent killer cells) by their ability .specifically to lyse target cells bearing the appr@riate (allogeneic or viral) antigens in the absence of detectable antibody and~or complement. The molecular events associated with cytolysis are irnperfectIy ~me]erstood2. In this article Robson MacDonald discusses what is known qf the precursors of C T L and their development into the @ctor and memory c~lls of the cytotoxic re@onse. Before considering the differentiation of CTL, it is necessary to define the model systems in which C T L can be generated and quantitated. Historically, the first detailed studies of C T L generation involved model systems in vivo, in which mice were sensitized by immunization with allogeneic tumor cells 3 or in graftv-host reactions 4. Although such systems proved extremely useful in establishing a n u m b e r of important properties of CTL, analysis of the cellular (and molecular) events associated with the acquisition of cytolytic function could not be approached in such a complex experimental situation. The fact that C T L could be generated in vitro by culturing lymphoid cells h'om genetically disparate strains of mice (mixed leukocyte culture, M L C ) ~ thus provided an experimental framework in which the differentiation pathway of C T L could be rigorously examined. In this brief article, I will restrict the discussion, for the most part, to results obtained in the M L C system in the mouse. This is the best characterized system at the present time, but recent studies suggest that the conclusions will hold good in man.

The i m m e d i a t e p r e c u r s o r of the CTL (CTL-P) C T L are derived from precursor cells (CTL-P) present in lymphoid tissues such as the spleen and lymph node as well as in peripheral blood. Since CTLP have no detectable cytolytic activity, it is necessary to define them operationally, by measuring their ability to differentiate into cytolytically active (and hence experirnentally detectable) CTL. Nevertheless, with the advent of improved tissue culture conditions supplemented with soluble growth-promoting substances such as T-cell growth factor (TCGF)% it has become possible recently to quantitatively assess the n u m b e r of C T L - P specific for a given antigen in a heterogeneous population of lymphoid cells. From such assays, which are based upon the application of Poisson statistics to the analysis of multiple replicate microculturcs under limiting dilution conditions, it

has become apparent that the frequency of C T L - P directed against any given set of alloantigens, or virusassociated antigens, is typically of the order of 0.02-0.2% (Refs. 7, 8). This low frequency, in combination with the indirect nature of the C T L - P assay, has led to cellular and molecular events which accompany the acquisition of cytolytic function (vide

i,¢~a ). Despite these problems, several properties of C T L P in the mouse have been inferred from physical and immunological separation methods. Velocity sedimentation at unit gravity - a technique which separates cells according to size - has been used to • demonstrate that C T L - P in various lymphoid tissues have a size distribution characteristic of small lymphocytes 9 (modal sedimentation velocity = 3.5 m m / h ) . Similarly, equilibrium density gradient centrifugation has established that C T L - P have a high buoyant density uj. W h e n irradiated with X- or "y-irradiation, C T L - P are highly radiosensitive with a Do of about 200 fads '~,lL. Taken together, these physical studies indicate that C T L - P are typical small lymphocytes and hence indistinguishable from a multitude of other functionally defined cells. The application of recent advances in the study of cell surface allo-antigens has provided a more useful approach in characterizing CTL-P. It has been known tbr some time that C T L - P express the Thy-I surface antigen (formerly known as 0) characteristic of all thymus-dependent lymphocytes in the mouse 4. More recently, Cantor and Boyse ~e have demonstrated that T lymphocytcs can be further subdivided according to their expression of the surface alloantigens l,yt-1 and Lyt-2. According to their original scheme, three sub-. populations of T lymphocytes could be defined, i.e. Lyt-l+2-,Lyt-l+2 + and Lyt-1 2 +. However, recent studies using monoclonal antibodies and sensitive detection systems such as flow microfluorometry have failed to verify the existence of the Lyt-1 2 + subpopulation ~. It is now clearly established that C T L - P

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immunology T o @ , vol. ,3, 2¢b. 7, 1,982

