viewpoint

The developmental relationship between NK cells and T cells Lewis L. Lanier, Hergen Spits and Joseph H. Phillips NK cells and T cells share many features, suggesting that they have a common origin. Here, Lewis Lanier and colleagues describe recent studies of fetal ontogeny and thymic development that provide support for this hypothesis, and propose an integrated scheme for NK-cell and T-cell development. Of the many types of hematopoietic cells, lymphocytes alone are responsible for antigen-specific immunity. As yet, however, a progenitor cell that can give rise exclusively to the three types of lymphocytes (T cells, B cells and natural killer (NK) cells) has not been identified. Commitment to the B and T lineages is marked by irreversible alterations in genotype, as a consequence of rearrangement of the immunoglobulin (Ig) and T-cell antigen receptor (TCR) genes, respectively. Individual B cells and T cells possess clonally distinct Igs or TCRs that are required for specific antigen recognition. The third type of lymphocyte, the NK cell, does not rearrange Ig or TCR genes 1 and apparently develops normally in immunodeficient scid mice that have a defect in the process required for rearrangement of TCR and Ig genes 2. This is underlined by the presence of apparently normal NK cells in RAG-2-deficient mice that fail to develop mature T and B cells "~. While TCR gene rearrangement clearly discriminates between these cell types, T cells and NK cells are remarkably similar with respect to expression of other membrane receptors and immune effector cell functions (Table 1). These similarities have given rise to the notion that NK cells and T cells are probably related, although there has been no direct evidence to support this hypothesis. A single scid patient has been described that lacks both T cells and NK cells, but possesses normal myeloid cells and B cells (R. Bacchetta, pers. commum). This finding is consistent with the existence of a common T-cell-NK-cell progenitor that is distinct from totipotent hematopoietic or lymphoid progenitors. In another case, normal T and B cells were present in an immunodeficient patient that had no NK cells4, arguing that at least the later stages of NK-cell differentiation are distinct from the T lineage. Recently, new clues to help resolve the relationship between T-cell and NKcell development have emerged from studies of lymphocyte differentiation during fetal ontogeny and from analysis of T-cell maturation in the thymus.

The CD3-TCR complex The TCR is composed of antigen-binding ix[3 or y8 heterodimers that are noncovalently associated with at least four other invariant proteins, designated CD37, 8,

~; and { (Ref. 5). Analysis of CD3 expression and TCR rearrangement provided the first unambiguous evidence that NK cells and T cells were distinct. While all mature T cells express CD3-TCR complexes on the cell surface, antibodies against the invariant CD3 subunits failed to react with the cell surface of NK cells. Moreover, NK cells do not rearrange TCR [3, ~' and 8 genes and do not transcribe TCR 0t genes 6-9 (although germline transcripts of TCR [~ and TCR iS are present in activated NK cells8'~°). Initially, mature mouse and human NK cells were reported to lack expression of CD3E, 8 and )' proteins or transcriptsl~,12; it was, therefore, assumed that lymphocytes in fetal tissues that did not express membrane (m)CD3 but did express CD7 and cytoplasmic CD3 proteins (mCD3-CD7 ÷) were pre-T cells ~3. However, it is now clear that mature, functional NK cells, identified as mCD3-CD7+CD45*CD56 + lymphocytes, are abundantly present in human fetal liver and spleen as early as six weeks gestation ~4. Surprisingly, the majority of these fetal NK cells express CD38 and 8 proteins in the cytoplasm but not on the cell surface. Fetal NK-cell clones stably express cytoplasmic CDM'y and CD3~8 complexes, and the ~ chain, but none rearranged TCR 7, 8 or [3 genes 14'~. Fetal NK cells can mediate major histocompatibility complex (MHC)-unrestricted cytotoxicity against NK-sensitive tumors, function in antibody-dependent cellular cytotoxicity (ADCC) assays, respond to interleukin 2 (IL-2), and produce regulatory cytokines, including gamma-interferon (IFNq'), granuIocyte-macrophage colony stimulating factor (GMCSF), and tumor necrosis factor ¢~ (TNF-~) after stimulation in vitro 14. A small number of fetal-type NK cells (expressing cytoplasmic CD38, 8 and y proteins) are present in cord blood, but none are detectable in peripheral blood after birth 14. CD38 or CD37 is not detectable in either freshly isolated or in vitro cultured adult peripheral blood NK cells. However, in vitro activation of adult NK cells results in transcription of CD38 (Ref. 12) and expression of cytoplasmic CD3e proteins ~s. These observations demonstrate that immunocompetent NK cells develop before T cells during fetal ontogeny. These early NK cells may regulate hematopoiesis by secreting

© 1992, Elsevier Science Publishers Itd, UK.