184 belong to the Lyt-1 +2 + subset. This conclusion is supported not only by negative selection experiments, in which treatment of T-cell populations with anti-Lyt-1 or anti-Lyt-2 antibodies plus complement eliminated CTL~P activity s, but also by more recent positive selection experiments in which essentially all C T L - P could be recovered in the Lyt-2 + fraction of T cells separated on a fluorescence-activated cell sorter. A crucial unresolved question about C T L - P is the nature of their antigen receptors. Whereas m u c h speculation has centred upon the notion that T cells recognize antigens via receptors which are structurally related to surface immunoglobulin molecules on B cells, recent studies using cloned DNA probes have failed to detect functional rearrangements of either heavy or light chain DNA sequences in cloned lines of functional T ceils. Thus, at the present time the molecular structure of antigen receptors on T cells remains a complete mystery. (For a recent review of this complex problem, see Krammerl4.) A c q u i s i t i o n of f u n c t i o n a l activity b y CTL-P. As outlined schematically in Fig. 1, the interaction of C T L - P with determinants on the surface of appropriate metabolically active antigen-presenting cells initiates the series of events which culminates in the expressior~ of cytolytic function. T h e precise nature of this interaction is unknown, but available evidence would suggest that specific antigen receptors //

CTL PRECURSOR ~ O - ( i" / ANTIGEN PRESENTING Small lymphocyte \\ CELL Lyt-I+2+

I

" DIFFERENTIATION (Independent of DNA synthesis)

/ HELPER CTL (r ~J~I--TCGF-I/ T CELL Lymphoblast ~ ~ Lyt_l+2_ Lyt-l÷ 2+ / ~ x PROLIFERATION

DIFFERENTIATION (Independentof DNAsynthesis) MEMORY CELL I mall lymphocyte Lyt- 1+2 +

@

Fig. 1. Minimal model for the induction and differentiation of cytolytic T lymphocytes (CTL) in vitro. (Modificd ti'om Engers, H. D. and MacDonald H. R. (1976) Cor~temp. Top. Imm~nobioL 5, 145)

on C T L - P reeognize molecules which are similar (if not identical) to the serologically defined H-2K and H-2D molecules encoded in the major histocompatibility complex. Whereas all cells express these antigenic: determinants, some cells (particularly macrophages and dendritic cells) function more efficiently to trigger CTL-P. T h e reasons for this a p p a r e n t discrepancy are unclear, but are assumed to reflect unique metabolic or antigen-presenting properties of these specialized cells 15. Following triggering by antigen, cytolytie activity is first detectable after about 24 h (Ref. 16). Detailed analysis of the cellular and molecular events occurring during this time period has been extremely difficult because homogeneous populations of C T L - P specific for a given antigen are not available. Nevertheless, it is clear from recent studies that both blast transformation of C T L - P and the concomitant appearance of cytolytic activity can occur in the absence of detectable DNA synthesis ~6. These findings, which are true irrespective of whether C T L induction occurs specifically (via stimulation with allo-antigen) or 'nonspecifically' (via stimulation with lectins such as Con A), impose certain constraints on the molecular mechanism of C T L - P differentiation. Evidence from a variety of developmental systems supports the hypothesis that thc derepression of genetic: information required for cellular differentiation in eukaryotic cells occurs preferentially during a round of DNA synthesis 17. Interference with this DNA synthesis (e.g. by incorporating the thymidine analogue 5-bromo-2-deoxyuridine into DNA in place of thymidine) results in a marked inhibition of differentiated functional In the context of these findings, the failure of inhibitors of D N A synthesis to interfere with the acquisition of cytolytic function by C T L - P raises the fundamental question of whether or not any derepression of genetic information is taking place. The expression of effeetor function in these cells may instead be more a matter of metabolic activation (or maturation) t h a n of differentiatkm per se. The question of whether' soluble factors play a role in the induction of C T L function remains open. Recent studies from Lalande e! aL~9"suggest that the cellular events accompanying the differentiation of C T L - P into C T L can be dissociated into at least two steps. Using a suboptimal concentration of a fluorescent DNA-binding dye (ttoechst 33342) to stain a heterogeneous population of M L C (:ells stimulated by alloantigen, these authors were able to identify a subpopulation o f ' a c t i v a t e d ' cells (as defined by increased fluorescence intensity measured on a flow cytometer) as early as 12 h after" exposure to alloantigen. These activated cells, which were assumed to be non-cytolytic, could be induced to differentiate into C T L following exposure to a crude preparation of growth factor(s) containing T C G F . These findings, and other evidence that C T L - P cultured under limiting dilution conditions require both antigen and

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growth factor(s) to give rise to CTL, have led to the widely accepted hypothesis that the activation of C T L - P to functionally active C T L requires two 'signals', namely specific antigen and T-cell growth factor (TCGF) (Ref. 20) ttowever, it is possible that the actual differentiation step from C T L - P to C T L does not depend upon soluble factors and that the role of such factors is merely to increase the number of C T L to detectable levels. Since the production of soluble factors by T cells is independent of DNA synthesis, the inhibition experiments discussed above shed little light on this problem.