Immu otogy r,,day

392

Vol

No

992

viewpoint essential cytokines and/or may provide protective immunity prior to the development of T cells, Anderson et al. ~ were first to report ~ chain ex . pression in mature human NK cells. Subsequent studies revealed that the ~ chain is associated with C D I 6 , an IgG Fc receptor involved in ADCC, in NK cellsC Most, if not all, NK cells express cytoplasmic { chain, irrespective of whether or not CD16 is expressed.

The thymus The thymus is the site of differentiation and education of most T cells. NK cells develop normally in athymic nude mice, demonstrating the thymus-independent maturation of this lineage ~s. NK cells are, however, present at a very low frequency ( and the mouse thymus 2°. The relative proportion of NK ceils is increased by isolation of 'triple negative' (CD3-CD4-CD8-) thymocytes, but still comprises only a minor fraction of this population (-0.1-5%). Thymic NK cells express the prototypic phenotype: mCD3e-NKI.1 + in appropriate mouse strains, and mCD3e-CD56 + in humans. (Note that the phenotype of human thymic NK cells is controversial. Moretta and colleagues ~ have reported that these cells are mCD3 CDI 6+CD56-; however, our studies indicate that most are mCD3 CD56 +, with heterogeneous expression of CD16. We agree that essentially all NKcell clones derived from thymus are mCD3-CD56 + and CD16- or CDI6".} It is unclear whether these thymic NK cells are resident ceils or are mature, recirculating lymph{}cytes. Recently, Reinherz and colleagues 2~ reported that the majority of thymocytes isolated from fetal mice of 14.5 days gestation express CD16. When CD16 + fetal thymocytes were transplanted by intravenous injection into recipient mice and the splenocytes of recipients grown m vitro with 1L-2, NKI.1 + NK cells of donor origin were detected in these cultures. Adoptive transplantation of the CDI6" fetal thymocytes by intrathymic injection resulted in the generation of CD4 + and CD8 + thymocytes. This means that either NK cells and T cells arise from a c o m m o n CD16 + progenitor present in day 14.5 fetal thymus or that, within the C D I 6 + fetal thymic population, two different cell types exist, a transplantable T-cell progenitor and a low frequency of mature NK cells that expand when in culture with IL-2. Therefore, these studies are consistent with, but do not prove, the existence of a c o m m o n T-cell-NK-cell progenitor. Earlier studies by Kumar and colleagues 2° showed that the thymus of posmatal scid mice contains two cell populations: early T lineage cells that resemble 1 4 - 1 5 day fetal thymocytes, and NK cells. However, scid thymocytes failed to reconstitute the NK lineage upon adoptive transfer into irradiated mice, whereas the bone marrow contained the transplantable NK progenitor. Mature, adult mouse NK cells are also unable to reconstitute the T lineage after intrathymic injection, indicating that committed NK cells lack T-ceN progenitor activity (B. Mathieson, pers. commun.). In studies of human fetal and postnatal thymus, we have shown that, within the CD3-CD4-CD8- popu-

;,,,,,,,o;ogy

Table 1. Expression of leukocyte differentiation antigens by NK cells and T cells . . . . . . . . . Antigen Fetal NK cells Adult NK cells Thymocytes T cells CDI CD2 cyCl)3y, 8, e mCD3 CD3~/FceRI-y TCR CD4 CD8~ CD813 CD5 CD7 CD1 Ia/CD18 CD1 Ib/CD18 CDI6 (FcyRIII) CD29 CD38 (]I)44 CD45RA CD45RO CD56 CD57 1L-2RI~ 1L-2R0~ LECAM- 1

+ + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + +' + + + + + + + + + +

Expression of selected differentiation antigens on freshly isolated T cells and NK cells from normal individuals. A + indicates that the antigen is expressed on at least a subset of NK cells or T cells within these populations. ~CD16 is expressed on a minor subset of T cells in certain individuals~. cvCD3: Cytoplasmic CD3; mCD3: membrane CD3; IL-2R: interleukin 2 receptor; LECAM-I: leucocvte endothelial cell adhesion molecule I. lation, more than >95% of these cells co-express CD2, CD5 and CD34, but not CD16 or CD56 (Ref. 22). These CI)2+CD3-CD4-CD8-CD5+CD7"CD 16 CD 19 CD33-- CD34*CD56 - thymocytes essentially all express CD3y, 8 and e chains in the cytoplasm, but no detectable TCR 13 or TCR 8 proteins (authors' unpublished observation). (CD19 and CD33 assessment is used to exclude B- and myeloid-lineage cells, respectively, which are also present at low frequencies in the thymus.) The CD2*CD3 CD4-CD8-CD5*CD7+CDI9 -CD33-CD34*CD56 - thymocyte population is the most likely candidate for a committed pre-T cell, a notion that is supported by the ability of these cells to acquire CD4 and CD8ix expression on culture with IL-7 in vitro (Ref. 22 and authors' unpublished observation). The small number of NK cells present within the CD3-CD4 CD8- thymocyte population are C D 5 CD7÷CD19-CD33-CD34-CD56 + and heterogeneous for CI)2 and C D I 6 expression. The cloning frequency of mCD3-CD4-(]D8 CD5-CD56 + fetal and posmatal thymocytes is similar to adult NK cells, and the chines that have been produced stably express the antigenic and functional characteristics of typical NK cells (Ref. 23 and authors' unpublished observation). Most NKcell chines derived from both fetal and posmatal

393

v,,/. ;

N,,. 1o

viewpoint

,iver,a,j,t one arro, f"Hematopoietic stem cell (CD34+)

"%

~

" ~ Lymphoidprogenitor ~

(CD34+)

Thymus B-cell progenitor/" v ,,~ T-celI-NK-cell progenitor /'~ ~ (cyCD3+ mCD3-CD4-CD8~ CD5-CD34+CD56 -)

Germline TCR

(~ ~ Committed T-cell Committed NK-cell progenitor ~'~" progenitor

I~ Committed

CD34-CD56+)

@

RearrangedTCR Pre-T

Common

T-cell (cyCD3- mCD3thymocyte progenitor CD4±CD8 ~ (cyCD3+ mCD3÷ (cyCD3+ mCD3- CD5+CD34+CD56-) CD4+CD8+CD5÷ CD4-CD8-CD5 + CD34-CD56-)

~K-ce~(mCD3+CD4+CD8-)

Adult NK@c~ell

--@

0°4+ T-cell ~

NK-cell (mCD3-CD56 +)

(mCD3+CD4+CD8)

(mCD3+CD4-CD8+

T-cell~k~__.,/-(mCD3+CD4-CD8+)

Fig. 1. Hypothetical scheme of NK-cell and T-cell differentiation. thymus express only cytoplasmic CD3e; however, a minority express CD3% 5 and ~ proteins 23, like most fetal liver and some cord blood NK cells 14. CD34 is a marker of immature progenitor cells of several cell lineages, including myeloid, erythroid, and B cell 24-26. Expression of CD34 on the CD2+CD3-CD4-CD8-CD5+CD7+CD16-CD19-CD33-CD56 - thymocytes supports the possibility that these cells are Tcell progenitors. Moreover, human CD34 ÷ progenitor cells from fetal liver or bone marrow can develop into T cells when transplanted into scid-hu mice z;. By contrast, we have failed to detect CD34" on CD56 ÷ thymocytes, which is consistent with the notion that NK cells within the thymus are more mature, lineage-committed cells. If a common progenitor for T and NK cells exists in fetal liver or thymus, the likely phenotype would be CD2-CD3-CD4-CD8-CD5-CD7+CD19-CD33-CD34 +CD56-. The frequency of these putative progenitors in both fetal and postnatal human thymus is less than 0.01%.