Differentiation pathway of CTL Once activated to functional activity, C T L undergo several rounds of proliferation in MLC. This requirement for proliferation is reflected in the radiosensitivity of C T L generation and the demonstration that inhibitors of DNA synthesis (such as hydroxyurea and cytosine arabinoside) block further increases in C T L activity occurring after 24 h (Ref. 21 ). Regulation of C T L proliferation is generally assumed to bc controlled by TCGF. Evidence in favor of this supposition comes mainly from recent studies of cloned C T L lines, which have been shown to depend absolutely upon T C G F for growth 22. It should be noted, however, that rigorous proof of an absolute requirement for this factor during the proliferative phase of CTL differentiation can be achieved only when purified sources of T C G F a n d / o r selective inhibitors of its action become available. Recent progress in T C G F purification and in the production of monoclonal antibodies against this molecule should aid in the resolution of this problem. Another unanswered question about this proliferative phase is whether or not the cytolytic activity (or 'efficiency') of individual C T L is a static property of these cells or, alternatively, can vary as a function of time. Since individual cells cannot be assayed quantitatively for lytic activity, this question must be approached by comparing the cytolytic activity of a culture (in terms of lyric units) at a given point in time with the number of C T L present at that time. The frequency of C T L is very low (<1 1%)s soon after activation in MI,C, so this parameter will depend critically on the accuracy of the measurement of C T L frequency. It is thus not surprising that early studies > (in which C T L frequency was estimated by conjugate formation) suggested a dramatic change in cytolytic efficiency as a function of time in MLC, whereas no such change was observed when C T L frequency was estimated by a limiting dilution approach t~'. At the peak of the primary in-vitro response (on day 5), CTt, have the physical properties of typical lymphoblasts. When separated by velocity sedimentation at unit gravity, C T L sediment more rapidly (6 m m / h ) than do CTL-P and exhibit a heterogeneous distribution which is consistent with a cycling population 9.21. Equilibrium density gradient centrif'u-

185 gation studies also show that CTL have a lighter buoyant density than do C T L - P (Ref. 10). C T L activity is highly radioresistant 9 (Do ~ 5000 fads). Although this property would appear to distinguish mature CTI, from highly radiosensitive CTL-P, it should bc noted that the measurement of radiosensitivity of CTL-P is dependent upon extensive proliferation, whereas C T L function is not. Clearly, measurements of the radiosensitivity of cloned CTL lines will be useful to establish whether there are intrinsic differences in the radiosensitivity of CTL-P and CTL. The surface phenotype of C T L is very similar {if not identical) to that of CTL-P. Like CTL-P, most CTI, cxprcss the Thy-1, Lyt-1 and Lyt-2 surface alloantigens. However, several groups have had considerable difticulty in eliminating C T L activity by treatment with anti-Lyt-i sera plus complement. At the moment, it is not clear whether this problem is related to low Lyt-I antigen density on C T L or to generalized problems in killing these activated cells in a complement-dependent assay. Many cloned C T L lines express low (or undetectable) amounts of Lyt-I antigen 24, but it is difficult to formally exclude inadvertent selection of Lyt-l-negative variants during the course of extended propagation in vz'lro. After the peak proliferative phase of CTL generation in MLC, there follows a period of several days in which cytolytic activity gradually disappears. Analysis of the physical properties of C T L during this time, by velocity sedimentation at unit gravity, indicates a shift in the size profile of C T L from large to small cells 21,aS. Direct evidence that this size shift represents a differentiation event (as opposed to the selective survival of smaller cells) was provided by experiments in which M L C cells were fi'actionated according to size on day 5 and recultured for various time periods 25. The quantitative analysis of C T L activity recovered indicated that only the large cell population was able to give rise to small C T L 3-6 days later. The molecular basis for the transition of large, highly active C T L to smaller inactive cells remains obscure. Experiments using metabolic inhibitors to block DNA synthesis and mitosis during this process have clearly demonstrated that cell division is not required 21. In the light of more recent findings, it is possible that the exhaustion of soluble growthpromoting factors (such as T C G F ) might influence the metabolic status and functional activity of C T L ; however, the failure to observe such functional effects on cloned TCGF-dependent C T L lines deprived of T C G F for periods of up to 30 h argue against such a possiblity. M e m o r y ~C T L responses As mentioned in the previous section, long-term MLC cultures lose their cytolytic activity with time. However, when restimulated by appropriate allogeneic cells, C T L quickly reappear 21,26. This