Speculation If NK and T cells do share a common pathway of differentiation, there are many unanswered questions regarding how the pathways diverge during development. A hypothetical model of the relationship

Immunology Today

between NK-cell and T-cell development is presented in Fig. 1. Beginning with the putative T-cell-NK-cell progenitors that are probably present in fetal liver and adult bone marrow, we postulate that these cells may be identified by the phenotype CD5-CD7-+CD19 -CD33-CD34+CD56-, and possibly express CD37, 5 and e proteins and the ~ chain in the cytoplasm. Cells destined for the T lineage migrate to the thymus, initiate TCR rearrangement, and acquire expression of CD1, CD2 and CD5. This is based on the observation that the vast majority of CD3-CD4-CD8- thymocytes express CD34, and essentially all CD34 ÷ cells bear CD2 and CD5. At this point, CD2*CD3-CD4-CD8-CD5÷CD34 * thymocytes are irreversibly committed to the T lineage and proceed along the well-defined pathway of CD4 and CD8 expression, rearrangement of the TCR genes, and the TCR receptor repertoire selection process. 'T-cell-NK-cell progenitor' cells in the fetal liver or bone marrow that either fail to receive signals for thymic homing and TCR rearrangement, or receive other signals, differentiate along the NK-cell pathway, as reflected by the expression of CD56, loss of CD34, and acquisition of cytolytic activity. While these committed NK-cell progenitors may traffic to the thymus, this is not obligatory since NK cells develop normally

394

Vol. 13 No. 101992

viewpoint in athymic individuals. The predominant NK-cell population in fetal liver and spleen constitutively expresses cytoplasmic CD3"f, 8 and e proteins ~4. By contrast, all postnatal peripheral blood NK cells express only cytoplasmic CD3e after activation. If fetal and adult NK cells arise from a linear developmental pathway, CD38 and 7 expression are lost during maturation. Alternatively, fetal and adult NK cells may be distinct subpopulations, similar to the 'waves' of TCR '/8* T cells displaying particular V gene products that predominate at different times during ontogeny2s,>. At present, it is not possible to distinguish between these alternatives, since NK-cell clones derived from fetal liver maintain expression of cytoplasmic CD33', 8 and proteins stably and do not, by these criteria, 'differentiate' in vitro. A proportion of both fetal and adult NK cells express CD16 (Ref. 14). While we have previously suggested that CD16- NK cells may represent immature NK cells in adult blood ~°,31, studies of ontogeny have not shown that CD16 NK cells develop before the appearance of CDI6 ÷ NK cells '4. Alternative possibilities are that CD16 correlates with the existence of functionally distinct NK-cell subsets, analogous to CD4 and CD8 expression in T cells, or that CD16 is an inducible receptor which is influenced by environmental factors or relative activation state. Previous studies have clearly demonstrated that CD16 is induced on myeloid cells as a consequence of exposure to transforming growth factor 131 (TGF-~I) -~z'3~, but TGF-131 does not modulate CD16 expression on T cells or NK cells ~z. In summary, recent studies support the concept that NK cells and T cells share common developmental pathways. It is important, however, to emphasize that with exception of TCR gene rearrangement, there is no single, unequivocal criterion to distinguish between NK cells and T cells. This is particularly true of cytolytic function since lysis of 'NK-sensitive' targets can be mediated not only by NK cells, but also by IL-2-actirated immature thymocytes and mature T cells expressing either TCR 0t[3 or ~'8 (Refs 34, 35). Definitive proof of a common T-cell-NK-cell progenitor will require positive identification by a variety of criteria, and confirmation by the demonstration of bipotential differentiation of this cell type at the clonal level.

3 Shinkai, Y., Rathbun, G., Lain, K-P. et al. (t992) Cell 68, 855-867 4 Biron, C.A., Byron, K.S. and Sullivan, J.L. (1989) New Engl. ]. Med. 320, 1731-1735 5 Clevers, H., Alarcon, B., Wileman, T. et al. (1988) Annu. Rez,. lmmunol. 6,629-662 6 Lanier. L.L., (iwirla, S., Federspiel, N. et al. (1986)I. Exp. Med. 163,209-214 7 Lanier, L.L., (;wirla, 5. and Phillips, .I.H. (1986) J. 1mmunol. 137, 3375-3377 8 Loh, E.Y., Cwirla, S., Serafini, A.T. et al. (1')88) Proc. Natl Acad. Sci. USA 85, 9714-9718 9 Tutt, M.M., 5chuler, W., Kuziel, W.A. et al. (I 987} J. Immunol. ! 38, 2338-2344 10 Ritz, J., Campen, T.J., Schmidt, R.E. et ,71. 11985) Science 228, 1540-1543 11 Biron, C.A., van den Elsen, P., Tutt, M.M. et al. (1987) J. Immunol. 139. 1704-1710 12 Biassoni, R., Ferrini, 5., Prigione, 1. et ,li. (1988) J. lmmunol. 140, 1685-1689 13 Haynes, B.F., Martin, M.E., Kay,,H.It. et al. (1988i J. Exp. Med. 168, 1061-1080 14 Phillips, J.H., Hori, T., Nagler, A. et al. (1992) 1. Exp. Med. 175, 1055-1066 15 Lanier, L.I.., Chang, C., Spits, H. et al..l. Immunol. (in press) 16 Anderson, P., Caligiuri, M., Ritz, J. et a/. (1989) Nature 341, 1.59-162 17 Lanier, L.L., Yu, G. and Phillips, J.H. (1989) Nature 342, 803-805 18 Herberman, R.B., Nunn, M.E., Holden, H.T. et al. (1975) Int. J. Cancer 16, 230-239 19 Mmgari, M.C., Poggi, A., Biassoni, R. et al. (1991) J. Exp. Med. 174, 21-26 20 Garni-Wagner, B.A., Witte, P.L., Tutt, M.M. et a/. (199(t) J. lmmunol. 144, 796-803 21 Rodewald, H-R., Moingeon, P., kucich, J.L. eta/. (1992) Cell 69, 139-150 22 Hori, T., Cupp, J., Wrighton, N. et al. ( 199 I1 J. lmmunol. 146, 4078-4084 23 Hori, T. and Spits, H. (1991)]. lmmunol. 146, 2116-2121 24 Ryan, D., Kossover, 5, Mitchell, S. et al. (1986) Blood 68,417 25 Andrews, R.G., Singer, J.W. and Bernstein, I.D. t1989) /. Exp. Med. 169, 1721 26 Andrews, R.G., Singer, J.W. and Bernstein, I.D. (199(/) J. Exp. Med. 172, 355-358 27 Peault, B., Weissman, I.L., Baum, C. et al. (1991)J. Exp. Med. 174, 1283-1286 28 Havran, W.L. and Allison, J.P. (1988) Nature 335, 443-445 We thank Dr Vinay Kumar for critical review of the manu- 29 Krangel, M.5., Yssel, H., Brocklehurst, C. et al. (I 990) script and Dan Finn for expert assistance with the illus.l. Exp. Med. 172, 847-859 trations. DNAX Research Institute is supported by the 30 Lanier, L.L., Le, A,M., Civin, C.I. etal. (19861 Schering-Plough Corporation. J. Immunol. 136, 4480-4486 31 Nagler, A., Lanier, L.L., Cwirla, 5. eta/. (1989) Lewis Lanier, Hergen Spits and Joseph Phillips are at J. lmmunol. 143, 3183-3191 32 Phillips, J.H., Chang, C. and Lanier, I,.L. (1991) Eur. ]. D N A X Research Institute for Cellular and Molecular lmmunol. 21,895-899 Biology, 901 California Avenue, Palo Alto, CA 94304, 33 Welch, G.R., Wong, H.L. and Wahl, 5.M. (1990) USA. J, ImnmnoI. 144, 3444-3448 34 Phillips, J.H. and Lanier, L.L. (1987)J. Immunol. 139, 683-687 References 35 Phillips, J.H., Weiss, A., Gemlo, B.T. et al. (1987) ]. Exp. 1 Lanier, L.L., Phillips, J.H., Hackett, J. Jr et al. (1986) Med. 166, 1579-1584 J. lmmunol. 137, 2735-2739 36 Lanier, L.L., Kipps, T.J. and Phillips, J.H. (1985).l. Exp. 2 Hackett, J. Jr, Bosma, G.C., Bosma, M.J. et al. (1986) Med. 162, 2089-2106 Proc. Natl Acad. Sci. USA 83, 3427-3431

Immunology Today

395

V,,t

N,, 1,) 1")92