186 phenomenon, which has been referred to operationally as 'memory', resembles a primary generation of CTL in a number of ways. Thus, the 'precursors' of C T L in long-term M L C cultures are small lymphocytes with a Lyt-l+2 + surface phenotype. After antigenic stimulation, C T L activity appears within 24 h and does not require 1)NA synthesis 27,2s. Subsequently, proliferation of CTL occurs reaching a peak after 3-4 days. It is important to note that this secondary CTL generation in M L C does not difter in any qualitative way from the primary C T L response. However, quantitative differences in the kinetics and peak levels of C T L activity are consistently observed. Recent studies using limiting dilution methods to assess the frequency of C T L - P in long-term MLC populations provide direct evidence that quantitative difterences between primary and secondary C T L responses are due (at least in part) to differences in CTL-P frequency 2'). Thus, whereas 1 in 500 normal C57BL/6 spleen ceils (or 1 in 200 T ceils) is a CTL-P directed against D B A / 2 alloantigens, this figure increases to about I in 10 as a result of priming in MLC. Various lines of evidence indicate that the 'memory' cell identified in long-term M L C is a lineal descendent of the C T L 2~,e6. Early studies using two cycles of velocity sedimentation cell separation and recuhure demonstrated that the fraction of large ceils (containing CTL) isolated from day 5 M L C gave rise ultimately to the small 'memory' cells capable of being restimulated on day 14 (Ref. 25). Furthermore, with the advent of elonal assays for CTL-P, it was shown that memory cells occurred only in limiting dilution microcultures where C T L had appeared during the primary response 3°. Thus, there can be little doubt that expansion of CTL-P can occur as a direct result of antigenic stimulation. It has been known for some time that the repeated stimulation of M L C cells gives rise to waves of C T L activity. /n an early study 3~, four sequential cycles of C T L generation were demonstrated at 2-week intervals with intervening periods in which cytolytic activity dramatically declined, fIowever, after a sufficient number of cycles of restimulation, C T L activity was invariably lost. Although these results could indicate that C T L have a finite lifespan, we now know fi'om studies of C T L clones that this is not the case. It rather appears that C T L are eventually overgrown by other non-cytolytic (Lyt-l+2-) T cells when heterogeneous cultures are multiply restimulated. Hence for all practical purposes, the cycle of differentiation from C T L - P to C T L and back to 'memory' CTL-P can apparently be repeated indefinitely.

Memory responses after in vivo priming Befbre leaving the subject of memory CTL, a brief comment on the effect of priming in vivo should be made. It has been known for some time that the magnitude of C T L responses in vitro can be substantially increased by prior exposure of lymphoid cells to the

hnmu,s/sgy 7?)day, vo/. 3, ,/Vo.7, L982

relevant antigens in vivo32; in fact, in the case of CTI. responses to many minor histocompatibility antigens and virus-associated antigens, such priming in vivo is an obligatory prerequisite for' the generation of a detectable CTL response in vitro. Although recent limiting dilution studies indicate that at least part of the effect of priming in vivo can be attributed to an increase in the frequency of CTL-P specific for the relevant antigen s,29, certain observations suggest that some qualitative differences may exist between CTL-P in normal and primed animals. Thus, a variety of 'suboptimal' antigenic preparations, including crude m e m b r a n e fragments and heat-killed or u.v.irradiated stimulating ceils, are capable of inducing CTL generation in lymphoid cell populations which have been primed in vivo but not in unprimcd controls 2t,33. It is particularly interesting that the memory CTL-P obtained after priming in vitro (in M L C ) do not respond significantly to stimulation by any of the suboptimal antigenic preparations which trigger primed cells in vivo. Although several explanations can be invoked to explain these discrepant findings (including less stringent antigenic requirements for the liberation of soluble factors by primed helper T cells in vivo), it is nevertheless possible that limitations in the accessibility a n d / o r presentation of antigen in vivo result in a selection for a subpopulation of C T L - P expressing high avidity antigen receptors. Unfbrtunately, the lack of availability of assay systems for measuring the relative avidity of C T L - P receptors precludes any direct testing of this hypothesis.

Conclusions and future directions A simplified model for the cellular events accompanying the differentiation of CTL-P into C T L and memory cells is summarized in Fig. 1. From this schema, it is apparent that our present knowledge of this pathway is very limited. As has been frequently mentioned in this article, a major problem in the analysis of early events in C T L differentiation has been the absence of homogeneous (i.e. antigenspecific) populations of undifferentiated CTL-P. One potential approach to solving this problem would be to purify CTL-P by absorbing and eluting them on monolayers of cells bearing the appropriate alloantigens. Such a method has recently been described 3'1, but these results are still controversial 35. In theory, another possible source of homogeneous material for the study of CTL difterentiation would be C T L clones. Recently, it has become possible to clone C T L expressing a variety of antigenic specificities and maintain these cloned lines in the presence of T C G F (Ref. 22). Unfortunately, for the purposes of this article, such cloned C T L do not modulate their cytolytic function either in response to T C G F or as a function of their growth phase 36. Thus, it will be necessary to look for other (perhaps less differentiated) cloned lines in order to study CTL differentiation at the cellular and molecular level.

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References 1 Cerottini, J.-C. and Brunner, K. T. (1974) A&. lrnmunol. 18, 67 2 Henney, C. S. (1980) Immunol. Today 1, 36 3 Brunner, K. T., MaueI,J. and Schindler, R. (1966) Iramur~ology 11,499 4 Cerottini, J.-C., Nordin, A. A. and Brunner, K. T. (1970) Salute (London) 228, 1308 5 H~-iyry,P. and Defendi, V. (1970) &ience 168, 133 6 Smith, K. A. (1980) lmmunol. Rev. 51,337 7 Miller, R. G., Teh, H.-S., Harley, E. and Phillips, R. A. (1977) Immunot. Rev. 35, 38 8 MacDonald, H. R., Cerottini, J.-C., Ryser, J.-E., Maryanski, J. L., Taswcll, C., Widmer, M. B. and Brunner, K. T. (1980) Immunol. Rev. 51, 93 9 MacDonald, H. R., Phillips, R. A. and Miller, R. G. (1973)J. lmmunol. 111, 575 10 Shortman, K., Brunner, K. T. and Cerottini, J.-C. (1972) J. Exp. Med. 135, 1375 11 Teh, H.-S., Harley, E., Phillips, R. A. and Miller, R. G. (1977) J. ImmunoI. 118, 1049 12 Cantor, H and Boyse, E. A. (1977) (.'old Spring Harbor Syrup. Quanl. Biol. 41,23 13 Ledbetter, J. A., Rouse, R. V., Micklem, H. S. and Herzenberg, L. A. (1980).7" Exp. Med. 152, 280 14 Krammer, P. H. (1981) G~rr. Top. Microbial. lmm~mol. 91, I79 15 Lafferty, K. J., Andrus, L. and Prowse, S. J. (1980) Imm,nol. Rev. 51,279 16 MacDonald, H. R. and Lees, R. K. (1980)J. ImmunoL 124, 1308 17 Holtzer, H., Weintraub, H., Mayne, R. and Mochan, B. (1972) C, rr. Top. Dev. Biol. 7,229 18 Rutter, W.J., Pictet, R. L. and Morris, P. W. (1973) Annu. Rev.

Bio&em. 42, 601 19 Lalande, M. E., McCutcheon, M.J. and Miller, R. G. (1980) J. Exp. Med. 151, 12 20 M61Ier, G. (ed.) (¼980) Imm*~nol. Rev. 51 (357 pp) 21 Engers, H. D. and MacDonald, H. R. (1976) (,'onlemp. Top. Immunobml. 5, 145 22 M/Jller, G. (ed.) (1981) Immunol. Rev. 54 23 Touton, M. H. and Clark, W. R. (1978)ff. lmmunol. 120, 1537 24 Glasebrook, A. L. and Fitch, F. W. (1980)ft. Exp. Med. 151, 876 25 MacDonald, H. R., Cerottini, J.-C. and Brunner, K T. (1974) J. Exp. Med. 140, 1511 26 H/iyry, P. (1976) Immunogenelicr 3, 417 27 MacDonald, H. R., Sordat, B., Cerottini, J.-C. and Brunner, K. T. (1975)J. Exp. Med. 142,622 28 Nedrud, J., Touton, M. and Clark, W. R. (1975)J. Exp. Med. 142, 960 29 Ryser, J.-E. and MacDonald, H. R. (1979) J. Immunol. 123, 128 30 Teh, H-S., Phillips, R. A. and Miller, R. G. (1977) J. Exp. Med. 146, 1280 31 MacDonald, H. R., Engers, H. D., Cerottini, J.-C. and Brunner, K. T. (1974) J. Exp. Med. 140, 718 32 Cerottini, J.-C., Engers, H. D., MacDonald, H. R. and Brunner, K. T. (1974)J. Exp. Med. 140, 703 33 Engers, H. D., Thomas, K., Cerottini, J.-C. and Brunner, K. T. (1975). 7. lmmunol. 115,356 34 Schnagl, H.-Y. and Boyle, W. (1979)Nalare (London) 279, 331 35 Kelso, A. (1981)J. lmmunol. 127, 1563 36 Sekaly, R. P., MacDonald, H. R., Zaech, P., Glasebrook, A. L. and Cerottini, J.-C. (1981)J. Evp. Med. 154, 575

The genetic basis of autoimmunity in murine lupus Toshikazu Shirai Department of Pathology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo / 13,.Japan Altho,gh all lhe trails o/-a~loimmunily are quantilalively expressed and as s,ch may nol be s,b]ecl to starzdard genelic analysis, evidence s,ggesls lhal the differenl az~toimmune trails of N e w Zealand Black ( N Z B ) miee and their @bri&" are under s@arate genetic conlrol. In Ibis arlicle Toshikazu Shirai reviews tkis evidence and the indications thai some genes are common Io d{f.ferenl lrail.~ zohile others show linkages and interactions. A .fi~rther complication is that lrails are ir¢t, enced to a greal degree by allelie or non-allelic genes which either enkance or Slit)press.

Since Bielschowsky el al. ~ first r e p o r t e d t h e developm e n t of a u t o i m m u n e d i s e a s e in the i n b r e d N e w Z e a l a n d Black ( N Z B ) m o u s e strain, the genetic basis for a u t o i m m u n i t y in N Z B mice h a s b e e n i n v e s t i g a t e d in m a n y laboratories. T h e a u t o i m m u n e d i s e a s e of N Z B mice is c h a r a c t e r i z e d by t h e s p o n t a n e o u s develo p m e n t of a u t o i m m u n e h e m o l y t i c a n e m i a , h y p e r g a m m a g l o b u l i n e m i a , h y p o c o m p l e m e n t e m i a , occasional I y m p h o p r o l i f e r a t i v e disorders, a n d i m m u n e complex-type glomerulonephritis resembling human l u p u s n e p h r i t i s a n d a s s o c i a t e d w i t h a positive L.E. cell test 2. T h e s e mice s p o n t a n e o u s l y p r o d u c e a variety of a u t o a n t i b o d i e s , with specificity for ( a m o n g o t h e r s ) e r y t h r o c y t e s , T cells a n d several nucleic acid antigens. T h e y also s h o w s p o n t a n e o u s polyclonal activation of B cells 3. All t h e s e a b n o r m a l i t i e s are genetically determined.

Initial interest in the genetic basis for this a u t o i m m u n e disease w a s e n c o u r a g e d by t h e discovery, by H e l y e r a n d H o w i e (see Ref. 2), t h a t in t h e F~ p r o g e n y of t h e N Z B a n d t h e n o n - a u t o i m m u n e N e w Z e a l a n d W h i t e ( N Z W ) strain, renal d i s e a s e was m o r e severe, h a d a n earlier onset, h i g h e r i n c i d e n c e a n d was a s s o c i a t e d with h i g h e r titers of a n t i n u c l e a r antibodies, t h a n w a s t h e disease in t h e p a r e n t a l N Z B strain. T h e s e findings are best e x p l a i n e d by t h e i n v o l v e m e n t of one or m o r e N Z W g e n e ( s ) w h i c h act to m o d i f y t h e e x p r e s s i o n of m a j o r a u t o i m m u n e N Z B gene(s). T h u s , t h e d a t a i m p l y t h a t m o r e t h a n two genetic loci c o n t r i b u t e to the full m a n i f c s t a t i o n of t h e severe renal d i s e a s e c h a r a c t e r i s t i c of t h e F~ h y b r i d . T w o newly developed m o u s e s t r a i n s , M R L / 1 a n d B X S B exhibit c e r t a i n a u t o i m m u n e traits of the N Z B a n d N Z B x N Z W ( B / W ) F I h y b r i d mice, while

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